The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner.

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein-protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

Synergistic Inhibition of Synbiotic Cultures among Lactobacilli and Plant Extracts against Vaginal Discharge Causing Candida albicans.

Vulvovaginal candidiasis (VVC) is the most common cause of vaginal discharge among women. The present study aimed to investigate the synergistic anticandidal effect of lactobacillus cultures supplemented with plant extracts. Among 600 isolates of lactic acid bacteria, 41 isolates exhibited inhibitory activity against Candida albicans ATCC10231. Six out of 41 cell-free supernatants demonstrated the most potent antibacterial and anticandidal activities. They also inhibited the clinical isolates of C. albicans, causing VVC and non-C. albicans. The synergistic effect between Lactobacillus crispatus 84/7 and Limosilactobacillus reuteri 89/4 was demonstrated by the lowest fractional inhibitory concentration index (FICI = 0.5). The synbiotic culture of bacterial combination, cultured with Jerusalem artichoke (H. tuberosus) extract, also exhibited the strongest inhibition against the tested C. albicans. Biofilm formation decreased after 12 h of incubation in the selected cell-free supernatants of this synbiotic culture. The anticandidal activity of crude extracts was lost after treatment with proteinase K and trypsin but not with heating conditions, suggesting that it may be a heat-stable substance. In conclusion, the combination of L. crispatus 84/7 and L. reuteri 89/4 with H. tuberosus may be a promising candidate for inhibiting Candida infection and biofilm formation, with the potential use as ingredients in vaginal biotherapeutic products.

Inhibitory actions of oxyresveratrol on the PI3K/AKT signaling cascade in cervical cancer cells.

The phosphatidyl inositol 3-kinase (PI3K)/AKT signaling plays a critical role in cancer cell proliferation, migration, and invasion. This signal transduction axis in HPV-positive cervical cancer has been proved to be directly activated by E6/E7 proteins of the virus enhancing cervical cancer progression. Hence, the PI3K/AKT pathway is one of the key therapeutic targets for HPV-positive cervical cancer. Here we discovered that oxyresveratrol (Oxy) at noncytotoxic concentration specifically suppressed the phosphorylation of AKT but not ERK1/2. This potent inhibitory effect of Oxy was still observed even when cells were stimulated with fetal bovine serum. Inhibition of AKT phosphorylation at serine 473 by Oxy resulted in a significant decrease in serine 9 phosphorylation of GSK-3ß, a downstream target of AKT. Dephosphorylation of GSK-3ß at this serine residue activates its function in promoting the degradation of MCL-1, an anti-apoptotic protein. Results clearly demonstrated that in association with GSK-3ß activation, Oxy preferentially downregulated the expression of anti-apoptotic protein MCL-1. Furthermore, results from the functional analyses revealed that Oxy inhibited cervical cancer cell proliferation, at least in part through suppressing nuclear expression of Ki-67. Besides, the compound retarded cervical cancer cell migration even the cells were exposed to a potent enhancer of epithelial-mesenchymal transition, TGF-ß1. In consistent with these data, Oxy reduced the expression of ß-catenin, N-cadherin, and vimentin. In conclusion, the study disclosed that Oxy specifically inhibits the AKT/GSK-3ß/MCL-1 axis resulting in reduction in cervical cancer cell viability, proliferation, and migration.

Combination of Pinocembrin and Epidermal Growth Factor Enhances the Proliferation and Survival of Human Keratinocytes.

Re-epithelialization is delayed in aged skin due to a slow rate of keratinocyte proliferation, and this may cause complications. Thus, there has been development of new therapies that increase treatment efficacy for skin wounds. Epidermal growth factor (EGF) has been clinically used, but this agent is expensive, and its activity is less stable. Therefore, a stable compound possessing EGF-like properties may be an effective therapy, especially when combined with EGF. The current study discovered that pinocembrin (PC) effectively synergized with EGF in increasing keratinocyte viability. The combination of PC and EGF significantly enhanced the proliferation and wound closure rate of the keratinocyte monolayer through activating the phosphorylation of ERK and Akt. Although these effects of PC were like those of EGF, we clearly proved that PC did not transactivate EGFR. Recent data from a previous study revealed that PC activates G-protein-coupled receptor 120 which further activates ERK1/2 and Akt phosphorylation. Therefore, this clearly indicates that PC possesses a unique property to stimulate the growth and survival of keratinocytes through activating a different receptor, which subsequently conveys the signal to cross-talk with the effector kinases downstream of the EGFR, suggesting that PC is a potential compound to be combined with EGF.

Resveratrol Enhances Cytotoxic Effects of Cisplatin by Inducing Cell Cycle Arrest and Apoptosis in Ovarian Adenocarcinoma SKOV-3 Cells through Activating the p38 MAPK and Suppressing AKT.

In the current study, we identified a mechanism of resveratrol (RES) underlying its anti-cancer properties against human ovarian adenocarcinoma SKOV-3 cells. We investigated its anti-proliferative and apoptosis-inducing effects in combination with cisplatin, using cell viability assay, flow cytometry, immunofluorescence study and Western blot analysis. We discovered that RES suppressed cancer cell proliferation and stimulated apoptosis, especially when combined with cisplatin. This compound also inhibited SKOV-3 cell survival, which may partly be due to its potential to inhibit protein kinase B (AKT) phosphorylation and induce the S-phase cell cycle arrest. RES in combination with cisplatin strongly induced cancer cell apoptosis through activating the caspase-dependent cascade, which was associated with its ability to stimulate nuclear phosphorylation of p38 mitogen-activated protein kinase (MAPK), well recognized to be involved in transducing environmental stress signals. RES-induced p38 phosphorylation was very specific, and the activation status of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was not mainly affected. Taken together, our study provides accumulated evidence that RES represses proliferation and promotes apoptosis in SKOV-3 ovarian cancer cells through activating the p38 MAPK pathway. It is interesting that this active compound may be used as an effective agent to sensitize ovarian cancer to apoptosis induced by standard chemotherapies.

Investigation of the activity of baicalein towards Zika virus.

BACKGROUND: Zika virus (ZIKV) is a mosquito transmitted virus spread primarily by Aedes species mosquitoes that can cause disease in humans, particularly when infection occurs in pregnancy where the virus can have a significant impact on the developing fetus. Despite this, there remains no prophylactic agent or therapeutic treatment for infection. Baicalein is a trihydroxyflavone, that is found in some traditional medicines commonly used in Asia, and has been shown to have several activities including antiviral properties. Importantly, studies have shown baicalein to be safe and well tolerated in humans, increasing its potential utilization. METHODS: This study sought to determine the anti-ZIKV activity of baicalein using a human cell line (A549). Cytotoxicity of baicalein was determined by the MTT assay, and the effect on ZIKV infection determined by treating A549 cells with baicalien at different time points in the infection process. Parameters including level of infection, virus production, viral protein expression and genome copy number were assessed by flow cytometry, plaque assay, western blot and quantitative RT-PCR, respectively. RESULTS: The results showed that baicalein had a half-maximal cytotoxic concentration (CC50) of > 800 µM, and a half-maximal effective concentration (EC50) of 124.88 µM. Time-of-addition analysis showed that baicalein had an inhibitory effect on ZIKV infection at the adsorption and post-adsorption stages. Moreover, baicalein also exerted a significant viral inactivation activity on ZIKV (as well as on dengue virus and Japanese encephalitis virus) virions. CONCLUSION: Baicalein has now been shown to possess anti-ZIKV activity in a human cell line.

Alpinetin Suppresses Zika Virus-Induced Interleukin-1ß Production and Secretion in Human Macrophages.

Zika virus (ZIKV) infection has been recognized to cause adverse sequelae in the developing fetus. Specially, this virus activates the excessive release of IL-1ß causing inflammation and altered physiological functions in multiple organs. Although many attempts have been invested to develop vaccine, antiviral, and antibody therapies, development of agents focusing on limiting ZIKV-induced IL-1ß release have not gained much attention. We aimed to study the effects of alpinetin (AP) on IL-1ß production in human macrophage upon exposure to ZIKV. Our study demonstrated that ZIKV stimulated IL-1ß release in the culture supernatant of ZIKV-infected cells, and AP could effectively reduce the level of this cytokine. AP exhibited no virucidal activities against ZIKV nor caused alteration in viral production. Instead, AP greatly inhibited intracellular IL-1ß synthesis. Surprisingly, this compound did not inhibit ZIKV-induced activation of NF-κB and its nuclear translocation. However, AP could significantly inhibit ZIKV-induced p38 MAPK activation without affecting the phosphorylation status of ERK1/2 and JNK. These observations suggest the possibility that AP may reduce IL-1ß production, in part, through suppressing p38 MAPK signaling. Our current study sheds light on the possibility of using AP as an alternative agent for treating complications caused by ZIKV infection-induced IL-1ß secretion.

Cell Type Variability in the Incorporation of Lipids in the Dengue Virus Virion.

A lipid bilayer produced from the host membrane makes up around 20% of the weight of the dengue virus (DENV) virion and is crucial for virus entry. Despite its significance, the virion's lipid composition is still poorly understood. In tandem with lipid profiles of the cells utilised to generate the virions, this work determined a partial lipid profile of DENV virions derived from two cell lines (C6/36 and LLC-MK2). The results showed distinctive profiles between the two cell types. In the mammalian LLC-MK2 cells, 30.8% (73/237 identified lipid species; 31 upregulated, 42 downregulated) of lipid species were altered in response to infection, whilst in insect C6/36 cells only 12.0% (25/208; 19 upregulated, 6 downregulated) of lipid species showed alterations in response to infection. For virions from LLC-MK2 cells, 14 lipids were detected specifically in virions with a further seven lipids being enriched (over mock controls). For virions from C6/36 cells, 43 lipids were detected that were not seen in mock preparations, with a further 16 being specifically enriched (over mock control). These results provide the first lipid description of DENV virions produced in mammalian and mosquito cells, as well as the lipid changes in the corresponding infected cells.

Synergistic Effects of Some Methoxyflavones Extracted from Rhizome of Kaempferia parviflora Combined with Gentamicin against Carbapenem-Resistant Strains of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

The present study aimed to investigate the antibacterial activity of ethanolic Kaempferia parviflora extracts and the combined effects of the plant's specific compounds with gentamicin against clinical strains of carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The minimal inhibitory concentrations (MIC) of gentamicin and Kaempferia parviflora extracts against the tested bacterial strains were determined by using broth microdilution. The combined effects of Kaempferia parviflora extract and gentamicin were investigated by using a checkerboard assay and expressed as a fractional inhibitory concentration index (FICI). Crude ethanolic extract of Kaempferia parviflora showed the lowest median values of MIC towards the tested isolates (n = 10) of these tested bacteria at doses of 64 µg/mL, compared to those of other Kaempferia extracts. Among the isolated compounds, only three compounds, namely 3,5,7-trimethoxyflavone, 3,5,7,3'4'-pentamethoxyflavone, and 5,7,4'-trimethoxyflavone, were identified by NMR structural analysis. According to their FICIs, the synergistic effects of gentamicin combined with 3,5,7,3'4'-pentamethoxyflavone were approximately 90%, 90%, and 80% of tested carbapenem-resistant Klebsiella pneumoniae (CRKP), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB), respectively. The present study concluded that 3,5,7,3'4'-pentamethoxyflavone extracted from Kaempferia parviflora potentiated the antibacterial action of gentamicin to combat bacterial resistance against the tested bacteria.

Alpinetin Suppresses Effects of TGF-ß1 on Stimulating the Production and Organization of Fibrotic Markers in Human Primary Dermal Fibroblasts.

Overgrowths of dermal fibroblasts and myofibroblast phenoconversion in response to TGF-ß stimulation are the hallmarks of skin fibrosis. Constitutive activation of dermal fibroblasts by TGF-ß induces the excessive production of extracellular matrix as well as certain key intracellular proteins which form a complex interaction network. Current therapies include monoclonal anti-bodies against TGF-ß and surgery, but these treatments generally elicit a limited effect on certain kinds of skin fibrosis. In the current study, we investigated the effects of alpinetin (AP) on human primary dermal fibroblasts (HPDFs) stimulated with TGF-ß1. Results demonstrated that AP exhibited strong inhibitory effects on TGF-ß1-induced proliferation and migration of HPDFs. AP also inhibited TGF-ß1-induced morphological changes of fibroblasts to myofibroblasts, and these were found to be from its effects on blocking actin stress fiber formation and organization. The expression of major fibrotic molecules including α-SMA and type I collagen upon TGF-ß1 stimulation was also inhibited by AP. In addition, AP attenuated TGF-ß1-induced production and organization of vimentin, ß-catenin, and N-cadherin, important for the pathophysiology of skin fibrosis. In conclusion, we revealed that AP has an ability to reverse the fibrotic effects of TGF-ß1 at the cellular level, and this discovery suggests the therapeutic potential of AP for skin fibrosis.

Molecular Targets of Pinocembrin Underlying Its Regenerative Activities in Human Keratinocytes.

Pinocembrin is one of the well-known compounds in the group of flavonoids. The pharmacological activities of pinocembrin in association with wound-healing activities have been reported. However, its effects on the aspect of cellular interaction underlying growth and survival are still unidentified in human keratinocytes. Our previous study reported that Boesenbergia rotunda potently stimulated survival and proliferation of a human keratinocyte cell line (HaCaT). On the basis that pinocembrin is revealed to be one of the major constituents of this plant, we aimed to define the survival- and proliferation-enhancing effects of this compound at the cellular level. Results from the current study confirmed that pinocembrin induced an increase in HaCaT cell number. At the signaling perspective, we identified that pinocembrin significantly triggered ERK1/2 and Akt activation. The stimulating effects of pinocembrin were clearly inhibited by MEK and PI3K inhibitors authenticating that proliferation- and survival-promoting activities of pinocembrin were mainly acted on these two signaling cascades. Altogether, we successfully identified that pinocembrin functions to induce keratinocyte proliferation and survival, at least by provoking MAPK and PI3K pathways. Our study encourages the fact that pinocembrin is one of the interesting natural flavonoid compounds to be developed as a wound closure-promoting agent.

The Leaf Extract of Mitrephora chulabhorniana Suppresses Migration and Invasion and Induces Human Cervical Cancer Cell Apoptosis through Caspase-Dependent Pathway.

Cervical cancer is rated to be the leading cause of cancer-related death in women worldwide. Since screening test and conventional treatments are less accessible for people in developing countries, an alternative use of medicinal plants exhibiting strong anticancer activities may be an affordable means to treat cervical cancer. Mitrephora chulabhorniana (MC) is the newly identified species; however, its biological functions including anticancer activities have been largely unexplored. Hence, in this study, we were interested in investigating anticancer effects of this plant on the human cervical cell line (HeLa). MC extract was profiled for phytochemicals by TLC. This plant was tested to contain alkaloids, flavonoids, and terpenes. HeLa cells were treated with MC extract to investigate the anticancer activities. Cytotoxicity and viability of cells treated with MC were determined by MTT assay and Trypan blue exclusion assay. Cell migration was tested by wound healing assay, and cell invasion was determined by Transwell assay. The level of caspase 7, caspase 9, and PARP was determined by western blot analysis. We found that the leaf extract of MC strongly reduced cancer cell survival rate. This finding was consistent with the discovery that the extract dramatically induced apoptosis of cervical cancer cells through the activation of caspase 7 and caspase 9 which consequently degraded PARP protein. Furthermore, MC extract at lower concentrations which were not cytotoxic to the cancer cells showed potent inhibitory activities against HeLa cervical cancer cell migration and invasion. Mitrephora chulabhorniana possesses its pharmacological properties in inhibiting cervical cancer cell migration/invasion and inducing apoptotic signaling. This accumulated information suggests that Mitrephora chulabhorniana may be a beneficial source of potential agents for cervical cancer treatment.

Oroxylin A shows limited antiviral activity towards dengue virus.

OBJECTIVE: The mosquito transmitted dengue virus (DENV) the causative agent of dengue fever (DF) remains a significant public health burden in many countries. Thailand, along with many countries in Asia and elsewhere, has a long history of using traditional medicines to combat febrile diseases such as DF. Screening bioactive compounds from traditional medicines reported to have antipyretic or anti-inflammatory activity may lead to the development of potent antivirals. In this study oroxylin A (OA), a flavonoid derivative found in Oroxylum indicum (commonly called the Indian trumpet flower or tree of Damocles), was screened for antiviral activity towards DENV. RESULTS: Cytotoxicity analysis in BHK-21 cells showed a 50% cytotoxic concentration (CC50) of 534.17 µM. The compound showed no direct virucidal activity towards DENV, and pre-treatment of cells had no effect on virus production. A deficit was seen in virus production when cells were post-infection treated with oroxylin A. Under conditions of post-infection treatment, the EC50 value was 201.1 µM, giving a selectivity index (SI) value of 2.66. Accumulation of DENV E protein inside the cell was seen under conditions of post-infection treatment, suggesting that oroxylin A may exert some effects at the virus assembly/egress stages of the replication cycle.

Anti-psoriatic and anti-inflammatory effects of Kaempferia parviflora in keratinocytes and macrophage cells.

Kaempferia parviflora (KP) has been used as folk medicine for curing various conditions, including anti-inflammatory diseases. However, anti-psoriatic effects in an aspect of suppression of NF-κB activation have not been explored. Therefore, our current study aimed to elucidate the anti-inflammation of KP in lipopolysaccharide (LPS)-induced RAW264.7 cells and anti-psoriatic effects of KP in cytokine-induced human keratinocytes, HaCaT cells. We discovered that KP extract significantly suppressed LPS-induced inflammation at both gene expression and protein production. Specifically, dramatic reduction of nitric oxide (NO) was explored by using Griess method. Consistently, data from RT-qPCR, ELISA, and western blot analysis confirmed that crucial inflammatory and psoriatic markers including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-17, IL-22, and IL-23 were significantly decreased by the action of KP. These events were associated with the results from immunofluorescence study and western blot analysis where the activation of NF-κB upon LPS stimulation was clearly inhibited by KP through its ability to suppress IκB-α degradation resulting in inhibition of NF-κB nuclear translocation. Furthermore, KP extract significantly inhibited LPS-stimulated phosphorylation of ERK1/2, JNK, and p38 in a dose-dependent manner, along with inhibition of ERK1/2 activation in both TNF-α- and EGF-induced HaCaT cells. Interestingly, HaCaT cells exposed to 15 µg/mL of KP also exhibited significant decrease of cell migration and proliferation. Our results revealed that KP extract has a potential to be developed as a promising agent for treating inflammation and psoriasis, in part through targeting the proliferation and the NF-κB pathways.

Berberine Inhibits Dengue Virus through Dual Mechanisms.

Mosquito transmitted viruses, particularly those of the genus Flavivirus, are a significant healthcare burden worldwide, especially in tropical and sub-tropical areas. However, effective medicines for these viral infections remains lacking. Berberine (BBR) is an alkaloid found in some plants used in traditional medicines in Southeast Asia and elsewhere, and BBR has been shown to possess anti-viral activities. During a screen for potential application to mosquito transmitted viruses, BBR was shown to have virucidal activity against dengue virus (DENV; IC50 42.87 µM) as well as against Zika virus (IC50 11.42 µM) and chikungunya virus (IC50 14.21 µM). BBR was shown to have cellular effects that lead to an increase in cellular DENV E protein without a concomitant effect on DENV nonstructural proteins, suggesting an effect on viral particle formation or egress. While BBR was shown to have an effect of ERK1/2 activation this did not result in defects in viral egress mechanisms. The primary effect of BBR on viral production was likely to be through BBR acting through AMPK activation and disruption of lipid metabolism. Combined these results suggest that BBR has a dual effect on DENV infection, and BBR may have the potential for development as an anti-DENV antiviral.

Essential Oil from Zingiber ottensii Induces Human Cervical Cancer Cell Apoptosis and Inhibits MAPK and PI3K/AKT Signaling Cascades.

Zingiber ottensii (ZO) is a local plant in Thailand and has been used as a Thai traditional therapy for many conditions. ZO has been reported to exhibit many pharmacological effects, including anti-cancer activity. Nevertheless, its anti-cancer effects explored at the signaling level have not been elucidated in cervical cancer, which is one of the leading causes of fatality in females. We discovered that the essential oil of ZO significantly increased the apoptosis of human cervical cancer cells (HeLa) after 24 h of treatment in a concentration-dependent manner. Our data also clearly demonstrated that ZO essential oil reduced IL-6 levels in the culture supernatants of the cancer cells. Moreover, Western blot analysis clearly verified that cells were induced to undergo apoptotic death via caspase activation upon treatment with ZO essential oil. Interestingly, immunofluorescence studies and Western blot analyses showed that ZO essential oil suppressed epidermal growth factor (EGF)-induced pAkt and pERK1/2 signaling pathway activation. Together, our study demonstrates that ZO essential oil can reduce the proliferation and survival signaling of HeLa cervical cancer cells. Our study provides convincing data that ZO essential oil suppresses the growth and survival of cervical cancer cells, and it may be a potential choice for developing an anti-cancer agent for treating certain cervical cancers.

Curcuma amarissima Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte.

Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of Curcuma amarissima (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that Curcuma amarissima can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.

A functional interaction between GRP78 and Zika virus E protein.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused significant public health concerns around the world, partly because of an association with microcephaly in babies born to mothers who were infected with ZIKV during pregnancy. As a recently emerging virus, little is known as to how the virus interacts with the host cell machinery. A yeast-2-hybrid screen for proteins capable of interacting with the ZIKV E protein domain III, the domain responsible for receptor binding, identified 21 proteins, one of which was the predominantly ER resident chaperone protein GRP78. The interaction of GRP78 and ZIKV E was confirmed by co-immunoprecipitation and reciprocal co-immunoprecipitation, and indirect immunofluorescence staining showed intracellular and extracellular co-localization between GRP78 and ZIKV E. Antibodies directed against the N-terminus of GRP78 were able to inhibit ZIKV entry to host cells, resulting in significant reductions in the levels of ZIKV infection and viral production. Consistently, these reductions were also observed after down-regulation of GRP78 by siRNA. These results indicate that GRP78 can play a role mediating ZIKV binding, internalization and replication in cells. GRP78 is a main regulator of the unfolded protein response (UPR), and the study showed that expression of GRP78 was up-regulated, and the UPR was activated. Increases in CHOP expression, and activation of caspases 7 and 9 were also shown in response to ZIKV infection. Overall these results indicate that the interaction between GRP78 and ZIKV E protein plays an important role in ZIKV infection and replication, and may be a potential therapeutic target.

Oxyresveratrol Inhibits TNF-α-Stimulated Cell Proliferation in Human Immortalized Keratinocytes (HaCaT) by Suppressing AKT Activation.

Psoriasis is a complex inflammatory disease characterized by hyperproliferative keratinocyte caused by active PI3K/AKT signaling. TNF-α concentrated in the psoriatic lesions stimulates AKT activation. We previously discovered that oxyresveratrol inhibited inflammation via suppressing AKT phosphorylation, therefore oxyresveratrol may possess a conserved property to block AKT activation and proliferation in keratinocyte in response to TNF-α. Our current study proved that oxyresveratrol exhibited potent anti-proliferative effects against TNF-α. These effects are explained by the findings that oxyresveratrol could potentially inhibit TNF-α-stimulated AKT and GSK3-ß activation in a dose-dependent manner, and its inhibitory pattern was comparable to that of a specific PI3K inhibitor. Results from immunofluorescence supported that oxyresveratrol effectively inhibited AKT and GSK3-ß activation in individual cells upon TNF-α stimulation. Furthermore, functional assay confirmed that oxyresveratrol repressed the expansion of the HaCaT colony over 3 days, and this was caused by the ability of oxyresveratrol to induce cell cycle arrest at S and G2/M phases and the reduction in the expression of a proliferative marker (Ki-67) and a survival marker (MCL-1). Given the importance of TNF-α and the PI3K/AKT pathway in the psoriatic phenotype, we anticipate that oxyresveratrol, which targets the TNF-α-stimulated PI3K/AKT pathway, would represent a promising psoriasis therapy in the near future.

Production of Zika Virus Virus-Like Particles.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus and thus present a similar antigenic profile. These properties make them good candidates for vaccine development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation of the VLP is achieved through western blotting and electron microscopy.