Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 µmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 µmol/L) nor artemisinin (50 µmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

Reversible Deacidification and Preventive Conservation of Paper-Based Cultural Relics by Mineralized Bacterial Cellulose.

Paper-based cultural relics experience irreversible aging and deterioration during long-term preservation. The most common process of paper degradation is the acid-catalyzed hydrolysis of cellulose. Nowadays, deacidification has been considered as a practical way to protect acidified literature; however, two important criteria of minimal intervention and reversibility should be considered. Inspired by the superior properties of bacterial cellulose (BC) and its structural similarity to paper, herein, the mineralized BC membranes are applied to deacidification and conservation of paper-based materials for the first time. Based on the enzyme-induced mineralization process, the homogeneous and high-loaded calcifications of hydroxyapatite (HAP) and calcium carbonate (CaCO3) nanoparticles onto the nanofibers of BC networks have been achieved, respectively. The size, morphology, structure of minerals, as well as the alkalinity and alkali reserve of BC membranes are well controlled by regulating enzyme concentration and mineralization time. Compared with HAP/CaCO3-immersed method, HAP/CaCO3-BC membranes show more efficient and sustained deacidification performance on paper. The weak alkalinity of mineralized BC membranes avoids the negative effect of alkali on paper, and the high alkali reserve implies a good sustained-release effect of alkali to neutralize the future generated acid. The multiscale nanochannels of the BC membrane provide ion exchange and acid/alkali neutralization channels between paper and the BC membrane, and the final pH of protected paper can be well stabilized in a certain range. Most importantly, this BC-deacidified method is reversible since the BC membrane can be removed without causing any damage to paper and the original structure and fiber morphology of paper are well preserved. In addition, the mineralized BC membrane provides excellent flame-retardant performance on paper thanks to its unique organic-inorganic composite structure. All of these advantages of the mineralized BC membrane indicate its potential use as an effective protection material for the reversible deacidification and preventive conservation of paper-based cultural relics.

Colloidal magnesium hydroxide Nanoflake: One-Step Surfactant-Assisted preparation and Paper-Based relics protection with Long-Term Anti-Acidification and Flame-Retardancy.

Enhancing the interfacial dispersion and suspension stability is crucial for magnesium hydroxide (Mg(OH)2) nanomaterials in the long-term deacidification of paper-based cultural relics. However, because of the low specific surface area and the poor solvent compatibility of as-prepared large-sized Mg(OH)2, it often tends to agglomerate and settle down during the usage and storage, that is harmful for paper protection due to its unevenly deacidification and nonuniformly distribution on paper cellulose. Herein, we propose a feasible preparation of colloidal Mg(OH)2 ultrathin nanoflakes with high dispersion stability via a simple one-step surfactant-assisted strategy. The surfactant acts as both a structure-direct agent to confine the growth of Mg(OH)2 with rich active sites and a surface modifier to enhance its solvent adaptability and dispersion stability, avoiding the common fussy procedure with additional steric stabilizer. Owing to the evenly interaction with free acid species therein and the uniformly distribution on the paper fiber as alkaline reserve, the as-obtained Mg(OH)2 presents the superior paper protection performance characterized by its safer pH of 7.29 for the original aged paper (pH = 5.03) and the excellent long-term anti-acidification effect with competitive pH of 5.47 after accelerated-aging at 105 °C for 5 months. Furthermore, Mg(OH)2 nanoflakes with surfactant-modified structure also endue them as an improved flame retardant for multifunctional paper protection. The protection with Mg(OH)2 has little effect on the paper surface properties and cellulose crystallinity, in line with the principle of least intervention. This work will put forward a feasible way toward colloidal Mg(OH)2 nanoflakes with excellent paper protection performance, shedding light on the development of emerging protection materials for paper-based cultural relics.

Supermolecular film crosslinked by polyoxometalate and chitosan with superior antimicrobial effect.

This paper reported a facile strategy to fabricate ionically crosslinked supramolecular film, in which an in situ formed chitosan ionic film was produced by post-crosslinking the chitosan chains using negatively charged anion polyoxometalate. The incorporation of polyoxometalate into the chitosan ionic system accelerated the crosslinked degree, as evidenced by an increase in surface wrinkle via scanning electron microscopy. Interestingly, the resultant supramolecular film realized the combination of prominent antimicrobial effect and excellent biocompatibility, which was considered to have enormous application potential range from biomedical to environmental science.

Coxsackievirus A6 Induces Cell Cycle Arrest in G0/G1 Phase for Viral Production.

Recent epidemiological data indicate that outbreaks of hand, foot, and mouth disease (HFMD), which can be categorized according to its clinical symptoms as typical or atypical, have markedly increased worldwide. A primary causative agent for typical HFMD outbreaks, enterovirus 71 (EV71), has been shown to manipulate the cell cycle in S phase for own replication; however, it is not clear whether coxsackievirus (CVA6), the main agent for atypical HFMD, also regulates the host cell cycle. In this study, we demonstrate for the first time that CVA6 infection arrests the host cell cycle in G0/G1-phase. Furthermore, synchronization in G0/G1 phase, but not S phase or G2/M phase, promotes viral production. To investigate the mechanism of cell cycle arrest induced by CVA6 infection, we analyzed cell cycle progression after cell cycle synchronization at G0/G1 or G2/M. Our results demonstrate that CVA6 infection promotes G0/G1 phase entry from G2/M phase, and inhibits G0/G1 exit into S phase. In line with its role to arrest cells in G0/G1 phase, the expression of cyclinD1, CDK4, cyclinE1, CDK2, cyclinB1, CDK1, P53, P21, and P16 is regulated by CVA6. Finally, the non-structural proteins of CVA6, RNA-dependent RNA polymerase 3D and protease 3C , are demonstrated to be responsible for the G0/G1-phase arrest. These findings suggest that CVA6 infection arrested cell cycle in G0/G1-phase via non-structural proteins 3D and 3C, which may provide favorable environments for virus production.

Caspase-3 Inhibition Attenuates the Cytopathic Effects of EV71 Infection.

Previous studies demonstrate that human enterovirus 71 (EV71), a primary causative agent for hand, foot, and mouth disease, activates caspase-3 through the non-structural viral 3C protein to induce host cell apoptosis; however, until now it was unclear how 3C activates caspase-3 and how caspase-3 activation affects viral production. Our results demonstrate that 3C binds caspase-8 and caspase-9 but does not directly bind caspase-3 to activate them, and that the proteolytic activity of 3C is required by the activation of caspase-8, caspase-9, and caspase-3. Inhibition of caspase-3 activity attenuates apoptosis in 3C-transfected cells. Furthermore, caspase-3 inhibitor protects host cells from the cytopathic effect of EV71 infection and prevents cell cycle arrest, which is known to be favored for EV71 viral replication. Inhibition of caspase-3 activity decreases EV71 viral protein expression and viral production, but has no effect on viral entry, replication, even polyprotein translation. Therefore, caspase-3 is exploited functionally by EV71 to facilitate its production, which suggests a novel therapeutic approach for the treatment and prevention of hand, foot, and mouth disease.

Zeolite nanoparticle modified microchip reactor for efficient protein digestion.

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.

Mechanism exploration of adsorption-immobilized enzymatic reactor using polymer-coated silica microbeads.

A verified mechanism of adsorption-immobilized enzymatic reactor for enhanced proteolysis is presented. Silica microbeads coated with poly (diallyldimethylammonium chloride) (PDDA) or poly (styrene sulfonate) (PSS) were used to trap trypsin and proteins on the surface through electrostatic interactions in order to improve digestion efficiency. Charge states measured by zeta-potentials showed their positively and negatively charged respectively. We found that high proteolytic efficiency could be achieved only if both proteases and proteins were adsorbed by materials. Once the proteins and proteases were confined together in a nanoscopic area, the enrichment of the substrate could lead to a high performance proteolytic effect. Electrostatic interactions were considered as the predominant adsorption factor rather than hydrophilic/hydrophobic interactions. In less than 5 min, in the presence of PSS-coated silica beads, 10 peptides digested from positively-charged cytochrome C were detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF), with the high sequence coverage up to 63%, while using PDDA-coated silica beads or conventional in-solution digestion yielded only 5 detectable peptides and 39% sequence coverage was obtained. Ovalbumin seemed incompatible with any kind of charged-material-aided tryptic digestion. The mechanism of adsorption-immobilized enzymatic processes has also been studied in detail. The adsorption equilibrium was proven to be attained in less than one minute, and the proteolytic procedure was regarded as the rate-determining step. This study provides a reasonable mechanism for an adsorption-material catalyzed proteolytic procedure and a promising guideline for designing the next generation of high-performance enzymatic reactors.