Apolipoprotein E but not B is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A

Apolipoprotein e mediates attachment of clinical hepatitis C virus to hepatocytes by binding to cell surface heparan sulfate proteoglycan receptors

Our previous studies demonstrated that the cell culture-grown hepatitis C virus of genotype 2a (HCVcc) uses apolipoprotein E (apoE) to mediate its attachment to the surface of human hepatoma Huh-7.5 cells. ApoE mediates HCV attachment by binding to the cell surface heparan sulfate (HS) which is covalently attached to the core proteins of proteoglycans (HSPGs). In the present study, we further determined the physiological importance of apoE and HSPGs in the HCV attachment using a clinical HCV of genotype 1b (HCV1b) obtained from hepatitis C patients and human embryonic stem cell-differentiated hepatocyte-like cells (DHHs). DHHs were found to resemble primary human hepatocytes. Similar to HCVcc, HCV1b was found to attach to the surface of DHHs by the apoE-mediated binding to the cell surface HSPGs. The apoE-specific monoclonal antibody, purified HSPGs, and heparin were all able to efficiently block HCV1b attachment to DHHs. Similarly, the removal of heparan sulfate from cell surface by treatment with heparinase suppressed HCV1b attachment to DHHs. More significantly, HCV1b attachment was potently inhibited by a synthetic peptide derived from the apoE receptor-binding region as well as by an HSPG-binding peptide. Likewise, the HSPG-binding peptide prevented apoE from binding to heparin in a dose-dependent manner, as determined by an in vitro heparin pull-down assay. Collectively, these findings demonstrate that HSPGs serve as major HCV attachment receptors on the surface of human hepatocytes to which the apoE protein ligand on the HCV envelope binds

Zhx2 (Zinc Fingers and Homeoboxes 2) Regulates Major Urinary Protein Gene Expression in the Mouse Liver

The mouse major urinary proteins (Mups) are encoded by a large family of highly related genes clustered on chromosome 4. Mups, synthesized primarily and abundantly in the liver and secreted through the kidneys, exhibit male-biased expression. Mups bind a variety of volatile ligands; these ligands, and Mup proteins themselves, influence numerous behavioral traits. Although urinary Mup protein levels vary between inbred mouse strains, this difference is most pronounced in BALB/cJ mice, which have dramatically low urinary Mup levels; this BALB/cJ trait had been mapped to a locus on chromosome 15. We previously identified Zhx2 (zinc fingers and homeoboxes 2) as a regulator of numerous liver-enriched genes. Zhx2 is located on chromosome 15, and a natural hypomorphic mutation in the BALB/cJ Zhx2 allele dramatically reduces Zhx2 expression. Based on these data, we hypothesized that reduced Zhx2 levels are responsible for lower Mup expression in BALB/cJ mice. Using both transgenic and knock-out mice along with in vitro assays, our data show that Zhx2 binds Mup promoters and is required for high levels of Mup expression in the adult liver. In contrast to previously identified Zhx2 targets that appear to be repressed by Zhx2, Mup genes are positively regulated by Zhx2. These data identify Zhx2 as a novel regulator of Mup expression and indicate that Zhx2 activates as well as represses expression of target genes

Evaluation of the communication delay in a hybrid real-time simulator for weak grids

Real-time Simulation (RTS) is one of the effective means via which to study device level or system level dynamics, such as power converter online testing, evaluation, and control, and power system stability analysis. The RTS -enabled design-chain offers a time -effective, low-cost, and fail-safe development process. As the penetration of renewable energy is becoming higher, the demand in hybrid system real-time simulation becomes imperative, where fast-dynamic device level power converters and slow -dynamic large -scale power systems are simulated at the same time. This paper introduces a novel hybrid real-time simulation architecture based on the central processing unit (CPU) and the field-programmable gate array (FPGA). Compared with the off-the-shelf power system real-time simulation system, it offers both wide time scale simulation and high accuracy. The multi-time scale model can perform electromechanical electromagnetic transient hybrid simulation, which can be applied to the research of power systems penetrated with power converters. In the proposed simulation platform, the communication delay is introduced when different RTS platforms exchange real-time data. The communication delay should be considered in the stability analysis of the grid-connected inverters in a weak grid environment. Based on the virtual impedance characteristic formed by the control loop with and without communication delay, the impedance characteristics are analyzed and inter-simulator delay impacts are revealed in this paper. Theoretical analysis indicates that the communication delay, contrary to expectation, can improve the virtual impedance characteristics of the system. With the same hardware simulation parameters, the grid-converter system is verified on both the Typhoon system alone and the Typhoon-dSPACE-SpaceR hybrid simulation platform. The THD value of grid current in a weak grid environment that works in the Typhoon system is 4.98%, and 2.38% in the Typhoon-dSPACE-SpaceR hybrid simulation platform. This study eventually reveals the fact that the inter-simulation delay creates the illusion that the control system built in the novel hybrid real-time simulation is more stable under weak grid conditions

Design and implementation of a low-power low-cost digital current-sink electronic load ‡

Electronic load (e-load) is essential equipment for power converter performance test, where a designated load profile is executed. Electronic load is usually implemented with the analog controller for fast tracking of the load profile reference. In this paper, a low-power low-cost electronic load is proposed. MOSFETs (metal-oxide-semiconductor field-effect transistors) are used as the power consumption devices, which are regulated to the active region as controlled current-sink. In order to achieve fast transient response using the low-cost digital signal controller (DSC) PWM peripherals, the interleaving PWM method is proposed to achieve active current ripple mitigation. To obtain the system open-loop gain for current-sink operation, an offline digital system identification method, followed by model reduction, is proposed by applying Pseudo-Random Binary Sequence (PRBS) excitation. Pole-zero cancelation method is used in the control system design and later implemented in a DSC. The prototype is built and tested, in which meaningful testing scenarios under constant current-sink mode, pulse current sink mode, and double line-frequency current mode are verified. The experimental results indicate that the proposed e-load can sink pre-programmed current profile with well-attenuated ripple for static and dynamic load testing, and is applicable to fully digitalized power testing equipment

A novel step current excitation control method to reduce the torque ripple of outer-rotor switched reluctance motors

Featured in low-speed and high-torque operation, outer-rotor switched reluctance motors (OSRMs) have the potential to be widely deployed in low-speed commuter and logistics vehicle applications. In this paper, a five-phase OSRM and the control method featuring torque ripple reduction has been proposed, which can be applied as the wheel hub motor in the electric vehicles. The simulation was carried out to analyze the OSRM operation. The electromagnetic characteristics of single-phase and two-phase hybrid excitation mode, as well as step current excitation mode, were compared and analyzed. To solve the problem of the large torque ripple of OSRMs under traditional excitation modes, the torque ripple suppression method based on step current excitation was also studied. The experiment design, including motor start-up control, speed control, and torque ripple reduction, are presented to verify the system torque ripple mitigation method

Autophagic Flux Modulation by Wnt/β-Catenin Pathway Inhibition in Hepatocellular Carcinoma

Autophagy targets cellular components for lysosomal-dependent degradation in which the products of degradation may be recycled for protein synthesis and utilized for energy production. Autophagy also plays a critical role in cell homeostasis and the regulation of many physiological and pathological processes and prompts this investigation of new agents to effect abnormal autophagy in hepatocellular carcinoma (HCC). 2,5-Dichloro-N-(2-methyl-4-nitrophenyl) benzenesulfonamide (FH535) is a synthetic inhibitor of the Wnt/β-catenin pathway that exhibits anti-proliferative and anti-angiogenic effects on different types of cancer cells. The combination of FH535 with sorafenib promotes a synergistic inhibition of HCC and liver cancer stem cell proliferation, mediated in part by the simultaneous disruption of mitochondrial respiration and glycolysis. We demonstrated that FH535 decreased HCC tumor progression in a mouse xenograft model. For the first time, we showed the inhibitory effect of an FH535 derivative, FH535-N, alone and in combination with sorafenib on HCC cell proliferation. Our study revealed the contributing effect of Wnt/β-catenin pathway inhibition by FH535 and its derivative (FH535-N) through disruption of the autophagic flux in HCC cells

The C-Terminal α-Helix Domain of Apolipoprotein E Is Required for Interaction with Nonstructural Protein 5A and Assembly of Hepatitis C Virus▿

We have recently demonstrated that human apolipoprotein E (apoE) is required for the infectivity and assembly of hepatitis C virus (HCV) (K. S. Chang, J. Jiang, Z. Cai, and G. Luo, J. Virol. 81:13783-13793, 2007; J. Jiang and G. Luo, J. Virol. 83:12680-12691, 2009). In the present study, we have determined the molecular basis underlying the importance of apoE in HCV assembly. Results derived from mammalian two-hybrid studies demonstrate a specific interaction between apoE and HCV nonstructural protein 5A (NS5A). The C-terminal third of apoE per se is sufficient for interaction with NS5A. Progressive deletion mutagenesis analysis identified that the C-terminal α-helix domain of apoE is important for NS5A binding. The N-terminal receptor-binding domain and the C-terminal 20 amino acids of apoE are dispensable for the apoE-NS5A interaction. The NS5A-binding domain of apoE was mapped to the middle of the C-terminal α-helix domain between amino acids 205 and 280. Likewise, deletion mutations disrupting the apoE-NS5A interaction resulted in blockade of HCV production. These findings demonstrate that the specific apoE-NS5A interaction is required for assembly of infectious HCV. Additionally, we have determined that using different major isoforms of apoE (E2, E3, and E4) made no significant difference in the apoE-NS5A interaction. Likewise, these three major isoforms of apoE are equally compatible with infectivity and assembly of infectious HCV, suggesting that apoE isoforms do not differentially modulate the infectivity and/or assembly of HCV in cell culture

Cell Consistency Evaluation Method Based on Multiple Unsupervised Learning Algorithms

Unsupervised learning algorithms can effectively solve sample imbalance. To address battery consistency anomalies in new energy vehicles, we adopt a variety of unsupervised learning algorithms to evaluate and predict the battery consistency of three vehicles using charging fragment data from actual operating conditions. We extract battery-related features, such as the mean of maximum difference, standard deviation, and entropy of batteries and then apply principal component analysis to reduce the dimensionality and record the amount of preserved information. We then build models through a collection of unsupervised learning algorithms for the anomaly detection of cell consistency faults. We also determine whether unsupervised and supervised learning algorithms can address the battery consistency problem and document the parameter tuning process. In addition, we compare the prediction effectiveness of charging and discharging features modeled individually and in combination, determine the choice of charging and discharging features to be modeled in combination, and visualize the multidimensional data for fault detection. Experimental results show that the unsupervised learning algorithm is effective in visualizing and predicting vehicle core conformance faults, and can accurately predict faults in real time. The “distance+boxplot” algorithm shows the best performance with a prediction accuracy of 80%, a recall rate of 100%, and an F1 of 0.89. The proposed approach can be applied to monitor battery consistency faults in real time and reduce the possibility of disasters arising from consistency faults