Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1␤ and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1␤/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1␤ in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigenspecific responses in vivo.Because many protein antigens do not elicit strong immune responses on their own, vaccines often contain stimulatory adjuvants that enhance cell-mediated and humoral immune responses to help confer stronger protection. However, despite widespread use, there is little known regarding the pathways affected by many adjuvants. A better understanding of the mechanisms involved in adjuvant-generated protection can assist in the design of better vaccines against infections that currently lack effective immunization.Adjuvants activate an innate immune response, which in turn determines the strength and quality of the adaptive immune response. This response is first mediated by activation of antigen-presenting cells (APCs) such as dendritic cells and macrophages. Engagement of pattern recognition receptors, such as extracellular, membrane-bound Toll-like receptors (TLRs) 2 and cytosolic inflammasome-stimulating Nod-like receptors (NLRs) by their ligands elicits an inflammatory milieu and can eventually lead to a honed adaptive immune response.The NLR inflammasomes are multiprotein complexes that upon activation license the proteolytic processing of the zymogen pro-caspase-1 into mature caspase-1 (1). caspase-1 can then activate pro-forms of the inflammatory cytokines IL-1␤ and IL-18 into mature proteins, which are then secreted through unknown pathways. IL-1␤ and IL-18 are potent proinflammatory cytokines that can, for instance, promote T helper 17 ...

SIRT1 is a member of a highly conserved gene family (sirtuins) encoding nicotinamide adenine dinucleotide (NAD)+-dependent deacetylases, originally found to deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals. SIRT1 has been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis and axonal degeneration. However, the role of SIRT1 in ultraviolet (UV) signalling pathways remains unknown. Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes. Both UV radiation and H2O2, two major inducers of skin cell damage, down-regulate SIRT1 in a time- and dose-dependent manner. We observed that reactive oxygen species-mediated JNK activation is involved in this SIRT1 down-regulation. SIRT1 activator, resveratrol, which has been considered as an important antioxidant, protects against UV- and H2O2-induced cell death, whereas SIRT inhibitors such as sirtinol and nicotinamide enhance cell death. Activation of SIRT1 negatively regulates UV- and H2O2-induced p53 acetylation, because nicotinamide and sirtinol as well as SIRT1 siRNA enhance UV- and H2O2-induced p53 acetylation, whereas SIRT1 activator resveratrol inhibits it. We also found that SIRT1 is involved in UV-induced AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC), phosphofructose kinase-2 (PFK-2) phosphorylation. Collectively, our data provide new insights into understanding of the molecular mechanisms of UV-induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti-skin aging agents.

One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin. Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). We find that UV induces aquaporin-3 (AQP3) down-regulation in human skin keratinocytes. MEK/ERK inhibitors PD98059 and U0126 inhibit UV-induced down-regulation of AQP3. Antioxidant N-acetyl-L-cysteine or NAC blocks UV-induced MEK/ERK activation and down-regulation of AQP3. All-trans retinoic acid or atRA, while alone inducing AQP3 expression, attenuates UV-induced down-regulation of AQP3 and water permeability. Using special inhibitors, we find that activation of EGFR and inhibition on ERK activation are involved in atRA's protective effects against UV-induced AQP3 down-regulation. Using specific AQP3's water transport inhibitors and siRNA knockdown, we observe that AQP3 is involved in cell migration and in vitro wound healing. UV-induced AQP3 down-regulation results in reduced water permeability, decreased cell migration, and delayed wound healing, which are attenuated by atRA pretreatment. We conclude that atRA protects against UV-induced down-regulation AQP3 and decrease in water permeability, reduction in cell migration and delayed in vitro wound healing via trans-activation of EGFR and inhibition on ROS-mediated MEK/ERK pathway. This novel finding provides evidence to support possible involvement of AQP3 in UV induced skin dehydration.

Collectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. The results that curcumin inhibits EGF-induced up-regulation of AQP3 and cell migration, provide a new explanation for the anticancer potential of curcumin.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies and the third leading cause of cancer-related deaths worldwide. Tumour metastasis is one of the major causes of high mortality. microRNAshave been implicated in HCC metastasis. In this study, we found that miR-625 was frequently downregulated in HCC samples. A decrease in miR-625 was significantly correlated with lymph node anddistance metastasis (P=0.013), the presence of portal venous invasion (P=0.036), tumor-node-metastasis (TNM) stage (P=0.027) and unfavourable overall survival (P=0.003). Compared with primary tumours, miR-625 expression was markedly reduced in portal venous metastatic tumours. Re-expression of miR-625 in HCC cells was remarkably effective in suppressing cell migration andinvasiveness in vitro and in vivo. Mechanistically, miR-625 was confirmed to downregulate IGF2 mRNA-binding protein 1(IGF2BP1) directly, the expression of which was inversely correlated with the level of miR-625 in HCC cell lines and tissues. High expression of IGF2BP1 was frequently found in HCC samples, and associated with poor prognosis. Knockdown of endogenous IGF2BP1 by siRNA exhibited similar effects as the overexpression of miR-625, whereas overexpression of IGF2BP1 (without the 3'-UTR) abrogated miR-625-mediated metastasis inhibition. Interference of the PTEN/HSP27 pathway contributed to miR-625-mediated metastasis inhibition. Taken together, our data suggest that miR-625 might function as an antimetastatic miRNA to have an important role in HCC progression by modulating the IGF2BP1/PTEN pathway. The newly identified miR-625/IGF2BP1 axis represents a new potential therapeutic target for HCC treatment.

DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses.

Aqueous solubility is an important physicochemical property of compounds in anti-cancer drug discovery. Artificial intelligence solubility prediction tools have scored impressive performances by employing regression, machine learning, and deep learning methods. The reported performances vary significantly partly because of the different datasets used. Solubility prediction on novel compounds needs to be improved, which may be achieved by going deeper with deep learning. We constructed deeper-net models of ∼20-layer modified ResNet convolutional neural network architecture, which were trained and tested with 9,943 compounds encoded by molecular fingerprints. Retrospectively tested by 62 recently-published novel compounds, one deeper-net model outperformed four established tools, shallow-net models, and four human experts. Deeper-net models also outperformed others in predicting the solubility values of a series of novel compounds newly-synthesized for anti-cancer drug discovery. Solubility prediction may be improved by going deeper with deep learning. Our deeper-net models are accessible at http://www.npbdb.net/solubility/index.jsp.

In recent years, various virtual screening (VS) tools have been developed, and many successful screening campaigns have been showcased. However, whether by conventional molecular docking or pharmacophore screening, the selection of virtual hits is based on the ranking of compounds by scoring functions or fit values, which remains the bottleneck of VS due to insufficient accuracy. As the limitations of individual methods persist, a comprehensive comparison and integration of different methods may provide insights into selecting suitable methods for VS. Here, we evaluated the performance of molecular docking, fingerprint-based 2D similarity and multicomplex pharmacophore in an individual and a combined manner, through a retrospective VS study on VEGFR-2 inhibitors. An integrated two-layer workflow was developed and validated through VS of VEGFR-2 inhibitors against the DUD-E database, which demonstrated improved VS performance through a ligand-based method ECFP_4, followed by molecular docking, and then a strict multicomplex pharmacophore. Through a retrospective comparison with six published papers, this integrated approach outperformed 43 out of 45 methods, indicating a great effectiveness. This kind of integrated VS approach can be extended to other targets for the screening and discovery of inhibitors.