Acetaminophen overdose-induced acute liver injury can be alleviated by static magnetic field.

Acetaminophen (APAP), the most frequently used mild analgesic and antipyretic drug worldwide, is implicated in causing 46% of all acute liver failures in the USA and between 40% and 70% in Europe. The predominant pharmacological intervention approved for mitigating such overdose is the antioxidant N-acetylcysteine (NAC); however, its efficacy is limited in cases of advanced liver injury or when administered at a late stage. In the current study, we discovered that treatment with a moderate intensity static magnetic field (SMF) notably reduced the mortality rate in mice subjected to high-dose APAP from 40% to 0%, proving effective at both the initial liver injury stage and the subsequent recovery stage. During the early phase of liver injury, SMF markedly reduced APAP-induced oxidative stress, free radicals, and liver damage, resulting in a reduction in multiple oxidative stress markers and an increase in the antioxidant glutathione (GSH). During the later stage of liver recovery, application of vertically downward SMF increased DNA synthesis and hepatocyte proliferation. Moreover, the combination of NAC and SMF significantly mitigated liver damage induced by high-dose APAP and increased liver recovery, even 24 h post overdose, when the effectiveness of NAC alone substantially declines. Overall, this study provides a non-invasive non-pharmaceutical tool that offers dual benefits in the injury and repair stages following APAP overdose. Of note, this tool can work as an alternative to or in combination with NAC to prevent or minimize liver damage induced by APAP, and potentially other toxic overdoses.

Exogenous modification of EL-4 T cell extracellular vesicles with miR-155 induce macrophage into M1-type polarization.

Extracellular vesicles (EVs) show promising potential to be used as therapeutics, disease biomarkers, and drug delivery vehicles. We aimed to modify EVs with miR-155 to modulate macrophage immune response that can be potentially used against infectious diseases. Primarily, we characterized T cells (EL-4) EVs by several standardized techniques and confirmed that the EVs could be used for experimental approaches. The bioactivities of the isolated EVs were confirmed by the uptake assessment, and the results showed that target cells can successfully uptake EVs. To standardize the loading protocol by electroporation for effective biological functionality, we chose fluorescently labelled miR-155 mimics because of its important roles in the immune regulations to upload them into EVs. The loading procedure showed that the dosage of 1 µg of miRNA mimics can be efficiently loaded to the EVs at 100 V, further confirmed by flow cytometry. The functional assay by incubating these modified EVs (mEVs) with in vitro cultured cells led to an increased abundance of miR-155 and decreased the expressions of its target genes such as TSHZ3, Jarid2, ZFP652, and WWC1. Further evaluation indicated that these mEVs induced M1-type macrophage polarization with increased TNF-α, IL-6, IL-1ß, and iNOS expression. The bioavailability analysis revealed that mEVs could be detected in tissues of the livers. Overall, our study demonstrated that EVs can be engineered with miR-155 of interest to modulate the immune response that may have implications against infectious diseases.

A safety study on ultra­high or moderate static magnetic fields combined with platycodin D against lung cancer.

Due to the serious side effects of chemotherapy drugs against lung cancer, and the antitumor properties and high safety of magnetic fields, the present study combined moderate or ultra-high intensity statics magnetic fields (SMFs) with platycodin D (PD) to explore the antitumor efficiency and biosafety. The antitumor effects of PD with or without moderate and ultra-high SMFs on A549 cells bearing mice were compared. Mouse body weight, food/water intake, hematology routine, blood biochemistry, tumor weight and tissues hematoxylin and eosin (H&E) staining were examined. Behavior was measured using the elevated plus maze, open field and vital signs tests. The combined targets of PD and SMFs were detected using RNA-sequencing (RNA-seq). The results showed that the antitumor effect of 22 Tesla (T) SMF group was 3.6-fold higher compared with that of the 2 mg/kg PD group (tumor growth inhibition=10.08%), while the antitumor effect of 150 mT SMF was only 1.56-fold higher compared with that of PD. Although PD reduced the food intake, there was no significant difference in body weight, water intake or food consumption among PD and SMF groups. Behavioral results indicated that PD ameliorated dysphoria in mice, but SMFs reduced this effect. However, no significant abnormalities were found in routine blood, blood biochemistry test, H&E staining or organ index, except renal index which was reduced by PD with or without SMFs. RNA-sequencing (RNA-seq) demonstrated that SMFs and PD synergistically targeted the expression of genes associated with tumor growth, inflammation and neurological disease. The present study showed the antitumor efficacy and biosafety of moderate or ultra-high SMF combined with PD, which exhibited only few side effects in the treatment of lung cancer, thus supporting further research for the clinical application of magnetic fields.

Proteomic characterization of Opisthorchis felineus exosome-like vesicles and their uptake by human cholangiocytes.

The epidemiologically important food-borne trematode Opisthorchis felineus infests the liver biliary tract of fish-eating mammals and causes disorders, including bile duct neoplasia. Many parasitic species release extracellular vesicles (EVs) that mediate host-parasite interaction. Currently, there is no information on O. felineus EVs. Using gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry, we aimed to characterize the proteome of EVs released by the adult O. felineus liver fluke. Differential abundance of proteins between whole adult worms and EVs was assessed by semiquantitative iBAQ (intensity-based absolute quantification). Imaging, flow cytometry, inhibitor assays, and colocalization assays were performed to monitor the uptake of the EVs by H69 human cholangiocytes. The proteomic analysis reliably identified 168 proteins (at least two peptides matched a protein). Among major proteins of EVs were ferritin, tetraspanin CD63, helminth defense molecule 1, globin 3, saposin B type domain-containing protein, 60S ribosomal protein, glutathione S-transferase GST28, tubulin, and thioredoxin peroxidase. Moreover, as compared to the whole adult worm, EVs proved to be enriched with tetraspanin CD63, saposin B, helminth defense molecule 1, and Golgi-associated plant pathogenesis-related protein 1 (GAPR1). We showed that EVs are internalized by human H69 cholangiocytes via clathrin-dependent endocytosis, whereas phagocytosis and caveolin-dependent endocytosis do not play a substantial role in this process. Our study describes for the first time proteomes and differential abundance of proteins in whole adult O. felineus worms and EVs released by this food-borne trematode. Studies elucidating the regulatory role of individual components of EVs of liver flukes should be continued to determine which components of EV cargo play the most important part in the pathogenesis of fluke infection and in a closely linked pathology: bile duct neoplasia. SIGNIFICANCE: The food-borne trematode Opisthorchis felineus is a pathogen that causes hepatobiliary disorders in humans and animals. Our study describes for the first time the release of EVs by the liver fluke O. felineus, their microscopic and proteomic characterization, and internalization pathways by human cholangiocytes. Differential abundance of proteins between whole adult worms and EVs was assessed. EVs are enriched with canonical EV markers as well as parasite specific proteins, i.e. tetraspanin CD63, saposin B, helminth defense molecule 1, and others. Our findings will form the basis of the search for potential immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases, as well as novel vaccine candidates.

Advanced Oxidation Nanoprocessing Boosts Immunogenicity of Whole Tumor Cells.

Whole tumor cells expressing a wide array of tumor antigens are considered as a highly promising source of antigens for cancer vaccines. However, simultaneously preserving the antigen diversity, improving immunogenicity, and eliminating the potential tumorigenic risk of whole tumor cells are highly challenging. Inspired by the recent progress in sulfate radical-based environmental technology, herein, an advanced oxidation nanoprocessing (AONP) strategy is developed for boosting the immunogenicity of whole tumor cells. The AONP is based on the activation of peroxymonosulfate by ZIF-67 nanocatalysts to produce SO4 -∙ radicals continuously, leading to sustained oxidative damage to tumor cells and consequently extensive cell death. Importantly, AONP causes immunogenic apoptosis as evidenced by the release of a series of characteristic damage associated molecular patterns and at the same time maintains the integrity of cancer cells, which is critical to preserve the cellular components and thus maximize the diversity of antigens. Finally, the immunogenicity of AONP-treated whole tumor cells is evaluated in a prophylactic vaccination model, demonstrating significantly delayed tumor growth and increased survival rate of live tumor-cell-challenged mice. It is expected that the developed AONP strategy would pave the way to develop effective personalized whole tumor cell vaccines in future.

The Effect of Long-Term Moderate Static Magnetic Field Exposure on Adult Female Mice.

Because of the high cost and safety of ultra-high magnetic resonance imaging (MRI), its application has certain limitations. Whereas 0.5−3 T MRI has been widely applied in hospitals, static magnetic fields (SMFs) have been shown to improve mice mental health and have anti-tumor potentials. Here, we compared the effects of the upward and downward 150 mT SMF groups with the sham group on C57BL/6J adult female mice. Locomotor and exploratory activity were also measured by behavioral tests, including the open field and elevated plus test. Additionally, physiology, pathology indicators and gut microbiota were examined. We found that 150 mT SMFs long-term exposure enhanced locomotive and exploratory activity of mice, especially the downward 150 mT SMF. Compared with the downward 150 mT SMF group, the movement speed and distance in the center area of the sham group were increased by 65.99% (p < 0.0001) and 68.58% (p = 0.0038), respectively. Moreover, compared to the sham group, downward 150 mT SMF increased the number of entrances to the center area by 67.0% (p = 0.0082) and time in the center area by 77.12% (p = 0.0054). Additionally, we observed that upward 150 mT SMF improved the number of follicles (~2.5 times, p = 0.0325) and uterine glands through increasing the total antioxidant capacity and reducing lipid peroxidation level in mice. Gut microbiome analysis showed that 150 mT SMFs long-term exposure improved the microbiota abundance (Clostridium, Bifidobacterium, Ralstonia and Yaniella) in the genus level, which may affect metabolism, anxiety and behavior in adult female mice. Our results demonstrated that 150 mT SMFs long-term exposure not only had good biosafety, but also improved athletic performance, emotion and the function of ovarian, uterine and gut microbiota abundance in adult female mice, which unraveled the potential of moderate long-term SMF exposure in clinical applications.

Spider Toxin Peptide-Induced NIR Gold Nanocluster Fabrication for GSH-Responsive Cancer Cell Imaging and Nuclei Translocation.

Goldnanoclusters (GNCs) have become a promising nanomaterial for bioimaging because of their unique optical properties and biocompatibility. In this study, lycosin-I peptide, which possesses a highly selective anticancer activity by affecting the permeability of cancer cell membrane, was firstly modified for constructing fluorescent GNCs (LGNCs) for bioimaging of tumor cells. The obtained LGNCs exhibited strong near-infrared (NIR) fluorescence, which can be further enhanced by the peptide-induced aggregation and selectively stained three cancerous cell lines over normal cell lines with low intrinsic toxicity. After uptake by tumor cells, LGNC aggregates can be depolymerized into ultrasmall nanoclusters by high-level glutathione (GSH) and realize the nuclear targeting translocation. Collectively, our work suggests the potential of natural active biomolecules in designing NIR fluorescent GNCs for bioimaging.

Targeted Long-Read Sequencing Reveals Comprehensive Architecture, Burden, and Transcriptional Signatures from Hepatitis B Virus-Associated Integrations and Translocations in Hepatocellular Carcinoma Cell Lines.

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden, and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5, and Hep3B. To generate high-coverage, long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1,000 bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations, and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV is a driver of genetic instability and provides a potential new mechanism for HCC development. IMPORTANCE HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture, and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay that reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the interchromosomal junctions. Incorporation of long-read transcriptome sequencing (RNA-Seq) data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

Antioxidants resveratrol and SkQ1 attenuate praziquantel adverse effects on the liver in Opisthorchis felineus infected hamsters.

Anthelmintic praziquantel (PZQ) is the drug of the choice for opisthorchiasis, schistosomiasis and other trematodiases therapy for several decades. Despite its good therapeutic performance and effective control of trematode infections, PZQ has some shortcomings; its inability to counteract disease sequelae necessitates novel therapeutic strategies. Testing of antioxidants that have proven themselves in clinical practice, in combination with this anthelmintic drug, offers new opportunities for developing alternatives to PZQ monotherapy. The effects of two antioxidants combined with PZQ on histological parameters of liver tissue were evaluated in a hamster model of opisthorchiasis felinea. Liver pathology including the parenchyma state, accumulation of neutral lipids and 4-Hydroxy-2-nonenal as a lipid peroxidation product, biochemical characteristics of hamster blood serum, and mRNA expression of inflammation- and fibrogenesis-associated genes were determined. PZQ and opisthorchiasis caused liver accumulation of lipids and glycogen. The combination of PZQ with resveratrol (RSV) or 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) significantly reduced hepatocyte changes (P = 0.009 and P = 0.009, respectively, Mann-Whitney U test) as compared with infected hamsters treated only with PZQ. RSV and SkQ1 significantly reduced cholangiocyte hyperplasia, bile duct proliferation, fibrosis, and lipid droplet and glycogen granule accumulation. The downregulation of 4-hydroxynonenal was also observed. The combinations of the anthelmintic drug with antioxidants RSV and SkQ1 ameliorate host oxidative stress and mitigate adverse effects of PZQ on hepatic parenchyma. The use of drug combinations may improve the action of standard anthelmintic agents, such as PZQ, which still remains the most effective agent against adult trematodes.

RNA sequencing analysis of altered expression of long noncoding RNAs associated with Schistosoma japonicum infection in the murine liver and spleen.

BACKGROUND: Schistosomiasis is a chronic, debilitating infectious disease caused by members of the genus Schistosoma. Previous findings have suggested a relationship between infection with Schistosoma spp. and alterations in the liver and spleen of infected animals. Recent reports have shown the regulatory role of noncoding RNAs, such as long noncoding RNAs (lncRNAs), in different biological processes. However, little is known about the role of lncRNAs in the mouse liver and spleen during Schistosoma japonicum infection. METHODS: In this study, we identified and investigated lncRNAs using standard RNA sequencing (RNA-Seq). The biological functions of the altered expression of lncRNAs and their target genes were predicted using bioinformatics. Ten dysregulated lncRNAs were selected randomly and validated in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) experiments. RESULTS: Our study identified 29,845 and 33,788 lncRNAs from the liver and spleen, respectively, of which 212 were novel lncRNAs. We observed that 759 and 789 of the lncRNAs were differentially expressed in the respective organs. The RT-qPCR results correlated well with the sequencing data. In the liver, 657 differentially expressed lncRNAs were predicted to target 2548 protein-coding genes, whereas in the spleen 660 differentially expressed lncRNAs were predicted to target 2673 protein-coding genes. Moreover, functional annotation showed that the target genes of the differentially expressed lncRNAs were associated with cellular processes, metabolic processes, and binding, and were significantly enriched in metabolic pathways, the cell cycle, ubiquitin-mediated proteolysis, and pathways in cancer. CONCLUSIONS: Our study showed that numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control liver and spleen; this suggested that lncRNAs may be involved in pathogenesis in the liver and spleen during S. japonicum infection.

Host miR-148 regulates a macrophage-mediated immune response during Schistosoma japonicum infection.

Extracellular vesicles are critical regulators of host-parasite interactions. We previously demonstrated that Schistosoma japonicum EVs contain a remarkably high abundance of host miR-148a. Here, we characterised the abundance of miR-148a in circulation, in peripheral immune cells, and in plasma EVs of S. japonicum-infected mice. The results suggested the high abundance of miR-148a in macrophages to be likely linked to S. japonicum EVs. Additionally, miR-148a was found to target PTEN through the PI3K/AKT pathway to regulate cytokine production in macrophages. Consequently, our findings suggest that high abundance of miR-148a in macrophages may be associated with S. japonicum EVs, and regulate the host immune response during schistosome infection.

Schistosoma japonicum extracellular vesicle miRNA cargo regulates host macrophage functions facilitating parasitism.

Schistosome infection persists for decades. Parasites are in close contact with host peripheral blood immune cells, yet little is known about the regulatory interactions between parasites and these immune cells. Here, we report that extracellular vesicles (EVs) released from Schistosoma japonicum are taken up primarily by macrophages and other host peripheral blood immune cells and their miRNA cargo transferred into recipient cells. Uptake of S. japonicum EV miR-125b and bantam miRNAs into host cells increased macrophage proliferation and TNF-α production by regulating the corresponding targets including Pros1, Fam212b, and Clmp. Mice infected with S. japonicum exhibit an increased population of monocytes and elevated levels of TNF-α. Reduction of host monocytes and TNF-α level in S. japonicum infected mice led to a significant reduction in worm and egg burden and pathology. Overall, we demonstrate that S. japonicum EV miRNAs can regulate host macrophages illustrating parasite modulation of the host immune response to facilitate parasite survival. Our findings provide valuable insights into the schistosome-host interaction which may help to develop novel intervention strategies against schistosomiasis.

In vitro resistance profile of hepatitis C virus NS5A inhibitor velpatasvir in genotypes 1 to 6.

Velpatasvir is a pan-genotypic hepatitis C virus (HCV) NS5A inhibitor, which is used with sofosbuvir for treatment of infection with HCV genotypes 1-6. In vitro resistance studies were performed to characterize NS5A changes that might confer reduced velpatasvir susceptibility in vivo. Resistance selection studies using HCV replicon cells for subtypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a identified NS5A resistance-associated substitutions (RASs) at nine positions, most often 28M/S/T, 31F/I/M/P/V and 93D/H/N/S. In subtype 1a, RASs were selected at positions 31 and/or 93, while in subtype 1b, replicons with two or more RASs at positions 31, 54 or 93 were selected. Y93H was selected in subtypes 1a, 1b, 2a, 3a and 4a. In subtype 5a or 6a, L31P or P32L/Q was selected, respectively. Velpatasvir susceptibility of 358 replicons from genotypes 1 to 6 containing one or more NS5A RASs was also evaluated. The majority (63%) of subtypes 1a and 1b single RAS-containing replicons retained susceptibility to velpatasvir (<2.5-fold change in EC50 ). High levels of resistance to velpatasvir were observed for six single mutants in subtype 1a, including M28G, A92K, Y93H/N/R/W and for one mutant, A92K, in subtype 1b. Most single mutants in subtypes 2a, 2b, 3a, 4a and 5a displayed low levels of reduced velpatasvir susceptibility. High-level resistance was observed for C92T and Y93H/N in subtype 2b, Y93H/S in 3a, and L31V and P32A/L/Q/R in 6a, and several double mutants in these subtypes. Overall, velpatasvir maintained activity against most common RASs that are known to confer resistance to first-generation NS5A inhibitors.

Molecular characterization, expression profile, and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum.

Schistosomes are the causative agents of human schistosomiasis, which is endemic in tropical and subtropical zones. CD63 is a member of the tetraspanin protein family widely expressed among eukaryotes. Previously, we identified a CD63 homolog from extracellular vesicles isolated from Schistosoma japonicum. In this study, we characterized this CD63 homolog using a molecular approach and evaluated the potential of its recombinant protein for the diagnosis of schistosomiasis. A sequence alignment indicated that S. japonicum CD63 (SjCD63) has sequence identities of 76 and 28% with S. mansoni and human CD63, respectively. A phylogenetic analysis displayed that S. japonicum CD63 is related to S. mansoni and Opisthorchis viverrini CD63. The cDNA of SjCD63 was 740 bp long with an expected protein size of 23.58 kDa. A RT-qPCR analysis revealed significantly higher expression of SjCD63 mRNA in adult worms on days 21, 28, and 35 than in 7-day schistosomula, cercariae, and eggs. In addition, recombinant SjCD63 protein detected by ELISA revealed significantly higher optical density values compared to that of the negative control in both S. japonicum-infected mouse and rabbit sera, providing preliminary evidence for its diagnostic potential. Overall, these results provide insight into the molecular properties of SjCD63, its expression profiles, and its preliminary diagnostic potential.

In silico analysis of endogenous siRNAs associated transposable elements and NATs in Schistosoma japonicum reveals their putative roles during reproductive development.

Schistosomiasis is a neglected tropical disease caused by trematode of the genus Schistosoma. Successful reproductive development is critical for the production of eggs, which are responsible for host pathology and disease dissemination. Endogenous small non-coding RNAs play important roles in many biological processes such as protection against foreign pathogens, cell differentiation, and chromosomal stability by regulating target gene expression at the transcriptional and post-transcriptional levels. In this study, we performed in silico analysis of endogenous small non-coding RNAs in different stages, and sex of S. japonicum focusing on endogenous small interfering RNAs (endo-siRNAs) generated from transposable elements (TEs) and natural antisense transcripts (NATs). Both total and unique siRNA populations show 18-30 nt in length, but the predominant size was 20 nt and the leading first base was adenosine. Sense TE-derived endo-siRNAs reads were higher than antisense reads at different relative positions of TEs, whereas no such difference was observed for NAT-derived endo-siRNAs. TE- and NAT-derived endo-siRNAs were more enriched in the male compared to female worms, with the higher relative expression in early phase of pairing. Putative targets of endo-siRNAs indicated more of them in males (106 and 66) than in females (6 and 23) for TE- and NAT-derived endo-siRNAs, respectively. Our preliminary study revealed vital role of endo-siRNAs during the reproductive development of S. japonicum and provide clues for putative novel targets to suppress worm reproduction and direction for effective anti-schistosomal drug development.

The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.

In Vitro Antiviral Activity and Resistance Profile Characterization of the Hepatitis C Virus NS5A Inhibitor Ledipasvir.

Ledipasvir (LDV; GS-5885), a component of Harvoni (a fixed-dose combination of LDV with sofosbuvir [SOF]), is approved to treat chronic hepatitis C virus (HCV) infection. Here, we report key preclinical antiviral properties of LDV, including in vitro potency, in vitro resistance profile, and activity in combination with other anti-HCV agents. LDV has picomolar antiviral activity against genotype 1a and genotype 1b replicons with 50% effective concentration (EC50) values of 0.031 nM and 0.004 nM, respectively. LDV is also active against HCV genotypes 4a, 4d, 5a, and 6a with EC50 values of 0.11 to 1.1 nM. LDV has relatively less in vitro antiviral activity against genotypes 2a, 2b, 3a, and 6e, with EC50 values of 16 to 530 nM. In vitro resistance selection with LDV identified the single Y93H and Q30E resistance-associated variants (RAVs) in the NS5A gene; these RAVs were also observed in patients after a 3-day monotherapy treatment. In vitro antiviral combination studies indicate that LDV has additive to moderately synergistic antiviral activity when combined with other classes of HCV direct-acting antiviral (DAA) agents, including NS3/4A protease inhibitors and the nucleotide NS5B polymerase inhibitor SOF. Furthermore, LDV is active against known NS3 protease and NS5B polymerase inhibitor RAVs with EC50 values equivalent to those for the wild type.

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

Elimination of schistosomiasis japonica from formerly endemic areas in mountainous regions of southern China using a praziquantel regimen.

Schistosomiasis japonica is a major public health problem in China. Domestic animals play a major role in the transmission of Schistosoma japonicum to humans. To better understand the epidemiology of schistosomiasis japonica in domestic animals in the mountainous areas of China, we performed a 5-year longitudinal study of schistosomiasis in cattle and horses in Yunnan Province from 2009 to 2013. We also performed a concurrent drug-based intervention study in three settlement groups in Yunnan Province aimed at developing an effective means of controlling transmission in this region. The prevalence of infection in cattle fluctuated between 1.67% and 3.05% from 2009 to 2011, and monthly treatments of schistosome-positive animals reduced the prevalence to 0% (P<0.05) from 2012 to 2013. Prior to the intervention, we found that schistosomiasis was prevalent from May to October, with the highest prevalence observed in June (10.00%). We surveyed for environmental schistosome contamination, and 94.29% of the miracidia found were from cattle. Our study showed that it is possible to eliminate schistosomiasis in domestic animals in the mountainous regions of China by monthly treating cattle and horses from schistosome-positive households from May to October.