FRET-based genetically encoded sensors allow high-resolution live cell imaging of Ca²⁺ dynamics

Temporally and spatially defined calcium signatures are integral parts of numerous signalling pathways. Monitoring calcium dynamics with high spatial and temporal resolution is therefore critically important to understand how this ubiquitous second messenger can control diverse cellular responses. Yellow cameleons (YCs) are fluorescence resonance energy transfer (FRET)-based genetically encoded Ca(2+) -sensors that provide a powerful tool to monitor the spatio-temporal dynamics of Ca(2+) fluxes. Here we present an advanced set of vectors and transgenic lines for live cell Ca(2+) imaging in plants. Transgene silencing mediated by the cauliflower mosaic virus (CaMV) 35S promoter has severely limited the application of nanosensors for ions and metabolites and we have thus used the UBQ10 promoter from Arabidopsis and show here that this results in constitutive and stable expression of YCs in transgenic plants. To improve the spatial resolution, our vector repertoire includes versions of YCs that can be targeted to defined locations. Using this toolkit, we identified temporally distinct responses to external ATP at the plasma membrane, in the cytosol and in the nucleus of neighbouring root cells. Moreover analysis of Ca(2+) dynamics in Lotus japonicus revealed distinct Nod factor induced Ca(2+) spiking patterns in the nucleus and the cytosol. Consequently, the constructs and transgenic lines introduced here enable a detailed analysis of Ca(2+) dynamics in different cellular compartments and in different plant species and will foster novel approaches to decipher the temporal and spatial characteristics of calcium signatures.     

Diversity and evolution of ectomycorrhizal host associations in the Sclerodermatineae (Boletales, Basidiomycota)

This study uses phylogenetic analysis of the Sclerodermatineae to reconstruct the evolution of ectomycorrhizal host associations in the group using divergence dating, ancestral range and ancestral state reconstructions. Supermatrix and supertree analysis were used to create the most inclusive phylogeny for the Sclerodermatineae. Divergence dates were estimated in BEAST. Lagrange was used to reconstruct ancestral ranges. BayesTraits was used to reconstruct ectomycorrhizal host associations using extant host associations with data derived from literature sources. The supermatrix data set was combined with internal transcribed spacer (ITS) data sets for Astraeus, Calostoma, and Pisolithus to produce a 168 operational taxonomic unit (OTU) supertree. The ensuing analysis estimated that basal Sclerodermatineae originated in the late Cretaceous while major genera diversified near the mid Cenozoic. Asia and North America are the most probable ancestral areas for all Sclerodermatineae, and angiosperms, primarily rosids, are the most probable ancestral hosts. Evolution in the Sclerodermatineae follows the biogeographic history of disjunct plant communities associated with early Cenozoic mesophytic forests and a boreotropical history. Broad geographic distributions are observed in the most promiscuous Sclerodermatineae (those with broad host ranges), while those with relatively limited distribution have fewer documented ectomycorrhizal associations. This suggests that ectomycorrhizal generalists have greater dispersal capabilities than specialists.     

Sutorius: a new genus for Boletus eximius

Sutorius is described as a new genus of Boletaceae to accommodate Boletus robustus originally named illegitimately by C.C. Frost from eastern North America. The legitimate name, Boletus eximius, provided by C.H. Peck, has been used since for a dark purple to chocolate brown bolete with finely scaly stipe and reddish brown spore deposit. This iconic taxon has been documented on five continents. Despite the straightforward species identification from morphology, the interpretation of stipe macro-morphology and spore color has led to equivocal generic placement. Phylogenetic analyses of genes encoding large subunit rRNA and translation elongation factor 1α confirm Sutorius as a unique generic lineage in the Boletaceae. Two species are recognized based on multiple accessions: S. eximius, represented by collections from North America, Costa Rica, Guyana, Indonesia and Japan (molecular data are lacking for only the Guyanan and Japanese material); and S. australiensis, represented by material from Queensland, Australia. Additional collections from Zambia and Thailand represent independent lineages, but sampling is insufficient to describe new species for these entities.     

Phenotypic detection of clonotypic B cells in multiple myeloma by specific immunoglobulin ligands reveals their rarity in multiple myeloma

In multiple myeloma, circulating "clonotypic" B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential "feeder" cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10(-3), this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology.     

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3? (GSK3?) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.     

Predictors of Remission in Depression to Individual and Combined Treatments (PReDICT): Study Protocol for a Randomized Controlled Trial

ABSTRACT: BACKGROUND: Limited controlled data exist to guide treatment choices for clinicians caring for patients with major depressive disorder (MDD). Although many putative predictors of treatment response have been reported, most were identified through retrospective analyses of existing datasets and very few have been replicated in a manner that can impact clinical practice. One major confound in previous studies examining predictors of treatment response is the patient's treatment history, which may affect both the predictor of interest and treatment outcomes. Moreover, prior treatment history provides an important source of selection bias, thereby limiting generalizability. Consequently, we initiated a randomized clinical trial designed to identify factors that moderate response to three treatments for MDD among patients never treated previously for the condition. METHODS: Treatment-naive adults aged 18-65 years with moderate-to-severe, non-psychotic MDD are randomized equally to one of three 12-week treatment arms: 1) cognitive behavior therapy (CBT, 16 sessions), 2) duloxetine (30-60 mg/d), or 3) escitalopram (10-20 mg/d). Prior to randomization, patients undergo multiple assessments, including resting state functional magnetic resonance imaging (fMRI), immune markers, DNA and gene expression products, and dexamethasone-corticotropin releasing hormone (Dex/CRH) testing. Prior to or shortly after randomization, patients also complete a comprehensive personality assessment. Repeat assessment of the biological measures (fMRI, immune markers, and gene expression products) occur at an early time-point in treatment, and upon completion of 12-week treatment, when a a second Dex/CRH test is also conducted, Patients remitting by the end of this acute treatment phase are then eligible to enter a 21-month follow-up phase, with quarterly visits to monitor for recurrence. Non-remitters are offered augmentation treatment for a second 12-week course of treatment, during which they receive a combination of CBT and antidepressant medication. Predictors of the primary outcome, remission, will be identified for overall and treatment-specific effects, and a statistical model incorporating multiple predictors will be developed to predict outcomes. DISCUSSION: The PReDICT study's evaluation of biological, psychological, and clinical factors that may differentially impact treatment outcomes represents a sizeable step toward developing personalized treatments for MDD. Identified predictors should help guide the selection of initial treatments, and identify those patients most vulnerable to recurrence, who thus warrant maintenance or combination treatments to achieve and maintain wellness.     

Targeting of malate synthase 1 to the peroxisomes of Saccharomyces cerevisiae cells depends on growth on oleic acid medium.

The eukaryotic glyoxylate cycle has been previously hypothesized to occur in the peroxisomal compartment, which in the yeast Saccharomyces cerevisiae additionally represents the sole site for fatty acid beta-oxidation. The subcellular location of the key glyoxylate-cycle enzyme malate synthase 1 (Mls1p), an SKL-terminated protein, was examined in yeast cells grown on different carbon sources. Immunoelectron microscopy in combination with cell fractionation showed that Mls1p was abundant in the peroxisomes of cells grown on oleic acid, whereas in ethanol-grown cells Mls1p was primarily cytosolic. This was reinforced using a green fluorescent protein (GFP)-Mls1p reporter, which entered peroxisomes solely in cells grown under oleic acid-medium conditions. Although growth of cells devoid of Mls1p on ethanol or acetate could be fully restored using a cytosolic Mls1p devoid of SKL, this construct could only partially alleviate the requirement for native Mls1p in cells grown on oleic acid. The combined results indicated that Mls1p remained in the cytosol of cells grown on ethanol, and that targeting of Mls1p to the peroxisomes was advantageous to cells grown on oleic acid as a sole carbon source.