Systems Biology Ireland

Progress in the Formulation and Delivery of Somatostatin Analogues for Acromegaly

Mon, 25 Sep 2017 00:00:00 GMT

Progress in the Formulation and Delivery of Somatostatin Analogues for Acromegaly Fattah, Sarinj; Brayden, David James A 14-amino acid cystin bridge-containing neuropeptide was discovered in 1973 and designated as "growth hormone-inhibiting hormone (GHIH)," i.e. somatostatin. Its discovery led to the synthesis of three analogues which were licenced for the treatment of acromegaly: octreotide, lanreotide, and pasireotide. Somatostatin analogues are currently approved only as either subcutaneous (s.c.) or intramuscular (i.m.) long-acting injections. We examine the challenges that must be overcome to create oral formulations of somatostatin analogues and examine selected clinical trial data. While octreotide has low intestinal permeability, similar to almost all other peptides, it has an advantage of being more stable against intestinal peptidases. The development of new oral formulation strategies may eventually allow for the successful oral administration of potent somatostatin analogues with high therapeutic indices.

In vitro study of the interaction of heregulin-functionalized magnetic-optical nanorods with MCF7 and MDA-MB- 231 cells

Mon, 01 Sep 2014 00:00:00 GMT

In vitro study of the interaction of heregulin-functionalized magnetic-optical nanorods with MCF7 and MDA-MB- 231 cells Lesniak, Anna; Kilinc, Devrim; Rashdan, Suad Ahmed; Kriegsheim, Alexander von; Ashall, B.; Zerulla, Dominic; Kolch, Walter; Lee, Gil U. Multifunctional nanoparticles that actively target specific cells are promising tools for cancer diagnosis and therapy. In this article we review the synthesis and surface chemistry of Fe–Au nanorods and their characterization using microscopy. The diameter of the rods used in this study was selected to be 150–200 nm so that they did not enter the cells. The 80 nm-long Au tips of the nanorods were functionalized with heregulin (HRG), and the micron-long Fe portion was coated with a poly(ethylene glycol) monolayer to minimize non-specific interactions. Nanorods functionalized with HRG were found to preferentially bind to MCF7 cells that express high levels of the receptor tyrosine-protein kinase ErbB2/3. Magnetic tweezers measurements were used to characterize the kinetic properties of the bond between the HRG on the rods and ErbB2/3 on the surface of the cells. The strong magnetization of Fe–Au nanorods makes them excellent candidates for in-vitro and in-vivo imaging, and magnetic therapeutic applications targeting cancer cells in circulation.

On the personalised modelling of cancer signalling

Wed, 12 Oct 2016 00:00:00 GMT

On the personalised modelling of cancer signalling Fey, Dirk; Kuehn, Axel; Kholodenko, Boris N. Dynamic modelling has long been used to understand fundamental principles of cell signalling and its dysregulation in cancer. More recently these models have also been used to understand the individual risks of cancer patients, and predict their survival probabilities. However, the current methodologies for integrating tumour data and generating patient-specific simulations suffer from the lack of general applicability; they only work for cell signalling models in which only posttranslational protein modifications are considered, so that the total protein concentrations are conserved. Here, we present novel, generally applicable method. The method is based on a simple theoretical framework for modelling gene-regulation, and the indirect estimation of patient-specific parameters from tumour data. Because our method does not require time-invariance of the total-protein concentrations, it can be applied to models of any nature, including the many cancer signalling models involving gene-regulation. 6th IFAC Conference on Foundations of Systems Biology in Engineering (FOSBE 2016), Magdeburg, Germany, 9-12 October 2016

A role for adrenergic receptors in the uterotonic effects of ergometrine in isolated human term non-laboring myometrium

Wed, 01 Feb 2017 00:00:00 GMT

A role for adrenergic receptors in the uterotonic effects of ergometrine in isolated human term non-laboring myometrium Fanning, Rebecca A.; Sheehan, Florike; Leyden, Claire; Duffy, Niamh; Iglesias-Martinez, Luis; Carey, Michael F,; Campion, Deirdre P.; O'Connor, J. J. Background: Ergometrine is a uterotonic agent that is recommended in the prevention and management of post partum hemorrhage. Despite its long-standing use the mechanism by which it acts in humans has never been fully elucidated. The objective of this study was to investigate the role of adrenoreceptors in ergometrine's mechanism of action in human myometrium. The study examined the hypothesis that alpha adrenoreceptor antagonism would result in the reversal of the uterotonic effects of ergometrine. Methods: Myometrial samples were obtained from women undergoing elective cesarean delivery. The samples were then dissected into strips and mounted in organ bath chambers. Following generation of an ergometrine concentration-response curve (10-15 to 10-5 M), strips were treated with increasing concentrations of ergometrine (10-15 to 10-7 M) alone and ergometrine (10-7 to 10-5 M) in the presence of phentolamine (10-7 M), prazosin (10-7 M), propranolol (10-6 M) or yohimbine (10-6 M). The effects of adding ergometrine and the effect of drug combinations were analysed using linear mixed effects models with measures of amplitude (g), frequency (contractions/10min) and motility index (g*contractions/10min). Results: A total of 157 experiments were completed on samples obtained from 33 women. There was a significant increase in the motility index (adding 0.342 g*counts/10min/µM; 95% CI from 0.253 to 0.431, P<0.001), amplitude (0.078 g/µM; 95% CI, from 0.0344 to 0.121, P=5e-04) and frequency (0.051 counts/10min/µM; 95% CI, 0.038 to 0.063, P<0.001) in the presence of ergometrine. The α adrenergic antagonist phentolamine and the more selective α1 adrenergic antagonist prazosin, inhibited the ergometrine mediated increase in motility index, amplitude and frequency (-1.63 g*counts/10mins/µM and -16.70 g*counts/10mins/µM for motility index, respectively). Conclusions: These results provide novel evidence for a role for α adrenergic signaling mechanisms in the action of ergometrine on human myometrial smooth muscle in the in vitro setting. Information that sheds light on the mechanism of action of ergometrine may have implications for the development of further uterotonic agents.

A dynamic model of the MYCN regulated DNA damage response in Neuroblastoma

Wed, 05 Nov 2014 00:00:00 GMT

A dynamic model of the MYCN regulated DNA damage response in Neuroblastoma Kuehn, Axel; Kholodenko, Boris N.; Fey, Dirk Neuroblastoma is the most common the most common cancer in infancy with an extremely heterogeneous phenotype that is mainly driven by the MYCN oncogene. The MYCN transcription factor and its amplification is commonly associated with poor prognosis in patients, although it has also been shown that elevated MYCN levels correlates with apoptosis sensitization in cells. HMGA1 is one of MYCN target genes and is involved in triggering apoptosis through a DNA Damage Response (DDR) by inducing ataxia-telangiectasia-mutated (ATM) gene expression. But HMGA1 is also involved in preventing apoptosis by directly binding HIPK2 and decreasing its presence in the nucleus, therefore decreasing phosphorylation of p53 at serine 46 which is required for the activation of p53 apoptotic targets. In this article, we propose a model in which MYCN protein regulates the HMGA1-ATM-p53 and HMGA1-HIPK2-p53 subsystems. Because the molecular details concerning the HMGA1-HMGA1 interaction are uncertain several possibilities were explored in simulations. Our model points towards an important role of MYCN-dependent regulation of HMGA1 expression levels and the subsequent HIPK2 nuclear/cytoplasmic re-localization and led to experimentally testable predictions that can discern between alternative model structures. IEEE International Conference on Bioinformatics and Biomedicine (BIBM 2014): Workshop on Empowering Systems Medicine Through Optimal Design of Experimentation and Computational Modeling, Belfast, Northern Ireland, 2-5 November 2014

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses

Wed, 25 Jun 2014 00:00:00 GMT

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses Dobrzyński, Maciej; Nguyen, Lan K.; Birtwistle, Marc R.; Kriegsheim, Alexander von; Fernández, Alfonso Blanco; Cheong, Alex; Kolch, Walter; Kholodenko, Boris N. We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input–output characteristics (the dose–response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose–response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose–response obtained experimentally.

The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-κB signaling pathways

Mon, 31 May 2010 00:00:00 GMT

The RASSF8 candidate tumor suppressor inhibits cell growth and regulates the Wnt and NF-κB signaling pathways Lock, Frances E.; Underhill-Day, Nicholas; Dunwel, Thomas; Matallanas, David; Kolch, Walter; et al. The Ras-assocation domain family (RASSF) of tumor suppressor proteins until recently contained six proteins named RASSF1–6. Recently, four novel family members, RASSF7–10, have been identified by homology searches for RA-domain-containing proteins. These additional RASSF members are divergent and structurally distinct from RASSF1–6, containing an N-terminal RA domain and lacking the Sav/RASSF/Hpo (SARAH) domain. Here, we show that RASSF8 is ubiquitously expressed throughout the murine embryo and in normal human adult tissues. Functionally, RNAi-mediated knockdown of RASSF8 in non-small-cell lung cancer (NSCLC) cell lines, increased anchorage-independent growth in soft agar and enhanced tumor growth in severe combined immunodeficiency (SCID) mice. Furthermore, EdU staining of RASSF8-depleted cells showed growth suppression in a manner dependent on contact inhibition. We show that endogenous RASSF8 is not only found in the nucleus, but is also membrane associated at sites of cell–cell adhesion, co-localizing with the adherens junction (AJ) component β- catenin and binding to E-cadherin. Following RASSF8 depletion in two different lung cancer cell lines using alternative small interfering RNA (siRNA) sequences, we show that AJs are destabilized and Ecadherin is lost from the cell membrane. The AJ components β-catenin and p65 are also lost from sites of cell–cell contact and are relocalized to the nucleus with a concomitant increase in β-catenindependent and nuclear factor-κB (NF-κB)-dependent signaling following RASSF8 depletion. RASSF8 may also be required to maintain actin -cytoskeletal organization since immunofluorescence analysis shows a striking disorganization of the actin- cytoskeleton following RASSF8 depletion. Accordingly, scratch wound healing studies show increased cellular migration in RASSF8-deficient cells. These results implicate RASSF8 as a tumor suppressor gene that is essential for maintaining AJs function in epithelial cells and have a role in epithelial cell migration.

Signaling pathway models as biomarkers: Patient-specific simulations of JNK activity predict the survival of neuroblastoma patients

Tue, 22 Dec 2015 00:00:00 GMT

Signaling pathway models as biomarkers: Patient-specific simulations of JNK activity predict the survival of neuroblastoma patients Fey, Dirk; Halasz, Melinda; Dreidax, Daniel; Kennedy, Sean P.; Rauch, Nora; Garcia Munoz, Amaya; Pilkington, Ruth; Fischer, Matthias; Kolch, Walter; Kholodenko, Boris N.; Croucher, David R.; et al. Signaling pathways control cell fate decisions that ultimately determine the behavior of cancer cells. Therefore, the dynamics of pathway activity may contain prognostically relevant information different from that contained in the static nature of other types of biomarkers. To investigate this hypothesis, we characterized the network that regulated stress signaling by the c-Jun N-terminal kinase (JNK) pathway in neuroblastoma cells. We generated an experimentally calibrated and validated computational model of this network and used the model to extract prognostic information from neuroblastoma patient–specific simulations of JNK activation. Switch-like JNK activation mediates cell death by apoptosis. An inability to initiate switch-like JNK activation in the simulations was significantly associated with poor overall survival for patients with neuroblastoma with or without MYCN amplification, indicating that patient-specific simulations of JNK activation could stratify patients. Furthermore, our analysis demonstrated that extracting information about a signaling pathway to develop a prognostically useful model requires understanding of not only components and disease-associated changes in the abundance or activity of the components but also how those changes affect pathway dynamics.

Systems biology embedded target validation: improving efficacy in drug discovery

Sat, 01 Feb 2014 00:00:00 GMT

Systems biology embedded target validation: improving efficacy in drug discovery Vandamme, Drieke; Minke, Benedikt A.; Fitzmaurice, William; Kholodenko, Boris N.; Kolch, Walter The pharmaceutical industry is faced with a range of challenges with the ever-escalating costs of drug development and a drying out of drug pipelines. By harnessing advances in -omics technologies and moving away from the standard, reductionist model of drug discovery, there is significant potential to reduce costs and improve efficacy. Embedding systems biology approaches in drug discovery, which seek to investigate underlying molecular mechanisms of potential drug targets in a network context, will reduce attrition rates by earlier target validation and the introduction of novel targets into the currently stagnant market. Systems biology approaches also have the potential to assist in the design of multidrug treatments and repositioning of existing drugs, while stratifying patients to give a greater personalization of medical treatment.

On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics

Wed, 16 Apr 2014 00:00:00 GMT

On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics Turriziani, Benedetta; Garcia Munoz, Amaya; Pilkington, Ruth; Raso, Cinzia; Kolch, Walter; Kriegsheim, Alexander von With the advent of the '-omics' era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

Mechanochemical Stimulation of MCF7 Cells with Rod-Shaped Fe-Au Janus Particles Induces Cell Death through Paradoxical Hyperactivation of ERK

Wed, 18 Feb 2015 00:00:00 GMT

Mechanochemical Stimulation of MCF7 Cells with Rod-Shaped Fe-Au Janus Particles Induces Cell Death through Paradoxical Hyperactivation of ERK Kilinc, Devrim; Lesniak, Anna; Rashdan, Suad Ahmed; Gandhi, Dhruv; Blasiak, Agata; Fannin, Paul C.; Kriegsheim, Alexander von; Kolch, Walter; Lee, Gil U. Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe–Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG–nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG–nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to 'active zones,' dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles.

GSK3 inhibitors regulate MYCN mRNA levels and reduce neuroblastoma cell viability through multiple mechanisms including p53 and Wnt signalling

Sat, 01 Feb 2014 00:00:00 GMT

GSK3 inhibitors regulate MYCN mRNA levels and reduce neuroblastoma cell viability through multiple mechanisms including p53 and Wnt signalling Duffy, David J.; Krstic, Aleksandar; Schwarzl, Thomas; Higgins, D. (Des); Kolch, Walter Neuroblastoma is an embryonal tumor accounting for approximately 15% of childhood cancer deaths. There exists a clinical need to identify novel therapeutic targets, particularly for treatment-resistant forms of neuroblastoma. Therefore, we investigated the role of the neuronal master regulator GSK3 in controlling neuroblastoma cell fate. We identified novel GSK3-mediated regulation of MYC (c-MYC and MYCN) mRNA levels, which may have implications for numerous MYC-driven cancers. In addition, we showed that certain GSK3 inhibitors induced large-scale cell death in neuroblastoma cells, primarily through activating apoptosis. mRNA-seq of GSK3 inhibitor–treated cells was performed and subsequent pathway analysis revealed that multiple signaling pathways contributed to the loss of neuroblastoma cell viability. The contribution of two of the signaling pathways highlighted by the mRNA-seq analysis was functionally validated. Inhibition of the p53 tumor suppressor partly rescued the cell death phenotype, whereas activation of canonical Wnt signaling contributed to the loss of viability, in a p53-independent manner. Two GSK3 inhibitors (BIO-acetoxime and LiCl) and one small-molecule Wnt agonist (Wnt Agonist 1) demonstrated therapeutic potential for neuroblastoma treatment. These inhibitors reduced the viability of numerous neuroblastoma cell lines, even those derived from high-risk MYCN-amplified metastatic tumors, for which effective therapeutics are currently lacking. Furthermore, although LiCl was lethal to neuroblastoma cells, it did not reduce the viability of differentiated neurons. Taken together our data suggest that these small molecules may hold potential as effective therapeutic agents for the treatment of neuroblastoma and other MYC-driven cancers.

A microfluidic dual gradient generator for conducting cell-based drug combination assays

Fri, 01 Jan 2016 00:00:00 GMT

A microfluidic dual gradient generator for conducting cell-based drug combination assays Kilinc, Devrim; Schwab, Jefrem; Rampini, Stefano; Ikpekha, Oshoke W.; Thampi, Ashwin; Blasiak, Agata; Li, Peng; Schwamborn, Robert; Kolch, Walter; Matallanas, David; Lee, Gil U. We present a microfluidic chip that generates linear concentration gradients of multiple solutes that are orthogonally-aligned to each other. The kinetics of gradient formation was characterized using a fluorescent tracer matching the molecular weight of small inhibitory drugs. Live-cell signalling and motility experiments were conducted to demonstrate the potential uses and advantages of the device. A431 epidermoid carcinoma cells, where EGF induces apoptosis in a concentration-dependent manner, were simultaneously exposed to gradients of MEK inhibitor and EGF receptor (EGFR) inhibitor. By monitoring live caspase activation in the entire chip, we were able to quickly assess the combinatorial interaction between MEK and EGFR pathways, which otherwise would require costly and time consuming titration experiments. We also characterized the motility and morphology of MDA-MB-231 breast cancer cells exposed to orthogonal gradients of EGF and EGFR inhibitor. The microfluidic chip not only permitted the quantitative analysis of a population of cells exposed to drug combinations, but also enabled the morphological characterization of individual cells. In summary, our microfluidic device, capable of establishing concentration gradients of multiple compounds over a group of cells, facilitates and accelerates in vitro cell biology experiments, such as those required for cell-based drug combination assays.

Problems, challenges and promises: perspectives on precision medicine

Sat, 01 Aug 2015 00:00:00 GMT

Problems, challenges and promises: perspectives on precision medicine Duffy, David J. The 'precision medicine (systems medicine)' concept promises to achieve a shift to future healthcare systems with a more proactive and predictive approach to medicine, where the emphasis is on disease prevention rather than the treatment of symptoms. The individualization of treatment for each patient will be at the centre of this approach, with all of a patient’s medical data being computationally integrated and accessible. Precision medicine is being rapidly embraced by biomedical researchers, pioneering clinicians and scientific funding programmes in both the European Union (EU) and USA. Precision medicine is a key component of both Horizon 2020 (the EU Framework Programme for Research and Innovation) and the White House’s Precision Medicine Initiative. Precision medicine promises to revolutionize patient care and treatment decisions. However, the participants in precision medicine are faced with a considerable central challenge. Greater volumes of data from a wider variety of sources are being generated and analysed than ever before; yet, this heterogeneous information must be integrated and incorporated into personalized predictive models, the output of which must be intelligible to non-computationally trained clinicians. Drawing primarily from the field of ‘oncology’, this article will introduce key concepts and challenges of precision medicine and some of the approaches currently being implemented to overcome these challenges. Finally, this article also covers the criticisms of precision medicine overpromising on its potential to transform patient care.

HGF induces epithelial-to-mesenchymal transition by modulating the mammalian Hippo/MST2 and ISG15 pathways

Fri, 06 Jun 2014 00:00:00 GMT

HGF induces epithelial-to-mesenchymal transition by modulating the mammalian Hippo/MST2 and ISG15 pathways Farrell, Jennifer; Kelly, Ciara; Rauch, Jens; Kida, Katarzyna; Garcia Munoz, Amaya; Monsefi, Naser; Turriziani, Benedetta; Doherty, Carolanne; Mehta, J. P.; Matallanas, David; Simpson, Jeremy C.; Kolch, Walter; Kriegsheim, Alexander von Epithelial to mesenchymal transition (EMT) is a fundamental cell differentiation/dedifferentiation process which is associated with dramatic morphological changes. Formerly polarized and immobile epithelial cells which form cell junctions and cobblestone-like cell sheets undergo a transition into highly motile, elongated, mesenchymal cells lacking cell-to-cell adhesions. To explore how the proteome is affected during EMT we profiled protein expression and tracked cell biological markers in Madin-Darby kidney epithelial cells undergoing hepatocyte growth factor (HGF) induced EMT. We were able to identify and quantify over 4000 proteins by mass spectrometry. Enrichment analysis of this revealed that expression of proteins associated with the ubiquitination machinery was induced, whereas expression of proteins regulating apoptotic pathways was suppressed. We show that both the mammalian Hippo/MST2 and the ISG15 pathways are regulated at the protein level by ubiquitin ligases. Inhibition of the Hippo pathway by overexpression of either ITCH or A-Raf promotes HGF-induced EMT. Conversely, ISG15 overexpression is sufficient to induce cell scattering and an elongated morphology without external stimuli. Thus, we demonstrate for the first time that the Hippo/MST2 and ISG15 pathways are regulated during growth-factor induced EMT.

Primary cilium-associated genes mediate bone marrow stromal cell response to hypoxia

Wed, 01 Jan 2014 00:00:00 GMT

Primary cilium-associated genes mediate bone marrow stromal cell response to hypoxia Brown, James A. L.; Santra, Tapesh; Owens, Peter; Morrison, Aline M.; Barry, Frank P. Currently there is intense interest in using mesenchymal stem cells (MSC) for therapeutic interventions in many diseases and conditions. To accelerate the therapeutic use of stem cells we must understand how they sense their environment. Primary cilia are an extracellular sensory organelle present on most growth arrested cells that transduce information about the cellular environment into cells, triggering signaling cascades that have profound effects on development, cell cycle, proliferation, differentiation and migration. Migrating cells likely encounter differing oxygen tensions, therefore we investigated the effect of oxygen tension on cilia. Using bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) we found that oxygen tension significantly affected the length of cilia in primary BMSCs. Chronic exposure to hypoxia specifically down-regulated genes involved in hedgehog signaling and re-localized the Smo and Gli2 proteins to cilia. Investigating the effects of chemotactic migration on cilia, we observed significantly longer cilia in migrating cells which was again, strongly influenced by oxygen tension. Finally, using computational modeling we identified links between migration and ciliation signaling pathways, characterizing the novel role of HSP90 and PI3K signaling in regulating BMSC cilia length. These findings enhance our current understanding of BMSC adaptions to hypoxia and advance our knowledge of BMSC biology and cilia regulation.

HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus

Thu, 18 Sep 2014 00:00:00 GMT

HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus Diggle, Christine P.; Moore, Daniel J.; Mali, Girish; Garcia Munoz, Amaya; Kriegsheim, Alexander von; et al. Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.

Relationship between serum response factor and androgen receptor in prostate cancer

Sun, 01 Nov 2015 00:00:00 GMT

Relationship between serum response factor and androgen receptor in prostate cancer Prencipe, Maria; O'Neill, Amanda; O'Hurley, Gillian; Nguyen, Lan K.; Fabre, Aurélie; Gallagher, William M.; Watson, R. William; et al. Background: Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. Materials and Methods: Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. Results: To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. Conclusion: Our data support SRF as a promising therapeutic target in combination with current treatments. Prostate 75:1704–1717, 2015. © 2015 Wiley Periodicals, Inc.

Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets

Fri, 11 Dec 2015 00:00:00 GMT

Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets Nagy, Zoltan; Wynne, Kieran; Kriegsheim, Alexander von; Gambaryan, Stepan; Smolenski, Albert P. Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells

Fri, 01 May 2015 00:00:00 GMT

Overexpression of the microRNA miR-433 promotes resistance to paclitaxel through the induction of cellular senescence in ovarian cancer cells Weiner-Gorzel, Karolina; Dempsey, Eugene; Milewska, Malgorzata; McGoldrick, Aloysius; Toh, Valerie; Walsh, Aoibheann; Lindsay, Sinead; Gubbins, Luke; Murphy, Madeline; McCann, Amanda; et al. Annually, ovarian cancer (OC) affects 240,000 women worldwide and is the most lethal gynecological malignancy. High-grade serous OC (HGSOC) is the most common and aggressive OC subtype, characterized by widespread genome changes and chromosomal instability and is consequently poorly responsive to chemotherapy treatment. The objective of this study was to investigate the role of the microRNA miR-433 in the cellular response of OC cells to paclitaxel treatment. We show that stable miR-433 expression in A2780 OC cells results in the induction of cellular senescence demonstrated by morphological changes, downregulation of phosphorylated retinoblastoma (p-Rb), and an increase in β-galactosidase activity. Furthermore, in silico analysis identified four possible miR-433 target genes associated with cellular senescence: cyclin-dependent kinase 6 (CDK6), MAPK14, E2F3, and CDKN2A. Mechanistically, we demonstrate that downregulation of p-Rb is attributable to a miR-433-dependent downregulation of CDK6, establishing it as a novel miR-433 associated gene. Interestingly, we show that high miR-433 expressing cells release miR-433 into the growth media via exosomes which in turn can induce a senescence bystander effect. Furthermore, in relation to a chemotherapeutic response, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that only PEO1 and PEO4 OC cells with the highest miR-433 expression survive paclitaxel treatment. Our data highlight how the aberrant expression of miR-433 can adversely affect intracellular signaling to mediate chemoresistance in OC cells by driving cellular senescence.

Multiscale Model of Dynamic Neuromodulation Integrating Neuropeptide-Induced Signaling Pathway Activity with Membrane Electrophysiology

Tue, 06 Jan 2015 00:00:00 GMT

Multiscale Model of Dynamic Neuromodulation Integrating Neuropeptide-Induced Signaling Pathway Activity with Membrane Electrophysiology Makadia, Hirenkumar K.; Anderson, Warren D.; Fey, Dirk; et al. We developed a multiscale model to bridge neuropeptide receptor-activated signaling pathway activity with membrane electrophysiology. Typically, the neuromodulation of biochemical signaling and biophysics have been investigated separately in modeling studies. We studied the effects of Angiotensin II (AngII) on neuronal excitability changes mediated by signaling dynamics and downstream phosphorylation of ion channels. Experiments have shown that AngII binding to the AngII receptor type-1 elicits baseline-dependent regulation of cytosolic Ca2+ signaling. Our model simulations revealed a baseline Ca2+-dependent response to AngII receptor type-1 activation by AngII. Consistent with experimental observations, AngII evoked a rise in Ca2+ when starting at a low baseline Ca2+ level, and a decrease in Ca2+ when starting at a higher baseline. Our analysis predicted that the kinetics of Ca2+ transport into the endoplasmic reticulum play a critical role in shaping the Ca2+ response. The Ca2+ baseline also influenced the AngII-induced excitability changes such that lower Ca2+ levels were associated with a larger firing rate increase. We examined the relative contributions of signaling kinases protein kinase C and Ca2+/Calmodulin-dependent protein kinase II to AngII-mediated excitability changes by simulating activity blockade individually and in combination. We found that protein kinase C selectively controlled firing rate adaptation whereas Ca2+/Calmodulin-dependent protein kinase II induced a delayed effect on the firing rate increase. We tested whether signaling kinetics were necessary for the dynamic effects of AngII on excitability by simulating three scenarios of AngII-mediated KDR channel phosphorylation: (1), an increased steady state; (2), a step-change increase; and (3), dynamic modulation. Our results revealed that the kinetics emerging from neuromodulatory activation of the signaling network were required to account for the dynamical changes in excitability. In summary, our integrated multiscale model provides, to our knowledge, a new approach for quantitative investigation of neuromodulatory effects on signaling and electrophysiology.

Sensitivity Analysis of Stochastic Models of Bistable Biochemical Reactions

Sun, 01 Jun 2008 00:00:00 GMT

Sensitivity Analysis of Stochastic Models of Bistable Biochemical Reactions Degasperi, Andrea; Gilmore, Stephen Sensitivity Analysis (SA) provides techniques which can be used to identify the parameters which have the greatest influence on the results obtained from a model. Classical SA methods apply to deterministic simulations of ODE models. We extend these to stochastic simulations and consider the analysis of models with bifurcation points and bistable behaviour. We consider local, global and screening SA methods applied to multiple runs of Gillespie’s Stochastic Simulation Algorithm (SSA) . We present an example of stochastic sensitivity analysis of a real pathway, the MAPK signalling pathway.

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

Mon, 27 Jan 2014 00:00:00 GMT

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data Degasperi, Andrea; Birtwistle, Marc R.; Volinsky, Natalia; Kolch, Walter; Kholodenko, Boris N. Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

Negative hysteresis in the behavioral dynamics of the affordance graspable

Fri, 08 Mar 2013 00:00:00 GMT

Negative hysteresis in the behavioral dynamics of the affordance graspable Lopresti-Goodman, Stacy M.; Turvey, Michael T.; Frank, Till D. One commonly perceives whether a visible object will afford grasping with one hand or with both hands. In experiments in which differently sized objects of a fixed type are presented, the transition from using one of these manual modes to the other depends on the ratio of object size to hand span and on the presentation sequence, with size increasing versus decreasing. Conventional positive hysteresis (i.e., a larger transition ratio for the increasing sequence) can be accommodated by the order parameter dynamics that typify self-organizing systems (Lopresti-Goodman, Turvey, and Frank, Attention, Perception, & Psychophysics 73:1948–1965, 2011). Here we identified and addressed conditions of unconventional negative hysteresis (i.e., a larger transition ratio for the decreasing sequence). They suggest a second control parameter in the self-organization of affordance perception, one that is seemingly regulated by inhibitory dynamics occurring in the agent–task–environment system. Our experimental results and modeling extend the investigation of affordance perception within dynamical systems theory.

High levels of ephrinB2 over-expression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix

Fri, 01 Feb 2013 00:00:00 GMT

High levels of ephrinB2 over-expression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix Tierney, Erica G.; McSorley, Kevin; Hastings, Conn L.; et al. Gene therapy can be combined with tissue engineering constructs to produce gene-activated matrices (GAMs) with enhanced capacity for repair. Polyethyleneimine (PEI), a non-viral vector, has previously been optimised for high efficiency gene transfer in rat mesenchymal stem cells (rMSCs). The use of PEI to transfect human MSCs (hMSCs) with ephrinB2 is assessed here. Recently a role for the ephrinB2 ligand and EphB4 receptor duo has been proposed in bone remodelling. Herein, over-expression of the ephrinB2 ligand resulted in increased osteogenic differentiation in hMSCs. As ephrinB2 is a cell surface anchored ligand which only interacts with cells expressing the cognate EphB4 receptor through direct contact, we have shown that direct cell–cell contact between two neighbouring cells is responsible for enhanced osteogenesis. In an effort to begin to elucidate the molecular mechanisms at play downstream of ephrinB2 over-expression, RT-PCR was performed on the GAMs which revealed no significant changes in runx2 or BMP2 expression but an upregulation of osterix (Osx) and Dlx5 expression prompting the belief that the mode of osteogenesis is independent of the BMP2 pathway. This select interaction, coupled with the transient gene expression profile of PEI, makes the PEI-ephrinB2 GAM an ideal candidate matrix for a bone targeted GAM.

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells

Wed, 01 Sep 2010 00:00:00 GMT

PI3K/Akt-sensitive MEK-independent compensatory circuit of ERK activation in ER-positive PI3K-mutant T47D breast cancer cells Aksamitiene, Edita; Kholodenko, Boris N.; Kolch, Walter; et al.

Extracellular Signal-Regulated Kinase Regulates RhoA Activation and Tumor Cell Plasticity by Inhibiting Guanine Exchange Factor H1 Activity

Mon, 16 Sep 2013 00:00:00 GMT

Extracellular Signal-Regulated Kinase Regulates RhoA Activation and Tumor Cell Plasticity by Inhibiting Guanine Exchange Factor H1 Activity Thun, A. von; Preisinger, C.; Rath, Oliver; et al. In certain Ras mutant cell lines, the inhibition of extracellular signal-regulated kinase (ERK) signaling increases RhoA activity and inhibits cell motility, which was attributed to a decrease in Fra-1 levels. Here we report a Fra-1-independent augmentation of RhoA signaling during short-term inhibition of ERK signaling. Using mass spectrometry-based proteomics, we identified guanine exchange factor H1 (GEF-H1) as mediating this effect. ERK binds to the Rho exchange factor GEF-H1 and phosphorylates it on S959, causing inhibition of GEF-H1 activity and a consequent decrease in RhoA activity. Knockdown experiments and expression of a nonphosphorylatable S959A GEF-H1 mutant showed that this site is crucial in regulating cell motility and invasiveness. Thus, we identified GEF-H1 as a critical ERK effector that regulates motility, cell morphology, and invasiveness.

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia

Sun, 01 Jul 2012 00:00:00 GMT

Development of a thermoresponsive chitosan gel combined with human mesenchymal stem cells and desferrioxamine as a multimodal pro-angiogenic therapeutic for the treatment of critical limb ischaemia Hastings, Conn L.; Kelly, Helena M.; Murphy, Mary J.; et al. Critical limb ischaemia (CLI) is a debilitating ischaemic disease caused by vascular occlusion. Pro-angiogenic therapeutics have the potential to produce collateral vasculature, delaying or negating the need for amputation or invasive revascularisation. Thermoresponsive hydrogels can provide an in situ depot for the sustained release of drugs and provide protection and cohesion for encapsulated cells. Human mesenchymal stem cells (hMSCs) have demonstrated strong angiogenic potential in vitro and angiogenic efficacy in vivo. Desferrioxamine (DFO), a pharmacological activator of the pro-angiogenic hypoxia inducible factor-1α pathway, has shown pro-angiogenic efficacy in vivo. This study combined hMSCs and DFO with a thermoresponsive chitosan/β-glycerophosphate (β-GP) gel, to function as an injectable, multimodal, pro-angiogenic therapeutic for the treatment of CLI. This gel underwent a thermogelation beginning at 33 °C, and provided a sustained, biologically active release of DFO over the space of seven days, whilst permitting the survival, proliferation and migration of encapsulated hMSCs. hMSCs encapsulated in gel containing a 100 μM concentration of DFO displayed an upregulation in VEGF expression. The combination of hMSCs and DFO within the gel resulted in a synergistic enhancement in bioactivity, as measured by increased VEGF expression in gel-exposed human umbilical vein endothelial cells. This formulation displays significant potential as an injectable pro-angiogenic therapeutic for the treatment of CLI.

Increased Baseline Proinflammatory Cytokine Production in Chronic Hepatitis C Patients with Rapid Virological Response to Peginterferon Plus Ribavirin

Tue, 09 Jul 2013 00:00:00 GMT

Increased Baseline Proinflammatory Cytokine Production in Chronic Hepatitis C Patients with Rapid Virological Response to Peginterferon Plus Ribavirin Par, Gabriella; Szereday, Laszlo; Berki, Timea; et al. Background: Chronic hepatitis C (CHC) patients achieving rapid virological response (RVR) on PEG-IFN/ribavirin (P/R) therapy have high chance of sustained virological response (SVR). To analyze host immunological factors associated with RVR, viral kinetics, phenotype distribution and Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) were studied prior to and during P/R therapy. Methods: TNF-α, IFN-γ, IL-2, IL-6, IL-4 and IL-10 production by PBMC were measured after Toll-like receptor 4 (TLR-4) or phorbol myristate acetate/Ionomycin stimulation in 20 healthy controls and in 50 CHC patients before receiving and during P/R therapy. RVR was achieved by 14, complete early virological response (cEVR) by 19 patients and 17 patients were null-responders (NR). Results: Patients with RVR showed an increased baseline TNF-α and IL-6 production by TLR-4 activated monocytes and increased IFN-γ, decreased IL-4 and IL-10 production by lymphocytes compared to non-RVR patients. SVR was also associated with increased baseline TNF-α production and decreased IL-10 levels compared to patients who did not achieve SVR. Baseline IL-2 production was higher in cEVR compared to NR patients. Antiviral treatment increased TNF-α, IL-6 production by monocytes and IFN-γ secretion by lymphocytes and decreased IL-4 and IL-10 production by lymphocytes in cEVR compared to NR patients. Conclusion: RVR was associated with increased baseline proinflammatory cytokine production by TLR-4 stimulated monocytes and by activated lymphocytes. In null-responders and in patients who did not achieve SVR both TLR-4 sensing function and proinflammatory cytokine production were impaired, suggesting that modulation of TLR activity and controlled induction of inflammatory cytokine production may provide further therapeutic strategy for CHC patients non-responding to P/R treatment.

The OARSI histopathology initiative – recommendations for histological assessments of osteoarthritis in sheep and goats

Fri, 01 Oct 2010 00:00:00 GMT

The OARSI histopathology initiative – recommendations for histological assessments of osteoarthritis in sheep and goats Little, C. B.; Smith, M. M.; Cake, M. A.; et al. Objective: Sheep and goats are commonly used large animal species for studying pathogenesis and treatment of osteoarthritis (OA). This review focuses on the macroscopic and microscopic criteria for assessing OA in sheep and goats and recommends particular assessment criteria to assist standardization in the conduct and reporting of preclinical trials of OA. Methods: A review was conducted of all published OA studies using sheep and goats and the most common macroscopic, microscopic, or ultrastructural scoring systems were summarised. General recommendations regarding methods of OA assessment in the sheep and goat have been made and a preliminary study of their reliability and utility was undertaken. Results: The modified Mankin scoring system is recommended for semiquantitative histological assessment of OA due to its already widespread adoption, ease of use, similarity to scoring systems used for OA in humans, and its achievable inter-rater reliability. Specific recommendations are also provided for histological scoring of synovitis and scoring of macroscopic lesions of OA. Conclusions: The proposed system for assessment of sheep and goat articular tissues appears to provide a useful versatile method to quantify OA change. It is hoped that by adopting more standardised quantitative outcome measures, better comparison between different studies and arthritis models will be possible. The suggested scoring systems can be modified in the future as our knowledge of disease pathophysiology advances.

Mesenchymal chondroprogenitor cell origin and therapeutic potential

Sat, 01 Jan 2011 00:00:00 GMT

Mesenchymal chondroprogenitor cell origin and therapeutic potential O'Sullivan, Janice; D'Arcy, Sinéad; Barry, Frank P.; et al. Mesenchymal progenitor cells, a multipotent adult stem cell population, have the ability to differentiate into cells of connective tissue lineages, including fat, cartilage, bone and muscle, and therefore generate a great deal of interest for their potential use in regenerative medicine. During development, endochondral bone is formed from a template of cartilage that transforms into bone; however, mature articular cartilage remains in the articulating joints, where its principal role is reducing friction and dispersing mechanical load. Articular cartilage is prone to damage from sports injuries or ageing, which regularly progresses to more serious joint disorders, such as osteoarthritis. Osteoarthritis is a degenerative joint disease characterized by the thinning and eventual wearing of articular cartilage, and affects millions of people worldwide. Due to low chondrocyte motility and proliferative rates, and complicated by the absence of blood vessels, cartilage has a limited ability to self-repair. Current pharmaceutical and surgical interventions fail to generate repair tissue with the mechanical and cellular properties of native host cartilage. The long-term success of cartilage repair will therefore depend on regenerative methodologies resulting in the restoration of articular cartilage that closely duplicates the native tissue. For cell-based therapies, the optimal cell source must be readily accessible with easily isolated, abundant cells capable of collagen type II and sulfated proteoglycan production in appropriate proportions. Although a cell source with these therapeutic properties remains elusive, mesenchymal chondroprogenitors retain their expansion capacity with the promise of reproducing the structural or biomechanical properties of healthy articular cartilage. As current knowledge regarding chondroprogenitors is relatively limited, this review will focus on their origin and therapeutic application

Mitochondrial reactive oxygen species enhance AMP-activated protein kinase activation in the endothelium of patients with coronary artery disease and diabetes

Tue, 27 Nov 2012 00:00:00 GMT

Mitochondrial reactive oxygen species enhance AMP-activated protein kinase activation in the endothelium of patients with coronary artery disease and diabetes Mackenzie, Ruth M.; Salt, Ian P.; Miller, William H.; et al. The aim of the present study was to determine whether the endothelial dysfunction associated with CAD (coronary artery disease) and T2D (Type 2 diabetes mellitus) is concomitant with elevated mtROS (mitochondrial reactive oxygen species) production in the endothelium and establish if this, in turn, regulates the activity of endothelial AMPK (AMP-activated protein kinase). We investigated endothelial function, mtROS production and AMPK activation in saphenous veins from patients with advanced CAD. Endothelium-dependent vasodilation was impaired in patients with CAD and T2D relative to those with CAD alone. Levels of mitochondrial H2O2 and activity of AMPK were significantly elevated in primary HSVECs (human saphenous vein endothelial cells) from patients with CAD and T2D compared with those from patients with CAD alone. Incubation with the mitochondria-targeted antioxidant, MitoQ10 significantly reduced AMPK activity in HSVECs from patients with CAD and T2D but not in cells from patients with CAD alone. Elevated mtROS production in the endothelium of patients with CAD and T2D increases AMPK activation, supporting a role for the kinase in defence against oxidative stress. Further investigation is required to determine whether pharmacological activators of AMPK will prove beneficial in the attenuation of endothelial dysfunction in patients with CAD and T2D.

Systems medicine: helping us understand the complexity of disease

Tue, 30 Jul 2013 00:00:00 GMT

Systems medicine: helping us understand the complexity of disease Vandamme, Drieke; Fitzmaurice, William; Kholodenko, Boris N.; et al. Advances in genomics and other -omic fields in the last decade have resulted in unprecedented volumes of complex data now being available. These data can enable physicians to provide their patients with care that is more personalized, predictive, preventive and participatory. The expertise required to manage and understand this data is to be found in fields outside of medical science, thus multidisciplinary collaboration coupled to a systems approach is key to unlocking its potential, with concomitant new ways of working. Systems medicine can build on the successes in the field of systems biology, recognizing the human body as the multidimensional network of networks that it is. While systems medicine can provide a conceptual and theoretical framework, its practical goal is to provide physicians the tools necessary for harnessing the rapid advances in basic biomedical science into their routine clinical arsenal.

Regulation of gene expression by carbon dioxide

Tue, 04 Jan 2011 00:00:00 GMT

Regulation of gene expression by carbon dioxide Taylor, Cormac T.; Cummins, Eoin P. Carbon dioxide (CO2) is a physiological gas found at low levels in the atmosphere and produced in cells during the process of aerobic respiration. Consequently, the levels of CO2 within tissues are usually significantly higher than those found externally. Shifts in tissue levels of CO2 (leading to either hypercapnia or hypocapnia) are associated with a number of pathophysiological conditions in humans and can occur naturally in niche habitats such as those of burrowing animals. Clinical studies have indicated that such altered CO2 levels can impact upon disease progression. Recent advances in our understanding of the biology of CO2 has shown that like other physiological gases such as molecular oxygen (O2) and nitric oxide (NO), CO2 levels can be sensed by cells resulting in the initiation of physiological and pathophysiological responses. Acute CO2 sensing in neurons and peripheral and central chemoreceptors is important in rapidly activated responses including olfactory signalling, taste sensation and cardiorespiratory control. Furthermore, a role for CO2 in the regulation of gene transcription has recently been identified with exposure of cells and model organisms to high CO2 leading to suppression of genes involved in the regulation of innate immunity and inflammation. This latter, transcriptional regulatory role for CO2, has been largely attributed to altered activity of the NF-κB family of transcription factors. Here, we review our evolving understanding of how CO2 impacts upon gene transcription.

Isolation and Phenotypic Characterisation of Stem Cells from Late Stage Osteoarthritic Mesenchymal Tissues

Wed, 01 Aug 2012 00:00:00 GMT

Isolation and Phenotypic Characterisation of Stem Cells from Late Stage Osteoarthritic Mesenchymal Tissues Labusca, Luminita; Zugun-Eloae, Florin; Shaw, Georgina; et al. Introduction: Osteoarthritis (OA) represents an increasing health issue worldwide. Regenerative medicine (RM) has raised the hope for introducing revolutionary therapies in clinical practice. Detection of autologus cell sources can improve accessibility to RM strategies. Objectives: To assess the presence and biological potential of mesehchymal stem cells in three tissues (subchondral bone, synovial layer, periarticular adipose tissue) in late stages osteoarthritic patients. Material and Methods: Samples were collected from subjects undergoing total knee replacement (TKR). MSCs were isolated and cultured in complete αMEM with β FGF. Cell morphology and growth potential was assessed. Flow cytometry was used for detection of several relevant cell surface markers. Quantitative and qualitative assessment of differentiation potential towards three mesenchymal lineages (osteogenesis adipogenesis chondrogenesis) was performed. Time lapse life cell imaging of nondiferentiated cells over 24 hours period was used to determine cell kinetics. Results: Mesenchymal cells derived from all donors and tissue types showed morphology, growth and surface cell markers associated with stemness. All cell types underwent differentiation toward three mesenchymal lineages with significant differences between tissues of origin, not between donors. Cell kinetics, as derived from life imaging records, was variable with tissue of origin, significant higher for adipose derived MSCS. Conclusion: Human late stage OA mesenchymal tissues, contain progenitors with proliferative and differentiation potential of MSCs. These populations can be used for research and autologus regenerative therapies. Further comparative studies with age matched non OA samples has the potential of contributing to deepening knowledge about disease occurrence and progression.

Loss of Prolyl Hydroxylase-1 Protects Against Colitis Through Reduced Epithelial Cell Apoptosis and Increased Barrier Function

Wed, 01 Dec 2010 00:00:00 GMT

Loss of Prolyl Hydroxylase-1 Protects Against Colitis Through Reduced Epithelial Cell Apoptosis and Increased Barrier Function Tambuwala, Murtaza M.; Cummins, Eoin P.; Lenihan, Colin R.; et al. Background & Aims: Hypoxia inducible factor (HIF) prolyl hydroxylase inhibitors are protective in mouse models of inflammatory bowel disease (IBD). Here, we investigated the therapeutic target(s) and mechanism(s) involved. Methods: The effect of genetic deletion of individual HIF-prolyl hydroxylase (PHD) enzymes on the development of dextran sulphate sodium (DSS)–induced colitis was examined in mice. Results: PHD1−/−, but not PHD2+/− or PHD3−/−, mice were less susceptible to the development of colitis than wild-type controls as determined by weight loss, disease activity, colon histology, neutrophil infiltration, and cytokine expression. Reduced susceptibility of PHD1−/− mice to colitis was associated with increased density of colonic epithelial cells relative to wild-type controls, which was because of decreased levels of apoptosis that resulted in enhanced epithelial barrier function. Furthermore, with the use of cultured epithelial cells it was confirmed that hydroxylase inhibition reversed DSS-induced apo tosis and barrier dysfunction. Finally, PHD1 levels were increased with disease severity in intestinal tissue from patients with IBD and in colonic tissues from DSS-treated mice. Conclusions: These results imply a role for PHD1 as a positive regulator of intestinal epithelial cell apoptosis in the inflamed colon. Genetic loss of PHD1 is protective against colitis through decreased epithelial cell apoptosis and consequent enhancement of intestinal epithelial barrier function. Thus, targeted PHD1 inhibition may represent a new therapeutic approach in IBD.

Hypoxia, innate immunity and infection in the lung

Wed, 01 Dec 2010 00:00:00 GMT

Hypoxia, innate immunity and infection in the lung Schaible, Bettina; Schaffer, Kirsten; Taylor, Cormac T. The mucosal surface of the lung is the key interface between the external atmosphere and the bloodstream. Normally, this well oxygenated tissue is maintained in state of sterility by a number of innate immune processes. These include a physical and dynamic mucus barrier, the production of microbiocidal peptides and the expression of specific pattern recognition receptors on alveolar epithelial cells and resident macrophages and dendritic cells which recognise microbial structures and initiate innate immune responses which promote the clearance of potentially infectious agents. In a range of diseases, the mucosal surface of the lung experiences decreased oxygen tension leading to localised areas of prominent hypoxia which can impact upon innate immune and subsequent infectious and inflammatory processes. Under these conditions, the lung is generally more susceptible to infection and subsequent inflammation. In the current review, we will discuss recent data pertaining to the role of hypoxia in regulating both host and pathogen in the lung during pulmonary disease and how this contributes to innate immunity, infection and inflammation.

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling

Tue, 06 Mar 2012 00:00:00 GMT

Hypercapnia Induces Cleavage and Nuclear Localization of RelB Protein, Giving Insight into CO2 Sensing and Signaling Oliver, K. M.; Lenihan, Colin R.; Bruning, Ulrike; et al. Carbon dioxide (CO2) is increasingly being appreciated as an intracellular signaling molecule that affects inflammatory and immune responses. Elevated arterial CO2 (hypercapnia) is encountered in a range of clinical conditions, including chronic obstructive pulmonary disease, and as a consequence of therapeutic ventilation in acute respiratory distress syndrome. In patients suffering from this syndrome, therapeutic hypoventilation strategy designed to reduce mechanical damage to the lungs is accompanied by systemic hypercapnia and associated acidosis, which are associated with improved patient outcome. However, the molecular mechanisms underlying the beneficial effects of hypercapnia and the relative contribution of elevated CO2 or associated acidosis to this response remain poorly understood. Recently, a role for the non-canonical NF-κB pathway has been postulated to be important in signaling the cellular transcriptional response to CO2. In this study, we demonstrate that in cells exposed to elevated CO2, the NF-κB family member RelB was cleaved to a lower molecular weight form and translocated to the nucleus in both mouse embryonic fibroblasts and human pulmonary epithelial cells (A549). Furthermore, elevated nuclear RelB was observed in vivo and correlated with hypercapnia-induced protection against LPS-induced lung injury. Hypercapnia-induced RelB processing was sensitive to proteasomal inhibition by MG-132 but was independent of the activity of glycogen synthase kinase 3β or MALT-1, both of which have been previously shown to mediate RelB processing. Taken together, these data demonstrate that RelB is a CO2-sensitive NF-κB family member that may contribute to the beneficial effects of hypercapnia in inflammatory diseases of the lung.

MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability

Mon, 12 Oct 2009 00:00:00 GMT

MST Kinases Monitor Actin Cytoskeletal Integrity and Signal via c-Jun N-Terminal Kinase Stress-Activated Kinase To Regulate p21Waf1/Cip1 Stability Densham, R. M.; E'Neill, Eric; Munro, J.; et al. As well as providing a structural framework, the actin cytoskeleton plays integral roles in cell death, survival, and proliferation. The disruption of the actin cytoskeleton results in the activation of the c-Jun N-terminal kinase (JNK) stress-activated protein kinase (SAPK) pathway; however, the sensor of actin integrity that couples to the JNK pathway has not been characterized in mammalian cells. We now report that the mammalian Ste20-like (MST) kinases mediate the activation of the JNK pathway in response to the disruption of the actin cytoskeleton. One consequence of actin disruption is the JNK-mediated stabilization of p21Waf1/Cip1 (p21) via the phosphorylation of Thr57. The expression of MST1 or MST2 was sufficient to stabilize p21 in a JNK- and Thr57-dependent manner, while the stabilization of p21 by actin disruption required MST activity. These data indicate that, in addition to being components of the Salvador-Warts-Hippo tumor suppressor network and binding partners of c-Raf and the RASSF1A tumor suppressor, MST kinases serve to monitor cytoskeletal integrity and couple via the JNK SAPK pathway to the regulation of a key cell cycle regulatory protein.

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor

Tue, 30 Aug 2011 00:00:00 GMT

Mesenchymal stem cell inhibition of T-helper 17 cell- differentiation is triggered by cell-cell contact and mediated by prostaglandin E2 via the EP4 receptor Duffy, Michelle M.; Pindjakova, Jana; Hanley, Shirley A.; et al. Mesenchymal stem cells (MSCs) inhibit T-cell activation and proliferation but their effects on individual T-cell-effector pathways and on memory versus naïve T cells remain unclear. MSC influence on the differentiation of naïve and memory CD4+ T cells toward the Th17 phenotype was examined. CD4+ T cells exposed to Th17-skewing conditions exhibited reduced CD25 and IL-17A expression following MSC co-culture. Inhibition of IL-17A production persisted upon re-stimulation in the absence of MSCs. These effects were attenuated when cell–cell contact was prevented. Th17 cultures from highly purified naïve- and memory-phenotype responders were similarly inhibited. Th17 inhibition by MSCs was reversed by indomethacin and a selective COX-2 inhibitor. Media from MSC/Th17 co-cultures contained increased prostaglandin E2 (PGE2) levels and potently suppressed Th17 differentiation in fresh cultures. MSC-mediated Th17 inhibition was reversed by a selective EP4 antagonist and was mimicked by synthetic PGE2 and a selective EP4 agonist. Activation-induced IL-17A secretion by naturally occurring, effector-memory Th17 cells from a urinary obstruction model was also inhibited by MSC co-culture in a COX-dependent manner. Overall, MSCs potently inhibit Th17 differentiation from naïve and memory T-cell precursors and inhibit naturally-occurring Th17 cells derived from a site of inflammation. Suppression entails cell-contact-dependent COX-2 induction resulting in direct Th17 inhibition by PGE2 via EP4.

Signalling ballet in space and time

Tue, 01 Jun 2010 00:00:00 GMT

Signalling ballet in space and time Kholodenko, Boris N.; Hancock, John F.; Kolch, Walter Although we have amassed extensive catalogues of signalling network components, our understanding of the spatiotemporal control of emergent network structures has lagged behind. Dynamic behaviour is starting to be explored throughout the genome, but analysis of spatial behaviours is still confined to individual proteins. The challenge is to reveal how cells integrate temporal and spatial information to determine specific biological functions. Key findings are the discovery of molecular signalling machines such as Ras nanoclusters, spatial activity gradients and flexible network circuitries that involve transcriptional feedback. They reveal design principles of spatiotemporal organization that are crucial for network function and cell fate decisions.

Signalling over a distance: gradient patterns and phosphorylation waves within single cells

Fri, 01 Oct 2010 00:00:00 GMT

Signalling over a distance: gradient patterns and phosphorylation waves within single cells Muñoz-García, Javier; Kholodenko, Boris N. Recent discoveries of phosphorylation gradients and microdomains with different protein activities have revolutionized our perception of information transfer within single cells. The different spatial localization of opposing reactions in protein-modification cycles has been shown to bring about heterogeneous stationary patterns and travelling waves of protein activities. We review spatial patterns and modes of signal transfer through phosphorylation/dephosphorylation and GDP/GTP exchange cycles and cascades. We show how switches between low-activity and high-activity states in a bistable activation–deactivation cycle can initiate the propagation of travelling protein-modification waves in the cytoplasm. Typically, an activation wave is initiated at the plasma membrane and propagates through the cytoplasm until it reaches the nucleus. An increase in deactivator activity is followed by the initiation of an inactivation wave that moves in the reverse direction from the nucleus. We show that the ratio of opposing enzyme rates is a key parameter that controls both the spread of activation through cascades and travelling waves.

Signalling by protein phosphatases and drug development: a systems-centred view

Wed, 14 Mar 2012 00:00:00 GMT

Signalling by protein phosphatases and drug development: a systems-centred view Nguyen, Lan K.; Matallanas, David; Croucher, David R.; et al. Protein modification cycles catalysed by opposing enzymes, such as kinases and phosphatases, form the backbone of signalling networks. Although, historically, kinases have been at the research forefront, a systems-centred approach reveals predominant roles for phosphatases in controlling the network response times and spatio-temporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for network function and cell-fate decisions. Protein phosphatases operate as both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasizes the need for systems biology approaches to understand cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.

NF-B Links CO2 Sensing to Innate Immunity and Inflammation in Mammalian Cells

Fri, 03 Sep 2010 00:00:00 GMT

NF-B Links CO2 Sensing to Innate Immunity and Inflammation in Mammalian Cells Cummins, Eoin P.; Oliver, K. M.; Lenihan, Colin R.; et al. Molecular O2 and CO2 are the primary substrate and product of aerobic metabolism, respectively. Levels of these physiologic gases in the cell microenvironment vary dramatically both in health and in diseases, such as chronic inflammation, ischemia, and cancer, in which metabolism is significantly altered. The identification of the hypoxia-inducible factor led to the discovery of an ancient and direct link between tissue O2 and gene transcription. In this study, we demonstrate that mammalian cells (mouse embryonic fibroblasts and others) also sense changes in local CO2 levels, leading to altered gene expression via the NF-κB pathway. IKKα, a central regulatory component of NF-κB, rapidly and reversibly translocates to the nucleus in response to elevated CO2. This response is independent of hypoxia-inducible factor hydroxylases, extracellular and intracellular pH, and pathways that mediate acute CO2-sensing in nematodes and flies and leads to attenuation of bacterial LPS-induced gene expression. These results suggest the existence of a molecular CO2 sensor in mammalian cells that is linked to the regulation of genes involved in innate immunity and inflammation.

Toggle switches, pulses and oscillations are intrinsic properties of the Src activation/deactivation cycle

Sat, 01 Aug 2009 00:00:00 GMT

Toggle switches, pulses and oscillations are intrinsic properties of the Src activation/deactivation cycle Kaimachnikov, Nikolai P.; Kholodenko, Boris N. Src-family kinases (SFKs) play a pivotal role in growth factor signaling, mitosis, cell motility and invasiveness. In their basal state, SFKs maintain a closed autoinhibited conformation, where the Src homology 2 domain interacts with an inhibitory phosphotyrosine in the C-terminus. Activation involves dephosphorylation of this inhibitory phosphotyrosine, followed by intermolecular autophosphorylation of a specific tyrosine residue in the activation loop. The spatiotemporal dynamics of SFK activation controls cell behavior, yet these dynamics remain largely uninvestigated. In the present study, we show that the basic properties of the Src activation/deactivation cycle can bring about complex signaling dynamics, including oscillations, toggle switches and excitable behavior. These intricate dynamics do not require imposed external feedback loops and occur at constant activities of Src inhibitors and activators, such as C-terminal Src kinase and receptor-type protein tyrosine phosphatases. We demonstrate that C-terminal Src kinase and receptor-type protein tyrosine phosphatase underexpression or their simultaneous overexpression can transform Src response patterns into oscillatory or bistable responses, respectively. Similarly, Src overexpression leads to dysregulation of Src activity, promoting sustained self-perpetuating oscillations. Distinct types of responses can allow SFKs to trigger different cell-fate decisions, where cellular outcomes are determined by the stimulation threshold and history. Our mathematical model helps to understand the puzzling experimental observations and suggests conditions where these different kinetic behaviors of SFKs can be tested experimentally.

Gremlin Plays a Key Role in the Pathogenesis of Pulmonary Hypertension

Fri, 13 Jan 2012 00:00:00 GMT

Gremlin Plays a Key Role in the Pathogenesis of Pulmonary Hypertension Cahill, Edwina; Costello, Christine M.; Rowan, Simon C.; et al. Background—Pulmonary hypertension occurs in chronic hypoxic lung diseases, significantly worsening morbidity and mortality. The important role of altered bone morphogenetic protein (BMP) signaling in pulmonary hypertension was first suspected after the identification of heterozygous BMP receptor mutations as the underlying defect in the rare heritable form of pulmonary arterial hypertension. Subsequently, it was demonstrated that BMP signaling was also reduced in common forms of pulmonary hypertension, including hypoxic pulmonary hypertension; however, the mechanism of this reduction has not previously been elucidated. Methods and Results—Expression of 2 BMP antagonists, gremlin 1 and gremlin 2, was higher in the lung than in other organs, and gremlin 1 was further increased in the walls of small intrapulmonary vessels of mice during the development of hypoxic pulmonary hypertension. Hypoxia stimulated gremlin secretion from human pulmonary microvascular endothelial cells in vitro, which inhibited endothelial BMP signaling and BMP-stimulated endothelial repair. Haplodeficiency of gremlin 1 augmented BMP signaling in the hypoxic mouse lung and reduced pulmonary vascular resistance by attenuating vascular remodeling. Furthermore, gremlin was increased in the walls of small intrapulmonary vessels in idiopathic pulmonary arterial hypertension and the rare heritable form of pulmonary arterial hypertension in a distribution suggesting endothelial localization. Conclusions—These findings demonstrate a central role for increased gremlin in hypoxia-induced pulmonary vascular remodeling and the increased pulmonary vascular resistance in hypoxic pulmonary hypertension. High levels of basal gremlin expression in the lung may account for the unique vulnerability of the pulmonary circulation to heterozygous mutations of BMP type 2 receptor in pulmonary arterial hypertension.

Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles

Fri, 16 Nov 2012 00:00:00 GMT

Cell Type-Specific Activation of AKT and ERK Signaling Pathways by Small Negatively-Charged Magnetic Nanoparticles Rauch, Jens; Kolch, Walter; Mahmoudi, Morteza The interaction of nanoparticles (NPs) with living organisms has become a focus of public and scientific debate due to their potential wide applications in biomedicine, but also because of unwanted side effects. Here, we show that superparamagnetic iron oxide NPs (SPIONs) with different surface coatings can differentially affect signal transduction pathways. Using isogenic pairs of breast and colon derived cell lines we found that the stimulation of ERK and AKT signaling pathways by SPIONs is selectively dependent on the cell type and SPION type. In general, cells with Ras mutations respond better than their non-mutant counterparts. Small negatively charged SPIONs (snSPIONs) activated ERK to a similar extent as epidermal growth factor (EGF), and used the same upstream signaling components including activation of the EGF receptor. Importantly, snSPIONs stimulated the proliferation of Ras transformed breast epithelial cells as efficiently as EGF suggesting that NPs can mimic physiological growth factors.

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells

Tue, 01 Nov 2011 00:00:00 GMT

Prolactin-stimulated activation of ERK1/2 mitogen-activated protein kinases is controlled by PI3-kinase/Rac/PAK signaling pathway in breast cancer cells Aksamitiene, Edita; Achanta, Sirisha; Kolch, Walter; et al. There is strong evidence that deregulation of prolactin (PRL) signaling contributes to pathogenesis and chemoresistance of breast cancer. Therefore, understanding cross-talk between distinct signal transduction pathways triggered by activation of the prolactin receptor (PRL-R), is essential for elucidating the pathogenesis of metastatic breast cancer. In this study, we applied a sequential inhibitory analysis of various signaling intermediates to examine the hierarchy of protein interactions within the PRL signaling network and to evaluate the relative contributions of multiple signaling branches downstream of PRL-R to the activation of the extracellular signal-regulated kinases ERK1 and ERK2 in T47D and MCF-7 human breast cancer cells. Quantitative measurements of the phosphorylation/activation patterns of proteins showed that PRL simultaneously activated Src family kinases (SFKs) and the JAK/STAT, phosphoinositide-3 (PI3)-kinase/Akt and MAPK signaling pathways. The specific blockade or siRNA-mediated suppression of SFK/FAK, JAK2/STAT5, PI3-kinase/PDK1/Akt, Rac/PAK or Ras regulatory circuits revealed that (1) the PI3-kinase/Akt pathway is required for activation of the MAPK/ERK signaling cascade upon PRL stimulation; (2) PI3-kinase-mediated activation of the c-Raf-MEK1/2-ERK1/2 cascade occurs independent of signaling dowstream of STATs, Akt and PKC, but requires JAK2, SFKs and FAK activities; (3) activated PRL-R mainly utilizes the PI3-kinase-dependent Rac/PAK pathway rather than the canonical Shc/Grb2/SOS/Ras route to initiate and sustain ERK1/2 signaling. By interconnecting diverse signaling pathways PLR may enhance proliferation, survival, migration and invasiveness of breast cancer cells.

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase

Thu, 24 Oct 2013 00:00:00 GMT

Tyr728 in the Kinase Domain of the Murine Kinase Suppressor of RAS 1 Regulates Binding and Activation of the Mitogen-activated Protein Kinase Kinase Sibilski, C.; Mueller, T.; Kollipara, L.; et al. In metazoans, the highly conserved MAPK signaling pathway regulates cell fate decision. Aberrant activation of this pathway has been implicated in multiple human cancers and some developmental disorders. KSR1 functions as an essential scaffold that binds the individual components of the cascade and coordinates their assembly into multiprotein signaling platforms. The mechanism of KSR1 regulation is highly complex and not completely understood. In this study, we identified Tyr728 as a novel regulatory phosphorylation site in KSR1. We show that Tyr728 is phosphorylated by LCK, uncovering an additional and unexpected link between Src kinases and MAPK signaling. To understand how phosphorylation of Tyr728 may regulate the role of KSR1 in signal transduction, we integrated structural modeling and biochemical studies. We demonstrate that Tyr728 is involved in maintaining the conformation of the KSR1 kinase domain required for binding to MEK. It also affects phosphorylation and activation of MEK by RAF kinases and consequently influences cell proliferation. Moreover, our studies suggest that phosphorylation of Tyr728 may affect the intrinsic kinase activity of KSR1. Together, we propose that phosphorylation of Tyr728 may regulate the transition between the scaffolding and the catalytic function of KSR1 serving as a control point used to fine-tune cellular responses.

Carbon dioxide-sensing in organisms and its implications for human disease

Wed, 18 Sep 2013 00:00:00 GMT

Carbon dioxide-sensing in organisms and its implications for human disease Cummins, Eoin P.; Selfridge, Andrew C.; Sporn, Peter H.; et al. The capacity of organisms to sense changes in the levels of internal and external gases and to respond accordingly is central to a range of physiologic and pathophysiologic processes. Carbon dioxide, a primary product of oxidative metabolism is one such gas that can be sensed by both prokaryotic and eukaryotic cells and in response to altered levels, elicit the activation of multiple adaptive pathways. The outcomes of activating CO2-sensitive pathways in various species include increased virulence of fungal and bacterial pathogens, prey-seeking behavior in insects as well as taste perception, lung function, and the control of immunity in mammals. In this review, we discuss what is known about the mechanisms underpinning CO2 sensing across a range of species and consider the implications of this for physiology, disease progression, and the possibility of developing new therapeutics for inflammatory and infectious disease.