Molecular Control of B Cell Activation and Immunological Synapse Formation

B cells form an essential part of the adaptive immune system by producing specific antibodies that can neutralize toxins and target infected or malignant cells for destruction. During B cell activation, a fundamental role is played by a specialized intercellular structure called the immunological synapse (IS). The IS serves as a platform for B cell recognition of foreign, often pathogenic, antigens on the surface of antigen‐presenting cells (APC). This recognition is elicited by highly specific B cell receptors (BCR) that subsequently trigger carefully orchestrated intracellular signaling cascades that lead to cell activation. Furthermore, antigen internalization, essential for full B cell activation and differentiation into antibody producing effector cells or memory cells, occurs in the IS. Recent developments especially in various imaging‐based methods have considerably advanced our understanding of the molecular control of B cell activation. Interestingly, the cellular cytoskeleton is emerging as a key player at several stages of B cell activation, including the initiation of receptor signaling. Here, we discuss the functions and molecular mechanisms of the IS and highlight the multifaceted role of the actin cytoskeleton in several aspects of B cell activation.      

ADRA2A is involved in neuro‐endocrine regulation of bone resorption

Adrenergic stimulation is important for osteoclast differentiation and bone resorption. Previous research shows that this happens through β2‐adrenergic receptor (AR), but there are conflicting evidence on presence and role of α2A‐AR in bone. The aim of this study was to investigate the presence of α2A‐AR and its involvement in neuro‐endocrine signalling of bone remodelling in humans. Real‐time polymerase chain reaction (PCR) and immunohistochemistry were used to investigate α2A‐AR receptor presence and localization in bone cells. Functionality of rs553668 and rs1800544 single nucleotide polymorphism SNPs located in α2A‐AR gene was analysed by qPCR expression on bone samples and luciferase reporter assay in human osteosarcoma HOS cells. Using real‐time PCR, genetic association study between rs553668 A>G and rs1800544 C>G SNPs and major bone markers was performed on 661 Slovenian patients with osteoporosis. α2A‐AR is expressed in osteoblasts and lining cells but not in osteocytes. SNP rs553668 has a significant influence on α2A‐AR mRNA level in human bone samples through the stability of mRNA. α2A‐AR gene locus associates with important bone remodelling markers (BMD, CTX, Cathepsin K and pOC). The results of this study are providing comprehensive new evidence that α2A‐AR is involved in neuro‐endocrine signalling of bone turnover and development of osteoporosis. As shown by our results the neurological signalling is mediated through osteoblasts and result in bone resorption. Genetic study showed association of SNPs in α2A‐AR gene locus with bone remodelling markers, identifying the individuals with higher risk of development of osteoporosis.     

84 PPC measurements of unfolding volumes of DNA stem-loop motifs

One focus of our research is to further our understanding of the physico-chemical properties of non-canonical nucleic acid structures. In this work, DNA hairpins are used to mimic a common motif present in RNA, i.e. a stem-loop motif with a bulge or internal loop in their stem. Specifically, we used a combination of temperature-dependent UV spectroscopy, differential scanning (DSC), and pressure perturbation (PPC) calorimetric techniques to determine complete thermodynamic profiles for the helix–coil transitions of two sets of hairpins with 5′–3′ sequences: d(GCGCT _n GTAACT₅GTTACGCGC) and d(GCGCT _n GTAACT₅GTTACT _n GCGC). “T _n ” is a variable loop of thymines, n?=?1, 3 or 5; and “T₅” is an end-loop of five thymines. Unfolding curves show monophasic transitions with TMs independent of strand concentration, confirming their intramolecular formation. DSC thermodynamic profiles indicate that the favorable folding of each hairpin results from the typical compensation of favorable enthalpy and unfavorable entropy contributions, while the DSC curves as a function of salt concentration yielded an uptake of cations and negative heat capacity effects. PPC melting curves yielded positive folding volumes ranging 12–31?cm³/mol, corresponding to releases of water molecules; in contrast, an uptake of water (ranging from 32 to 63?mol of H₂O/mol) is observed from osmotic stress experiments using ethylene glycol as the osmolyte. Overall, the increase in the size of the variable bulge or internal-loop yielded lower TMs and slightly more favorable enthalpies, corresponding to less favorable free energy contributions of ～0.7?kcal/mol per thymine residue. The volume measurements will be correlated with the unfolding entropies and discussed in terms of the type of water that is hydrating these stem-loop motifs structures.     

Chilling and forcing temperatures interact to predict the onset of wood formation in Northern Hemisphere conifers

The phenology of wood formation is a critical process to consider for predicting how trees from the temperate and boreal zones may react to climate change. Compared to leaf phenology, however, the determinism of wood phenology is still poorly known. Here, we compared for the first time three alternative ecophysiological model classes (threshold models, heat‐sum models and chilling‐influenced heat‐sum models) and an empirical model in their ability to predict the starting date of xylem cell enlargement in spring, for four major Northern Hemisphere conifers (Larix decidua, Pinus sylvestris, Picea abies and Picea mariana). We fitted models with Bayesian inference to wood phenological data collected for 220 site‐years over Europe and Canada. The chilling‐influenced heat‐sum model received most support for all the four studied species, predicting validation data with a 7.7‐day error, which is within one day of the observed data resolution. We conclude that both chilling and forcing temperatures determine the onset of wood formation in Northern Hemisphere conifers. Importantly, the chilling‐influenced heat‐sum model showed virtually no spatial bias whichever the species, despite the large environmental gradients considered. This suggests that the spring onset of wood formation is far less affected by local adaptation than by environmentally driven plasticity. In a context of climate change, we therefore expect rising winter–spring temperature to exert ambivalent effects on the spring onset of wood formation, tending to hasten it through the accumulation of forcing temperature, but imposing a higher forcing temperature requirement through the lower accumulation of chilling.     

Signalling Network Construction for Modelling Plant Defence Response

Plant defence signalling response against various pathogens, including viruses, is a complex phenomenon. In resistant interaction a plant cell perceives the pathogen signal, transduces it within the cell and performs a reprogramming of the cell metabolism leading to the pathogen replication arrest. This work focuses on signalling pathways crucial for the plant defence response, i.e., the salicylic acid, jasmonic acid and ethylene signal transduction pathways, in the Arabidopsis thaliana model plant. The initial signalling network topology was constructed manually by defining the representation formalism, encoding the information from public databases and literature, and composing a pathway diagram. The manually constructed network structure consists of 175 components and 387 reactions. In order to complement the network topology with possibly missing relations, a new approach to automated information extraction from biological literature was developed. This approach, named Bio3graph, allows for automated extraction of biological relations from the literature, resulting in a set of (component1, reaction, component2) triplets and composing a graph structure which can be visualised, compared to the manually constructed topology and examined by the experts. Using a plant defence response vocabulary of components and reaction types, Bio3graph was applied to a set of 9,586 relevant full text articles, resulting in 137 newly detected reactions between the components. Finally, the manually constructed topology and the new reactions were merged to form a network structure consisting of 175 components and 524 reactions. The resulting pathway diagram of plant defence signalling represents a valuable source for further computational modelling and interpretation of omics data. The developed Bio3graph approach, implemented as an executable language processing and graph visualisation workflow, is publically available at http://ropot.ijs.si/bio3graph/and can be utilised for modelling other biological systems, given that an adequate vocabulary is provided.     

Enterocyte-Specific Inactivation of SIRT1 Reduces Tumor Load in the APC+/min Mouse Model

SIRT1 is a mammalian NAD⁺-dependent histone deacetylase implicated in metabolism, development, aging and tumorigenesis. Prior studies that examined the effect of enterocyte-specific overexpression and global deletion of SIRT1 on polyp formation in the intestines of APC^+/min mice, a commonly used model for intestinal tumorigenesis, yielded conflicting results, supporting either tumor-suppressive or tumor-promoting roles for SIRT1, respectively. In order to resolve the controversy emerging from these prior in vivo studies, in the present report we examined the effect of SIRT1 deficiency confined to the intestines, avoiding the systemic perturbations such as growth retardation seen with global SIRT1 deletion. We crossed APC^+/min mice with mice bearing enterocyte-specific inactivation of SIRT1 and examined polyp development in the progeny. We found that SIRT1-inactivation reduced total polyp surface (9.3 mm² vs. 23.3 mm², p = 0.01), average polyp size (0.24 mm² vs. 0.51 mm², p = 0.005) and the number of polyps >0.5 mm in diameter (14 vs. 23, p = 0.04), indicating that SIRT1 affects both the number and size of tumors. Additionally, tumors in SIRT1-deficient mice exhibited markedly increased numbers of cells undergoing apoptosis, suggesting that SIRT1 contributes to tumor growth by enabling survival of tumor cells. Our results indicate that SIRT1 acts as a tumor promoter in the APC^+/min mouse model of intestinal tumorigenesis.     

A critical thermal transition driving spring phenology of Northern Hemisphere conifers

Despite growing interest in predicting plant phenological shifts, advanced spring phenology by global climate change remains debated. Evidence documenting either small or large advancement of spring phenology to rising temperature over the spatio-temporal scales implies a potential existence of a thermal threshold in the responses of forests to global warming. We collected a unique data set of xylem cell-wall-thickening onset dates in 20 coniferous species covering a broad mean annual temperature (MAT) gradient (−3.05 to 22.9°C) across the Northern Hemisphere (latitudes 23°–66° N). Along the MAT gradient, we identified a threshold temperature (using segmented regression) of 4.9 ± 1.1°C, above which the response of xylem phenology to rising temperatures significantly decline. This threshold separates the Northern Hemisphere conifers into cold and warm thermal niches, with MAT and spring forcing being the primary drivers for the onset dates (estimated by linear and Bayesian mixed-effect models), respectively. The identified thermal threshold should be integrated into the Earth-System-Models for a better understanding of spring phenology in response to global warming and an improved prediction of global climate-carbon feedbacks.     

Erythropoietin unfolding: thermodynamics and its correlation with structural features

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.