Conventional protein kinase C-α (PKC-α) and PKC-β negatively regulate RIG-I antiviral signal transduction

Retinoic acid-inducible gene I (RIG-I) is a key sensor for viral RNA in the cytosol, and it initiates a signaling cascade that leads to the establishment of an interferon (IFN)-mediated antiviral state. Because of its integral role in immune signaling, RIG-I activity must be precisely controlled. Recent studies have shown that RIG-I CARD-dependent signaling function is regulated by the dynamic balance between phosphorylation and TRIM25-induced K₆₃-linked ubiquitination. While ubiquitination of RIG-I is critical for RIG-I''s ability to induce an antiviral IFN response, phosphorylation of RIG-I at S₈ or T₁₇₀ suppresses RIG-I signal-transducing activity under normal conditions. Here, we not only further define the roles of S₈ and T₁₇₀ phosphorylation for controlling RIG-I activity but also identify conventional protein kinase C-α (PKC-α) and PKC-β as important negative regulators of the RIG-I signaling pathway. Mutational analysis indicated that while the phosphorylation of S₈ or T₁₇₀ potently inhibits RIG-I downstream signaling, the dephosphorylation of RIG-I at both residues is necessary for optimal TRIM25 binding and ubiquitination-mediated RIG-I activation. Furthermore, exogenous expression, gene silencing, and specific inhibitor treatment demonstrated that PKC-α/β are the primary kinases responsible for RIG-I S₈ and T₁₇₀ phosphorylation. Coimmunoprecipitation showed that PKC-α/β interact with RIG-I under normal conditions, leading to its phosphorylation, which suppresses TRIM25 binding, RIG-I CARD ubiquitination, and thereby RIG-I-mediated IFN induction. PKC-α/β double-knockdown cells exhibited markedly decreased S₈/T₁₇₀ phosphorylation levels of RIG-I and resistance to infection by vesicular stomatitis virus. Thus, these findings demonstrate that PKC-α/β-induced RIG-I phosphorylation is a critical regulatory mechanism for controlling RIG-I antiviral signal transduction under normal conditions.     

Forest Remnants Along Urban-Rural Gradients: Examining Their Potential for Global Change Research

Over the next century, ecosystems throughout the world will be responding to rapid changes in climate and rising levels of carbon dioxide, inorganic N and ozone. Because people depend on biological systems for water, food and other ecosystem services, predicting the range of responses to global change for various ecosystem types in different geographic locations is a high priority. Modeling exercises and manipulative experimentation have been the principle approaches used to place upper and lower bounds on community and ecosystem responses. However, each of these approaches has recognized limitations. Manipulative experiments cannot vary all the relevant factors and are often performed at small spatio-temporal scales. Modeling is limited by data availability and by our knowledge of how current observations translate into future conditions. These weaknesses would improve if we could observe ecosystems that have already responded to global change factors and thus presage shifts in ecosystem structure and function. Here we consider whether urban forest remnants might offer this ability. As urban forests have been exposed to elevated temperature, carbon dioxide, nitrogen deposition and ozone for many decades, they may be ahead of the global change “response curve” for forests in their region. Therefore, not only might forests along urbanization gradients provide us with natural experiments for studying current responses to global change factors, but their legacy of response to past urbanization may also constitute space-for-time substitution experiments for predicting likely regional forest responses to continued environmental change. For this approach to be successful, appropriate criteria must be developed for selecting forest remnants and plots that would optimize our ability to detect incipient forest responses to spatial variation in global change factors along urbanization gradients, while minimizing artifacts associated with remnant size and factors other than those that simulate global change. Studying forests that meet such criteria along urban-to-rural gradients could become an informative part of a mixed strategy of approaches for improving forecasts of forest ecosystem change at the regional scale.     

Rapid,reliable, and reproducible cell fusion assay to quantify SARS-Cov-2 spike interaction with hACE2

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.     

Lithotrophic growth ofSulfurospirillum deleyianum with sulfide as electron donor coupled to respiratory reduction of nitrate to ammonia

Sulfurospirillum deleyianum grew in batch culture under anoxic conditions with sulfide (up to 5 mM) as electron donor, nitrate as electron acceptor, and acetate as carbon source. Nitrate was reduced to ammonia via nitrite, a quantitatively liberated intermediate. Four moles of sulfide were oxidized to elemental sulfur per mole nitrate converted to ammonia. The molar growth yield per mole sulfide consumed, Y_m, was 1.5 ± 0.2 g mol^–1 for the reduction of nitrate to ammonia. By this type of metabolism, S. deleyianum connected the biogeochemical cycles of sulfur and nitrogen. The sulfur reductase activity in S. deleyianum was inducible, as the activity depended on the presence of sulfide or elemental sulfur during cultivation with nitrate or fumarate as electron acceptor. Hydrogenase activity was always high, indicating that the enzyme is constitutively expressed. The ammonia-forming nitrite reductase was an inducible enzyme, expressed when cells were cultivated with nitrate, nitrite, or elemental sulfur, but repressed after cultivation with fumarate. Received: 13 March 1995 / Accepted: 29 May 1995     

Patterns in potassium dynamics in forest ecosystems

The biotic cycling of potassium (K) in forest systems has been relatively understudied in comparison with nitrogen (N) and phosphorus (P) despite its critical roles in maintaining the nutrition of primary production in forests. We investigated the ecological significance of K in forests from a literature review and data synthesis. We focused on (1) describing patterns of the effects of K availability on aboveground growth and change in foliar tissue of tree species from a variety of forests; and (2) documenting previously unreported relationships between hydrologic losses of K and N in forested watersheds from the Americas. In a review of studies examining tree growth under K manipulations/fertilizations, a high percentage (69% of studies) showed a positive response to increases in K availability in forest soils. In addition, 76% of the tree studies reviewed showed a positive and significant increase in K concentrations in plant tissue after soil K manipulation/fertilization. A meta-analysis on a subset of the reviewed studies was found to provide further evidence that potassium effects tree growth and increased tissue [K] with an effect size of 0.709 for growth and an overall effect size of 0.56. In our review of watershed studies, we observed that concentrations of K typically decreased during growing seasons in streams draining forested areas in the Temperate Zones and were responsive to vegetation disturbance in both temperate and tropical regions. We found a strong relationship (r2 = 0.42-0.99) between concentrations of K and N (another critical plant nutrient) in stream water, suggesting that similar mechanisms of biotic retention may control the flow of these nutrients. Furthermore, K dynamics appear to be unique among the base cations, e.g. calcium, magnesium, and sodium, because the others do not show similar seasonal patterns to K. We suggest that K may be important to the productivity and sustenance of many forests, and its dynamics and ecological significance warrant further study. We suggest that knowledge about the dynamics of this understudied element is imperative for our understanding patterns and processes in forest ecosystems.     

The ephrin receptor tyrosine kinase A2 is a cellular receptor for Kaposi's sarcoma–associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma(1), a highly vascularized tumor originating from lymphatic endothelial cells, and of at least two different B cell malignancies(2,3). A dimeric complex formed by the envelope glycoproteins H and L (gH-gL) is required for entry of herpesviruses into host cells(4). We show that the ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH-gL. EphA2 co-precipitated with both gH-gL and KSHV virions. Infection of human epithelial cells with a GFP-expressing recombinant KSHV strain, as measured by FACS analysis, was increased upon overexpression of EphA2. Antibodies against EphA(2) and siRNAs directed against EphA2 inhibited infection of endothelial cells. Pretreatment of KSHV with soluble EphA2 resulted in inhibition of KSHV infection by up to 90%. This marked reduction of KSHV infection was seen with all the different epithelial and endothelial cells used in this study. Similarly, pretreating epithelial or endothelial cells with the soluble EphA2 ligand ephrinA4 impaired KSHV infection. Deletion of the gene encoding EphA2 essentially abolished KSHV infection of mouse endothelial cells. Binding of gH-gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry(5,6). Quantitative RT-PCR and in situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from human Kaposi's sarcoma lesions or unaffected human lymphatic endothelium, and in situ in Kaposi's sarcoma specimens, respectively. Taken together, our results identify EphA2, a tyrosine kinase with known functions in neovascularization and oncogenesis, as an entry receptor for KSHV.     

Kaposi's sarcoma-associated herpesvirus gH/gL: glycoprotein export and interaction with cellular receptors

The attachment, entry, and fusion of Kaposi's sarcoma-associated herpesvirus (KSHV) with target cells are mediated by complex machinery containing, among others, viral glycoprotein H (gH) and its alleged chaperone, gL. We observed that KSHV gH, in contrast to its homologues in several other herpesviruses, is transported to the cytoplasm membrane independently from gL, but not vice versa. Mutational analysis revealed that the N terminus of gH is sufficient for gL interaction. However, the entire extracellular part of gH is required for efficient gL secretion. The soluble ectodomain of gH was sufficient to interact with the surfaces of potential target cells in a heparin-dependent manner, and binding was further enhanced by coexpression of gL. Surface plasmon resonance revealed a remarkably high affinity of gH for glycosaminoglycans. Heparan sulfate (HS) proteoglycans of the syndecan family act as cellular receptors for the gH/gL complex. They promoted KSHV infection, and expression of gH/gL on target cells inhibited subsequent KSHV infection. Whereas gH alone was able to bind to HS, we observed that only the gH/gL complex adhered to heparan sulfate-negative cells at lamellipodium-like structures.     

Consequence of salinity and excess boron on growth, evapotranspiration and ion uptake in date palm (Phoenix dactylifera L., cv. Medjool)

Patches of common juniper (Juniperus communis L.) shrubs potentially facilitate the formation of fertile islands in heath tundra ecosystems thereby influencing the long-term resilience of these ecosystems. Although the role of juniper in the formation of such ‘islands of fertility’ has been studied in semiarid landscapes, there has been little attention paid to the importance of juniper in other ecosystems. In this study we contrast the soil fertility and rates of N fixation under juniper shrubs with that in open heath tundra in northern Sweden. Plots were established at several individual sites in alpine heath tundra in Northern Sweden and mineral soils to a depth of 10 cm were characterized for available N and P and total C, N, P, Ca, Mg, K, Fe, Mn, Zn, and Cu. Nitrogen fixation rates were measured by acetylene reduction in feather mosses under juniper canopies and contrasted with N fixation in both feather mosses and surface soils in the open heath. Soils under juniper had concentrations of total P greatly in excess of P in open heath, furthermore, juniper islands had the highest concentrations of bioavailable P. Nitrogen fixation rates in the feather moss Pleurozium schreberi (Bird.) Mitt were approximately 150 μmol acetylene reduced m⁻² d⁻¹ under the juniper canopy compared to less than 10 μmol acetylene reduced m⁻² d⁻¹ in the open heath. Feather mosses under the juniper canopy also fixed N at a significantly higher rate (on an aerial basis) than that of surface cores from the open heath that included lichen, mosses, and soil crusts. Juniper facilitates the formation of islands of soil fertility that may in turn facilitate the growth of other plants and positively influence the long term recovery of heath tundra ecosystems following disturbance.