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.

A panel of qPCR assays to detect and quantify soybean soil-borne pathogens.

Fusarium oxysporum,F. graminearum,F. acuminatum,F. equiseti,F. proliferatum,F. solani, and Rhizoctonia solani are soil-borne fungal pathogens that cause substantial yield loss in a widespread list of crops worldwide. The objective of this study was to develop a panel of TaqMan assays for the detection and quantification of these six widespread soil-borne fungal species using real-time polymerase chain reaction (qPCR). The primers and probes were designed based on the intergenic spacer ribosomal RNA and translation elongation factor 1-alpha gene (tef1). These assays, although not multiplexed, can be performed simultaneously as they have similar reaction conditions, allowing more efficiency when targeting multiple pathogens in a sample. The assays presented high efficiency (94.3%-108.9%) and sensitivity, with a limit of detection of 0.05 picograms (50 femtograms) of target DNA. Results from an assay targeting 19 non-target and closely related species confirmed the specificity of the developed assays. The assays were also evaluated to detect the target species in different matrices, such as soil and plant material. This panel of qPCR assays is an additional tool that can be used by plant pathologists, microbiologists, plant breeders, diagnostic clinics, and other researchers interested in these fungal species.

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.

Ecology and diversity of culturable fungal species associated with soybean seedling diseases in the Midwestern United States.

AIMS: To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. METHODS AND RESULTS: Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. CONCLUSION: Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.

Microbial Communities Associated With Long-Term Tillage and Fertility Treatments in a Corn-Soybean Cropping System.

Tillage and fertilization are common practices used to enhance soil fertility and increase yield. Changes in soil edaphic properties associated with different tillage and fertility regimes have been widely examined, yet, the microbially mediated pathways and ecological niches involved in enhancing soil fertility are poorly understood. The effects of long-term conventional tillage and no-till in parallel with three fertility treatments (No fertilization, N-only, and NPK) on soil microbial communities were investigated in a long-term field study that was established in the 1970's. Here, we used high-throughput sequencing of bacterial, fungal and oomycetes markers, followed by community-level functional and ecological assembly to discern principles governing tillage and fertility practices' influence on associated soil microbiomes. Both tillage and fertilizer significantly altered microbial community structure, but the tillage effect was more prominent than the fertilizer effect. Tillage significantly affected bacteria, fungi, fusaria, and oomycete beta-diversity, whereas fertilizer only affected bacteria and fungi beta-diversity. In our study different tillage and fertilizer regimes favored specific networks of metabolic pathways and distinct ecological guilds. No-till selected for beneficial microbes that translocate nutrients and resources and protect the host against pathogens. Notably, ecological guilds featuring arbuscular mycorrhizae, mycoparasites, and nematophagous fungi were favored in no-till soils, while fungal saprotrophs and plant pathogens dominated in tilled soils. Conventional till and fertilizer management shifted the communities toward fast growing competitors. Copiotrophic bacteria and fusarium species were favored under conventional tillage and in the presence of fertilizers. The analysis of the metagenomes revealed a higher abundance of predicted pathways associated with energy metabolism, translation, metabolism of cofactors and vitamins, glycan biosynthesis and nucleotide metabolism in no-till. Furthermore, no specific pathways were found to be enriched under the investigated fertilization regimes. Understanding how tillage and fertilizer management shift microbial diversity, structure and ecological niches, such as presented here, can assist with designing farming systems that can maintain high crop yield, while reducing soil erosion and nutrient losses.

Trichoderma Isolates Inhibit Fusarium virguliforme Growth, Reduce Root Rot, and Induce Defense-Related Genes on Soybean Seedlings.

Sudden death syndrome (SDS) caused by Fusarium virguliforme is among the most important diseases affecting soybean in the United States. The use of biological control agents (BCAs) such as Trichoderma spp. can be a valuable resource to suppress F. virguliforme populations. Therefore, this research focused on screening possible BCAs against F. virguliforme and evaluating mycoparasitism and the induction of systemic resistance as mechanisms underlying the antagonistic activity of selected BCAs against F. virguliforme. In total, 47 potential BCAs, including 41 Trichoderma isolates and 6 Mortierella isolates, were screened in a dual-plate assay. The most effective isolates belonged to the Trichoderma harzianum species and were able to inhibit F. virguliforme radial growth by up to 92%. Selected Trichoderma isolates were tested in the greenhouse and in a microplot study. They reduced root rot caused by F. virguliforme when the plants were coinoculated with the pathogen and the BCA. The tested BCA's ability to reduce F. virguliforme growth may be related to several mechanisms of action, including mycoparasitism and induction of defense-related genes in plants, as revealed by monitoring the expression of defense-related genes in soybean. Our results highlight the potential of native Trichoderma isolates to inhibit F. virguliforme growth and reduce SDS severity, providing the basis for future implementation of biological control in soybean production. More efforts are needed to implement the use of these approaches in production fields, and to deepen the current knowledge on the biology of these highly antagonistic isolates.

Targeting Aflatoxin Biosynthetic Genes.

Chemical detoxification and physical destruction of aflatoxins in foods and feed commodities are mostly unattainable in a way that preserves the edibility of the food. Therefore, preventing mycotoxins in general and aflatoxins in particular from entering the food chain is a better approach. This requires early detection of the aflatoxin-causing organisms. Detection and quantification of aflatoxin-producing fungi has always been a challenge, especially within species of Aspergillus and Penicillium. Culture-based methods require a high level of expertise and a list of sophisticated equipment. Furthermore, even for a trained taxonomist, species that are identical in morphology, physiology, and nutritional aspects can be challenging to classify. Fungal taxonomy has changed over the past few decades; more species are being reclassified, and new species are being described due to advances in sequencing and genome assembly. These developments make the use of PCR-based approaches practical, rapid, and more reliable for the identification of fungi to the species level. This chapter presents a variety of protocols to detect and quantify aflatoxin-producing fungi using mycotoxin biosynthesis pathway genes.

Multilaboratory Comparison of Quantitative PCR Assays for Detection and Quantification of Fusarium virguliforme from Soybean Roots and Soil.

The ability to accurately detect and quantify Fusarium virguliforme, the cause of sudden death syndrome (SDS) in soybean, in samples such as plant root tissue and soil is extremely valuable for accurate disease diagnoses and to address research questions. Numerous quantitative real-time polymerase chain reaction (qPCR) assays have been developed for this pathogen but their sensitivity and specificity for F. virguliforme have not been compared. In this study, six qPCR assays were compared in five independent laboratories using the same set of DNA samples from fungi, plants, and soil. Multicopy gene-based assays targeting the ribosomal DNA intergenic spacer (IGS) or the mitochondrial small subunit (mtSSU) showed relatively high sensitivity (limit of detection [LOD] = 0.05 to 5 pg) compared with a single-copy gene (FvTox1)-based assay (LOD = 5 to 50 pg). Specificity varied greatly among assays, with the FvTox1 assay ranking the highest (100%) and two IGS assays being slightly less specific (95 to 96%). Another IGS assay targeting four SDS-causing fusaria showed lower specificity (70%), while the two mtSSU assays were lowest (41 and 47%). An IGS-based assay showed consistently highest sensitivity (LOD = 0.05 pg) and specificity and inclusivity above 94% and, thus, is suggested as the most useful qPCR assay for F. virguliforme diagnosis and quantification. However, specificity was also above 94% in two other assays and their selection for diagnostics and research will depend on objectives, samples, and materials used. These results will facilitate both fundamental and disease management research pertinent to SDS.

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.

Association between serotonin transporter polymorphisms and problem behavior in adult males with intellectual disabilities.

Research on aggression over the past two decades has focused on gene-environment interaction models to explain the relative contribution of each to this behavioral phenotype in various clinical populations. Recent investigations suggest a link between aggression in people with intellectual disabilities the functionality of the serotonin transporter. The aims in this study were to examine the possible association of the STin2 and/or the 5-HTTLPR serotonin transporter polymorphisms in adult males with and without intellectual disabilities, and to examine the association of these polymorphisms with aggression in people with intellectual disabilities. DNA samples and behavioral records were obtained from adult males with intellectual disabilities, distinguished only by the presence or absence of aggression. No association was found between either transporter polymorphism for aggression. However, the long 5-HTTLPR allele, and not the short allele or the heterozygous state, was associated with the severity of aggression. The association with aggression appears to be genetically complex, suggesting there may be other genes, interactions between genes, and/or environmental relations occasioning aggression in people with intellectual disabilities.

Monoamine oxidase a promoter gene associated with problem behavior in adults with intellectual/developmental disabilities.

A functional polymorphism in the promoter of the gene encoding monoamine oxidase A has been associated with problem behavior in various populations. We examined the association of MAOA alleles in adult males with intellectual/developmental disabilities with and without established histories of problem behavior. These data were compared with a gender, ethnicity, and age-matched contrast sample. About 43% (15/35) of adults with intellectual/developmental disabilities and problem behavior possessed the low-efficiency version of the MAOA gene. In comparison, 20% (7/35) of adults with intellectual/developmental disabilities and no problem behavior and 20% (7/35) of the contrast group had the short-allele MAOA polymorphism. Therefore, a common variant in the MAOA gene may be associated with problem behavior in adults with intellectual/developmental disabilities.

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.