Causal genetic regulation of DNA replication on immune microenvironment in colorectal tumorigenesis: Evidenced by an integrated approach of trans-omics and GWAS

The interplay between DNA replication stress and immune microenvironment alterations is known to play a crucial role in colorectal tumorigenesis,but a comprehensive understanding of their association with and relevant biomarkers involved in colorectal tumorigenesis is lacking.To address this gap,we conducted a study aiming to investigate this association and identify relevant biomarkers.We analyzed transcriptomic and proteomic profiles of 904 colorectal tumor tissues and 342 normal tissues to examine pathway enrichment,biological activity,and the immune microenvironment.Additionally,we evaluated genetic effects of single variants and genes on colorectal cancer susceptibility using data from genome-wide association studies(GWASs)involving both East Asian(7062 cases and 195745 controls)and European(24476 cases and 23073 controls)populations.We employed mediation analysis to infer the causal pathway,and applied multiplex immunofluorescence to visualize colocalized biomarkers in colorectal tumors and immune cells.Our findings revealed that both DNA replication activity and the flap structure-specific endonuclease 1(FEN1)gene were significantly enriched in colorectal tumor tissues,compared with normal tissues.Moreover,a genetic variant rs4246215 G>T in FEN1 was associated with a decreased risk of colorectal cancer(odds ratio=0.94,95%confidence interval:0.90–0.97,P_(meta)=4.70×10^(-9)).Importantly,we identified basophils and eosinophils that both exhibited a significantly decreased infiltration in colorectal tumors,and were regulated by rs4246215 through causal pathways involving both FEN1 and DNA replication.In conclusion,this trans-omics incorporating GWAS data provides insights into a plausible pathway connecting DNA replication and immunity,expanding biological knowledge of colorectal tumorigenesis and therapeutic targets.

Regulation of eukaryotic DNA replication and nuclear structure

In eukaryote, nuclear structure is a key component forthe functions of eukaryotic cells. More and more evidencesshow that the nuclear structure plays important role in re-gulating DNA replication. The nuclear structure providesa physical barrier for the replication licensing, participatesin the decision where DNA replication initiates, and orga-nizes replication proteins as replication factory for DNAreplication. Through these works, new concepts on theregulation of DNA replication have emerged, which willbe discussed in this minireview.

FRS:Replication Driver Layer-Based Data Replication System on FreeBSD

Data replication is a key way to design a disaster tolerance system. This paper presents a replication driver layer-based data replication system on FreeBSD （FRS）. The system is embedded into the replication device driver layer, does not depend on specific storage devices and logical volume manager, and can achieve replication on data block level. The design considerations and decisions in defining FRS are described in detail.

SQL Server CE中RDA与Replication的研究

介绍移动数据库SQL Server CE及其特点,详细分析RDA(远程数据访问)和Replica-tion(合并复制)数据同步技术,探讨两种技术的各自特点和应用环境,描述两种数据同步技术的实现过程,比较这两种技术适合的环境。

一种基于Active Replication的TCP连接容错技术

本文提出一种基于ActiveReplication的TCP连接容错技术(ARTCP),采用两层的Cluster结构,并通过前端的分发器和聚合网关机维持多个同步的TCP连接副本,达到了相互容错的效果。测试结果表明,该技术不仅保证网络服务连接级的高可用性,而且具有恢复时间短、开销小的优势。

Replication Server数据复制技术及实例

介绍Replication Server的复制模型和应用程序体系结构,并举例如何利用Sybase Workspace构建一个完整的Replication Server系统应用,希望能给开发和管理Replication Server系统的管理员一些借鉴。

Replication Server数据复制技术及实例

3实例下面介绍在Sybase Workspace 1.5开发平台上,如何开发一个完整的Replication Server系统应用。实例是大型超市的POS系统数据库到经理办公室的销售数据分析数据库的复制。超市的销售数据在数据库（server_1.source）中,进行数据分析的是数据仓库（server_2.IQ）。各个超市的销售数据在数据库中是相当繁多的,但对于提供决策支持来说,只需要分析部分数据就够了。

Replication Strategy with Comprehensive Data Center Selection Method in Cloud Environments

As the amount of data continues to grow rapidly,the variety of data produced by applications is becoming more affluent than ever.Cloud computing is the best technology evolving today to provide multi-services for the mass and variety of data.The cloud computing features are capable of processing,managing,and storing all sorts of data.Although data is stored in many high-end nodes,either in the same data centers or across many data centers in cloud,performance issues are still inevitable.The cloud replication strategy is one of best solutions to address risk of performance degradation in the cloud environment.The real challenge here is developing the right data replication strategy with minimal data movement that guarantees efficient network usage,low fault tolerance,and minimal replication frequency.The key problem discussed in this research is inefficient network usage discovered during selecting a suitable data center to store replica copies induced by inadequate data center selection criteria.Hence,to mitigate the issue,we proposed Replication Strategy with a comprehensive Data Center Selection Method(RS-DCSM),which can determine the appropriate data center to place replicas by considering three key factors:Popularity,space availability,and centrality.The proposed RS-DCSM was simulated using CloudSim and the results proved that data movement between data centers is significantly reduced by 14%reduction in overall replication frequency and 20%decrement in network usage,which outperformed the current replication strategy,known as Dynamic Popularity aware Replication Strategy(DPRS)algorithm.

Dynamic Replication Strategies in Data Grid

Dynamic data replication is a technique used in data grid environments that helps to reduce access latency and network bandwidth utilization. Replication also increases data availability thereby enhancing system reliability. In this paper we discuss the issues with single-location strategies in large-scale data integration applications, and examine potential multiple-location schemes. Dynamic multiple-location replication is NP-complete in nature. We therefore transform the multiple-location problem into several classical mathematical problems with different parameter settings, to which efficient approximation algorithms apply experimental results indicate that unlike single-location strategies our multiple-location schemes are efficient with respect to access latency and bandwidth consumption, especially when the requesters of a data set are distributed over a large scale of locations.

基于MySQL Replication的数据库集群解决方案

随着电子商务的快速发展,人们对商务系统数据库的稳定性、可用性、可拓展性等提出了更高的要求,而单一的数据库服务器往往达不到这些要求。数据库集群技术的出现,为我们解决这个问题提供了很好的方法。比较了MySQL簇和MySQL Replication两种MySQL的数据库集群技术,并设计了一种基于MySQL Replication的商务系统数据库集群的解决方案。

Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.

Hepatitis B virus and micro RNAs:Complex interactions affecting hepatitis B virus replication and hepatitis B virusassociated diseases

Chronic infection with the hepatitis B virus(HBV) is the leading risk factor for the development of hepatocellular carcinoma(HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, micro RNAs(mi RNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of mi RNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between mi RNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some mi RNAs, such as mi R-122, and mi R-125 and mi R-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and mi RNAs, including how HBV affects cellular mi RNAs, how these mi RNAs impact HBV replication, and the relationship between HBV-mediated mi RNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and mi RNAs, and proposepotential applications of mi RNA-related techniques that could enhance our understanding of the role mi RNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.

HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

Establishment and primary application of a mouse model with hepatitis B virus replication

AIM： To establish a rapid and convenient animal model with hepatitis B virus （HBV） replication.METHODS： A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen （HBcAg） and hepatitis B surface antigen （HBsAg） in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen （HBeAg） was detected by enzyme- linked immunosorbent assay （ELISA）. Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid （polyIC） or phosphate-buffered saline （PBS）.RESULTS： After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS.CONCLUSION： A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome.

Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression

Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.

A Novel Replication-competent Adenovirus CNHK500 in the Treatment of Heptocellular Carcinoma In Vitro

Objective: To evaluate the therapeutic efficacy of replicative adenovirus CNHK500 in the treatment of hepatocellular carcinoma. Methods: Virus proliferation assay, cell viability assay and Western blot were performed to assess the selective replication and cytolysis of CNHK500 in telomerase positive liver cancer cells Hep3B, HepGII, SMMC7721 and in normal cells. Results: The replicative multiples of CNHK500 in HepGII, Hep3B and SMMC7221 after 96 h of virus proliferation were 52 000, 396 984.9 and 632 911.3 fold respectively, similar to those of wtAd5. However, CNHK500 demonstrated more significant attenuated replicative ability in normal cell lines than wtAd5. CNHK500 replicated only 3.1-100 fold at 96 h, while the wtAd5 still reached 3160-17 357 fold. CNHK500 could cause half of HepGII cells death within 7 days at MOI 2, in Hep3B cell lines the IC50 was as low as MOI 0.01, whereas the IC50 in BJ cell was as high as MOI 1000. CNHK500 E1A protein could only be detected in hepatocellular cancer cells but not in normal cells under normoxia. E1B protein could only be detected under hypoxia condition at a MOI of 1. Conclusion: CNHK500 can efficiently replicate in and kill liver cancer cells as well as wtAd5 do while it is severely attenuated in proliferation and cytolysis among normal cells. It would be a prominsing strategy for liver cancer tratment.

Animal models for the study of hepatitis C virus infection and replication

Hepatitis C virus (HCV) hepatitis, initially termed non-A, non-B hepatitis, has become one of the leading causes of cirrhosis and hepatocellular carcinoma worldwide. With the help of animal models, our understanding of the virus has grown substantially from the time of initial discovery. There is a paucity of available animal models for the study of HCV, mainly because of the selective susceptibility limited to humans and primates. Recent work has focused modification of animals to permit HCV entry, replication and transmission. In this review, we highlight the currently available models for the study of HCV including chimpanzees, tupaia, mouse and rat models. Discussion will include methods of model design as well as the advantages and disadvantages of each model. Particular focus is dedicated to knowledge of pathophysiologic mechanisms of HCV infection that have been elucidated through animal studies. Research within animal models is critically important to establish a complete understanding of HCV infection, which will ultimately form the basis for future treatments and prevention of disease.

Replication potentials of HIV-1/HSIV in PBMCs from northern pigtailed macaque （Macaca leonina）

The northern pig-tailed macaque （Macaca leonina） has been identified as an independent species of Old World monkey, and we previously found that PBMCs from M. leonina were susceptible to human immunodeficiency virus type 1 （HIV-1）, which may be due to the absence of a TRIM5 protein restricting HIV-1 replication. Here we investigated the infection potentials of six laboratory adapted HIV-1 strains and three primary HIV-1 isolates in PBMCs from M. leonina. The results indicate that these strains are characterized by various but low replication levels, and among which, HIV-INL4-3 shows the highest replication ability. Based on the abundant evidence of species-specific interactions between restriction factors APOBEC3 and HIV/SIV-derived Vif protein, we subsequently examined the replication potentials of v/f-substituted HIV-1 （HSIV） in M. leonina PBMCs. Notably, HSIV-vifmac and stHIV-lsv chimeras, two HIV-1Ni.4-3-derived viruses encoding the viral infectivity factor （Vif） protein from SIVmac239, replicated robustly in cells from M. leonina, which suggests that HSIV could effectively antagonize the antiviral activity of APOBEC3 proteins expressed in cells of M. leonina. Therefore, our data demonstrate that M. leonina has the potential to be developed into a promising animal model for human AIDS.

mi R-29a promotes hepatitis B virus replication and expression by targeting SMARCE1 in hepatoma carcinoma

AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.

Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution

AIM:To explore the function of Nonstructural 5A(NS5A) protein of genotype 2a(JFH1)in the replication and infection of hepatitis C virus(HCV).METHODS:Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR)method and the restriction enzyme reaction.In vitro RNA transcripts of chimera,prototype J6JFH and negative control J6JFH1(GND)were prepared and transfected into Huh-7.5 cells with liposomes.Immunofluorescence assay(IFA),fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.RESULTS:The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point(2.58 ±5.97×106 vs 4.27±1.72×104,P=0.032).The maximal level of HCV RNA in chimera was 5.6±1.8× 104 GE/μg RNA at day 34 after transfection,while the wild type reached a peak level at day 13 which was 126 folds higher(70.65±14.11×105 vs 0.56±0.90 ×105,P=0.028).HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level.Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles,ranging from 10±5 ffu/mL to 78.3±23.6 ffu/mL,while that of FL-J6JFH1 ranged from 5.8±1.5×102 ffu/mL to 2.5±1.4×104 ffu/mL.CONCLUSION:JFH1 NS5A might play an important role in the robust replication of J6JFH1.The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.