A Marine Natural Product, Harzianopyridone, as an Anti-ZIKV Agent by Targeting RNA-Dependent RNA Polymerase.

The Zika virus (ZIKV) is a mosquito-borne virus that already poses a danger to worldwide human health. Patients infected with ZIKV generally have mild symptoms like a low-grade fever and joint pain. However, severe symptoms can also occur, such as Guillain-Barré syndrome, neuropathy, and myelitis. Pregnant women infected with ZIKV may also cause microcephaly in newborns. To date, we still lack conventional antiviral drugs to treat ZIKV infections. Marine natural products have novel structures and diverse biological activities. They have been discovered to have antibacterial, antiviral, anticancer, and other therapeutic effects. Therefore, marine products are important resources for compounds for innovative medicines. In this study, we identified a marine natural product, harzianopyridone (HAR), that could inhibit ZIKV replication with EC50 values from 0.46 to 2.63 µM while not showing obvious cytotoxicity in multiple cellular models (CC50 > 45 µM). Further, it also reduced the expression of viral proteins and protected cells from viral infection. More importantly, we found that HAR directly bound to the ZIKV RNA-dependent RNA polymerase (RdRp) and suppressed its polymerase activity. Collectively, our findings provide HAR as an option for the development of anti-ZIKV drugs.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

Enterovirus A71 (EV-A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand-foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon-stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad-spectrum antiviral activity, it remains elusive whether SHFL inhibits EV-A71. In this work, we validated that EV-A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71-infected cells suggested that this factor could markedly suppress EV-A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV-A71 and promoted the degradation of 3Dpol through the ubiquitin-proteasome pathway. Furthermore, the zinc-finger domain and the 36 amino acids (164-199) of SHFL were crucial to the interaction between SHFL and EV-A71 3Dpol . Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV-A71.

Akt Phosphorylation Influences Persistent Chlamydial Infection and Chlamydia-Induced Golgi Fragmentation Without Involving Rab14.

Chlamydia trachomatis is an obligate intracellular bacterium that causes multiple diseases involving the eyes, gastrointestinal tract, and genitourinary system. Previous studies have identified that in acute chlamydial infection, C. trachomatis requires Akt pathway phosphorylation and Rab14-positive vesicles to transmit essential lipids from the Golgi apparatus in survival and replication. However, the roles that Akt phosphorylation and Rab14 play in persistent chlamydial infection remain unclear. Here, we discovered that the level of Akt phosphorylation was lower in persistent chlamydial infection, and positively correlated with the effect of activating the development of Chlamydia but did not change the infectivity and 16s rRNA gene expression. Rab14 was found to exert a limited effect on persistent infection. Akt phosphorylation might regulate Chlamydia development and Chlamydia-induced Golgi fragmentation in persistent infection without involving Rab14. Our results provide a new insight regarding the potential of synergistic repressive effects of an Akt inhibitor with antibiotics in the treatment of persistent chlamydial infection induced by penicillin.

Antibiotic fidaxomicin is an RdRp inhibitor as a potential new therapeutic agent against Zika virus.

BACKGROUND: Zika virus (ZIKV) infection is a global health problem, and its complications, including congenital Zika syndrome and Guillain-Barré syndrome, constitute a continued threat to humans. Unfortunately, effective therapeutics against ZIKV infection are not available thus far. METHODS: We screened the compounds collection consisting of 1789 FDA-approved drugs by a computational docking method to obtain anti-ZIKV candidate compounds targeting ZIKV RNA-dependent RNA polymerase (RdRp). SPR (BIAcore) assay was employed to demonstrate the candidate compounds' direct binding to ZIKV RdRp, and polymerase activity assay was used to determine the inhibitory effect on ZIKV RdRp-catalyzed RNA synthesis. The antiviral effects on ZIKV in vitro and in vivo were detected in infected cultured cells and in Ifnar1-/- mice infected by ZIKV virus using plaque assay, western blotting, tissue immunofluorescence, and immunohistochemistry. RESULTS: Here, we report that a first-in-class macrocyclic antibiotic, which has been clinically used to treat Clostridium difficile infection, fidaxomicin, potently inhibits ZIKV replication in vitro and in vivo. Our data showed that fidaxomicin was effective against African and Asian lineage ZIKV in a wide variety of cell lines of various tissue origins, and prominently suppressed ZIKV infection and significantly improved survival of infected mice. In addition, fidaxomicin treatment reduced the virus load in the brains and testes, and alleviated ZIKV-associated pathological damages, such as paralysis, hunching, and neuronal necrosis in the cerebra. Furthermore, our mechanistic study showed that fidaxomicin directly bound ZIKV NS5 protein and inhibited the RNA synthesis-catalyzing activity of ZIKV RdRp. CONCLUSIONS: Our data suggest that fidaxomicin may represent an effective anti-ZIKV agent. In the light that fidaxomicin is already a clinically used drug, there might be a promising prospect in the development of fidaxomicin to be an antiviral therapeutic.

MYEOV functions as an amplified competing endogenous RNA in promoting metastasis by activating TGF-ß pathway in NSCLC.

Non-small cell lung cancer (NSCLC) remains a major cause of death worldwide. As metastatic disease is primarily responsible for the poor clinical outcome of NSCLC, it is important to understand the process, and its underlying molecular mechanism as well, via which NSCLC cells disseminate. In this study, we identified a new competing endogenous RNA (ceRNA), namely, the MYEOV transcript, and found that it is upregulated in NSCLC and associated with a poor prognosis of the disease. We further uncovered that the MYEOV ceRNA plays a critical role in the invasion and metastasis of NSCLC cells. Intriguingly, the MYEOV ceRNA exerted its pro-metastatic function independent of its protein-coding capacity, but in a miR-30c-2-3p binding-dependent manner. Further experiments demonstrated that the MYEOV ceRNA sequestered miR-30c-2-3p from binding its targets TGFBR2 and USP15 mRNAs, which in turn leading to constitutive activation of TGF-ß signaling and tumor progression in NSCLC. By identifying a new layer of regulatory modality for TGF-ß signaling, our findings extend the current understanding on the molecular mechanism mediating NSCLC progression and highlight a potential role of MYEOV transcript to serve as the therapeutic target.

EGF-induced nuclear localization of SHCBP1 activates ß-catenin signaling and promotes cancer progression.

Aberrant activation of EGFR represents a common event in non-small cell lung carcinoma (NSCLC) and activates various downstream signaling pathways. While EGFR activation of ß-catenin signaling was previously reported, the mediating mechanism remains unclear. Our current study found that EGFR activation in NSCLC cells releases SHC-binging protein 1 (SHCBP1) from SHC adaptor protein 1 (SHC1), which subsequently translocates into the nucleus and directly promotes the transactivating activity of ß-catenin, consequently resulting in development of NSCLC cell stemness and malignant progression. Furthermore, SHCBP1 promotes ß-catenin activity through enhancing the CBP/ß-catenin interaction, and most interestingly, a candidate drug that blocks the CBP/ß-catenin binding effectively abrogates the aforementioned biological effects of SHCBP1. Clinically, SHCBP1 level in NSCLC tumors was found to inversely correlate with patient survival. Together, our study establishes a novel convergence between EGFR and ß-catenin pathways and highlights a potential significance of SHCBP1 as a prognostic biomarker and a therapeutic target.

LncRNA DUXAP9-206 directly binds with Cbl-b to augment EGFR signaling and promotes non-small cell lung cancer progression.

Long noncoding RNAs (lncRNAs) are involved in the pathology of various tumours, including non-small cell lung cancer (NSCLC). However, the underlying molecular mechanisms of their specific association with NSCLC have not been fully elucidated. Here, we report that a cytoplasmic lncRNA, DUXAP9-206 is overexpressed in NSCLC cells and closely related to NSCLC clinical features and poor patient survival. We reveal that DUXAP9-206 induced NSCLC cell proliferation and metastasis by directly interacting with Cbl-b, an E3 ubiquitin ligase, and reducing the degradation of epidermal growth factor receptor (EGFR) and thereby augmenting EGFR signaling in NSCLC. Notably, correlations between DUXAP9-206 and activated EGFR signaling were also validated in NSCLC patient specimens. Collectively, our findings reveal the novel molecular mechanisms of DUXAP9-206 in mediating the progression of NSCLC and DUXAP9-206 may serve as a potential target for NSCLC therapy.

Negative HBcAg in immunohistochemistry assay of liver biopsy is a predictive factor for the treatment of patients with nucleos(t)ide analogue therapy.

The hepatitis B core antigen (HBcAg) is an important target for antiviral response in chronic hepatitis B (CHB) patients. However, the correlation between HBcAg in the hepatocyte nucleus and nucleos(t)ide analogue (NA) therapeutic response is unclear. We sought to evaluate the role of HBcAg by analysing liver biopsies for viral response in NA-naïve hepatitis B e antigen (HBeAg) positive (+) CHB patients via immunohistochemistry (IHC). A total of 48 HBcAg-negative (-) patients and 48 HBcAg (+) patients with matching baseline characteristics were retrospectively analysed for up to 288 weeks. Virological response (VR) rates of patients in the HBcAg (-) group were significantly higher at week 48 and 96 than the HBcAg (+) group (77.1% versus 45.8% at week 48, respectively, P = 0.002 and 95.3% versus 83.3% at week 96, respectively, P = 0.045). The serological negative conversion rate of HBeAg was significantly higher in the HBcAg (-) than in the HBcAg (+) group from week 96 to 288 (35.4 % versus 14.6% at week 96, respectively, P = 0.018; 60.4% versus 14.6%, respectively, P < 0.001 at week 144; 72.9% versus 35.4%, respectively, P < 0.001 at week 288). The cumulative frequencies of VR and lack of HBeAg were higher in the HBcAg (-) group (both P < 0.05). Binary logistic regression analysis showed that HBcAg (-) was the predictor for the lack of HBeAg (OR 4.482, 95% CI: 1.58-12.68). In summary, the absence of HBcAg in the hepatocyte nucleus could be an independent predictor for HBeAg seroconversion rates during NA-naïve treatment in HBeAg (+) CHB patients.

Dengue Virus Subverts Host Innate Immunity by Targeting Adaptor Protein MAVS.

UNLABELLED: Dengue virus (DENV) is the most common mosquito-borne virus infecting humans and is currently a serious global health challenge. To establish infection in its host cells, DENV must subvert the production and/or antiviral effects of interferon (IFN). The aim of this study was to understand the mechanisms by which DENV suppresses IFN production. We determined that DENV NS4A interacts with mitochondrial antiviral signaling protein (MAVS), which was previously found to activate NF-κB and IFN regulatory factor 3 (IRF3), thus inducing type I IFN in the mitochondrion-associated endoplasmic reticulum membranes (MAMs). We further demonstrated that NS4A is associated with the N-terminal CARD-like (CL) domain and the C-terminal transmembrane (TM) domain of MAVS. This association prevented the binding of MAVS to RIG-I, resulting in the repression of RIG-I-induced IRF3 activation and, consequently, the abrogation of IFN production. Collectively, our findings illustrate a new molecular mechanism by which DENV evades the host immune system and suggest new targets for anti-DENV strategies. IMPORTANCE: Type I interferon (IFN) constitutes the first line of host defense against invading viruses. To successfully establish infection, dengue virus (DENV) must counteract either the production or the function of IFN. The mechanism by which DENV suppresses IFN production is poorly understood and characterized. In this study, we demonstrate that the DENV NS4A protein plays an important role in suppressing interferon production through binding MAVS and disrupting the RIG-I-MAVS interaction in mitochondrion-associated endoplasmic reticulum membranes (MAMs). Our study reveals that MAVS is a novel host target of NS4A and provides a molecular mechanism for DENV evasion of the host innate immune response. These findings have important implications for understanding the pathogenesis of DENV and may provide new insights into using NS4A as a therapeutic and/or prevention target.

PSAT1 regulates cyclin D1 degradation and sustains proliferation of non-small cell lung cancer cells.

Multiple nodes in the one-carbon metabolism pathway play important regulatory roles in cancer cell growth and tumorigenesis. The specific biological functions of metabolic enzymes in regulating the signaling pathways that are associated with tumor cell growth and survival, however, remain unclear. Our current study found that phosphoserine aminotransferase 1 (PSAT1), an enzyme catalyzing serine biosynthesis, was significantly up-regulated in non-small cell lung cancer (NSCLC) and was involved in the regulation of E2F activity. Loss- and gain-of-function experiments demonstrated that PSAT1 promoted cell cycle progression, cell proliferation and tumorigenesis. Mechanistic study suggested that elevated PSAT1 led to inhibition of cyclin D1 degradation and subsequently an alteration in Rb-E2F pathway activity, which in turn enhanced G1 progression and proliferation of NSCLC cells. Moreover, phosphorylation of cyclin D1 at threonine 286 by GSK-3ß was required for PSAT1-induced blockage of cyclin D1 degradation. We also found that the activity of p70S6K mediated the effects of PSAT1 on GSK-3ß phosphorylation and cyclin D1 degradation. We further identified that PSAT1 was over-expressed in NSCLC and predicted poor clinical outcome of patients with the disease. Correlation analysis showed that PSAT1 expression positively correlated with the levels of phosphorylated GSK-3ß, cyclin D1 and phosphorylated Rb in NSCLC primary tumors. These findings uncover a mechanism for constitutive activation of E2F via which unrestrained cell cycle progression occurs in NSCLC and may represent a prognostic biomarker and therapeutic target.

Tetravalent recombinant dengue virus-like particles as potential vaccine candidates: immunological properties.

BACKGROUND: Currently, a licensed vaccine for Dengue Virus (DENV) is not yet available. Virus-like particles (VLP) have shown considerable promise for use as vaccines and have many advantages compared to many other types of viral vaccines. VLPs have been found to have high immunogenic potencies, providing protection against various pathogens. RESULTS: In the current study, four DENV-VLP serotypes were successfully expressed in Pichia pastoris, based on co-expression of the prM and E proteins. The effects of a tetravalent VLP vaccine were also examined. Immunization with purified, recombinant, tetravalent DENV1-4 VLPs induced specific antibodies against all DENV1-4 antigens in mice. The antibody titers were higher after immunization with the tetravalent VLP vaccine compared to titers after immunization with any of the dengue serotype VLPs alone. Indirect immunofluorescence assay (IFA) results indicated that sera from VLP immunized mice recognized the native viral antigens. TNF-α and IL-10 were significantly higher in mice immunized with tetravalent DENV-VLP compared to those mice received PBS. The tetravalent VLP appeared to stimulate neutralizing antibodies against each viral serotype, as shown by PRNT50 analysis (1:32 against DENV1 and 2, and 1:16 against DENV3 and 4). The highest titers with the tetravalent VLP vaccine were still a little lower than the monovalent VLP against the corresponding serotype. The protection rates of tetravalent DENV-VLP immune sera against challenges with DENV1 to 4 serotypes in suckling mice were 77, 92, 100, and 100%, respectively, indicating greater protective efficacy compared with monovalent immune sera. CONCLUSIONS: Our results provide an important basis for the development of the dengue VLP as a promising non-infectious candidate vaccine for dengue infection.

Epidemiology characteristics of respiratory viruses found in children and adults with respiratory tract infections in southern China.

BACKGROUND: The World Health Organization (WHO) ranks respiratory tract infection (RTI) as the second leading cause of death worldwide for children under 5 years of age. The aim of this work was to evaluate the epidemiology characteristics of respiratory viruses found in children and adults with RTI from July 2009 to June 2012 in southern China. METHODS: In this work, a total of 14 237 nasopharyngeal swabs (14 237 patients from 25 hospitals) were analyzed, and seven respiratory viruses (influenza virus, respiratory syncytial virus, parainfluenza virus, adenovirus, human metapneumovirus, human coronavirus, human bocavirus) were detected using PCR/RT-PCR from nasopharyngeal swabs. RESULTS: The demographic characteristics, viral prevalence, age distribution, seasonal distribution, and pathogen spectrum of the patients with RTIs were analyzed. Co-infection was observed in 483 specimens, but it was more common in male patients, inpatients, children, and young adults. It varied by season, being more prevalent in the spring and summer and less so in the winter. Human coronavirus and human bocavirus were the most common pathogens, tending to occur in co-infection with other respiratory viruses. CONCLUSIONS: This work adds to our knowledge of the epidemiology characteristics of these seven common respiratory viruses among patients with RTI in southern China. The detection of the specific viral causes of infection provides a useful starting point for an understanding of illness attributable to respiratory infection, and might also provide data relevant to the development of prevention strategies.

A marine anthraquinone SZ-685C overrides adriamycin-resistance in breast cancer cells through suppressing Akt signaling.

Breast cancer remains a major health problem worldwide. While chemotherapy represents an important therapeutic modality against breast cancer, limitations in the clinical use of chemotherapy remain formidable because of chemoresistance. The HER2/PI-3K/Akt pathway has been demonstrated to play a causal role in conferring a broad chemoresistance in breast cancer cells and thus justified to be a target for enhancing the effects of anti-breast cancer chemotherapies, such as adriamycin (ADR). Agents that can either enhance the effects of chemotherapeutics or overcome chemoresistance are urgently needed for the treatment of breast cancer. In this context, SZ-685C, an agent that has been previously shown, as such, to suppress Akt signaling, is expected to increase the efficacy of chemotherapy. Our current study investigated whether SZ-685C can override chemoresistance through inhibiting Akt signaling in human breast cancer cells. ADR-resistant cells derived from human breast cancer cell lines MCF-7, MCF-7/ADR and MCF-7/Akt, were used as models to test the effects of SZ-685C. We found that SZ-685C suppressed the Akt pathway and induced apoptosis in MCF-7/ADR and MCF-7/Akt cells that are resistant to ADR treatment, leading to antitumor effects both in vitro and in vivo. Our data suggest that use of SZ-685C might represent a potentially promising approach to the treatment of ADR-resistant breast cancer.

Dual function of RGD-modified VEGI-192 for breast cancer treatment.

Identification of endogenous angiogenesis inhibitors has led to development of an increasingly attractive strategy for cancer therapy and other angiogenesis-driven diseases. Vascular endothelial growth inhibitor (VEGI), a potent and relatively nontoxic endogenous angiogenesis inhibitor, has been intensively studied, and this work shed new light on developing promising anti-angiogenic strategies. It is well-documented that the RGD (Arg-Gly-Asp) motif exhibits high binding affinity to integrin α(v)ß(3), which is abundantly expressed in cancer cells and specifically associated with angiogenesis on tumors. Here, we designed a fusion protein containing the special RGD-4C motif sequence and VEGI-192, aimed at offering more effective multiple targeting to tumor cells and tumor vasculature, and higher anti-angiogenic and antitumor efficacy. Functional tests demonstrated that the purified recombinant human RGD-VEGI-192 protein (rhRGD-VEGI-192) potently inhibited endothelial growth in vitro and suppressed neovascularization in chicken chorioallantoic membrane in vivo, to a higher degree as compared with rhVEGI-192 protein. More importantly, rhRGD-VEGI-192, but not rhVEGI-192 protein, could potentially target MDA-MB-435 breast tumor cells, significantly inhibiting growth of MDA-MB-435 cells in vitro, triggered apoptosis in MDA-MB-435 cells by activation of caspase-8 as well as caspase-3, which was mediated by activating the JNK signaling associated with upregulation of pro-apoptotic protein Puma, and consequently led to the observed significant antitumor effect in vivo against a human breast cancer xenograft. Our study indicated that the RGD-VEGI-192 fusion protein might represent a novel anti-angiogenic and antitumor strategy.

A novel adriamycin analogue derived from marine microbes induces apoptosis by blocking Akt activation in human breast cancer cells.

1403P-3 is a novel anthracenedione derivative isolated from the secondary metabolites of endophytic fungus from the South China Sea. In previous studies, 1403P-3 was found to exhibit potent cytotoxicity against human cancer cells, but its molecular target and the mechanisms mediating its cytotoxicity remain unknown. In this study, we showed that 1403P-3 markedly inhibited the survival of the human breast cancer cells MCF-7 and MDA-MB-435 in a dose-dependent manner, with an IC50 of 9.5 and 7.6 µM, respectively. Apoptosis induced by 1403P-3 was detected, as indicated by Annexin V-FITC/PI staining, elevated activated caspase-8 and -9, and cleavaged PARP determined by Western blot analysis. It is of note that the phosphorylation level of Akt was significantly reduced in 1403P-3-treated cells in a dose- and time-dependent manner. Taken together, our data demonstrated that 1403P-3 induced breast cancer cell apoptosis by blocking Akt activation, suggesting that 1403P-3 may be a promising candidate compound for anti-tumor drug development.

SZ-685C, a marine anthraquinone, is a potent inducer of apoptosis with anticancer activity by suppression of the Akt/FOXO pathway.

BACKGROUND AND PURPOSE: The aims of this study were to investigate the anti-cancer activity of SZ-685C, an anthracycline analogue isolated from marine-derived mangrove endophytic fungi, and to explore the molecular mechanisms underlying such activity. EXPERIMENTAL APPROACH: The effect of SZ-685C on the viability of cancer cell lines was investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SZ-685C-induced apoptosis was assessed by Annexin V-fluorescein isothiocyanate/propidium iodide staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay and analysis of caspase activation. The effect of SZ-685C on the Akt/FOXO pathway was studied using Western blotting analysis, and the in vivo anti-tumour efficacy was examined in an MDA-MB-435 breast cancer xenograft model. KEY RESULTS: SZ-685C suppressed the proliferation of six cancer cell lines derived from human breast cancer, prostate cancer, glioma and hepatoma (IC(50) values ranged from 3.0 to 9.6 microM) and the growth of breast cancer xenografts in mice. SZ-685C had a direct apoptosis-inducing effect through both the extrinsic and intrinsic apoptotic pathways, as shown by activation of caspase-8 and 9 as well as effector caspase-3 and poly (ADP-ribose) polymerase. Phosphorylation of Akt and its downstream effectors, forkhead box protein O1 and forkhead box protein O3a, was down-regulated in SZ-685C-treated cancer cells. Furthermore, the pro-apoptotic protein Bim was up-regulated by SZ-685C treatment consistent with FOXO dephosphorylation. CONCLUSIONS AND IMPLICATIONS: SZ-685C could induce apoptosis through the Akt/FOXO pathway, which consequently leads to the observed anti-tumour effect both in vitro and in vivo. Our data suggest that SZ-685C may be a potentially promising Akt inhibitor and anti-cancer drug candidate.

Approaches to efficient production of recombinant angiogenesis inhibitor rhVEGI-192 and characterization of its structure and antiangiogenic function.

Methods to prepare pure, bioactive recombinant human vascular endothelial growth inhibitor (rhVEGI), a potent inhibitor of angiogenesis potentially applicable in antiangiogenic cancer therapy, are in urgent demand for preclinical investigation as well as future clinical trials of the protein. Here, we report expression and purification of rhVEGI-192, a recombinant VEGI isoform, comparatively using host strains BL21 (DE3) pLysS and Origami B (DE3) with IPTG-induction and autoinduction techniques. Our study identified that a combined use of Origami B (DE3) strain and autoinduction expression system gave rise to a high yield of purified rhVEGI-192 at 105.38 mg/L culture by immobilized-metal affinity chromatography on Ni-NTA column. The antiangiogenic activity was effectively restored after the insoluble fractions being dissolved in 8M urea and subsequently subjected to a gradient-dialysis refolding process. Functional tests demonstrated that the purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane, indicating that the developed method allows preparation of rhVEGI-192 with high yield, solubility, and bioactivity. Most importantly, our study also demonstrates that VEGI-192 is capable of forming polymeric structure, which is possibly required for its antiangiogenic activity.