Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus.

The human ß-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer's disease (AD). In this study, Urechis unicinctus (U. unicinctus), which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential ß-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one, cholesta-4,6-dien-3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, ¹H, and 13C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11) and published values. Hecogenin and cholest-4-en-3-one showed significant inhibitory activity against BACE1 with EC50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6-dien-3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli), Salmonella enterica (S. enterica), Pasteurella multocida (P. multocida), and Physalospora piricola (P. piricola), with minimal inhibitory concentration (MIC) ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

Trachelogenin, a novel inhibitor of hepatitis C virus entry through CD81.

Although much progress has been made in antiviral agents against hepatitis C virus (HCV) in recent years, novel HCV inhibitors with improved efficacy, optimized treatment duration and more affordable prices are still urgently needed. Here, we report the identification of a natural plant-derived lignan, trachelogenin (TGN), as a potent entry inhibitor of HCV without genotype specificity, and with low cytotoxicity. TGN was extracted and purified from Caulis trachelospermi, a traditional Chinese herb with anti-inflammatory and analgesic effects. A crucial function of TGN was the inhibition of HCV entry during a post-binding step without affecting virus replication, translation, assembly and release. TGN blocked virus infection by interfering with the normal interactions between HCV glycoprotein E2 and the host entry factor CD81, which are key processes for valid virus entry. In addition, TGN diminished HCV cell-to-cell spread and exhibited additional synergistic effects when combined with IFN or telaprevir. In conclusion, this study highlights the effect of a novel HCV entry inhibitor, TGN, which has a target that differs from those of the current antiviral agents. Therefore, TGN is a potential candidate for future cocktail therapies to treat HCV-infected patients.

The role of lipid rafts in the early stage of Enterovirus 71 infection.

BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. METHODS: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. RESULTS: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. CONCLUSION: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.

Potential Dermal Exposure to Flonicamid and Risk Assessment of Applicators During Treatment in Apple Orchards.

Exposure and risk assessments of flonicamid for applicators were performed in apple orchards in Korea. Fifteen experiments were done with two experienced applicators under typical field conditions using a speed sprayer. In this study, cotton gloves, socks, masks, and dermal patches were used to monitor potential dermal exposure to flonicamid, and personal air samplers with XAD-2 resin and glass fiber filter were used to monitor potential inhalation exposure. The analytical methods were validated for the limit of detection, limit of quantitation, reproducibility, linearity of the calibration curve, and recovery of flonicamid from various exposure matrices. The results were encouraging and acceptable for an exposure study. The applicability of XAD-2 resin was evaluated via a trapping efficiency and breakthrough test. During the mixing/loading, the average total dermal exposure was 22.6 µg of flonicamid, corresponding to 4.5×10(-5)% of the prepared amount. For the spraying, the potential dermal exposure was 9.32 mg, and the ratio to applied amount was 1.9 × 10(-2%). The primary exposed body parts were the thigh (2.90 mg), upper arm (1.75 mg), and lower leg (1.66 mg). By comparison, absorbable quantity of exposure was small, only 1.62 µg (3.2×10(-6)%). The margin of safety (MOS) were calculated for risk assessment, in all sets of trials, MOS > 1, indicating the exposure level of flonicamid was considered to be safe in apple orchards. Although this was a limited study, it provided a good estimate of flonicamid exposure for orchard applicators.

Multiresidue method for the determination of 227 pesticides in hot pepper (Capsicum annuum L.) by liquid chromatography with tandem mass spectrometry.

A high-throughput, rapid, and efficient modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method with a simple cleanup procedure has been developed for simultaneously determining 227 pesticides in pepper samples by liquid chromatography with tandem mass spectrometry (running time: 10 min). Pesticide residues were extracted/partitioned with an acetonitrile/DisQuE QuEChERS pouch, and the resulting samples were cleaned up with different methods: dispersive solid-phase extraction with primary secondary amines or multiwalled carbon nanotubes and graphitized carbon solid mini cartridge column. The results indicated that multiwalled carbon nanotubes dispersive sorbents achieved the best recoveries and had less matrix interference. The numbers of pesticides with a recovery in the range of 70-120% were 199 at a spiked level of 40 µg/kg. The correlation coefficients (r(2)) for 227 pesticides were above 0.99, while the limits of quantitation of pesticides in pepper samples ranged from 0.13 to 13.51 µg/kg (S/N = 10), and the limits of detection ranged from 0.04 to 4.05 µg/kg (S/N = 3). The relative standard deviations of approximately 197 pesticides were below 20% at spiked levels of 40 µg/kg. Based on these results, the proposed method was chosen as the most suitable cleanup procedure for the determination of multiresidue pesticides in pepper samples.

Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells.

Japanese encephalitis virus (JEV) is an enveloped flavivirus and the most common agent of viral encephalitis. It enters cells through receptor-mediated endocytosis and low pH-triggered membrane fusion. Although lipid rafts, cholesterol-enriched lipid-ordered membrane domains, have been shown to participate in JEV entry, the mechanisms of the early events of JEV infection, including the cellular receptors of JEV, remain largely unknown. In the current study, it was demonstrated that heat-shock protein 70 (HSP70), rather than other members of the HSP70 family, was required for JEV entry into a human cell line. Cell-surface expression of HSP70 and a direct interaction between JEV envelope (E) protein and HSP70 were observed. Biochemical fractionation showed that HSP70 clearly migrated into the raft fraction after virus infection and co-fractioned with E protein. Depletion of cholesterol shifted the E protein and HSP70 to a non-raft membrane and decreased JEV entry without affecting virus binding to host cells. Notably, recruitment of HSP70 into lipid rafts was required for activation of the phosphoinositide 3-kinase/Akt signalling pathway in the early stage of JEV infection. These results indicate that lipid rafts facilitate JEV entry, possibly by providing a convenient platform to concentrate JEV and its receptors on the host-cell membrane.

Structure-inhibitory activity relationships of pyrrolnitrin analogues on its biosynthesis.

Pyrrolnitrin is a bacterial metabolite, served as a natural lead of agricultural fungicides. In a previous study, fenpiclonil was proven to inhibit the oxidative transformation of aminopyrrolnitrin to pyrrolnitrin, catalyzed by aminopyrrolnitrin oxidase (PrnD). This monooxygenase has an interesting catalytic activity of selective oxidation of aromatic amines, rather than aliphatic amines. However, its structural details are not well understood. In this study, various analogues of pyrrolnitrin were prepared to elucidate the structures of active site of PrnD through structure-activity relationships. In vivo pyrrolnitrin biosynthesis inhibition was determined with Burkholderia sp. O33 and Pseudomonas fluorescens Pf-5. Quantitative analysis of pyrrolnitrin and precursors indicates that 2,3-disubstituted phenyl at 3rd carbon and small substituents at 4th carbon of pyrrole are strictly required to give strong inhibitory effects. In addition, dissociable proton of pyrrole is also critical for inhibitory activity. Molecular simulation with homology-based PrnD model suggests a highly restricted conformational space in active site. The results may help more detailed understanding of this unusual enzyme. In addition, the information will be useful for the development of novel fungicide, compatible with pyrrolnitrin-producing bacterium.

Metabolism of insecticide diazinon by Cunninghamella elegans ATCC36112.

The fungal metabolism of diazinon was investigated and the microbial model (Cunninghamella elegans ATCC36112) could effectively degrade the organophosphorus pesticide (diazinon) mediated by cytochrome P450, which was mainly involved in oxidation and hydrolysis of phase I metabolism. Approximately 89% of diazinon was removed within 7 days and was not observed after 13 days with concomitant accumulation of eight metabolites. Structures of the metabolites were fully or tentatively identified with GC-MS and 1H, 13C NMR. The major metabolites of diazinon were diethyl (2-isopropyl-6-methylpyrimidin-4-yl) phosphate (diazoxon) and 2-isopropyl-6-methyl-4-pyrimidinol (pyrimidinol), and formation of minor metabolites was primarily the result of hydroxylation. To determine the responsible enzymes in diazinon metabolism, piperonyl butoxide and methimazole were treated, and the kinetic responses of diazinon and its metabolites by Cunninghamella elegans were measured. Results indirectly demonstrated that cytochrome P450 and flavin monooxygenase were involved in the metabolism of diazinon, but methimazole inhibited the metabolism less effectively. Based on the metabolic profiling, a possible metabolic pathway involved in phase I metabolism of diazinon was proposed, which would contribute to providing insight into understanding the toxicological effects of diazinon and the potential application of fungi on organophosphorus pesticides.

Endophilin-A2-mediated endocytic pathway is critical for enterovirus 71 entry into caco-2 cells.

Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.

Isolation and Characterization of 2,4-D Butyl Ester Degrading Acinetobacter sp. ZX02 from a Chinese Ginger Cultivated Soil.

Strain ZX02 was isolated from Chinese ginger cultivated soil contaminated with various pesticides, which could utilize 2,4-dichlorophenoxyacetic acid butyl ester (2,4-D butyl ester) as the sole carbon source. On the basis of the sequence analysis of 16S rRNA gene as well as the morphological, biochemical, and physiological characteristics of strain ZX02, the organism belonged to Gram-negative bacterium and was identified as Acinetobacter sp. ZX02. The strain ZX02 showed a remarkable performance in 2,4-D butyl ester degradation (100% removal in <96 h) in pure culture. Strain ZX02 was sensitive to tetracycline and resistant to amoxicillin and chloramphenicol in an antibiotic sensitivity test. The curing study indicates that the gene for degradation of 2,4-D butyl ester was encoded on a single plasmid of 23 kb. The gene encoding resistance to polymixin B sulfate was also located on this plasmid. On the basis of its greater biodegradation activity, this bacterium is a potential candidate as a bioremediation agent in soils contaminated with 2,4-D butyl ester.

Metabolism of an Insecticide Fenitrothion by Cunninghamella elegans ATCC36112.

In this study, the detailed metabolic pathways of fenitrothion (FNT), an organophosphorus insecticide by Cunninghamella elegans, were investigated. Approximately 81% of FNT was degraded within 5 days after treatment with concomitant accumulation of four metabolites (M1-M4). The four metabolites were separated by high-performance liquid chromatography, and their structures were identified by mass spectroscopy and/or nuclear magnetic resonance. M3 is confirmed to be an initial precursor of others and identified as fenitrothion-oxon. On the basis of their metabolic profiling, the possible metabolic pathways involved in phase I and II metabolism of FNT by C. elegans was proposed. We also found that C. elegans was able to efficiently and rapidly degrade other organophosphorus pesticides (OPs). Thus, these results will provide insight into understanding of the fungal degradation of FNT and the potential application for bioremediation of OPs. Furthermore, the ability of C. elegans to mimic mammalian metabolism would help us elucidate the metabolic fates of organic compounds occurring in mammalian liver cells and evaluate their toxicity and potential adverse effects.

Entry inhibitors: New advances in HCV treatment.

Hepatitis C virus (HCV) infection affects approximately 3% of the world's population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry.

A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes.

Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations.

How hepatitis C virus invades hepatocytes: the mystery of viral entry.

Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.

Multi-residue method for determination of 238 pesticides in Chinese cabbage and cucumber by liquid chromatography-tandem mass spectrometry: comparison of different purification procedures.

This paper describes the comparison of five sample cleanup procedures for the determination of 238 pesticides via triple quadrupole liquid chromatography-tandem mass spectrometry (LC-MS/MS, with only 10 min of chromatographic running time) in Chinese cabbage and cucumber. Samples were extracted with a quick, easy, cheap, effective, rugged, and safe (QuECHERS) preparation method and cleanup with different sorbents, including primary secondary amine (PSA), multi-walled carbon nanotubes (MWCNTs), and polystyrene (PLS), to find out the most suitable cleanup methods for Chinese cabbage and cucumber. The recovery and matrix effect were evaluated by monitoring the main parameters in one group of 238 pesticides at the spiked level of 8 and 40 µg/kg. In Chinese cabbage, when PSA dispersive solid-phase extraction (D-SPE) was applied, recoveries of 183 pesticides ranged between 70 and 120% with relative standard deviation (RSD) values lower than 20% at a spiked level of 40 µg/kg, indicating the effectiveness of the purification step. In cucumber, 203 pesticides were in the 70-120% recovery range with good reproducibility by PSA mini-cartridge column cleanup at a spiked level of 40 µg/kg and RSD values were generally below 20%. The limits of quantitation [LOQs; signal-to-noise (S/N) = 10] were in the range of 0.16-10.20 µg/kg for Chinese cabbage and 0.06-21.06 µg/kg for cucumber, while the limits of detection (LODs; S/N = 3) were between 0.05 and 3.06 µg/kg and between 0.02 and 6.32 µg/kg in Chinese cabbage and cucumber, respectively. The proposed methods that might be applied for the multi-residue analysis in Chinese cabbage and cucumber are contributed to their rapid speed and good recoveries.

Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry.

CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.

Metabolism of a fungicide mepanipyrim by soil fungus Cunninghamella elegans ATCC36112.

Mepanipyrim is a fungicide against several plant pathogens. However, no metabolic details have been established in fungi, which is the most important biomass in the natural environment. Cunninghamella elegans is a well-known fungal species with its strong resemblance to the mammalian xenobiotic metabolism. In this study, the detailed metabolic pathways of mepanipyrim were investigated with C. elegans. Approximately 87% of mepanipyrim was removed within 12 h with concomitant accumulation of nine metabolites. Structures of the metabolites were fully or tentatively identified with GC-MS and (1)H NMR. To determine the possible role of representative oxidative enzymes, piperonyl butoxide and methimazole were treated, and the kinetic responses of mepanipyrim and its metabolites were measured. Dose-dependent inhibition of metabolism was observed with piperonyl butoxide, while methimazole also inhibited the metabolism less effectively. The results indicate the possible involvement of cytochrome P450 and flavin-dependent monooxygenase in mepanipyrim metabolism. Comprehensive metabolic pathways can be deduced from the detailed analysis of metabolite profiles in control and inhibitor assays.