Dahai Yu

Evaluation of the anti-neuraminidase activity of the traditional Chinese medicines and determination of the anti-influenza A virus effects of the neuraminidase inhibitory TCMs in vitro and in vivo.

UNLABELLED ETNOPHARMACOLOGICAL RELEVANCE: Neuraminidase (NA) inhibitors are currently the most effective drugs to treat influenza A viruses infection. Many traditional Chinese medicines (TCMs) have been used in the clinics to treat influenza. The anti-viral mechanisms of these TCMs and their inhibitory effects towards NA need to be systematically tested. AIM OF THE STUDY To evaluate the anti-NA activity of the TCMs and the anti-influenza A virus effects of the NA inhibitory TCMs in vitro and in vivo. MATERIAL AND METHODS We tested the inhibitory activity of water extracts from 439 TCMs towards NA. The in vitro anti-influenza virus activities of the 5 TCMs were evaluated using the strain A/California/7/2009 (H1N1) NYMC X-179A of influenza A virus. A randomly selected TCM with NA inhibitory activity, Melia toosendan extract, was further evaluated using a mouse model infected with influenza A virus. RESULTS Five TCMs, Duchesnea indica (Andr.) Focke [Fragaria indica Andr.], Liquidambar formosana Hance., Lithospermum erythrorhizon Sieb. et Zucc., Melia toosendan Sieb. et Zucc., and Prunella vulgaris L., exerted potent inhibitory activity towards NA. These TCMs in the range of 25-250 μg/mL had the ability to reduce virus-induced cytopathic effect (CPE) and the virus yield in MDCK cells. Melia toosendan significantly reduced death rate and prolonged mean day to death (MDD) of the viral infected mice. CONCLUSIONS This study describes five TCMs exerted strong inhibitory activities towards NA, and exhibited antiviral effect against influenza A virus by reducing viral reproduction and reduced CPE of the viral infected cells. Melia toosendan, significantly reduced death rate and prolonged survival of the H1N1 viral infected mice.

Enhanced catalytic activity and thermal stability of 2,4-dichlorophenol hydroxylase by using microwave irradiation and imidazolium ionic liquid for 2,4-dichlorophenol removal

Enzymatic removal of 2,4-dichlorophenol (2,4-DCP) has become more attractive recently due to its high efficiency, low cost and environmental benefits. A highly active 2,4-DCP hydroxylase for 2,4-DCP removal was obtained and used in 2,4-DCP removal by employing microwave irradiation as a heating mode and ionic liquid (IL) as an additive. Both [EMIM][PF6] (1-ethyl-3-methylimidazolium hexafluorophosphate) and microwave irradiation were found to increase the 2,4-DCP removal efficiency and the thermal stability of 2,4-DCP hydroxylase, and a further incremental effect of microwave irradiation and [EMIM][PF6] on improving the 2,4-DCP removal efficiency and enzymatic thermal stability was observed. Conditions for 2,4-DCP removal were optimized and the removal of 2,4-DCP was completed in 15 min at 25 °C with 0.66 ± 0.015 U mg−1 enzyme activity under the optimum conditions, much faster than the present enzymatic removal route, which took several hours for complete 2,4-DCP removal. Only 30 min were required for complete 2,4-DCP removal by using the 2,4-DCP hydroxylase at 4 °C, indicating its psychrotrophic adaptability. These results showed that the use of 2,4-DCP hydroxylase under microwave in [EMIM][PF6] is a fast, efficient and environmentally benign method for the removal of 2,4-DCP, and this method can be used over a wide temperature range.

2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity

Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments.

Safflomin A inhibits neuraminidase activity and influenza virus replication

Neuraminidase (NA) is a glycoprotein on the surface of the influenza virus that plays an important role in the early processes of virus infection and viral release from the infected cells. NA inhibitors are currently the most effective drugs to treat influenza virus infection. Many traditional Chinese medicines (TCMs) in various formulations have been used in Chinese clinics to treat influenza, however, the effective constituents and the mechanism of action are mostly unknown. In this paper, we have tested almost 300 natural compounds from a Chinese medicinal herbal compound library to evaluate their anti-NA activities in vitro. Safflomin A (SA) was one of the compound detected with NA inhibitory activities. It showed inhibition against neuraminidases from H1N1 and H3N2 of type A and neuraminidase of type B influenza viruses. Enzyme kinetic tests using SA revealed that the types of inhibition against N1 and N2 neuraminidases were noncompetitive. The interaction of SA with the N1 and N2 neuraminidases was analyzed using molecular simulation and docking, which showed that SA was bound in the non-active sites of N1 and N2. SA was also analyzed for its cytotoxicity and anti-viral activities in cell culture. It showed inhibitory effect for viral replication of H1N1 and H3N2 influenza viruses in MDCK cells. Enzymatic analysis indicated that a SA and oseltamivir carboxylate combination treatment was synergistic with combination index (CI) values ranging between 0.49 and 0.52 against neuraminidase of A/New Caledonia/20/1999 (H1N1), and ranging between 0.56 and 0.88 against neuraminidase of A/Fujian/411/2002 (H3N2). These results suggest that an herbal formulation containing SA in combination with oseltamivir carboxylate may serve as a potential therapeutic option to currently available anti-influenza therapeutics.

Computational study on new natural polycyclic compounds of H1N1 influenza virus neuraminidase

A new strain of influenza A (H1N1) virus is a major cause of morbidity and mortality around the world. The neuraminidase of the influenza virus has been the most potential target for the anti-influenza drugs such as oseltamivir and zanamivir. However, the emergence of drug-resistant variants of these drugs makes a pressing need for the development of new neuraminidase inhibitors for controlling illness and transmission. Here a 3D structure model of H1N1 avian influenza virus neuraminidase type 1 (N1) was constructed based on the structure of the template H5N1 avian influenza virus N1. Upon application of virtual screening technique for N1 inhibitors, two novel compounds (ZINC database ID: ZINC02128091, ZINC02098378) were found as the most favorable interaction energy with N1. Docking results showed that the compounds bound not only in the active pocket, but also in a new hydrophobic cave which contains Arg368, Trp399, Ile427, Pro431 and Lys432 of N1. Our result suggested that both of the screened compounds containing the hydrophobic group bring a strong conjugation effect with Arg293, Arg368 Lys432 of N1 by pi-pi interaction. However, the control inhibitors zanamivir and oseltamivir do not have this effect. The details of N1-compound binding structure obtained will be valuable for the development of a new anti-influenza virus agent.

Exploring substrate promiscuity of chlorophenol hydroxylase against biphenyl derivatives

A 2,4-dichlorophenol hydroxylase, whose gene was derived from the metagenomic library of polychlorinated biphenyl (PCB)-contaminated soil has been found to exhibit a broad range of activity for single ring aromatic contaminants including chlorophenols (CPs) and their homologues. In this study, we intended to explore its activity to aromatic bicyclic compounds such as biphenyl and its derivatives which are also important persistent environmental contaminants. Results demonstrated that the enzyme exhibited broad substrate specificity to selected biphenyl derivatives including hydroxylated biphenyls, halogenated biphenyls, PCBs and hydroxylated PCBs, which extended its substrate promiscuity apart from CPs and their homologues. The enzymatic activities against these aromatic bicyclic compounds were congener dependent and the position and type of the substituent on biphenyl derivatives greatly affected the substrate priority of this enzyme. The hypothesis of the catalysis preference of the enzyme on the aromatic ring was preliminarily proposed on the basis of the analyses of the enzymatic activities against biphenyl derivatives. The high activity and removal ability of this enzyme against selected aromatic contaminants would make it a very promising catalyst for bioremediation of biphenyl derivatives.

Simultaneous efficient removal of high‐strength ammonia nitrogen and chemical oxygen demand from landfill leachate by using an extremely high ammonia nitrogen–resistant strain

Bioaugmentation is a promising technology for pollutant elimination from stressed environments, and it would provide an efficient way to solve challenges in traditional biotreatment of wastewater with high strength of ammonia nitrogen (NH4+‐N). A high NH4+‐N‐resistant bacteria strain, identified as Bacillus cereus (Jlu BC), was domesticated and isolated from the bacteria consortium in landfill leachate. Jlu BC could survive in 100 g/L NH4+‐N environment, which indicated its extremely high NH4+‐N tolerance than the stains found before. Jlu BC was employed in the bioaugmented system to remove high strength of NH4+‐N from landfill leachate, and to increase the removal efficiency, response surface methodology (RSM) was used for optimizing bioaugmentation degradation conditions. At the optimum condition (initial pH 7.33, 4.14 days, initial chemical oxygen demand [COD] concentration [18,000 mg/L], 3.5 mL inoculated domesticated bacteria strain, 0.3 mg/mL phosphorus supplement, 30 °C, and 170 rpm), 94.74 ± 3.8% removal rate of NH4+‐N was obtained, and the experiment data corresponded well with the predicted removal rate of the RSM models (95.50%). Furthermore, COD removal rate of 81.94 ± 1.4% was obtained simultaneously. The results presented are promising, and the screened strain would be of great practical importance in mature landfill leachate and other NH4+‐N enrichment wastewater pollution control.

DNA binding ability of histone-like protein HPhA is negatively affected by interaction with Pb2+.

The histone-like protein (HPhA) is highly homologous to the eukaryotic histones and has the ability to bind to the DNA molecules. In this study, we tested divalent metal ions Mg2+, Ca2+, Zn2+, Pb2+ for their effect on the recombinant HPhA (rHPhA)-DNA binding. We found that only Pb2+ was able to reduce the formation of rHPhA-DNA complex using gel mobility shift assays. Equilibrium dialysis showed that Pb2+ bound to rHPhA by a 2:1 ratio. The interaction of Pb2+ and rHPhA was further studied by spectroscopic method including fluorescence, ultraviolet visible (UV–Vis) absorption, and circular dichroism (CD) spectroscopies. Fluorescent spectroscopy results suggested that Pb2+ and rHPhA formed a complex that caused internal quenching of the fluorescence of the protein at the ground state, and the quenching is predominately static and mixed with dynamic quenching. UV–Vis absorption spectrum results showed Pb2+ caused a slightly blue shift of the maximum absorption wavelength of rHPhA which indicated the reduction of the protein’s hydrophobicity. The CD spectrum further indicated that a reduction of the α-helix content of rHPhA occurred upon binding to Pb2+. Synchronous fluorescence spectrometry analysis revealed that Pb2+ was able to affect the polarity of the amino acids that near the Trp and Tyr residues. These results together showed that Pb2+ interact with the recombinant rHPhA and this interaction negatively affect the ability of rHPhA to form complex with DNA molecules.

Potent selective inhibition of MMP-14 by chloroauric acid and its inhibitory effect on cancer cell invasion

Matrix metalloproteinases (MMPs) are a family of zinc-containing proteases with vital roles in extracellular matrix remodeling. The regulation of MMPs can modulate a number of cellular activities. The therapeutic potential of MMP inhibitors has been shown for diseases such as arthritis and cancer. This paper is the first to demonstrate that HAuCl4 inhibits the activity of MMP-2 and MMP-13 as well as having a more specific inhibitory effect on MMP-14. The effect of HAuCl4 on MMP-14 involves a non-competitive reversible inhibitory mechanism. Moreover, 0–50 μM HAuCl4 did not affect the cell viability of HT-1080 human fibrosarcoma cells. However, HAuCl4 at these concentrations showed significant inhibitory effects on the invasion of the HT-1080 cells, thereby suggesting that HAuCl4 may modulate tumor cell behaviors by inhibiting MMP-14. These findings provide initial clues to further elucidate the biological activity of HAuCl4 and its potential therapeutic value for related diseases.

Potent and selective inhibition of matrix metalloproteinases by lanthanide trichloride

Matrix metalloproteinases (MMPs) are a family of Zn-containing and Ca-dependent proteases with vital roles in extracellular matrix remodeling. Deregulation of MMPs occurs in many pathological conditions such as cardiovascular diseases, inflammation, and cancer. The therapeutic potential of MMP inhibitors has been demonstrated in diseases such as arthritis and cancer. Here we demonstrated that the 3-valent lanthanide compounds LaCl3, TbCl3, GdCl3, YbCl3, and EuCl3 inhibit MMPs such as MMP-2, MMP-13, and MMP-14 (MT1-MMP). The inhibition is more potent and selective toward MT1-MMP compared to the other MMPs. EuCl3 was further selected to study the enzyme kinetics of the MT1-MMP inhibition. The results showed that the inhibition is a mixed type with anti-competition and non-competitive types, which indicated that inhibition was achieved by the compound bound to the non-active center of MT1-MMP and changing the enzyme conformation. The interaction between EuCl3 and MT1-MMP was further studied by UV-visible (UV-vis) light absorption. EuCl3 caused a slight blue shift of the maximum absorption wavelength of MT1-MMP, indicating the interaction reduced protein hydrophobicity. Moreover, EuCl3 exerted substantial inhibitory effects on the migration of HT-1080 cells. Thus, EuCl3 may play a role in modulating tumor cell behavior by inhibiting MMPs activities especially the MT1-MMP activity. These findings provide initial insight into the biological activity and potential therapeutic value of EuCl3.