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Feraru E.,Vlaams Instituut Voor Biotechnologie | Feraru E.,Ghent University | Feraru M.I.,Vlaams Instituut Voor Biotechnologie | Feraru M.I.,Ghent University | And 10 more authors.
Current Biology | Year: 2011

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development [1]. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes [2-4]. In addition, plant cells have a complex extracellular matrix, the cell wall [5, 6], whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters [7] in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix. © 2011 Elsevier Ltd.

Willenborg S.,University of Cologne | Lucas T.,University of Cologne | Van Loo G.,Vlaams Instituut Voor Biotechnologie | Van Loo G.,Ghent University | And 9 more authors.
Blood | Year: 2012

Monocytes/macrophages are critical in orchestrating the tissue-repair response. However, the mechanisms that govern macrophage regenerative activities during the sequential phases of repair are largely unknown. In the present study, we examined the dynamics and functions of diverse monocyte/macrophage phenotypes during the sequential stages of skin repair. By combining the analysis of a new CCR2-eGFP reporter mouse model with conditional mouse mutants defective in myeloid cell-restricted CCR2 signaling or VEGF-A synthesis, we show herein that among the large number of inflammatory CCR2 +Ly6C + macrophages that dominate the early stage of repair, only a small fraction strongly expresses VEGF-A that has nonredundant functions for the induction of vascular sprouts. The switch of macrophage-derived VEGF-A during the early stage of tissue growth toward epidermal-derived VEGF-A during the late stage of tissue maturation was critical to achieving physiologic tissue vascularization and healing progression. The results of the present study provide new mechanistic insights into CCR2- mediated recruitment of blood monocyte subsets into damaged tissue, the dynamics and functional consequences of macrophage plasticity during the sequential repair phases, and the complementary role of macrophage-derived VEGF-A in coordinating effective tissue growth and vascularization in the context of tissue-resident wound cells. Our findings may be relevant for novel monocyte-based therapies to promote tissue vascularization.© 2012 by The American Society of Hematology.

Weimer A.K.,University of Strasbourg | Nowack M.K.,Vlaams Instituut voor Biotechnologie | Nowack M.K.,Ghent University | Bouyer D.,University of Strasbourg | And 11 more authors.
Plant Cell | Year: 2012

Formative, also called asymmetric, cell divisions produce daughter cells with different identities. Like other divisions, formative divisions rely first of all on the cell cycle machinery with centrally acting cyclin-dependent kinases (CDKs) and their cyclin partners to control progression through the cell cycle. However, it is still largely obscure how developmental cues are translated at the cellular level to promote asymmetric divisions. Here, we show that formative divisions in the shoot and root of the flowering plant Arabidopsis thaliana are controlled by a common mechanism that relies on the activity level of the Cdk1 homolog CDKA;1, with medium levels being sufficient for symmetric divisions but high levels being required for formative divisions. We reveal that the function of CDKA;1 in asymmetric cell divisions operates through a transcriptional regulation system that is mediated by the Arabidopsis Retinoblastoma homolog RBR1. RBR1 regulates not only cell cycle genes, but also, independent of the cell cycle transcription factor E2F, genes required for formative divisions and cell fate acquisition, thus directly linking cell proliferation with differentiation. This mechanism allows the implementation of spatial information, in the form of high kinase activity, with intracellular gating of developmental decisions. © 2012 American Society of Plant Biologists. All rights reserved.

Beck F.,Leibniz Institute for Analytical Sciences | Geiger J.,Universitatsklinikum Wurzburg | Gambaryan S.,Universitatsklinikum Wurzburg | Gambaryan S.,RAS Sechenov Institute of Evolutionary Physiology and Biochemistry | And 10 more authors.
Blood | Year: 2014

One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of platelet inhibition and activation in 3 independent biological replicates. We quantified over 2700 different phosphorylated peptides of which 360 were significantly regulated upon stimulation. This comprehensive and time-resolved analysis indicates that platelet inhibition is a multipronged process involving different kinases and phosphatases aswell asmany previously unanticipated proteins and pathways. © 2014 by The American Society of Hematology.

Zaki Md.H.,St Jude Childrens Research Hospital | Zaki Md.H.,University of Texas Southwestern Medical Center | Man S.M.,St Jude Childrens Research Hospital | Vogel P.,St Jude Childrens Research Hospital | And 3 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2014

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 12 (NLRP12) plays a protective role in intestinal inflammation and carcinogenesis, but the physiological function of this NLR during microbial infection is largely unexplored. Salmonella enterica serovar Typhimurium (S. typhimurium) is a leading cause of food poisoning worldwide. Here, we show that NLRP12-deficient mice were highly resistant to S. typhimurium infection. Salmonella-infected macrophages induced NLRP12-dependent inhibition of NF-κB and ERK activation by suppressing phosphorylation of IκBα and ERK. NLRP12-mediated downregulation of proinflammatory and antimicrobial molecules prevented efficient clearance of bacterial burden, highlighting a role for NLRP12 as a negative regulator of innate immune signaling during salmonellosis. These results underscore a signaling pathway defined by NLRP12-mediated dampening of host immune defenses that could be exploited by S. typhimurium to persist and survive in the host.

Van Damme P.,Vlaams Instituut voor Biotechnologie | Van Damme P.,Ghent University | Staes A.,Vlaams Instituut voor Biotechnologie | Staes A.,Ghent University | And 12 more authors.
Nature Methods | Year: 2010

We describe a positional proteomics approach to simultaneously analyze N-and C-terminal peptides and used it to screen for human protein substrates of granzyme B and carboxypeptidase A4 in human cell lysates. This approach allowed comprehensive proteome studies, and we report the identification of 965 database-annotated protein C termini, 334 neo-C termini resulting from granzyme B processing and 16 neo-C termini resulting from carboxypeptidase A4 processing. © 2010 Nature America, Inc. All rights reserved.

Ohman D.,Swedish University of Agricultural Sciences | Demedts B.,Vlaams Instituut voor Biotechnologie | Demedts B.,Ghent University | Kumar M.,Swedish University of Agricultural Sciences | And 10 more authors.
Plant Journal | Year: 2013

The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103. © 2012 The Authors.

Marroni F.,Instituto Of Genomica Applicata | Pinosio S.,Instituto Of Genomica Applicata | Pinosio S.,University of Udine | Di Centa E.,Instituto Of Genomica Applicata | And 7 more authors.
Plant Journal | Year: 2011

Common variants, such as those identified by genome-wide association scans, explain only a small proportion of trait variation. Growing evidence suggests that rare functional variants, which are usually missed by genome-wide association scans, play an important role in determining the phenotype. We used pooled multiplexed next-generation sequencing and a customized analysis workflow to detect mutations in five candidate genes for lignin biosynthesis in 768 pooled Populus nigra accessions. We identified a total of 36 non-synonymous single nucleotide polymorphisms, one of which causes a premature stop codon. The most common variant was estimated to be present in 672 of the 1536 tested chromosomes, while the rarest was estimated to occur only once in 1536 chromosomes. Comparison with individual Sanger sequencing in a selected sub-sample confirmed that variants are identified with high sensitivity and specificity, and that the variant frequency was estimated accurately. This proposed method for identification of rare polymorphisms allows accurate detection of variation in many individuals, and is cost-effective compared to individual sequencing. © 2011 Blackwell Publishing Ltd.

Lupfer C.R.,St Jude Childrens Research Hospital | Anand P.K.,St Jude Childrens Research Hospital | Liu Z.,St Jude Childrens Research Hospital | Stokes K.L.,St Jude Childrens Research Hospital | And 4 more authors.
PLoS Pathogens | Year: 2014

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3- and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway. © 2014 Lupfer et al.

Montelione G.T.,Rutgers University | Nilges M.,CNRS Institute of Pharmacology and Structural Biology | Nilges M.,French National Center for Scientific Research | Bax A.,U.S. National Institute of Diabetes and Digestive and Kidney Diseases | And 12 more authors.
Structure | Year: 2013

Summary As methods for analysis of biomolecular structure and dynamics using nuclear magnetic resonance spectroscopy (NMR) continue to advance, the resulting 3D structures, chemical shifts, and other NMR data are broadly impacting biology, chemistry, and medicine. Structure model assessment is a critical area of NMR methods development, and is an essential component of the process of making these structures accessible and useful to the wider scientific community. For these reasons, the Worldwide Protein Data Bank (wwPDB) has convened an NMR Validation Task Force (NMR-VTF) to work with wwPDB partners in developing metrics and policies for biomolecular NMR data harvesting, structure representation, and structure quality assessment. This paper summarizes the recommendations of the NMR-VTF, and lays the groundwork for future work in developing standards and metrics for biomolecular NMR structure quality assessment. © 2013 Elsevier Ltd.

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