Comparison of Predictive Immunohistochemical Marker Expression of Primary Breast Cancer and Paired Distant Metastasis using Surgical Material: A Practice-Based Study

Parallel studies of primary breast carcinomas and corresponding distant metastases samples reveal considerable differences. Our aim was to highlight this issue from another perspective and provide further data based on 98 patient samples: 69 primary breast carcinoma and 85 distant metastases from bone, central nervous system (CNS) and lung (56 paired). Two independent series of immunohistochemical reactions with different antibodies for estrogen receptor (ER), progesterone receptor (PgR) and human epidermal growth factor receptor 2 (Her2), along with HER2 fluroscence in situ hybridization (FISH) were performed on tissue microarrays to classify breast carcinoma and distant metastases samples into Luminal A, Luminal B-proliferating, Luminal B-HER2+, HER2+ and triple negative (TNBC) surrogate breast cancer groups. Correlation and agreement between the two assessments of ER and PgR were fair-to-moderate, and almost perfect for HER2 and Ki67. There was 40% discordance concerning immunophenotype between breast carcinomas and distant metastases. Most common metastatic site of ER+ breast carcinoma was the skeletal system (59.2%), whereas that of TNBCs was the CNS (58.8%) and lungs (23.5%). Distant metastases in bones were mostly luminal (54.3%), in the CNS, Luminal B (53.2%), and in the lung, TNBC (37.5%). The change of drugable properties of primary breast cancers in the respective bone and CNS metastases suggests that characterization of the metastasis is necessary for appropriate treatment planning.     

Temperature Dependence of Soil Respiration Modulated by Thresholds in Soil Water Availability Across European Shrubland Ecosystems

Soil respiration (SR) is a major component of the global carbon cycle and plays a fundamental role in ecosystem feedback to climate change. Empirical modelling is an essential tool for predicting ecosystem responses to environmental change, and also provides important data for calibrating and corroborating process-based models. In this study, we evaluated the performance of three empirical temperature–SR response functions (exponential, Lloyd–Taylor and Gaussian) at seven shrublands located within three climatic regions (Atlantic, Mediterranean and Continental) across Europe. We investigated the performance of SR models by including the interaction between soil moisture and soil temperature. We found that the best fit for the temperature functions depended on the site-specific climatic conditions. Including soil moisture, we identified thresholds in the three different response functions that improved the model fit in all cases. The direct soil moisture effect on SR, however, was weak at the annual time scale. We conclude that the exponential soil temperature function may only be a good predictor for SR in a narrow temperature range, and that extrapolating predictions for future climate based on this function should be treated with caution as modelled outputs may underestimate SR. The addition of soil moisture thresholds improved the model fit at all sites, but had a far greater ecological significance in the wet Atlantic shrubland where a fundamental change in the soil CO₂ efflux would likely have an impact on the whole carbon budget.     

Induction of Abiotic Stress Tolerance by Salicylic Acid Signaling

The role of salicylic acid (SA) as a key molecule in the signal transduction pathway of biotic stress responses has already been well described. Recent studies indicate that it also participates in the signaling of abiotic stresses. The application of exogenous SA could provide protection against several types of stresses such as high or low temperature, heavy metals, and so on. Although SA may also cause oxidative stress to plants, partially through the accumulation of hydrogen peroxide, the results published so far show that the preliminary treatment of plants with low concentrations of SA might have an acclimation-like effect, causing enhanced tolerance toward most kinds of abiotic stresses due primarily to enhanced antioxidative capacity. The effect of exogenous SA depends on numerous factors such as the species and developmental stage of the plant, the mode of application, and the concentration of SA and its endogenous level in the given plant. Recent results show that not only does exogenous SA application moderate stress effects, but abiotic stress factors may also alter the endogenous SA levels in the plant cells. This review compares the roles of SA during different abiotic stresses.     

Full Design Automation of Multi-State RNA Devices to Program Gene Expression Using Energy-Based Optimization

Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 5′ untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design.     

In vivo direct interaction of the antibiotic primycin on a Candida albicans clinical isolate and its ergosterol-less mutant

Interaction of primycin antibiotic with plasma membrane, and its indirect biological effects were investigated in this study. The antifungal activity of primycin against 13 human pathogenic Candida ATCC and CBS reference species and 74 other Candida albicans clinical isolates was investigated with a microdilution technique. No primycin-resistant strain was detected. Direct interaction of primycin with the plasma membrane was demonstrated for the first time by using an ergosterol-producing strain 33erg+ and its ergosterol-less mutant erg-2. In growth inhibition tests, the 33erg+ strain proved to be more sensitive to primycin than its erg-2 mutant, indicating the importance of the plasma membrane composition in primycin-induced processes. The 64 μg ml-1 (56.8 nM) primycin treatment induced an enhanced membrane fluidity and altered plasma membrane dynamics, as measured by steady-state fluorescence anisotropy applying a trimethylammonium-diphenylhexatriene (TMA-DPH) fluorescence polarization probe. The following consequences were detected. The plasma membrane of the cells lost its barrier function, and the efflux of 260-nm-absorbing materials from treated cells of both strains was 1.5-1.8 times more than that for the control. Depending on the primycin concentration, the cells exhibited unipolar budding, pseudohyphae formation, and a rough cell surface visualized by scanning electron microscopy.     

A comprehensive study on the 5-hydroxytryptamine3A receptor binding of agonists serotonin and m-chlorophenylbiguanidine

Serotonin type 3 receptors (5-HT₃R) are members of the ligand gated ion channel receptor family. In this study, the interactions of the agonists serotonin (5-HT) and m-chlorophenylbiguanidine (mCPBG) at the binding site of the 5-HT_3AR were investigated at an atomic level. Site-directed mutagenesis studies in Loop B and E along with our earlier published results from mutations within Loops A, C, and D provide comprehensive data on the interaction of 5-HT and mCPBG with 5-HT_3ARs. Using this data we have constructed a refined homology model of the 5-HT_3AR that considers all of the available experimental data. 5-HT and mCPBG were docked into the newly constructed homology model and the amino acid residues critical in binding of these agonists were compared and analyzed. Our docking results reveal many similar binding interactions for 5-HT and mCPBG. Namely, residues THR181, TRP183, PHE226, ILE228, TYR234 and GLU129 were all found to play key roles in binding of both 5-HT and mCPBG. However, the results also revealed two important differences that exist between the interactions of the two agonists. In our model, a hydrogen bond is formed between the indole hydrogen of 5-HT and the residue TYR153. This interaction is not present in the case of mCPBG. Conversely, a hydrogen bond exists between SER182 and a protonated nitrogen of mCPBG, which does not exist in 5-HT. Our modeling results were found to be in accordance with experimental data.     

Detection of protein complex interactions via a Blue Native-PAGE retardation assay

We describe the Blue Native (BN)-PAGE retardation assay for the detection of interactions of biomolecules with protein complexes. Potential interactors of proteins are included in the BN gel matrix, resulting in retardation of proteins that interact with the added molecule. After validation using the T-cell antigen receptor, we applied the assay for a general identification of dextran interactors in combination with mass spectroscopy. The proteomic screen revealed triosephosphate isomerase oligomer as a dextran-binding, high M_R complex.