Immunological regulation and the role of autophagy in preeclampsia.

Autophagy is a bulk degradation system that maintains cellular homeostasis by producing energy and/or recycling excess proteins. During early placentation, extravillous trophoblasts invade the decidua and uterine myometrium, facing maternal immune cells, which participate in the immune suppression of paternal and fetal antigens. Regulatory T cells will likely increase in response to a specific antigen before and during early pregnancy. Insufficient expansion of antigen-specific Treg cells, which possess the same T cell receptor, is associated with the pathophysiology of preeclampsia, suggesting sterile systemic inflammation. Autophagy is involved in reducing inflammation through the degradation of inflammasomes and in the differentiation and function of regulatory T cells. Autophagy dysregulation induces protein aggregation in trophoblasts, resulting in placental dysfunction. In this review, we discuss the role of regulatory T cells in normal pregnancies. In addition, we discuss the association between autophagy and regulatory T cells in the development of preeclampsia based on reports on the role of autophagy in autoimmune diseases.

The Role of Autophagy in the Female Reproduction System: For Beginners to Experts in This Field.

Autophagy is a fundamental process involved in regulating cellular homeostasis. Autophagy has been classically discovered as a cellular process that degrades cytoplasmic components non-selectively to produce energy. Over the past few decades, this process has been shown to work in energy production, as well as in the reduction of excessive proteins, damaged organelles, and membrane trafficking. It contributes to many human diseases, such as neurodegenerative diseases, carcinogenesis, diabetes mellitus, development, longevity, and reproduction. In this review, we provide important information for interpreting results related to autophagic experiments and present the role of autophagy in this field.

Chloroquine is a safe autophagy inhibitor for sustaining the expression of antioxidant enzymes in trophoblasts.

Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.

Silver nanoparticles suppress forskolin-induced syncytialization in BeWo cells.

Opportunities for the exposure of pregnant women to engineered nanoparticles have been increasing with the expanding use of these materials. Therefore, there are concerns that nanoparticles could have adverse effects on the establishment and maintenance of pregnancy. The effects of nanoparticles on the mother and fetus have been evaluated from this perspective, but there is still little knowledge about the effects on placentation and function acquisition, which are essential for the successful establishment and maintenance of pregnancy. Formation of the syncytiotrophoblast is indispensable for the acquisition of placental function, and impairment of syncytialization inevitably affects pregnancy outcomes. Here, we assessed the effect of nanoparticles on placental formation by using forskolin-treated BeWo cells, a typical in vitro model of trophoblast syncytialization. Immunofluorescence staining analysis revealed that silver nanoparticles with a diameter of 10 nm (nAg10) (at 0.156 µg/mL) significantly decreased the proportion of syncytialized BeWo cells, but gold nanoparticles with a diameter of 10 nm did not. Consistently, only nAg10 (at 0.156 µg/mL) significantly suppressed forskolin-induced elevation of CGB and SDC1 mRNA expression levels and human chorionic gonadotropin ß production in a dose-dependent manner; these molecules are all markers of syncytialization. Besides, nAg10 significantly decreased the expression of ERVFRD-1, which encodes proteins associated with cell fusion. Moreover, nAg10 tended to suppress the expression of sFlt-1 e15a, a placental angiogenesis marker. Collectively, our data suggest that nAg10 could suppress formation of the syncytiotrophoblast and that induce placental dysfunction and the following poor pregnancy outcomes.

Aggrephagy Deficiency in the Placenta: A New Pathogenesis of Preeclampsia.

Aggrephagy is defined as the selective degradation of aggregated proteins by autophagosomes. Protein aggregation in organs and cells has been highlighted as a cause of multiple diseases, including neurodegenerative diseases, cardiac failure, and renal failure. Aggregates could pose a hazard for cell survival. Cells exhibit three main mechanisms against the accumulation of aggregates: protein refolding by upregulation of chaperones, reduction of protein overload by translational inhibition, and protein degradation by the ubiquitin-proteasome and autophagy-lysosome systems. Deletion of autophagy-related genes reportedly contributes to intracellular protein aggregation in vivo. Some proteins recognized in aggregates in preeclamptic placentas include those involved in neurodegenerative diseases. As aggregates are derived both intracellularly and extracellularly, special endocytosis for extracellular aggregates also employs the autophagy machinery. In this review, we discuss how the deficiency of aggrephagy and/or macroautophagy leads to poor placentation, resulting in preeclampsia or fetal growth restriction.

Four Aromatic Sulfates with an Inhibitory Effect against HCV NS3 Helicase from the Crinoid Alloeocomatella polycladia.

Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 µM, respectively.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

A fluorescence-based screening assay for identification of hepatitis C virus NS3 helicase inhibitors and characterization of their inhibitory mechanism.

Hepatitis C virus (HCV) can establish a chronic infection in the majority of individuals infected, resulting in liver cirrhosis and hepatocellular carcinoma. Because the current standard treatment for HCV infection has limitations in terms of severe side effects, the emergence of drug resistance, and drug-drug interactions, it is desirable to develop novel antivirals that target viral proteins involved in viral replication. HCV nonstructural protein 3 (NS3) helicase, which unwinds double-stranded nucleic acids to yield single-stranded nucleic acids, is one possible target for new drug development, because it plays an essential role in viral replication. In this chapter, we describe a helicase assay based on fluorescence resonance energy transfer (FRET) that can be used for high-throughput screening of HCV NS3 helicase inhibitors. The assay uses a double-stranded RNA (dsRNA) substrate with a fluorophore-labeled strand hybridized to a quencher-labeled strand and monitors the increase in fluorescence intensity resulting from helicase-catalyzed unwinding of the dsRNA substrate. We further describe radioactive assays to directly visualize RNA strands unwound by helicase and to evaluate the ATPase and RNA-binding activities of NS3, which are linked to helicase activity, for characterization of the inhibitory mechanism.

PBDE: structure-activity studies for the inhibition of hepatitis C virus NS3 helicase.

The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

Identification and biochemical characterization of halisulfate 3 and suvanine as novel inhibitors of hepatitis C virus NS3 helicase from a marine sponge.

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase.

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

Psammaplin A inhibits hepatitis C virus NS3 helicase.

Hepatitis C virus (HCV) is the causative agent of hepatitis C, a chronic infectious disease that can lead to development of hepatocellular carcinoma. The NS3 nucleoside triphosphatase (NTPase)/helicase has an essential role in HCV replication, and is therefore an attractive target for direct-acting antiviral strategies. In this study, we employed high-throughput screening using a photo-induced electron transfer (PET) system to identify an inhibitor of NS3 helicase from marine organism extracts. We successfully identified psammaplin A as a novel NS3 inhibitor. The dose-response relationship clearly demonstrates the inhibition of NS3 RNA helicase and ATPase activities by psammaplin A, with IC50 values of 17 and 32 µM, respectively. Psammaplin A has no influence on the apparent Km value (0.4 mM) of NS3 ATPase activity, and acts as a non-competitive inhibitor. Additionally, it inhibits the binding of NS3 to single-stranded RNA in a dose-dependent manner. Furthermore, psammaplin A shows an inhibitory effect on viral replication, with EC50 values of 6.1 and 6.3 µM in subgenomic replicon cells derived from genotypes 1b and 2a, respectively. We postulate that psammaplin A is a potential anti-viral agent through the inhibition of ATPase, RNA binding and helicase activities of NS3.

Inhibition of both protease and helicase activities of hepatitis C virus NS3 by an ethyl acetate extract of marine sponge Amphimedon sp.

Combination therapy with ribavirin, interferon, and viral protease inhibitors could be expected to elicit a high level of sustained virologic response in patients infected with hepatitis C virus (HCV). However, several severe side effects of this combination therapy have been encountered in clinical trials. In order to develop more effective and safer anti-HCV compounds, we employed the replicon systems derived from several strains of HCV to screen 84 extracts from 54 organisms that were gathered from the sea surrounding Okinawa Prefecture, Japan. The ethyl acetate-soluble extract that was prepared from marine sponge Amphimedon sp. showed the highest inhibitory effect on viral replication, with EC50 values of 1.5 and 24.9 µg/ml in sub-genomic replicon cell lines derived from genotypes 1b and 2a, respectively. But the extract had no effect on interferon-inducing signaling or cytotoxicity. Treatment with the extract inhibited virus production by 30% relative to the control in the JFH1-Huh7 cell culture system. The in vitro enzymological assays revealed that treatment with the extract suppressed both helicase and protease activities of NS3 with IC50 values of 18.9 and 10.9 µg/ml, respectively. Treatment with the extract of Amphimedon sp. inhibited RNA-binding ability but not ATPase activity. These results suggest that the novel compound(s) included in Amphimedon sp. can target the protease and helicase activities of HCV NS3.

Inhibition of hepatitis C virus replication and viral helicase by ethyl acetate extract of the marine feather star Alloeocomatella polycladia.

Hepatitis C virus (HCV) is a causative agent of acute and chronic hepatitis, leading to the development of hepatic cirrhosis and hepatocellular carcinoma. We prepared extracts from 61 marine organisms and screened them by an in vitro fluorescence assay targeting the viral helicase (NS3), which plays an important role in HCV replication, to identify effective candidates for anti-HCV agents. An ethyl acetate-soluble fraction of the feather star Alloeocomatella polycladia exhibited the strongest inhibition of NS3 helicase activity, with an IC(50) of 11.7 µg/mL. The extract of A. polycladia inhibited interaction between NS3 and RNA but not ATPase of NS3. Furthermore, the replication of the replicons derived from three HCV strains of genotype 1b in cultured cells was suppressed by the extract with an EC(50) value of 23 to 44 µg/mL, which is similar to the IC(50) value of the NS3 helicase assay. The extract did not induce interferon or inhibit cell growth. These results suggest that the unknown compound(s) included in A. polycladia can inhibit HCV replication by suppressing the helicase activity of HCV NS3. This study may present a new approach toward the development of a novel therapy for chronic hepatitis C.

Inhibition of hepatitis C virus NS3 helicase by manoalide.

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC(50) values of 15 and 70 µM, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K(m) value (0.31 mM) of NS3 ATPase activity, suggesting that 1 acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by 1. Manoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.

Quantitative detection of Cryptosporidium oocyst in water source based on 18S rRNA by alternately binding probe competitive reverse transcription polymerase chain reaction (ABC-RT-PCR).

We describe an assay for simple and cost-effective quantification of Cryptosporidium oocysts in water samples using a recently developed quantification method named alternately binding probe competitive PCR (ABC-PCR). The assay is based on the detection of 18S rRNA specific for Cryptosporidium oocysts. The standard curve of the ABC-PCR assay had a good fitting to a rectangular hyperbola with a correlation coefficient (R) of 0.9997. Concentrations of Cryptosporidium oocysts in real river water samples were successfully quantified by the ABC-reverse transcription (RT)-PCR assay. The quantified values by the ABC-RT-PCR assay very closely resemble those by the real-time RT-PCR assay. In addition, the quantified concentration in most water samples by the ABC-RT-PCR assay was comparable to that by conventional microscopic observation. Thus, Cryptosporidium oocysts in water samples can be accurately and specifically determined by the ABC-RT-PCR assay. As the only equipment that is needed for this end-point fluorescence assay is a simple fluorometer and a relatively inexpensive thermal cycler, this method can markedly reduce time and cost to quantify Cryptosporidium oocysts and other health-related water microorganisms.

Real-time monitoring of RNA helicase activity using fluorescence resonance energy transfer in vitro.

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput.