Quantifying the phosphorylation timescales of receptor-ligand complexes: a Markovian matrix-analytic approach.

Cells interact with the extracellular environment by means of receptor molecules on their surface. Receptors can bind different ligands, leading to the formation of receptor-ligand complexes. For a subset of receptors, called receptor tyrosine kinases, binding to ligand enables sequential phosphorylation of intra-cellular residues, which initiates a signalling cascade that regulates cellular function and fate. Most mathematical modelling approaches employed to analyse receptor signalling are deterministic, especially when studying scenarios of high ligand concentration or large receptor numbers. There exist, however, biological scenarios where low copy numbers of ligands and/or receptors need to be considered, or where signalling by a few bound receptor-ligand complexes is enough to initiate a cellular response. Under these conditions stochastic approaches are appropriate, and in fact, different attempts have been made in the literature to measure the timescales of receptor signalling initiation in receptor-ligand systems. However, these approaches have made use of numerical simulations or approximations, such as moment-closure techniques. In this paper, we study, from an analytical perspective, the stochastic times to reach a given signalling threshold for two receptor-ligand models. We identify this time as an extinction time for a conveniently defined auxiliary absorbing continuous time Markov process, since receptor-ligand association/dissociation events can be analysed in terms of quasi-birth-and-death processes. We implement algorithmic techniques to compute the different order moments of this time, as well as the steady-state probability distribution of the system. A novel feature of the approach introduced here is that it allows one to quantify the role played by each kinetic rate in the timescales of signal initiation, and in the steady-state probability distribution of the system. Finally, we illustrate our approach by carrying out numerical studies for the vascular endothelial growth factor and one of its receptors, the vascular endothelial growth factor receptor of human endothelial cells.

Heuristics-Based Trust Estimation in Multiagent Systems Using Temporal Difference Learning.

The application of multiagent system (MAS) is becoming increasing popular as it allows agents in a system to pool resources together to achieve a common objective. A vital part of the MAS is the teamwork cooperation through the sharing of information and resources among the agents to optimize their efforts in accomplishing given objectives. A critical part of the teamwork effort is the ability to trust each other when executing any task to ensure efficient and successful cooperation. This paper presents the development of a trust estimation model that could empirically evaluate the trust of an agent in MAS. The proposed model is developed using temporal difference learning by incorporating the concept of Markov games and heuristics to estimate trust. Simulation experiments are conducted to test and evaluate the performance of the developed model against some of the recently reported model in the literature. The simulation experiments indicate that the developed model performs better in terms of accuracy and efficiency in estimating trust.

Chapter Eight - Ubiquitin-Mediated Regulation of Cellular Responses to Vascular Endothelial Growth Factors.

Vascular endothelial growth factors (VEGFs) bind receptor tyrosine kinases (VEGFRs) to regulate vascular and lymphatic development and homeostasis. Such interactions are also implicated in pathological conditions ranging from cancer to heart disease. Increasingly, it is evident that ubiquitination plays a central role in regulating VEGFR function and the cellular response to VEGFs. E1, E2, and E3 ubiquitination enzymes deliver ubiquitin-specific modifications to protein substrates but there is much debate on the exact enzymes involved. The deubiquitinase (DUB) enzymes remove such modifications and are attracting increasing interest as potential therapeutic targets in a host of different disease states. Understanding how these enzyme families regulate VEGFR function in different cells and tissues is a major challenge. An understanding of the fundamental mechanisms underlying such biochemical regulation is needed for providing new therapeutics that target diseases such as cancer and heart disease.

Heuristics for multiobjective optimization of two-sided assembly line systems.

Products such as cars, trucks, and heavy machinery are assembled by two-sided assembly line. Assembly line balancing has significant impacts on the performance and productivity of flow line manufacturing systems and is an active research area for several decades. This paper addresses the line balancing problem of a two-sided assembly line in which the tasks are to be assigned at L side or R side or any one side (addressed as E). Two objectives, minimum number of workstations and minimum unbalance time among workstations, have been considered for balancing the assembly line. There are two approaches to solve multiobjective optimization problem: first approach combines all the objectives into a single composite function or moves all but one objective to the constraint set; second approach determines the Pareto optimal solution set. This paper proposes two heuristics to evolve optimal Pareto front for the TALBP under consideration: Enumerative Heuristic Algorithm (EHA) to handle problems of small and medium size and Simulated Annealing Algorithm (SAA) for large-sized problems. The proposed approaches are illustrated with example problems and their performances are compared with a set of test problems.

PCB drill path optimization by combinatorial cuckoo search algorithm.

Optimization of drill path can lead to significant reduction in machining time which directly improves productivity of manufacturing systems. In a batch production of a large number of items to be drilled such as printed circuit boards (PCB), the travel time of the drilling device is a significant portion of the overall manufacturing process. To increase PCB manufacturing productivity and to reduce production costs, a good option is to minimize the drill path route using an optimization algorithm. This paper reports a combinatorial cuckoo search algorithm for solving drill path optimization problem. The performance of the proposed algorithm is tested and verified with three case studies from the literature. The computational experience conducted in this research indicates that the proposed algorithm is capable of efficiently finding the optimal path for PCB holes drilling process.

Experimental Investigation of the Effect of the Driving Voltage of an Electroadhesion Actuator.

This paper investigates the effect of driving voltage on the attachment force of an electroadhesion actuator, as the existing literature on the saturation of the adhesive force at a higher electric field is incomplete. A new type of electroadhesion actuator using normally available materials, such as aluminum foil, PVC tape and a silicone rubber sheet used for keyboard protection, has been developed with a simple layered structure that is capable of developing adhesive force consistently. The developed actuator is subjected to the experiment for the evaluation of various test surfaces; aluminum, brick, ceramic, concrete and glass. The driving high voltage is varied in steps to determine the characteristics of the output holding force. Results show a quadratic relation between F (adhesion force) and V (driving voltage) within the 2 kV range. After this range, the F-V responses consistently show a saturation trend at high electric fields. Next, the concept of the leakage current that can occur in the dielectric material and the corona discharge through air has been introduced. Results show that the voltage level, which corresponds to the beginning of the supply current, matches well with the beginning of the force saturation. With the confirmation of this hypothesis, a working model for electroadhesion actuation is proposed. Based on the experimental results, it is proposed that such a kind of actuator can be driven within a range of optimum high voltage to remain electrically efficient. This practice is recommended for the future design, development and characterization of electroadhesion actuators for robotic applications.

The LOX-1 Scavenger Receptor and Its Implications in the Treatment of Vascular Disease.

Cardiovascular disease is the leading cause of death. The disease is due to atherosclerosis which is characterized by lipid and fat accumulation in arterial blood vessel walls. A key causative event is the accumulation of oxidised low density lipoprotein particles within vascular cells, and this is mediated by scavenger receptors. One such molecule is the LOX-1 scavenger receptor that is expressed on endothelial, vascular smooth muscle, and lymphoid cells including macrophages. LOX-1 interaction with OxLDL particles stimulates atherosclerosis. LOX-1 mediates OxLDL endocytosis via a clathrin-independent internalization pathway. Transgenic animal model studies show that LOX-1 plays a significant role in atherosclerotic plaque initiation and progression. Administration of LOX-1 antibodies in cellular and animal models suggest that such intervention inhibits atherosclerosis. Antiatherogenic strategies that target LOX-1 function using gene therapy or small molecule inhibitors would be new ways to address the increasing incidence of vascular disease in many countries.

Indolinones and anilinophthalazines differentially target VEGF-A- and basic fibroblast growth factor-mediated responses in primary human endothelial cells.

BACKGROUND AND PURPOSE: The potent pro-angiogenic growth factors VEGF-A and basic fibroblast growth factor (bFGF) exert their effects by binding VEGF receptor 2 and FGF receptor tyrosine kinases, respectively. Indolinones (e.g. SU5416 and Sutent) and anilinophthalazines (e.g. PTK787) are potent small molecule inhibitors of VEGFR2 and other tyrosine kinases, but their effects on VEGF-A- and bFGF-stimulated endothelial responses are unclear. Here we assess the ability of these compounds to inhibit pro-angiogenic responses through perturbation of receptor activity and endothelial function(s). EXPERIMENTAL APPROACH: We used in silico modelling, in vitro tyrosine kinase assays, biochemistry and microscopy to evaluate the effects of small molecules on receptor tyrosine kinase activation and intracellular signalling. Primary human endothelial cells were used to assess intracellular signalling, cell migration, proliferation and tubulogenesis. KEY RESULTS: We predicted that the anilinophthalazine PTK787 binds the tyrosine kinase activation loop whereas indolinones are predicted to bind within the hinge region of the split kinase domain. Sutent is a potent inhibitor of both VEGFR2 and FGFR1 tyrosine kinase activity in vitro. The compounds inhibit both ligand-dependent and -independent VEGFR2 trafficking events, are not selective for endothelial cell responses and inhibit both VEGF-A- and bFGF-mediated migration, wound healing and tubulogenesis at low concentrations. CONCLUSIONS AND IMPLICATIONS; We propose that these compounds have novel properties including inhibition of bFGF-mediated endothelial responses and perturbation of VEGFR2 trafficking. Differential inhibitor binding to receptor tyrosine kinases translates into more potent inhibition of bFGF- and VEGF-A-mediated intracellular signalling, cell migration and tubulogenesis. Indolinones and anilinophthalazines thus belong to a class of multi-kinase inhibitors that show clinical efficacy in disease therapy.

Gene therapy in the treatment of peripheral arterial disease.

BACKGROUND: Peripheral arterial disease remains a significant global health burden despite revolutionary improvements in endovascular techniques over the past decade. The durability of intervention for critical limb ischaemia is poor, and the condition is associated with high morbidity and mortality rates. To address this deficiency, alternative therapeutic options are being explored. Advances in the fields of gene therapy and therapeutic angiogenesis have led to these being advocated as potential future treatments. METHODS: Relevant medical literature from PubMed, Embase, the Cochrane Library and Google Scholar from the inception of these databases to June 2011 was reviewed. RESULTS: Encouraging outcomes in preclinical trials using a variety of proangiogenic growth factors have led to numerous efficacy and safety studies. However, no clinical study has shown significant benefit for gene therapy over placebo. CONCLUSION: Identifying the optimal site for gene delivery, choice of vector and duration of treatment is needed if gene therapy is to become a credible therapeutic option for peripheral arterial disease.

A combinatorial in silico and cellular approach to identify a new class of compounds that target VEGFR2 receptor tyrosine kinase activity and angiogenesis.

BACKGROUND AND PURPOSE: Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target for the treatment of diseases such as cancer. Small-molecule VEGFR2 inhibitors of a variety of chemical classes are currently under development or in clinical use. In this study, we describe the de novo design of a new generation pyrazole-based molecule (JK-P3) that targets VEGFR2 kinase activity and angiogenesis. EXPERIMENTAL APPROACH: JK-P compound series were designed using de novo structure-based identification methods. Compounds were tested in an in vitro VEGFR2 kinase assay. Using primary endothelial cells, JK-P compounds were assessed for their ability to inhibit VEGF-A-stimulated VEGFR2 activation and intracellular signalling. We tested these compounds in cell migration, proliferation and angiogenesis assays. KEY RESULTS: JK-P3 and JK-P5 were predicted to bind the VEGFR2 kinase domain with high affinity, and both compounds showed pronounced inhibition of endogenous VEGFR2 kinase activity in primary human endothelial cells. Only JK-P3 inhibited VEGF-A-stimulated VEGFR2 activation and intracellular signalling. Interestingly, JK-P3 inhibited endothelial monolayer wound closure and angiogenesis but not endothelial cell proliferation. Both compounds inhibited fibroblast growth factor receptor kinase activity in vitro, but not basic fibroblast growth factor-mediated signalling in endothelial cells. CONCLUSIONS AND IMPLICATIONS: This is the first report that describes an anti-angiogenic inhibitor based on such a pyrazole core. Using a de novo structure-based identification approach is an attractive method to aid such drug discovery. These results thus provide an important basis for the development of multi-tyrosine kinase inhibitors for clinical use in the near future.

Cytosolic phospholipase A2-alpha mediates endothelial cell proliferation and is inactivated by association with the Golgi apparatus.

Arachidonic acid and its metabolites are implicated in regulating endothelial cell proliferation. Cytosolic phospholipase A2-alpha (cPLA2alpha) is responsible for receptor-mediated arachidonic acid evolution. We tested the hypothesis that cPLA2alpha activity is linked to endothelial cell proliferation. The specific cPLA2alpha inhibitor, pyrrolidine-1, inhibited umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner. Exogenous arachidonic acid addition reversed this inhibitory effect. Inhibition of sPLA2 did not affect HUVEC proliferation. The levels of cPLA2alpha did not differ between subconfluent and confluent cultures of cells. However, using fluorescence microscopy we observed a novel, confluence-dependent redistribution of cPLA2alpha to the distal Golgi apparatus in HUVECs. Association of cPLA2alpha with the Golgi was linked to the proliferative status of HUVECs. When associated with the Golgi apparatus, cPLA2alpha activity was seen to be 87% inhibited. Relocation of cPLA2alpha to the cytoplasm and nucleus, and cPLA2alpha enzyme activity were required for cell cycle entry upon mechanical wounding of confluent monolayers. Thus, cPLA2alpha activity and function in controlling endothelial cell proliferation is regulated by reversible association with the Golgi apparatus.

The trans Golgi network is lost from cells infected with African swine fever virus.

The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249-278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.

Characterization and regulation of constitutive transport intermediates involved in trafficking from the trans-Golgi network.

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans-Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans-Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. An in vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200 nm in length. These membrane-enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

The effect of ceruloplasmin on iron release from placental (BeWo) cells; evidence for an endogenous Cu oxidase.

The mechanism of iron release from the placenta into the fetal circulation is not well understood. Ceruloplasmin, a plasma ferroxidase, has been implicated in iron efflux from a variety of cell types. The hypothesis is that circulating ceruloplasmin facilitates iron efflux by oxidizing the released Fe(II) to Fe(III) for incorporation into transferrin. We tested whether this mechanism mediates iron release from placental cells into the fetal circulation, using the BeWo cell line, a choriocarcinoma which can differentiate into a syncytium.(59)Fe release from undifferentiated or differentiated cells and from cells grown on porous filters was not stimulated by extracellular ceruloplasmin. Instead, we found that BeWo cells express an endogenous ferroxidase. The protein is membrane bound and cross-reacts with an anti-ceruloplasmin antibody, but has a different size; 100 and 140 kDa. Similar immunoreactivity was identified in first- and third-trimester human placentae. In BeWo cells, the protein has a perinuclear localization but does not entirely co-localize with markers for the endoplasmic reticulum or Golgi apparatus. We propose that this oxidase performs the same function as serum ceruloplasmin and is involved in iron release into the fetal circulation.

The manganese cation disrupts membrane dynamics along the secretory pathway.

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.

Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane.

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.