Cell Systems Biology Research Articles

An Introduction to the Mathematical Modeling in the Study of Cancer Systems Biology.

Background:Frequently occurring in cancer are the aberrant alterations of regulatory onco-metabolites, various oncogenes/epigenetic stochasticity, and suppressor genes, as well as the deficient mismatch repair mechanism, chronic inflammation, or those deviations belonging to the other cancer characteristics. How these aberrations that evolve overtime determine the global phenotype of malignant tumors remains to be completely understood. Dynamic analysis may have potential to reveal the mechanism of carcinogenesis and can offer new therapeutic intervention.Aims:We introduce simplified mathematical tools to model serial quantitative data of cancer biomarkers. We also highlight an introductory overview of mathematical tools and models as they apply from the viewpoint of known cancer features.Methods:Mathematical modeling of potentially actionable genomic products and how they proceed overtime during tumorigenesis are explored. This report is intended to be instinctive without being overly technical.Results:To date, many mathematical models of the common features of cancer have been developed. However, the dynamic of integrated heterogeneous processes and their cross talks related to carcinogenesis remains to be resolved.Conclusions:In cancer research, outlining mathematical modeling of experimentally obtained data snapshots of molecular species may provide insights into a better understanding of the multiple biochemical circuits. Recent discoveries have provided support for the existence of complex cancer progression in dynamics that span from a simple 1-dimensional deterministic system to a stochastic (ie, probabilistic) or to an oscillatory and multistable networks. Further research in mathematical modeling of cancer progression, based on the evolving molecular kinetics (time series), could inform a specific and a predictive behavior about the global systems biology of vulnerable tumor cells in their earlier stages of oncogenesis. On this footing, new preventive measures and anticancer therapy could then be constructed.

Protocol for eukaryotic plasma membrane isolation using superparamagnetic nanoparticles

Abstract Superparamagnetic nanoparticles (SPMNP) are used in cell biology for various applications such as subcellular fractionation and for high pure organelle isolation. We simplified previously established methodology with the use of SPMNP for isolation of high pure and high yield plasma membrane from adherent eukaryotic cell line (Thimiri Govinda Raj et al., 2011, 2012, 2016; Tharkeshwar et al., 2014). Here, we show a step by step protocol for SPMNP based plasma membrane fractionation. Our research thus could be used as an alternative method to isolate plasma membrane fraction for cell biology, system biology and immunology research.

TBK1 Provides Context-Selective Support of the Activated AKT/mTOR Pathway in Lung Cancer.

Emerging observations link dysregulation of TANK-binding kinase 1 (TBK1) to developmental disorders, inflammatory disease, and cancer. Biochemical mechanisms accounting for direct participation of TBK1 in host defense signaling have been well described. However, the molecular underpinnings of the selective participation of TBK1 in a myriad of additional cell biological systems in normal and pathophysiologic contexts remain poorly understood. To elucidate the context-selective role of TBK1 in cancer cell survival, we employed a combination of broad-scale chemogenomic and interactome discovery strategies to generate data-driven mechanism-of-action hypotheses. This approach uncovered evidence that TBK1 supports AKT/mTORC1 pathway activation and function through direct modulation of multiple pathway components acting both upstream and downstream of the mTOR kinase itself. Furthermore, we identified distinct molecular features in which mesenchymal, Ras-mutant lung cancer is acutely dependent on TBK1-mediated support of AKT/mTORC1 pathway activation for survival. Cancer Res; 77(18); 5077-94. ©2017 AACR.

Dye-Labeled Polyacryloyl Hydrazide-Ag Nanoparticle Fluorescent Probe for Ultrasensitive and Selective Detection of Au Ion.

The efficiency of a fluorescence sensing device based on metal-enhanced fluorescence (MEF) is dependent on the optimization of interaction between the fluorophore and the metal nanoparticle (NP). Herewith, ultrasensitive and selective turn-on sensing of Au3+ is achieved by using a suitable combination of fluorophore and metal NP system through sequential MEF effect. Dansyl hydrazide-tagged Ag NPs in the polyacryloyl hydrazide cavity are utilized to sense the picomolar concentration of Au3+ in aqueous media. We demonstrated that the selective Au3+ sensing is due to the selective deposition of Au on the Ag NP surface over the 16 other metal ions studied. The sensitivity is assigned to the strong overlapping of the emission band of the fluorophore with the surface plasmon band of the Au and improvement of fluorescence signal through successive MEF by Ag and Au colloids. The sensing is associated with a fivefold increase in fluorescence intensity and appearance of violet color of the solution. These luminescent Ag–Au bimetallic NPs may be utilized to trace cancer cells in biological systems and for cell imaging applications.

Role of the β Common (βc) Family of Cytokines in Health and Disease.

The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease.

Distinct Mechanisms Underlying Resveratrol-Mediated Protection from Types of Cellular Stress in C6 Glioma Cells.

The polyphenolic phytostilbene, trans-resveratrol, is found in high amounts in several types and tissues of plants, including grapes, and has been proposed to have beneficial effects in the central nervous system due to its activity as an antioxidant. The objective of the present study was to identify the mechanisms underlying the protective effects of resveratrol under conditions of oxidative stress or DNA damage, induced by the extracellularly applied oxidant, tert-butyl hydrogen peroxide, or UV-irradiation, respectively. In C6 glioma cells, a model system for glial cell biology and pharmacology, resveratrol was protective against both types of insult. Prevention of tau protein cleavage and of the formation of neurofibrillary tangles were identified as mechanisms of action of resveratrol-mediated protection in both paradigms of cellular damage. However, depending on the type of insult, resveratrol exerted its protective activity differentially: under conditions of chemically induced oxidative stress, inhibition of caspase activity, while with DNA damage, resveratrol regulated tau phosphorylation at Ser422. Results advance our understanding of resveratrol’s complex impact on cellular signaling pathway and contribute to the notion of resveratrol’s role as a pleiotropic therapeutic agent.

Semantics and Efficient Simulation Algorithms of an Expressive Multilevel Modeling Language

The domain-specific modeling and simulation language ML-Rules is aimed at facilitating the description of cell biological systems at different levels of organization. Model states are chemical solutions that consist of dynamically nested, attributed entities. The model dynamics are described by rules that are constrained by arbitrary functions, which can operate on the entities’ attributes, (nested) solutions, and the reaction kinetics. Thus, ML-Rules supports an expressive hierarchical, variable structure modeling of cell biological systems. The formal syntax and semantics of ML-Rules show that it is firmly rooted in continuous-time Markov chains. In addition to a generic stochastic simulation algorithm for ML-Rules, we introduce several specialized algorithms that are able to handle subclasses of ML-Rules more efficiently. The algorithms are compared in a performance study, leading to conclusions on the relation between expressive power and computational complexity of rule-based modeling languages.

Robust microbial cell segmentation by optical-phase thresholding with minimal processing requirements.

High‐throughput imaging with single‐cell resolution has enabled remarkable discoveries in cell physiology and Systems Biology investigations. A common, and often the most challenging step in all such imaging implementations, is the ability to segment multiple images to regions that correspond to individual cells. Here, a robust segmentation strategy for microbial cells using Quantitative Phase Imaging is reported. The proposed method enables a greater than 99% yeast cell segmentation success rate, without any computationally‐intensive, post‐acquisition processing. We also detail how the method can be expanded to bacterial cell segmentation with 98% success rates with substantially reduced processing requirements in comparison to existing methods. We attribute this improved performance to the remarkably uniform background, elimination of cell‐to‐cell and intracellular optical artifacts, and enhanced signal‐to‐background ratio—all innate properties of imaging in the optical‐phase domain. © 2017 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research.

Coordination of Metabolism Through Metabolite-Protein Interactions

The omics revolution has vastly expanded our ability to monitor intracellular molecular events. This technological advance has typically not been matched with conceptual advances in our ability to interpret and understand such data or to systematically generate hypotheses from them. The iterative systems biology cycle of experiment and computational analysis is a great promise, but turns in practise typically only one or twice per project. A particular challenge for all cell types, from bacteria to humans, are the multiple, overlapping regulatory mechanisms that coordinate adaptation to changing environments. Myriads of regulatory interactions have been identified at the level of transcription, translation, post-translational modification and metabolite feedback, but our understanding which of them govern a given adaptation and actually control a given biological function is very limited. For metabolism this function is the flux of small molecules that is coordinated through a variety of overlapping regulation mechanism. Although the specific molecular implementation might differ, the regulatory logic is usually highly similar in different microbes (1). In this lecture I will focus on the key role of metabolite-protein interactions in achieving this coordination in E. coli, both as input into transcriptional regulation and by directly affecting enzyme activity, and outline how we delineate regulation events that actively control the coordination of metabolic fluxes by combining various omics methods with computational analysis (2, 3). Out of the bewildering complexity of possible regulatory interactions, surprisingly few regulation events appear to be required for a given transition, typically involving less than a handful of active regulators. 1. Chubukov, Gerosa, Kochanowski & Sauer. Nat Rev Microbiol 12: 327 (2014) 2. Gerosa, Haverkorn, Christodoulou, Kochanowski, Schmidt, Noor & Sauer. Cell Systems Biology. 1: 270-282 (2015) 3. Link, Fuhrer, Gerosa, Zamboni & Sauer. Nature Methods 12: 1091-1079 (2015).

Muscle Stem Cells: A Model System for Adult Stem Cell Biology.

Skeletal muscle stem cells, originally termed satellite cells for their position adjacent to differentiated muscle fibers, are absolutely required for the process of skeletal muscle repair and regeneration. In the last decade, satellite cells have become one of the most studied adult stem cell systems and have emerged as a standard model not only in the field of stem cell-driven tissue regeneration but also in stem cell dysfunction and aging. Here, we provide background in the field and discuss recent advances in our understanding of muscle stem cell function and dysfunction, particularly in the case of aging, and the potential involvement of muscle stem cells in genetic diseases such as the muscular dystrophies.

Fractional-order model of the disease Psoriasis: A control based mathematical approach

Autoimmune diseases are generated through irregular immune response of the human body. Psoriasis is one type of autoimmune chronic skin diseases that is differentiated by T-Cells mediated hyper-proliferation of epidermal Keratinocytes. Dendritic Cells and CD8+ T-Cells have a significant role for the occurrence of this disease. In this paper, the authors have developed a mathematical model of Psoriasis involving CD4+ T-Cells, Dendritic Cells, CD8+ T-Cells and Keratinocyte cell populations using the fractional differential equations with the effect of Cytokine release to observe the impact of memory on the cell-biological system. Using fractional calculus, the authors try to explore the suppressed memory, associated with the cell-biological system and to locate the position of Keratinocyte cell population as fractional derivative possess non-local property. Thus, the dynamics of Psoriasis can be predicted in a better way using fractional differential equations rather than its corresponding integer order model. Finally, the authors introduce drug into the system to obstruct the interaction between CD4+ T-Cells and Keratinocytes to restrict the disease Psoriasis. The authors derive the Euler-Lagrange conditions for the optimality of the drug induced system. Numerical simulations are made through Matlab by developing iterative schemes.

Exploring Quantitative Yeast Phenomics with Single-Cell Analysis of DNA Damage Foci

A significant challenge of functional genomics is todevelop methods for genome-scale acquisition and analysis of cell biological data. Here, we present an integrated method that combines genome-wide genetic perturbation of Saccharomyces cerevisiae with high-content screening to facilitate the genetic description of sub-cellular structures and compartment morphology. As proof of principle, we used a Rad52-GFP marker to examine DNA damage foci in ∼20 million single cells from ∼5,000 different mutant backgrounds in the context of selected genetic or chemical perturbations. Phenotypes were classified using a machine learning-based automated image analysis pipeline. 345 mutants were identified that had elevated numbers of DNA damage foci, almost half of which were identified only in sensitized backgrounds. Subsequent analysis of Vid22, a protein implicated in the DNA damage response, revealed that it acts together with the Sgs1 helicase at sites of DNA damage and preferentially binds G-quadruplex regions of the genome. This approach is extensible to numerous other cell biological markers and experimental systems.

Abstract 2486: Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling

Abstract Both normal organogenesis and tumor development follow a Gompertzian growth curve. An understanding of the mechanisms operating in these apparently discordant situations, in which growth rate slows as mass increases, may be useful for the treatment of cancer. We have previously shown that encapsulating murine renal adenocarcinoma (RENCA) cells in a double layer of agarose to form spherical macrobeads, undergo Gompertzian growth regulation as tumor colonies within the confines of the inner agarose matrix. As the colonies reach maximal size, the RENCA macrobeads (MBs) secrete factors that also inhibit the proliferation of freely growing target cells outside the MB. In the current study, we report the ability of the tumor colonies within the MB to re-form following the debulking of tumor mass with docetaxel (0.5 μg/ml or 5.0 μg/ml). Docetaxel-resistant cells were assessed for the stem cell marker OCT4 using immunohistochemistry (Abcam ab19857), RT-PCR (Qiagen Cancer Stem Cell PCR Array) and cell migration/invasion (Corning Cell Migration Chemotaxis and Invasion Assay). Docetaxel-resistant cells are shown to be a discrete population of cells that exhibit stem cell-like properties, including prolonged quiescence (>16 wk), OCT4 staining, stem cell gene expression, increased migration and invasion, and in vivo tumor induction. These cancer stem-like properties are dose and time post-treatment dependent with the greatest expression observed in the few surviving cells at 6 wk post-treatment with 5 μg/ml. Tumor growth is observed in syngeneic BALB/c mice following transplantation of a single docetaxel-resistant cell at higher frequencies (≥ 6 of 9) as compared to the grafting of single monolayer cells (0 of 8) at 5 wk when using cells recovered at 6 wk from the 5 μg/ml dose treated MBs. To understand the mechanism of colony growth regulation within the MB, we hypothesized that tumor cells external to the MB would use similar growth control signaling when exposed to RENCA MBs or MB conditioned media. We have previously shown an increase in the activity of the transcription factor MEF2 in cells exposed to RENCA MB conditioned media. To investigate PI3/Akt signaling, a known pathway for MEF2 regulation, in human DU145 prostate tumor cells exposed to MB conditioned media, we used In-Cell and Western blotting. Exposure to RENCA MBs resulted in Akt hyperphosphorylation (≥ 48 hours) and de-phosphorylation of MEF2D in DU145 target cells. These findings support the hypothesis that RENCA MBs, as biological cell systems with the in vitro capability of inhibiting cancer cell proliferation, achieve this effect, at least in part, by signaling through Akt to regulate MEF2D. This effect is being investigated in colorectal patients with progressive disease, following informed consent, who underwent laparoscopic intraperitoneal implantations of RENCA MBs as part of ongoing clinical trials. Citation Format: Prithy C. Martis, Melissa A. Laramore, Hunter L. Gazda, Atira Dudley, Pradeep R. Dumpala, Allyson J. Ocean, Nathaniel Berman, Tapan Parikh, Zoe P. Andrada, Angelica Nazarian, Joanne Thomas, Eugene Akahoho, George Stoms, Alex Yaroshinsky, Thomas J. Fahey, David J. Wolf, Lawrence S. Gazda, Barry H. Smith. Docetaxel resistant cells display cancer stem cell properties and regulate growth via PI3/Akt signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2486.

Micrasterias as a Model System in Plant Cell Biology.

The unicellular freshwater alga Micrasterias denticulata is an exceptional organism due to its complex star-shaped, highly symmetric morphology and has thus attracted the interest of researchers for many decades. As a member of the Streptophyta, Micrasterias is not only genetically closely related to higher land plants but shares common features with them in many physiological and cell biological aspects. These facts, together with its considerable cell size of about 200 μm, its modest cultivation conditions and the uncomplicated accessibility particularly to any microscopic techniques, make Micrasterias a very well suited cell biological plant model system. The review focuses particularly on cell wall formation and composition, dictyosomal structure and function, cytoskeleton control of growth and morphogenesis as well as on ionic regulation and signal transduction. It has been also shown in the recent years that Micrasterias is a highly sensitive indicator for environmental stress impact such as heavy metals, high salinity, oxidative stress or starvation. Stress induced organelle degradation, autophagy, adaption and detoxification mechanisms have moved in the center of interest and have been investigated with modern microscopic techniques such as 3-D- and analytical electron microscopy as well as with biochemical, physiological and molecular approaches. This review is intended to summarize and discuss the most important results obtained in Micrasterias in the last 20 years and to compare the results to similar processes in higher plant cells.

A Single-Cell Assay for Time Lapse Studies of Exosome Secretion and Cell Behaviors.

To understand the inhomogeneity of cells in biological systems, there is a growing demand on the capability of characterizing the properties of individual single cells. Since single-cell studies require continuous monitoring of the cell behaviors, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and, proliferation of single cells and convenient, noninvasive tests of single-cell behaviors from molecular markers. Here, a highly versatile single-cell assay is presented that can accommodate different cellular types, enable easy and efficient single-cell loading and culturing, and be suitable for the study of effects of in vitro environmental factors in combination with drug screening. One salient feature of the assay is the noninvasive collection and surveying of single-cell secretions at different time points, producing unprecedented insight of single-cell behaviors based on the biomarker signals from individual cells under given perturbations. Above all, the acquired information is quantitative, for example, measured by the number of exosomes each single-cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single-cell properties.

Abstract B36: RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells

Abstract We have previously reported that suppression of the isoforms of myocyte enhancer factor (MEF2) in target tumor cells in vitro reduces the demonstrated proliferation-inhibitory effect of agarose-agarose encapsulated mouse renal adenocarcinoma cells (RENCA macrobeads), suggesting that it is a critical component of a pathway by which the inhibitory action, now being evaluated also in Phase IIb human clinical trials, is mediated. Importantly, MEF2 originally described as active in myogenesis, has been found to be a key regulator of many developmental pathways and to have both pro- and anti-growth regulatory functions in murine and human tumor cells. Recent studies have supported a molecular intersection between components of the PI3K/Akt and the canonical Wnt signaling (carcinogenic) pathways, converging at the glycogen synthase kinase 3β (GSK3β) node. In skeletal and cardiac muscle cells, GSK3β regulates MEF2 activity indirectly through reciprocal regulation of the p38 mitogen-activated protein kinase (MAPK) pathway. To determine, therefore, whether PI3K/Akt is also involved in the anti-proliferative effect of the RENCA macrobeads, we evaluated signaling through PI3K/Akt in response to macrobead-conditioned media and examined post-translational modification of MEF2D in two human cell lines DU145 (prostate cancer) and MCF7 (breast adenocarcinoma). Using In-Cell Western analysis, we determined the expression of phospho-Akt T308 and S473 in parallel with total Akt in cells exposed to naïve or conditioned media. Nuclear extracts for phospho-MEF2D and total MEF2D were assessed using western blotting. In the DU145 cells, exposure to RENCA macrobead-conditioned medium resulted in Akt hyperphosphorylation (≥ 48 hours) in parallel with the increasing age and inhibitory capacity of macrobeads. Further, the growth inhibition of DU145 cells exposed to RENCA conditioned medium (36-40% at 12-16 weeks of age; 45-48% at 20-24 weeks of age) was accompanied by de-phosphorylation of MEF2D at Serine 444, supporting a role for increased MEF2D transcriptional activity in the inhibitory effect. In the MCF7 cells, which are less sensitive to RENCA macrobead inhibition (8-12% at 12-16 weeks of age; 17-21% at 20-24 weeks of age), Akt phosphorylation diminished one hour following exposure to conditioned media, and MEF2D remained phosphorylated at Serine 444. These findings support the hypothesis that RENCA macrobeads, as biological cell systems with the in vitro and in vivo (model and clinical) capability of inhibiting cancer cell proliferation and tumor growth, achieve this effect, at least in part, by signaling through Akt to differentially regulate MEF2D. Citation Format: Prithy C. Martis, Melissa A. Laramore, Atira T. Dudley, Barry H. Smith, Lawrence S. Gazda. RENCA macrobead-induced AKT hyperphosphorylation leads to MEF2 activation and inhibition of the proliferation of human DU145 prostate carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B36.

Cellular and molecular etiology of hepatocyte injury in a murine model of environmentally induced liver abnormality.

Exposures to a wide variety of environmental substances are negatively associated with many biological cell systems both in humans and rodents. Trichloroethane (TCE), a ubiquitous environmental toxicant, is used in large quantities as a dissolvent, metal degreaser, chemical intermediate, and component of consumer products. This increases the likelihood of human exposure to these compounds through dermal, inhalation and oral routes. The present in vivo study was aimed to investigate the possible cellular and molecular etiology of liver abnormality induced by early exposure to TCE using a murine model. The results showed a significant increase in liver weight. Histopathological examination revealed a TCE-induced hepatotoxicity which appeared as heavily congested central vein and blood sinusoids as well as leukocytic infiltration. Mitotic figures and apoptotic changes such as chromatin condensation and nuclear fragments were also identified. Cell death analysis demonstrates hepatocellular apoptosis was evident in the treated mice compared to control. TCE was also found to induce oxidative stress as indicated by an increase in the levels of lipid peroxidation, an oxidative stress marker. There was also a significant decrease in the DNA content of the hepatocytes of the treated groups compared to control. Agarose gel electrophoresis also provided further biochemical evidence of apoptosis by showing internucleosomal DNA fragmentation in the liver cells, indicating oxidative stress as the cause of DNA damage. These results suggest the need for a complete risk assessment of any new chemical prior to its arrival into the consumer market.

Live Cell Microscopy-Based RNAi Screening in the Moss Physcomitrella patens.

RNA interference (RNAi) is a powerful technique enabling the identification of the genes involved in a certain cellular process. Here, we discuss protocols for microscopy-based RNAi screening in protonemal cells of the moss Physcomitrella patens, an emerging model system for plant cell biology. Our method is characterized by the use of conditional (inducible) RNAi vectors, transgenic moss lines in which the RNAi vector is integrated, and time-lapse fluorescent microscopy. This method allows for effective and efficient screening of >100 genes involved in various cellular processes such as mitotic cell division, organelle distribution, or cell growth.

Stress Signaling Responses in Plants.

Stress Signaling Responses in Plants.