Machine learning-based identification of symptomatic carotid atherosclerotic plaques with dual-energy computed tomography angiography.

OBJECTIVE: This study aimed to develop and validate a machine learning model incorporating both dual-energy computed tomography (DECT) angiography quantitative parameters and clinically relevant risk factors for the identification of symptomatic carotid plaques to prevent acute cerebrovascular events. METHODS: The data of 180 patients with carotid atherosclerosis plaques were analysed from January 2017 to December 2021; 110 patients (64.03±9.58 years old, 20 women, 90 men) were allocated to the symptomatic group, and 70 patients (64.70±9.89 years old, 50 women, 20 men) were allocated to the asymptomatic group. Overall, five machine learning models using the XGBoost algorithm, based on different CT and clinical features, were developed in the training cohort. The performances of all five models were assessed in the testing cohort using receiver operating characteristic curves, accuracy, recall rate, and F1 score. RESULTS: The shapley additive explanation (SHAP) value ranking showed fat fraction (FF) as the highest among all CT and clinical features and normalised iodine density (NID) as the 10th. The model based on the top 10 features from the SHAP measurement showed optimal performance (area under the curve [AUC] .885, accuracy .833, recall rate .933, F1 score .861), compared with the other four models based on conventional CT features (AUC .588, accuracy .593, recall rate .767, F1 score .676), DECT features (AUC .685, accuracy .648, recall rate .667, F1 score .678), conventional CT and DECT features (AUC .819, accuracy .740, recall rate .867, F1 score .788), and all CT and clinical features (AUC .878, accuracy .833, recall rate .867, F1 score .852). CONCLUSION: FF and NID can serve as useful imaging markers of symptomatic carotid plaques. This tree-based machine learning model incorporating both DECT and clinical features could potentially comprise a non-invasive method for identification of symptomatic carotid plaques to guide clinical treatment strategies.

[Cardiac Structural and Functional Features in Patients With Type 2 Diabetes Mellitus and Heart Failure With Preserved Ejection Fraction:A Study Based on Propensity Score Matching].

Objective To investigate the cardiac structural and functional characteristics in the patients with heart failure with preserved ejection fraction (HFpEF) and type 2 diabetes mellitus (T2DM),and predict the factors influencing the characteristics. Methods A total of 783 HFpEF patients diagnosed in the Department of Geriatric Cardiology,the First Hospital of Lanzhou University from April 2009 to December 2020 were enrolled in this study.Echocardiography and tissue Doppler technique were employed to evaluate cardiac structure and function.According to the occurrence of T2DM,the patients were assigned into a HFpEF+T2DM group (n=332) and a HFpEF group (n=451).Propensity score matching (PSM)(in a 1∶1 ratio) was adopted to minimize confounding effect.According to urinary albumin excretion rate (UAER),the HFpEF+T2DM group was further divided into three subgroups with UAER<20 µg/min,of 20-200 µg/min,and>200 µg/min,respectively.The comorbidities,symptoms and signs,and cardiac structure and function were compared among the groups to clarify the features of diabetes related HFpEF.Multivariate linear regression was conducted to probe the relationship of systolic blood pressure,blood glucose,glycosylated hemoglobin,and UARE with cardiac structural and functional impairment. Results The HFpEF+T2DM group had higher prevalence of hypertension (P=0.001) and coronary heart disease (P=0.036),younger age (P=0.020),and larger body mass index (P=0.005) than the HFpEF group,with the median diabetic course of 10 (3,17) years.After PSM,the prevalence of hypertension and coronary heart disease,body mass index,and age had no significant differences between the two groups(all P>0.05).In addition,the HFpEF+T2DM group had higher interventricular septal thickness (P=0.015),left ventricular posterior wall thickness (P=0.040),and left ventricular mass (P=0.012) and lower early diastole velocity of mitral annular septum (P=0.030) and lateral wall (P=0.011) than the HFpEF group.Compared with the HFpEF group,the HFpEF+T2DM group showed increased ratio of early diastolic mitral filling velocity to early diastolic mitral annular velocity (E/e') (P=0.036).Glycosylated hemoglobin was correlated with left ventricular mass (P=0.011),and the natural logarithm of UAER with interventricular septal thickness (P=0.004),left ventricular posterior wall thickness (P=0.006),left ventricular mass (P<0.001),and E/e' ratio (P=0.049). Conclusion The patients with both T2DM and HFpEF have thicker left ventricular wall,larger left ventricular mass,more advanced left ventricular remodeling,severer impaired left ventricular diastolic function,and higher left ventricular filling pressure than the HFpEF patients without T2DM.Elevated blood glucose and diabetic microvascular diseases might play a role in the development of the detrimental structural and functional changes of the heart.

New insight into the response and transport of nitrate in karst groundwater to rainfall events.

Although numerous studies focused on nitrate source, transformation and transport of river water in karst area have been reported, it's still unclear in understanding nitrate main source and transformation in karst groundwater system and how nitrate transport from soil to water during rainfall events in karst critical zone. In order to explore the response and transport of nitrate in karst groundwater to rainfall events, different depths of well water before, during and after rainfall event were sampled, and hillslope runoff, surface runoff of different land-use types during rainfall event were sampled synchronously at a typical karst agricultural catchment in Southwest China. Results showed that fluctuations of EC, pH and DO in deep borehole well (W1) and artesian well (W2) were small, on the contrary, variations of EC and DO in shallow well (W3) were large during sampling period. The nitrate concentrations and isotopic values indicated that nitrate in karst groundwater mainly originated from chemical fertilizer (CF), and influenced by denitrification process. High intensity of denitrification was observed in deep groundwater (87%) and artesian well water (almost 100%). Extremely high dual nitrate isotope values up to 46.8 ± 1.5‰ and 24.7 ± 0.5‰ were found in the deep artesian well. The small variation of water chemistry (EC, DO and pH), nitrate concentration and dual nitrate isotope values in deep wells during sampling period suggested that newly supplied nitrogen in deep groundwater during rainfall events also comes from deep groundwater. Low nitrogen concentrations in hillslope subsurface flow and surface runoff suggests that nitrogen transport process leading to increase of water nitrogen content mainly occur in depression. Nitrogen in depression soil is mainly transported to groundwater through fissures, fractures and conduits, rather than through vertical migration processes in the soil during rainfall events.

The prognostic value of soluble Klotho in patients with haemodialysis: a systematic review and meta-analysis.

AIM: The correlation between soluble Klotho (sKlotho) levels and clinical outcomes remains inconclusive for patients undergoing maintenance haemodialysis (MHD). We aimed to evaluate the potential predictive significance of sKlotho in this population by conducting a meta-analysis. METHODS: PubMed, Embase, Web of Science and Cochrane Library were comprehensively searched for studies concerning the association between sKlotho level and clinical outcomes including cardiovascular (CV) events and all-cause mortality. The pooled hazard ratios (HR) and 95% confidence intervals (CI) were generated using either random or fixed effects models. Sensitivity and subgroup analyses were used to explore heterogeneity sources. RESULTS: Eight prospective studies with 992 MHD participants were included and reduced sKlotho levels predicted more adverse outcomes in this meta-analysis. The pooled HRs and 95% CIs related to CV events, mortality, or composite outcomes were 1.73 (95% CI 1.08-2.76, p = 0.02), 2.34 (95% CI 1.34-2.07, p = 0.003) or 1.75 (95% CI 1.19-2.57, p = 0.005). Moderate heterogeneity was observed in the composite adverse outcomes (I 2 = 57%, p = 0.05). Age and sKlotho level were the main sources of heterogeneities in the subgroup analysis. CONCLUSION: Lower sKlotho levels were associated with more CV events and all-cause mortality, suggesting that sKlotho may have predictive value in CKD patients receiving haemodialysis.

Antihypertensive Effect of Tianmu Jiangya Powder on SHR Rats and Its Mechanism / 中国实验方剂学杂志

Objective::To investigate the antihypertensive effect of Tianmu Jiangya powder and its related antihypertensive mechanism by using SHR rats as a model, and protein expressions provide an experimental basis for the clinical application of Tianmu Jiangya powder in the treatment of hypertension. Method::Sixty male SHR rats were randomly divided into six groups according to body weight after one week of adaptive feeding: model group, valsartan group (12 mg·kg-1), captopril group (9 mg·kg-1), hydrochlorothiazide group (6 mg·kg-1), Tianmu Jiangya powder low and high-dose group (0.36, 1.44 g·kg-1), WKY rats were used as the normal group, and the intragastric administration lasted for 16 weeks. Softron BP-2010A intelligent non-invasive blood pressure meter was used to measure the systolic blood pressure (SBP)and heart rate (HR) of rat tail arteries. Adobe Photoshop CS5 software was used to analyze the left auricle and claw fixed selected areas to evaluate the effect on blood stasis syndrome. Vevo 2100 small animal ultrasound imaging system detects left ventricular ejection fraction (LVEF), left ventricular shortening (FS), left ventricular end-systolic volume (LVESV), left ventricular end-diastolic volume (LVEDV), left ventricular end-systole dimension (LVIDs), left ventricular end-diastole dimension (LVIDd), interventricular septum end-systolic depth (IVSs), and interventricular septum end-diastolic depth (IVSd). Then the rats were sacrificed and the materials were taken (blood, heart, aorta, liver, kidney, tibia), and the weight of heart, liver, kidney and tibia length were measured and recorded. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of the heart and thoracic aorta. Separation of serum and plasma, and determination of nitric oxide (NO) in serum by nitrate reductase method. Radioimmunoassay was used to detect plasma adrenaline/3 methoxyadrenaline (MN), urea (UREA), and uric acid (UA) contents. The expression of nitric oxide synthase(iNOS) and vascular endothelial growth factor(VEGF) protein in thoracic aorta of each group was detected and analyzed by immunohistochemical method. Result::Compared with normal group, the SBP and HR of the rats in model group were significantly increased (P<0.05). The r value of the claw was significantly reduced and the g value was significantly increased at 8 and 16 weeks (P<0.05). LVEF and FS significantly decreased, LVESV, LVIDs, IVSd increased significantly (P<0.05). Heart weight, heart weight /tibia length, liver weight and liver weight /tibia length, plasma of MN, UREA, and UA contents significantly increased, and promoted the expression of iNOS and VEGF proteins in the aortic (P<0.05). Compared with the model group, the Tianmu Jiangya powder administration group could continuously reduce SBP in SHR rats, maintain HR stability (P<0.05), significantly increase the claw of r value, lower the claw of g value(P<0.05). LVEF, FS significantly increased, LVEDV, LVESV, LVIDd and LVIDs significantly decreased (P<0.05), significantly increased serum NO content, decreased liver weight, liver weight/tibia length, plasma MN, UREA, UA content (P<0.05), and down-regulated the expression of iNOS and VEGF protein in the aorta(P<0.05). Conclusion::Tianmu Jiangya powder has a certain antihypertensive effect, and its mechanism may be mainly related to protecting heart function, improving vascular endothelial function, reducing catecholamines and sedative analgesia.

Salvianolic Acid A Protects Neonatal Cardiomyocytes Against Hypoxia/Reoxygenation-Induced Injury by Preserving Mitochondrial Function and Activating Akt/GSK-3β Signals / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role.@*METHODS@#An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting.@*RESULTS@#SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002.@*CONCLUSION@#SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.

Research and development of safety management information system for neonatal bathroom and its clinical application: (A STROBE-compliant article).

We developed a bathroom safety management information system to decrease adverse nursing events, and observed the application of the self-developed safety management information system in neonatal bathroom.A total of 3482 newborns receiving neonatal bath and rooming in between May 2015 and May 2017, were enrolled in this study. Of the 3482 newborns, 1727 that did not use the safety management information system from May 1, 2015 to May 31, 2016, were considered as control group; and other 1755 that used the safety management information system from June 1, 2016 to May 31, 2017 were entered in observation group. The accident rate of adverse nursing events, the duration to check wristbands, response time of urgency call, quantitative data recording for nursing procedures, and pregnant women's and their families' satisfaction degree were compared between the 2 groups.The management information system possesses 4 functions including personal identification, nursing operation quantification, monitoring alarm and music function, and guidance on specialized knowledge and skills. The accident rate of adverse nursing events was significantly lower in the observation than in the control group (P < .05). The duration to check wristbands and the response time of urgency call were all significantly shorter in the observation group than in the control group (all P < .05). Quantitative data recording was significantly better in the observation than in the control group (P < .05). Satisfaction degree was significantly higher in the observation group (96.47%) than in the control group (89.69%) (P < .05). The wireless transmission information was exact and safe, and the system was sensitive and reliable.The system not only is clinically practical but also can enhance the safety of newborns and improve pregnant women's and their families' satisfaction degree.

Contribution of Borneolum syntheticum to the Intervention Effect of Liuwei Dihuang Pill () on Experimental Retinal Degeneration / 中国结合医学杂志

<p><b>OBJECTIVE</b>To observe the contribution of Borneolum syntheticum to the intervention effect of Liuwei Dihuang Pill (, LDP) on experimental retinal degeneration, and initially investigate the mechanism of Borneolum syntheticum as meridian-lead-in drug.</p><p><b>METHODS</b>A total of 180 sodium iodateinduced retinital degeneration rats were randomly divided into three groups, including distilled water group, LDP group, and LDP+Borneolum syntheticum (LDP+BS) group. Twenty normal rats were fed regularly without any treatment as normal control. On day 7 and 14 after treatment, histopathological study and transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) test were performed to evaluate the retinopathy. Claudin-5 expression at blood-retina barrier (BRB) was detected by Western blot at different time points from 0.5 to 8 h after gavage.</p><p><b>RESULTS</b>On day 7 and 14 after treatment, the retinal lesion grades were significantly different among the three groups (P<0.05). The grade in the LDP+BS group was significantly less than the LDP and distilled water groups (both P<0.05), no significant difference was observed between the LDP and distilled water groups (P>0.05). The apoptosis rates in the LDP+BS group was significantly less than the distilled water and LDP groups (both P<0.05), while there was no significant difference between LDP and distilled water groups (P>0.05). Expression of claudin-5 in LDP+BS group was significantly less than the other two groups at 0.5, 1 and 2 h after gavage (P<0.05). There was no apparent difference among the three groups at 4 and 8 h after gavage (P>0.05).</p><p><b>CONCLUSION</b>Borneolum syntheticum could strengthen the effect of LDP on experimental retinal degeneration, indicated that Borneolum syntheticum might play the role of meridian-lead-in drug in the formula. The mechanism may be due to Borneolum syntheticum could promote the physiologically openness of bloodretina barrier through transiently affecting the expression of claudin-5.</p>

Segmentation of the prostate on magnetic resonance images using an ellipsoidal shape prior constraint algorithm / 南方医科大学学报

We propose a novel strategy for multi-atlas-based image segmentation of the prostate on magnetic resonance (MR) images using an ellipsoidal shape prior constraint algorithm. An ellipsoidal shape prior constraint was incorporated into the process of multi-atlas based segmentation to restrict the regions of interest on the prostate images and avoid the interference by the surrounding tissues and organs in atlas selection. In the subsequent process of atlas fusion, the ellipsoidal shape prior constraint calibrated and compensated for the shape prior obtained by the registration technique to avoid incorrect segmentation caused by registration errors. Evaluation of this proposed method on prostate images from 50 subjects showed that this algorithm was effective and yielded a mean Dice similarity coefficients of 0.8812, suggesting its high accuracy and robustness to segment the prostate on MR images.

Protective effect of rosmarinic acid on hypoxia/reoxygenation injury in cardiomyocytes / 中国中药杂志

<p><b>OBJECTIVE</b>To study the protective effect of rosmarinic acid (Ros A) on the primary cardiomyocyte hypoxia/reoxygenation (H/R) injury.</p><p><b>METHOD</b>Primary cardiomyocytes of rats were cultured in vitro to establish the H/R injury of cardiomyocytes and observe the changes in the cell viability and LDH leakage. The changes in ATP content and ROS in cardiomyocytes were measured by using chemiluminescence and fluorescent probe technique. The effects of rosmarinic acid on the apoptosis of cardiomyocytes, cleaved-caspase 3, Akt and p-Akt protein expression were further detected by flow cytometry and western blot analysis.</p><p><b>RESULT</b>According to the experimental results, Ros A at doses of 25, 50, 100 mg x L(-1) could inhibit the decrease in H/R-induced cell viability, LDH leakage and excessive ROS generation, and maintain the ATP level in cells. Ros A at doses of 50, 100 mg x L(-1) could remarkably inhibit the H/R-induced cardiomyocyte apoptosis and down-regulate the expression of cleaved caspase-3. Moreover, Ros A at doses of 100 mg x L(-1) could significantly up-regulate the expression of p-Akt.</p><p><b>CONCLUSION</b>Ros A has the significant effect in resisting the cardiomyocyte H/R injury, improve cardiomyocyte energy metabolism and reduce cell apoptosis, which is related to the activation of Akt pathway.</p>

[Microscopic Raman spectral characteristics and diagnostic value of pathological lip minor salivary glands affected in primary Sjögren's syndrome].

OBJECTIVE: To investigate the Raman spectral characteristics of the pathological lip minor salivary glands affected in primary Sjögren's syndrome. METHODS: Thirty pathological samples and 30 normal samples were collected in this study. The samples were examined by Raman microscope.Support vector machine(SVM) was employed to analyze the data and establish the classification model. RESULTS: The spectra of pathological tissues was different from the controls in proteins, nucleic acids, lipids and glycogen skeleton. The sensitivity, specificity and accuracy of the model established by SVM on the training sets were all 92.0% (92/100), but the sensitivity, specificity and accuracy of the model established by SVM on the testing sets were 69.2% (37/53), 100.0% (37/37) and 82.0% (73/89) respectively. CONCLUSIONS: There was significant difference in Raman spectra between the pathological and normal lip salivary glands, and the classification model established by SVM could discriminate the pathological glands from the normal ones.

[Gingival retraction paste versus gingival retraction cord for fixed prosthodontics: a systematic review].

PURPOSE: To assess the efficacy of gingival retraction paste versus gingival retraction cord in fixed prosthodontics on gingiva protection, tooth preparation, clarity of the impression and plaster model, and aptness of the prosthesis. METHODS: Chinese BioMedical Literature Database, VIP, China National Knowledge Infrastructure (CNKI) and Wangfang database were searched on November 15th 2012 for eligible studies. Hand-searching included references of the included studies and Chinese dental journals. Risk of bias of the included studies was assessed by 2 reviewers independently using Cochrane Collaboration's tool, and data extraction was done by the 2 reviewers. Meta analysis was performed with Revman 5.1 software. RESULTS: Nine randomized controlled trials, involving 1153 participants, were included. All of them had moderate risk of bias. Meta analysis revealed that gingival retraction paste had better effect on gingival health [RR=1.05, 95%CI (1.00, 1.11), P=0.04], tooth preparation [RR=1.17, 95%CI (1.07, 1.28), P=0.0008] and clarity of the impression and plaster model [RR=1.08, 95%CI (1.03, 1.13), P=0.0009] than gingival retraction cord, and the aptness of the prosthesis was as good as the cord [RR=1.07, 95%CI (0.96, 1.19), P=0.21]. CONCLUSIONS: The gingival retraction paste has better effect on gingival health, tooth preparation and clarity of the impression and plaster model, while the aptness of the prosthesis is as good as the cord and can be considered as a good candidate of the gingival retraction cord, but more randomized controlled trials are needed.

Effect of shuangshen ningxin formula on energy metabolism of myocardial ischemia/reperfusion rats / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of Shuangshen Ningxin (SSNX) formula on energy metabolism of myocardial ischemia/reperfusion rats.</p><p><b>METHOD</b>The myocardial ischemia/reperfusion model of Wistar rats was established through the ligation of left anterior descending branch of coronary artery of for 40 min and the reperfusion for 2 h. The Wistar rats were randomly divided into six groups: the sham operation group, the model group, the Trimetazidine group (10 mg x kg(-1)) and SSNX groups (22.5, 45, 90 mg x kg(-1)). Preventive administration was conducted for 5 d. The operation was performed at 1 h on the day of the last administration. CK-MB assay kit was adopted to detect the activity of serum CK-MB. HPLC was used to determine ATP, ADP and AMP contents in myocardial tissues and calculate TAN and EC.</p><p><b>RESULT</b>The preventive administration with SSNX could reduce the activity of serum CK-MB and increase ATP content and EC level in myocardial tissues (P < 0.01 or P < 0.05 vs. the model group).</p><p><b>CONCLUSION</b>SSNX formula can maintain energy charge in cardiomyocytes and relieve ischemia/reperfusion injury by preserving ischemic myocardium ATP.</p>

Construction and functional characterization of a monocistronic replicon based on the HCV genotype 2a promotor / 中华肝脏病杂志

To construct a hepatitis C virus (HCV) genotype 2a monocistronic replicon and investigate its replication capabilities in the human hepatocarcinoma cell lines, Huh7.5 and Huh7.1, in order to determine its potential as a molecular tool for future in vitro studies of HCV replication and selection studies for putative anti-HCV drugs. Site-directed mutagenesis was used to delete the Core-E1-E2-p7-NS2 fragment (about 3090 bp) from plasmid pJ6JFH1BlaRL. The resultant trianglepJ6JFH1BlaRL plasmid was digested with AgeI and AvrII to release the cDNA fragment (hereafter, referred to as fragment L) containing partial 5'-untranslated region (UTR), the first 12 amino acid (aa) of HCV Core coding sequence, full-length coding sequences for the blasticidin-resistance gene, Renilla luciferase, foot-and-mouth disease virus (FMDV) 2a antiprotease and ubiquitin, and partial coding sequence for HCV NS3. To generate the monocistronic replicon, pSGRmJFH1BlaRL, fragment L was ligated into the pSGR-JFH1 vector that had been digested with AgeI and AvrII to remove the partial 5'-UTR, the first 19 aa of HCV Core coding sequence, the full-length coding sequence for the neomycin phosphotransferase II gene, the internal ribosomal entry site from encephalomyocarditis virus, and partial HCV NS3 coding sequence. A replication-defective mutant replicon, pSGRmJFH1BlaRL/GND, was constructed by a similar procedure using the pSGR-JFH1/GND vector. Fragment L was confirmed in both constructs by sequencing. Replicon RNAs were prepared from XbaI-linearized plasmid DNA templates with Invitrogen's T7 MEGAscript kit, and were purified by DNase I treatment and LiCl precipitation. RNAs were quanti?ed by optical density, and the quality and concentration were con?rmed by agarose gel electrophoresis. Replicon RNAs were transfected into Huh7.5 and Huh7.1 cells using Invitrogen's DMRIE-C transfection reagent at a ratio of 5 mug of lipid to 1mug of RNA. Time course assay of Renilla luciferase activity indicated the replicon's replication function. The pSGRmJFH1BlaRL monocistronic replicon and pSGRmJFH1BlaRL/GND replication-defective mutant replicon were successfully constructed. The pSGRmJFH1BlaRL replicon was replication-proficient in Huh7.5 and Huh7.1 cells, with replication peaking at 72 hours post-transfection and decreasing after 96 hours. No replication was detected at any time point post-transfection for the defective mutant replicon. A monocistronic replicon of HCV genotype 2a was constructed and shown to be replication-proficient in human hepatocarcinoma cell lines.