A Flexible Multidomain Structure Drives the Function of the Urokinase-type Plasminogen Activator Receptor (uPAR)

The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.     

AB-QTL analysis reveals new alleles associated to proline accumulation and leaf wilting under drought stress conditions in barley (Hordeum vulgareL.)

Background

Phytophthora infestans (Mont.) de Bary, the causal organism of late blight, is economically the most important pathogen of potato and resistance against it has been one of the primary goals of potato breeding. Some potentially durable, broad-spectrum resistance genes against this disease have been described recently. However, to obtain durable resistance in potato cultivars more genes are needed to be identified to realize strategies such as gene pyramiding or use of genotype mixtures based on diverse genes.

Results

A major resistance gene, Rpi-rzc1, against P. infestans originating from Solanum ruiz-ceballosii was mapped to potato chromosome X using Diversity Array Technology (DArT) and sequence-specific PCR markers. The gene provided high level of resistance in both detached leaflet and tuber slice tests. It was linked, at a distance of 3.4 cM, to violet flower colour most likely controlled by the previously described F locus. The marker-trait association with the closest marker, violet flower colour, explained 87.1% and 85.7% of variance, respectively, for mean detached leaflet and tuber slice resistance. A genetic linkage map that consisted of 1,603 DArT markers and 48 reference sequence-specific PCR markers of known chromosomal localization with a total map length of 1204.8 cM was constructed.

Conclusions

The Rpi-rzc1 gene described here can be used for breeding potatoes resistant to P. infestans and the breeding process can be expedited using the molecular markers and the phenotypic marker, violet flower colour, identified in this study. Knowledge of the chromosomal localization of Rpi-rzc1 can be useful for design of gene pyramids. The genetic linkage map constructed in this study contained 1,149 newly mapped DArT markers and will be a valuable resource for future mapping projects using this technology in the Solanum genus.     

Assessment of association between BRAF-V600E mutation status in melanomas and clinical response to ipilimumab

Ipilimumab, a fully human monoclonal antibody against cytotoxic T lymphocyte antigen-4, has demonstrated significant improvement in overall survival in previously treated advanced melanoma patients. The BRAF inhibitor, vemurafenib, has shown up to 78% objective response rates in melanoma patients harboring the BRAF-V600E mutation but not in patients lacking the mutation. As an immune potentiator, the mechanism of action of ipilimumab may not be dependent of the activity of the BRAF pathway. To test this, we investigated whether the clinical activity of ipilimumab would be affected by the BRAF-V600E mutation status of the tumors. Thus, this retrospective analysis was carried using a set of tumor biopsies from a completed phase II clinical trial. CA184004 was a randomized, double-blind, multicenter trial of 82 previously treated or untreated patients with unresectable stage III/IV melanoma. Patients received ipilimumab 3 or 10 mg/kg every 3 weeks for four doses followed by maintenance dosing in eligible patients. The BRAF-V600E mutation status for 80 patients was determined in tumor biopsies by PCR-based assays. Data on disease control were available for 69 patients with evaluated BRAF-V600E mutation status. Rates of objective responses and stable disease in patients with BRAF-V600E mutation positive tumors (30%) were comparable to those in patients with the wild-type gene (~33%). Eleven patients displayed Durable Disease Control (DDC) of which 55% had BRAF-V600E mutation positive tumors and 45% did not. In the 48 patients showing no DDC, the mutation frequency was 50%. In this study, no association between BRAF-V600E mutation status of melanoma tumors and DDC after treatment with ipilimumab was detected.     

Landscape genomics and a common garden trial reveal adaptive differentiation to temperature across Europe in the tree species Alnus glutinosa

The adaptive potential of tree species to cope with climate change has important ecological and economic implications. Many temperate tree species experience a wide range of environmental conditions, suggesting high adaptability to new environmental conditions. We investigated adaptation to regional climate in the drought‐sensitive tree species Alnus glutinosa (Black alder), using a complementary approach that integrates genomic, phenotypic and landscape data. A total of 24 European populations were studied in a common garden and through landscape genomic approaches. Genotyping‐by‐sequencing was used to identify SNPs across the genome, resulting in 1990 SNPs. Although a relatively low percentage of putative adaptive SNPs was detected (2.86% outlier SNPs), we observed clear associations among outlier allele frequencies, temperature and plant traits. In line with the typical drought avoiding nature of A. glutinosa, leaf size varied according to a temperature gradient and significant associations with multiple outlier loci were observed, corroborating the ecological relevance of the observed outlier SNPs. Moreover, the lack of isolation by distance, the very low genetic differentiation among populations and the high intrapopulation genetic variation all support the notion that high gene exchange combined with strong environmental selection promotes adaptation to environmental cues.     

Predictive Mapping of Plant Species and Communities Using GIS and Landsat Data in a Southern Mongolian Mountain Range

We assessed presence/absence prediction of plant species and communities in a southern Mongolian mountain range from geospatial data using a randomized sampling approach. One hundred randomized vegetation samples (3?×?3 m) were collected within the 2?×?2 km summit region of the Dund Saykhan range, which forms part of the core zone of the Gobi Gurvan Saykhan National Park in arid southern Mongolia. Using logistic regression, habitat preference models for all abundant species (n?=?52) and communities (n?=?5) were constructed; predictors were derived from Landsat 5 imagery and a digital elevation model. Nagelkerkes r ² was used for an initial data mining, and all significant models were validated by splitting the data and using one half for accuracy assessment based on the AUC (Area Under the receiver operating characteristic Curve)-values. Significant models could be built for half of the species. Altitude proved to be the most important predictor followed by variables derived from Landsat data. The clear altitudinal distribution patterns most definitely reflect precipitation; overall biodiversity in this arid environment is widely controlled by moisture availability. The chosen approach may prove valuable for applied studies wherever spatial data on species distributions are required for conservation efforts.     

Non-cultured adipose-derived CD45- side population cells are enriched for progenitors that give rise to myofibres in vivo

Side population (SP) cells are highly able to exclude the Hoechst 33342 dye through membrane transporters, a feature associated with cell immaturity and therefore proposed as a marker of stem cells. Herein we demonstrate that the adipose tissue derived stromal vascular fraction (SVF) contains a novel population of non-haematopoietic “side population” (SPCD45⁻) cells. Simultaneous qRT-PCR of 64 genes revealed that the freshly isolated SPCD45⁻ was highly enriched for cells expressing genes related to stem cells, the Notch pathway, and early vascular precursors. Notably, the expression of smooth muscle actin, C-met and Cd34 together with Angpt2, Flk1, VE-cadherin, and Cd31 suggested a phenotypic resemblance to pericytes and aorta-derived mesoangioblasts. Recent evidence suggests that cells residing within the vascular niche may participate in regeneration of skeletal muscle and although skeletal muscle repair mainly relies on the satellitecell, several reports have shown that vessel-associated cells may adopt a myogenic phenotype when exposed to a muscle environment. In accordance with these findings, we also observed invitro myogenic specification of SPCD45⁻ cells when cocultured with myoblasts. Furthermore, immediate intramuscular engraftment of non-cultured SPCD45⁻ cells gave rise to myofibres andcells lining blood vessels, whereas the SVF only provided donor derived mononuclear cells. We therefore conclude that the SPCD45⁻ fraction of adipose-derived SVF is enriched for cells expressing vascular associated markers and that the myogenic differentiation potential of these cells does not depend on prior in vitro expansion.     

RNA helicase-mediated regulation of snoRNP dynamics on pre-ribosomes and rRNA 2′-O-methylation

RNA helicases play important roles in diverse aspects of RNA metabolism through their functions in remodelling ribonucleoprotein complexes (RNPs), such as pre-ribosomes. Here, we show that the DEAD box helicase Dbp3 is required for efficient processing of the U18 and U24 intron-encoded snoRNAs and 2′-O-methylation of various sites within the 25S ribosomal RNA (rRNA) sequence. Furthermore, numerous box C/D snoRNPs accumulate on pre-ribosomes in the absence of Dbp3. Many snoRNAs guiding Dbp3-dependent rRNA modifications have overlapping pre-rRNA basepairing sites and therefore form mutually exclusive interactions with pre-ribosomes. Analysis of the distribution of these snoRNAs between pre-ribosome-associated and ‘free’ pools demonstrated that many are almost exclusively associated with pre-ribosomal complexes. Our data suggest that retention of such snoRNPs on pre-ribosomes when Dbp3 is lacking may impede rRNA 2′-O-methylation by reducing the recycling efficiency of snoRNPs and by inhibiting snoRNP access to proximal target sites. The observation of substoichiometric rRNA modification at adjacent sites suggests that the snoRNPs guiding such modifications likely interact stochastically rather than hierarchically with their pre-rRNA target sites. Together, our data provide new insights into the dynamics of snoRNPs on pre-ribosomal complexes and the remodelling events occurring during the early stages of ribosome assembly.     

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.