An alternative to radiation hybrid mapping for large-scale genome analysis in barley

The presence of a monosomic gametocidal chromosome (GC) in a barley chromosome addition line of common wheat generates structural aberrations in the barley chromosome as well as in the wheat chromosomes of gametes lacking the GC. A collection of structurally aberrant barley chromosomes is analogous to a panel of radiation hybrid (RH) mapping and is valuable for high-throughput physical mapping. We developed 90 common wheat lines (GC lines) containing aberrant barley 7H chromosomes induced by a gametocidal chromosome, 2C. DNAs isolated from these GC lines provided a panel of 7H chromosomal fragments in a wheat genetic background, comparable with RH mapping panels in mammals. We used this 7H GC panel and the methodology for RH mapping to physically map PCR-based barley markers, SSRs and AFLPs, onto chromosome 7H, relying on polymorphism between the 7H chromosome and the wheat genome. We call this method GC mapping. This study describes a novel adaptation and combination of methods of inducing chromosomal rearrangements to produce physical maps of markers. The advantages of the presented method are similar to RH mapping in that non-polymorphic markers can be used and the mapping panels can be relatively easily obtained. In addition, mapping results are cumulative when using the same mapping set with new markers. The GC lines will be available from the National Bioresources Project-KOMUGI (). Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Landscape of nuclear transport receptor cargo specificity

Nuclear transport receptors (NTRs) recognize localization signals of cargos to facilitate their passage across the central channel of nuclear pore complexes (NPCs). About 30 different NTRs constitute different transport pathways in humans and bind to a multitude of different cargos. The exact cargo spectrum of the majority of NTRs, their specificity and even the extent to which active nucleocytoplasmic transport contributes to protein localization remains understudied because of the transient nature of these interactions and the wide dynamic range of cargo concentrations. To systematically map cargo–NTR relationships in situ, we used proximity ligation coupled to mass spectrometry (BioID). We systematically fused the engineered biotin ligase BirA* to 16 NTRs. We estimate that a considerable fraction of the human proteome is subject to active nuclear transport. We quantified the specificity and redundancy in NTR interactions and identified transport pathways for cargos. We extended the BioID method by the direct identification of biotinylation sites. This approach enabled us to identify interaction interfaces and to discriminate direct versus piggyback transport mechanisms. Data are available via ProteomeXchange with identifier PXD007976.     

Search for Cellular Stress Biomarkers in Lymphocytes from Patients with Multiple Sclerosis: A Pilot Study

Multiple Sclerosis (MS) is a chronic disease of the central nervous system, the etiology of which, although not completely known, involves inflammation and autoimmunity. In the present study we aimed at identifying molecular markers of apoptosis, cellular stress and DNA damage in isolated peripheral blood mononuclear cells (PBMCs) of MS patients. The analysis was carried on 19 relapsing-remitting untreated MS patients and 13 healthy individuals. We investigated the emergency-driven synthesis of poly(ADP-ribose) (PAR), the expression level of the constitutive enzyme poly(ADP-ribose) polymerase-1 (PARP-1) and the DNA damage-induced phosphorylation of histone H2AX. PAR accumulation, PARP-1 and phosphorylated H2AX (γH2AX) were detected by immunofluorescence experiments on PBMCs isolated from 19 patients and 13 healthy volunteers. Our results show for the first time a net increased amount in PAR and γH2AX in MS patients compared to healthy individuals. Patients were further subdivided in three groups, according to the neuroimaging (MRI)-based classification of disease phase. Remarkably, we found a positive correlation between the level of γH2AX and MS aggressiveness. In addition, apoptosis in PBMCs was monitored by flow cytometry of both phosphatidylserine exposure (revealed by Annexin V-FITC labeling) and membrane permeability to propidium iodide. Our observations provide the evidence that the number of apoptotic cells was significantly higher in patients compared to healthy individuals, thus suggesting that apoptosis could affect MS lymphocyte function.     

Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium

Fibroblasts are a diverse cell type and display clear topographic differentiation and positional memory. In a screen for fibroblast specific markers we have characterized four monoclonal antibodies to endosialin (TEM1/CD248). Previous studies have reported that endosialin is a tumour endothelium marker and is localized intracellularly. We demonstrate conclusively that endosialin is a cell surface glycoprotein and is predominantly expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium. These novel antibodies will facilitate the isolation and classification of fibroblast and pericyte lineages as well as the further functional analysis of endosialin.     

TAS2R38 and Its Influence on Smoking Behavior and Glucose Homeostasis in the German Sorbs

Background

Genetic variants within the bitter taste receptor gene TAS2R38 are associated with sensitivity to bitter taste and are related to eating behavior in the Amish population. Sensitivity to bitter taste is further related to anthropometric traits in an genetically isolated Italian population. We tested whether the TAS2R38 variants (rs713598; rs1726866 and rs10246939) may be related to eating behavior, anthropometric parameters, metabolic traits and consumer goods intake in the German Sorbs.

Materials and Methods

The three SNPs were genotyped in a total cohort of 1007 individuals (male/female: 405/602). The German version of the three-factor eating questionnaire was completed by 548 individuals. Genetic association analyses for smoking behavior, alcohol and coffee intake, eating behavior factors (restraint, disinhibition and hunger) and other metabolic traits were analyzed. Further, by combining the three SNPs we applied comparative haplotype analyses categorizing PAV (proline-alanine-valine) carriers (tasters) vs. homozygous AVI (alanin-valine-isoleucine) carriers (non-tasters).

Results

Significant associations of genetic variants within TAS2R38 were identified with percentage of body fat, which were driven by associations in women. In men, we observed significant associations with 30 min plasma glucose, and area under the curve for plasma glucose (0–120 min) (all adjusted P≤0.05). Further, we found that carriers of at least one PAV allele show significantly lower cigarette smoking per day (P = 0.002) as well as, albeit non-significant, lower alcohol intake. We did not confirm previously reported associations between genetic variants of TAS2R38 and eating behavior.

Conclusion

Our data suggest that genetic variation in TAS2R38 is related to individual body composition measures and may further influence consumer goods intake in the Sorbs possibly via individual sensitivity to bitter taste.