Diagnostic Accuracy of Serum Hyaluronan for Detecting HCV Infection and Liver Fibrosis in Asymptomatic Blood Donors

Background: The disease caused by hepatitis C virus (HCV) is asymptomatic, silent, and progressive liver disease. In HCV-infected patients the increase in serum HA is associated with the development of hepatic fibrosis and disease progression. Methods: HCV-RNA detection was performed in all serological samples of blood donors that tested positive using HCV Ultra ELISA. Determination of hyaluronan (HA) was performed in positive HCV samples using ELISA-like fluorometric method. The HA content was compared to HCV viral load, genotype of the virus, liver fibrosis as well as ALT and GGT liver biomarkers. Results: Persistently normal ALT (<40 U/L) and GGT (<50 U/L) serum levels were detected in 75% and 69% of the HCV-Infected blood donors, respectively. Based on ROC analysis, the HA value < 34.2 ng/mL is an optimal cut-off point to exclude HCV viremia (specificity = 91%, NPV = 99%). Applying HA value ≥34.2 ng/mL significant liver fibrosis (≥F2) can be estimated in 46% of the HCV-infected blood donors. HA serum level (≥34.2 ng/mL) associated with a high ALT level (>40 U/mL) can correctly identify HCV infection and probable liver fibrosis (sensitivity = 96% and specificity = 90%) in asymptomatic blood donors. Conclusions: A high level of HA (≥34.2 ng/mL) in association with ALT (≥40 U/L) in serum can provide a good clinical opportunity to detect HCV-infected asymptomatic persons that potentially require a liver biopsy confirmation and antiviral treatment to prevent the development of advanced liver fibrosis or cirrhosis.     

Synthesis and biological evaluation of echinocystic acid derivatives as HCV entry inhibitors

A series of echinocystic acid (EA) 28-COOH derivatives was synthesized, and their anti-HCV entry activity was evaluated by HCVpp and VSVpp entry assay. It was found that some of them showed moderate anti-HCV entry activity, especially compound 12, and these modifications also removed the undesired hemolytic effect.     

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimer-size self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)(20), suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.     

Inhibitor Development against p7 Channel in Hepatitis C Virus

Hepatitis C Virus (HCV) is the key cause of chronic and severe liver diseases. The recent direct-acting antiviral agents have shown the clinical success on HCV-related diseases, but the rapid HCV mutations of the virus highlight the sustaining necessity to develop new drugs. p7, the viroporin protein from HCV, has been sought after as a potential anti-HCV drug target. Several classes of compounds, such as amantadine and rimantadine have been testified for p7 inhibition. However, the efficacies of these compounds are not high. Here, we screened some novel p7 inhibitors with amantadine scaffold for the inhibitor development. The dissociation constant (Kd) of 42 ARD-series compounds were determined by nuclear magnetic resonance (NMR) titrations. The efficacies of the two best inhibitors, ARD87 and ARD112, were further confirmed using viral production assay. The binding mode analysis and binding stability for the strongest inhibitor were deciphered by molecular dynamics (MD) simulation. These ARD-series compounds together with 49 previously published compounds were further analyzed by molecular docking. Key pharmacophores were identified among the structure-similar compounds. Our studies suggest that different functional groups are highly correlated with the efficacy for inhibiting p7 of HCV, in which hydrophobic interactions are the dominant forces for the inhibition potency. Our findings provide guiding principles for designing higher affinity inhibitors of p7 as potential anti-HCV drug candidates.     

Cheminformatics study of some indole compounds through QSAR modeling,ADME prediction,molecular docking,and molecular dynamic simulation to identify novel inhibitors of HCV NS5B protease

The hepatitis C virus is a viral disease that causes cirrhosis and hepatocellular carcinoma in the liver. Since the virus's discovery, significant therapeutic advances have been made. Notwithstanding, its upsurge necessitates the development of novel approaches to combating this disease. Recent studies have seen a rise in the value of plant compounds in the establishment of novel, efficient, and cost-efficient anti-HCV medications. These factors have motivated us to continuously search for novel, potent anti-HCV inhibitors, ideally with mechanisms different from those used in conventional medicine. Several filtrations processes including ADMET, molecular docking, and molecular dynamics (MD) simulation were used to choose potent hits from plant molecules deposited in the PubChem database. The binding kinetics were explored using MD simulation studies performed for 100 ns using GROMACS-2018.1. which was done to validate and supplement the findings of the virtual screening. The selected hits (with PubChem CID of 3,560,948 and 4,754,183) had preferable molecular traits that suggested they might work well as HCV inhibitors and are term as effective potential hits. The MD study confirmed the stability of the identified hit with a binding energy of ?111.724 ( ± 81.668) kj/mol and mechanisms different from the FDA-approved drug (sofosbuvir) used as a reference with a binding energy of ?106.132 ( ± 78.008) kj/mol. This approach could aid in the establishment and identification of novel inhibitors in pharmaceutical discovery. Experimental assessments could indeed ascertain as to if the identified molecule can be employed as an anti-HCV drug to address the ailment and confirm the reliability of our in-silico studies.     

Theoretical Study and NBO Analysis of the Decomposition Kinetics of Oxetane, 2-Methyloxetane and 2,2-Dimethyloxetane

A theoretical study of the thermal decomposition kinetics of oxetane (1), 2-methyloxetane (2), and 2,2-dimethyloxetane (3) has been carried out at the B3LYP/6-311+G**, B3PW91/6-311+G**, and MPW1PW91/6-311+G** levels of theory. The MPW1PW91/6-311+G** method was found to give a reasonable good agreement with the experimental kinetics and thermodynamic parameters. The decomposition reaction of compounds 1～3 yields formaldehyde and the corresponding substituted olefin. Based on the optimized ground state geometries using MPW1PW91/6-311+G** method, the natural bond orbital (NBO) analysis of donor-acceptor (bond-antibond) interactions revealed that the stabilization energies associated with the electronic delocalization from σC3-C4 bonding to σ*O1-C2 antibonding orbitals decrease from compounds 1 to 3. The σC3-C4→σO1-C2 resonance energies for compounds 1～3 are 2.63, 2.59 and 2.45 kcal mol-1, respectively. Further, the results showed that the energy gaps between σC3-C4 bonding and σ*O1-C2 antibonding orbitals decrease from compounds 1 to 3. Also, the decomposition process in these compounds are controlled by σ→σ* resonance energies. Moreover, the obtained order of energy barriers could be explained by the number of electron-releasing methyl groups substituted to the Csp3 atom (which is attached to oxygen atom). NBO analysis shows that the occupancies of σCsp3-O bonds decrease for compounds 1～3 as 3<2<1, and those of σCsp3-O bonds increase in the opposite order (3 > 2 > 1). This fact illustrates a comparatively easier thermal decomposition of the sCsp3-O bond in compound 3 compared to compound 2, and in compound 2 compared to compound 1. NBO results indicate that these reactions are occurring through a concerted and asynchronous four-membered cyclic transition state type of mechanism.     

Antiviral phytochemicals identification from Azadirachta indica leaves against HCV NS3 protease: an in silico approach

Hepatitis C virus (HCV) is a major health problem across the world affecting the people of all age groups. It is the main cause of hepatitis and at chronic stage causes liver cirrhosis and hepatocellular carcinoma. Various therapeutics are made against HCV but still there is a need to find out potential therapeutics to combat the virus. The goal of this study is to identify the phytochemicals of Azadirachta indica leaves having antiviral activity against HCV NS3 protease through molecular docking and simulation approach. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses good binding properties with HCV NS3/4A protease. It can be concluded from this study that Deacetyl-3-cinnamoyl-azadirachtin may serve as a potential inhibitor against NS3/4A protease.     

Fabrication of Robust High‐Quality ORMOCER® Inverse Opals

Summary: The nanostructuring of ORMOCER® to form inverse opals is described. For this purpose a polymer opal is used as a template and infiltrated with liquid ORMOCER®. After photopolymerization of the resin the host opal is dissolved in tetrahydrofuran and an ORMOCER® inverse opal is obtained. It shows excellent periodicity (by SEM) and optical properties to reveal a high degree of face centered cubic order. This replication process leads to a nanostructured photonic crystal with the outstanding mechanical properties of ORMOCER® and high temperature stability up to 350 °C.

ORMOCER® inverse opal annealed at 200 °C for 10 h.     

CLONING AND SEQUENCE ANALYSIS OF 3''''-END REGION OF POTATO VIRUS Y GENOME

The entire coat protein (CP) gene and part of the 3'-noncoding sequence of the potatovirus Y (PVY, the Chinese isolate) genome were synthesized with polymerase chain reaction(PCR) using cDNA of its genomic RNA as a template. A restriction endonuclease site Ncoland the initiation codon AUG were included in primer Y5 while the SalI site was includedin primer Y3. After being double digested with Ncol and SalI enzymes, the PCR product wascloned into a pGEM derivative plasmid, and the CP gene in one of the clones, pPCY6, wassequenced. Several clones were selected from the cDNA library by using the CP gene frag-ment of pPCY6 as a probe and the sequences of these clones were determined. These se-quences included part of the NIb gene, entire CP gene and 3'-noncoding region, 1317 bp alltogether.Sequence analysis indicated that the nucleotide sequence homology of the CP geneof this strain with that of the 0 strain (94.2%) was a little higher than with that of the Nstrain (89.6%), but the homology of amino acid se     

Identification of Potential HCV Inhibitors Based on the Interaction of Epigallocatechin-3-Gallate with Viral Envelope Proteins

Hepatitis C is affecting millions of people around the globe annually, which leads to death in very high numbers. After many years of research, hepatitis C virus (HCV) remains a serious threat to the human population and needs proper management. The in silico approach in the drug discovery process is an efficient method in identifying inhibitors for various diseases. In our study, the interaction between Epigallocatechin-3-gallate, a component of green tea, and envelope glycoprotein E2 of HCV is evaluated. Epigallocatechin-3-gallate is the most promising polyphenol approved through cell culture analysis that can inhibit the entry of HCV. Therefore, various in silico techniques have been employed to find out other potential inhibitors that can behave as EGCG. Thus, the homology modelling of E2 protein was performed. The potential lead molecules were predicted using ligand-based as well as structure-based virtual screening methods. The compounds obtained were then screened through PyRx. The drugs obtained were ranked based on their binding affinities. Furthermore, the docking of the topmost drugs was performed by AutoDock Vina, while its 2D interactions were plotted in LigPlot+. The lead compound mms02387687 (2-[[5-[(4-ethylphenoxy) methyl]-4-prop-2-enyl-1,2,4-triazol-3-yl] sulfanyl]-N-[3(trifluoromethyl) phenyl] acetamide) was ranked on top, and we believe it can serve as a drug against HCV in the future, owing to experimental validation.     

Understanding of the drug resistance mechanism of hepatitis C virus NS3/4A to paritaprevir due to D168N/Y mutations: A molecular dynamics simulation perspective

Hepatitis C virus (HCV) NS3/4A protease is an attractive target for the development of antiviral therapy. However, the evolution of antiviral drug resistance is a major problem for treatment of HCV infected patients. Understanding of drug-resistance mechanisms at molecular level is therefore very important for the guidance of further design of antiviral drugs with high efficiency and specificity. Paritaprevir is a potent inhibitor against HCV NS3/4A protease genotype 1a. Unfortunately, this compound is highly susceptible to the substitution at D168 in the protease. In this work, molecular dynamics simulations of paritaprevir complexed with wild-type (WT) and two mutated strains (D168 N and D168Y) were carried out. Due to such mutations, the inhibitor-protein hydrogen bonding between them was weakened and the salt-bridge network among residues R123, R155 and D168 responsible for inhibitor binding was disrupted. Moreover, the per-residue free energy decomposition suggested that the main contributions from key residues such as Q80, V132, K136, G137 and R155 were lost in the D168 N/Y mutations. These lead to a lower binding affinity of paritaprevir for D168 N/Y variants of the HCV NS3/4A protease, consistent with the experimental data. This detailed information could be useful for further design of high potency anti-HCV NS3/4A inhibitors.     

Design,Synthesis and Biological Evaluation of Pentacyclic Triterpene Dimers as HCV Entry Inhibitors

A series of triterpene dimers bearing different scaffold were designed and synthesized via CuAAC reaction. Their anti‐HCV entry activities were evaluated by HCVpp and VSVpp entry assays. It was found that echinocystic acid (EA) and its dimer were still necessary for maintaining anti‐HCV entry activity, and replacement of EA by other triterpenes might significantly decrease its anti‐viral activities. Using a linker bearing a piperazine group, compound 14 dramatically increased its potency with IC₅₀ at 2.87 nmol/L. In addition, the undesired hemolytic effect of all these compounds was removed.     

Fast DNA and protein microarray tests for the diagnosis of hepatitis C virus infection on a single platform

Hepatitis C virus (HCV) is a major cause of chronic liver disease and liver cancer, and remains a large health care burden to the world. In this study we developed a DNA microarray test to detect HCV RNA and a protein microarray to detect human anti-HCV antibodies on a single platform. A main focus of this study was to evaluate possibilities to reduce the assay time, as a short time-to-result (TTR) is a prerequisite for a point-of-care test. Significantly reducing hybridisation and washing times did not impair the assay performance. This was confirmed first using artificial targets and subsequently using clinical samples from an HCV seroconversion panel derived from a HCV-infected patient. We were able to reduce the time required for the detection of human anti-HCV antibodies to only 14 min, achieving nanomolar sensitivity. The protein microarray exhibited an analytical sensitivity comparable to that of commercial systems. Similar results were obtained with the DNA microarray using a universal probe which covered all different HCV genotypes. It was possible to reduce the assay time after PCR from 150 min to 16 min without any loss of sensitivity. Taken together, these results constitute a significant step forward in the design of rapid, microarray-based diagnostics for human infectious disease, and show that the protein microarray is currently the most favourable candidate to fill this role.     

AntiHepatitis C Virus Activity of Alectryon serratus Leaves Extract

Hepatitis C Virus (HCV) has infected approximately 2-3% (130-170 million) of the world's population. No vaccine is available to prevent HCV infection. Investigation of anti-HCV agent is thus deemed necessary. Various plants have been explored for their anti-HCV activity. A. serratus is a member of Sapindaceae family, which fruit and seed were traditionally used as insecticide. Anti-HCV activity tested on A.serratus leaves extract has been done. The result showed that leaves extract exhibited anti-HCV with IC₅₀ value of 14.9 μg/ml and 9.8 μg/ml against HCV J6/JFH1 and JFH1a, respectively. The cytotoxicity assay results showed that A.serratus leaves extract was not toxic and has CC₅₀ >100 μg/ml. Mode of action experiment results suggested that A.serratus extract inhibited HCV at the post-entry step. Further fractionation of leaves extract by open column chromatography resulted in 4 fractions. Only Fraction 1 (AP-5F.1) exhibited anti-HCV with IC₅₀ value of 1.2 μg/ml against HCV JFH1a. Separation of AP-5F.1 by open column chromatography resulted in 15 fractions. Fraction number 13 (AP-5F.1.13) exhibited anti-HCV with IC₅₀ value of 0.43 μg/ml against HCV JFH1a. Separation of AP-5F.1.13 by semi preparative-HPLC resulted in isolate identified by TLC and LC-MS method as chlorophyll derivate. There was a possibility that chlorophyll derivate has participated in performing the anti-HCV activity of fractions and extract besides the other compounds contained. In this study, we concluded that A. serratus leaves extract, AP-5F.1, and AP-5F.1.13 exhibited anti-HCV activity against JFH1a virus.     

Synthesis and molecular docking studies of some new thiosemicarbazone derivatives as HCV polymeraseinhibitors

Series of new thiosemicarbazones was prepared and molecular studied as inhibitors of HCV 4WTG polymerase. Thus, the thiosemicarbazone derivatives (3a–k) were synthesized by two different ways, from reacting thiosemicarbazides with aldehydes and by one-pot three component reaction using ZnCl₂/SiO₂ as a catalyst under solvent free conditions. Molecular docking analysis of the synthesized products predicted that the thiosemicarbazone derivatives 3c, 3g, and 3k were the most promised as a highly inhibitors for HCV 4WTG polymerase in comparison with Sofosbuvir drug.     

丙型肝炎病毒抑制剂的三维药效团和构效关系

通过ＣＡＴＡＬＹＳＴ软件包得到了两类ＨＣＶ ＮＳ３丝氨酸蛋白酶抑制剂的三维药效团模型。尽管这两类抑制剂具有完全不同的骨架结构,但得到的药效团却具有共同的特性。这当这两类抑制剂和受体发生相互作用时,可能采用了相似的结合模式。根据药效团模型,进行了三维构效关系的研究。结果表明,得到的药效团模型具有良好的预测能力（线性回归系数Ｒ＝０．８９）。     

Evaluation of Water Ammonium Ion Test Kit and Its Feasibility for the Analysis of Protein in Food

The traditional method for the determination of protein in food needs the operations of digestion, distillation, absorption, and titration; therefore, it is complicated and time-consuming and requires professional personnel. Is there a more convenient and faster detection method that can directly determine the ammonium ions in protein digestion solution to obtain the protein content of food and avoid the distillation–absorption–titration process? The feasibility of water ammonium ion test kits for food protein rapid detection was discussed here. After digestion, the protein in food transforms into ammonium ions in the digestion solution. Because of the variety of food, there are many different inorganic ions left in the food digestion solution, and at the same time, digestion agents are added in the digestion process and become potential interference factors in ammonium determination. Therefore, the detection accuracy of ammonium test kits needs to be evaluated first, including their anti-interference ability. The standard curve of ammonium was established by the test kit. When the ammonium concentration was 0.00–2.50 mg/L, the absorbance at 620 nm was linearly related to the ammonium concentration, the determination coefficient R² was 0.9995, and the detection limit of this method was 0.01 mg/L. The influences of temperature, pH value, and reaction time on the test kit method were discussed. The precision was 0.90–3.33%; the repeatability was 1.71–4.86%; and the recovery rate of tap water, river water, and sea water was controlled within 90–103%. The anti-interference ability of the evaluated test kit was better than that of the national standard detection method. The test kit, combined with sample pretreatment and protein conversion formula, was used to detect protein in different types of food (milk powder, rice flour, wheat flour, soy, banana, milk, fish food, chicken food, and dog food). The results showed that there were no significant differences (ρ > 0.05) between the national method and the test kit method. The ammonium ion test kit method shortened the determination time and had higher sensitivity, showing its potential for the rapid determination of food protein.     

Affinity-Based Screening Technology and HCV Drug Discovery

NS5A is one of the non-structural gene products encoded by Hepatitis C virus (HCV) and related viruses that are essential for viral replication. The amino acid sequence of NS5A is conserved between different HCV genotypes and the primary amino acid sequence of NS5A is unique to HCV and closely related viruses. Importantly, NS5A is unrelated to any human protein. This indicates that drugs designed to block the actions of NS5A could inhibit the replication of HCV without showing toxic side effects in human host cells, thus making NS5A inhibitors ideal anti-viral drugs. However, there are presently no functional assays for this essential viral protein. Therefore, conventional high throughput screening (HTS) approaches can not be used to discover antiviral drugs against NS5A.