Human Golgi Antiapoptotic Protein Modulates Intracellular Calcium Fluxes

Golgi antiapoptotic protein (GAAP) is a novel regulator of cell death that is highly conserved in eukaryotes and present in some poxviruses, but its molecular mechanism is unknown. Given that alterations in intracellular Ca²⁺ homeostasis play an important role in determining cell sensitivity to apoptosis, we investigated if GAAP affected Ca²⁺ signaling. Overexpression of human (h)-GAAP suppressed staurosporine-induced, capacitative Ca²⁺ influx from the extracellular space. In addition, it reduced histamine-induced Ca²⁺ release from intracellular stores through inositol trisphosphate receptors. h-GAAP not only decreased the magnitude of the histamine-induced Ca²⁺ fluxes from stores to cytosol and mitochondrial matrices, but it also reduced the induction and frequency of oscillatory changes in cytosolic Ca²⁺. Overexpression of h-GAAP lowered the Ca²⁺ content of the intracellular stores and decreased the efficacy of IP₃, providing possible explanations for the observed results. Opposite effects were obtained when h-GAAP was knocked down by siRNA. Thus, our data demonstrate that h-GAAP modulates intracellular Ca²⁺ fluxes induced by both physiological and apoptotic stimuli.     

Conjugated eicosapentaenoic acid inhibits human topoisomerase IB with a mechanism different from camptothecin

Conjugated eicosapentaenoic acid (cEPA) has been found to have antitumor effects which has been ascribed to their ability to inhibit DNA topoisomerases and DNA polymerases. We here show that cEPA inhibits the catalytic activity of human topoisomerase I, but unlike camptothecin it does not stabilize the cleavable complex, indicating a different mechanism of action. cEPA inhibits topoisomerase by impeding the catalytic cleavage of the DNA substrate as demonstrated using specific oligonucleotide substrates, and prevents the stabilization of the cleavable complex by camptothecin. Preincubation of the inhibitor with the enzyme is required to obtain complete inhibition. Molecular docking simulations indicate that the preferred cEPA binding site is proximal to the active site with the carboxylic group strongly interacting with the positively charged K443 and K587. Taken together the results indicate that cEPA inhibitor does not prevent DNA binding but inhibits DNA cleavage, binding in a region close to the topoisomerase active site.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21^Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient''s selection and monitoring response, as well as targets for improved combination therapy.     

Intra-aortic Filtration in Cardiac Surgery: An Effective Method to Reduce Neurologic Injury in High-Risk Patients

Cardiac surgery is associated with a significant risk of adverse outcomes, particularly neurologic and renal. Embolic events are the primary source of these deleterious consequences. Intraaortic filtration is the only current technology shown to effectively capture particulates released during cardiac procedures and decrease morbidity and mortality. Although most surgical candidates may potentially benefit from intraaortic filtration, some patients are more likely to experience improved outcomes. Based on the evidence reported in the literature and the extensive experience of the authors, the following opinion details the authors' rationale and recommendations for patient selection for intraaortic filtration during cardiac surgery.     

Investigating the Conformational Stability of Prion Strains through a Kinetic Replication Model

Prion proteins are known to misfold into a range of different aggregated forms, showing different phenotypic and pathological states. Understanding strain specificities is an important problem in the field of prion disease. Little is known about which PrP^Sc structural properties and molecular mechanisms determine prion replication, disease progression and strain phenotype. The aim of this work is to investigate, through a mathematical model, how the structural stability of different aggregated forms can influence the kinetics of prion replication. The model-based results suggest that prion strains with different conformational stability undergoing in vivo replication are characterizable in primis by means of different rates of breakage. A further role seems to be played by the aggregation rate (i.e. the rate at which a prion fibril grows). The kinetic variability introduced in the model by these two parameters allows us to reproduce the different characteristic features of the various strains (e.g., fibrils' mean length) and is coherent with all experimental observations concerning strain-specific behavior.     

Wild grapevine: silvestris,hybrids or cultivars that escaped from vineyards? Molecular evidence in Sardinia

Vitis vinifera ssp. silvestris, the spontaneous subspecies of V. vinifera L., is believed to be the ancestor of present grapevine cultivars. In this work, polymorphism at 13 SSR loci was investigated to answer the following key question: are wild plants (i) true silvestris, (ii) hybrids between wild and cultivated plants or (iii) or ‘escapes’ from vineyards? In particular, the objective of the present study was to identify truly wild individuals and to search for possible hybridization events. The study was performed in Sardinia, the second largest island in the Mediterranean Sea, which is characterized by a large and well‐described number of both grape cultivars and wild populations. This region was ideal for the study because of its spatial isolation and, consequently, limited contamination from outside material. The results of this study show that domesticated and wild grapevine germplasms are genetically divergent and thus are real silvestris. Pure lineages (both domesticated and wild) show very high average posterior probabilities of assignment to their own clusters, with a low level of introgression.     

Integrin alphaIIbbeta3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site-were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the alphaIIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming--i.e., ectodomain conformational changes and oligomerization induced by incubation at 35 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.     

The threshold bootstrap clustering: a new approach to find families or transmission clusters within molecular quasispecies

Background

Phylogenetic methods produce hierarchies of molecular species, inferring knowledge about taxonomy and evolution. However, there is not yet a consensus methodology that provides a crisp partition of taxa, desirable when considering the problem of intra/inter-patient quasispecies classification or infection transmission event identification. We introduce the threshold bootstrap clustering (TBC), a new methodology for partitioning molecular sequences, that does not require a phylogenetic tree estimation.

Methodology/Principal Findings

The TBC is an incremental partition algorithm, inspired by the stochastic Chinese restaurant process, and takes advantage of resampling techniques and models of sequence evolution. TBC uses as input a multiple alignment of molecular sequences and its output is a crisp partition of the taxa into an automatically determined number of clusters. By varying initial conditions, the algorithm can produce different partitions. We describe a procedure that selects a prime partition among a set of candidate ones and calculates a measure of cluster reliability. TBC was successfully tested for the identification of type-1 human immunodeficiency and hepatitis C virus subtypes, and compared with previously established methodologies. It was also evaluated in the problem of HIV-1 intra-patient quasispecies clustering, and for transmission cluster identification, using a set of sequences from patients with known transmission event histories.

Conclusion

TBC has been shown to be effective for the subtyping of HIV and HCV, and for identifying intra-patient quasispecies. To some extent, the algorithm was able also to infer clusters corresponding to events of infection transmission. The computational complexity of TBC is quadratic in the number of taxa, lower than other established methods; in addition, TBC has been enhanced with a measure of cluster reliability. The TBC can be useful to characterise molecular quasipecies in a broad context.