Poxvirus Antigen Staining of Immune Cells as a Biomarker to Predict Disease Outcome in Monkeypox and Cowpox Virus Infection in Non-Human Primates

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1–2×10⁷ PFU) or CPXV (>10² PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.     

Cellular alteration and differential protein profile explain effects of GA3 and ABA and their inhibitor on Trichocline catharinensis (Asteraceae) seed germination

Seed physiology of wild species has not been studied as deeply as that of domesticated crop species. Trichocline catharinensis (Asteraceae) is an endemic wildflower species from the high-altitude fields of southern Brazil. This species is of interest as a source of genes to improve cultivated Asteraceae because of its ornamental features, disease resistance and ability to tolerate drought and poor soil conditions. We studied the effects of abscisic acid (ABA) and gibberellic acid (GA₃) and their inhibitors, fluridone (FLU) and paclobutrazol (PAC), on seed germination. We individually assessed ultrastructural changes and differential protein accumulation. The principal component analysis explained 69.66% of differential accumulation for 32 proteins at phase II of seed germination in response to hormone and inhibitor treatment. GA₃-imbibed seed germination (98.75%) resulted in increased protein accumulation to meet energy demand, redox regulation, and reserve metabolism activation. FLU-imbibed seeds showed a higher germination speed index as a consequence of metabolism activation. ABA-imbibed seeds (58.75%) showed osmotolerance and flattened cells in the hypocotyl-radicular axis, suggesting that ABA inhibits cell expansion. PAC-imbibed seeds remained at phase II for 300 h, and germination was suppressed (7.5%) because of the increased signaling proteins and halted reserve mobilization. Therefore, our findings provide insight into the behavior of Asteraceae non-dormant seed germination, which broadens our knowledge of seed germination in a wild and endemic plant species from a threatened ecosystem.     

Inhibition of histone acetylation and deacetylation enzymes affects longevity,development, and fecundity in the pea aphid (Acyrthosiphon pisum)

Histone acetylation is an evolutionarily conserved epigenetic mechanism of eukaryotic gene regulation which is tightly controlled by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In insects, life-history traits such as longevity and fecundity are severely affected by the suppression of HAT/HDAC activity, which can be achieved by RNA-mediated gene silencing or the application of chemical inhibitors. We used both experimental approaches to investigate the effect of HAT/HDAC inhibition in the pea aphid (Acyrthosiphon pisum) a model insect often used to study complex life-history traits. The silencing of HAT genes (kat6b, kat7, and kat14) promoted survival or increased the number of offspring, whereas targeting rpd3 (HDAC) reduced the number of viviparous offspring but increased the number of premature nymphs, suggesting a role in embryogenesis and eclosion. Specific chemical inhibitors of HATs/HDACs showed a remarkably severe impact on life-history traits, reducing survival, delaying development, and limiting the number of offspring. The selective inhibition of HATs and HDACs also had opposing effects on aphid body weight. The suppression of HAT/HDAC activity in aphids by RNA interference or chemical inhibition revealed similarities and differences compared to the reported role of these enzymes in other insects. Our data suggest that gene expression in A. pisum is regulated by multiple HATs/HDACs, as indicated by the fitness costs triggered by inhibitors that suppress several of these enzymes simultaneously. Targeting multiple HATs or HDACs with combined effects on gene regulation could, therefore, be a promising approach to discover novel targets for the management of aphid pests.     

Expression of Betapapillomavirus Oncogenes Increases the Number of Keratinocytes with Stem Cell-Like Properties

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a “stem cell-like” state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44^high EpCAM^high cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.     

Micrometric segregation of fluorescent membrane lipids: relevance for endogenous lipids and biogenesis in erythrocytes

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.     

The floral transition is not the developmental switch that confers competence for the Arabidopsis age-related resistance response to Pseudomonas syringae pv. tomato

Age-related resistance (ARR) is a plant defense response characterized by enhanced resistance to certain pathogens in mature plants relative to young plants. In Arabidopsis thaliana the transition to flowering is associated with ARR competence, suggesting that this developmental event is the switch that initiates ARR competence in mature plants (Rusterucci et al. in Physiol Mol Plant Pathol 66:222–231, 2005). The association of ARR and the floral transition was examined using flowering-time mutants and photoperiod-induced flowering to separate flowering from other developmental events that occur as plants age. Under short-day conditions, late-flowering plant lines ld-1 (luminidependens-1), soc1-2 (suppressor of overexpression of co 1-2), and FRI ⁺ (FRIGIDA) displayed ARR before the transition to flowering occurred. Early-flowering svp-31, svp-32 (short vegetative phase), and Ws-2 were ARR-defective, whereas early-flowering tfl1-14 (terminal flower 1-14) displayed ARR at the same time as Col-0. While svp-31, svp-32 and Ws-2 produced few rosette leaves, tfl1-14 produced a rosette leaf number similar to Col-0, suggesting that the development of a minimum number of rosette leaves is necessary to initiate ARR competence under short-day conditions. Photoperiod-induced transient expression of FT (FLOWERING LOCUS T) caused precocious flowering in short-day-grown Col-0 but this was not associated with ARR competence. Under long-day conditions co-9 (constans-9) mutants did not flower but displayed an ARR response at the same time as Col-0. This study suggests that SVP is required for the ARR response and that the floral transition is not the developmental event that regulates ARR competence.     

The catalytic domain CysPc of the DEK1 calpain is functionally conserved in land plants

DEK1, the single calpain of land plants, is a member of the ancient membrane bound TML–CysPc–C2L calpain family that dates back 1.5 billion years. Here we show that the CysPc–C2L domains of land plant calpains form a separate sub‐clade in the DEK1 clade of the phylogenetic tree of plants. The charophycean alga Mesostigma viride DEK1‐like gene is clearly divergent from those in land plants, suggesting that a major evolutionary shift in DEK1 occurred during the transition to land plants. Based on genetic complementation of the Arabidopsis thaliana dek1‐3 mutant using CysPc–C2L domains of various origins, we show that these two domains have been functionally conserved within land plants for at least 450 million years. This conclusion is based on the observation that the CysPc–C2L domains of DEK1 from the moss Physcomitrella patens complements the A. thaliana dek1‐3 mutant phenotype. In contrast, neither the CysPc–C2L domains from M. viride nor chimeric animal–plant calpains complement this mutant. Co‐evolution analysis identified differences in the interactions between the CysPc–C2L residues of DEK1 and classical calpains, supporting the view that the two enzymes are regulated by fundamentally different mechanisms. Using the A. thaliana dek1‐3 complementation assay, we show that four conserved amino acid residues of two Ca²⁺‐binding sites in the CysPc domain of classical calpains are conserved in land plants and functionally essential in A. thaliana DEK1.     

Indicator selection in life cycle assessment to enable decision making: issues and solutions

Purpose

With an ever increasing list of indicators available, life cycle assessment (LCA) practitioners face the challenge of effectively communicating results to decision makers. Simplification of LCA is often limited to an arbitrary selection of indicators, use of single scores by using weighted values or single attribute indicators. These solutions are less attractive to decision makers, since value judgments are introduced or multi-indicator information is lost. Normalization could be a means to narrow the list of indicators by ranking indicators vs. a reference system. This paper shows three different normalization approaches that produce very different ranking of indicators. It is explained how normalization helps maintain a multi-indicator approach while keeping the most relevant indicators, allowing effective decision making.

Methods

The approaches are illustrated on a hand dishwashing case study, using ReCiPe as the impact assessment method and taking the European population (year 2000) as the reference situation. Indicators are ranked using midpoint normalization factors, and compared to the ranking from endpoint normalization broken down by midpoint contribution.

Results and discussion

Endpoint normalization shows Resources as the most relevant area of protection for this case, closely followed by Human Health and Ecosystem. Broken down by their key driving midpoints, fossil depletion, climate change and, to a lesser extent, particulate matter formation and metal depletion, are most relevant. Midpoint normalization, however, indicates Freshwater Eutrophication, Natural Land Transformation and Toxicity indicators (marine and freshwater ecotoxicity and human toxicity) are most relevant.

Conclusions

A three-step approach based on endpoint normalization is recommended to present only the most relevant indicators, allowing more effective decision making instead of communicating all LCA indicators. The selection process breaks out the normalized endpoint results into the most contributing midpoints (relevant indicators) and reports results with midpoint level units. Bias due to lack of data completeness is less of an issue in the endpoint normalization process (compared to midpoint normalization), while midpoint results are less subject to uncertainty (compared to endpoint results). Focusing on the relevant indicators and key contributing unit processes has proven to be effective for non-LCA expert decision makers to understand, use, and communicate complex LCA results.     

Multivariate Models of Adult Pacific Salmon Returns

Most modeling and statistical approaches encourage simplicity, yet ecological processes are often complex, as they are influenced by numerous dynamic environmental and biological factors. Pacific salmon abundance has been highly variable over the last few decades and most forecasting models have proven inadequate, primarily because of a lack of understanding of the processes affecting variability in survival. Better methods and data for predicting the abundance of returning adults are therefore required to effectively manage the species. We combined 31 distinct indicators of the marine environment collected over an 11-year period into a multivariate analysis to summarize and predict adult spring Chinook salmon returns to the Columbia River in 2012. In addition to forecasts, this tool quantifies the strength of the relationship between various ecological indicators and salmon returns, allowing interpretation of ecosystem processes. The relative importance of indicators varied, but a few trends emerged. Adult returns of spring Chinook salmon were best described using indicators of bottom-up ecological processes such as composition and abundance of zooplankton and fish prey as well as measures of individual fish, such as growth and condition. Local indicators of temperature or coastal upwelling did not contribute as much as large-scale indicators of temperature variability, matching the spatial scale over which salmon spend the majority of their ocean residence. Results suggest that effective management of Pacific salmon requires multiple types of data and that no single indicator can represent the complex early-ocean ecology of salmon.