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Graphical abstract, Objectives To investigate the association between risk of VTE with 30-day mortality in COVID-19 patients. Methods 1030 COVID-19 patients were retrospective collected, with baseline data on demographics, Sequential Organ Failure Assessment (SOFA) score, and VTE risk assessment models (RAMs) including Padua Prediction Score (PPS), International Medical Prevention Registry (IMPROVE) and Caprini RAM. Results Thirty-day mortality increased progressively from 2% in patients at low risk of VTE to 63% in those at high risk defined by PPS. Similar findings were also observed by IMPROVE and Caprini score. Progressive increases in VTE risk also were associated with higher SOFA score. The presence of high risk of VTE was independently associated with mortality regardless of adjusted gender, smoking status and some comorbidities with hazard ratios of 29.19, 37.37, 20.60 for PPS, IMPROVE and Caprini RAM, respectively (P

AIMS: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), infection has been deemed as a global pandemic by the World Health Organisation. While diffuse alveolar damage (DAD) is recognised to be the primary manifestation of COVID-19 pneumonia, there has been little emphasis on the progression to the fibrosing phase of DAD. This topic is of great interest, due to growing concerns regarding the potential long-term complications in prolonged survivors. METHODS AND RESULTS: Here we report a detailed histopathological study of 30 autopsy cases with COVID-19 virus infection, based on minimally invasive autopsies performed between February and March, 2020. The mean age was 69 years, with 20 (67%) males and 10 (33%) females and frequent (70.0%) underlying comorbidities. The duration of illness ranged from 16 to 82 (median = 42) days. Histologically, the most common manifestation was diffuse alveolar damage (DAD) in 28 (93.3%) cases which showed predominantly acute (32%), organising (25%) and/or fibrosing (43%) patterns. Patients with fibrosing DAD were one decade younger (P = 0.034) and they had a longer duration of illness (P = 0.033), hospitalisation (P = 0.037) and mechanical ventilation (P = 0.014) compared to those with acute DAD. Patients with organising DAD had a longer duration of illness (P = 0.032) and hospitalisation (P = 0.023) compared to those with acute DAD. CONCLUSIONS: COVID-19 pneumonia patients who develop DAD can progress to the fibrosing pattern. While we observed fibrosing DAD in fatal cases, whether or not surviving patients are at risk for developing pulmonary fibrosis and the frequency of this complication will require further clinical and radiological follow-up studies.

It is estimated that ∼1% of European ancestry patients clinically diagnosed with type 1 diabetes (T1D) actually have monogenic forms of the disease. Because of the much lower incidence of true T1D in East Asians, we hypothesized that the percentage would be much higher. To test this, we sequenced the exome of 82 Chinese Han patients clinically diagnosed with T1D but negative for three autoantibodies. Analysis focused on established or proposed monogenic diabetes genes. We found credible mutations in 18 of the 82 autoantibody-negative patients (22%). All mutations had consensus pathogenicity support by five algorithms. As in Europeans, the most common gene was HNF1A (MODY3), in 6 of 18 cases. Surprisingly, almost as frequent were diallelic mutations in WFS1, known to cause Wolfram syndrome but also described in nonsyndromic cases. Fasting C-peptide varied widely and was not predictive. Given the 27.4% autoantibody negativity in Chinese and 22% mutation rate, we estimate that ∼6% of Chinese with a clinical T1D diagnosis have monogenic diabetes. Our findings support universal sequencing of autoantibody-negative cases as standard of care in East Asian patients with a clinical T1D diagnosis. Nonsyndromic diabetes with WSF1 mutations is not rare in Chinese. Its response to alternative treatments should be investigated.

Radiotherapy is an effective treatment for lung cancer but lacks a reliable prediction method. Cell-free nucleic acids in plasma have been reported as a novel tumor marker. Here, we evaluate circulating succinate dehydrogenase 5 (SDH5) mRNA in plasma and SDH5 protein in tumors, assess their predictive value in lung cancer patients undergoing radiotherapy, and explore the underlying mechanisms. Methods: SDH5 expression was measured in peripheral blood samples and fresh tumor specimens from 208 non-small cell lung cancer (NSCLC) patients and correlated with clinical outcomes. SDH5 knockout mice and human xenograft mice were used to evaluate radiosensitivity. Cell growth, apoptosis, and the DNA damage response were assessed. Relevant RNA and protein levels were analyzed by qRT-PCR and Western blotting. Immunoprecipitation and GST pulldown assays were performed to detect protein-protein interactions. Polyubiquitination of p53 was examined by an in vitro ubiquitination assay. Results: Plasma and tumor SDH5 mRNA levels were positively correlated (rho=0.894, P

14‐3‐3ζ has been reported to function as critical regulators of diverse cellular responses. However, the role of 14‐3‐3ζ in gliomas progression remains largely unknown. The expression level of 14‐3‐3ζ and Snail was detected by Western blot analysis and quantitative polymerase chain reaction in different grades of human gliomas. The effect of 14‐3‐3ζ on gliomas progression was measured using cell migration and invasion assay, the colony formation experiment, and CCK‐8 assay. The effect of 14‐3‐3ζ on PI3K/AKT/Snail signaling protein expression levels was tested by Western blotting. Firstly, 14‐3‐3ζ was often up‐regulated in high‐grade gliomas relative to low‐grade gliomas, and this overexpression was significantly related to tumor size, Karnofsky Performance Scale score and weaker disease‐free survival. Secondly, the overexpression of 14‐3‐3ζ promoted gliomas cells proliferation, migration, and invasion. Conversely, the knockdown of 14‐3‐3ζ suppressed gliomas cells proliferation, migration, and invasion. Furthermore, subsequent mechanistic studies showed that 14‐3‐3ζ could activate PI3K/AKT /Snail signaling pathway to facilitate gliomas cells proliferation, migration, and invasion. This study shows that the overexpression of 14‐3‐3ζ can promote remarkably gliomas cells proliferation, migration, and invasion by regulating the Snail protein expression through activating PI3K/AKT signaling, and it may serve as a potential prognostic marker and therapeutic target for gliomas.

Omethoate, an organophosphorous pesticide, can cause a variety of health effects, especially the decrease of cholinesterase activity. The aim of this study is to explore the association of genetic polymorphisms of telomere binding proteins with cholinesterase activity in omethoate-exposed population. Cholinesterase activities in whole blood, red blood cell and plasma were detected using acetylthiocholine and dithio-bis-(nitrobenzoic acid) method; Genetic Genotyping of POT1 rs1034794, POT1 rs10250202, TERF1 rs3863242 and TERT rs2736098 were performed with PCR-RFLP. The cholinesterase activities of whole blood, red blood cells and plasma in exposure group are significantly lower than that of the control group (P 0.001). Multivariate analysis indicates that exposure group (b = - 1.016, P 0.001), agender (b = 0.365, P 0.001), drinking (b = 0.271, P = 0.004) and TERF1rs3863242 (b = - 0.368, P = 0.016) had an impact on cholinesterase activities. The results suggest that individual carrying AG+GG genotypes in TERF1 gene rs3863242 polymorphism were susceptible to damage in cholinesterase induced by omethoate.

Objective— Thoracic aortic aneurysm and dissection (TAAD) are severe vascular conditions. Dysfunctional transforming growth factor-β (TGF-β) signaling in vascular smooth muscle cells and elevated angiotensin II (AngII) levels are implicated in the development of TAAD. In this study, we investigated whether these 2 factors lead to TAAD in a mouse model and explored the possibility of using microRNA-21 ( miR-21 ) for the treatment of TAAD. Approach and Results— TAAD was developed in Smad3 (mothers against decapentaplegic homolog 3) heterozygous (S3 +/− ) mice infused with AngII. We found that p-ERK (phosphorylated extracellular regulated protein kinases)– and p-JNK (phosphorylated c-Jun N-terminal kinase)–associated miR-21 was higher in TAAD lesions. We hypothesize that downregulation of miR-21 mitigate TAAD formation. However, Smad3 +/− :miR-21 −/− (S3 +/− 21 −/− ) mice exhibited conspicuous TAAD formation after AngII infusion. The vascular wall was dilated, and aortic rupture occurred within 23 days during AngII infusion. We then examined canonical and noncanonical TGF-β signaling and found that miR-21 knockout in S3 +/− mice increased SMAD7 and suppressed canonical TGF-β signaling. Vascular smooth muscle cells lacking TGF-β signals tended to switch from a contractile to a synthetic phenotype. The silencing of Smad7 with lentivirus prevented AngII-induced TAAD formation in S3 +/− 21 −/− mice. Conclusions— Our study demonstrated that miR-21 knockout exacerbated AngII-induced TAAD formation in mice, which was associated with TGF-β signaling dysfunction. Therapeutic strategies targeting TAAD should consider unexpected side effects associated with alterations in TGF-β signaling.

// Xiaoran Duan 1 , Yongli Yang 2 , Sihua Wang 3 , Xiaolei Feng 1 , Tuanwei Wang 1 , Pengpeng Wang 1 , Suxiang Liu 4 , Lei Li 4 , Guoyu Li 4 , Wu Yao 1 , Liuxin Cui 1 and Wei Wang 1 1 Department of Occupational and Environmental Health, College of Public Health, Zhengzhou University, Zhengzhou, China 2 Department of Epidemiology and Biostatistics, College of Public Health, Zhengzhou University, Zhengzhou, China 3 Department of Occupational Health, Henan Institute for Occupational Medicine, Zhengzhou, China 4 Clinical Department, Zhengzhou Institute of Occupational Health, Zhengzhou, China Correspondence to: Wei Wang, email: ww375@126.com Yongli Yang, email: ylyang377@126.com Keywords: omethoate, telomere, polymorphism, metabolic enzymes Received: June 27, 2017 Accepted: August 29, 2017 Published: September 18, 2017 ABSTRACT Purpose: This study aimed to explore the effects of genetic polymorphisms in metabolic enzymes on relative telomere length changes and explore the mechanism of canceration induced by omethoate. Materials and Methods: 180 long-term omethoate-exposed workers and 115 healthy controls were recruited. Real-time PCR method was applied to determine the relative telomere length in peripheral blood leukocytes DNA, and Six polymorphic loci of GSTT1 (+/−), GSTM1 (+/−), GSTP1 rs1695, CYP2E1 rs6413432, CYP2E1 rs3813867 and PON2 rs12026 were detected by polymerase chain reaction and restriction fragment length polymorphism method; Multiple linear regression was conducted to explore the effects of omethoate exposure and genetic polymorphisms on the telomere length. Results: The relative telomere lengths in the control group (0.94 [0.76, 1.32]) were significantly shorter than that in the exposure group (1.50 [1.11, 2.57]) ( Z = 7.910, P < 0.001). Univariate analysis showed that the relative telomere lengths of the GSTM1 -deletion individuals were significantly longer than that of the non - deletion genotype in the control group ( Z = 2.911, P = 0.004), and the relative telomere lengths of GSTP1 rs1695 polymorphism locus (GG+AG) genotype individuals were longer than that of AA genotype in the exposure group. The difference was statistically significant ( Z = 2.262, P = 0.024). Multivariate analysis found that pesticide-exposure ( b = 0.524, P < 0.001) and GSTM1 polymorphism ( b = −0.136, P = 0.029) had an impact on telomere length. Conclusions: The relative telomere lengths of omethoate-exposure workers were longer than that in the control population. Also GSTM1 genetic polymorphism may influence the changes of the telomere length induced by omethoate.

Programmed death-ligand 1 (PD-L1) is a crucial target for lung cancer immunotherapy. In lung cancer patients with high PD-L1 expression, blocking or reducing its expression can inhibit tumor growth. PD-L1 is regulated by signaling pathways, transcription factors and epigenetic factors, such as the GSK3β/β-catenin pathway, P53 protein and EMT. In our previous study, succinate dehydrogenase 5 (SDH5) was reported to regulate ZEB1 expression, induce EMT and lead to lung cancer metastasis via the GSK3β/β-catenin pathway. It is possible that SDH5 is involved in the mechanisms of PD-L1 regulation.In the present study, we observed a negative correlation between the expression of PD-L1 and SDH5 in vivo and in vitro. The examination of patient tissues also confirmed our results. Furthermore, we also found that SDH5 could reverse PD-L1 expression by the GSK3β/β-catenin/ZEB1 pathways. All these results reveal that SDH5 regulates PD-L1 expression and suggest that SDH5 can be used as a marker to predict tumor immune micro-states and provide guidance for clinical immunotherapy.

Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3'-end of the forward primer was next to the polymorphic site, and the third base from the 3'-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

Background In recent research, it has been shown that rs2289487 within the PLIN1 gene has different variants that have been associated with obesity, type 2 diabetes, and other diseases. However, the isochizomers such as the BsmI enzyme required for detection of this polymorphism through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method are expensive. In this study, we aimed to explore a novel PCR-RFLP method for identifying the single-nucleotide polymorphism (SNP) of rs2289487 of PLIN1 gene. Methods A new restriction enzyme site was created through created restriction site PCR. In the forward primer, a deoxynucleotide A was substituted with C, and after PCR a new restriction enzyme site for BstUI was introduced into the PCR products. A total of 108 samples from Han Chinese were tested to evaluate this new method. Results Allele frequencies in the Asian population were 0.326 for allele A and 0.674 for allele G, and the genotype frequencies were 12.8% for AA, 39.5% for AG, and 47.7% for GG. Conclusion The PCR-RFLP with new site for detecting the SNP of rs2289487 is an improved method with low cost and high accuracy.

Background/Aims: Cardiac fibrosis is the primary cause of deteriorated cardiac function in various cardiovascular diseases. Numerous studies have demonstrated that microRNAs (miRNAs) are critical regulators of myocardial fibrosis. Specifically, many studies have reported that miR-150 is downregulated in cardiovascular diseases, such as acute myocardial infarction (AMI), myocardial hypertrophy and myocardial fibrosis. However, the exact role of miR-150 in these pathological processes remains unknown. Methods: We used the transverse aortic constriction (TAC) mouse model to study the role of miR-150 in cardiac fibrosis induced by pressure overload. After the TAC operation, qRT-PCR was used to measure the expression profiles of miR-150 in left ventricle tissues and populations of primary heart cell types. Then, we used both miR-150 knockout mice and wild type (WT) mice in the TAC model. Changes in cardiac function and pathology were measured using transthoracic echocardiography and pathological analysis, respectively. Furthermore, we predicted the target of miR-150 in cardiac fibroblasts (CFs) and completed in vitro CF transfection experiments using miR-150 analogs and siRNA corresponding to the predicted target. Results: We observed decreased expression levels of miR-150 in hearts suffering pressure overload, and these levels decreased more sharply in CFs than in cardiomyocytes. In addition, the degrees of cardiac function deterioration and cardiac fibrosis in miR-150-/- mice were more severe than were those in WT mice. By transfecting CFs with an miR-150 analog in vitro, we observed that miR-150 inhibited cardiac fibroblast activation. We predicted that the transcription factor c-Myb was the target of miR-150 in CFs. Transfecting CFs with c-Myb siRNA eliminated the effects of an miR-150 inhibitor, which promoted CF activation. Conclusion: These findings reveal that miR-150 acts as a pivotal regulator of pressure overload-induced cardiac fibrosis by regulating c-Myb.

Highlights • Patients in the experimental group received intravenous ribavirin versus supportive care only in the control group. • Ribavirin was not associated with reduced negative conversion time of SARS-CoV-2 PCR test compared with the control group. • Ribavirin did not reduce the mortality rate compared with the control group., The aim of this study was to compare ribavirin therapy versus supportive therapy only for patients with severe coronavirus disease 2019 (COVID-19). A total of 115 patients with laboratory-confirmed COVID-19 were retrospectively analysed. All patients received supportive care as well as regular laboratory and clinical monitoring. The 115 patients comprised 44 patients who received intravenous ribavirin (treatment group) and 71 who did not (control group). Baseline laboratory and clinical characteristics were similar between the two groups. The negative conversion time for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR in the ribavirin group was 12.8 ± 4.1 days compared with 14.1 ± 3.5 days in the control group (P = 0.314). Moreover, 7/41 patients (17.1%) in the ribavirin group died compared with 17/69 (24.6%) in the control group (P = 0.475). Adverse effects were similar between the two groups. In conclusion, in patients with severe COVID-19, ribavirin therapy is not associated with improved negative conversion time for SARS-CoV-2 test and is not associated with an improved mortality rate. Further assessment in designed randomised controlled trials is recommended.

Background Programmed cell death 1 (PD-1) functions as an immune checkpoint in the process of anti-tumor immune response. The PD-1 blockade is now becoming a fundamental part in cancer immunotherapy. So it’s essential to elicit the PD-1 related immune process in different types of cancer. Methods The Cancer Genome Atlas was used to collect the RNA-seq data of 33 cancer types. The microenvironment cell populations-counter was used to analyze the immune cell infiltrates. KEGG and GO analysis were performed to investigate PD-1 associated biological process. Kaplan–Meier survival curves and Cox’s proportional hazards model were performed for prognostic value analysis. Results We demonstrated that PD-1 expression varied in different cancer types. The uveal melanoma had a low PD-1 expression and poor infiltrated with immune cells. But it showed the strong correlation of PD-1 with the most types of immune cells. The PD-1 demonstrated a robust relationship with other immunomodulators and showed its involvement in critical functions correlated with anti-tumor immune pathways. Survival analysis indicated the PD-1 expression suggested different prognosis in different cancer types. Conclusions Our investigations promote a better understanding of the PD-1 blockade and provide PD-1 related personized combined immunotherapy for different types of cancer patients. Electronic supplementary material The online version of this article (10.1186/s12935-018-0712-y) contains supplementary material, which is available to authorized users.

Recently, pseudogenes have emerged as critical regulators in the onset of human neoplasia. Here, we present a comprehensive analysis of pseudogene alterations at transcriptional levels in lung adenocarcinoma (LUAD) from The Cancer Genome Atlas. By combinations of differential expression analysis, survival analysis, and univariate and multivariable Cox proportional hazards regression models, we identified four dysregulated pseudogenes, whose expression level was closely related to LUAD patients' prognosis and the four pseudogene signature could act as an independent prognostic indicator for LUAD patients. We further characterized CHIAP2, one of those four pseudogenes, whose expression level was the most closely linked to LUAD patients' prognosis. Consistent with our analysis, the expression of CHIAP2 was abnormally downregulated in LUAD tissues compared with that in normal tissues in our 50 pairs of clinical samples. Functional assays demonstrated that upregulation of CHIAP2 significantly impaired cell proliferation and invasion. After performing RNA sequencing (RNA-seq) and small RNA-seq between CHIAP2 overexpression and negative control LUAD cell lines, we identified differentially expressed messenger RNAs and microRNAs (miRNAs), among which six miRNAs were downregulated. Target genes of six downregulated miRNAs were predicted with online miRNA target prediction tools and significant pathways including the WNT signal pathway were identified with Gene Set Enrichment Analysis. By combining predictor genes of six downregulated miRNAs and dysregulated genes of the WNT pathway, we inferred that overexpression of CHAP2 may inhibit LUAD cell proliferation and invasion via modulation of NFATC2 or GSK3B (WNT signal pathway) targeted by miR-3614-5p or miR-873-3p.

Cholinesterase activity (ChA), the effective biomarker for organophosphate pesticide exposure, is possibly affected by single nucleotide polymorphisms (SNPs) in cell-cycle-related genes. One hundred and eighty workers with long-term exposure to omethoate and 115 healthy controls were recruited to explore the gene–gene and gene–environment interactions. The acetylthiocholine and dithio-bis-(nitrobenzoic acid) method was used to detect the cholinesterase activities in whole blood, erythrocytes and plasma. Genetic polymorphisms were determined by the PCR-RFLP and direct PCR electrophoresis methods. Statistical results showed that the cholinesterase activities of whole blood, erythrocytes and plasma in the exposure group were significantly lower than those in the control group ( p p p21 rs1801270 locus in the control group ( p = 0.033), but not in the exposure group. A significant interaction between genes and environmental factors (i.e. p53 , p21 , mdm2, gender, smoking and drinking) affecting ChA was found through a generalized multifactor dimensionality reduction analysis. These obtained markers will be useful in further marker-assisted selection in workers with exposure to omethoate.

Graft arteriosclerosis (GA) is the leading cause of late cardiac allograft dysfunction. The innate immune system plays a major role in GA, paprticularly Toll-like receptor 4 (TLR4) signaling. Here we characterized the role of TLR4 and its antagonist TAK-242 in a mouse model of GA. BALB/c (H-2d) donor aortas were transplanted into C57BL/6 (H-2b) recipients, and the mice received intraperitoneal injection of 3 or 10 mg/kg of TAK-242 or vehicle every other day for 1, 2, 4, 6, 8 and 12 weeks. With TAK-242 administration, intimal hyperplasia initially appeared at 2 weeks after transplantation, and TAK-242 postponed the progression of neointimal formation in allogeneic aortic grafts. TAK-242 treatment reduced CD68+ macrophage accumulation in the allografts, reduced the levels of ly-6Chi monocytes in peripheral blood, bone marrow and spleen, and downregulated proinflammatory cytokine and chemokine levels. Ex vivo we observed that TAK-242 could improve the graft microenvironment by interfering the Tck/Mφ IL12p70 and IFNγ axis, reducing CCL2-mediated migration of vascular smooth cells.

Organophosphorous pesticides (OPs), with high efficiency, broad-spectrum and low residue, are widely used in China. Omethoate is a broad category of organophosphorous pesticides and is more domestically utilized which has chronic toxic effect on human health caused by long-term, low-dose exposure to Ops, recently its potential genotoxicity has attracted wide attention which can cause chromosomal DNA damage. Thus, the aim of this study is screen susceptible biomarkers and explore the mechanism of canceration induced by omethoate. 180 long-term organophosphorus pesticide–exposed workers and 115 healthy controls were recruited. Quantitative polymerase chain reaction method was applied to determine the relative telomere length in peripheral lymphocyte DNA as well as p53 and p21 gene expression levels. Genetic polymorphisms were determined by the polymerase chain reaction–restriction fragment length polymorphism method. Multiple linear regression was conducted to explore the effects of exposure, expression levels, and polymorphisms in genes on the telomere length. The results showed the relative telomere lengths in the exposure group were significantly longer than that in the control group. The messenger RNA expression levels of p53 and p21 in exposure group were significantly lower than that in the control group; telomere lengths of the CA genotype individuals of p21 rs1801270 polymorphism locus were significantly longer than that of the CC genotype in the control group that were estimated using the Bonferroni method; and bivariate correlation analysis showed that the messenger RNA expression level of gene p53 was negatively correlated with telomere length, and the messenger RNA expression level of gene p21 was positively correlated with telomere length. Multivariate analysis found that p53 messenger RNA and p21 messenger RNA had an impact on telomere length. These results demonstrated that the messenger RNA expression levels of p53 and p21 may have a relationship with the changes in telomere length induced by omethoate and provided strong evidence for the mechanism of canceration induced by poison.

Background Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a common and mature method of detecting the single nucleotide polymorphism (SNP). But, for the polymorphism site rs3863242 of telomeric repeat binding factor 1(TERF1) gene, there is no appropriate restriction enzyme to recognize it, which limits the research between the variants of rs3863242 and human diseases. Methods The reverse primer was designed based on turning the 3rd base T into the mismatch base G. After PCR amplification, a new restriction enzyme site was introduced into the TERF1 gene amplification products. Two hundred forty samples from Chinese Han individuals were genotyped to evaluate this method. Results A new restriction enzyme site for CviQI was introduced into the PCR products. The genotype frequencies of 240 samples from Chinese Han individuals were 4.17% for A/A, 29.58% for A/G, 66.25% for G/G respectively. The allele frequencies were 18.96% for A and 81.04% for G respectively. The genotyping results of PCR products were consistent with the gene sequencing result. Conclusions We developed a simple, direct and economical technique for analyzing the polymorphism of TERF1 rs3863242. It may be applied to the colony screening of other SNPs, mutation-screening of tumor-related gene or mutations in some specific genes on a large scale, in the future.

Objective To identify the significance of a support vector machine (SVM) model and a decision tree (DT) model for the diagnosis of lung cancer combined with the detection of fragile histidine triad (FHIT), RAS association domain family 1 (RASSF1A) and cyclin-dependent kinase inhibitor 2A (p16) promoter methylation levels and relative telomere length (RTL) of white blood cells from peripheral blood DNA. Methods The levels of p16, RASSF1A and FHIT promoter methylation and the RTL of white blood cells in peripheral blood DNA of 200 healthy individuals and 200 lung cancer patients were analyzed by SYBR Green-based quantitative methylation-specific PCR and quantitative PCR. Based on the 4 biomarkers, SVM and DT models were developed. Results The levels of FHIT, RASSF1A and p16 promoter methylation were 3.33 (1.86-6.40) and 2.85 (1.39-5.44) (p = 0.002); 27.62 (9.09-52.86) and 17.17 (3.86-50.87) (p = 0.038); and 0.59 (0.16-4.50) and 0.36 (0.06-4.00) (p = 0.008) in cases and controls, respectively. RTL was 0.93 ± 0.32 and 1.16 ± 0.57 (pConclusions The SVM and DT models for diagnosing lung cancer were successfully developed through the combined detection of p16, RASSF1A and FHIT promoter methylation and RTL, which provided useful tools for screening lung cancer.

Previous studies mainly focused on the role of the epidermal growth factor receptor (EGFR) in tumor cells, whereas the effects of the EGFR on immune responses has not been determined. Our study shows that the EGFR signaling pathway play a role in the regulation of regulatory T cells (Treg cells) in cancer patients. The EGF-like growth factor Amphiregulin (AREG) protein was frequently up-regulated in a tissue microarray, which was associated with worse overall survival. Additionally, in sera, tissue specimens, and effusions of lung or gastric cancer patients, up-regulated AREG protein enhanced the suppressive function of Treg cells. AREG maintained the Treg cell suppressive function via the EGFR/GSK-3β/Foxp3 axis in vitro and in vivo. Furthermore, inhibition of EGFR by the tyrosine kinase inhibitor gefitinib restored the activity of GSK-3β and attenuated Treg cell function. β-TrCP was involved in GSK-3β-mediated Foxp3 degradation, and mass spectrometry identified Lys356 as the ubiquitination site of Foxp3 by β-TrCP. These findings demonstrate the posttranslational regulation of Foxp3 expression by AREG in cancer patients through AREG/EGFR/GSK-3β signaling, which could lead to Foxp3 protein degradation in Treg cells and a potential therapeutic target for cancer treatment.

Human protection of telomeres 1 (POT1) gene is a single stranded telomere binding proteins with a critical role in ensuring chromosome stability. There have been variants of POT1 gene, and the polymorphisms of POT1 gene were associated with some diseases. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) is a traditional method to detect the single nucleotide polymorphism (SNP), and it can be used to detect the polymorphism of rs10250202. But the restriction enzymes required for the detection of the polymorphism of rs10250202 are expensive. So we designed a novel PCR–RFLP assay for genotyping the POT1 rs10250202 SNP. In the study, a new restriction enzyme cutting site was created by created restriction site PCR (CRS-PCR), and the restriction enzyme BclI for CRS-PCR was cheaper than other enzymes. After detecting Han Chinese workers, Allele frequencies were found to be 51.54 % for allele A and 48.46 % for allele C respectively. The PCR results were confirmed by DNA sequencing. CRS-PCR provides a simple, low-cost, practical, and reproducible method.

The purpose of the study was to explore the application of artificial neural network model in the auxiliary diagnosis of lung cancer and compare the effects of back-propagation (BP) neural network with Fisher discrimination model for lung cancer screening by the combined detections of four biomarkers of p16, RASSF1A and FHIT gene promoter methylation levels and the relative telomere length. Real-time quantitative methylation-specific PCR was used to detect the levels of three-gene promoter methylation, and real-time PCR method was applied to determine the relative telomere length. BP neural network and Fisher discrimination analysis were used to establish the discrimination diagnosis model. The levels of three-gene promoter methylation in patients with lung cancer were significantly higher than those of the normal controls. The values of Z(P) in two groups were 2.641 (0.008), 2.075 (0.038) and 3.044 (0.002), respectively. The relative telomere lengths of patients with lung cancer (0.93 ± 0.32) were significantly lower than those of the normal controls (1.16 ± 0.57), t = 4.072, P