Exploring the Impact of Ring Mobility on the Macroscopic Properties of Doubly Threaded Slide-Ring Gel Networks.

The integration of mechanically interlocked molecules (MIMs) into polymeric materials has led to the development of mechanically interlocked polymers (MIPs). One class of MIPs that have gained attention in recent years are slide-ring gels (SRGs), which are generally accessed by crosslinking rings on a main-chain polyrotaxane. The mobility of the interlocked crosslinking moieties along the polymer backbone imparts enhanced properties onto these networks. An alternative synthetic approach to SRGs is to use a doubly threaded ring as the crosslinking moiety, yielding doubly threaded slide-ring gel networks (dt-SRGs). In this study, a photo-curable ligand-containing thread was used to assemble a series of metal-templated pseudo[3]rotaxane crosslinkers that allow access to polymer networks that contain doubly threaded interlocked rings. The physicochemical and mechanical properties of these dt-SRGs with varying size of the ring crosslinking moieties were investigated and compared to an entangled gel (EG) prepared by polymerizing the metal complex of the photo-curable ligand-containing thread, and a corresponding covalent gel (CG). Relative to the EG and CG, the dt-SRGs exhibit enhanced swelling behavior, viscoelastic properties, and stress relaxation characteristics. In addition, the macroscopic properties of dt-SRGs could be altered by "locking" ring mobility in the structure through remetalation, highlighting the impact of the mobility of the crosslinks.

Doubly Threaded Slide-Ring Polycatenane Networks.

Crosslinking in polymer networks leads to intrinsic structural inhomogeneities that result in brittle materials. Replacing fixed covalent crosslinks with mobile ones in mechanically interlocked polymers (MIPs), such as in slide-ring networks (SRNs) in which interlocked crosslinks are formed when polymer chains are threaded through crosslinked rings, can lead to tougher, more robust networks. An alternative class of MIPs is the polycatenane network (PCN), in which the covalent crosslinks are replaced with interlocked rings that introduce the unusual catenane's mobility elements (elongation, rotation, and twisting) as connections between polymer chains. A slide-ring polycatenane network (SR-PCN), with doubly threaded rings embedded as crosslinks in a covalent network, combines the mobility features of both the SRNs and PCNs, where the catenated ring crosslinks can slide along the polymer backbone between the two limits of network bonding (covalent and interlocked). This work explores using a metal ion-templated doubly threaded pseudo[3]rotaxane (P3R) crosslinker, combined with a covalent crosslinker and a chain extender, to access such networks. A catalyst-free nitrile-oxide/alkyne cycloaddition polymerization was used to vary the ratio of P3R and covalent crosslinker to yield a series of SR-PCNs that vary in the amount of interlocked crosslinking units. Studies on their mechanical properties show that metal ions fix the rings in the network, leading to similar behavior as the covalent PEG gels. Removal of the metal ion frees the rings resulting in a high-frequency transition attributed to the additional relaxation of polymer chains through the catenated rings while also increasing the rate of poroelastic draining at longer timescales.

Balancing ring and stopper group size to control the stability of doubly threaded [3]rotaxanes.

Synthesizing doubly threaded [3]rotaxanes requires the use of larger rings than more traditional singly threaded [2]rotaxanes. A key challenge in accessing stable doubly threaded [3]rotaxanes with large rings is finding the right combination of ring to stopper size. In this study, a series of doubly threaded [3]rotaxanes derived from five different sized macrocycles in the size range of 40-48 atoms and two different stopper groups, which contain 1 or 2 tris(p-t-butylbiphenyl)methyl moieties, were prepared and their kinetic stability examined. These interlocked compounds were synthesized using a metal-templated approach and fully characterized utilizing a combination of mass spectrometry, NMR spectroscopy, and size-exclusion chromatography techniques. The effect of ring size on the stability of the doubly threaded [3]rotaxane was investigated via kinetic stability tests monitored using 1H-NMR spectroscopy. By tightening the macrocycle systematically every 2 atoms from 48 to 40 atoms, a wide range of doubly threaded interlocked molecules could be accessed in which the rate of room temperature slippage of the macrocycle from the dumbbells could be tuned. Using the larger stopper group with a 48-atom ring results in no observable rotaxane, 46-44 atom macrocycles result in metastable rotaxane species with a slippage half-life of ∼5 weeks and ∼9 weeks, respectively, while macrocycles of 42 atoms or smaller yield a stable rotaxane. The smaller sized stopper is not able to fully stabilize any of the [3]rotaxane structures but metastable [3]rotaxanes are obtained with slippage half-lives of 25 ± 2 hours and 13 ± 1 days using macrocycles with 42 or 40 atoms, respectively. These results highlight the dramatic effect that relatively small ring size changes can have on the structure of doubly threaded [3]rotaxanes and lay the synthetic groundwork for a range of higher order doubly threaded interlocked architectures.

Dynamic multimodal holograms of conjugated organogels via dithering mask lithography.

Polymeric materials have been used to realize optical systems that, through periodic variations of their structural or optical properties, interact with light-generating holographic signals. Complex holographic systems can also be dynamically controlled through exposure to external stimuli, yet they usually contain only a single type of holographic mode. Here, we report a conjugated organogel that reversibly displays three modes of holograms in a single architecture. Using dithering mask lithography, we realized two-dimensional patterns with varying cross-linking densities on a conjugated polydiacetylene. In protic solvents, the organogel contracts anisotropically to develop optical and structural heterogeneities along the third dimension, displaying holograms in the form of three-dimensional full parallax signals, both in fluorescence and bright-field microscopy imaging. In aprotic solvents, these heterogeneities diminish as organogels expand, recovering the two-dimensional periodicity to display a third hologram mode based on iridescent structural colours. Our study presents a next-generation hologram manufacturing method for multilevel encryption technologies.

Anisotropic Electrical Conductivity of a Single-Crystalline Oxo-Bridged Cr4IIIMo2VI Heterometallic Complex.

A new oxo-bridged chromium-molybdenum heterometallic complex, O-CrMoHC ([Cr4(MoO4)2O2(OAc)4(DMF)4]·2DMF), was synthesized by using a simple solvothermal reaction. In this complex, the octahedrally coordinated Cr(III) and tetrahedrally coordinated Mo(VI) metal centers are bridged by oxo ligands. O-CrMoHC has in-plane π-conjugation systems, which are interconnected by noncoordinating DMF molecules. The crystals show anisotropic conductivity with respect to the crystal planes, and theoretical calculations were used to study their origins. The O-CrMoHC single crystals exhibited that a relatively high electrical conductivity with an average value of 5.37 × 10-7 S/cm was observed along the [01-1] direction, but the current level was very low along the [100] direction. This is the first report of anisotropic conductivity observed in the single crystal of a monomeric heterometallic complex.

Regulation of La/SSB-dependent viral gene expression by pre-tRNA 3' trailer-derived tRNA fragments.

tRNA-derived RNA fragments (tRFs) have emerged as a new class of functional RNAs implicated in cancer, metabolic and neurological disorders, and viral infection. Yet our understanding of their biogenesis and functions remains limited. In the present study, through analysis of small RNA profile we have identified a distinct set of tRFs derived from pre-tRNA 3' trailers in the hepatocellular carcinoma cell line Huh7. Among those tRFs, tRF_U3_1, which is a 19-nucleotide-long chr10.tRNA2-Ser(TGA)-derived trailer, was expressed most abundantly in both Huh7 and cancerous liver tissues, being present primarily in the cytoplasm. We show that genetic loss of tRF_U3_1 does not affect cell growth and it is not involved in Ago2-mediated gene silencing. Using La/SSB knockout Huh7 cell lines, we demonstrate that this nuclear-cytoplasmic shuttling protein directly binds to the 3' U-tail of tRF_U3_1 and other abundantly expressed trailers and plays a critical role in their stable cytoplasmic accumulation. The pre-tRNA trailer-derived tRFs capable of sequestering the limiting amounts of La/SSB in the cytoplasm rendered cells resistant to various RNA viruses, which usurp La/SSB with RNA chaperone activity for their gene expression. Collectively, our results establish the trailer-derived tRF-La/SSB interface, regulating viral gene expression.

What We Should Consider in Point of Care Blood Glucose Test; Current Quality Management Status of a Single Institution.

Background and Objectives: Point of care test (POCT) is generally performed by non-laboratory staff who often lack an understanding on the quality control and quality assurance programs. The purpose of this study was to understand the current status of quality management of point of care (POC) blood glucose testing in a single institution where non-laboratory staff perform the tests. Materials and Methods: From July to August 2020, management status of glucometer, test strips, quality control (QC) materials, quality assurance program, and operators' response to processing of displayed results was monitored in all Soonchunhyang University Bucheon hospital departments that performed POC blood glucose test. Results of the POC blood glucose test conducted from January 2019 to May 2020 were analyzed retrospectively. Results: A total 124 glucometers were monitored in 47 departments. Insufficient management of approximately 50% of blood sugar, test strips, and QC materials was observed. Although daily QC was conducted by 95.7% of the departments, the QC records were inaccurate. The method of recording test results varied with departments and operators. Various judgments and troubleshooting were performed on the unexpected or out of measurable range results, including some inappropriate processes. In POC blood glucose test results review, 4568 atypical results were identified from a total of 572,207 results. Conclusions: Sufficient training of the non-laboratory staff and ongoing assessment of competency through recertification is needed to maintain acceptable levels of POCT quality. In this study, various problems were identified in glucometer and reagent management, QC and post-analytic phase. We believe that these results provide meaningful basal information for planning effective operators' training and competency evaluation, and the development of an efficient POCT quality management system.

Performance evaluation of the QMAC-dRAST for staphylococci and enterococci isolated from blood culture: a comparative study of performance with the VITEK-2 system.

Objectives: To evaluate the performance of a rapid antimicrobial susceptibility testing (AST) platform based on microfluidic chip technology, the QMAC-dRAST, which enables AST from colony isolates or positive blood culture broth (PBCB), and to compare the performance of the QMAC-dRAST for staphylococci and enterococci with that of the VITEK-2 system based on reference broth microdilution (BMD). Methods: A total of 110 staphylococcal and enterococcal isolates from blood cultures were included. AST was performed directly using the QMAC-dRAST with PBCB. Thereafter, colony isolates derived from subculture of PBCB were used for the QMAC-dRAST, the VITEK-2 system and BMD. Results: The overall agreement between the QMAC-dRAST with PBCB and BMD was 91.5%. There were 1.2% very major errors (VMEs), 4.3% major errors (MEs) and 5.4% minor errors (mEs). The QMAC-dRAST with colony isolates yielded 94.6% agreement and error rates of 1.0% VMEs, 1.8% MEs and 4.0% mEs. The VITEK-2 system showed 96.2% agreement and error rates of 2.3% VMEs, 0.5% MEs and 2.6% mEs. The incubation time in the QMAC-dRAST was significantly shorter than in the VITEK-2 system (median of 6 versus 10 h; P < 0.0001). Conclusions: The QMAC-dRAST system provided rapid results and represents an alternative to conventional AST methods. The QMAC-dRAST with colony isolates produced more reliable results for staphylococci and enterococci than the QMAC-dRAST with PBCB. The QMAC-dRAST system also performed comparably to BMD and the VITEK-2 system.

Hepatitis C Virus Core Protein Promotes miR-122 Destabilization by Inhibiting GLD-2.

The liver-specific microRNA miR-122, which has essential roles in liver development and metabolism, is a key proviral factor for hepatitis C virus (HCV). Despite its crucial role in the liver and HCV life cycle, little is known about the molecular mechanism of miR-122 expression regulation by HCV infection. Here, we show that the HCV core protein downregulates the abundance of miR-122 by promoting its destabilization via the inhibition of GLD-2, a non-canonical cytoplasmic poly(A) polymerase. The decrease in miR-122 expression resulted in the dysregulation of the known functions of miR-122, including its proviral activity for HCV. By high-throughput sequencing of small RNAs from human liver biopsies, we found that the 22-nucleotide (nt) prototype miR-122 is modified at its 3' end by 3'-terminal non-templated and templated nucleotide additions. Remarkably, the proportion of miR-122 isomers bearing a single nucleotide tail of any ribonucleotide decreased in liver specimens from patients with HCV. We found that these single-nucleotide-tailed miR-122 isomers display increased miRNA activity and stability over the 22-nt prototype miR-122 and that the 3'-terminal extension is catalyzed by the unique terminal nucleotidyl transferase activity of GLD-2, which is capable of adding any single ribonucleotide without preference of adenylate to the miR-122 3' end. The HCV core protein specifically inhibited GLD-2, and its interaction with GLD-2 in the cytoplasm was found to be responsible for miR-122 downregulation. Collectively, our results provide new insights into the regulatory role of the HCV core protein in controlling viral RNA abundance and miR-122 functions through miR-122 stability modulation.

Inhibition of hepatitis C virus in mouse models by lipidoid nanoparticle-mediated systemic delivery of siRNA against PRK2.

Host-targeting antivirals have an advantage over direct-acting antivirals in that they have a high genetic barrier to resistance. Here, we describe in vivo anti-hepatitis C virus (HCV) efficacy of a potent siRNA targeting the protein kinase C-related kinase 2 (PRK2), which phosphorylates HCV NS5B RNA-dependent RNA polymerase and promotes HCV replication. PRK2-silencing reduced the phosphorylated NS5B level and resulted in inhibition of NS5B RdRp activity to decrease HCV genome abundance. Systemic administration of lipidoid nanoparticle-formulated PRK2 siRNA (once every three days for a total of three injections at a dose of 3mgkg(-1)) resulted in a 3.72 and 1.96 log10 reduction in serum HCV RNA titer, in mouse subcutaneous and orthotopic xenograft models for HCV replication, respectively. Our results verify the essential role of PRK2 in HCV replication and offer a host-targeting anti-HCV siRNA therapy that might be beneficial for non-responders to current treatment regimens.

Phosphorylation of hepatitis C virus RNA polymerases ser29 and ser42 by protein kinase C-related kinase 2 regulates viral RNA replication.

UNLABELLED: Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE: While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the Δ1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

Liver extracellular matrix providing dual functions of two-dimensional substrate coating and three-dimensional injectable hydrogel platform for liver tissue engineering.

Decellularization of tissues or organs can provide an efficient strategy for preparing functional scaffolds for tissue engineering. Microstructures of native extracellular matrices and their biochemical compositions can be retained in the decellularized matrices, providing tissue-specific microenvironments for efficient tissue regeneration. Here, we report the versatility of liver extracellular matrix (LEM) that can be used for two-dimensional (2D) coating and three-dimensional (3D) hydrogel platforms for culture and transplantation of primary hepatocytes. Collagen type I (Col I) has typically been used for hepatocyte culture and transplantation. In this study, LEM was compared with Col I in terms of biophysical and mechanical characteristics and biological performance for enhancing cell viability, differentiation, and hepatic functions. Surface properties of LEM coating and mechanical properties and gelation kinetics of LEM hydrogel could be manipulated by adjusting the LEM concentration. In addition, LEM hydrogel exhibited improved elastic properties, rapid gelation, and volume maintenance compared to Col I hydrogel. LEM coating significantly improved hepatocyte functions such as albumin secretion and urea synthesis. More interestingly, LEM coating upregulated hepatic gene expression of human adipose-derived stem cells, indicating enhanced hepatic differentiation of these stem cells. The viability and hepatic functions of primary hepatocytes were also significantly improved in LEM hydrogel compared to Col I hydrogel both in vitro and in vivo. Albumin and hepatocyte transcription factor expression was upregulated in hepatocytes transplanted in LEM hydrogels. In conclusion, LEM can provide functional biomaterial platforms for diverse applications in liver tissue engineering by promoting survival and maturation of hepatocytes and hepatic commitment of stem cells. This study demonstrates the feasibility of decellularized matrix for both 2D coating and 3D hydrogel in liver tissue engineering.

Phenolic phytochemical displaying SARS-CoV papain-like protease inhibition from the seeds of Psoralea corylifolia.

Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro) is a key enzyme that plays an important role in SARS virus replication. The ethanol extract of the seeds of Psoralea corylifolia showed high activity against the SARS-CoV PLpro with an IC50 of value of 15 µg/ml. Due to its potency, subsequent bioactivity-guided fractionation of the ethanol extract led to six aromatic compounds (1-6), which were identified as bavachinin (1), neobavaisoflavone (2), isobavachalcone (3), 4'-O-methylbavachalcone (4), psoralidin (5) and corylifol A (6). All isolated flavonoids (1-6) inhibited PLpro in a dose-dependent manner with IC50 ranging between 4.2 and 38.4 µM. Lineweaver-Burk and Dixon plots and their secondary replots indicated that inhibitors (1-6) were mixed inhibitors of PLpro. The analysis of KI and KIS values proved that the two most promising compounds (3 and 5) had reversible mixed type I mechanisms.

Functional polymeric DNA nanostructure-decorated cellulose nanocrystals for targeted and stimuli-responsive drug delivery.

Targeted and stimuli-responsive drug delivery enhances therapeutic efficacy and minimizes undesirable side effects of cancer treatment. Although cellulose nanocrystals (CNCs) are used as drug carriers because of their robustness, spindle shape, biocompatibility, renewability, and nontoxicity, the lack of programmability and functionality of CNCs-based platforms hampers their application. Thus, high adaptability and the capacity to form dynamic 3D nanostructures of DNA may be advantageous, as they can provide functionalities such as target-specific and stimuli-responsive drug release. Using DNA nanotechnology, the functional polymeric form of DNA nanostructures can be replicated using rolling circle amplification (RCA), and the biologically and physiologically stable DNA nanostructures may overcome the challenges of CNCs. In this study, multifunctional polymeric DNAs produced with RCA were strongly complexed with surface-modified CNCs via electrostatic interactions to form polymeric DNA-decorated CNCs (pDCs). Particle size, polydispersity, zeta potential, and biostability of the nanocomplexes were analyzed. As a proof of concept, the dynamic structural functionalities of DNA nanostructures were verified by observing cancer-targeted intracellular delivery and pH-responsive drug release. pDCs showed anticancer properties without side effects in vitro, owing to their aptamer and i-motif functionalities. In conclusion, pDCs exhibited multifunctional anticancer activities, demonstrating their potential as a promising hybrid nanocomplex platform for targeted cancer therapy.

SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics to induce robust virus propagation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a 'highly transmissible respiratory pathogen, leading to severe multi-organ damage. However, knowledge regarding SARS-CoV-2-induced cellular alterations is limited. In this study, we report that SARS-CoV-2 aberrantly elevates mitochondrial bioenergetics and activates the EGFR-mediated cell survival signal cascade during the early stage of viral infection. SARS-CoV-2 causes an increase in mitochondrial transmembrane potential via the SARS-CoV-2 RNA-nucleocapsid cluster, thereby abnormally promoting mitochondrial elongation and the OXPHOS process, followed by enhancing ATP production. Furthermore, SARS-CoV-2 activates the EGFR signal cascade and subsequently induces mitochondrial EGFR trafficking, contributing to abnormal OXPHOS process and viral propagation. Approved EGFR inhibitors remarkably reduce SARS-CoV-2 propagation, among which vandetanib exhibits the highest antiviral efficacy. Treatment of SARS-CoV-2-infected cells with vandetanib decreases SARS-CoV-2-induced EGFR trafficking to the mitochondria and restores SARS-CoV-2-induced aberrant elevation in OXPHOS process and ATP generation, thereby resulting in the reduction of SARS-CoV-2 propagation. Furthermore, oral administration of vandetanib to SARS-CoV-2-infected hACE2 transgenic mice reduces SARS-CoV-2 propagation in lung tissue and mitigates SARS-CoV-2-induced lung inflammation. Vandetanib also exhibits potent antiviral activity against various SARS-CoV-2 variants of concern, including alpha, beta, delta and omicron, in in vitro cell culture experiments. Taken together, our findings provide novel insight into SARS-CoV-2-induced alterations in mitochondrial dynamics and EGFR trafficking during the early stage of viral infection and their roles in robust SARS-CoV-2 propagation, suggesting that EGFR is an attractive host target for combating COVID-19.

Lrig1-expression confers suppressive function to CD4+ cells and is essential for averting autoimmunity via the Smad2/3/Foxp3 axis.

Regulatory T cells (Treg) are CD4+ T cells with immune-suppressive function, which is defined by Foxp3 expression. However, the molecular determinants defining the suppressive population of T cells have yet to be discovered. Here we report that the cell surface protein Lrig1 is enriched in suppressive T cells and controls their suppressive behaviors. Within CD4+ T cells, Treg cells express the highest levels of Lrig1, and the expression level is further increasing with activation. The Lrig1+ subpopulation from T helper (Th) 17 cells showed higher suppressive activity than the Lrig1- subpopulation. Lrig1-deficiency impairs the suppressive function of Treg cells, while Lrig1-deficient naïve T cells normally differentiate into other T cell subsets. Adoptive transfer of CD4+Lrig1+ T cells alleviates autoimmune symptoms in colitis and lupus nephritis mouse models. A monoclonal anti-Lrig1 antibody significantly improves the symptoms of experimental autoimmune encephalomyelitis. In conclusion, Lrig1 is an important regulator of suppressive T cell function and an exploitable target for treating autoimmune conditions.

Destabilization of PDK1 by Hsp90 inactivation suppresses hepatitis C virus replication through inhibition of PRK2-mediated viral RNA polymerase phosphorylation.

Heat shock protein 90 (Hsp90), which chaperones multiple client proteins, has been shown to be implicated in HCV replication. Pharmacological inhibitors of Hsp90 display an anti-HCV activity. However, little is known about the mechanisms of regulation of HCV replication by Hsp90. Here, we show that Hsp90 inhibition by 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) destabilizes phosphoinositide-dependent kinase-1 (PDK1), an upstream kinase of the protein kinase C-related kinase 2 (PRK2) responsible for phosphorylation of HCV RNA polymerase, through the proteosome pathway. Destabilization of PDK1 led to inhibition of phosphorylation of the viral RNA polymerase through a decrease in the abundance of active form PRK2 level. Consequently, Hsp90 inhibition resulted in suppression of HCV replication both in human hepatoma Huh7 cells harboring an HCV subgenomic replicon and in HCV-infected cells. 17-DMAG treatment did not interfere with HCV internal ribosome entry site-mediated translation and the cell cycle in Huh7 cells. Co-treatment of 17-DMAG with interferon-α or HA1077, an inhibitor of PRK2, enhanced the anti-HCV activity of 17-DMAG. Taken together, these findings suggest that Hsp90 plays a critical role in the regulation of HCV RNA polymerase phosphorylation via the PDK1-PRK2 signaling pathway.

Inhibition of hepatitis C virus replication by Monascus pigment derivatives that interfere with viral RNA polymerase activity and the mevalonate biosynthesis pathway.

OBJECTIVES: Hepatitis C virus (HCV) infection causes chronic liver disease and is a major public health problem worldwide. The aim of this study was to evaluate the potential of Monascus pigment derivatives, which were derived from a microbial secondary metabolite synthesized from polyketides by Monascus spp., as HCV antiviral agents. METHODS: We performed an in vitro RNA-dependent RNA polymerase (RdRp) assay to screen for HCV RdRp inhibitors. The anti-HCV activity of RdRp inhibitors in HCV-replicating cells was evaluated by quantification of the RNA viral genome. Molecular docking analysis was performed to predict the binding sites of the selected RdRp inhibitors. RESULTS: We have identified a Monascus pigment and its derivatives as inhibitors of the HCV NS5B RdRp. A group of Monascus orange pigment (MOP) amino acid derivatives, in which the reactive oxygen moiety was changed to amino acids, significantly inhibited HCV replication. Further, combination of the MOP derivatives (Phe, Val or Leu conjugates) with interferon (IFN)-α inhibited HCV replication more than IFN-α treatment alone. Lastly, molecular docking studies indicate the inhibitors may bind to a thumb subdomain allosteric site of NS5B. The antiviral activity of the MOP derivatives was related to a modulation of the mevalonate pathway, since the mevalonate-induced increase in HCV replication was suppressed by the MOP compounds. CONCLUSIONS: Our results identify amino acid derivatives of MOP as potential anti-HCV agents and suggest that their combination with IFN-α might offer an alternative strategy for the control of HCV replication.

Biochemical characterization of a recombinant SARS coronavirus nsp12 RNA-dependent RNA polymerase capable of copying viral RNA templates.

The severe acute respiratory syndrome coronavirus (SARS-CoV) RNA genome is replicated by a virus-encoded RNA replicase, the key component of which is the nonstructural protein 12 (nsp12). In this report, we describe the biochemical properties of a full-length recombinant SARS-CoV nsp12 RNA-dependent RNA polymerase (RdRp) capable of copying viral RNA templates. The purified SARS-CoV nsp12 showed both primer-dependent and primer-independent RNA synthesis activities using homopolymeric RNA templates. The RdRp activity was strictly dependent on Mn(2+). The nsp12 preferentially copied homopolymeric pyrimidine RNA templates in the absence of an added oligonucleotide primer. It was also able to initiate de novo RNA synthesis from the 3'-ends of both the plus- and minus-strand genome of SARS-CoV, using the 3'-terminal 36- and 37-nt RNA, respectively. The in vitro RdRp assay system established with a full-length nsp12 will be useful for understanding the mechanisms of coronavirus replication and for the development of anti-SARS-CoV agents.

A Simple and High-Throughput LC-MS-MS Method for Simultaneous Measurement of Nicotine, Cotinine, 3-OH Cotinine, Nornicotine and Anabasine in Urine and Its Application in the General Korean Population.

Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography--tandem mass spectrometry (LC-MS-MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine and anabasine for population-based studies using LC-MS-MS. Each 30 µL of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column, and LC-MS-MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect and carryover according to guidelines. Our methods required a short preparation time (∼20 min) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run coefficient of variation (CV) 2.9-9.4%, between-run CV 4.8-8.7% and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine and anabasine; 2-5,000 ng/mL for cotinine and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carryover was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine and anabasine were detected in 97.6, 99.2, 98.4, 96.8 and 87.2%, respectively. No markers were detected in 93.9% of 609 nonsmokers. The overlapping detection of multiple markers made it possible to identify the smoking status even in current smokers with a low concentration of cotinine. Our LC-MS-MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.