Perturbation Of Signaling Pathways Research Articles

Application of NMR-based Metabolomics to the Study of Gut Microbiota in Obesity

Lifestyle habits, host gene repertoire, and alterations in the intestinal microbiota concur to the development of obesity. A great deal of research has recently been focused on investigating the role gut microbiota plays in the pathogenesis of metabolic dysfunctions and increased adiposity. Altered microbiota can affect host physiology through several pathways, including enhanced energy harvest, and perturbations in immunity, metabolic signaling, and inflammatory pathways. A broad range of "omics" technologies is now available to help decipher the interactions between the host and the gut microbiota at detailed genetic and functional levels. In particular, metabolomics--the comprehensive analysis of metabolite composition of biological fluids and tissues--could provide breakthrough insights into the links among the gut microbiota, host genetic repertoire, and diet during the development and progression of obesity. Here, we briefly review the most insightful findings on the involvement of gut microbiota in the pathogenesis of obesity. We also discuss how metabolomic approaches based on nuclear magnetic resonance spectroscopy could help understand the activity of gut microbiota in relation to obesity, and assess the effects of gut microbiota modulation in the treatment of this condition.

Lentiviral Arrays for Live-cell Dynamic Monitoring of Gene and Pathway Activity During Stem Cell Differentiation

Uncovering the complexity of mesenchymal stem cell (MSC) differentiation requires novel methods to capture the dynamics of the process in a quantitative and high-throughput manner. To this end, we developed a lentiviral array (LVA) of reporters to capture the dynamics of gene and pathway activity during MSC differentiation into adipogenic, chondrogenic, and osteogenic lineages. Our results identified signature promoters and pathways with unique activation profile for each MSC lineage. In combination with chemical inhibitors, lineage-specific reporters predicted the effects of signaling pathway perturbations on MSC differentiation. Interestingly, some pathways were critical for differentiation into all lineages, while others had differential effects on each lineage. Our study suggests that when combined with large chemical or siRNA libraries, the reporter LVA can be used to uncover novel genes and signaling pathways affecting complex biological processes such as stem cell differentiation or reprogramming.

Pharmacological suppression of the Ras/MAPK pathway in thyroid carcinoma cells can provoke opposite effects on cell migration and proliferation: The appearance of yin-yang effects and the need of combinatorial treatments.

A major challenge in tumor therapy is the decrease or even the halting of cell proliferation and migration of cancerous cells. In the present study, we have analyzed the impact of a pharmacological blockade of the PI3K/Akt and MAPK/ERK1/2 signaling pathways on cell migration, proliferation and cell death in three human thyroid tumor cell lines that represent the main types of malignant thyroid carcinomas (B-CPAP, follicular; Cal-62, anaplastic; FTC-133, papillary thyroid carcinoma cells) and in which these pathways are constitutively activated. In general, pharmacological perturbation of PI3/Akt (application of MK-2206) and MEK/ERK1/2 (application of PD0325901 or U0126) signaling led to a cell line and drug-specific decrease in the proliferation and migration potential of thyroid carcinoma cells, although to a varying extent. However, one exception became apparent: in Cal-62 cells inhibition of the MEK/ERK1/2 module increased the migration rate up to 50%. This effect could be prevented by a simultaneous suppression of the PI3/Akt pathway, but also by application of the multiple kinase inhibitor sorafenib, a treatment that did not change the activation state of Akt. Thus, a pharmacological perturbation of canonical signaling pathways in thyroid carcinoma may induce drug-dependent yin-yang effects that are characterized by a simultaneous suppression of one (i.e., proliferation) and the activation of another (i.e., migration) cellular process. The appearance of such phenomena should be taken into account when therapy plans are established.

EU-OPENSCREEN-chemical tools for the study of plant biology and resistance mechanisms.

EU-OPENSCREEN is an academic research infrastructure initiative in Europe for enabling researchers in all life sciences to take advantage of chemical biology approaches to their projects. In a collaborative effort of national networks in 16 European countries, EU-OPENSCREEN will develop novel chemical compounds with external users to address questions in, among other fields, systems and network biology (directed and selective perturbation of signalling pathways), structural biology (compound-target interactions at atomic resolution), pharmacology (early drug discovery and toxicology) and plant biology (response of wild or crop plants to environmental and agricultural substances). EU-OPENSCREEN supports all stages of a tool development project, including assay adaptation, high-throughput screening and chemical optimisation of the ‘hit’ compounds. All tool compounds and data will be made available to the scientific community. EU-OPENSCREEN integrates high-capacity screening platforms throughout Europe, which share a rationally selected compound collection comprising up to 300,000 (commercial and proprietary compounds collected from European chemists). By testing systematically this chemical collection in hundreds of assays originating from very different biological themes, the screening process generates enormous amounts of information about the biological activities of the substances and thereby steadily enriches our understanding of how and where they act.

A pro‐inflammatory signalome is constitutively activated by C33Y mutant TNF receptor 1 in TNF receptor‐associated periodic syndrome (TRAPS)

Mutations in TNFRSF1A encoding TNF receptor 1 (TNFR1) cause the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS): a systemic autoinflammatory disorder. Misfolding, intracellular aggregation, and ligand-independent signaling by mutant TNFR1 are central to disease pathophysiology. Our aim was to understand the extent of signaling pathway perturbation in TRAPS. A prototypic mutant TNFR1 (C33Y), and wild-type TNFR1 (WT), were expressed at near physiological levels in an SK-Hep-1 cell model. TNFR1-associated signaling pathway intermediates were examined in this model, and in PBMCs from C33Y TRAPS patients and healthy controls. In C33Y-TNFR1-expressing SK-Hep-1 cells and TRAPS patients’ PBMCs, a subtle, constitutive upregulation of a wide spectrum of signaling intermediates and their phosphorylated forms was observed; these were associated with a proinflammatory/antiapoptotic phenotype. In TRAPS patients’ PBMCs, this upregulation of proinflammatory signaling pathways was observed irrespective of concurrent treatment with glucocorticoids, anakinra or etanercept, and the absence of overt clinical symptoms at the time that the blood samples were taken. This study reveals the pleiotropic effect of a TRAPS-associated mutant form of TNFR1 on inflammatory signaling pathways (a proinflammatory signalome), which is consistent with the variable and limited efficacy of cytokine-blocking therapies in TRAPS. It highlights new potential target pathways for therapeutic intervention.

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in AtminGpg6/Gpg6 kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including β-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, AtminGpg6/Gpg6 mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between AtminGpg6/+ and Vangl2Lp/+ mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

VEGF promotes osteogenic differentiation of ASCs on ordered fluorapatite surfaces.

Vascular endothelial growth factor (VEGF) has been reported to mediate both osteogenesis and angiogenesis in bone regeneration. We previously found an upregulation of VEGF in adipose-derived stem cells (ASCs) when obvious mineralization occurred on a novel fluorapatite (FA)-coated surfaces. This study investigated the effect of FA and VEGF on the growth, differentiation and mineralization of (ASC) grown on ordered FA surfaces. Cells grown on FA and treated with VEGF demonstrated osteogenic differentiation as measured with ALP staining, and obvious mineralization as measured by Alizarin red staining. A combined stimulating effect of FA and VEGF was seen using both indicators. VEGF signaling pathway perturbation using a specific VEGF receptor inhibitor showed the lowest levels of ALP and Alizarin red staining, which was partially rescued when the cells were grown on FA and/or treated with the addition of VEGF. The osteogenic differentiation of ASCs stimulated by these FA surfaces as well as VEGF has been shown to be mediated through, but probably not only, the VEGF signaling pathway. The enhancement of osteogenic differentiation and mineralization supports the potential use of therapeutic VEGF and FA coatings in bone regeneration.

Perturbation of specific pro-mineralizing signalling pathways in human and murine pseudoxanthoma elasticum.

BackgroundPseudoxanthoma elasticum (PXE) is characterized by skin (papular lesions), ocular (subretinal neovascularisation) and cardiovascular manifestations (peripheral artery disease), due to mineralization and fragmentation of elastic fibres in the extracellular matrix (ECM). Caused by mutations in the ABCC6 gene, the mechanisms underlying this disease remain unknown. The knowledge on the molecular background of soft tissue mineralization largely comes from insights in vascular calcification, with involvement of the osteoinductive Transforming Growth Factor beta (TGFβ) family (TGFβ1-3 and Bone Morphogenetic Proteins [BMP]), together with ectonucleotides (ENPP1), Wnt signalling and a variety of local and systemic calcification inhibitors. In this study, we have investigated the relevance of the signalling pathways described in vascular soft tissue mineralization in the PXE knock-out mouse model and in PXE patients.MethodsThe role of the pro-osteogenic pathways BMP2-SMADs-RUNX2, TGFβ-SMAD2/3 and Wnt-MSX2, apoptosis and ER stress was evaluated using immunohistochemistry, mRNA expression profiling and immune-co-staining in dermal tissues and fibroblast cultures of PXE patients and the eyes and whiskers of the PXE knock-out mouse. Apoptosis was further evaluated by TUNEL staining and siRNA mediated gene knockdown. ALPL activity in PXE fibroblasts was studied using ALPL stains.ResultsWe demonstrate the upregulation of the BMP2-SMADs-RUNX2 and TGFβ-2-SMAD2/3 pathway, co-localizing with the mineralization sites, and the involvement of MSX2-canonical Wnt signalling. Further, we show that apoptosis is also involved in PXE with activation of Caspases and BCL-2. In contrast to vascular calcification, neither the other BMPs and TGFβs nor endoplasmic reticulum stress pathways seem to be perturbed in PXE.ConclusionsOur study shows that we cannot simply extrapolate knowledge on cell signalling in vascular soft tissue calcification to a multisystem ectopic mineralisation disease as PXE. Contrary, we demonstrate a specific set of perturbed signalling pathways in PXE patients and the knock-out mouse model. Based on our findings and previously reported data, we propose a preliminary cell model of ECM calcification in PXE.

Sphingosine analog fingolimod (FTY720) increases radiation sensitivity of human breast cancer cells in vitro

Radiotherapy is one of the most effective therapeutic strategies for breast cancer patients, although its efficacy may be reduced by intrinsic radiation resistance of cancer cells. Recent investigations demonstrate a link between cancer cell radio-resistance and activation of sphingosine kinase (SphK1), which plays a key role in the balance of lipid signaling molecules. Sphingosine kinase (SphK1) activity can alter the sphingosine-1-phosphate (S1P)/ceramide ratio leading to an imbalance in the sphingolipid rheostat. Fingolimod (FTY720) is a novel sphingosine analog and a potent immunosuppressive drug that acts as a SphK1 antagonist, inhibits the growth, and induces apoptosis in different human cancer cell lines. We sought to investigate the in vitro radiosensitizing effects of FTY720 on the MDA-MB-361 breast cancer cell line and to assess the effects elicited by radiation and FTY720 combined treatments. We found that FTY720 significantly increased anti-proliferative and pro-apoptotic effects induced by a single dose of ionizing radiation while causing autophagosome accumulation. At the molecular level, FTY720 significantly potentiated radiation effects on perturbation of signaling pathways involved in regulation of cell cycle and apoptosis, such as PI3K/AKT and MAPK. In conclusion, our data highlight a potent radiosensitizing effect of FTY720 on breast cancer cells and provide the basis of novel therapeutic strategies for breast cancer treatment.

Structural basis for the inhibition of HIV-1 Nef by a high-affinity binding single-domain antibody

BackgroundThe HIV-1 Nef protein is essential for AIDS pathogenesis by its interaction with host cell surface receptors and signaling factors. Despite its critical role as a virulence factor Nef is not targeted by current antiviral strategies.ResultsWe have determined the crystal structure of the complex formed by a camelid single-domain antibody fragment, termed sdAb19, bound to HIV-1 Nef together with a stabilizing SH3 domain. sdAb19 forms a stoichiometric 1:1 complex with Nef and binds to a conformationally conserved surface at the C-terminus of Nef that overlaps with functionally important interaction sites involved in Nef-induced perturbations of signaling and trafficking pathways. The antibody fragment binds Nef with low nanomolar affinity, which could be attenuated to micromolar affinity range by site-directed mutagenesis of key interaction residues in sdAb19. Fusion of the SH3 domain to sdAb19, termed Neffin, leads to a significantly increased affinity for Nef and formation of a stoichiometric 2:2 Nef–Neffin complex. The 19 kDa Neffin protein inhibits all functions of Nef as CD4 and MHC-I downregulation, association with Pak2, and the increase in virus infectivity and replication.ConclusionsTogether, sdAb19 and Neffin thus represent efficient tools for the rational development of antiviral strategies against HIV-1 Nef.

Phytophthora infestans RXLR Effector PexRD2 Interacts with Host MAPKKKε to Suppress Plant Immune Signaling

Mitogen-activated protein kinase cascades are key players in plant immune signaling pathways, transducing the perception of invading pathogens into effective defense responses. Plant pathogenic oomycetes, such as the Irish potato famine pathogen Phytophthora infestans, deliver RXLR effector proteins to plant cells to modulate host immune signaling and promote colonization. Our understanding of the molecular mechanisms by which these effectors act in plant cells is limited. Here, we report that the P. infestans RXLR effector PexRD2 interacts with the kinase domain of MAPKKKε, a positive regulator of cell death associated with plant immunity. Expression of PexRD2 or silencing MAPKKKε in Nicotiana benthamiana enhances susceptibility to P. infestans. We show that PexRD2 perturbs signaling pathways triggered by or dependent on MAPKKKε. By contrast, homologs of PexRD2 from P. infestans had reduced or no interaction with MAPKKKε and did not promote disease susceptibility. Structure-led mutagenesis identified PexRD2 variants that do not interact with MAPKKKε and fail to support enhanced pathogen growth or perturb MAPKKKε signaling pathways. Our findings provide evidence that P. infestans RXLR effector PexRD2 has evolved to interact with a specific host MAPKKK to perturb plant immunity-related signaling.

Tailoring chimeric ligands for studying and biasing ErbB receptor family interactions.

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways.

Long-Term Differential Changes in Mouse Intestinal Metabolomics after γ and Heavy Ion Radiation Exposure

Tissue consequences of radiation exposure are dependent on radiation quality and high linear energy transfer (high-LET) radiation, such as heavy ions in space is known to deposit higher energy in tissues and cause greater damage than low-LET γ radiation. While radiation exposure has been linked to intestinal pathologies, there are very few studies on long-term effects of radiation, fewer involved a therapeutically relevant γ radiation dose, and none explored persistent tissue metabolomic alterations after heavy ion space radiation exposure. Using a metabolomics approach, we report long-term metabolomic markers of radiation injury and perturbation of signaling pathways linked to metabolic alterations in mice after heavy ion or γ radiation exposure. Intestinal tissues (C57BL/6J, female, 6 to 8 wks) were analyzed using ultra performance liquid chromatography coupled with electrospray quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) two months after 2 Gy γ radiation and results were compared to an equitoxic 56Fe (1.6 Gy) radiation dose. The biological relevance of the metabolites was determined using Ingenuity Pathway Analysis, immunoblots, and immunohistochemistry. Metabolic profile analysis showed radiation-type-dependent spatial separation of the groups. Decreased adenine and guanosine and increased inosine and uridine suggested perturbed nucleotide metabolism. While both the radiation types affected amino acid metabolism, the 56Fe radiation preferentially altered dipeptide metabolism. Furthermore, 56Fe radiation caused upregulation of ‘prostanoid biosynthesis’ and ‘eicosanoid signaling’, which are interlinked events related to cellular inflammation and have implications for nutrient absorption and inflammatory bowel disease during space missions and after radiotherapy. In conclusion, our data showed for the first time that metabolomics can not only be used to distinguish between heavy ion and γ radiation exposures, but also as a radiation-risk assessment tool for intestinal pathologies through identification of biomarkers persisting long after exposure.

Abstract CN06-03: Cell plasticity promotes drug resistance and pathway redundancy.

Abstract Multiple signal transduction pathways can be concurrently active within a single cell, and extensive crosstalk can occur between RTKs. Additionally, tumor tissues can be comprised of a heterogeneous collection of cell states utilizing distinct RTKs for maintenance of tumor cell growth and survival. As a consequence of this complexity, many tumors may be only partially sensitive to single agent therapies and would require the interdiction of multiple RTKs and other protein signaling targets for optimal anticancer therapy. Understanding pathway crosstalk is vital to guide the rational combination of approved and experimental anticancer agents. Resistance to targeted therapies can result from de novo pathway redundancy, from acquired feedback activation of parallel pathways and from de novo or acquired cell plasticity. It is clear that metastatic cancers are increasingly heterogeneous, that is many different cell types and states are present within cancer tissues, and importantly metastatic cancers show increased resistance to radiation, chemotherapies, and targeted therapies. Cellular plasticity in epithelial cancers has been associated with progression and resistant to anticancer therapies. Several forms of plasticity have been documented, including epithelial mesenchymal transition (EMT), endothelial-mesenchymal transition, and neuroendocrine-epithelial transition. Epithelial mesenchymal transition (EMT) is an important cellular change promoting metastatic cell survival. We have shown mesenchymal-like tumors are relatively resistant to EGFR-TKi (Thomson, Cancer Res 2005), to IGF1R TKi (Buck, Cancer Res 2008), and to a broad spectrum of chemotherapy agents. A similar resistance of EMT-derived cells to anticancer therapies has been shown clinically in breast cancer patients (Creighton PNAS 2009), in prostate cancers (Domingo-Domenech, Cancer Cell 2012), and in NSCLC patients (Sequist, Cancer Cell 2012). Finally clinical EMT biomarkers predict response to EGFR inhibitors in NSCLC patients (Yauch, Clin. Cancer Res 2005; Byers et al., Clin Cancer Res 2012). New therapeutics are needed to target the new spectrum of tumor survival signals now present within EMT-derived cells and within related cancer-stem cells Through the generation and molecular characterization of EMT models, coupled with RNAi-based target validation studies and follow-on pharmacology studies, new targets and compounds which promote death of metastatic cells can be identified and validated in panels of cell lines and in animal models. Cell models for in vitro and in vivo target progression will broadly reflect both inducible and epigenetically-fixed mesenchymal cell states. In two cases (H358 and A549), TGFbeta was used to induce a reversible metastable mesenchymal cell state. The role of TGFbeta as both tumor suppressor and promoter of metastatic progression highlight the altered signal transduction cascades downstream of TGFbeta receptor activation in comparing epithelial and mesenchymal cell states. Here we show that mesenchymal-like tumor cells derived by EMT are preferentially sensitive to perturbations in integrin, focal adhesion and MAPK signaling pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN06-03. Citation Format: John D. Haley. Cell plasticity promotes drug resistance and pathway redundancy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN06-03.

The Role of Protein Kinase A Regulation of the E6 PDZ-Binding Domain during the Differentiation-Dependent Life Cycle of Human Papillomavirus Type 18

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

Hexavalent Chromium-induced Alteration of Proteomic Landscape in Human Skin Fibroblast Cells

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K₂Cr₂O₇). We were able to quantify ∼4600 unique proteins, among which ∼400 exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 μM K₂Cr₂O₇. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K₂Cr₂O₇ treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K₂Cr₂O₇ treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells.

Systems biology analysis of the ceca and bursa revealed unique innate immune responses to Campylobacter jejuni in two genetically distinct lines of chickens (P1269)

Abstract Campylobacter jejuni (C. jejuni) is one of the most commonly reported causes of foodborne illness worldwide, although C. jejuni is a commensal in chickens. Two genetically distinct broiler lines, either resistant (line A) or susceptible (line B) to C. jejuni colonization, , were used to understand innate immune response to C. jejuni in a time-course (1, 4, 24, and 36 h post-inoculation) study. A total of 128 1-d-old chicks (64 per line), were either orally challenged with C. jejuni or inoculated with PBS. Total RNA were isolated from ceca and bursa, and a chicken 44K Agilent microarray was performed on all samples. Microarray data was then analyzed using a dynamic Bayesian network machine learning approach. Bursa samples from infected line A birds showed significantly more perturbations in carbohydrate and lipid metabolism compared to non-infected controls while susceptible line B birds responded with perturbations in signal transduction and amino acid biosynthesis. Differences were also observed in the ceca where apoptosis, MAPK and Toll-like receptor signaling pathways were significantly perturbed in line A birds while cell adhesion molecules, Jak-STAT signaling and fatty acid metabolism were significantly perturbed in line B birds. The distinct perturbed signaling pathways between these two lines and between two tissues provide new insights into the early immune response of chickens to C.jejuni and will allow for in silico hypothesis testing for future functional studies.

Pyrvinium pamoate does not activate protein kinase CK1, but promotes Akt/PKB down-regulation and GSK3 activation

It has been reported that pyrvinium pamoate (PyrPam), an FDA (U.S. Food and Drug Administration)-approved anthelminthic drug, is a potent inhibitor of Wnt signalling by a mechanism which implies the direct activation of protein kinase CK1α. In the present paper, we provide data ruling out any direct stimulatory effect of PyrPam on CK1, by showing that the catalytic activity of CK1α and those of its isoforms δ and γ1 are not significantly affected by PyrPam when tested with the aid of specific peptide and protein substrates. Accordingly, cell treatment with PyrPam has no significant effect on the phosphorylation of β-catenin Ser(45). By contrast, the phosphorylation of β-catenin Thr(41) is increased upon cell treatment with PyrPam, through a mechanism that implies the upstream dephosphorylation of Akt/PKB (protein kinase B) and of GSK3 (glycogen synthase kinase 3). It can be concluded from the present study that PyrPam is not a bona fide activator of CK1, its perturbation of cell signalling pathways being mediated by a complex mechanism initiated by a fall in Akt phosphorylation whose down-regulation promotes reduced phosphorylation and activation of GSK3. Consistent with this, lysates of cells treated with PyrPam display enhanced protein phosphorylation which is unaffected by CK1 inhibition, while disappearing upon inhibition of GSK3. Our data are consistent with the observation that PyrPam ultimately inhibits Wnt signalling despite its lack of efficacy on CK1.

Nanoparticle Technology as a Double-Edged Sword: Cytotoxic, Genotoxic and Epigenetic Effects on Living Cells

Nanoparticles are considered as powerful tools in nanotechnological applications. Due to their unique physicochemical properties, their interactions with different biological systems have been shown. Nanomaterials have been successfully used as coating materials or treatment and diagnosis tools. Nevertheless, toxic effects of nanoparticles in vitro and in vivo have also been reported. Here, we summarize the current state of knowledge on exposure routes, cellular uptake and toxicological activities of the commonly used nanoparticles. In this context, we discuss the mechanisms of toxicity of nanoparticles involving perturbation of redox milieu homeostasis and cellular signaling pathways.

Magnetic Manipulation of Intracellular Signaling

In vivo imaging has shown how the establishment and maintenance of cell polarity relies on complex mechanisms by which signaling cascades become regulated at sub-cellular levels. How signaling networks are spatio-temporally coordinated into a polarized cell is not elucidated. In this context, we developed a new tool to locally probe and perturb signaling pathways inside living cells.In our approach, magnetic nanoparticles (MNPs) functionalized with active proteins are inserted in the cytosol of mammalian cells where they behave as solid signaling platforms. The precise control of MNPs surface chemistry allows to probe the self assembly of macromolecular complexes directly inside the cell. By exerting magnetic forces, MNPs are then manipulated in the cytosol to position their signaling activity at different subcellular locations. The cellular response to this spatially resolved biochemical perturbation is finally quantified in term of effector recruitment and cytoskeleton/membrane dynamics.We show that MNPs of different sizes, from 50nm to 500nm in diameter, can be used to generate different spatial perturbation patterns. While the biggest MNPs are trapped in internal structures of the cell and require large forces (>10 pN) to be displaced, they allow us to create precise point-like perturbations at the cell periphery. At the opposite, smallest MNPs diffuse fast in the cytosol (∼1μm²/s) and are used to create gradient of signaling activity spanning over the whole cell.We apply our technique to the Rho-GTPase signaling network which orchestrate cell polarity and migration. MNPs were functionnalized with GEF or active GTPase. The effect of intracellular GTPase gradient (rac, cdc42) on cell polarity was analyzed. We demonstrated that the pathway linking Rac1 to actin polymerization is spatially restricted to the protrusive areas of the cell by transporting TIAM1 particles at different subcellular locations while monitoring GTPase activation and actin polymerization.