GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae)

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm‐isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)‐like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria‐colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high‐affinity sucrose‐specific proton‐dependent importer. Plasma‐membrane/tonoplast localization of GeSUT4‐GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant‐microbe interactions.     

Rare Variants in PLXNA4 and Parkinson’s Disease

Approximately 20% of individuals with Parkinson’s disease (PD) report a positive family history. Yet, a large portion of causal and disease-modifying variants is still unknown. We used exome sequencing in two affected individuals from a family with late-onset familial PD followed by frequency assessment in 975 PD cases and 1014 ethnically-matched controls and linkage analysis to identify potentially causal variants. Based on the predicted penetrance and the frequencies, a variant in PLXNA4 proved to be the best candidate and PLXNA4 was screened for additional variants in 862 PD cases and 940 controls, revealing an excess of rare non-synonymous coding variants in PLXNA4 in individuals with PD. Although we cannot conclude that the variant in PLXNA4 is indeed the causative variant, these findings are interesting in the light of a surfacing role of axonal guidance mechanisms in neurodegenerative disorders but, at the same time, highlight the difficulties encountered in the study of rare variants identified by next-generation sequencing in diseases with autosomal dominant or complex patterns of inheritance.     

Area-Level Attributes and Esophageal Adenocarcinoma in Surveillance,Epidemiology and End Results Registries

Purpose

To examine the associations between area-level socioeconomic attributes and stage of esophageal adenocarcinoma diagnoses in 16 SEER cancer registries during 2000-2007.

Methods

Odds ratios (OR) and 95% confidence intervals (CI) were calculated using multivariable logistic regression models to assess the relationship between distant-stage esophageal adenocarcinoma and individual, census tract, and county-level attributes.

Results

Among cases with data on birthplace, no significant association was seen between reported birth within versus outside the United States and distant-stage cancer (adjusted OR=1.02, 95% CI: 0.85-1.22). Living in an area with a higher percentage of residents born outside the United States than the national average was associated with distant-stage esophageal adenocarcinoma; census tract level: >11.8%, (OR=1.10, 95% CI:1.01–1.19), county level: >11.8%, (OR=1.14, 95% CI:1.05-1.24). No association was observed between median household income and distant-stage cancer at either census tract or county levels.

Conclusion

The finding of greater odds of distant-stage esophageal adenocarcinoma among cases residing in SEER areas with higher proportion of non-U.S. Natives suggests local areas where esophageal cancer control efforts might be focused. Missing data at the individual level was a limitation of the present study. Furthermore, inconsistent associations with foreign birth at individual- versus area-levels cautions against using area-level attributes as proxies for case attributes.     

High tolerance of aluminum in the grass species Cynodon aethiopicus

Concentrations of aluminum (Al) were determined in leaves of native terrestrial plants, macrophytes and fruit parts (watermelon and tomato) using inductively coupled plasma mass spectrometry. Al concentrations in water and soil were determined by inductively coupled plasma optical emission spectrometry. Potamogeton thunbergii (macrophyte) and Cynodon aethiopicus (terrestrial grass) had the highest leaf Al concentrations (2 and 1 g kg^?1 dw, respectively). Transfer factors (mg kg^?1 dw plants/mg kg^?1 dw soil) based on total Al concentrations in soil varied from 2 × 10^?3 to 0.05 and from 1.9 to 78 based on mobile Al concentrations determined after sequential extraction. Bioconcentration factors (mg kg^?1 dw plants/mg L^?1 water) varied from 19 to 9.5 × 10³ L kg^?1 dw. Plants can accumulate high concentrations of Al when growing in neutral pH soils and slightly alkaline lakes in the Ethiopian Rift Valley. Controlled experiments showed that C. aethiopicus can accumulate high levels of Al both in root and shoot. Compared to Arabidopsis thaliana, C. aethiopicus was more tolerant to Al exposure as ≥400 μM AlCl₃ was needed to inhibit root growth compared to 200 μM in A. thaliana. After exposing C. aethiopicus and A. thaliana in 800 μM AlCl_3, alkaline comet assay indicates significant DNA (deoxyribonucleic acid) damage in A. thaliana while C. aethiopicus was unaffected. No significant induction of reactive oxygen species (ROS), in terms of leaf H₂O₂ levels, could be observed in C. aethiopicus. C. aethiopicus has mechanisms to suppress both Al-induced ROS and DNA damage, thereby increasing tolerance of the species to high Al concentrations.     

Optimization of allosteric MEK inhibitors. Part 1: Venturing into underexplored SAR territories

Using PD325901 as a starting point for identifying novel allosteric MEK inhibitors with high cell potency and long-lasting target inhibition in vivo, truncation of its hydroxamic ester headgroup was combined with incorporation of alkyl and aryl ethers at the neighboring ring position. Whereas alkoxy side chains did not yield sufficient levels of cell potency, specifically substituted aryloxy groups allowed for high enzymatic and cellular potencies. Sulfamide 28 was identified as a highly potent MEK inhibitor with nanomolar cell potency against B-RAF (V600E) as well as Ras-mutated cell lines, high metabolic stability and resulting long half-lives. It was efficacious against B-RAF as well as K-Ras driven xenograft models and showed—despite being orally bioavailable and not a P-glycoprotein substrate—much lower brain/plasma exposure ratios than PD325901.     

Lymphatic and Angiogenic Candidate Genes Predict the Development of Secondary Lymphedema following Breast Cancer Surgery

The purposes of this study were to evaluate for differences in phenotypic and genotypic characteristics in women who did and did not develop lymphedema (LE) following breast cancer treatment. Breast cancer patients completed a number of self-report questionnaires. LE was evaluated using bioimpedance spectroscopy. Genotyping was done using a custom genotyping array. No differences were found between patients with (n = 155) and without LE (n = 387) for the majority of the demographic and clinical characteristics. Patients with LE had a significantly higher body mass index, more advanced disease and a higher number of lymph nodes removed. Genetic associations were identified for four genes (i.e., lymphocyte cytosolic protein 2 (rs315721), neuropilin-2 (rs849530), protein tyrosine kinase (rs158689), vascular cell adhesion molecule 1 (rs3176861)) and three haplotypes (i.e., Forkhead box protein C2 (haplotype A03), neuropilin-2 (haplotype F03), vascular endothelial growth factor-C (haplotype B03)) involved in lymphangiogensis and angiogenesis. These genetic associations suggest a role for a number of lymphatic and angiogenic genes in the development of LE following breast cancer treatment.     

The invasive fish Acestrorhynchus pantaneiro Menezes, 1992 in Southern Brazil: Occurrence and food niche overlap with two native species

Acestrorhynchus pantaneiro is an invasive species in the Patos Lagoon system in South Brazil. The aim of the study was to document the expansion of the occurrence range of A. pantaneiro in southern Brazil and to analyze the diet and food feeding overlap with the two native species Oligosarcus robustus and O. jenynsii. Occurrence data were based on fish collections databases and collected material. Fish for the stomach analysis were captured in the Jacuí and Sinos rivers from 2007 to 2009 by gillnetting and electrofishing. Food uptake was quantified by frequency of occurrence and relative abundance of the prey items. Food niche overlap was estimated by the Pianka Index. Database records indicated a rapid expansion of the occurrence range in all principal basins of the southernmost state of Rio Grande do Sul in Brazil. Food niche overlap was greatest between A. pantaneiro and O. robustus (O_jk = .970). Both species fed predominantly on small Characids. O. jenynsii ingested predominantly insects and crustaceans and, consequently, had low food niche overlap when compared with the two other species. Results indicate that the expansion of A. pantaneiro occurrence range may principally affect O. robustus populations.     

rRNA and rDNA based assessment of sea ice protist biodiversity from the central Arctic Ocean

Sea ice is a large and diverse ecosystem contributing significantly to primary production in ice-covered regions. In the Arctic Ocean, sea ice consists of mixed multi-year ice (MYI), often several metres thick, and thinner first-year ice (FYI). Current global warming is most severe in Arctic regions; as a consequence, summer sea ice cover is decreasing and MYI is disappearing at an alarming rate. Despite its apparent hostility, sea ice is inhabited by a diverse microbial community of bacteria and protists, many of which are photosynthetic. Here we present an assessment of eukaryotic biodiversity in MYI and FYI from the central Arctic Ocean using high-throughput 454 sequencing of 18S rRNA and rDNA amplicons. We compared the rDNA-based ‘total’ biodiversity with the ‘active’ biodiversity from rRNA amplicons and found differences between them including an over-representation of Ciliophora, Bicosoecida and Bacillariophyceae operational taxonomic units (OTUs) in the active part of the community. Differences between the two libraries are more pronounced at the lower taxonomic level: certain genera, such as Melosira, are more abundant in the rRNA library, indicating activity of these genera. Furthermore, we found that one FYI station showed a higher activity of potential grazers which was probably due to the advanced stage of melt evident by higher ice temperatures and highly porous ice compared with the other stations.     

Enlightening energy parasitism by analysis of an ATP/ADP transporter from chlamydiae

Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.     

Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.