Polycation-π Interactions Are a Driving Force for Molecular Recognition by an Intrinsically Disordered Oncoprotein Family

Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness", often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs. © 2013 Song et al

Anisotropic Charge Distribution and Anisotropic van der Waals Radius Leading to Intriguing Anisotropic Noncovalent Interactions

Although group (IV-VII) nonmetallic elements do not favor interacting with anionic species, there are counterexamples including the halogen bond. Such binding is known to be related to the charge deficiency because of the adjacent atom's electron withdrawing effect, which creates s/p-holes at the bond-ends. However, a completely opposite behavior is exhibited by N-2 and O-2, which have electrostatically positive/negative character around cylindrical-bond-surface/bond-ends. Inspired by this, here we elucidate the unusual features and origin of the anisotropic noncovalent interactions in the ground and excited states of the 2nd and 3rd row elements belonging to groups IV-VII. The anisotropy in charge distributions and van der Waals radii of atoms in such molecular systems are scrutinized. This provides an understanding of their unusual molecular configuration, binding and recognition modes involved in new types of molecular assembling and engineering. This work would lead to the design of intriguing molecular systems exploiting anisotropic noncovalent interactions.open

Reliability of cyclin A assessment on tissue microarrays in breast cancer compared to conventional histological slides

Cyclin A has in some studies been associated with poor breast cancer survival, although all studies have not confirmed this. Its prognostic significance in breast cancer needs evaluation in larger studies. Tissue microarray (TMA) technique allows a simultaneous analysis of large amount of tumours on a single microscopic slide. This makes a rapid screening of molecular markers in large amount of tumours possible. Because only a small tissue sample of each tumour is punched on an array, the question has arisen about the representativeness of TMA when studying markers that are expressed in only a small proportion of cells. For this reason, we wanted to compare cyclin A expression on TMA and on traditional large sections. Two breast cancer TMAs were constructed of 200 breast tumours diagnosed between 1997–1998. TMA slides and traditional large section slides of these 200 tumours were stained with cyclin A antibody and analysed by two independent readers. The reproducibility of the two readers' results was good or even very good, with kappa values 0.71–0.87. The agreement of TMA and large section results was good with kappa value 0.62–0.75. Cyclin A overexpression was significantly (P<0.001) associated with oestrogen receptor and progesterone receptor negativity and high grade both on TMA and large sections. Cyclin A overexpression was significantly associated with poor metastasis-free survival both on TMA and large sections. The relative risks for metastasis were similar on TMA and large sections. This study suggests that TMA technique could be useful to study histological correlations and prognostic significance of cyclin A on breast cancer on a large scale

Winter Time Concentrations and Size Distribution of Bioaerosols in Different Residential Settings in the UK

The total concentration and size distribution of bioaerosols in three different types of housing (single room in shared accommodation [type I], single bedroom flat in three-storey building [type II] and two- or threebedroom detached houses [type III]) was assessed during the winter. This research was an extension of a previous study carried out in the summer. The measurement campaign was undertaken in winter 2008 and 30 houses were sampled. Samples were taken from kitchens, living rooms, corridors (only in housing type I) and outdoors with an Anderson 6 stage viable impactor. In housing type I, the total geometric mean concentration was highest in the corridor for both bacteria and fungi (3,171 and 1,281 CFU/m3, respectively). In type II residences, both culturable bacteria and fungi were greatest in the living rooms (3,487 and 833 CFU/m3, respectively). The living rooms in type III residences had largest number of culturable bacteria (1,361 CFU/m3) while fungi were highest in kitchens (280 CFU/m3). The concentrations of culturable bacteria and fungi were greater in mouldy houses than non-mouldy houses. A considerable variation was seen in the size distribution of culturable bacteria in type I residences compared to types II and III. For all housing types more than half of culturable bacterial and fungal aerosol were respirable (<4.7 μm) and so have the potential to penetrate into lower respiratory system. Considerable variation in concentration and size distribution within different housing types in the same geographical region highlights the impact of differences in design, construction, use and management of residential built environment on bioaerosols levels and consequent varied risk of population exposure to airborne biological agents. © Springer Science+Business Media B.V. 2012

Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T

Теневой человек как социокультурный феномен

Background: Cardiorespiratory fitness (CRF) is a physical condition that is now well established as a predictor of numerous adverse health outcomes, independent of physical activity levels. In order to be able to improve CRF at the population level and to develop effective interventions and public health programmes, it is important to understand why some people are more fit than others. Therefore, the primary aim of the systematic review described in this protocol is to examine individual and interpersonal factors that are correlated with or determine CRF among adults. Methods: The review will focus on quantitative studies that investigate any personal and interpersonal correlates and/or determinants of objectively measured CRF among the general, non-symptomatic, non-institutionalized adult population (aged 18–65 years) worldwide. The databases MEDLINE, Embase, and Cochrane Library will be searched to identify all relevant published journal articles, and Google Scholar and Scopus will be searched for grey literature. Studies where CRF is not an outcome variable and experimental studies where participants specifically receive a fitness intervention that increases CRF will be excluded. For each study, data extracted will include, among other variables, study characteristics, methodology for selecting participants into the study as well as the participants’ demographic characteristics, types of correlates and determinants of CRF investigated and their measurement methods, the objective measure of CRF used and its measurement method and validity, and the main reported results on the association between the correlates or determinants and CRF. In addition, observational studies will be assessed for methodological quality and risk of bias using a customized version of the Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies by the National Heart, Lung, and Blood Institute. Experimental studies will be assessed using the 27-item Downs and Black “Checklist for Measuring Study Quality”. The final results will be presented as a narrative synthesis of the main findings of all included studies. Discussion: By consolidating and synthesizing the current research on possible individual and interpersonal correlates and determinants of CRF among adults worldwide, we aim to aid future public health actions, as well as identify gaps in our full understanding of what influences CRF

Integrated Analysis of Gene Expression, CpG Island Methylation, and Gene Copy Number in Breast Cancer Cells by Deep Sequencing

We used deep sequencing technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER− cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER− cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5 kb of the 5′ end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER− breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations

Testing the thrifty gene hypothesis: the Gly482Ser variant in PPARGC1A is associated with BMI in Tongans

<p>Abstract</p> <p>Background</p> <p>The thrifty gene hypothesis posits that, in populations that experienced periods of feast and famine, natural selection favoured individuals carrying thrifty alleles that promote the storage of fat and energy. Polynesians likely experienced long periods of cold stress and starvation during their settlement of the Pacific and today have high rates of obesity and type 2 diabetes (T2DM), possibly due to past positive selection for thrifty alleles. Alternatively, T2DM risk alleles may simply have drifted to high frequency in Polynesians. To identify thrifty alleles in Polynesians, we previously examined evidence of positive selection on T2DM-associated SNPs and identified a T2DM risk allele at unusually high frequency in Polynesians. We suggested that the risk allele of the Gly482Ser variant in the <it>PPARGC1A </it>gene was driven to high frequency in Polynesians by positive selection and therefore possibly represented a thrifty allele in the Pacific.</p> <p>Methods</p> <p>Here we examine whether <it>PPARGC1A </it>is a thrifty gene in Pacific populations by testing for an association between Gly482Ser genotypes and BMI in two Pacific populations (Maori and Tongans) and by evaluating the frequency of the risk allele of the Gly482Ser variant in a sample of worldwide populations.</p> <p>Results</p> <p>We find that the Gly482Ser variant is associated with BMI in Tongans but not in Maori. In a sample of 58 populations worldwide, we also show that the 482Ser risk allele reaches its highest frequency in the Pacific.</p> <p>Conclusion</p> <p>The association between Gly482Ser genotypes and BMI in Tongans together with the worldwide frequency distribution of the Gly482Ser risk allele suggests that <it>PPARGC1A </it>remains a candidate thrifty gene in Pacific populations.</p

Mouse Hepatitis Coronavirus RNA Replication Depends on GBF1-Mediated ARF1 Activation

Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected