Astragalus Polysaccharides Attenuate Postburn Sepsis via Inhibiting Negative Immunoregulation of CD4+CD25high T Cells

BACKGROUND: Astragalus polysaccharides (APS) isolated from one of the Chinese herbs, Astragalus mongholicus, are known to have a variety of immunomodulatory activities. However, it is not yet clear whether APS can exert an effect on the immune functions of regulatory T cells (Tregs). This study was carried out to investigate the effect of APS on the immune function of peripheral blood Tregs in postburn sepsis. METHODOLOGY/PRINCIPAL FINDINGS: BALB/C mice were randomly divided into six groups as follows: sham burn group, burn control (burn without infection animals) group, burn plus P. aeruginosa group, burn plus P. aeruginosa with APS (50 mg/kg) treatment group, burn plus P. aeruginosa with APS (100 mg/kg) treatment group, and burn plus P. aeruginosa with APS (200 mg/kg) treatment group, and they were sacrificed on postburn day 1, 3, 5, and 7, respectively, with seven animals at each time point. Magnetic microbeads were used to isolate peripheral blood Tregs and CD4(+) T cells. Phenotypes were analyzed by flow cytometry, and cytokine levels were determined with ELISA. In the burn plus P. aeruginosa group, forkhead/winged helix transcription factor p3 (Foxp3) expression on CD4(+)CD25(+)Tregs were strongly enhanced in comparison to the sham group, and the capacity of CD4(+)CD25(+)Tregs to produce interleukin (IL)-10 was markedly increased. Administration of APS to inhibit CD4(+)CD25(+)Tregs could significantly decrease expression of Foxp3 on CD4(+)CD25(+)Tregs, and IL-10 production in burned mice with P. aeruginosa infection. At the same time, proliferative activity and expression of IL-2 and IL-2Rα on CD4(+) T cells were restored. In contrast, anti-Toll-like receptor 4 (TLR4) antibody could block the effect of APS on Tregs immune function. CONCLUSION: APS might suppress CD4(+)CD25(+)Treg activity, at least in part, via binding TLR4 on Tregs and trigger a shift of Th2 to Th1 with activation of CD4(+) T cells in burned mice with P. aeruginosa infection

Novel Insights to Be Gained From Applying Metacommunity Theory to Long-Term, Spatially Replicated Biodiversity Data

Global loss of biodiversity and its associated ecosystem services is occurring at an alarming rate and is predicted to accelerate in the future. Metacommunity theory provides a framework to investigate multi-scale processes that drive change in biodiversity across space and time. Short-term ecological studies across space have progressed our understanding of biodiversity through a metacommunity lens, however, such snapshots in time have been limited in their ability to explain which processes, at which scales, generate observed spatial patterns. Temporal dynamics of metacommunities have been understudied, and large gaps in theory and empirical data have hindered progress in our understanding of underlying metacommunity processes that give rise to biodiversity patterns. Fortunately, we are at an important point in the history of ecology, where long-term studies with cross-scale spatial replication provide a means to gain a deeper understanding of the multiscale processes driving biodiversity patterns in time and space to inform metacommunity theory. The maturation of coordinated research and observation networks, such as the United States Long Term Ecological Research (LTER) program, provides an opportunity to advance explanation and prediction of biodiversity change with observational and experimental data at spatial and temporal scales greater than any single research group could accomplish. Synthesis of LTER network community datasets illustrates that long-term studies with spatial replication present an under-utilized resource for advancing spatio-temporal metacommunity research. We identify challenges towards synthesizing these data and present recommendations for addressing these challenges. We conclude with insights about how future monitoring efforts by coordinated research and observation networks could further the development of metacommunity theory and its applications aimed at improving conservation efforts

The dual nature of metacommunity variability

There is increasing interest in measuring ecological stability to understand how communities and ecosystems respond to broad-scale global changes. One of the most common approaches is to quantify the variation through time in community or ecosystem aggregate attributes (e.g. total biomass), referred to as aggregate variability. It is now widely recognized that aggregate variability represents only one aspect of communities and ecosystems, and compositional variability, the changes in the relative frequency of species in an assemblage, is equally important. Recent contributions have also begun to explore ecological stability at regional spatial scales, where interconnected local communities form metacommunities, a key concept in managing complex landscapes. However, the conceptual frameworks and measures of ecological stability in space have only focused on aggregate variability, leaving a conceptual gap. Here, we address this gap with a novel framework for quantifying the aggregate and compositional variability of communities and ecosystems through space and time. We demonstrate that the compositional variability of a metacommunity depends on the degree of spatial synchrony in compositional trajectories among local communities. We then provide a conceptual framework in which compositional variability of 1) the metacommunity through time and 2) among local communities combine into four archetype scenarios: spatial stasis (low/low), spatial synchrony (high/low), spatial asynchrony (high/high) and spatial compensation (low/high). We illustrate this framework based on numerical examples and a case study of a macroalgal metacommunity in which low spatial synchrony reduced variability in aggregate biomass at the metacommunity scale, while masking high spatial synchrony in compositional trajectories among local communities. Finally, we discuss the role of dispersal, environmental heterogeneity, species interactions and suggest future avenues. We believe this framework will be helpful for considering both aspects of variability simultaneously, which is important to better understand ecological stability in natural and complex landscapes in response to environmental changes

Diversity–stability relationships across organism groups and ecosystem types become decoupled across spatial scales

The relationship between biodiversity and stability, or its inverse, temporal variability, is multidimensional and complex. Temporal variability in aggregate properties, like total biomass or abundance, is typically lower in communities with higher species diversity (i.e., the diversity–stability relationship or DSR). At broader spatial extents, regional-scale aggregate variability is also lower with higher regional diversity (in plant systems) and with lower spatial synchrony. However, focusing exclusively on aggregate properties of communities may overlook potentially destabilizing compositional shifts. It is not yet clear how diversity is related to different components of variability across spatial scales, nor whether regional DSRs emerge across a broad range of organisms and ecosystem types. To test these questions, we compiled a large collection of long-term metacommunity data spanning a wide range of taxonomic groups (e.g., birds, fish, plants, invertebrates) and ecosystem types (e.g., deserts, forests, oceans). We applied a newly developed quantitative framework for jointly analyzing aggregate and compositional variability across scales. We quantified DSRs for composition and aggregate variability in local communities and metacommunities. At the local scale, more diverse communities were less variable, but this effect was stronger for aggregate than compositional properties. We found no stabilizing effect of γ-diversity on metacommunity variability, but β-diversity played a strong role in reducing compositional spatial synchrony, which reduced regional variability. Spatial synchrony differed among taxa, suggesting differences in stabilization by spatial processes. However, metacommunity variability was more strongly driven by local variability than by spatial synchrony. Across a broader range of taxa, our results suggest that high γ-diversity does not consistently stabilize aggregate properties at regional scales without sufficient spatial β-diversity to reduce spatial synchrony

The Amino-Acid Substituents of Dipeptide Substrates of Cathepsin C Can Determine the Rate-Limiting Steps of Catalysis

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for <i>k</i>_cat/<i>K</i>_a and <i>k</i>_cat values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for <i>k</i>_cat/<i>K</i>_a (1.6 ± 0.09 μM^–1 s^–1) and <i>k</i>_cat (255 ± 6 s^–1), an inverse sKIE on <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.6 ± 0.15), a modest, normal sKIE on <i>k</i>_cat (^D<i>k</i>_cat = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s^–1). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (<i>k</i>_ac) and deacylation (<i>k</i>_dac) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) <i>Biochemistry 47</i>, 8697–8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective <i>k</i>_cat/<i>K</i>_a values of 0.49 ± 0.07 μM^–1s^–1 and 5.3 ± 0.5 μM^–1s^–1, and <i>k</i>_cat values of 28 ± 1 s^–1 and 25 ± 0.5 s^–1. Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.74 ± 0.05) and normal for <i>k</i>_cat (^D<i>k</i>_cat = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s^–1 and 390 s^–1, respectively), from which it was determined that <i>k</i>_ac ≫ <i>k</i>_dac ∼ <i>k</i>_cat. The third substrate type, Gly-Ile-AMC, gave very low values of <i>k</i>_cat/<i>K</i>_a (0.0015 ± 0.0001 μM^–1 s^–1) and <i>k</i>_cat (0.33 ± 0.02 s^–1), no sKIEs, (^D(<i>k</i>_cat/<i>K</i>_a) = 1.05 ± 0.5 and ^D<i>k</i>_cat = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which <i>k</i>_dac > <i>k</i>_ac ∼ <i>k</i>_cat, and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P₁ position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases

The Amino-Acid Substituents of Dipeptide Substrates of Cathepsin C Can Determine the Rate-Limiting Steps of Catalysis

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for <i>k</i>_cat/<i>K</i>_a and <i>k</i>_cat values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for <i>k</i>_cat/<i>K</i>_a (1.6 ± 0.09 μM^–1 s^–1) and <i>k</i>_cat (255 ± 6 s^–1), an inverse sKIE on <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.6 ± 0.15), a modest, normal sKIE on <i>k</i>_cat (^D<i>k</i>_cat = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s^–1). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (<i>k</i>_ac) and deacylation (<i>k</i>_dac) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) <i>Biochemistry 47</i>, 8697–8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective <i>k</i>_cat/<i>K</i>_a values of 0.49 ± 0.07 μM^–1s^–1 and 5.3 ± 0.5 μM^–1s^–1, and <i>k</i>_cat values of 28 ± 1 s^–1 and 25 ± 0.5 s^–1. Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.74 ± 0.05) and normal for <i>k</i>_cat (^D<i>k</i>_cat = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s^–1 and 390 s^–1, respectively), from which it was determined that <i>k</i>_ac ≫ <i>k</i>_dac ∼ <i>k</i>_cat. The third substrate type, Gly-Ile-AMC, gave very low values of <i>k</i>_cat/<i>K</i>_a (0.0015 ± 0.0001 μM^–1 s^–1) and <i>k</i>_cat (0.33 ± 0.02 s^–1), no sKIEs, (^D(<i>k</i>_cat/<i>K</i>_a) = 1.05 ± 0.5 and ^D<i>k</i>_cat = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which <i>k</i>_dac > <i>k</i>_ac ∼ <i>k</i>_cat, and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P₁ position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases

The Amino-Acid Substituents of Dipeptide Substrates of Cathepsin C Can Determine the Rate-Limiting Steps of Catalysis

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for <i>k</i>_cat/<i>K</i>_a and <i>k</i>_cat values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for <i>k</i>_cat/<i>K</i>_a (1.6 ± 0.09 μM^–1 s^–1) and <i>k</i>_cat (255 ± 6 s^–1), an inverse sKIE on <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.6 ± 0.15), a modest, normal sKIE on <i>k</i>_cat (^D<i>k</i>_cat = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s^–1). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (<i>k</i>_ac) and deacylation (<i>k</i>_dac) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) <i>Biochemistry 47</i>, 8697–8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective <i>k</i>_cat/<i>K</i>_a values of 0.49 ± 0.07 μM^–1s^–1 and 5.3 ± 0.5 μM^–1s^–1, and <i>k</i>_cat values of 28 ± 1 s^–1 and 25 ± 0.5 s^–1. Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for <i>k</i>_cat/<i>K</i>_a (^D(<i>k</i>_cat/<i>K</i>_a) = 0.74 ± 0.05) and normal for <i>k</i>_cat (^D<i>k</i>_cat = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s^–1 and 390 s^–1, respectively), from which it was determined that <i>k</i>_ac ≫ <i>k</i>_dac ∼ <i>k</i>_cat. The third substrate type, Gly-Ile-AMC, gave very low values of <i>k</i>_cat/<i>K</i>_a (0.0015 ± 0.0001 μM^–1 s^–1) and <i>k</i>_cat (0.33 ± 0.02 s^–1), no sKIEs, (^D(<i>k</i>_cat/<i>K</i>_a) = 1.05 ± 0.5 and ^D<i>k</i>_cat = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which <i>k</i>_dac > <i>k</i>_ac ∼ <i>k</i>_cat, and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P₁ position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases