ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings

Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway of E. coli α2 Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit β of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in β to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-β, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit β(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. 2H electron–nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed

The thermal stability of topologically close-packed phases in the single-crystal Ni-base superalloy ERBO/1

In Ni-base superalloys, the addition of refractory elements such as Cr, Mo, Co, W, and Re is necessary to increase the creep resistance. Nevertheless, these elements induce the formation of different kinds of intermetallic phases, namely, the topologically close-packed (TCP) phases. This work focuses on intermetallic phases present in the second-generation single-crystal (SX) Ni-base superalloy ERBO/1. In the as-cast condition, the typical γ/γ′ structure is accompanied by undesirable intermetallic phases located in the interdendritic regions. The nature of these precipitates as well as their thermal stability between 800 and 1200 °C has been investigated by isothermal heat treatments. The investigation techniques include DSC, SEM, EDX, and TEM. The experimental information is complemented by (1) comparison with a structure map to link the local chemical composition with phase stability, as well as (2) thermodynamic calculations based on the CALPHAD method to determine the occurrence and composition of phases during solidification and in equilibrium conditions. The TCP phases Laves, µ and σ were identified in various temperature/time ranges. © 2015, Springer Science+Business Media New York

CALCULATING LEAF BOUNDARY LAYER PARAMETERS WITH THE TWO-DIMENSIONAL MODEL 2DLEAF COMPARING TRANSPIRATION RATES OF NORMAL (CV. DESIREE) AND TRANSGENIC (SUCROSE TRANSPORT ANTISENSE) POTATO PLANTS

The leaf boundary layer, i. e. the layer of air adjacent to a leaf surface in which gas flow is significantly influenced by the leaf, considerably affects leaf gas exchange. Numerous factors, both external conditions and leaf properties, have a strong influence on boundary layer characteristics and the challenge to develop a reliable model of this link in the leaf gas exchange pathway has persisted for decades. Two parameters, the boundary layer thickness, d, and the ratio, B, of the diffusion coefficients of gases in the boundary layer and in the intercellular space, were shown to be sufficient to represent the effect of the boundary layer in a two-dimensional leaf gas exchange model 2DLEAF. An algorithm for calculation of these parameters is described and applied to simulate the transpiration rate of leaves in normal (cv. Desiree) and transgenic (expressing a mRNA antisense construct targeted to the cp-fructose-6-bisphosphate phosphatase) potato plants (Solanum tuberosum). For these leaves, both gas exchange and leaf anatomy have been studied. Parameters d and B were different for normal and transgenic leaves, and they expressed real differences in anatomy and surface properties

The role of local chemical composition for TCP phase precipitation in Ni-base and Co-base superalloys

The precipitation of topologically close-packed (TCP) phases in single-crystal superalloys is highly undesirable due to the detrimental effect on the mechanical properties. The TCP phases bind atoms responsible for the solid solution strengthening of the γ′ phase (Re, W, Mo), as well as elements that are important for the formation of the γ phase (Ti, Ta). A thorough understanding of TCP phase precipitation is therefore a prerequisite for the design of future superalloys. The thermodynamic stability of TCP phases as bulk material is meanwhile well understood. However, little is known about the factors that govern the stability of the experimentally observed precipitates of TCP phases within the superalloy matrix. The focus of this paper is the role of the local chemical composition for the stability of TCP phase precipitates. We combine experimental measurements of the local chemical composition of TCP phase precipitates in the Ni-base superalloy ERBO-1 and the Co-base superalloy ERBO-Co0 with a theoryguided interpretation of the structural stability of bulk TCP phases. The experimental characterization of the microstructure and the crystal structure of the precipitates are based on scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The local chemical composition is determined by energydispersive X-ray spectroscopy (EDX) and electron probe microanalysis (EPMA). The measured local chemical compositions are assessed regarding the likelihood of TCP phase formation by determining their location in a structure map of the stability of bulk TCP phases. This establishes a direct link between the measured local chemical composition of TCP phase precipitates and the phase stability of bulk TCP phases at this composition. By converting the measured local chemical composition into structure-map coordinates, we effectively construct a compound map that indicates the regions in the microstructure that are prone to TCP phase precipitation. Analyzing the intermetallic precipitates in more detail, we find that the chemical compositions of the TCP phase precipitates would be expected to form the same TCP phase as bulk material. This suggests that the observed precipitates of TCP phases can be regarded to be in a local thermodynamic equilibrium. The challenge of predicting TCP phase precipitation in superalloys is hence reduced to the prediction of the local chemical composition during casting, heat treatment and service

Single-cell transcriptomics reveal different maturation stages and sublineage commitment of human thymic invariant natural killer T cells

Invariant natural killer T cells are a rare, heterogeneous T-cell subset with cytotoxic and immunomodulatory properties. During thymic development, murine invariant natural killer T cells go through different maturation stages differentiating into distinct sublineages, namely, invariant natural killer T1, 2, and 17 cells. Recent reports indicate that invariant natural killer T2 cells display immature properties and give rise to other subsets, whereas invariant natural killer T1 cells seem to be terminally differentiated. Whether human invariant natural killer T cells follow a similar differentiation model is still unknown. To define the maturation stages and assess the sublineage commitment of human invariant natural killer T cells during thymic development, in this study, we performed single-cell RNA sequencing analysis on human Vα24+Vβ11+ invariant natural killer T cells isolated from thymocytes. We show that these invariant natural killer T cells displayed heterogeneity, and our unsupervised analysis identified 5 clusters representing different maturation stages, from an immature profile with high expression of genes important for invariant natural killer T cell development and proliferation to a mature, fully differentiated profile with high levels of cytotoxic effector molecules. Evaluation of expression of sublineage-defining gene sets revealed mainly cells with an invariant natural killer T2 signature in the most immature cluster, whereas the more differentiated ones displayed an invariant natural killer T1 signature. Combined analysis with a publicly available single-cell RNA sequencing data set of human invariant natural killer T cells from peripheral blood suggested that the 2 main subsets exist both in thymus and in the periphery, while a third more immature one was restricted to the thymus. Our data point to the existence of different maturation stages of human thymic invariant natural killer T cells and provide evidence for sublineage commitment of invariant natural killer T cells in the human thymus