DNA intercalative 4-butylaminopyrimido4',5':4,5]thieno(2,3-b)quinoline induces cell cycle arrest and apoptosis in leukemia cells

DNA intercalators are one of the interesting groups in cancer chemotherapy. The development of novel anticancer small molecule has gained remarkable interest over the last decade. In this study, we synthesized and investigated the ability of a tetracyclic-condensed quinoline compound, 4-butylaminopyrimido4',5':4,5]thieno(2,3-b)quinoline (BPTQ), to interact with double-stranded DNA and inhibit cancer cell proliferation. Circular dichroism, topological studies, molecular docking, absorbance, and fluorescence spectral titrations were employed to study the interaction of BPTQ with DNA. Cytotoxicity was studied by performing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assay. Further, cell cycle analysis by flow cytometry, annexin V staining, mitochondrial membrane potential assay, DNA fragmentation, and western blot analysis were used to elucidate the mechanism of action of BPTQ at the cellular level. Spectral, topological, and docking studies confirmed that BPTQ is a typical intercalator of DNA. BPTQ induces dose-dependent inhibitory effect on the proliferation of cancer cells by arresting cells at S and G2/M phase. Further, BPTQ activates the mitochondria-mediated apoptosis pathway, as explicated by a decrease in mitochondrial membrane potential, increase in the Bax:Bcl-2 ratio, and activation of caspases. These results confirmed that BPTQ is a DNA intercalative anticancer molecule, which could aid in the development of future cancer therapeutic agents.

Novel anti IGFBP2 single chain variable fragment inhibits glioma cell migration and invasion

Insulin like growth factor binding protein 2 (IGFBP2) is highly up regulated in glioblastoma (GBM) tissues and has been one of the prognostic indicators. There are compelling evidences suggesting important roles for IGFBP2 in glioma cell proliferation, migration and invasion. Extracellular IGFBP2 through its carboxy terminal arginine glycine aspartate (RGD) motif can bind to cell surface alpha 5 beta 1 integrins and activate pathways downstream to integrin signaling. This IGFBP2 activated integrin signaling is known to play a crucial role in IGFBP2 mediated invasion of glioma cells. Hence a molecular inhibitor of carboxy terminal domain of IGFBP2 which can inhibit IGFBP2-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of IGFBP2, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I (Library size 1.47 x 10(8)) and Tomlinson J (Library size 1.37 x 10(8)) using human recombinant IGFBP2. After screening we obtained three IGFBP2 specific binders out of which one scFv B7J showed better binding to IGFBP2 at its carboxy terminal domain, blocked IGFBP2-cell surface association, reduced activity of matrix metalloprotease 2 in the conditioned medium of glioma cells and inhibited IGFBP2 induced migration and invasion of glioma cells. We demonstrate for the first time that in vitro inhibition of extracellular IGFBP2 activity by using human scFv results in significant reduction of glioma cell migration and invasion. Therefore, the inhibition of IGFBP2 can serve as a potential therapeutic strategy in the management of GBM.

A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells

Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two-and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model.

Recent advances in engineering topography mediated antibacterial surfaces

The tendency of bacterial cells to adhere and colonize a material surface leading to biofilm formation is a fundamental challenge underlying many different applications including microbial infections associated with biomedical devices and products. Although, bacterial attachment to surfaces has been extensively studied in the past, the effect of surface topography on bacteria-material interactions has received little attention until more recently. We review the recent progress in surface topography based approaches for engineering antibacterial surfaces. Biomimicry of antibacterial surfaces in nature is a popular strategy. Whereas earlier endeavors in the field aimed at minimizing cell attachment, more recent efforts have focused on developing bactericidal surfaces. However, not all such topography mediated bactericidal surfaces are necessarily cytocompatible thus underscoring the need for continued efforts for research in this area for developing antibacterial and yet cytocompatible surfaces for use in implantable biomedical applications. This mini-review provides a brief overview of the current strategies and challenges in the emerging field of topography mediated antibacterial surfaces.

Definition of a serum marker panel for glioblastoma discrimination and identification of Interleukin 1 beta in the microglial secretome as a novel mediator of endothelial cell survival induced by C-reactive protein

Glioblastoma (GBM) is the most common malignant adult primary brain tumor. We profiled 724 cancer-associated proteins in sera of healthy individuals (n = 27) and GBM (n = 28) using antibody microarray. While 69 proteins exhibited differential abundance in GBM sera, a three-marker panel (LYAM1, BHE40 and CRP) could discriminate GBM sera from that of healthy donors with an accuracy of 89.7% and p < 0.0001. The high abundance of C-reactive protein (CRP) in GBM sera was confirmed in 264 independent samples. High levels of CRP protein was seen in GBM but without a change in transcript levels suggesting a non-tumoral origin. Glioma-secreted Interleukin 6 (IL6) was found to induce hepatocytes to secrete CRP, involving JAK-STAT pathway. The culture supernatant from CRP-treated microglial cells induced endothelial cell survival under nutrient-deprivation condition involving CRP-Fc gamma RIII signaling cascade. Transcript profiling of CRP-treated microglial cells identified Interleukin 1 beta (IL1 beta) present in the microglial secretome as the key mediator of CRP-induced endothelial cell survival. IL1 beta neutralization by antibody-binding or siRNA-mediated silencing in microglial cells reduced the ability of the supernatant from CRP-treated microglial cells to induce endothelial cell survival. Thus our study identifies a serum based three-marker panel for GBM diagnosis and provides leads for developing targeted therapies. Biological significance A complex antibody microarray based serum marker profiling identified a three-marker panel - LYAM1, BHE40 and CRP as an accurate discriminator of glioblastoma sera from that of healthy individuals. CRP protein is seen in high levels without a concomitant increase of CRP transcripts in glioblastoma. Glioma-secreted IL6 induced hepatocytes to produce CRP in a JAK-STAT signaling dependent manner. CRP induced microglial cells to release IL1 beta which in turn promoted endothelial cell survival. This study, besides defining a serum panel for glioblastoma discrimination, identified IL1 beta as a potential candidate for developing targeted therapy. (C) 2015 Elsevier B.V. All rights reserved.

Microhomology-mediated end joining is the principal mediator of double-strand break repair during mitochondrial DNA lesions

Mitochondrial DNA (mtDNA) deletions are associated with various mitochondrial disorders. The deletions identified in humans are flanked by short, directly repeated mitochondrial DNA sequences; however, the mechanism of such DNA rearrangements has yet to be elucidated. In contrast to nuclear DNA (nDNA), mtDNA is more exposed to oxidative damage, which may result in double-strand breaks (DSBs). Although DSB repair in nDNA is well studied, repair mechanisms in mitochondria are not characterized. In the present study, we investigate the mechanisms of DSB repair in mitochondria using in vitro and ex vivo assays. Whereas classical NHEJ (C-NHEJ) is undetectable, microhomology-mediated alternative NHEJ efficiently repairs DSBs in mitochondria. Of interest, robust microhomology-mediated end joining (MMEJ) was observed with DNA substrates bearing 5-, 8-, 10-, 13-, 16-, 19-, and 22-nt microhomology. Furthermore, MMEJ efficiency was enhanced with an increase in the length of homology. Western blotting, immunoprecipitation, and protein inhibition assays suggest the involvement of CtIP, FEN1, MRE11, and PARP1 in mitochondrial MMEJ. Knock-down studies, in conjunction with other experiments, demonstrated that DNA ligase III, but not ligase IV or ligase I, is primarily responsible for the final sealing of DSBs during mitochondrial MMEJ. These observations highlight the central role of MMEJ in maintenance of mammalian mitochondrial genome integrity and is likely relevant for deletions observed in many human mitochondrial disorders.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Peptide-utilizing carbon starvation gene yjiY is required for flagella-mediated infection caused by Salmonella

Peptide metabolism forms an important part of the metabolic network of Salmonella and to acquire these peptides the pathogen possesses a number of peptide transporters. Whilst various peptide transporters known in Salmonella are well studied, very little is known about the carbon starvation (cst) genes cstA and yjiY, which are also predicted to be involved in peptide metabolism. We investigated the role of these genes in the metabolism and pathogenesis of Salmonella, and demonstrated for the first time, to the best of our knowledge, that cst genes actually participate in transport of specific peptides in Salmonella. Furthermore, we established that the carbon starvation gene yjiY affects the expression of flagella, leading to poor adhesion of the bacterium to host cells. In contrast to the previously reported role of cstA in virulence of Salmonella in Caenorhabditis elegans, we showed that yjiY is required for successful colonization of Salmonella in the mouse gut. Thus, cst genes not only contribute to the metabolism of Salmonella, but also influence its virulence.

Anaesthetic impairment of immune function is mediated via GABA(A) receptors.

BACKGROUND: GABA(A) receptors are members of the Cys-loop family of neurotransmitter receptors, proteins which are responsible for fast synaptic transmission, and are the site of action of wide range of drugs. Recent work has shown that Cys-loop receptors are present on immune cells, but their physiological roles and the effects of drugs that modify their function in the innate immune system are currently unclear. We are interested in how and why anaesthetics increase infections in intensive care patients; a serious problem as more than 50% of patients with severe sepsis will die. As many anaesthetics act via GABA(A) receptors, the aim of this study was to determine if these receptors are present on immune cells, and could play a role in immunocompromising patients. PRINCIPAL FINDINGS: We demonstrate, using RT-PCR, that monocytes express GABA(A) receptors constructed of α1, α4, β2, γ1 and/or δ subunits. Whole cell patch clamp electrophysiological studies show that GABA can activate these receptors, resulting in the opening of a chloride-selective channel; activation is inhibited by the GABA(A) receptor antagonists bicuculline and picrotoxin, but not enhanced by the positive modulator diazepam. The anaesthetic drugs propofol and thiopental, which can act via GABA(A) receptors, impaired monocyte function in classic immunological chemotaxis and phagocytosis assays, an effect reversed by bicuculline and picrotoxin. SIGNIFICANCE: Our results show that functional GABA(A) receptors are present on monocytes with properties similar to CNS GABA(A) receptors. The functional data provide a possible explanation as to why chronic propofol and thiopental administration can increase the risk of infection in critically ill patients: their action on GABA(A) receptors inhibits normal monocyte behaviour. The data also suggest a potential solution: monocyte GABA(A) receptors are insensitive to diazepam, thus the use of benzodiazepines as an alternative anesthetising agent may be advantageous where infection is a life threatening problem.

Nonlinear Mechanical Modeling Of Cell Adhesion

Cell adhesion, which is mediated by the receptor-ligand bonds, plays an essential role in various biological processes. Previous studies often described the force-extension relationship of receptor-ligand bond with linear assumption. However, the force-extension relationship of the bond is intrinsically nonlinear, which should have significant influence on the mechanical behavior of cell adhesion. In this work, a nonlinear mechanical model for cell adhesion is developed, and the adhesive strength was studied at various bond distributions. We find that the nonlinear mechanical behavior of the receptor-ligand bonds is crucial to the adhesive strength and stability. This nonlinear behavior allows more bonds to achieve large bond force simultaneously, and therefore the adhesive strength becomes less sensitive to the change of bond density at the outmost periphery of the adhesive area. In this way, the strength and stability of cell adhesion are soundly enhanced. The nonlinear model describes the cell detachment behavior better than the linear model. (C) 2007 Elsevier Ltd. All rights reserved.

Influence of nutrient intake on antioxidant capacity, muscle damage and white blood cell count in female soccer players

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Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.

Ca2+ Influx and Tyrosine Kinases Trigger Bordetella Adenylate Cyclase Toxin (ACT) Endocytosis. Cell Physiology and Expression of the CD11b/CD18 Integrin Major Determinants of the Entry Route

Humans infected with Bordetella pertussis, the whooping cough bacterium, show evidences of impaired host defenses. This pathogenic bacterium produces a unique adenylate cyclase toxin (ACT) which enters human phagocytes and catalyzes the unregulated formation of cAMP, hampering important bactericidal functions of these immune cells that eventually cause cell death by apoptosis and/or necrosis. Additionally, ACT permeabilizes cells through pore formation in the target cell membrane. Recently, we demonstrated that ACT is internalised into macrophages together with other membrane components, such as the integrin CD11b/CD18 (CR3), its receptor in these immune cells, and GM1. The goal of this study was to determine whether ACT uptake is restricted to receptor-bearing macrophages or on the contrary may also take place into cells devoid of receptor and gain more insights on the signalling involved. Here, we show that ACT is rapidly eliminated from the cell membrane of either CR3-positive as negative cells, though through different entry routes, which depends in part, on the target cell physiology and characteristics. ACT-induced Ca2+ influx and activation of non-receptor Tyr kinases into the target cell appear to be common master denominators in the different endocytic strategies activated by this toxin. Very importantly, we show that, upon incubation with ACT, target cells are capable of repairing the cell membrane, which suggests the mounting of an anti-toxin cell repair-response, very likely involving the toxin elimination from the cell surface.

Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active "soluble AC''. The calpain-mediated ACT processing allows trafficking of the "soluble AC'' domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP "pools'', which would play different roles in the cell pathophysiology.