Characterization of acute-on-chronic liver diseases: A multicenter prospective cohort study

BACKGROUND Acute-on-chronic liver disease(AoCLD)accounts for the majority of patients hospitalized in the Department of Hepatology or Infectious Diseases.AIM To explore the characterization of AoCLD to provide theoretical guidance for the accurate diagnosis and prognosis of AoCLD.METHODS Patients with AoCLD from the Chinese Acute-on-Chronic Liver Failure(ACLF)study cohort were included in this study.The clinical characteristics and outcomes,and the 90-d survival rate associated with each clinical type of AoCLD were analyzed,using the Kaplan-Meier method and the log-rank test.RESULTS A total of 3375 patients with AoCLD were enrolled,including 1679(49.7%)patients with liver cirrhosis acute decompensation(LC-AD),850(25.2%)patients with ACLF,577(17.1%)patients with chronic hepatitis acute exacer-bation(CHAE),and 269(8.0%)patients with liver cirrhosis active phase(LC-A).The most common cause of chronic liver disease(CLD)was HBV infection(71.4%).The most common precipitants of AoCLD was bacterial infection(22.8%).The 90-d mortality rates of each clinical subtype of AoCLD were 43.4%(232/535)for type-C ACLF,36.0%(36/100)for type-B ACLF,27.0%(58/215)for type-A ACLF,9.0%(151/1679)for LC-AD,3.0%(8/269)for LC-A,and 1.2%(7/577)for CHAE.CONCLUSION HBV infection is the main cause of CLD,and bacterial infection is the main precipitant of AoCLD.The most common clinical type of AoCLD is LC-AD.Early diagnosis and timely intervention are needed to reduce the mortality of patients with LC-AD or ACLF.

Application of Structural Equation Model to Evaluate the Perception of Service Quality of Medical Staffs of infectious Disease Department in Chinese Hospitals

Objective To enhance the quality of medical service for Chinese patients through research of service quality from Chinese medical personnel. Methods Serv Qual scale was used for infection medical staffs randomly by sampling questionnaire in Beijing, Shanghai, Chengdu, Chongqing, Guangzhou and Nanning. The data collected were entered and analyzed using SPSS 20.0. Statistical methods included frequency, factor analysis, reliability analysis, correlation analysis, independent samples t test, one-way analyses of variance, simultaneous regression analysis and structural equation model analysis. Results The Kaiser-Meyer-Olkin value for the factor analysis of the scale was 0.970. The Cronbach's α for the reliability analysis was 0.975. The Pearson correlation coefficients were 0.624-0.874 and statistically significant. Undergraduates felt good, Ph D students felt bad; the doctors felt bad; managers felt good. Standard 5 dimensions of the regression coefficients were positive, including empathy(β = 0.288), reliability(β = 0.241) impacting on perceived service quality mostly. The control ability and stability of the standard error of perceived service quality directly effected value were 0.646 and 0.382, respectively. Conclusions Medical staffs of infectious disease department have poor perception of service quality. Hospitals should improve awareness and of clinicians and deepen the reform of the medical care system.

Licorice-saponin A3 is a broad-spectrum inhibitor for COVID-19 by targeting viral spike and anti-inflammation

Currently,human health due to corona virus disease 2019(COVID-19)pandemic has been seriously threatened.The coronavirus severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike(S)protein plays a crucial role in virus transmission and several S-based therapeutic approaches have been approved for the treatment of COVID-19.However,the efficacy is compromised by the SARS-CoV-2 evolvement and mutation.Here we report the SARS-CoV-2 S protein receptor-binding domain(RBD)inhibitor licorice-saponin A3(A3)could widely inhibit RBD of SARS-CoV-2 variants,including Beta,Delta,and Omicron BA.1,XBB and BQ1.1.Furthermore,A3 could potently inhibit SARS-CoV-2 Omicron virus in Vero E6 cells,with EC50 of 1.016μM.The mechanism was related to binding with Y453 of RBD determined by hydrogen-deuterium exchange mass spectrometry(HDX-MS)analysis combined with quantum mechanics/molecular mechanics(QM/MM)simulations.Interestingly,phosphoproteomics analysis and multi fluorescent immunohistochemistry(mIHC)respectively indicated that A3 also inhibits host inflammation by directly modulating the JNK and p38 mitogen-activated protein kinase(MAPK)pathways and rebalancing the corresponding immune dysregulation.This work supports A3 as a promising broad-spectrum small molecule drug candidate for COVID-19.

Expression level of interferon-stimulated genes PKR,OAS1,MX1,and ISG15 in peripheral blood mononuclear cells of COVID-19 patients:A retrospective study

Objective:To explore expression level of interferon-stimulated genes PKR,OAS1,MX1,and ISG15 in peripheral blood mononuclear cells of COVID-19 patients.Methods:In this study,changes in the expression of four interferon-stimulated genes(ISGs),including PKR,OAS1,MX1,and ISG15,in peripheral blood mononuclear cells of 45 COVID-19 patients with different severities were evaluated by real-time PCR method.Results:OAS1,MX1,PKR,and ISG15 were differently expressed in COVID-19 patients with different severity.The results showed that the expression of OAS1,MX1,PKR,and ISG15 genes was significantly(P=0.001)lower in severe patients.Conclusions:Weak and defective IFN response and subsequent disruption of ISGs may be associated with COVID-19 severity.

Deciphering resistancemechanisms and novel strategies to overcome drug resistance in ovarian cancer:a comprehensive review

Ovarian cancer is among the most lethal gynecological cancers,primarily due to the lack of specific symptoms leading to an advanced-stage diagnosis and resistance to chemotherapy.Drug resistance(DR)poses the most significant challenge in treating patients with existing drugs.The Food and Drug Administration(FDA)has recently approved three new therapeutic drugs,including two poly(ADP-ribose)polymerase(PARP)inhibitors(olaparib and niraparib)and one vascular endothelial growth factor(VEGF)inhibitor(bevacizumab)for maintenance therapy.However,resistance to these new drugs has emerged.Therefore,understanding the mechanisms of DR and exploring new approaches to overcome them is crucial for effective management.In this review,we summarize the major molecular mechanisms of DR and discuss novel strategies to combat DR.

sTREM-1 as promising prognostic biomarker for acute-on-chronic liver failure and mortality in patients with acute decompensation of cirrhosis

BACKGROUND Acute decompensation(AD)of cirrhosis is associated with high short-term mortality,mainly due to the development of acute-on-chronic liver failure(ACLF).Thus,there is a need for biomarkers for early and accurate identification of AD patients with high risk of development of ACLF and mortality.Soluble triggering receptor expressed on myeloid cells-1(sTREM-1)is released from activated innate immune cells and correlated with various inflammatory processes.AIM To explore the prognostic value of sTREM-1 in patients with AD of cirrhosis.METHODS A multicenter prospective cohort of 442 patients with cirrhosis hospitalized for AD was divided into a study cohort(n=309)and validation cohort(n=133).Demographic and clinical data were collected,and serum sTREM-1 was measured at admission.All enrolled patients were followed-up for at least 1 year.RESULTS In patients with AD and cirrhosis,serum sTREM-1 was an independent prognosis predictor for 1-year survival and correlated with liver,coagulation,cerebral and kidney failure.A new prognostic model of AD(P-AD)incorporating sTREM-1,blood urea nitrogen(BUN),total bilirubin(TBil),international normalized ratio(INR)and hepatic encephalopathy grades was established and performed better than the model for end-stage liver disease(MELD),MELD-sodium(MELD-Na),chronic liver failure-consortium(CLIF-C)ACLF and CLIF-C AD scores.Additionally,sTREM-1 was increased in ACLF and predicted the development of ACLF during first 28-d follow-up.The ACLF risk score incorporating serum sTREM-1,BUN,INR,TBil and aspartate aminotransferase levels was established and significantly superior to MELD,MELD-Na,CLIF-C ACLF,CLIF-C AD and P-AD in predicting risk of ACLF development.CONCLUSION Serum sTREM-1 is a promising prognostic biomarker for ACLF development and mortality in patients with AD of cirrhosis.

Immunodiagnosis of human hydatid disease: Where do we stand?

Cystic echinococcosis(CE) is a zoonotic parasitic infection caused by the larval stage of Echinococcus granulosus. Diagnosis of CE mainly relies on a combination of serological testing along with imaging approaches. A variety of serological methods, mainly based on hydatid cyst fluid, antigen B(Ag B) and antigen 5, have been developed and used for immunodiagnosis of CE, yet their performances are not satisfactory. Although utilizing of recombinant or synthetic antigens, improved the performance of serological tests, it has not applicably overcome the problem of low sensitivity and cross reactivity, seen in the diagnosis of CE. Performances of immunodiagnostic tests based on Ag B subunits are promising. The 8 k Da subunit of Ag B is the most studied antigen in native, synthetic or recombinant form for diagnosis of CE. From the 5 subunits of Ag B, antigen B8/1 and B8/2 provided the highest diagnostic sensitivity and specificity. Moreover, detecting of specific antibodies of Ig G subclasses has improved the efficacy of immunodiagnostic tests. Among the Ig G subclasses, both Ig G2 and Ig G4 are considered as good markers for diagnosis and Ig G4 as a suitable marker for follow up of the patients. In this review an overview of immunodiagnostic methods, related antigens and their performances in the diagnosis of CE are given. The paper highlights pitfall and challenges in the serological diagnosis of CE. Moreover, limitation of currently available immunodiagnostic tests and the most recent development in the designing and application of serological assays for diagnosis of CE in human are addressed.

Prevalence and clinical characteristics of autoimmune liver disease in hospitalized patients with cirrhosis and acute decompensation in China

BACKGROUND Autoimmune liver disease(AILD)has been considered a relatively uncommon disease in China,epidemiological data for AILD in patients with cirrhosis and acute decompensation(AD)is sparse.AIM To investigate the prevalence,outcome and risk factors for AILD in cirrhotic patients complicated with AD in China.METHODS We collected data from patients with cirrhosis and AD from two prospective,multicenter cohorts in hepatitis B virus endemic areas.Patients were regularly followed up at the end of 28-d,90-d and 365-d,or until death or liver transplantation(LT).The primary outcome in this study was 90-d LTfree mortality.Acute-on-chronic liver failure(ACLF)was assessed on admission and during 28-d hospitalization,according to the diagnostic criteria of the European Association for the Study of the Liver(EASL).Risk factors for death were analyzed with logistic regression model.RESULTS In patients with cirrhosis and AD,the overall prevalence of AILD was 9.3%(242/2597).Prevalence of ACLF was significantly lower in AILD cases(14%)than those with all etiology groups with cirrhosis and AD(22.8%)(P<0.001).Among 242 enrolled AILD patients,the prevalence rates of primary biliary cirrhosis(PBC),autoimmune hepatitis(AIH)and PBC-AIH overlap syndrome(PBC/AIH)were 50.8%,28.5%and 12.0%,respectively.In ACLF patients,the proportions of PBC,AIH and PBC/AIH were 41.2%,29.4% and 20.6%.28-d and 90-d mortality were 43.8% and 80.0% in AILD-related ACLF.The etiology of AILD had no significant impact on 28-d,90-d or 365-d LTfree mortality in patients with cirrhosis and AD in both univariate and multivariate analysis.Total bilirubin(TB),hepatic encephalopathy(HE)and blood urea nitrogen(BUN)were independent risk factors for 90-d LT-free mortality in multivariate analysis.The development of ACLF during hospitalization only independently correlated to TB and international normalized ratio.CONCLUSION AILD was not rare in hospitalized patients with cirrhosis and AD in China,among which PBC was the most common etiology.90-d LT-free mortality were independently associated with TB,HE and BUN.

Tenofovir disoproxil fumarate in Chinese chronic hepatitis B patients:Results of a multicenter,double-blind,double-dummy,clinical trial at 96 weeks

BACKGROUND Tenofovir disoproxil fumarate(TDF)is a prodrug of a nucleotide analogue.As an antiviral drug,TDF has been proposed in the first-line treatment of chronic hepatitis B(CHB).Qingzhong,a brand name of TDF,commercialized by Jiangsu Chia-tai Tianqing Pharmaceutical Co Ltd.,and Viread,another brand name of TDF,commercialized by GlaxoSmithKline,have both been approved by the State Food and Drug Administration,China.AIM To investigate the efficacy and safety of the two TDF agents in the treatment of Chinese CHB patients.METHODS This trial was registered at ClinicalTrials.gov with the identifier number of NCT02287857.A total of 330 Chinese CHB patients,among which 232 were hepatitis B e antigen(HBeAg)-positive,were included in this 5-year-long,multicenter,double-blinded,double-dummy,randomized-controlled,noninferiority phase III trial.The participants were initially randomized into two groups:Group A(n=161),in which the participants received 300 mg Qingzhong once a day for 48 wk;and Group B,in which the participants received 300 mg Viread once a day for 48 wk.Starting from week 49,all the participants in Groups A and B received 300 mg Qingzhong once a day until the 96th week.In this study,the primary endpoint was the decrease in plasma level of hepatitis B virus(HBV)DNA at the 96th week,while the secondary endpoints were suppression of HBV replication,alanine aminotransferase(ALT)normalization,HBeAg loss,and HBeAg seroconversion rates.RESULTS For the participants with HBeAg-positive CHB,the decrease in mean HBV DNA level relative to the baseline value was comparable between Groups A and B(5.77 vs 5.73 log10 IU/mL,P>0.05)at the 96th week.In addition,similar percentages of HBeAg-positive participants in the two groups exhibited undetectable levels of HBV DNA,HBeAg loss,and HBeAg seroconversion(71.05%vs 77.97%,31.00%vs 27.27%,and 20.22%vs 15.79%,respectively,in Group A vs Group B;P>0.05).For the participants with HBeAg-negative CHB,the decrease in mean HBV DNA level relative to the baseline value was also comparable between Groups A and B(4.46 vs 4.70 log10 IU/mL,P>0.05)at the 96th week.In addition,similar percentages of HBeAg-negative participants in the two groups exhibited undetectable levels of HBV DNA(87.23%vs 94.12%in Group A vs Group B,respectively;P>0.05).Finally,similar percentages of CHB patients(HBeAg-positive or HBeAg-negative)in the two groups exhibited normalization of ALT(80.14%vs 84.57%in Group A vs Group B,respectively;P>0.05),and similar incidences of adverse events were observed(106 vs 104 in Group A vs Group B,respectively;P>0.05).CONCLUSION Both Qingzhong and Viread are effective and safe in the treatment of Chinese CHB patients according to the results of our clinical trial.

Investigation on Infectious Agents of Aborted Pig Fetuses and Its Correlation with PRRSV MLV Vaccine

Infectious agents causing aborted fetus problems in domestic pigs were investigated in this study. More than 10 different infectious agents were known to cause abortion in swine and the major eight viruses among them were inspected. One hundred twelve samples of aborted fetuses from nine provinces in South Korea were collected during April to November, 2013 in this study for the diagnosis of infectious agents causing abortions in pigs. Eight major infection viruses were examined in this study mainly using various diagnostic kits and reverse transcriptase polymerase chain reaction （RT-PCR）. Positive rate of the detection differed from each viruses. In this study, the main focus was the porcine reproductive and respiratory syndrome virus （PRRSV）, which took the second large portion in the positive rate of detection, and then its ORF5 gene was compared with modified live virus （MLV） vaccine strain to figure out the influence of vaccine on disease. Between four positive samples＇ sequence, two of them were 99.9%-100% similar to MLV vaccine strain and two other samples were 88.6%-92.7% similar. Similarity rate of the sequences between the vaccine and virus from aborted fetuses are very crucial, because it implies that abortion in swine can be made due to the usage of vaccine not only by the infection of field virus, and if MLV vaccine actually do have an impact on the infection, usage of the vaccine should be reconsidered.

Circulating tumor cells and circulating tumor DNA in breast cancer diagnosis and monitoring

Liquid biopsy,including both circulating tumor cells and circulating tumor DNA,is becoming more popular as a diagnostic tool in the clinical management of breast cancer.Elevated concentrations of these biomarkers during cancer treatment may be used as markers for cancer progression as well as to understand the mechanisms underlying metastasis and treatment resistance.Thus,these circulating markers serve as tools for cancer assessing and monitoring through a simple,non-invasive blood draw.However,despite several study results currently noting a potential clinical impact of ctDNA mutation tracking,the method is not used clinically in cancer diagnosis among patients and more studies are required to confirm it.This review focuses on understanding circulating tumor biomarkers,especially in breast cancer.

血清N-聚糖生物标志物诊断ALT水平正常慢性乙型肝炎患者显著肝纤维化和肝硬化的临床意义

本研究目的是探讨血清N-聚糖模型在285例丙氨酸转移酶(alanine aminotransferase,ALT)水平正常(<40 U·L^(-1))的慢性乙型肝炎(慢性乙肝)患者中诊断显著肝纤维化和肝硬化的临床意义。入组患者均进行肝组织活检,并使用Ishak评分系统评估患者肝组织纤维化程度。应用基于DNA测序仪的荧光糖电泳技术检测患者血清N-聚糖图谱,每例患者的血清样本中共鉴定出9个N-聚糖峰。利用机器学习算法,即随机森林(random forest,RF)构建更理想的血清N-聚糖模型,以诊断显著肝纤维化(≥F3)和肝硬化(≥F5),并比较血清N-聚糖模型和其他纤维化标志物的诊断效能。肝组织活检结果显示,有显著肝纤维化和肝硬化患者分别占63.86%(182/285)和16.49%(47/285),有显著炎症患者为4.91%(14/285)。血清N-聚糖RF-A模型具有很好的诊断显著肝纤维化(≥F3)的效能,其受试者工作特征曲线下面积(area under receiver operating characteristic curve,AUROC)为0.94,与肝活检的符合率为90.45%。在诊断肝硬化(≥F5)时,血清N-聚糖RF-B模型的AUROC为0.97,与肝组织活检的符合率为88.94%。血清N-聚糖模型(RF-A和RF-B)的诊断效能优于肝硬度值测量(liver stiffness measurement,LSM)、基于4因子的纤维化指数(fibrosis index based on the four factors,FIB-4)和天冬氨酸转氨酶与血小板比率指数(aspartate aminotransferase-to-platelet ratio index,APRI)。在ALT水平正常的慢性乙肝患者中,血清N-聚糖模型可作为诊断显著肝纤维化或肝硬化的潜在生物标志物。

草酸盐摄入量及个人习惯与肾结石风险的关系

肾结石是一种易复发的泌尿系统疾病。大多数肾结石是钙结石,通常由草酸钙或磷酸钙组成。尿液中可溶性钙、草酸盐、磷酸盐和柠檬酸盐过饱和是钙结石形成的基础。遗传、饮食、缺乏体力活动和个人习惯都会导致肾结石的形成。在这篇综述中,我们总结了肾结石风险与草酸盐摄入量和一些个人习惯(如吸烟、饮酒和吸毒)的关系。

Biomaterial-enhanced treg cell immunotherapy:A promising approach for transplant medicine and autoimmune disease treatment

Regulatory T cells(Tregs)are crucial for preserving tolerance in the body,rendering Treg immunotherapy a promising treatment option for both organ transplants and autoimmune diseases.Presently,organ transplant recipients must undergo lifelong immunosuppression to prevent allograft rejection,while autoimmune disorders lack definitive cures.In the last years,there has been notable advancement in comprehending the biology of both antigen-specific and polyclonal Tregs.Clinical trials involving Tregs have demonstrated their safety and effectiveness.To maximize the efficacy of Treg immunotherapy,it is essential for these cells to migrate to specific target tissues,maintain stability within local organs,bolster their suppressive capabilities,and ensure their intended function’s longevity.In pursuit of these goals,the utilization of biomaterials emerges as an attractive supportive strategy for Treg immunotherapy in addressing these challenges.As a result,the prospect of employing biomaterial-enhanced Treg immunotherapy holds tremendous promise as a treatment option for organ transplant recipients and individuals grappling with autoimmune diseases in the near future.This paper introduces strategies based on biomaterial-assisted Treg immunotherapy to enhance transplant medicine and autoimmune treatments.

1995-2005年青海省棘球蚴病流行病学调查分析

目的分析1995-2005年青海省人与动物棘球蚴病的流行病学调查结果。方法人群棘球蚴病感染情况以Bu-ELISA、EM18-ELISA和B超、X线进行检查和评价。动物棘球蚴病/棘球绦虫感染调查采用解剖学方法和寄生虫学方法。结果①女性人群的血清阳性率和患病率显著高于男性人群(14.41%和4.81%、10.22%和3.25%);随年龄增长,人群血清阳性率和患病率升高(见表5);以牧民、喇嘛的血清阳性率和患病率最高(17.73%和9.33%、18.56%和10.0%)。②青海省人与动物棘球蚴病/棘球绦虫的血清阳性率和感染/患病率以青南高原的果洛、玉树、黄南三州最高(见表1、7、8),祁连山地和河湟谷地的海南、海北两州次之,海东地区、西宁及柴达木盆地的海西州较低。③青海省是以囊型棘球蚴病/细粒棘球绦虫为主的囊型和泡型棘球蚴病的混合流行区,并在其南部发现存在石渠棘球绦虫的动物感染。结论青海省棘球绦虫的生活史循环链十分复杂,家养动物相互之间、野生动物相互之间、家养动物和野生动物之间均参与其中。

隐孢子虫SSU rRNA基因的巢式PCR-RFLP分析

目的 建立用巢式聚合酶链反应 (NestedPCR) 限制片段长度多态性 (Restrictionfragmentlength polymor phism ,RFLP)检测、鉴别微小隐孢子虫 (Cryptospordium parvum)和安氏隐孢子虫 (C .andersoni)的方法。 方法 用巢式PCR技术扩增长春市人和牛粪便中C .parvum与C .andersoni的Small SubunitrRNA (SSUrRNA)部分基因 ,并克隆和测序。扩增产物分别用SspⅠ和VspⅠ单酶切 ,进行RFLP分析 ,并与GenBank上的 17株隐孢子虫相应序列进行对比分析和系统发育分析。 结果 C .parvumHCC1、C .parvumBOCC1和C .andersoniBOCC1扩增产物片段大小分别为 83 0bp、83 0bp和 82 8bp ,经SspⅠ和VspⅠ分别单酶切后形成 2种不同的RFLP图谱 ,根据RFLP图谱可鉴别C .parvum与C .andersoni。序列分析结果显示 ,C .parvumHCC1与国外牛源C .parvumBOH 6、C .parvumGCH1、鹿源C .parvumDR1相应序列同源性均为 99.3 % ;C .parvumBOCC1与国外牛源C .parvumBOH6、C .parvumGCH1、鹿源C .parvumDR1相应序列同源性均为 99.5 % ;C .andersoniBOCC1与国外C .andersoni相应序列同源性为 99.9%。系统发育分析结果显示 ,隐孢子虫虫种形成了 2个群 ,1个群包括C .parvum、C .baileyi、C .meleagridis、C .felis和C .wrairi,另 1个群包括C .

应用巢式PCR-RFLP方法鉴别微小隐孢子虫、安氏隐孢子虫和火鸡隐孢子虫的研究

应用巢式聚合酶链反应(NestedPCR)-限制片段长度多态性(restrictionfragmentlengthpolymor-phism,RFLP)方法对微小隐孢子虫(C.parvum)、安氏隐孢子虫(C.andersoni)和火鸡隐孢子虫(C.melea-grides)的鉴别进行了研究。结果显示C.parvumBOCC2、C.andesoniBOCC2和C.meleagridesCHCC1扩增产物片段大小分别为830bp、828bp和828bp,扩增产物分别经VspⅠ酶切后形成3种不同的RFLP图谱,根据RFLP图谱可鉴别C.parvum、C.andersoni和C.meleagrides。本研究为我国隐孢子虫的分类和隐孢子虫病的分子流行病学研究打下了良好基础。

美国耐甲氧西林金黄色葡萄球菌感染治疗临床治疗指南(一)

美国感染病学会（IDSA）制订了治疗耐甲氧西林金黄色葡萄球菌（MRSA）患者的循证指南。该指南用于指导MRSA感染患者的治疗。该指南就多种与MRSA疾病相关的临床症状的治疗进行了论述,包括：皮肤和软组织感染（SSTI）、菌血症和感染性心内膜炎、肺炎、骨和关节感染、中枢神经系统（CNS）感染等。该指南重点介绍了关于万古霉素用量及监测,以及对万古霉素低敏的耐甲氧西林金黄色葡萄球菌菌株感染的治疗方案及治疗失败的处置。

Integrative analysis of aberrant Wnt signaling in hepatitis B virus-related hepatocellular carcinoma

AIM: To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma(HCC).METHODS: This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer(COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function(GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of Cp G islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nestedreverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ2 test as appropriate.RESULTS: Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC(P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies(P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates thatthe mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC.CONCLUSION: In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation.

Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication

AIM: To screen and investigate the effective g RNAs against hepatitis B virus(HBV) of genotypes A-D.METHODS: A total of 15 g RNAs against HBV of genotypes A-D were designed. Eleven combinations of two above g RNAs(dual-g RNAs) covering the regulatory region of HBV were chosen. The efficiency of each g RNA and 11 dual-g RNAs on the suppression of HBV(genotypes A-D) replication was examined by the measurement of HBV surface antigen(HBs Ag) or e antigen(HBe Ag) in the culture supernatant. The destruction of HBV-expressing vector was examined in Hu H7 cells co-transfected with dual-g RNAs and HBVexpressing vector using polymerase chain reaction(PCR) and sequencing method, and the destruction of ccc DNAwas examined in Hep AD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase(PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these g RNAs was assessed by a mitochondrial tetrazolium assay.RESULTS: All of g RNAs could significantly reduce HBs Ag or HBe Ag production in the culture supernatant, which was dependent on the region in which g RNA against. All of dual g RNAs could efficiently suppress HBs Ag and/or HBe Ag production for HBV of genotypes A-D, and the efficacy of dual g RNAs in suppressing HBs Ag and/or HBe Ag production was significantly increased when compared to the single g RNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual g RNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used g RNAs. Most importantly, g RNA-5 and g RNA-12 combination not only could efficiently suppressing HBs Ag and/or HBe Ag production, but also destroy the ccc DNA reservoirs in Hep AD38 cells.CONCLUSION: These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates(genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV ccc DNA in chronic HBV infection patients.