Background: A few epidemiologic surveys and experimental studies observed the synergistic carcinogenic effect of nitrosamines and High-Risk Human Papillomavirus (HPV) in partial digestive tract cancers. They mainly focused on the co-carcinogenesis of HPV E6 and E7 in cancer development. HPV E5 plays an important role in early-stage cancer but has limited capacity as an independent carcinogen. Objective: To investigate whether nitrosamines enhance the carcinogenesis of E5, a rapid in vitro rodent cell transformation assay was established. Materials and Methods: We have successfully established BALB/c 3T3-E5 cells expressing HPV16 E5 using a lentivirus vector. Six foodborne N-nitroso Compounds (NOCs) are selected to induce malignant cell transformation. Results: We have screened two compounds with strong transformation capability, N-nitrosodimethylamine and N-nitrosodiethylamine alone. The combined action of NOCs and E5 is highly efficient, with more transformed foci than a single NOC or E5 oncoprotein, and the synergistic effect becomes more evident with increasing concentrations of NOCs, especially N-nitrosodipropylamine. Furthermore, we also confirm the working concentration range of 0.05-0.1 ng/mL of NOCs to meet the demands for cell viability in carcinogenicity tests. Conclusion: In conclusion, we provide cell models for studying the interaction mechanism of HPV16 E5 and NOCs, as well as assessing preventive or therapeutic candidates for HPV-positive tumors in the future.

Under the catalysis of Rh 2 (esp) 2 (10 mol%) and BINAP (20 mol%) in 1,2‐dichloroethane at 80 °C, a (3+3) cascade cyclization between diazobarbiturates and 2 H ‐azirines proceeds smoothly, yielding spirooxazinopyrimidinediones in 24–92% yield with >20:1 diastereoselectivity. The chemical structure of one compound has been confirmed by X‐ray diffraction analysis, and the others are suggested by inference.

Under the catalysis of Rh2(OAc)4 (10 mol%) and (+/-)-Me-Pybox(20 mol%) in 1,2-DCE at 80 degrees C, the homocoupling/[4+1] cycloaddition cascade of diazobarbiturates with diazopyrazolones proceeded readily and provided spirobarbiturates in 32-88% chemical yields.The chemical structure of the obtained spirobarbiturates was identified by X-ray diffraction analysis. image

Monodispersed metal sites are commonly considered as active centers that dominate the high catalytic activity and selectivity of a catalyst. Photocatalysis is a complex series of photophysical and electrocatalytic process, in which, it still remains challenges to obtain a well-defined profile on the functions of the monodispersed metal sites. Here we report that monodispersed aluminum (Al) metal sites are not active centers but they create abundant nitrogen non-metal active centers in carbon nitrides. Density functional theory (DFT) calculations show that Al metal sites serve as the bridges among isolated carbon nitride layers to establish diverse transfer channels for electrons, thus achieving rapid carrier separation. These findings provide a new perspective on understanding the important role of monodispersed metal sites for highly effective catalytic reactions.

Abstract Background The occurrence and progression of various solid tumors are associated with the melanoma-associated antigen A (MAGE-A) family. Although it was demonstrated that demethylation at the promoter region usually causes the over-expression of the MAGE-A family, there has been very few research about the detailed mechanisms of how the genetic modification of promoter region promotes MAGE-A expression.Methods A new non-coding RNA (ncRNA) with the ability of binding with melanoma-associated antigen-A6 (MAGE-A6) promoter region was discovered. The expression consistency between MAGE-A6 and this novel ncRNA in different MAGE-A6 highly expressed malignant cell lines was analyzed by RT-qPCR. The full length of this ncRNA was acquired through RACE and were subsequently named as MAGEA6-DT1. Then up- and down-regulation of MAGEA6-DT1 in human malignant melanoma cells were achieved by lentivirus transduction and siRNA transfection respectively and the transcription and expression of MAGE-A6 was detected by RT-qPCR and Western Blot for verifying MAGE-A6 expression regulating function of MAGEA6-DT1. The exact binding site of MAGEA6-DT1 in MAGE-A6 promoter region was analyzed by dual-luciferase reporter system assay after MAGEA6-DT1 transfection in 293T cells. Moreover, by DNA methylation analysis, we tested whether MAGEA6-DT1 has the ability of MAGE-A6 expression regulation by manipulating its promoter region’s methylation. Finally, RNA pull-down assay was performed to identify the functional binding partner of MAGEA6-DT1.Results MAGEA6-DT1 was identified as a long non-coding RNA (lncRNA) with the length of 771 nucleotides and was abnormally expressed in consistency with MAGE-A6 among various cancer cell lines. Manipulation of MAGEA6-DT1 expression level would positively regulates MAGE-A6 expression. Specific binding site of MAGEA6-DT1 located near the enhancer of MAGE-A6, and its function was revealed to demethylate DNA near its binding site, probably with the assistance of relevant binding partners.Conclusion MAGEA6-DT1, as a lncRNA abnormally expressed in different malignant cell lines, could positively regulate MAGE-A6 expression via specifically combining with and subsequently demethylating MAGE-A6 enhancer. This function may be assisted by some of its binding protein such as DNA (cytosine-5)-methyltransferase 1 (DNMT1).

Objective The correlative cathodoluminescence (CL) imaging is a high-resolution fluorescence imaging technique using electron beam as excitation source. However, the sensitivity of biomaterials to electron irradiation limits the wide application of CL technique in life science. In order to study and develop the application of CL technology in biological samples, this paper aims to deeply understand the excitation characteristics of organic fluorophore by electron source by exploring the structural damage of carbon substrate, degradation of organic groups and fluorescence quenching caused by electron irradiation. Methods We used a CL and electron microscopy (CCLEM) technique in a field emission scanning electron microscope (SEM) to observe organic fluorophores and fluorescently labeled cells. Results We studied excitation and emitting characteristics of organic fluorophores by electron beam. Under the irradiation of low energy symbolscript keV) and low current (similar to 10 pA), the fluorescent microbeads had the stronger and stable CL emission, the resolution of CL image reached similar to 30 nm. After 12 min irradiation, fluorescence intensity of microbeads decreased by 25%, and CL image still maintained acceptable fluorescence intensity and adequate signal to noise ratio. Further, fluorescently labeled subcellular structures were identified from cell surface to a certain depth inside the cell. The nuclei and organelles were well observed with a bulk-sample imaging setup, while membrane proteins, that are widely localized on the cell surface, were clearly identified with a home-made imaging setup of thin-sample. Conclusion Results provide data and technical support for the application of CCLEM in the study of biological structures. CCLEM bioimaging can also be used as an important supplement to correlative light and electron microscopy (CLEM) technique.

In this study, we prepared a streptavidin magnetic bead based on graphene-coated iron nitride magnetic beads (G@FeN-MB) and tried to use it for the enrichment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The outer shell of our magnetic bead was wrapped with multiple graphene sheets, and there is no report on the application of graphene to the magnetic-bead-coating material. First, the graphene shell of G@FeN-MB was oxidized by a modified Hummer method so as to generate the carboxyl groups required for the coupling of streptavidin (SA) on the surface of the magnetic beads. X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM) were used to characterize the oxidized G@FeN-MB (GO@FeN-MB). Streptavidin was then linked to the surface of the GO@FeN-MB by coupling the amino of the streptavidin with the carboxyl on the magnetic beads by carbodiimide method; thus, the streptavidin magnetic beads (SAMBs) were successfully prepared. To prove the practicality of the SAMBs, biotinylated SARS-CoV-2 S1 antibody was linked with it to respectively capture SARS-CoV-2 Spike-protein-coupled polystyrene beads (S-PS) and pseudovirus with S-protein expressed. Microplate reader and fluorescence microscope results show that the SAMBs can effectively enrich viruses. In conclusion, the preparation of SAMBs with G@FeN-MB is feasible and has potential for application in the field of virus enrichment.

The melanoma antigen gene family A (MAGEA) family of proteins comprises of cancer-testis antigens that are highly expressed in a number of tumours but are minimally expressed in normal cells. Due to its expression characteristics, this protein family has become a popular target for anti-cancer drugs and immunotherapy research over recent years. Although, elevated expression levels of MAGEA6 has been found in different types of tumours, there remains to be insufficient information on the function of MAGEA6 and its associated gene regulation pathways. The present study used Transwell, Cell Counting Kit-8 and wound healing assays to analyse the effects of MAGEA6 on Eca109 cell invasion, migration and proliferation. The main functions and pathways involved in MAGEA6 were predicted by Illumina Hiseq screening for mutually regulated genes and core genes. Eca109 cell line with a high expression of MAGEA6 was a stable cell line obtained by transfection in the early stage, and this cell line was used in subsequent experiments. Transcriptome sequencing was performed on this cell line and the Eca109 cell line that normally expressed MAGEA6. It was revealed that a high expression of MAGEA6 conferred a significant stimulating effect on cell proliferation whilst also significantly increasing cell invasion and migration. Transcriptomic analysis identified 14 differentially expressed genes and 13 core regulatory genes closely associated with MAGEA6 expression regulation, such as methylsterol monooxygenase 1 (MSMO1). The present study suggest that MAGEA6 positively regulated MSMO1 expression, which may serve an oncogenic role in cells through this regulatory effect. Overall, this provided a novel route of investigation for an in-depth study of the regulatory function of MAGEA6.

Objective:The NS5 protein of dengue virus (DENV) mainly acts as the viral RNA-dependent RNA polymerase (RdRp). The aim of the present study was to obtain potent antiviral pro-drugs using the conformational phage display peptide library.Methods:The soluble recombinant DENV-2 NS5 protein was used as the target for screening oligopeptide (OP) with high specificity with the conformational phage display peptide library. Molecular docking was used to verify the interaction between OP and RdRp. DENV infected C6/36 cells were treated with different concentrations of OP. The antiviral effect of OP was assessed by virus titre detecmined by RT-qPCR. Meanwhile, C6/36 cells were culture with exposure to higher concentrations of OP. Cell viability was measured by CCK-8 assay to evaluate the cytotoxicity of OP.Results:The molecular docking indicated that OP bound specifically to the RdRp domain of DENV NS5 protein. Inhibitory effect on DENV-2 replication without cytotoxicity was observed in vitro. In addition, based on the high conservation of the RdRp domain in single-stranded plus RNA (+ssRNA) viruses and the preliminary results of molecular docking, OP may also bind to the RdRp active cavity of a number of +ssRNA viruses. Conclusions:OP may have the potential to inhibit RdRp of certain +ssRNA viruses, and is a potential broad-spectrum antiviral prodrug.

Purpose Different types of HPV have been associated with cancer in humans, but the role of HPV in esophageal cancer (EC) is controversial. The purpose of this study was to evaluate the correlation between HPV infection and EC in the Chinese population and to provide the scientific basis for the future prevention, control, early diagnosis, and treatment strategies of EC in China. Methods PCR detected HPV infection in 1112 esophageal cancer tissue samples, and 89 HPV-positive samples were detected by genotyping. Proximity ligation assays (PLAs) and immunohistochemistry were used to detect the expression of HPV E6 and E7 proteins. Real-time fluorescent quantitative PCR was used to detect the integration of HPV16 E6. The level of HPV-specific antibody IgG in serum was detected by ELISA and PLA. Results The positive rates of HPV L1, HPV16, HPV18, hpv16 + 18 E6 and hpv16/18 E6 in 1,112 EC tissue samples were 77.6%, 41.4%, 27.2%, 14.2% and 55.4% respectively. Multiple HPV subtypes were detected in HPV-positive EC samples. PLA showed that E6 and E7 were expressed in EC109 and formed complexes with p53 and pRb, respectively. Immunohistochemistry showed that the positive rates of hpv16 + 18 E6 and E7 in HPV-positive EC samples were 56.4% and 37.0%, respectively. HPV-DNA integration rate in HPV-positive EC tissues (88.79%) was higher than that in adjacent tissues (54.17%). HPV antibody was found in the serum of EC patients by a serological test. Conclusion The study suggests that HPV, especially HPV16 and HPV18, the infection may be a risk factor for EC in the Chinese population and that the E6 protein may play a key role in HPV-associated malignancies. These results may be important for the prevention and treatment of HPV-positive EC in China.

PURPOSE:We aimed to investigate RF-EMR-induced cell malignant transformation.METHODS:We divided Balb/c-3T3 cells into sham and expo groups. The expo groups were exposed to a 1800 MHz RF continuous wave for 40 and 60 days, for 4 h per day. The sham group was sham-exposed. Cells were harvested for a cell transformation assay, transplantation in severe combined immune deficient (SCID) mice, soft agar clone formation detection, and a transwell assay. The mRNA microarray assay was used to declare key genes and pathways.RESULTS:The exposed Balb/c-3T3 cells showed a strong increase in cell proliferation and migration. Malignant transformation was observed in expo Balb/c-3T3 cells exposed for 40 days and 60 days, which was symbolized with visible foci and clone formation. Expo Balb/c-3T3 cells that were exposed for 40 days and 60 days produced visible tumors in the SCID mice. Lipid metabolism was the key biological process and pathway involved. The mevalonate (MVA) pathway was the key metabolic pathway. The interacted miRNAs could be further research targets to examine the molecular mechanism of the carcinogenic effects of long-term exposure.CONCLUSION:Exposure for 40 and 60 days to 1800 MHz RF-EMR induced malignant transformation in Balb/c-3T3 cells at the SAR of 8.0 W/kg. We declared that lipid metabolism was the pivotal biological process and pathway. The MVA pathway was the key metabolic pathway.

目的 比较临床常用中药制剂对OVCAR3细胞OVCAR3的药效学.方法 以采用实时无标记细胞分析(RTCA)检测榄香烯注射液、参芪扶正注射液、艾迪注射液、复方苦参注射液、消癌平注射液、康艾注射液、康莱特注射液对OVCAR3细胞增殖、迁移、侵袭的抑制作用,JC-1检测上述药物对肿瘤细胞线粒体膜电位的影响,CCK8法检测它们对正常细胞系MRC-5的毒性作用.结果 榄香烯注射液、消癌平注射液、复方苦参注射液、艾迪注射液抑制OVCAR3细胞增殖的 IC50值分别为(44.98±0.13)μg/mL、(258.78±6.53)mg/mL、(121.73±1.23)mg/mL、(51.73±0.51)mg/mL,中、高剂量榄香烯注射液有明显抑制OVCAR3细胞迁移的作用;除康莱特注射液外,其他药物都有一定程度抑制OVCAR3细胞侵袭的作用.榄香烯注射液、消癌平注射液在IC50剂量下作用24 h后,可检测到线粒体膜电位的改变.榄香烯注射液、艾迪注射液IC50(MRC-5)/IC50(OVCAR3)均大于1.结论 以OVCAR3细胞为对象时,榄香烯注射液抗肿瘤作用最好,其次是艾迪注射液.

Oesophageal cancer is one of the deadliest cancers in the world. Oesophageal squamous cell carcinoma (ESCC) is the most prevalent histological type of oesophageal cancer. Oesophageal cancer has a poor prognosis because of its invasiveness. Thus, it is especially important to seek effective treatment methods. Research indicates that long non-coding RNAs (lncRNAs) play a significant role in the occurrence and development of oesophageal cancer. The aim of this study was to describe the role of LINC00958 in ESCC. Bioinformatics and real-time quantitative polymerase chain reaction (RT-qPCR) methods were utilized to predict and verify the expression of LINC00958 in ESCC. Related functional experiments, including cell proliferation, migration and invasion, were performed. In addition, a western blot and a dual luciferase reporter gene experiment were used to study the detailed carcinogenic mechanism of LINC00958. The results indicated there was a high expression of LINC00958 in ESCC, which promoted proliferation, migration, invasion and Epithelial-Mesenchymal Transition (EMT) of ESCC cells, and this effect may be via regulating miR-510-5p.

Liquid-phase electron microscopy is highly desirable for observing biological samples in their native liquid state at high resolution. We developed liquid imaging approaches for biological cells using scanning electron microscopy. Novel approaches included scanning transmission electron imaging using a liquid-cell apparatus (LC-STEM), as well as correlative cathodoluminescence and electron microscopy (CCLEM) imaging. LC-STEM enabled imaging at a ∼2 nm resolution and excellent contrast for the precise recognition of localization, distribution, and configuration of individually labeled membrane proteins on the native cells in solution. CCLEM improved the resolution of fluorescent images down to 10 nm. Liquid SEM technologies will bring unique and wide applications to the study of the structure and function of cells and membrane proteins in their near-native states at the monomolecular level.

Immunomagnetic beads (IMBs) for cell sorting are universally used in medical and biological fields. At present, the IMBs on the market are ferrite coated with a silicon shell. Based on a new type of magnetic material, the graphene coated iron nitride magnetic particle (G@FeN-MP), which we previously reported, we prepared a novel IMB, a graphene oxide coated iron nitride immune magnetic bead (GO@FeN-IMBs), and explored its feasibility for cell sorting. First, the surface of the G@FeN-MP was oxidized to produce oxygen-containing groups as carboxyl, etc. by the optimized Hummers’ method, followed by a homogenization procedure to make the particles uniform in size and dispersive. The carboxy groups generated were then condensed and coupled with anti-CD3 antibodies by the carbodiimide method to produce an anti-CD3-GO@FeN-IMB after the coupling efficacy was proved by bovine serum albumin (BSA) and labeled antibodies. Finally, the anti-CD3-GO@FeN-IMBs were incubated with a cell mixture containing human T cells. With the aid of a magnetic stand, the T cells were successfully isolated from the cell mixture. The isolated T cells turned out to be intact and could proliferate with the activation of the IMBs. The results show that the G@FeN-MP can be modified for IMB preparation, and the anti-CD3-GO@FeN-IMBs we prepared can potentially separate T cells.

Tobacco smoke and human papillomavirus (HPV) are both crucial causes of cancer, and their cooperative carcinogenesis has drawn more attention in recent years. Apigenin (AP), a typical flavonoid abundantly found in flowers of plants, vegetables, and fruits, has been demonstrated to exert an anti-carcinogenic effect on various types of cancer. In this study, we investigated the capability of AP against malignant transformation and DNA damage of immortalized human esophageal epithelial (SHEE) cells induced by the synergism of HPV18 and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The results indicated that the enhancement of migration, invasion, and proliferation ability of SHEE cells induced by HPV and NNK could be effectively inhibited by AP. Moreover, the levels of pyridyloxybutylated (POB)-DNA adducts induced by NNK via P450-catalyzed metabolic activation could also be significantly suppressed by AP. Further analyses on the molecular mechanism revealed that AP inhibited the synergistic carcinogenesis of NNK and HPV on SHEE cells by reducing the expression of mutp53, CDK4, Cyclin D1, and p-Rb (Ser 780), increasing caspase-3 activity, thereby arresting the cell cycle at G1 phase and promoting apoptosis of SHEE cells. We hypothesize that the decrease in NNK-induced POB-DNA adduct levels is related to the deactivation of P450 by AP, which needs to be confirmed in future studies. This study highlights that AP may be employed as a promising chemopreventive agent against cancers in smokers with an HPV infection.

There is an increased public concern about potential health hazards of exposure to electromagnetic radiation (EMR). To declare the carcinogenic effects of 1800 MHz EMR. In this study, Balb/c-3T3 cells were exposed to 1800 MHz EMR for 80 days. The cells were harvested for cell proliferation detection, cell cycle assay, plate clone, and soft agar formation assay, transwell assay, and mRNA microarray detection. 1800 MHz EMR promoted Balb/c-3T3 proliferation. No clones were observed in both plate clone and soft agar clone formation assay. The percentage of cells in S phase in Balb/c-3T3 cells of 80d Expo was obviously higher than the percetage in 80d Sham cells. 80d Expo Balb/c-3T3 cells had stronger migration ability than Sham cells. The mRNA microarray results indicated that cell cycle, cell division, and DNA replication were the main biological processes the significant genes enriched, with higher expression of RPs and Mcms. 1800 MHz EMR promoted Balb/c-3T3 cells proliferation and migration. The mRNA microarray results indicated that cell cycle, cell division, and DNA replication were the main biological processes the significant genes enriched.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important tobacco-specific nitrosamine (TSNA) that induces malignant tumors in rodents. High-risk human papillomavirus (hr-HPV) infection is an important cause of several human cancers. Epidemiological evidence has shown that HPV cooperatively induces carcinogenesis with tobacco smoke. In the present study, the synergistic carcinogenesis of NNK and HPV18 was investigated. Immortalized human esophageal epithelial SHEE cells containing the HPV18 E6E7 gene were constructed by lentiviral transfection. SHEE-E6E7 cells were exposed to NNK along with SHEE-V cells without HPV18 E6E7 as a negative control. The cooperation of NNK and HPV was examined by wound-healing, transwell, and colony-forming assays. The results showed that NNK exposure promoted the migration, invasion, and proliferation abilities of both SHEE-E6E7 and SHEE-V cells; however, the changes in these phenotypic features were remarkably stronger in SHEE-E6E7 cells than those in SHEE-V cells. Our findings indicate that NNK promotes malignant transformation of human esophageal epithelial cells and suggest a synergistic carcinogenesis with the HPV18 E6E7 oncogene. As reported previously, the formation of pyridyloxybutylated DNA adducts is a crucial step in NNK-mediated carcinogenesis. In order to clarify the influence of HPV on the formation of NNK-induced DNA adducts, the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts were determined using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. We observed that the levels of HPB-releasing adducts in SHEE-E6E7 cells were significantly higher (p < 0.01) than those of SHEE-V cells, which was in line with results of the phenotypic assays. In conclusion, this study provides direct evidence that NNK and HPV18 exhibit a synergistic effect on formation of DNA adducts, resulting in malignant transformation of esophageal epithelial cells. Such knowledge on the interaction between infection and smoking habits in the development of cancers informs cancer-prevention strategies. Further studies to delineate the molecular mechanism and to identify specific intervention targets are worthwhile.

目的 利用人乳头瘤病毒(Human papillomavirus,HPV)58突变型E6E7(Mutant type E6E7,mE6E7)融合基因为靶点,巨细胞病毒株(Towne株)细菌人工染色体(SW102 Towne bacterial artificial chromosome,SW102-T-BAC)为载体,构建携带HPV58 mE6E7融合基因的重组病毒,探讨HPV58mE6E7的转化活性.方法 PCR扩增带有50 bp Towne开放读码框(open reading frame,ORF)75左右同源臂的GalK、mE6E7及野生型E6E7(Wild type E6E7,wE6E7)融合基因片段,切胶回收纯化;将GalK片段电转到(SW102-T-BAC)感受态细胞,经过同源重组、GalK及氯霉素抗性筛选,获得SW102-T-ORF75-Galk-BAC克隆;然后分别将纯化的mE6E7及wE6E7电转到SW102-T-ORF75-Galk-BAC感受态细胞,经脱GalK及氯霉素抗性筛选,获得SW 102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC克隆;提取所得克隆质粒DNA,转染ARPE-19细胞(以转染SW102-T-BAC的细胞及未转染细胞为对照),逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达状况;观察重组病毒T-ORF75-mE6E7及T-ORF75-wE6E7对转染ARPE-19细胞的影响,通过软琼脂克隆分析mE6E7及wE6E7转化活性.结果 获得了SW102-T-ORF75-mE6E7-BAC及SW102-T-ORF75-wE6E7-BAC的克隆.逆转录PCR及测序分析验证转染细胞中mE6E7及wE6E7的表达正确.转染SW102-T-ORF75-wE6E7-BAC的细胞生长失去接触抑制,出现重叠生长现象,其形态由原来梭形变为变圆、肿胀、胞浆颗粒增多;并且在软琼脂中能够形成克隆;而转染SW102-T-ORF75-mE6E7-BAC、SW102-T-BAC及未转染细胞未出现上述细胞的特点.结论 获得了T-ORF75-mE6E7及T-ORF75-wE6E7重组病毒,并且重组病毒T-ORF75-mE6E7的转化活性已消除,为HPV58型治疗性疫苗的研究奠定了基础.