The Myeloid LSECtin Is a DAP12-Coupled Receptor That Is Crucial for Inflammatory Response Induced by Ebola Virus Glycoprotein

Fatal Ebola virus infection is characterized by a systemic inflammatory response similar to septic shock. Ebola glycoprotein (GP) is involved in this process through activating dendritic cells (DCs) and macrophages. However, the mechanism is unclear. Here, we showed that LSECtin (also known as CLEC4G) plays an important role in GP-mediated inflammatory responses in human DCs. Anti-LSECtin mAb engagement induced TNF-α and IL-6 production in DCs, whereas silencing of LSECtin abrogated this effect. Intriguingly, as a pathogen-derived ligand, Ebola GP could trigger TNF-α and IL-6 release by DCs through LSECtin. Mechanistic investigations revealed that LSECtin initiated signaling via association with a 12-kDa DNAX-activating protein (DAP12) and induced Syk activation. Mutation of key tyrosines in the DAP12 immunoreceptor tyrosine-based activation motif abrogated LSECtin-mediated signaling. Furthermore, Syk inhibitors significantly reduced the GP-triggered cytokine production in DCs. Therefore, our results demonstrate that LSECtin is required for the GP-induced inflammatory response, providing new insights into the EBOV-mediated inflammatory response.     

Honey Bee Inhibitory Signaling Is Tuned to Threat Severity and Can Act as a Colony Alarm Signal

Alarm communication is a key adaptation that helps social groups resist predation and rally defenses. In Asia, the world’s largest hornet, Vespa mandarinia, and the smaller hornet, Vespa velutina, prey upon foragers and nests of the Asian honey bee, Apis cerana. We attacked foragers and colony nest entrances with these predators and provide the first evidence, in social insects, of an alarm signal that encodes graded danger and attack context. We show that, like Apis mellifera, A. cerana possesses a vibrational “stop signal,” which can be triggered by predator attacks upon foragers and inhibits waggle dancing. Large hornet attacks were more dangerous and resulted in higher bee mortality. Per attack at the colony level, large hornets elicited more stop signals than small hornets. Unexpectedly, stop signals elicited by large hornets (SS _{large hornet}) had a significantly higher vibrational fundamental frequency than those elicited by small hornets (SS _{small hornet}) and were more effective at inhibiting waggle dancing. Stop signals resulting from attacks upon the nest entrance (SS _nest) were produced by foragers and guards and were significantly longer in pulse duration than stop signals elicited by attacks upon foragers (SS _forager). Unlike SS _forager, SS _nest were targeted at dancing and non-dancing foragers and had the common effect, tuned to hornet threat level, of inhibiting bee departures from the safe interior of the nest. Meanwhile, nest defenders were triggered by the bee alarm pheromone and live hornet presence to heat-ball the hornet. In A. cerana, sophisticated recruitment communication that encodes food location, the waggle dance, is therefore matched with an inhibitory/alarm signal that encodes information about the context of danger and its threat level.     

pH dependence of ligand-induced human epidermal growth factor receptor activation investigated by molecular dynamics simulations

The activation of human epidermal growth factor receptor (hEGFR) involves a large conformational change in its soluble extracellular domains (sECD, residues 1–620), from a tethered to an extended conformation upon binding of ligands, such as EGF. It has been reported that this dynamic process is pH-dependent, that is, hEGFR can be activated by EGF at high pH to form an extended dimer but remains as an inactive monomer at low pH. In this paper, we perform all-atom molecular dynamics (MD) simulations starting from the tethered conformation of sECD:EGF complex, at pH 5.0 and 8.5, respectively. Simulation results indicate that sECD:EGF shows different dynamic properties between the two pHs, and the complex may have a higher tendency of activation at pH 8.5. Twenty residues, including 13 histidines, in sECD:EGF have different protonation states between the two pHs (calculated by the H++ server). The charge distribution at pH 8.5 is more favorable for forming an extended conformation toward the active state of sECD than that at pH 5.0. Our study may shed light on the mechanism of pH dependence of hEGFR activation.

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Graphical abstract pH dependence of ligand-induced human epidermal growth factor receptor activation

     

Dihydroartemisinin attenuates lipopolysaccharide-induced osteoclastogenesis and bone loss via the mitochondria-dependent apoptosis pathway

Dihydroartemisinin (DHA) is a widely used antimalarial drug isolated from the plant Artemisia annua. Recent studies suggested that DHA has antitumor effects utilizing its reactive oxygen species (ROS) yielding mechanism. Here, we reported that DHA is inhibitory on lipopolysaccharide (LPS)-induced osteoclast (OC) differentiation, fusion and bone-resorption activity in vitro. Intracellular ROS detection revealed that DHA could remarkably increase ROS accumulation during LPS-induced osteoclastogenesis. Moreover, cell apoptosis was also increased by DHA treatment. We found that DHA-activated caspase-3 increased Bax/Bcl-2 ratio during LPS-induced osteoclastogenesis. Meanwhile, the translocation of apoptotic inducing factor (AIF) and the release of cytochrome c from the mitochondria into the cytosol were observed, indicating that ROS-mediated mitochondrial dysfunction is crucial in DHA-induced apoptosis during LPS-induced osteoclastogenesis. In vivo study showed that DHA treatment decreased OC number, prevents bone loss, rescues bone microarchitecture and restores bone strength in LPS-induced bone-loss mouse model. Together, our findings indicate that DHA is protective against LPS-induced bone loss through apoptosis induction of osteoclasts via ROS accumulation and the mitochondria-dependent apoptosis pathway. Therefore, DHA may be considered as a new therapeutic candidate for treating inflammatory bone loss.Bone is a dynamic organ that undergoes continuous remodeling throughout life. Bone homeostasis is maintained by a balanced bone-resorbing and bone-forming process. In this process, hematopoietic stem cells or monocytes/macrophage progenitor cell-derived osteoclasts (OCs) are mainly responsible for bone resorption.¹ Abnormal OC function is associated with numerous diseases, and most of them are due to excessive osteoclastic activity. These diseases include osteoporosis, rheumatoid arthritis and periodontitis.^{2, 3} Two of the most important regulating factors during OC differentiation are receptor activator of nuclear factor κB ligand (RANKL) and macrophage-colony-stimulating factor (M-CSF).^{4, 5} Binding of RANKL to RANK results in the initiation of the TNF receptor-associated factor 6 signaling, which activates nuclear factor-κB, Akt and MAP kinase (ERk, JNK and p-38), and eventually leads to the proliferation, differentiation and maturation of OCs.^{6, 7}Lipopolysaccharide (LPS) is an important component of the outer membrane of Gram-negative bacteria. In LPS-induced bone loss, many factors are involved including local host response, prostanoids and cytokine production, inflammatory cell recruitment and OC activation.^{8, 9, 10} Experimental evidence have shown that LPS-mediated inflammation is highly dependent on reactive oxygen species (ROS) and the associated downstream MAPK signaling pathways including ERK, JNK and p-38.^{11, 12} ROS has been shown having an important role in the process of OC differentiation, survival, activation and bone resorption.^{13, 14, 15, 16} It has also been proved that ROS production in OC and intracellular hydrogen peroxide accumulation is critical for osteoclastogenesis and skeletal homeostasis.¹⁷ Recently, a study reported that LPS induces OC formation via the ROS-mediated JNK and STAT3 pathway, which could be blocked by peroxiredoxin II.¹⁸Dihydroartemisinin (DHA) is the main active metabolite isolated from the plant Artemisia annua. DHA has been widely used as first-line therapeutics against falciparum malaria.¹⁹ Recent evidence suggested that DHA has antitumor effects because of its unique cytotoxicity mechanism.²⁰ In particular, studies reported that DHA is pro-apoptotic in tumor cell lines regarding breast and prostate cancer.^{21, 22} Although the detailed mechanism of DHA cytotoxicity and pro-apoptotic effects is not fully understood, DHA-mediated ROS production has a central role.^{23, 24} However, the effect of DHA on bone health has not been studied.In the present study, we reported that DHA could attenuate LPS-induced OC differentiation, fusion and bone-resorption activity in vitro. Our data showed that DHA-induced cell apoptosis during LPS-induced osteoclastogenesis via intracellular ROS generation and mitochondria-mediated pathways. DHA administration in LPS-induced mouse models decreased OC number and reversed bone loss in vivo.     

Regulation of Inflammation by IL-17A and IL-17F Modulates Non-Alcoholic Fatty Liver Disease Pathogenesis

Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. While it is well-accepted that inflammation is central to NAFLD pathogenesis, the immune pathway(s) orchestrating disease progression are poorly defined. Notably, IL-17RA signaling, via IL-17A, plays an important role in obesity-driven NAFLD pathogenesis. However, the role of the IL-17F, another IL-17RA ligand, in NAFLD pathogenesis has not been examined. Further, the cell types expressing IL-17RA and producing IL-17RA ligands in the pathogenesis of NAFLD have not been defined. Here, IL-17RA^-/-, IL-17A^-/-, IL-17F^-/- and wild-type (WT) mice were fed either standard chow diet or methionine and choline deficient diet (MCDD)—a diet known to induce steatosis and hepatic inflammation through beta-oxidation dysfunction—and hepatic inflammation and NAFLD progression were subsequently quantified. MCDD feeding augmented hepatic IL-17RA expression and significantly increased hepatic infiltration of macrophages and IL-17A and IL-17F producing CD4⁺ and CD8⁺ T cells in WT mice. In contrast, IL-17RA^-/-, IL-17A^-/-, and IL-17F^-/- mice, despite increased steatosis, exhibited significant protection from hepatocellular damage compared to WT controls. Protection from hepatocellular damage correlated with decreased levels of hepatic T-cell and macrophage infiltration and decreased expression of inflammatory mediators associated with NAFLD. In sum, our results indicate that the IL-17 axis also plays a role in a MCDD-induced model of NAFLD pathogenesis. Further, we show for the first time that IL-17F, and not only IL-17A, plays an important role in NAFLD driven inflammation.     

VA-Index: Quantifying Assortativity Patterns in Networks with Multidimensional Nodal Attributes

Network connections have been shown to be correlated with structural or external attributes of the network vertices in a variety of cases. Given the prevalence of this phenomenon network scientists have developed metrics to quantify its extent. In particular, the assortativity coefficient is used to capture the level of correlation between a single-dimensional attribute (categorical or scalar) of the network nodes and the observed connections, i.e., the edges. Nevertheless, in many cases a multi-dimensional, i.e., vector feature of the nodes is of interest. Similar attributes can describe complex behavioral patterns (e.g., mobility) of the network entities. To date little attention has been given to this setting and there has not been a general and formal treatment of this problem. In this study we develop a metric, the vector assortativity index (VA-index for short), based on network randomization and (empirical) statistical hypothesis testing that is able to quantify the assortativity patterns of a network with respect to a vector attribute. Our extensive experimental results on synthetic network data show that the VA-index outperforms a baseline extension of the assortativity coefficient, which has been used in the literature to cope with similar cases. Furthermore, the VA-index can be calibrated (in terms of parameters) fairly easy, while its benefits increase with the (co-)variance of the vector elements, where the baseline systematically over(under)estimate the true mixing patterns of the network.     

Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria

Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.     

Anti-Tumor Effects of Second Generation β-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression

Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate β-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA “knockdown” and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of β-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo.