Risk factors for high CAD-RADS scoring in CAD patients revealed by machine learning methods: a retrospective study.

Objective: This study aimed to investigate a variety of machine learning (ML) methods to predict the association between cardiovascular risk factors and coronary artery disease-reporting and data system (CAD-RADS) scores. Methods: This is a retrospective cohort study. Demographical, cardiovascular risk factors and coronary CT angiography (CCTA) characteristics of the patients were obtained. Coronary artery disease (CAD) was evaluated using CAD-RADS score. The stenosis severity component of the CAD-RADS was stratified into two groups: CAD-RADS score 0-2 group and CAD-RADS score 3-5 group. CAD-RADS scores were predicted with random forest (RF), k-nearest neighbors (KNN), support vector machines (SVM), neural network (NN), decision tree classification (DTC) and linear discriminant analysis (LDA). Prediction sensitivity, specificity, accuracy and area under the curve (AUC) were calculated. Feature importance analysis was utilized to find the most important predictors. Results: A total of 442 CAD patients with CCTA examinations were included in this study. 234 (52.9%) subjects were CAD-RADS score 0-2 group and 208 (47.1%) were CAD-RADS score 3-5 group. CAD-RADS score 3-5 group had a high prevalence of hypertension (66.8%), hyperlipidemia (50%) and diabetes mellitus (DM) (35.1%). Age, systolic blood pressure (SBP), mean arterial pressure, pulse pressure, pulse pressure index, plasma fibrinogen, uric acid and blood urea nitrogen were significantly higher (p < 0.001), and high-density lipoprotein (HDL-C) lower (p < 0.001) in CAD-RADS score 3-5 group compared to the CAD-RADS score 0-2 group. Nineteen features were chosen to train the models. RF (AUC = 0.832) and LDA (AUC = 0.81) outperformed SVM (AUC = 0.772), NN (AUC = 0.773), DTC (AUC = 0.682), KNN (AUC = 0.707). Feature importance analysis indicated that plasma fibrinogen, age and DM contributed most to CAD-RADS scores. Conclusion: ML algorithms are capable of predicting the correlation between cardiovascular risk factors and CAD-RADS scores with high accuracy.

Radiation-induced glymphatic dysfunction in patients with nasopharyngeal carcinoma: a study using diffusion tensor image analysis along the perivascular space.

Radiation encephalopathy (RE) refers to radiation-induced brain necrosis and is a life-threatening complication in patients with nasopharyngeal carcinoma (NPC) after radiotherapy (RT), and radiation-induced pre-symptomatic glymphatic alterations have not yet been investigated. We used diffusion tensor image analysis along the perivascular space (DTI-ALPS) index to examine the pre-symptomatic glymphatic alterations in NPC patients following RT. A total of 109 patients with NPC consisted of Pre-RT (n = 35) and Post-RT (n = 74) cohorts were included. The post-RT NPC patients, with normal-appearing brain structure at the time of MRI, were further divided into Post-RT-RE- (n = 58) and Post-RT-RE+ (n = 16) subgroups based on the detection of RE in follow-up. We observed lower DTI-ALPS left index, DTI-ALPS right index and DTI-ALPS whole brain index in post-RT patients than that in pre-RT patients (p < 0.05). We further found that post-RT-RE+ patients demonstrated significantly lower DTI-ALPS right (p = 0.013), DTI-ALPS whole brain (p = 0.011) and marginally lower DTI-ALPS left (p = 0.07) than Post-RT non-RE patients. Significant negative correlations were observed between the maximum dosage of radiation-treatment (MDRT) and DTI-ALPS left index (p = 0.003) as well as DTI-ALPS whole brain index (p = 0.004). Receiver operating characteristic (ROC) curve analysis showed that DTI-ALPS whole brain index exhibited good performance (AUC = 0.706) in identifying patients more likely developing RE. We concluded that glympathic function was impaired in NPC patients following RT and DTI-ALPS index may serve as a novel imaging biomarker for diagnosis of RE.

A hierarchical data reconciliation based on multiple time-delay interval estimation for industrial processes.

With the increasing demand for energy conversation and high efficiency, data quality is of great important to the operation management and monitoring in industrial applications. Data reconciliation, as a data processing technology, provides great potential to improve quality of process data, and is widely used to reduce measurement error and estimate unmeasured parameters. However, there are reactors connected in series in the long-running industrial processes so that liquid material information is difficult to mark and trace, and the liquid material has different residence times in each reactor due to the differences in the internal structure and operation mode. The time-delay in different reactors may be various and time-varying. In this paper, to solve these problems, a multiple time-delay interval estimation based hierarchical data reconciliation method is put forward. First, the multiple time-delay interval estimation is developed according to the process mechanism analysis and modeling. Then, an improved discrete state transition solution approach is presented to solve the data time-matching with multiple time-delay interval estimation for different reactors. Finally, a hierarchical data reconciliation frame is built by data characteristics. The feasible of the proposed data reconciliation method is verified utilizing the industrial application results.

A mouse model for studying the clearance of hepatitis B virus in vivo using a luciferase reporter.

Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.

Detection of hepatitis B surface antigen by target-induced aggregation monitored by dynamic light scattering.

In the current work, a one-step, washing-free, homogeneous nanosensor assay has been constructed to sensitively detect hepatitis B surface antigen (HBsAg) based on the light scattering property of gold nanoparticles (GNPs) through a sandwich model. The two nanoprobes in this study were designed by conjugating monoclonal and polyclonal hepatitis B surface antibody (HBsAb) onto the GNPs of different diameters. First, the detection behavior of the combinations of different sizes of GNPs was evaluated and the optimized combination was determined. In analyzing HBsAg in Tris-HCl buffer, such bioassay composed of GNPs of approximately 50 and 100 nm has a limit of detection (LOD) as high as 0.005 IU/ml and a dose-dependent response ranging from 0.005 to 1 IU/ml, which indicates its good diagnostic capability and provides a useful means to analyze protein biomarkers with low virus loads. Observation with transmission electron microscopy (TEM) provides direct evidence that the increase of hydrodynamic diameters resulted from the aggregation induced by immunological reactions. The bioassay also exhibits satisfactory specificity in analyzing HBsAg in serum media. Therefore, with its simple preparation, easy readout, and good stability, this bioassay has the potential to be developed into an automated and widely used biosensor assay.

High levels of gene expression in the hepatocytes of adult mice, neonatal mice and tree shrews via retro-orbital sinus hydrodynamic injections of naked plasmid DNA.

Hydrodynamic-based gene delivery has emerged as an efficient and simple method for the intracellular transfection of naked plasmid DNA (pDNA) in vivo. In this system, a hydrodynamic injection via the tail vein is the most effective non-viral method of liver-targeted gene delivery. However, this injection is often technically challenging when used in animals whose tail veins are difficult to visualize or too small to operate on. To overcome this limitation, an alternative in vivo gene delivery method, the rapid injection of large volume of pDNA solution through retro-orbital sinus, was established. Using this technique, we successfully delivered pDNA to the tissue of adult mice, neonatal mice and tree shrews. The efficient expression of exogenous genes was specifically detected in the liver of test animals treated with this gene delivery method. This study demonstrates for the first time that the hydrodynamic gene delivery via the retro-orbital sinus can not only reach the same transgene efficiency as a tradition hydrodynamic-based intravascular injection but also be used in animals that are difficult to inject via the tail vein. This method could open up new areas in gene function studies and gene therapy disease treatment.

A novel, universal and sensitive lateral-flow based method for the detection of multiple bacterial contamination in platelet concentrations.

In the present study, we aimed to develop a nucleic acid lateral-flow method for the rapid and sensitive detection of multiple bacteria that contaminate platelet concentrations (PCs). Polymerase chain reaction (PCR) amplicons were produced by a set of board-range primers that recognize the conserved region of bacteria 16S rDNA, followed by hybridization with both an FITC (fluorescein isothiocyanate)-labelled probe and biotin-labelled probe, and then a nucleic acid lateral-flow dipstick (LFD) assay. The LFD accurately identified 7 species of bacteria, but had no cross-reactivity with human genomic DNA. The limit of detection (LOD) of the LFD assay was as low as 10(1) copies/µL of 16S rDNA for plasmid. In the case of spiked PCs without enrichment, the detection limit of LFD for K. pneumonia was 5 CFU/mL, 6.5 × 10(4) CFU/mL for the S. epidermidis and 35 CFU/mL for P. aeruginosa.

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system.

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.

Bioluminescence imaging allows monitoring hepatitis C virus core protein inhibitors in mice.

BACKGROUND: The development of small molecule inhibitors of hepatitis C virus (HCV) core protein as antiviral agents has been intensively pursued as a viable strategy to eradicate HCV infection. However, lack of a robust and convenient small animal model has hampered the assessment of in vivo efficacy of any antiviral compound. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop a novel method to screen anti-core protein siRNA in the mouse liver by bioluminescence imaging. The inhibitory effect of two shRNAs targeting the highly conserved core region of the HCV genome, shRNA452 and shRNA523, was examined using this method. In the transient mouse model, the effect of shRNA-523 was detectable at as early as 24 h and became even more pronounced at later time points. The effect of shRNA-452 was not detectable until 48 h post-transduction. In a stable mouse model, shRNA523 reduced luciferase levels by up to 76.4±26.0% and 91.8±8.0% at 6 h and 12 h after injection respectively, and the inhibitory effect persisted for 1 day after a single injection while shRNA-Scramble did not seem to have an effect on the luciferase activity in vivo. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a simple and quantitative assay for real-time monitoring of HCV core protein inhibitors in mice.

Screening compounds against HCV based on MAVS/IFN-ß pathway in a replicon model.

AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-ß promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.

Gold nanorod-based localized surface plasmon resonance biosensor for sensitive detection of hepatitis B virus in buffer, blood serum and plasma.

A novel gold nanorods (GNRs) biosensor based on the localized surface plasmon resonance (LSPR) behavior was designed to detect the hepatitis B surface antigen (HBsAg) which indicates active viral replication of hepatitis B virus (HBV). The surface of GNRs was modified with monoclonal hepatitis B surface antibody (HBsAb) through physical adsorption. Raman spectrum, dynamic light scattering (DLS) and zeta potential measurement were conducted to access the nature of the GNRs after antibody modification. The binding of analyte to the molecular probe was monitored by the longitudinal wavelength shift of LSPR peak in the UV-Vis extinction spectrum resulting from the changes of local refractive index induced by the immunological reaction. The biosensor could be utilized in quantitative analysis in Tris buffers, which has dose-dependence response ranging from 0.01 IU/mL to 1 IU/mL. Further, the biosensor was suited for qualitative analysis of HBsAg in the actual media of blood serum and plasma. The ease of operation, high sensitivity, and its generality offer specific advantages over other GNRs-based immunoassay methods.

Bioluminescence imaging of Hepatitis C virus NS3/4A serine protease activity in cells and living animals.

The lack of robust small animal models has been an obstacle to the screening of Hepatitis C virus (HCV) NS3/4A protease inhibitors in vivo. Here, we described a reporter assay system for in vivo noninvasive imaging of NS3/4A serine protease activity using split firefly luciferase complementation strategy. The reporter construct ANluc(NS5A/B)BCluc constitutes the split N- and C-terminal fragments of luciferase, fused to interacting peptides, pepA and pepB, respectively, with an intervening HCV NS3/4A cleavage motif of NS5A/B. We proved that the reporter molecule could be proteolytically cleaved by NS3/4A at the NS5A/B motif in cells and living animals. Association of pepA and pepB brought inactive fragments of luciferase into close proximity, thereby restoring bioluminescence activity. The increase in luciferase activity was proportional to the dose of active NS3/4A protease. The ANluc(NS5A/B)BCluc reporter also could be used to detect the activity of NS3/4A-specific shRNA and IFN-alpha. Therefore, the reporter assay system using split firefly luciferase complementation strategy should prove useful for evaluating NS3/4A protease activity in cells and living animals.

Long-term hepatitis C internal ribosome entry site-dependent gene expression mediated by phage phiC31 integrase in mouse model.

BACKGROUND: The lack of a robust small animal model for hepatitis C virus (HCV) has hindered the development of novel drugs, including internal ribosome entry site (IRES) inhibitors. Phage phiC31 integrase has emerged as a potent tool for achieving long-term gene expression in vivo. This study utilized phiC31 integrase to develop a stable, reproducible and easily accessible HCV IRES mouse model. METHODS: phiC31 integrase plasmid and the reporter vector, HCV-IRES-luciferase expression cassette (containing an attB site), was codelivered to murine livers using high pressure tail vein injection. HCV IRES-dependent translation reflected by luciferase expression was accurately monitored in vivo by bioluminescence imaging. Genomic integration of the transgene was confirmed by partial hepatectomy and nested PCR. An HCV IRES-targeted short hairpin RNA (shRNA) expression plasmid, sh184, was hydrodynamically transfected into mouse liver to study its inhibition efficacy in vivo. RESULTS: phiC31 integrase mediated intramolecular recombination between wild-type attB and attP sites in mice. The expression of luciferase was stable after 30 days post-transfection and remained so for 300 days only in the livers of mice that were coinjected with the integrase-encoding plasmid. Luciferase levels reduced dramatically after hydrodynamic transfection of sh184. CONCLUSIONS: These results indicate that this mouse model provides a powerful tool for accurate and long-term evaluation of potential anti-IRES compounds in vivo.

Bioluminescence imaging of hepatitis B virus enhancer and promoter activities in mice.

By bioluminescence imaging and hydrodynamic gene transfer technology, the activities of hepatitis B virus (HBV) promoters and the effects of HBV enhancers on these promoters in mice under true physiological conditions have been assessed. Our studies reveal that either of the two HBV enhancers can stimulate HBV major promoter activity in hepa 1-6 cells (in vitro) and in mouse liver (in vivo), and the enhancer effects on the three promoters (S1, S2 and X promoter) are markedly greater in vivo than in vitro. The two HBV enhancers have no cooperative action on HBV promoters in vitro or in vivo.

Bioluminescent imaging of hepatocellular carcinoma in live mice.

A non-invasive orthotopic hepatocellular carcinoma (HCC) model was created with human HCC cells (HepG-Luc) constitutively expressing luciferase (Luc) in nude mice. Development of tumor growth and response to anti-tumor therapy combined with 5-fluorouracil and cisplatin was monitored by whole-body bioluminescent imaging (BLI). Luciferase activity in the tumor, determined by BLI, correlated with the tumor volume and weight. The anti-tumor therapy proved effective by BLI monitoring. In conclusion, BLI by luciferase provides a non-invasive method of monitoring tumor activities that can prove useful for therapeutic intervention studies.