Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.     

CD14 controls the LPS-induced endocytosis of Toll-like receptor 4

The transport of Toll-like Receptors (TLRs) to various organelles has emerged as an essential means by which innate immunity is regulated. While most of our knowledge is restricted to regulators that promote the transport of newly synthesized receptors, the regulators that control TLR transport after microbial detection remain unknown. Here, we report that the plasma membrane localized Pattern Recognition Receptor (PRR) CD14 is required for the microbe-induced endocytosis of TLR4. In dendritic cells, this CD14-dependent endocytosis pathway is upregulated upon exposure to inflammatory mediators. We identify the tyrosine kinase Syk and its downstream effector PLCγ2 as important regulators of TLR4 endocytosis and signaling. These data establish that upon microbial detection, an upstream PRR (CD14) controls the trafficking and signaling functions of a downstream PRR (TLR4). This innate immune trafficking cascade illustrates how pathogen detection systems operate to induce both membrane transport and signal transduction.     

Simulation of LV pacemaker lead in marginal vein: potential risk factors for acute dislodgement

Although left ventricular (LV) coronary sinus lead dislodgement remains a problem, the risk factors for dislodgement have not been clearly defined. In order to identify potential risk factors for acute lead dislodgement, we conducted dynamic finite element simulations of pacemaker lead dislodgement in marginal LV vein. We considered factors such as mismatch in lead and vein diameters, velocity of myocardial motion, branch angle between the insertion vein and the coronary sinus, degree of slack, and depth of insertion. The results show that large lead-to-vein diameter mismatch, rapid myocardial motion, and superficial insertion are potential risk factors for lead dislodgement. In addition, the degree of slack presents either a positive or negative effect on dislodgement risk depending on the branch angle. The prevention of acute lead dislodgment can be enforced by inducing as much static friction force as possible at the lead-vein interface, while reducing the external force. If the latter exceeds the former, dislodgement will occur. The present findings underscore the major risk factors for lead dislodgment, which may improve implantation criterion and future lead design.     

Listeria monocytogenes Behaviour in Presence of Non-UV-Irradiated Titanium Dioxide Nanoparticles

Listeria monocytogenes is the agent of listeriosis, a food-borne disease. It represents a serious problem for the food industry because of its environmental persistence mainly due to its ability to form biofilm on a variety of surfaces. Microrganisms attached on the surfaces are a potential source of contamination for environment and animals and humans. Titanium dioxide nanoparticles (TiO₂ NPs) are used in food industry in a variety of products and it was reported that daily exposure to these nanomaterials is very high. Anti-listerial activity of TiO₂ NPs was investigated only with UV-irradiated nanomaterials, based on generation of reactive oxigen species (ROS) with antibacterial effect after UV exposure. Since both Listeria monocytogenes and TiO₂ NPs are veicolated with foods, this study explores the interaction between Listeria monocytogenes and non UV-irradiated TiO₂ NPs, with special focus on biofilm formation and intestinal cell interaction. Scanning electron microscopy and quantitative measurements of biofilm mass indicate that NPs influence both production and structural architecture of listerial biofilm. Moreover, TiO₂ NPs show to interfere with bacterial interaction to intestinal cells. Increased biofilm production due to TiO₂ NPs exposure may favour bacterial survival in environment and its transmission to animal and human hosts.     

Functional characterization of the insulin-like growth factor I receptor on Jurkat T cells

Insulin-like growth factor I (IGF-I) has been shown to be important in the maintenance, development, and proliferation of various types of leukocytes, particularly T cells. Radio-receptor binding assays demonstrate that Jurkat T cells bind ¹²⁵I-IGF-I with an affinity of 1.77 nM (K_d) and express approximately 230 receptors/cell. Specificity studies show insulin also binds the IGF-I receptor with an affinity 20-fold lower than that of IGF-I. Interaction of IGF-I with its receptor on Jurkat T cells induces the phosphorylation of tyrosine kinase which is detectable by Western blotting. The 95,000 MW protein detected is equivalent to the molecular weight of the β chain of the IGF-I receptor described in other types of cells. These studies characterize the binding of IGF-I to its receptor on Jurkat T cells, demonstrate that IGF-I binding induces tyrosine phosphorylation, and support the hypothesis that IGF-I is important in the induction of T cell activation.     

Potential immunoregulatory role of heme oxygenase-1 in human milk: a combined biochemical and molecular modeling approach

Human milk contains biological factors that are involved in a newborn's growth and immune system regulation. By integrating standard biochemical experimental protocols with computational methods, the present study investigates the presence of heme oxygenase-1 (HO-1), a cytoprotective enzyme, in human milk at different levels of maturation and in milk formulae. Furthermore, we evaluated cytokine and glutathione S-transferase (GSH) levels. Samples were collected from colostrum (on Day 1 after birth), from transition milk (on Postdelivery Days 7 and 14) and from mature milk (on Day 30 after delivery) in 14 healthy women. HO-1 protein, GSH and cytokines levels were measured using enzyme-linked immunosorbent assay and flow cytometry. HO-1 protein levels were significantly higher in colostrum (1.33 ng/ml; 5th centile 0.92; 95th centile 2.38) and in transition milk at 14 days (0.97 ng/ml; 5th centile 0.87; 95th centile 1.45) than in mature milk (0.9 ng/ml; 5th centile 0.8; 95th centile 1.38). Levels of HO-1 in milk formulae were similar to those in colostrum. No significant differences in GSH content were observed in mature milk, transition milk and colostrum, whereas significantly higher GSH levels were observed in milk formulae. No significant levels of cytokines, with the exception of interleukin-8, were found. Computational studies on the possible interactions between HO-1 and CD91 were carried out by a battery of softwares, namely, GRAMM (version 1.03), DALI, CLUSTALW (version 2.0), PatchDock and FireDock, mutually counterchecking and validating each other. The computational results, the strong convergence (to the same “solution”) of which finally leads to an “experimental-like” character, showed that HO-1 may bind to CD91, thus suggesting its major role as a new chaperokine in immune response regulation. These findings, which connect and integrate biochemical data and computational data interpretation, represent a synergistic and powerful means of conducting biological research.     

Combining NMR and molecular modelling in a drug delivery context: investigation of the multi-mode inclusion of a new NPY-5 antagonist bromobenzenesulfonamide into beta-cyclodextrin

NMR spectroscopic and molecular modelling methods have been employed to describe the complexation of trans-N-4-[N'-(4-chlorobenzoyl)hydrazinocarbonyl]cyclohexylmethyl-4-bromobenzenesulfonamide, a new chemotype of NPY-5 antagonist, and beta-cyclodextrin, revealing the coexistence of two different kinds of 1:1 complexes where conformational changes of the guest compound with respect to the free state are also detected.     

The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis.     

Simulation of mechanical environment in active lead fixation: effect of fixation helix size

The risk of myocardial penetration due to active-fixation screw-in type pacing leads has been reported to increase as the helix electrodes become smaller. In order to understand the contributing factors for lead penetration, we conducted finite element analyses of acute myocardial micro-damage induced by a pacemaker lead screw-in helix electrode. We compared the propensity for myocardial micro-damage of seven lead designs including a baseline model, three modified designs with various helix wire cross-sectional diameters, and three modified designs with different helix diameters. The comparisons show that electrodes with a smaller helix wire diameter cause more severe micro-damage to the myocardium in the early stage. The damage severity, represented by the volume of failed elements, is roughly the same in the middle stage, whereas in the later stage the larger helix wire diameter generally causes more severe damage. The onset of myocardial damage is not significantly affected by the helix diameter. As the helix diameter increases, however, the extent of myocardial damage increases accordingly. The present findings identified several of the major risk factors for myocardial damage whose consideration for lead use and design might improve acute and chronic lead performance.