Network Physiology reveals relations between network topology and physiological function

The human organism is an integrated network where complex physiologic systems, each with its own regulatory mechanisms, continuously interact, and where failure of one system can trigger a breakdown of the entire network. Identifying and quantifying dynamical networks of diverse systems with different types of interactions is a challenge. Here, we develop a framework to probe interactions among diverse systems, and we identify a physiologic network. We find that each physiologic state is characterized by a specific network structure, demonstrating a robust interplay between network topology and function. Across physiologic states the network undergoes topological transitions associated with fast reorganization of physiologic interactions on time scales of a few minutes, indicating high network flexibility in response to perturbations. The proposed system-wide integrative approach may facilitate the development of a new field, Network Physiology.Comment: 12 pages, 9 figure

Adaptive Response of a Gene Network to Environmental Changes by Fitness-Induced Attractor Selection

Cells switch between various stable genetic programs (attractors) to accommodate environmental conditions. Signal transduction machineries efficiently convey environmental changes to the gene regulation apparatus in order to express the appropriate genetic program. However, since the number of environmental conditions is much larger than that of available genetic programs so that the cell may utilize the same genetic program for a large set of conditions, it may not have evolved a signaling pathway for every environmental condition, notably those that are rarely encountered. Here we show that in the absence of signal transduction, switching to the appropriate attractor state expressing the genes that afford adaptation to the external condition can occur. In a synthetic bistable gene switch in Escherichia coli in which mutually inhibitory operons govern the expression of two genes required in two alternative nutritional environments, cells reliably selected the “adaptive attractor” driven by gene expression noise. A mathematical model suggests that the “non-adaptive attractor” is avoided because in unfavorable conditions, cellular activity is lower, which suppresses mRNA metabolism, leading to larger fluctuations in gene expression. This, in turn, renders the non-adaptive state less stable. Although attractor selection is not as efficient as signal transduction via a dedicated cascade, it is simple and robust, and may represent a primordial mechanism for adaptive responses that preceded the evolution of signaling cascades for the frequently encountered environmental changes

Phenotypic Variation and Bistable Switching in Bacteria

Microbial research generally focuses on clonal populations. However, bacterial cells with identical genotypes frequently display different phenotypes under identical conditions. This microbial cell individuality is receiving increasing attention in the literature because of its impact on cellular differentiation, survival under selective conditions, and the interaction of pathogens with their hosts. It is becoming clear that stochasticity in gene expression in conjunction with the architecture of the gene network that underlies the cellular processes can generate phenotypic variation. An important regulatory mechanism is the so-called positive feedback, in which a system reinforces its own response, for instance by stimulating the production of an activator. Bistability is an interesting and relevant phenomenon, in which two distinct subpopulations of cells showing discrete levels of gene expression coexist in a single culture. In this chapter, we address techniques and approaches used to establish phenotypic variation, and relate three well-characterized examples of bistability to the molecular mechanisms that govern these processes, with a focus on positive feedback.

In vitro suppression of the MMP-3 gene in normal and cytokine-treated human chondrosarcoma using small interfering RNA

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinase (MMPs) synthesized and secreted from connective tissue cells have been thought to participate in degradation of the extracellular matrix. Increased MMPs activities that degrade proteoglycans have been measured in osteoarthritis cartilage. This study aims to suppress the expression of the <it>MMP-3 </it>gene in <it>in vitro </it>human chondrosarcoma using siRNA.</p> <p>Methods</p> <p>Cells were categorized into four groups: control (G.1); transfection solution treated (G.2); negative control siRNA treated (G.3); and <it>MMP-3 </it>siRNA treated (G.4). All four groups were further subdivided into two groups - treated and non-treated with IL-1β- following culture for 48 and 72 h. We observed the effects of gene suppression according to cell morphology, glycosaminoglycan (GAG) and hyaluronan (HA) production, and gene expression by using real-time polymerase chain reaction (PCR).</p> <p>Results</p> <p>In IL-1β treated cells the apoptosis rate in G.4 was found to be lower than in all other groups, while viability and mitotic rate were higher than in all other groups (<it>p </it>< 0.05). The production of GAG and HA in G.4 was significantly higher than the control group (<it>p </it>< 0.05). <it>MMP-3 </it>gene expression was downregulated significantly (<it>p </it>< 0.05).</p> <p>Conclusion</p> <p><it>MMP-3 </it>specific siRNA can inhibit the expression of <it>MMP-3 </it>in chondrosarcoma. This suggests that <it>MMP-3 </it>siRNA has the potential to be a useful preventive and therapeutic agent for osteoarthritis.</p

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation

Background: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. Methods: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. Results: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and alpha-1-antichymotrypsin, as well as complement C1q. Conclusions: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.Peer reviewe

Effects of Hydrogen Peroxide on Wound Healing in Mice in Relation to Oxidative Damage

10.1371/journal.pone.0049215PLoS ONE711

Increased Levels of Leukocyte-Derived MMP-9 in Patients with Stable Angina Pectoris

Objective: There is a growing interest for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in plasma as novel biomarkers in coronary artery disease (CAD). We aimed to identify the sources of MMP-8, MMP-9, TIMP-1 and TIMP-2 among peripheral blood cells and further explore whether gene expression or protein release was altered in patients with stable angina pectoris (SA). Methods: In total, plasma MMP-9 was measured in 44 SA patients and 47 healthy controls. From 10 patients and 10 controls, peripheral blood mononuclear cells (PBMC) and neutrophils were isolated and stimulated ex vivo. MMPs, TIMPs and myeloperoxidase were measured in plasma and supernatants by ELISA. The corresponding gene expression was measured by real-time PCR. Results: Neutrophils were the dominant source of MMP-8 and MMP-9. Upon moderate stimulation with IL-8, the neutrophil release of MMP-9 was higher in the SA patients compared with controls (p,0.05). In PBMC, the TIMP-1 and MMP-9 mRNA expression was higher in SA patients compared with controls, p,0.01 and 0.05, respectively. There were no differences in plasma levels between patients and controls except for TIMP-2, which was lower in patients, p,0.01. Conclusion: Measurements of MMPs and TIMPs in plasma may be of limited use. Despite similar plasma levels in SA patients and controls, the leukocyte-derived MMP-9 and TIMP-1 are significantly altered in patients. The findings indicate that th

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552