BACKGROUND: P16 inactivation is frequently accompanied by telomerase reverse transcriptase ( TERT ) amplification in human cancer genomes. P16 inactivation by DNA methylation often occurs automatically during immortalization of normal cells by TERT . However, direct evidence remains to be obtained to support the causal effect of epigenetic changes, such as P16 methylation, on cancer development. This study aimed to provide experimental evidence that P16 methylation directly drives cancer development. METHODS: A zinc finger protein-based P16 -specific DNA methyltransferase (P16-Dnmt) vector containing a "Tet-On" switch was used to induce extensive methylation of P16 CpG islands in normal human fibroblast CCD-18Co cells. Battery assays were used to evaluate cell immortalization and transformation throughout their lifespan. Cell subcloning and DNA barcoding were used to track the diversity of cell evolution. RESULTS: Leaking P16-Dnmt expression (without doxycycline-induction) could specifically inactivate P16 expression by DNA methylation. P16 methylation only promoted proliferation and prolonged lifespan but did not induce immortalization of CCD-18Co cells. Notably, cell immortalization, loss of contact inhibition, and anchorage-independent growth were always prevalent in P16-Dnmt&TERT cells, indicating cell transformation. In contrast, almost all TERT cells died in the replicative crisis. Only a few TERT cells recovered from the crisis, in which spontaneous P16 inactivation by DNA methylation occurred. Furthermore, the subclone formation capacity of P16-Dnmt&TERT cells was two-fold that of TERT cells. DNA barcoding analysis showed that the diversity of the P16-Dnmt&TERT cell population was much greater than that of the TERT cell population. CONCLUSION P16 methylation drives TERT -mediated immortalization and transformation of normal human cells that may contribute to cancer development.
Humans ; Telomerase/genetics* ; DNA Methylation/physiology* ; Fibroblasts/cytology* ; Cyclin-Dependent Kinase Inhibitor p16/metabolism* ; Cell Line ; Cell Transformation, Neoplastic/genetics*

Objective:To analyze the detection strategy and basic detection situation of markers of infectious diseases transmitted by transfusion in blood testing laboratories of blood stations in China.Methods:Based on the data of practice comparison working party of Blood Stations in Mainland of China from 2017 to 2021, the data on the testing strategies and the basic detection information of the markers for the transmission of infectious diseases through transfusion in the member laboratories of the practice comparison working party of Blood Stations in Mainland of China from 2017 to 2021 were collected, and the situation of the selection for testing markers, testing strategy and the testing method and other relevant aspects were sorted out and analyzed by charts.Results:The selection of the testing markers was consistent, but HTLV testing item was added in some member laboratories. The detection strategy of using two ELISA reagents and one nucleic acid testing (NAT) reagent simultaneously was adopted in 47 member blood stations; 3) NAT method was dominated by mini pool-NAT in member laboratories. The number of members adopting mini-pools of 8 (MP8)-NAT decreased from 17 in 2017 to 14 in 2021, while the number of members adopting mini-pools of 6 (MP6)-NAT increased from 13 in 2017 to 22 in 2021; Roche NAT system accounted for the largest proportion.Conclusions:In order to ensure blood safety and avoid missing detection, the blood stations still adopt the detection strategy of using two ELISA reagents and one nucleic acid testing (NAT) reagent simultaneously; Meanwhile, in order to increase the NAT positive rate, the proportion of mini pool-NAT mainly decreased year by year despite its dominating role, while the proportion of individual donation-NAT increased year by year; NAT method is transiting from mini-pools of 8 (MP8) to mini-pools of 6 (MP6); The proportion of imported NAT system used in NAT laboratory is relatively large.

Objective:To automatically synthesize Al 18F-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (NOTA)-fibroblast activation protein inhibitor (FAPI)-04, perform PET/CT imaging in vivo, and evaluate its diagnostic efficacy on tumors. Methods:Al 18F-NOTA-FAPI-04 was produced in All-in-one automatic synthesis module and its quality control was conducted by high performance liquid chromatography (HPLC) equipped with a radioactive detector. Al 18F-NOTA-FAPI-04 PET/CT imaging was performed in normal BALB/c mice ( n=3) and 4T1 breast cancer models ( n=3) to determine its biodistribution. Then Al 18F-NOTA-FAPI-04 and 18F-FDG PET/CT imaging were performed in a hepatocellular carcinoma patient (male, 51 years old). Results:The synthesis time of Al 18F-NOTA-FAPI-04 was about 35 min, and the radiochemical yield was (25.2±1.9)% (attenuation correction, n=3). The product was colorless transparent solution with pH value of 7.0-7.5, and the specific activity was (46.11±3.07) GBq/μmol (attenuation correction, n=3). The radiochemical purity was above 99.0% and was still above 98.0% at room temperature after 6 h. PET/CT imaging in mice showed that physiological uptake of Al 18F-NOTA-FAPI-04 was mainly in biliary system and bladder, and Al 18F-NOTA-FAPI-04 highly concentrated in tumor xenografts. PET/CT imaging in the patient showed that Al 18F-NOTA-FAPI-04 obtained high tumor background ratio (TBR) values of 4.1, 8.9, 5.4, 4.8, 2.2 in parasternal lymph nodes, anterior diaphragmatic lymph nodes, hilar lymph nodes, pancreaticoduodenal ligament lymph nodes, abdominal aortic lymph nodes, respectively, while TBR values were 1.0, 2.8, 5.0, 2.1, 1.1 by 18F-FDG. Conclusions:Al 18F-NOTA-FAPI-04 can be synthesized with short time, high radiochemical yield and good stability using All-in-one module. Al 18F-NOTA-FAPI-04 PET/CT imaging has high contrast and excellent diagnostic efficacy on tumors.

Objective:To evaluate the effects of different density rat fibroblasts on the expression of conjunctin 43 (Cx43) in cardiomyocytes and cell viability.Methods:Cardiomyocytes and fibroblasts were co-cultured using Transwell, cardiomyocytes were inoculated into the lower chamber of Transwell and fibroblasts into the upper chamber of Transwell.The cells were divided into 3 groups ( n=12 each) by a random number table method: fibroblast density 0.5×10 5 cells/ml group (group C 0.5), fibroblast density 1×10 5 cells/ml group (group C 1), and fibroblast density 2×10 5 cells/ml group (group C 2), with the density of cardiomyocytes 1×10 5 cells/ml in three groups.Cardiomyocytes and fibroblasts were co-cultured for 20 h in three groups.Cardiomyocytes were collected after co-culture for determination of cell viability (by CCK8 method), apoptosis rate (by flow cytometry), and expression of Cx43 mRNA (by quantitative real-time polymerase chain reaction) and expression of Cx43 and phosphorylated Cx43 (p-Cx43) (by Western blot). Results:There was no significant difference in the apoptosis rate of cardiomyocytes among the three groups ( P>0.05). Compared with group C 0.5, the expression of Cx43 protein and mRNA and p-Cx43 was significantly up-regulated in group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Compared with group C 1, the cardiomyocyte viability was significantly increased, and the expression of Cx43 protein and mRNA and p-Cx43 was up-regulated in group C 2 ( P<0.05). Conclusion:Rat fibroblasts up-regulate the expression of Cx43 and enhance the activity of Cx43 in cardiomyocytes and enhance cell viability in a density-dependent manner in a certain range.

Many viruses, enveloped or non-enveloped, remodel host membrane structures for their replication, assembly and escape from host cells. Herpesviruses are important human pathogens and cause many diseases. As large enveloped DNA viruses, herpesviruses undergo several complex steps to complete their life cycles and produce infectious progenies. Firstly, herpesvirus assembly initiates in the nucleus, producing nucleocapsids that are too large to cross through the nuclear pores. Nascent nucleocapsids instead bud at the inner nuclear membrane to form primary enveloped virions in the perinuclear space followed by fusion of the primary envelopes with the outer nuclear membrane, to translocate the nucleocapsids into the cytoplasm. Secondly, nucleocapsids obtain a series of tegument proteins in the cytoplasm and bud into vesicles derived from host organelles to acquire viral envelopes. The vesicles are then transported to and fuse with the plasma membrane to release the mature virions to the extracellular space. Therefore, at least two budding and fusion events take place at cellular membrane structures during herpesviruses assembly and egress, which induce membrane deformations. In this review, we describe and discuss how herpesviruses exploit and remodel host membrane structures to assemble and escape from the host cell.