Visualization of Allostery in P-Selectin Lectin Domain Using MD Simulations

Allostery of P-selectin lectin (Lec) domain followed by an epithelial growth factor (EGF)-like domain is essential for its biological functionality, but the underlying pathways have not been well understood. Here the molecular dynamics simulations were performed on the crystallized structures to visualize the dynamic conformational change for state 1 (S1) or state 2 (S2) Lec domain with respective bent (B) or extended (E) EGF orientation. Simulations illustrated that both S1 and S2 conformations were unable to switch from one to another directly. Instead, a novel S1' conformation was observed from S1 when crystallized B-S1 or reconstructed “E-S1” structure was employed, which was superposed well with that of equilibrated S1 Lec domain alone. It was also indicated that the corresponding allosteric pathway from S1 to S1' conformation started with the separation between residues Q30 and K67 and terminated with the release of residue N87 from residue C109. These results provided an insight into understanding the structural transition and the structure-function relationship of P-selectin allostery

Mechanical features of endothelium regulate cell adhesive molecule-induced calcium response in neutrophils

Atherosclerosis is caused by chronic inflammation associated with the adhesion of neutrophils and endothelial cells (ECs) that is mediated by their respective cellular adhesive molecules to stiffened blood vessel walls. However, the stiffness dependence of calcium flux on neutrophils remains unclear yet. Here, the effect of substrate stiffness by ECs on neutrophils' calcium spike was quantified when the individual neutrophils that adhered to the human umbilical vascular endothelial cell (HUVEC) monolayer were pre-placed onto a stiffness-varied polyacrylamide substrate (5 or 34.88 kPa) or glass surface. Our data indicated that E-/P-selectins and intercellular adhesion molecule 1 (ICAM-1) on HUVECs and β2-integrins, P-selectin glycoprotein ligand 1 (PSGL-1), and CD44s on neutrophils were all involved in mediating neutrophil calcium spike in a stiffness-dependent manner, in which the increase in substrate stiffness enhanced the calcium intensity and the oscillation frequency (spike number). Such stiffness-dependent calcium response is associated with the induced selectin related to β2-integrin activation through the Syk/Src signaling pathway, and F-actin/myosin II are also involved in this. Moreover, tension-activated calcium ion channels displayed critical roles in initiating stiffness-dependent calcium spike. These results provide an insight into understanding how the stiffening of vascular walls could regulate the calcium flux of adhered neutrophils, and thus the immune responses in atherosclerosis

E-selectin negatively regulates polymorphonuclear neutrophil transmigration through altered endothelial junction integrity

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM

Most Global Gauging Stations Present Biased Estimations of Total Catchment Discharge

Abstract Stream gauging stations provide critical streamflow measurements for hydrological applications; however, they may not accurately capture total catchment discharge due to unmonitored regional groundwater flow. Here, we evaluate the effectiveness of streamflow data from gauging stations worldwide to represent total catchment discharge through a modified hydrological model that includes baseflow signatures to constrain groundwater flow processes. We find that approximately 70% of gauging stations present biased estimations of total catchment discharge (bias >10%). This result implies that hydrology‐related processes may not be fully understood, and misleading conclusions may be drawn owing to the low streamflow measurement effectiveness. By influencing subsurface hydrological processes, catchment factors, including catchment area, topography, climate, and geological features, are linked to the effectiveness of streamflow measurements. Our findings highlight the importance of accurate streamflow measurement effectiveness for obtaining a reliable understanding of catchment hydrological processes to support sustainable water resource management

2,4,6-Trinitrotoluene Reduction by Carbon Monoxide Dehydrogenase from Clostridium thermoaceticum

Purified CO dehydrogenase (CODH) from Clostridium thermoaceticum catalyzed the transformation of 2,4,6-trinitrotoluene (TNT). The intermediates and reduced products of TNT transformation were separated and appear to be identical to the compounds formed by C. acetobutylicum, namely, 2-hydroxylamino-4,6-dinitrotoluene (2HA46DNT), 4-hydroxylamino-2,6-dinitrotoluene (4HA26DNT), 2,4-dihydroxylamino-6-nitrotoluene (24DHANT), and the Bamberger rearrangement product of 2,4-dihydroxylamino-6-nitrotoluene. In the presence of saturating CO, CODH catalyzed the conversion of TNT to two monohydroxylamino derivatives (2HA46DNT and 4HA26DNT), with 4HA26DNT as the dominant isomer. These derivatives were then converted to 24DHANT, which slowly converted to the Bamberger rearrangement product. Apparent K(m) and k(cat) values of TNT reduction were 165 ± 43 μM for TNT and 400 ± 94 s(−1), respectively. Cyanide, an inhibitor for the CO/CO(2) oxidation/reduction activity of CODH, inhibited the TNT degradation activity of CODH

Most High Mountainous Areas Around the World Present Elevation‐Dependent Aridification After the 1970s

Abstract The research on the trends of mountainous aridity and the issue of elevation‐dependent aridity (EDA) has long been hindered by limitations in data validity. In this study, we present global monthly merged data sets of Vapor Pressure Deficit and Aridity Index (AI) from 1960 to 2020, aiming to detect and attribute elevation‐dependent patterns of atmospheric and soil aridity in six representative high mountainous areas worldwide. Our findings reveal that most of the mountainous regions experienced significant aridification after the 1970s (p < 0.05). The Rocky Mountains and Ethiopian Highlands are identified as the most vulnerable areas, with both atmospheric and soil aridity increasing by 13% across all elevation gradients. Significant humidification has been observed only in High Asian Regions at 4,000 m above sea level with an approximate decrease of 11% in soil AI. In both temporal and spatial patterns, soil aridity exhibits stronger heterogeneity compared to atmospheric aridity, with certain regions and seasons showing humidification, despite the overall aridification trend. Elevation‐dependent aridity is observed in two‐thirds of the mountains, but whether high altitude alleviates or amplifies aridity depends on the pattern of precipitation changes at different elevations. The rise in air temperature is the primary driving factor for soil and atmospheric aridification, contributing to over 50% of each. In two‐thirds of the regions, changes in precipitation exacerbate soil aridity. Simulations show that human activities are closely related to the ongoing prolonged atmospheric aridification. This study contributes to a comprehensive understanding of global mountainous aridity evolution projected under climate change

Divalent Folate Modification on PEG: An Effective Strategy for Improving the Cellular Uptake and Targetability of PEGylated Polyamidoamine–Polyethylenimine Copolymer

The stability and targeting ability of nanocarrier gene delivery systems are necessary conditions to ensure the good therapeutic effect and low nonspecific toxicity of cancer treatment. Poly­(ethylene glycol) (PEG) has been widely applied for improving stability and as a spacer for linking ligands and nanocarriers to improve targetability. However, the cellular uptake and endosomal escape capacity of nanocarriers has been seriously harmed due to the introduction of PEG. In the present study, we synthesized a new gene delivery vector by coupling divalent folate-PEG (PEG_3.4k-FA₂) onto polyamidoamine–polyethylenimine (PME) copolymer (PME–(PEG_3.4k-FA₂)_1.72). Both PEG and monovalent folate-PEG (PEG_3.4k-FA₁) modified PME were prepared as control polymers, which were named as PME–(PEG_3.5k)_1.69 and PME–(PEG_3.4k-FA₁)_1.66, respectively. PME–(PEG_3.4k-FA₂)_1.72 exhibited strong DNA condensation capacity like parent polymer PME which was not significantly influenced by PEG. PME–(PEG_3.4k-FA₂)_1.72/DNA complexes at N/P = 10 had a diameter ∼143 nm and zeta potential ∼13 mV and showed the lowest cytotoxicity and hemolysis and the highest transfection efficiency among all tested polymers. In folate receptor positive (FR-positive) cells, the cellular uptake and transfection efficiency were increased with the increase in the number of folates coupled on PEG; the order was PME–(PEG_3.4k-FA₂)_1.72 > PME–(PEG_3.4k-FA₁)_1.66 > PME–(PEG_3.5k)_1.69. Folate competition assays showed that PME–(PEG_3.4k-FA₂)_1.72 complexes had stronger targeting ability than PME–(PEG_3.5k)_1.69 and PME–(PEG_3.4k-FA₁)_1.66 complexes due to their higher folate density per PEG molecule. Cellular uptake mechanism study showed that the folate density on PEG could change the endocytosis pathway of PME–(PEG_3.5k)_1.69 from clathrin-mediated endocytosis to caveolae-mediated endocytosis, leading to less lysosomal degradation. Distribution and uptake in 3D multicellular spheroid assays showed that divalent folate could offer PME–(PEG_3.4k-FA₂)_1.72 complexes stronger penetrating ability and higher cellular uptake. With these advantages, PME–(PEG_3.4k-FA₂)_1.72 may be a promising nonviral vector candidate for efficient gene delivery. This study also indicates that divalent folate modification on PEG can serve as an efficient strategy to improve the cellular uptake and targeting ability of PEGylated cationic polymers for gene delivery