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Agent-based models have been employed to describe numerous processes in immunology. Simulations based on these types of models have been used to enhance our understanding of immunology and disease pathology. We review various agent-based models relevant to host-pathogen systems and discuss their contributions to our understanding of biological processes. We then point out some limitations and challenges of agent-based models and encourage efforts towards reproducibility and model validation., LaTeX, 12 pages, 1 EPS figure, uses document class REVTeX 4, and packages hyperref, xspace, graphics, amsmath, verbatim, and SIunits

SUMMARYAn underground nuclear explosion (UNE) couples mechanical energy into crustal rock, which propagates as seismic and acoustic waves. These different physical phenomena transport, by different pathways, to standoff detectors at varying distances. The transport pathways attenuate the original signal but in different ways. Enabled by correct statistical weighting, signal attenuation models can be used to combine these disparate sensor data to estimate the yield of an UNE. Contemporaneous statistical models, used in yield estimation, can be improved with an advanced partition of error for these physical signal propagation models. We present an advanced multivariate approach to error modelling of multiphenomenology physical signatures. In addition to measurement error, our error model represents physical model biases as random with a physics-based covariance structure. To illustrate this proposed framework, we demonstrate the estimation of explosion yield using openly available seismic and acoustic data from chemical single-point explosions.

Cancer invasion and metastasis depend on tumor-induced angiogenesis, the means by which cancer cells attract and maintain a blood supply. During angiogenesis, cellular processes are tightly coordinated by signaling molecules and their receptors. Understanding how endothelial cells synthesize multiple biochemical signals can catalyze the development of novel therapeutic strategies to combat cancer. This study is the first to propose a signal transduction model highlighting the cross-talk between key receptors involved in angiogenesis, namely the VEGF, integrin, and cadherin receptors. From experimental data, we construct a network model of receptor cross-talk and analyze its dynamics. We identify relationships between receptor activation combinations and cellular function, and show that cross-talk is crucial to phenotype determination. The network converges to a unique set of output states that correspond to known cell phenotypes: migratory, proliferating, quiescent, apoptotic, and it predicts one phenotype that challenges the “go or grow” hypothesis. Finally, we use the model to study protein inhibition and to suggest molecular targets for anti-angiogenic therapies.

This paper investigates the theoretical stability bound of a Lagrangian staggered scheme used to solve hydrodynamics equations. We present the two-dimensional (2D) wave equation as a possible model for this study and, by using the numerical radius of the amplification matrix, we prove that the family of schemes defined with two time-centering parameters is limited by a nonclassical stability bound limit defined with an analytical curve. We further show that 2D numerical experiments agree with this theoretical result.

Tuberculosis is the leading cause of death due to infectious diseases in the world to- day, and it is increasing due to co-infection with HIV-1, the causative agent of AIDS. Here, we examine the impact that HIV-1 infection has on persons with latent tuberculosis. Based on previ- ous work, we develop a mathematical model of an adaptive immune response in the lung which considers relevant immune effectors such as macrophages, various sub-populations of T-cells, and key cytokines to predict which mechanisms are important to HIV-1 infection induced reactivation of tuberculosis. Our results indicate that persons latently infected with TB who are subsequently co-infected with HIV-1 will suffer reactive TB. The mechanisms that contribute to this are essen- tially related to a completely different cytokine environment at the onset of HIV-1 infection due to the presence of Mycobacterium tuberculosis. Our analysis suggests that macrophages play an important role during co-infection and decreases in macrophage counts are coupled to a decline in CD4 + T-cells and increased viral loads. These mechanisms are also coupled to lower recruitment of T-cells and macrophages, compromising protective immunity in the lung and eventually leading to TB reactivation. These results point to potential targets for drug and vaccine therapies.

The current study evaluated the interpersonal circumplex as a theoretical model of companion animal personality and companion animal attachment. To this end, the study surveyed 266 companion animal guardians (owners)—89 reporting their most recent pet a cat and 177 reporting their most recent pet a dog—to assess the relationships between interpersonal complementarity and companion animal attachment. The study used MANOVA to evaluate differences in interpersonal traits for cats, dogs, and people who self-identified that cats or dogs were their ideal pets. Results indicated that cats—and people who identified cats as their ideal pet—were more hostile in their orientation than were dogs or people who preferred dogs. In hierarchical regression-analysis, the study also confirmed the positive relationship between interpersonal complementarity and companion-animal attachment.

A thorough understanding of determining factors in angiogenesis is a necessary step to control the development of new blood vessels. Extracellular matrix density is known to have a significant influence on cellular behaviors and consequently can regulate vessel formation. The utilization of experimental platforms in combination with numerical models can be a powerful method to explore the mechanisms of new capillary sprout formation. In this study, using an integrative method, the interplay between the matrix density and angiogenesis was investigated. Owing the fact that the extracellular matrix density is a global parameter that can affect other parameters such as pore size, stiffness, cell-matrix adhesion and cross-linking, deeper understanding of the most important biomechanical or biochemical properties of the ECM causing changes in sprout morphogenesis is crucial. Here, we implemented both computational and experimental methods to analyze the mechanisms responsible for the influence of ECM density on the sprout formation that is difficult to be investigated comprehensively using each of these single methods. For this purpose, we first utilized an innovative approach to quantify the correspondence of the simulated collagen fibril density to the collagen density in the experimental part. Comparing the results of the experimental study and computational model led to some considerable achievements. First, we verified the results of the computational model using the experimental results. Then, we reported parameters such as the ratio of proliferating cells to migrating cells that was difficult to obtain from experimental study. Finally, this integrative system led to gain an understanding of the possible mechanisms responsible for the effect of ECM density on angiogenesis. The results showed that stable and long sprouts were observed at an intermediate collagen matrix density of 1.2 and 1.9 mg/ml due to a balance between the number of migrating and proliferating cells. As a result of weaker connections between the cells and matrix, a lower collagen matrix density (0.7 mg/ml) led to unstable and broken sprouts. However, higher matrix density (2.7 mg/ml) suppressed sprout formation due to the high level of matrix entanglement, which inhibited cell migration. This study also showed that extracellular matrix density can influence sprout branching. Our experimental results support this finding.

This work explores the somewhat subtle meaning and consequences of the salient properties of the discrete, compatible formulation of Lagrangian hydrodynamics. In particular, since this formulation preserves total energy to roundoff error, the amount of error in the conservation of total energy cannot be used to gauge the internal consistency of calculations, as is often done with the older forms of this algorithm. However, the compatible formulation utilizes two definitions of zone volume: the first is the usual definition whereby the volume of a zone is defined as some prescribed function of the coordinates of the points that define it; the second is given as the integration in time of the continuity equation for zone volume as expressed in Lagrangian form. It is the use of this latter volume in the specific internal energy equation that enables total energy to be exactly conserved. These two volume definitions are generally not precisely equal. It is the analysis of this difference that forms the first part of this study. It is shown that this difference in zone volumes can be used to construct a practical internal consistency measure that not only takes the place of the lack of total energy conservation of the older forms of Lagrangian hydrodynamics, but is more general in that it can be defined on a single zone basis. It can also be used to ascertain the underlying spatial and temporal order of accuracy of any given set of calculations. The difference in these two definitions of zone volume may be interpreted as a type of entropy error. However, this entropy error is found to be significant only when a given calculation becomes numerically unstable, otherwise it remains at or far beneath truncation error levels. In fact, it can be utilized to provide an upper bound on the size of the spatial truncation error for a stable computation. It is also shown how this volume difference can be used as an indicator of numerical difficulties, since exact local conservation of total energy does not guarantee numerical stability or the quality of any numerical calculation. The discrete, compatible formulation of Lagrangian hydrodynamics utilizes a two level predictor/corrector-type of time integration scheme; a stability analysis, both analytical and numerical, is given. This analysis reveals a novel stability diagram that has not been heretofore published, and gives definitive information as to how the stabilizing corrector step should be centered in time.

To study the change in intrapulmonary bacterial growth rate over time during Gram-negative pneumonia, a two-hit model of recurrent bacterial aspiration was developed in mice. A mutant of Klebsiella pneumoniae was isolated that could be distinguished from the wild type when cultured on appropriate media. These strains were intranasally administered, 4 h apart, to mice whose lungs were quantitatively cultured 24 h later. The relative burden of each aspirated inoculum was determined, and, using the administered dose and the number of bacteria from each inoculum present at the end of the experiment, first-order growth constants for each inoculum were calculated. Results indicate that after an initial aspiration of this organism, subsequently aspirated bacteria proliferate more slowly. When two aspirations occurred 4 h apart, the bacteria aspirated first represented 96% of total lung burden at 24 h. The growth constant of the second inoculum was related to the magnitude of the first inoculum in an inverse, nonlinear fashion. When parallel experiments were performed in complement C3-deficient mice, no suppression of the second inoculum was noted, suggesting that early upregulation of antibacterial activity in the lung is a C3-mediated event.

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen-sensitive cells of the carotid body and pheochromocytoma-derived PC12 cells. TH is also regulated by the von Hippel-Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia-inducible transcription factors (HIFs) that interact with a specific hypoxia-responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFalpha ubiquitination, increased accumulation of HIFalpha proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter-reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen-sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

The ocean provides food, economic activity, and cultural value for a large proportion of humanity. Our knowledge of marine ecosystems lags behind that of terrestrial ecosystems, limiting effective protection of marine resources. We describe the outcome of 2 workshops in 2011 and 2012 to establish a list of important questions, which, if answered, would substantially improve our ability to conserve and manage the world’s marine resources. Participants included individuals from academia, government, and nongovernment organizations with broad experience across disciplines, marine ecosystems, and countries that vary in levels of development. Contributors from the fields of science, conservation, industry, and government submitted questions to our workshops, which we distilled into a list of priority research questions. Through this process, we identified 71 key questions. We grouped these into 8 subject categories, each pertaining to a broad component of marine conservation: fisheries, climate change, other anthropogenic threats, ecosystems, marine citizenship, policy, societal and cultural considerations, and scientific enterprise. Our questions address many issues that are specific to marine conservation, and will serve as a road map to funders and researchers to develop programs that can greatly benefit marine conservation. Setenta y Un Preguntas Importantes para la Conservación de la Biodiversidad Marina Resumen Los océanos proporcionan alimento, actividad económica y valor cultural para una gran porción de la humanidad. Nuestro conocimiento de los ecosistemas marinos está atrasado con respecto al que tenemos de los ecosistemas terrestres, lo que limita la protección efectiva de los recursos naturales. Describimos el resultado de dos talleres en 2011 y 2012 para establecer una lista de preguntas importantes, las cuales al ser respondidas, mejorarían sustancialmente nuestra habilidad de conservar y manejar los recursos marinos del mundo. Entre los participantes se incluyeron a individuos de la docencia, el gobierno y organizaciones no-gubernamentales, con una amplia experiencia que atraviesa disciplinas, ecosistemas marinos y países que varían en el nivel de desarrollo. Los contribuyentes de los campos de la ciencia, la conservación, la industria y el gobierno, presentaron preguntas a nuestros talleres, las cuales separamos en una lista de preguntas de investigación prioritarias. Por medio de este proceso, identificamos 71 preguntas clave. Las agrupamos en ocho categorías temáticas, cada una perteneciente a un componente amplio de la conservación marina: pesquerías, cambio climático, otras amenazas antropogénicas, ecosistemas, ciudadanía marina, política, consideraciones sociales y culturales, y la iniciativa científica. Nuestras preguntas se dirigen a muchas cuestiones que son específicas de la conservación marina, y servirán como una ruta a seguir para patrocinadores e investigadores que busquen desarrollar programas que puedan beneficiar ampliamente a la conservación marina.

Physicochemical properties of DNA, such as shape, affect protein-DNA recognition. However, the properties of DNA that are most relevant for predicting the binding sites of particular transcription factors (TFs) or classes of TFs have yet to be fully understood. Here, using a model that accurately captures the melting behavior and breathing dynamics (spontaneous local openings of the double helix) of double-stranded DNA, we simulated the dynamics of known binding sites of the TF and nucleoid-associated protein Fis in Escherichia coli. Our study involves simulations of breathing dynamics, analysis of large published in vitro and genomic datasets, and targeted experimental tests of our predictions. Our simulation results and available in vitro binding data indicate a strong correlation between DNA breathing dynamics and Fis binding. Indeed, we can define an average DNA breathing profile that is characteristic of Fis binding sites. This profile is significantly enriched among the identified in vivo E. coli Fis binding sites. To test our understanding of how Fis binding is influenced by DNA breathing dynamics, we designed base-pair substitutions, mismatch, and methylation modifications of DNA regions that are known to interact (or not interact) with Fis. The goal in each case was to make the local DNA breathing dynamics either closer to or farther from the breathing profile characteristic of a strong Fis binding site. For the modified DNA segments, we found that Fis-DNA binding, as assessed by gel-shift assay, changed in accordance with our expectations. We conclude that Fis binding is associated with DNA breathing dynamics, which in turn may be regulated by various nucleotide modifications., Author Summary Cellular transcription factors (TFs) are proteins that regulate gene expression, and thereby cellular activity and fate, by binding to specific DNA segments. The physicochemical determinants of protein-DNA binding specificity are not completely understood. Here, we report that the propensity of transient opening and re-closing of the double helix, resulting from thermal fluctuations, aka “DNA breathing” or “DNA bubbles,” can be associated with binding affinity in the case of Fis, a well-studied nucleoid-associated protein in Escherichia coli. We found that a particular breathing profile is characteristic of high-affinity Fis binding sites and that DNA fragments known to bind Fis in vivo are statistically enriched for this profile. Furthermore, we used simulations of DNA breathing dynamics to guide design of gel-shift experiments aimed at testing the idea that local breathing influences Fis binding. As a result, we show that via nucleotide modifications but without modifying nucleotides that directly contact Fis, we were able to transform a low-affinity Fis binding site into a high-affinity site and vice versa. The nucleotide modifications were designed only based on DNA breathing simulations. Our study suggests that strong Fis-DNA binding depends on DNA breathing - a novel physicochemical characteristic that could be used for prediction and rational design of TF binding sites.

Cells are a natural unit of abstraction for tissue morphogenesis. Cell-based theoretical models provide a platform for determining key elements of cell behavior responsible for multicellular and tissue-level phenomena, and for integrating molecular and tissue/organ scales. This chapter reviews a limited selection of recent developments in cell-based modeling approaches to angiogenesis. We offer an introduction to building a cell-based model with an emphasis on model validation, and illustrate this model building process in tumor angiogenesis using the cellular Potts model.

Signal transduction is part of a complex communication system responsible for regulating fundamental cellular activities, including metabolism, protein synthesis, division, migration, and survival. The capacity of cells to perceive and precisely respond to their environment is critical to development, tissue repair, and normal tissue homeostasis. Vast amounts of disparate, postgenomic data facilitated by the development of high-throughput technologies are now available and computational modeling is needed to synthesize and understand these data within their systems biology context. In this chapter, we discuss computational approaches available to model signal transduction and present several types of Boolean network models of cell signaling during tumor angiogenesis. Using these models, we investigate the role of cross-talk between chemical and stromal factors in endothelial cell phenotype determination. Understanding the principal mechanisms underpinning the functions of signalling networks will enable identification of targets for pharmacological interventions in the treatment of cancer and other angiogenesis-dependent diseases.

Cancer is arguably the ultimate complex biological system. Solid tumors are microstructured soft matter that evolves as a consequence of spatiotemporal events at the intracellular (e.g., signaling pathways, macromolecular trafficking), intercellular (e.g., cell‐cell adhesion/communication), and tissue (e.g., cell‐extracellular matrix interactions, mechanical forces) scales. To gain insight, tumor and developmental biologists have gathered a wealth of molecular, cellular, and genetic data, including immunohistochemical measurements of cell type-specific division and death rates, lineage tracing, and gain-of-function/lossof-function mutational analyses. These data are empirically extrapolated to a diagnosis/prognosis of tissue-scale behavior, e.g., for clinical decision. Integrative physical oncology (IPO) is the science that develops physically consistent mathematical approaches to address the significant challenge of bridging the nano (nm)‐micro (µm) to macro (mm, cm) scales with respect to tumor development and progression. In the current literature, such approaches are referred to as multiscale modeling. In the present article, we attempt to assess recent modeling approaches on each separate scale and critically evaluate the current ‘hybridmultiscale’ models used to investigate tumor growth in the context of brain and breast cancers.Finally, weprovide ourperspective on the further development and the impact of IPO.  2011 John Wiley & Sons, Inc. WIREs Syst Biol Med2011 DOI: 10.1002/wsbm.158

An important step in understanding gene regulation is to identify the DNA binding sites recognized by each transcription factor (TF). Conventional approaches to prediction of TF binding sites involve the definition of consensus sequences or position-specific weight matrices and rely on statistical analysis of DNA sequences of known binding sites. Here, we present a method called SiteSleuth in which DNA structure prediction, computational chemistry, and machine learning are applied to develop models for TF binding sites. In this approach, binary classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. These features are determined via molecular dynamics calculations in which we consider each base in different local neighborhoods. For each of 54 TFs in Escherichia coli, for which at least five DNA binding sites are documented in RegulonDB, the TF binding sites and portions of the non-coding genome sequence are mapped to feature vectors and used in training. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth outperforms three conventional approaches: Match, MATRIX SEARCH, and the method of Berg and von Hippel. SiteSleuth also outperforms QPMEME, a method similar to SiteSleuth in that it involves a learning algorithm. The main advantage of SiteSleuth is a lower false positive rate., Author Summary An important step in characterizing the genetic regulatory network of a cell is to identify the DNA binding sites recognized by each transcription factor (TF) protein encoded in the genome. Current computational approaches to TF binding site prediction rely exclusively on DNA sequence analysis. In this manuscript, we present a novel method called SiteSleuth, in which classifiers are trained to discriminate between true and false binding sites based on the sequence-specific chemical and structural features of DNA. According to cross-validation analysis and a comparison of computational predictions against ChIP-chip data available for the TF Fis, SiteSleuth predicts fewer estimated false positives than any of four other methods considered. A better understanding of gene regulation, which plays a central role in cellular responses to environmental changes, is a key to manipulating cellular behavior for a variety of useful purposes, as in metabolic engineering applications.

The extracellular matrix plays a critical role in orchestrating the events necessary for wound healing, muscle repair, morphogenesis, new blood vessel growth, and cancer invasion. In this study, we investigate the influence of extracellular matrix topography on the coordination of multi-cellular interactions in the context of angiogenesis. To do this, we validate our spatio-temporal mathematical model of angiogenesis against empirical data, and within this framework, we vary the density of the matrix fibers to simulate different tissue environments and to explore the possibility of manipulating the extracellular matrix to achieve pro- and anti-angiogenic effects. The model predicts specific ranges of matrix fiber densities that maximize sprout extension speed, induce branching, or interrupt normal angiogenesis, which are independently confirmed by experiment. We then explore matrix fiber alignment as a key factor contributing to peak sprout velocities and in mediating cell shape and orientation. We also quantify the effects of proteolytic matrix degradation by the tip cell on sprout velocity and demonstrate that degradation promotes sprout growth at high matrix densities, but has an inhibitory effect at lower densities. Our results are discussed in the context of ECM targeted pro- and anti-angiogenic therapies that can be tested empirically., Author Summary A cell migrating in the extracellular matrix environment has to pull on the matrix fibers to move. When the matrix is too dense, the cell secretes enzymes to degrade the matrix proteins in order to get through. And when the matrix is too sparse, the cell produces matrix proteins to locally increase the “foothold”. How cells interact with the extracellular matrix is important in many processes from wound healing to cancer invasion. We use a computational model to investigate the topography of the matrix on cell migration and coordination in the context of tumor induced new blood vessel growth. The model shows that the density of the matrix fibers can have a strong effect on the extension speed and the morphology of a new blood vessel. Further results show that matrix degradation by the cells can enhance vessel sprout extension at high matrix density, but impede sprout extension at low matrix density. These results can potentially point to new targets for pro- and anti-angiogenesis therapies.

This work describes the first cell-based model of tumor-induced angiogenesis. At the extracellular level, the model describes diffusion, uptake, and decay of tumor-secreted pro-angiogenic factor. At the cellular level, the model uses the cellular Potts model based on system-energy reduction to describe endothelial cell migration, growth, division, cellular adhesion, and the evolving structure of the stroma. Numerical simulations show: 1), different tumor-secreted pro-angiogenic factor gradient profiles dramatically affect capillary sprout morphology; 2), average sprout extension speeds depend on the proximity of the proliferating region to the sprout tip, and the coordination of cellular functions; and 3), inhomogeneities in the extravascular tissue lead to sprout branching and anastomosis, phenomena that emerge without any prescribed rules. This model provides a quantitative framework to test hypotheses on the biochemical and biomechanical mechanisms that control tumor-induced angiogenesis.

Hypoxia-inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their alpha subunits. We have demonstrated that hypoxia stimulates translation of HIF-1alpha and -2alpha proteins by distributing HIF-alpha mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C-alpha (cPKC-alpha), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to approximately 40-50% of HIF-alpha proteins accumulation after 3 h of 1% O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC-alpha inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap-mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium-induced activation of cPKC-alpha hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia-stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.

Reduced oxygen tension (hypoxia) in the environment stimulates oxygen-sensitive cells in the carotid body (CB). Upon exposure to hypoxia, the CB immediately triggers a reflexive physiological response, thereby increasing respiration. Adaptation to hypoxia involves changes in the expression of various CB genes, whose products are involved in the transduction and modulation of the hypoxic signal to the central nervous system (CNS). Genes encoding neurotransmitter-synthesizing enzymes and receptors are particularly important in this regard. The cellular response to hypoxia correlates closely with the release and biosynthesis of catecholamines. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is regulated by hypoxia in the CB and in the oxygen-sensitive cultured PC12 cell line. Recently, genomic microarray studies have identified additional genes regulated by hypoxia. Patterns of gene expression vary, depending on the type of applied hypoxia, e.g., intermittent vs. chronic. Construction of a hypoxia-regulated, CB-specific, subtractive cDNA library will enable us to further characterize regulation of gene expression in the CB.

Loss of von Hippel-Lindau (VHL) gene function leads to VHL disease, which is characterized by vascular tumors of the central nervous system, renal clear cell carcinomas, and pheochromocytomas. Pheochromocytomas express high levels of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis. PC12 cells that express VHL antisense RNA had 5-10-fold reduced levels of endogenous pVHL and 2-3-fold increased levels of TH protein and mRNA. Nuclear run-on analysis revealed an augmentation of TH gene transcription with enhanced efficiency of transcript elongation in the 3' region of the gene. Transient coexpression of the VHL antisense RNA with a TH promoter reporter construct increased TH promoter activity by 2-3-fold. A decrease in pVHL accumulation also resulted in an increase in TH mRNA accumulation and transcription of the TH gene during hypoxia. This is the first evidence that endogenous pVHL is a physiological regulator of the catecholaminergic phenotype. Thus, loss of pVHL function may be causative in pheochromocytoma-associated hypercatecholaminemia and arterial hypertension.

Loss of von Hippel-Lindau (VHL) gene function leads to a familial cancer syndrome, VHL disease, characterized by CNS and retinal hemangioblastomas, renal clear cell carcinomas, and pheochromocytomas. Pheochromocytomas, tumors of the chromaffin cells of the adrenal medulla, secrete large amounts of catecholamines. This results from enhanced expression of TH, the rate-limiting enzyme in catecholamine biosynthesis. Overexpression of exogenous pVHL in PC12 cells lead to a substantial repression of TH protein and mRNA [1]. Here we examined the role of endogenous pVHL in the regulation of TH gene expression. We developed PC12 clonal cell lines that stably express VHL antisense RNA and have reduced levels of endogenous pVHL by 5–10 fold. In these cells, TH protein and mRNA were increased 2–3 fold as compared to control cells. Nuclear run-on analysis revealed an increase in the overall TH gene transcription. In addition, we measured augmented efficiency of the TH transcript elongation in the distal part of the gene. Transient co-transfections of -773 kb TH-CAT promoter-reporter constructs with the VHL antisense RNA resulted in an approximately 2–3 fold increase in reporter gene activity. TH is a hypoxia-inducible gene. The overall accumulation of THmRNA and the rate of TH gene transcription were substantially enhanced in cells with decreased levels of pVhl exposed to 1% O2 for 16 h. These results indicate that endogenous pVhl constitutively represses TH gene expression in PC12 cells. TH promoter contains a functional hypoxia-responsive element (HRE) - like sequence. Transient co-transfections of the HIF1α and HIF2α expression vectors with the wild type, but not mutated TH promoter revealed significant 3–4 fold induction of the reporter gene activity. Gel-shift analysis showed presence of the hypoxia-inducible complexes associated with the wild type but not mutated THHRE. Electroelution of protein complexes from the hypoxia-inducible complex showed presence of the HIF2α, and to lesser extent of the HIF1 subunit. pVHL is part of the E3 ubiquitin ligase complex that targets the HIF alphas for ubiquitination and degradation. Changes in the levels of the pVHL in PC12 cells by either overexpression of pVHL or expression of VHL antisense RNA resulted in respectively decreased and increased HIF-and hypoxia-mediated activation of the TH promoter. Stable changes in the pVHL levels in PC12 cells resulted in accumulation of HIF alphas during normoxia in cells with decreased amounts of pVHL, and in decreased expression of HIF alphas during hypoxia in cells overex-pressing pVHL. The changes in HIFα protein accumulation induced by pVHL resulted from ubiquitination of HIF alphas by pVHL.

Background Intermediate-conductance, calcium-activated potassium channels (IKs) modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1) expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D). Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is stimulated by calcium mobilization and influx, and differentiation stimuli coordinately upregulate mRNA levels of the calcium-activated hIK1 channel. This upregulation is paradoxical in that functional hIK1 channels are not observed in cultured keratinocytes. It appears, therefore, that hIK1 does not contribute to the functional electrophysiology of primary human keratinocytes, nor intact human skin. Further, the results indicate caution is required when interpreting experiments utilizing pharmacological hIK1 modulators in human keratinocytes.