Conditional transgene expression mediated by the mouse beta-actin locus

Transgenic mice are an effective model to study gene function in vivo; however, position effects can complicate tissue-specific transgene analysis. To facilitate precise targeting of a transgenic construct into the mouse genome, we combined the Cre/lox and Flp/FRT recombination systems to allow for rapid transgene replacement and conditional transgene expression from the endogenous beta-actin locus. Flp/FRT recombination was used to rapidly exchange FRT-flanked transgene cassettes by recombinase-mediated cassette exchange in embryonic stem cells, while transgene expression can be activated in mice after Cre-mediated excision of a floxed STOP cassette. To validate our system, we analyzed the expression profile of an EGFP reporter gene after integration into the beta-actin locus and Cre-mediated excision of the floxed STOP cassette. Breeding of EGFP reporter mice with various Cre mouse lines resulted in the expected expression profiles, demonstrating the feasibility of the model to facilitate predictable and strong transgene expression in a spatially and temporally controlled manner.     

TreeQ-VISTA: an interactive tree visualization tool with functional annotation query capabilities

We describe a general multiplatform exploratory tool called TreeQ-Vista, designed for presenting functional annotations in a phylogenetic context. Traits, such as phenotypic and genomic properties, are interactively queried from a user-provided relational database with a user-friendly interface which provides a set of tools for users with or without SQL knowledge. The query results are projected onto a phylogenetic tree and can be displayed in multiple color groups. A rich set of browsing, grouping and query tools are provided to facilitate trait exploration, comparison and analysis. AVAILABILITY: The program, detailed tutorial and examples are available online (http:/genome.lbl.gov/vista/TreeQVista).     

PepSplice: cache-efficient search algorithms for comprehensive identification of tandem mass spectra

MOTIVATION: Tandem mass spectrometry allows for high-throughput identification of complex protein samples. Searching tandem mass spectra against sequence databases is the main analysis method nowadays. Since many peptide variations are possible, including them in the search space seems only logical. However, the search space usually grows exponentially with the number of independent variations and may therefore overwhelm computational resources. RESULTS: We provide fast, cache-efficient search algorithms to screen large peptide search spaces including non-tryptic peptides, whole genomes, dozens of posttranslational modifications, unannotated point mutations and even unannotated splice sites. All these search spaces can be screened simultaneously. By optimizing the cache usage, we achieve a calculation speed that closely approaches the limits of the hardware. At the same time, we control the size of the overall search space by limiting the combinations of variations that can co-occur on the same peptide. Using a hypergeometric scoring scheme, we applied these algorithms to a dataset of 1 420 632 spectra. We were able to identify a considerable number of peptide variations within a modest amount of computing time on standard desktop computers.     

An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.     

Fluorescently labeled adrenomedullin allows real‐time monitoring of adrenomedullin receptor trafficking in living cells

The human adrenomedullin (ADM) is a 52 amino acid peptide hormone belonging to the calcitonin family of peptides, which plays a major role in the development and regulation of cardiovascular and lymphatic systems. For potential use in clinical applications, we aimed to investigate the fate of the peptide ligand after binding and activation of the adrenomedullin receptor (AM₁), a heterodimer consisting of the calcitonin receptor‐like receptor (CLR), a G protein‐coupled receptor, associated with the receptor activity‐modifying protein 2 (RAMP2). Full length and N‐terminally shortened ADM peptides were synthesized using Fmoc/tBu solid phase peptide synthesis and site‐specifically labeled with the fluorophore carboxytetramethylrhodamine (Tam) either by amide bond formation or copper(I)‐catalyzed azide alkyne cycloaddition. For the first time, Tam‐labeled ligands allowed the observation of co‐internalization of the whole ligand‐receptor complex in living cells co‐transfected with fluorescent fusion proteins of CLR and RAMP2. Application of a fluorescent probe to track lysosomal compartments revealed that ADM together with the CLR/RAMP2‐complex is routed to the degradative pathway. Moreover, we found that the N‐terminus of ADM is not a crucial component of the peptide sequence in terms of AM₁ internalization behavior. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Piwil 2 Expression Is Correlated with Disease-Specific and Progression-Free Survival of Chemotherapy-Treated Bladder Cancer Patients

Piwi-like 2 (Piwil 2) belongs to the family of Argonaute genes/proteins. The expression of Piwil 2 is associated with stem cells. A role in tumorigenesis and/or tumor progression is proposed for different cancers but not yet for bladder cancer (BCa). We investigated Piwil 2 expression by immunohistochemistry in a cohort of 202 BCa patients treated by cystectomy and adjuvant chemotherapy. The association between Piwil 2 expression and disease-specific (DSS) or progression-free survival (PFS) was calculated using Kaplan-Meier analyses and univariate/multivariate Cox regression hazard models. In a multivariate Cox regression analysis, Piwil 2 expression, either in the cytoplasm or the nucleus, was significantly associated with DSS and PFS. A weak cytoplasmic staining pattern was associated with poor DSS and tumor progression (relative risk [RR] = 2.7, P = 0.004, and RR = 2.4, P = 0.027). Likewise, absent nuclear Piwil 2 immunoreactivity was associated with poor DSS and tumor progression (RR = 2.3, P = 0.023, and RR = 2.2, P = 0.022). BCa patients whose tumors exhibited a combination of weak cytoplasmic and absent nuclear immunoreactivity had a 6-fold increased risk of tumor-related death (P = 0.005) compared with patients with strong expression. Considering only patients with high-grade G3 tumors, a 7.8-fold risk of tumor-associated death and a 3.6-fold risk of tumor progression were detected independently of the histologic tumor subtype or the chemotherapy regimen. In summary, a combination of weak cytoplasmic and absent nuclear expression of Piwil 2 is significantly associated with an increased risk of DSS and tumor progression. This indicates that Piwil 2 could be a valuable prognostic marker for high-risk BCa patients.     

The structure of the SOLE element of oskar mRNA

mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem–loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.     

Patterns of CO<Subscript>2</Subscript> exchange and productivity of the herbaceous vegetation and trees in a humid savanna in western Kenya

Factors governing the dynamics between woody and herbaceous vegetation in the savanna are of ecological interest since they determine ecosystem productivity and stability. Field measurements were conducted in a humid savanna in the Lambwe valley, western Kenya, to compare CO2 exchange of the herbaceous vegetation and trees and its regulation. Soil characteristics and root distribution patterns under tree canopies and in the open locations dominated by the herbaceous vegetation were profiled in 1-m-deep soil layers. Soil water content (SWC) was measured at 30 cm depth both in the herbaceous vegetation and also under the tree canopies. The mean maximum monthly gross primary production (GPPmax) in the herbaceous vegetation was determined from chamber measurements, while daily GPP (GPPday) in both the grass and tree canopies was simulated using the PIXGRO model. The highest mean GPPmax in the herbaceous vegetation was 26.2 ± 3.7 μmol m-2 s-1 during April. Seasonal fluctuations of GPP in the herbaceous vegetation were explained by soil water availability (R ² = 0.78) within the upper 30-cm soil profile. Seasonal GPPday fluctuations were larger (between 1 gC m-2 d-1 and 10 gC m-2 d-1) in the herbaceous vegetation compared to the trees, which fluctuated around 4.3 ± 0.3 gC m-2 d-1 throughout most of the measurement period. Daily tree canopy transpiration (Ec), canopy conductance (Gc), and GPPday were decoupled from SWC in the top 30-cm soil profile. On average, ecosystem GPPday (mean of tree and herbaceous vegetation) was 14.3 ± 1.2 gC m-2 d-1 during the wet period and 6.1 ± 0.9 gC m-2 d-1 during drought. Differences between the herbaceous and tree canopy responses were attributed to soil moisture availability.