Detection of glycocalyx degradation in real time: A conceptual model of thromboelastography.

BACKGROUND: The endothelial glycocalyx is a critical component of the vascular barrier; its disruption after shock states may contribute to coagulopathy in a variety of conditions. Measurement of glycocalyx components in plasma have been used to index glycocalyx degradation but are not available as a point of care test. Heparanoids, such as heparan sulfate, may affect coagulation which may be detected by either thromboelastography or activated clotting time. METHODS: Endothelial glycocalyx components syndecan-1 and heparan sulfate were added to blood samples at clinically relevant concentrations. Thromboelastography values included clot reaction time, clot amplification and fibrinogen values, and maximum clot strength (maximum amplitude, platelets). The heparinase thromboelastography cartridge was used to detect a heparin-like effect. The activated clotting time test was performed subsequently using the heparan sulfate blood samples to compare a standard coagulation test with thromboelastography clot reaction times. RESULTS: Both thromboelastography clot reaction time (with comparison to heparinase) and activated clotting time were useful to detect effects of coagulation. Thromboelastography also detected platelet and fibrinogen abnormalities at higher heparan sulfate concentrations. Studies using thromboelastography or even activated clotting time may be useful to detect glycocalyx degradation after shock states and may guide clinical decision making. CONCLUSION: Thromboelastography and or activated clotting time may be useful to detect glycocalyx degradation as a point of care test in patients in the acute setting. Additionally, these assays may detect previous undisclosed coagulopathy due to glycocalyx degradation.

Preservation of organs related to future sexual function during prostate stereotactic body radiotherapy: feasibility analysis through a re-optimization process.

Preserving the quality of life and sexual function of patients with a localized prostate cancer remains a challenge for physicians and a major issue for patients. The present study aimed at demonstrating the feasibility of a dosimetric preservation of the sexual organs during prostate stereotactic radiotherapy planning. Patients from a single centre were retrospectively included in the RPAH-2 trial and randomized in Arm B if they presented with either a low- or intermediate- risk prostate cancer. A 37.5Gy in 5 fractions stereotactic body radiotherapy was delivered on the prostate gland. The corpus cavernosum, penile bulb and internal pudental arteries were retrospectively delineated before a re-optimization process. During this process, RPAH-2 trial dose constraints were respected on Gross Tumor Volume (GTV), Planning Target Volume and usual organs at risk. Pre-defined dose setting delivered to corpus cavernosum, penile bulb and internal pudental arteries were collected and compared before and after the re-optimization process. Nine patients were included in the study. A decrease of the median of each investigated dose setting (except D90% for corpus cavernosum) was reported after the re-optimization for corpus cavernosum, penile bulb and internal pudental arteries. Our study demonstrated the feasibility of a dosimetric preservation of structures considered as relevant to preserve sexual function after prostate stereotactic radiotherapy.

Homo-dimerization and ligand binding by the leucine-rich repeat domain at RHG1/RFS2 underlying resistance to two soybean pathogens.

BACKGROUND: The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) was a receptor like kinase (RLK) encoded within the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). Previously the leucine rich repeat (LRR) domain was expressed in Escherichia coli. RESULTS: The aims here were to evaluate the LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. Western analysis suggested homo-dimers could form after protein extraction from roots. The purified LRR domain, from residue 131-485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N region was close (<11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20-142 nM for peptides found in plant and nematode secretions were found. Effects on plant phenotypes including wilting, stem bending and resistance to infection by SCN were observed when roots were treated with 50 pM of the peptides. Far-Western analyses followed by MS showed methionine synthase and cyclophilin bound strongly to the LRR domain. A second LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions. CONCLUSIONS: The LRR domain of the GmRLK18-1 protein formed both a monomer and a homo-dimer. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase. The CLE peptides GmTGIF, GmCLE34, GmCLE3 and HgCLE were previously reported to be involved in root growth inhibition but here GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN. One of several models from homology and ab-initio modeling was partially validated by cross-linking. The effect of the 3 amino acid replacements present among RLK allotypes, A87V, Q115K and H274N were predicted to alter domain stability and function. Therefore, the LRR domain of GmRLK18-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN.

The receptor like kinase at Rhg1-a/Rfs2 caused pleiotropic resistance to sudden death syndrome and soybean cyst nematode as a transgene by altering signaling responses.

BACKGROUND: Soybean (Glycine max (L. Merr.)) resistance to any population of Heterodera glycines (I.), or Fusarium virguliforme (Akoi, O'Donnell, Homma & Lattanzi) required a functional allele at Rhg1/Rfs2. H. glycines, the soybean cyst nematode (SCN) was an ancient, endemic, pest of soybean whereas F. virguliforme causal agent of sudden death syndrome (SDS), was a recent, regional, pest. This study examined the role of a receptor like kinase (RLK) GmRLK18-1 (gene model Glyma_18_02680 at 1,071 kbp on chromosome 18 of the genome sequence) within the Rhg1/Rfs2 locus in causing resistance to SCN and SDS. RESULTS: A BAC (B73p06) encompassing the Rhg1/Rfs2 locus was sequenced from a resistant cultivar and compared to the sequences of two susceptible cultivars from which 800 SNPs were found. Sequence alignments inferred that the resistance allele was an introgressed region of about 59 kbp at the center of which the GmRLK18-1 was the most polymorphic gene and encoded protein. Analyses were made of plants that were either heterozygous at, or transgenic (and so hemizygous at a new location) with, the resistance allele of GmRLK18-1. Those plants infested with either H. glycines or F. virguliforme showed that the allele for resistance was dominant. In the absence of Rhg4 the GmRLK18-1 was sufficient to confer nearly complete resistance to both root and leaf symptoms of SDS caused by F. virguliforme and provided partial resistance to three different populations of nematodes (mature female cysts were reduced by 30-50%). In the presence of Rhg4 the plants with the transgene were nearly classed as fully resistant to SCN (females reduced to 11% of the susceptible control) as well as SDS. A reduction in the rate of early seedling root development was also shown to be caused by the resistance allele of the GmRLK18-1. Field trials of transgenic plants showed an increase in foliar susceptibility to insect herbivory. CONCLUSIONS: The inference that soybean has adapted part of an existing pathogen recognition and defense cascade (H.glycines; SCN and insect herbivory) to a new pathogen (F. virguliforme; SDS) has broad implications for crop improvement. Stable resistance to many pathogens might be achieved by manipulation the genes encoding a small number of pathogen recognition proteins.

Recombination suppression at the dominant Rhg1/Rfs2 locus underlying soybean resistance to the cyst nematode.

Host resistance to "yellow dwarf" or "moonlight" disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24-48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred.

Conformational changes associated with template commitment in ATP-dependent chromatin remodeling by ISW2.

Distinct stages in ATP-dependent chromatin remodeling are found as ISW2, an ISWI-type complex, forms a stable and processive complex with nucleosomes upon hydrolysis of ATP. There are two conformational changes of the ISW2-nucleosome complex associated with binding and hydrolysis of ATP. The initial binding of ISW2 to extranucleosomal DNA, to the entry site, and near the dyad axis of the nucleosome is enhanced by ATP binding, whereas subsequent ATP hydrolysis is required for template commitment and causes ISW2 to expand its interactions with nucleosomal DNA to an entire gyre of the nucleosome and a short approximately 3-4 bp site on the other gyre. The histone-fold-like subunit Dpb4 associates with nucleosomal DNA approximately 15 bp from the ATPase domain as part of this change and may help to disrupt histone-DNA interactions. These additional contacts are independent of the ATPase domain tracking along nucleosomal DNA and are maintained as ISW2 moves nucleosomes on DNA.