Broad Antiviral Activity of Carbohydrate-Binding Agents against the Four Serotypes of Dengue Virus in Monocyte-Derived Dendritic Cells

BACKGROUND: Dendritic cells (DC), present in the skin, are the first target cells of dengue virus (DENV). Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs) against all four described serotypes of DENV replication in Raji/DC-SIGN(+) cells and in monocyte-derived DC (MDDC). METHODOLOGY/PRINCIPAL FINDINGS: A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA), Galanthus nivalis (GNA) and Urtica dioica (UDA), but not actinohivin (AH) was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold) than in Raji/DC-SIGN(+) cells. Pradimicin-S (PRM-S), a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN(+) cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection. CONCLUSIONS/SIGNIFICANCE: The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN(+) cells and in primary MDDC

Mitochondrial Calcium Buffering Contributes to the Maintenance of Basal Calcium Levels in Mouse Taste Cells

Taste stimuli are detected by taste receptor cells present in the oral cavity using diverse signaling pathways. Some taste stimuli are detected by G protein–coupled receptors (GPCRs) that cause calcium release from intracellular stores, whereas other stimuli depolarize taste cells to cause calcium influx through voltage-gated calcium channels (VGCCs). Although taste cells use two distinct mechanisms to transmit taste signals, increases in cytosolic calcium are critical for normal responses in both pathways. This creates a need to tightly control intracellular calcium levels in all transducing taste cells. To date, however, the mechanisms used by taste cells to regulate cytosolic calcium levels have not been identified. Studies in other cell types have shown that mitochondria can be important calcium buffers, even during small changes in calcium loads. In this study, we used calcium imaging to characterize the role of mitochondria in buffering calcium levels in taste cells. We discovered that mitochondria make important contributions to the maintenance of resting calcium levels in taste cells by routinely buffering a constitutive calcium influx across the plasma membrane. This is unusual because in other cell types, mitochondrial calcium buffering primarily affects large evoked calcium responses. We also found that the amount of calcium that is buffered by mitochondria varies with the signaling pathways used by the taste cells. A transient receptor potential (TRP) channel, likely TRPV1 or a taste variant of TRPV1, contributes to the constitutive calcium influx

Insights into (U)HP metamorphism of the Western Gneiss Region, Norway: A high-spatial resolution and high-precision zircon study

Combining high-spatial resolution and high-precision geochronology and geochemistry of zircon provides constraints on the timing and duration of ultrahigh-pressure (UHP) metamorphism resulting from the collision of Baltica–Avalonia and Laurentia during the Scandian orogeny in the Western Gneiss Region of Norway. Zircons were extracted from a layered eclogite in the Saltaneset region (southern UHP domain) and from an eclogite in the Ulsteinvik region (central UHP domain). Zircons were first analyzed for U–Pb and trace element compositions by laser ablation split-stream (LASS) inductively coupled plasma mass spectrometry (ICP-MS), followed by analysis of those same zircons that yielded Scandian dates by integrated U–Pb isotope dilution–thermal ionization mass spectrometry and Trace Element Analysis (TIMS–TEA). LASS results from a garnet–quartz layer within the Saltaneset eclogite give Scandian dates of ca. 413–397 Ma, with subsequent ID–TIMS analyses ranging from 408.9 ± 0.4 Ma to 401.4 ± 0.2 Ma (2σ). An omphacite-rich layer from the same eclogite yields LASS dates of ca. 414–398 Ma and a single ID–TIMS date of 396.7 ± 1.4 Ma. In comparison, the Ulsteinvik eclogite LASS results give dates spanning ca. 413–397 Ma, whereas ID–TIMS analyses range from 409.6 ± 0.6 Ma to 401.3 ± 0.4 Ma. ID–TIMS zircon data from the eclogites reveals two age populations: 1) ca. 409–407 Ma and 2) ca. 402 Ma. Both in situ and solution trace element data show a distinct pattern for Scandian zircons, with strongly-depleted HREE and weakly-negative Eu anomalies (Eu/Eu*), whereas inherited zircon REE patterns are distinguished by steep HREE slopes and marked negative Eu/Eu*. When coupled with partition coefficients calculated for zircon and garnet, these REE patterns indicate that zircon (re)crystallized during eclogite-facies metamorphism at ca. 409–407 Ma and ca. 402 Ma at two widely separated UHP localities

Transgenic overexpression of the SUR2A-55 splice variant in mouse heart reduces infract size and promotes protective mitochondrial function

ATP-sensitive potassium channels found in both the sarcolemma (sarcKATP) and mitochondria (mitoKATP) of cardiomyocytes are important mediators of cardioprotection during ischemic heart disease. Sulfonylurea receptor isoforms (SUR2), encoded by Abcc9, an ATP-binding cassette family member, form regulatory subunits of the sarcKATP channel and are also thought to regulate mitoKATP channel activity. A short-form splice variant of SUR2 (SUR2A-55) was previously shown to target mitochondria and display diaxoxide and ATP insensitive KATP activity when co-expressed with the inward rectifier channels Kir6.2 and Kir6.1. We hypothesized that mice with cardiac specific overexpression of SUR2A-55 would mediate cardioprotection from ischemia by altering mitoKATP properties. Mice overexpressing SUR2A-55 (TGSUR2A-55) in cardiomyocytes were generated and showed no significant difference in echocardiographic measured chamber dimension, percent fractional shortening, heart to body weight ratio, or gross histologic features compared to normal mice at 11–14 weeks of age. TGSUR2A-55 had improved hemodynamic functional recovery and smaller infarct size after ischemia reperfusion injury compared to WT mice in an isolated hanging heart model. The mitochondrial membrane potential of TGSUR2A-55 mice was less sensitive to ATP, diazoxide, and Ca2+ loading. These data suggest that the SUR2A-55 splice variant favorably affects mitochondrial function leading to cardioprotection. These data support a role for the regulation of mitoKATP activity by SUR2A-55

Potent STING activation stimulates immunogenic cell death to enhance antitumor immunity in neuroblastoma

BackgroundNeuroblastoma (NB) is a childhood cancer for which new treatment options are needed. The success of immune checkpoint blockade in the treatment of adult solid tumors has prompted the exploration of immunotherapy in NB; however, clinical evidence indicates that the vast majority of NB patients do not respond to single-agent checkpoint inhibitors. This motivates a need for therapeutic strategies to increase NB tumor immunogenicity. The goal of this study was to evaluate a new immunotherapeutic strategy for NB based on potent activation of the stimulator of interferon genes (STING) pathway.MethodsTo promote STING activation in NB cells and tumors, we utilized STING-activating nanoparticles (STING-NPs) that are designed to mediate efficient cytosolic delivery of the endogenous STING ligand, 2’3’-cGAMP. We investigated tumor-intrinsic responses to STING activation in both MYCN-amplified and non-amplified NB cell lines, evaluating effects on STING signaling, apoptosis, and the induction of immunogenic cell death. The effects of intratumoral administration of STING-NPs on CD8+ T cell infiltration, tumor growth, and response to response to PD-L1 checkpoint blockade were evaluated in syngeneic models of MYCN-amplified and non-amplified NB.ResultsThe efficient cytosolic delivery of 2’3’-cGAMP enabled by STING-NPs triggered tumor-intrinsic STING signaling effects in both MYCN-amplified and non-amplified NB cell lines, resulting in increased expression of interferon-stimulated genes and pro-inflammatory cytokines as well as NB cell death at concentrations 2000-fold to 10000-fold lower than free 2’3’-cGAMP. STING-mediated cell death in NB was associated with release or expression of several danger associated molecular patterns that are hallmarks of immunogenic cell death, which was further validated via cell-based vaccination and tumor challenge studies. Intratumoral administration of STING-NPs enhanced STING activation relative to free 2’3’-cGAMP in NB tumor models, converting poorly immunogenic tumors into tumoricidal and T cell-inflamed microenvironments and resulting in inhibition of tumor growth, increased survival, and induction of immunological memory that protected against tumor re-challenge. In a model of MYCN-amplified NB, STING-NPs generated an abscopal response that inhibited distal tumor growth and improved response to PD-L1 immune checkpoint blockade.ConclusionsWe have demonstrated that activation of the STING pathway, here enabled by a nanomedicine approach, stimulates immunogenic cell death and remodels the tumor immune microenvironment to inhibit NB tumor growth and improve responses to immune checkpoint blockade, providing a multifaceted immunotherapeutic approach with potential to enhance immunotherapy outcomes in NB