Enhanced ultrafast X-ray diffraction by transient resonances

Diffraction-before-destruction imaging with single ultrashort X-ray pulses has the potential to visualise non-equilibrium processes, such as chemical reactions, at the nanoscale with sub-femtosecond resolution in the native environment without the need of crystallization. Here, a nanospecimen partially diffracts a single X-ray flash before sample damage occurs. The structural information of the sample can be reconstructed from the coherent X-ray interference image. State-of-art spatial resolution of such snapshots from individual heavy element nanoparticles is limited to a few nanometers. Further improvement of spatial resolution requires higher image brightness which is ultimately limited by bleaching effects of the sample. We compared snapshots from individual 100 nm Xe nanoparticles as a function of the X-ray pulse duration and incoming X-ray intensity in the vicinity of the Xe M-shell resonance. Surprisingly, images recorded with few femtosecond and sub-femtosecond pulses are up to 10 times brighter than the static linear model predicts. Our Monte-Carlo simulation and statistical analysis of the entire data set confirms these findings and attributes the effect to transient resonances. Our simulation suggests that ultrafast form factor changes during the exposure can increase the brightness of X-ray images by several orders of magnitude. Our study guides the way towards imaging with unprecedented combination of spatial and temporal resolution at the nanoscale

Functional Consequences of the Macrophage Stimulating Protein 689C Inflammatory Bowel Disease Risk Allele

<div><p>Background</p><p>Macrophage stimulating protein (MSP) is a serum growth factor that binds to and activates the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON). A non-synonymous coding variant in MSP (689C) has been associated with genetic susceptibility to both Crohn's disease and ulcerative colitis, two major types of inflammatory bowel disease (IBD) characterized by chronic inflammation of the digestive tract. We investigated the consequences of this polymorphism for MSP-RON pathway activity and IBD pathogenesis.</p><p>Methods</p><p>RON expression patterns were examined on mouse and human cells and tissues under normal and disease conditions to identify cell types regulated by MSP-RON. Recombinant MSP variants were tested for their ability to bind and stimulate RON and undergo proteolytic activation. MSP concentrations were quantified in the serum of individuals carrying the MSP 689R and 689C alleles.</p><p>Results</p><p>In intestinal tissue, RON was primarily expressed by epithelial cells under normal and disease conditions. The 689C polymorphism had no impact on the ability of MSP to bind to or signal through RON. In a cohort of normal individuals and IBD patients, carriers of the 689C polymorphism had lower concentrations of MSP in their serum.</p><p>Conclusions</p><p>By reducing the quantities of circulating MSP, the 689C polymorphism, or a variant in linkage disequilibrium with this polymorphism, may impact RON ligand availability and thus receptor activity. Given the known functions of RON in regulating wound healing and our analysis of RON expression patterns in human intestinal tissue, these data suggest that decreased RON activity may impact the efficiency of epithelial repair and thus underlie the increased IBD susceptibility associated with the MSP 689C allele.</p></div

TGFβ2 and TGFβ3 isoforms drive fibrotic disease pathogenesis.

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade

The 689C polymorphism does not affect MSP signaling through RON.

<p>(A) Western blot analysis of total Akt and pAkt in A2780-hRON and BxPC3 cells treated as indicated. Blot was performed in triplicate and mean of the pAkt/total ratios is shown. (B) Quantitation of pAkt by MSD analysis in 3T3-hRON cells treated with scMSP or MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of three independent experiments. (C) Quantification of pAkt by MSD analysis in HCT15 cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (D) Quantitation of pAkt by MSD analysis in human primary colon cells treated with MSP variants. Mean +/− SD of triplicate wells is shown. Data are representative of two independent experiments. (E) Images from scratch wound assay of 3T3-hRON cells treated with scMSP or MSP variants. Images are from the same cell culture at the indicated times after scratch wounding. Dashed lines represent position of initial scratch. (F) Quantitation of scratch wound assay from 3T3-hRON cells treated with medium-alone, scMSP, or MSP variants. Mean +/− SD of three treatments is shown. Data are representative of three independent experiments. ^#not significant, **p≤0.0003 for comparisons between medium and MSP variants.</p

The 689C polymorphism in MSP does not affect binding to RON.

<p>(A) Overview of recombinant human MSP proteins used in this study. MSP α- and β-chains, PAN domain (N), kringle domains (K), serine protease-like domain (SPL) are indicated. scMSP is inactive due to the R483E mutation at the proteolytic cleavage site (arrow). 689R and 689C versions of all MSP proteins were generated. (B) Cell-free binding assay consisting of plate bound RON-Fc and soluble MSP. Means of three replicates per group are shown. Lines represent dose-response curves fit to a 4 parameter equation, which yielded EC₅₀ values of 0.1, 0.2, 0.05 and 0.1 nM for MSP 689R, 689C, MSP β 689R and MSP β 689C, respectively. (C) SPR analysis of MSP proteins binding to immobilized RON Sema/PSI, showing relative response in response units. Data are representative of three independent experiments. (D) Radioligand binding assay of MSP binding to RON. Competition binding to 3T3-hRON cells (large graphs) used to generate affinities and Scatchard plots (inset graphs) are shown. Mean +/− SD of three independent experiments is shown. (E) Homology model of the structure of RON Sema/PSI (PDB code 4FWW) in beige bound to MSP β (PDB code 2ASU) in blue. RON and MSP β were globally aligned to the Met/HGF β complex (PDB code 1SHY). Predicted locations of the MSP β contact residues within 4 Å of RON are highlighted in magenta. MSP β residue arginine 689 was mutated to a cysteine and is shown in yellow.</p

Carriers of the 689C polymorphism have lower quantities of serum MSP.

<p>(A) Analysis of rs3197999 alleles in DNA samples from normal, UC, and CD patients. The C allele encodes MSP 689R and T allele encodes MSP 689C. Allele frequency and number are indicated for each group. (B) MSP concentrations in matched serum samples of EMBARK subjects determined by ELISA, with results grouped by rs3197999 genotype. *p<0.005, **p<0.0005. (C) Serum MSP concentrations grouped by genotype and disease status. *p<0.05, **p<0.005, ***p<0.0005.</p