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  • 1.
    Achour, Cyrinne
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Canonical and non-canonical functions of METTL3 in breast cancer2022Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Gene expression is spatially and temporally regulated at multiple levels. N6-methyladenosine (m6A) is the most prevalent internal modification in messenger RNA (mRNA) and long noncoding RNA (lncRNAs). m6A plays important roles in multiple cellular processes including stem cell pluripotency, adipogenesis, spermatogenesis, neurogenesis, circadian rhythm and development by modulating RNA splicing, export, stability, degradation and translation. Although aberrant m6A methylation has been reported in various types of cancer, the underlying molecular functions of METTL3, the solely catalytic subunit of the m6A-methylase complex, has yet to be defined.

    m6A has been recently identified in nascent pre-mRNA, and more specifically intronic m6A has been linked to exon skipping events. The occurrence of impaired alternative splicing (AS) is frequently found during the development of cancer. We performed transcriptome wide analysis in breast cancer cell lines and explored AS events. Our results define an AS signature for breast tumorigenesis. We found that METTL3 modulates AS directly through m6A deposition at the intron-exon junctions or indirectly by the m6A deposition in transcripts encoding for splicing factors and transcription factors. In particular, we show that MYC mRNA harbours the m6A mark, suggesting that METTL3 regulates AS indirectly via the regulation of MYC expression. Indeed, the targets of MYC overlapped with METTL3-associated AS events. Importantly, five of the AS events identified and validated in vitro, are linked to a worse prognosis in breast cancer patients. Additionally, we show that METTL3 enhances the breast cancer phenotype through a dual mechanism depending on its sub-cellular localization. We find that the canonical nuclear function of METTL3 decorates transcripts that are involved in cell proliferation and migration. We observe that METTL3 is highly expressed in the cytoplasmic compartment of breast cancer cells from patients. Remarkably, we uncover that the cytoplasmic METTL3 interacts with subunits of the exocyst, whose subunit EXOC7 has been linked to cell adhesion, migration and invasion. Notably, we show that breast cancer cell lines depleted of METTL3 display less gelatinase activity and invadopodia formation, supporting the role of METTL3 in cell invasion via exocytosis.

    m6A is a reversible modification, which can be demethylated by the erasers FTO and ALKBH5. Depletion of FTO has been shown to increase the level of m6A in mRNA, however recent studies have reported that FTO could demethylate N6,2´-O-dimethyladenosine (m6Am), adjacent to the 7-methylguanosine cap on mRNA. In the cellular model of colorectal cancer CRC1, depletion of FTO leads to a cancer stem cell phenotype and confers chemotherapy resistance. By performing m6A-RNA immunoprecipitation followed by sequencing (MeRIP), we show that knockdown of FTO in CRC1 cells does not affect the global level of m6A in mRNA but of m6Am level.

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  • 2.
    Achour, Cyrinne
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Bhattarai, Devi Prasad
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Esteva-Socias, Margalida
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rodriguez-Barrueco, Ruth
    Malla, Sandhya
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Seier, Kerstin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Marchand, Virginie
    Motorine, Yuri
    Lundin, Eva
    Umeå University, Faculty of Medicine, Department of Medical Biosciences.
    Gilthorpe, Jonathan D.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Marzese, Diego Matias
    Bally, Marta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Roman, Angel-Carlos
    Pich, Andreas
    Aguilo, Francesca
    Reshaping the role of METTL3 in breast tumorigenesisManuscript (preprint) (Other academic)
  • 3.
    Aguilo, Francesca
    et al.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
    Walsh, Martin J.
    The N6-Methyladenosine RNA modification in pluripotency and reprogramming2017In: Current Opinion in Genetics and Development, ISSN 0959-437X, E-ISSN 1879-0380, Vol. 46, p. 77-82Article, review/survey (Refereed)
    Abstract [en]

    Chemical modifications of RNA provide a direct and rapid way to manipulate the existing transcriptome, allowing rapid responses to the changing environment further enriching the regulatory capacity of RNA. N-6-Methyladenosine(m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes, linking external stimuli to an intricate network of transcriptional, post-transcriptional and translational processes. M(6)A modification affects a broad spectrum of cellular functions, including maintenance of the pluripotency of embryonic stem cells (ESCs) and the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this review, we summarize the most recent findings on m(6)A modification with special focus on the different studies describing how m(6)A is implicated in ESC self-renewal, cell fate specification and iPSC generation.

  • 4.
    Alakpa, Enateri V.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Bahrd, Anton
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Novikov, Lev N.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Ljungberg, Christina
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
    Kelk, Peyman
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Bioprinted schwann and mesenchymal stem cell co-cultures for enhanced spatial control of neurite outgrowth2023In: Gels, E-ISSN 2310-2861, Vol. 9, no 3, article id 172Article in journal (Refereed)
    Abstract [en]

    Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration.

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  • 5.
    Alakpa, Enateri V.
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Saeed, Anwer
    Chung, Peter
    Riehle, Mathis O.
    Gadegaard, Nikolaj
    Dalby, Matthew J.
    Cusack, Maggie
    The Prismatic Topography of Pinctada maxima Shell Retains Stem Cell Multipotency and Plasticity In Vitro2018In: Advanced Biosystems, ISSN 2366-7478, Vol. 2, no 6, article id 1800012Article in journal (Refereed)
    Abstract [en]

    The shell of the bivalve mollusc Pinctada maxima is composed of the calcium carbonate polymorphs calcite and aragonite (nacre). Mother-of-pearl, or nacre, induces vertebrate cells to undergo osteogenesis and has good osteointegrative qualities in vivo. The calcite counterpart, however, is less researched in terms of the response of vertebrate cells. This study shows that isolation of calcite surface topography from the inherent chemistry allows viable long-term culture of bone marrow derived mesenchymal stem cells (MSCs). Self-renewal is evident from the increased gene expression of the self-renewal markers CD63, CD166, and CD271 indicating that cells cultured on the calcite topography maintain their stem cell phenotype. MSCs also retain their multipotency and can undergo successful differentiation into osteoblasts and adipocytes. When directed to adipogenesis, MSCs cultured on prism replicas are more amenable to differentiation than MSCs cultured on tissue culture polystyrene indicating a higher degree of plasticity in MSCs growing on calcite P. maxima prismatic topography. The study highlights the potential of the calcite topography of P. maxima as a biomimetic design for supporting expansion of MSC populations in vitro, which is of fundamental importance if it meets the demands for autologous MSCs for therapeutic use.

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  • 6.
    Al-Furoukh, Natalie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Ianni, Alessandro
    Nolte, Hendrik
    Hölper, Soraya
    Krüger, Marcus
    Wanrooij, Sjoerd
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Braun, Thomas
    ClpX stimulates the mitochondrial unfolded protein response (UPRmt) in mammalian cells2015In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1853, no 10, p. 2580-2591Article in journal (Refereed)
    Abstract [en]

    Proteostasis is crucial for life and maintained by cellular chaperones and proteases. One major mitochondrial protease is the ClpXP complex, which is comprised of a catalytic ClpX subunit and a proteolytic ClpP subunit. Based on two separate observations, we hypothesized that ClpX may play a leading role in the cellular function of ClpXP. Therefore, we analyzed the effect of ClpX overexpression on a myoblast proteome by quantitative proteomics. ClpX overexpression results in the upregulation of markers of the mitochondria( proteostasis pathway, known as the "mitochondrial unfolded protein response" (UPRmt). Although this pathway is described in detail in Caenorhabditis elegans, it is not clear whether it is conserved in mammals. Therefore, we compared features of the classical nematode UPRmt with our mammalian ClpX-triggered UPRmt dataset. We show that they share the same retrograde mitochondria-to-nucleus signaling pathway that involves the key UPRmt transcription factor CHOP (also known as Ddit3, CEBPZ or GADD153). In conclusion, our data confirm the existence of a mammalian UPRmt that has great similarity to the C elegans pathway. Furthermore, our results illustrate that ClpX overexpression is a good and simple model to study the underlying mechanisms of the UPRmt in mammalian cells.

  • 7. Andersson, Rebecca
    et al.
    Eisele-Burger, Anna Maria
    Hanzen, Sarah
    Vielfort, Katarina
    Department of Microbiology and Immunology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Oling, David
    Eisele, Frederik
    Johansson, Gustav
    Gustafsson, Tobias
    Kvint, Kristian
    Nystrom, Thomas
    Differential role of cytosolic Hsp70s in longevity assurance and protein quality control2021In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 17, no 1, article id e1008951Article in journal (Refereed)
    Abstract [en]

    70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1 Delta ssa2 Delta mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast.

    Author summary: All organisms have proteins that network together to stabilize and protect the cell throughout its lifetime. One of these types of proteins are the Hsp70s (heat shock protein 70). Hsp70 proteins take part in folding other proteins to their functional form, untangling proteins from aggregates, organize aggregates inside the cell and ensure that damaged proteins are destroyed. In this study, we investigated three closely related Hsp70 proteins in yeast; Ssa1, 2 and 4, in an effort to describe the functional difference of Ssa4 compared to Ssa1 and 2 and to answer the question: What types of cellular stress protection are necessary to reach a normal lifespan? We show that Ssa4 can perform many of the same tasks as Ssa1 and 2, but Ssa4 doesn't interact in the same manner as Ssa1 and 2 with other types of proteins. This leads to a delay in removing protein aggregates created after heat stress. Ssa4 also cannot ensure that misfolded proteins aggregate correctly inside the nucleus of the cell. However, this turns out not to be necessary for yeast cells to achieve a full lifespan, which shows us that as long as cells can prevent aggregates from forming in the first place, they can reach a full lifespan.

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  • 8.
    Anerillas, Luis Oliveros
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Wiberg, Mikael
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB). Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery.
    Kingham, Paul J.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Kelk, Peyman
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs2023In: Regenerative Therapy, E-ISSN 2352-3204, Vol. 24, p. 298-310Article in journal (Refereed)
    Abstract [en]

    Background: The use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems.

    Methods: We evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining.

    Results: Cell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT.

    Conclusions: PLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.

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  • 9.
    Bagge, Johan
    et al.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Danielson, Patrik
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    Forsgren, Sture
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.
    In situ hybridization studies favouring the occurrence of a local production of BDNF in the human Achilles tendon2012In: Histology and Histopathology, ISSN 0213-3911, E-ISSN 1699-5848, Vol. 27, no 9, p. 1239-1246Article in journal (Refereed)
    Abstract [en]

    Brain derived neurotrophic factor (BDNF) is a multipotent neurotrophin known for its growth-influencing and apoptosis-modulating functions, as well as for its function to interact with neurotransmitters/neuromodulators. BDNF is reported to be mainly produced in the brain. BDNF can be absorbed into peripheral tissue from the blood stream. Expression of this neurotrophin at the protein level, as well as of the neurotrophin receptor p75, has been previously shown for the principal cells (tenocytes) of the Achilles tendon. However, there is no proof at the mRNA level that BDNF is produced by the tenocytes. As the Achilles tendon tenocytes show "neuronal-like" characteristics, in the form of expressions favouring synthesis of several neuromodulators/neurotransmitters, and as BDNF especially is produced in neurons, it is of interest to confirm this. In the present study, therefore, in situ hybridization for demonstration of BDNF mRNA was performed on biopsies from Achilles tendons of patients with tendinosis and pain-free non-tendinosis individuals. The results showed that the tenocytes of both groups exhibited BDNF mRNA reactions. These observations indeed favour the idea that BDNF is produced by tenocytes in the human Achilles tendon, why Achilles tendon tissue is a tissue in which BDNF can be locally produced. BDNF can have modulatory functions for the tenocytes, including apoptosis-modifying effects via actions on the p75 receptor and interactive effects with neurotransmitters/neuromodulators produced in these cells. This possibility should be further studied for Achilles tendon tissue.

  • 10. Bagnato, Paola
    et al.
    Castagnino, Alessia
    Cortese, Katia
    Bono, Maria
    Grasso, Silvia
    Bellese, Grazia
    Daniele, Tiziana
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Defilippi, Paola
    Castagnola, Patrizio
    Tacchetti, Carlo
    Cooperative but distinct early co-signaling events originate from ERBB2 and ERBB1 receptors upon trastuzumab treatment in breast cancer cells2017In: Oncotarget, E-ISSN 1949-2553, Vol. 8, no 36, p. 60109-60122Article in journal (Refereed)
    Abstract [en]

    ERBB2 receptor belongs to the ERBB tyrosine kinase receptor family. At variance to the other family members, ERBB2 is a constitutively active orphan receptor. Upon ligand binding and activation, ERBB receptors form homo-or hetero-dimers with the other family members, including ERBB2, promoting an intracellular signaling cascade. ERBB2 is the preferred dimerization partner and ERBB2 heterodimers signaling is stronger and longer acting compared to heterodimers between other ERBB members. The specific contribution of ERBB2 in heterodimer signaling is still undefined. Here we report the formation of circular dorsal ruffles (CDRs) upon treatment of the ERBB2-overexpressing breast cancer cell lines SK-BR-3 and ZR751 with Trastuzumab, a therapeutic humanized monoclonal antibody directed against ERBB2. We found that in SK-BR-3 cells Trastuzumab leads to surface redistribution of ERBB2 and ERBB1 in CDRs, and that the ERBB2-dependent ERK1/2 phosphorylation and ERBB1 expression are both required for CDR formation. In particular, in these cells CDR formation requires activation of both the protein regulator of actin polymerization N-WASP, mediated by ERK1/2, and of the actin depolymerizing protein cofilin, mediated by ERBB1. Furthermore, we suggest that this latter event may be inhibited by the negative cell motility regulator p140Cap, as we found that p140Cap overexpression led to cofilin deactivation and inhibition of CDR formation. In conclusion, here we show for the first time an ERBB2-specific signaling contribution to an ERBB2/ERBB1 heterodimer, in the activation of a complex biological process such as the formation of CDRs.

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  • 11.
    Baranov, Vladimir
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology. Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hammarström, Sten
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Immunology/Immunchemistry.
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Lipids are a constitutive component of cytolytic granules.2000In: Histochemistry and Cell Biology, ISSN 0948-6143, E-ISSN 1432-119X, Vol. 114, no 2, p. 167-71Article in journal (Refereed)
    Abstract [en]

    Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.

  • 12. Bassères, Eugénie
    et al.
    Coppotelli, Giuseppe
    Pfirrmann, Thorsten
    Andersen, Jens B
    Masucci, Maria
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    The ubiquitin C-terminal hydrolase UCH-L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton2010In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 12, no 11, p. 1622-1633Article in journal (Refereed)
    Abstract [en]

    Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C-terminal hydrolase UCH-L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH-L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH-L1-negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH-L1 controls the early membrane-associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c-cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2- and AKT-dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH-L1(C90S). These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH-L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria-based drug delivery systems.

  • 13. Beck, Carole
    et al.
    Rodriguez-Vargas, José Manuel
    Boehler, Christian
    Robert, Isabelle
    Heyer, Vincent
    Hanini, Najat
    Gauthier, Laurent R.
    Tissier, Agnès
    Schreiber, Valérie
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    San Martin, Bernardo Reina
    Dantzer, Françoise
    PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers2019In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 26, no 9, p. 1615-1630Article in journal (Refereed)
    Abstract [en]

    PARP3 has been shown to be a key driver of TGF beta-induced epithelial-to-mesenchymal transition (EMT) and sternness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3(-/- )BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9(D10A) gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3 beta, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

  • 14.
    Beier, Frank
    et al.
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany; Department of Medical Biochemistry, University of Calgary, Calgary, Canada.
    Vornehm, Silvia
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    Pöschl, Ernst
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    von der Mark, Klaus
    Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, Erlangen, Germany.
    Lammi, Mikko
    Department of Anatomy, University of Kuopio, Kuopio, Finland.
    Localization of silencer and enhancer elements in the human type X collagen gene.1997In: Journal of Cellular Biochemistry, ISSN 0730-2312, E-ISSN 1097-4644, Vol. 66, no 2, p. 210-218, article id 9213222Article in journal (Refereed)
    Abstract [en]

    Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.

  • 15.
    Benedict, Catherine
    et al.
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Skinner, J. S.
    Meng, R.
    Chang, Y.
    Bhalerao, R.
    Finn, C.
    Chen, T. H. H.
    Umeå University, Faculty of Science and Technology.
    Hurry, Vaughan
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    The Role of the CBF-dependent Signalling Pathway in Woody Perennials2006In: Cold Hardiness in Plants: Molecular Genetics, Cell Biology and Physiology / [ed] T Chen, M Uemura, S Fujikawa, Wallingford: CABI Publishing, 2006, p. 167-180Chapter in book (Other academic)
  • 16.
    Benstein, Ruben M.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Schmid, Markus
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Plant Biology, Linnean Centre for Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    You, Yuan
    Center for Plant Molecular Biology (ZMBP), Department of General Genetics, Eberhard Karls University of Tübingen, Tübingen, Germany; Department of Molecular Life Sciences, Technical University of Munich, Freising, Germany.
    Isolation of nuclei tagged in specific cell types (INTACT) in Arabidopsis2023In: Flower development: methods and protocols / [ed] José Luis Riechmann; Cristina Ferrándiz, New york: Humana Press, 2023, 2, Vol. 2686, p. 313-328Chapter in book (Refereed)
    Abstract [en]

    Many functionally distinct plant tissues have relatively low numbers of cells that are embedded within complex tissues. For example, the shoot apical meristem (SAM) consists of a small population of pluripotent stem cells surrounded by developing leaves and/or flowers at the growing tip of the plant. It is technically challenging to collect enough high-quality SAM samples for molecular analyses. Isolation of Nuclei Tagged in specific Cell Types (INTACT) is an easily reproducible method that allows the enrichment of biotin-tagged cell-type-specific nuclei from the total nuclei pool using biotin-streptavidin affinity purification. Here, we provide a detailed INTACT protocol for isolating nuclei from the Arabidopsis SAM. One can also adapt this protocol to isolate nuclei from other tissues and cell types for investigating tissue/cell-type-specific transcriptome and epigenome and their changes during developmental programs at a high spatiotemporal resolution. Furthermore, due to its low cost and simple procedures, INTACT can be conducted in any standard molecular laboratory.

  • 17. Bento-Abreu, Andre
    et al.
    Jager, Gunilla
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Swinnen, Bart
    Rué, Laura
    Hendrickx, Stijn
    Jones, Ashley
    Staats, Kim A.
    Taes, Ines
    Eykens, Caroline
    Nonneman, Annelies
    Nuyts, Rik
    Timmers, Mieke
    Silva, Lara
    Chariot, Alain
    Nguyen, Laurent
    Ravits, John
    Lemmens, Robin
    Cabooter, Deirdre
    Van Den Bosch, Ludo
    Van Damme, Philip
    Al-Chalabi, Ammar
    Bystrom, Anders
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Robberecht, Wim
    Elongator subunit 3 (ELP3) modifies ALS through tRNA modification.2018In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 27, no 7, p. 1276-1289Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disorder of which the progression is influenced by several disease-modifying factors. Here, we investigated ELP3, a subunit of the elongator complex that modifies tRNA wobble uridines, as one of such ALS disease modifiers. ELP3 attenuated the axonopathy of a mutant SOD1, as well as of a mutant C9orf72 ALS zebrafish model. Furthermore, the expression of ELP3 in the SOD1G93A mouse extended the survival and attenuated the denervation in this model. Depletion of ELP3 in vitro reduced the modified tRNA wobble uridine mcm5s2U and increased abundance of insoluble mutant SOD1, which was reverted by exogenous ELP3 expression. Interestingly, the expression of ELP3 in the motor cortex of ALS patients was reduced and correlated with mcm5s2U levels. Our results demonstrate that ELP3 is a modifier of ALS and suggest a link between tRNA modification and neurodegeneration.

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  • 18.
    Bergonzini, Anna
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Avila-Cariño, Javier
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Lopez Chiloeches, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Frisan, Teresa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The challenge of establishing immunocompetent human intestinal 3D modelsManuscript (preprint) (Other academic)
    Abstract [en]

    Expression of typhoid toxin in Salmonella Typhimurium causes DNA damage, activating the DNA damage response (DDR), in absence of an inflammatory response in the colonic mucosa of infected mice. The anti-inflammatory effect is tissue specific and is not observed in the liver, suggesting that the local immune microenvironment modulates the DDR outcome.

    To assess the role of the immune cells in the DDR outcome induced by the genotoxigenic Salmonella, we have initiated the development of an immunocompetent 3D colonic mucosal model based on a collagen matrix containing colonic fibroblasts and different subtypes of immune cells, overlayed with colonic epithelial cells.

    Embedding of peripheral blood mononuclear cells in the collagen matrix did not influenced either the tissue integrity or the activation of the DDR, observed exclusively upon infection with the genotoxigenic strain. However, embedding of T cells, monocytes, or non-polarized macrophages altered the pattern of the DDR and the toxin specific effect was lost. Presence of macrophages was further associated with alteration of the epithelial layer integrity. This effect was infection-dependent, but not toxin specific.

    Our data demonstrated that addition of immune cells to a 3D mucosal model altered the DDR induced by a genotoxigenic bacterium, highlighting the need to develop and optimize immunocompetent in vitro models.

  • 19. Bergstrand, Sofie
    et al.
    Böhm, Stefanie
    Malmgren, Helena
    Norberg, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Medical and Clinical Genetics.
    Sundin, Mikael
    Nordgren, Ann
    Farnebo, Marianne
    Biallelic mutations in WRAP53 result in dysfunctional telomeres, Cajal bodies and DNA repair, thereby causing Hoyeraal-Hreidarsson syndrome2020In: Cell Death and Disease, E-ISSN 2041-4889, Vol. 11, no 4, article id 238Article in journal (Refereed)
    Abstract [en]

    Approximately half of all cases of Hoyeraal-Hreidarsson syndrome (HHS), a multisystem disorder characterized by bone marrow failure, developmental defects and very short telomeres, are caused by germline mutations in genes related to telomere biology. However, the varying symptoms and severity of the disease indicate that additional mechanisms are involved. Here, a 3-year-old boy with HHS was found to carry biallelic germline mutations in WRAP53 (WD40 encoding RNA antisense to p53), that altered two highly conserved amino acids (L283F and R398W) in the WD40 scaffold domain of the protein encoded. WRAP53 beta (also known as TCAB1 or WDR79) is involved in intracellular trafficking of telomerase, Cajal body functions and DNA repair. We found that both mutations cause destabilization, mislocalization and faulty interactions of WRAP53 beta, defects linked to misfolding by the TRiC chaperonin complex. Consequently, WRAP53 beta HHS mutants cannot elongate telomeres, maintain Cajal bodies or repair DNA double-strand breaks. These findings provide a molecular explanation for the pathogenesis underlying WRAP53 beta-associated HHS and highlight the potential contribution of DNA damage and/or defects in Cajal bodies to the early onset and/or severity of this disease.

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  • 20. Best, Myron G.
    et al.
    Sol, Nik
    Kooi, Irsan
    Tannous, Jihane
    Westerman, Bart A.
    Rustenburg, Francois
    Schellen, Pepijn
    Verschueren, Heleen
    Post, Edward
    Koster, Jan
    Ylstra, Bauke
    Ameziane, Najim
    Dorsman, Josephine
    Smit, Egbert F.
    Verheul, Henk M.
    Noske, David P.
    Reijneveld, Jaap C.
    Nilsson, R. Jonas A.
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology.
    Tannous, Bakhos A.
    Wesseling, Pieter
    Wurdinger, Thomas
    RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics2015In: Cancer Cell, ISSN 1535-6108, E-ISSN 1878-3686, Vol. 28, no 5, p. 666-676Article in journal (Refereed)
    Abstract [en]

    Tumor-educated blood platelets (TEPs) are implicated as central players in the systemic and local responses to tumor growth, thereby altering their RNA profile. We determined the diagnostic potential of TEPs by mRNA sequencing of 283 platelet samples. We distinguished 228 patients with localized and metastasized tumors from 55 healthy individuals with 96% accuracy. Across six different tumor types, the location of the primary tumor was correctly identified with 71% accuracy. Also, MET or HER2-positive, and mutant KRAS, EGFR, or PIK3CA tumors were accurately distinguished using surrogate TEP mRNA profiles. Our results indicate that blood platelets provide a valuable platform for pan-cancer, multiclass cancer, and companion diagnostics, possibly enabling clinical advances in blood-based "liquid biopsies".

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  • 21. Bhalerao, Rishikesh P
    et al.
    Eklöf, Jan
    Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Sciences, SE-901 83, Umeå, Sweden.
    Ljung, Karin
    Marchant, Alan
    Bennett, Malcolm
    Sandberg, Göran
    Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings2002In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 29, no 3, p. 325-332Article in journal (Refereed)
    Abstract [en]

    Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.

  • 22. Bieling, Peter
    et al.
    Laan, Liedewij
    Schek, Henry
    Munteanu, E Laura
    Sandblad, Linda
    European Mol Biol Lab, Cell Biol & Biophys Unit, D-69117 Heidelberg, Germany .
    Dogterom, Marileen
    Brunner, Damian
    Surrey, Thomas
    Reconstitution of a microtubule plus-end tracking system in vitro2007In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 450, no 7172, p. 1100-1105Article in journal (Refereed)
    Abstract [en]

    The microtubule cytoskeleton is essential to cell morphogenesis. Growing microtubule plus ends have emerged as dynamic regulatory sites in which specialized proteins, called plus-end-binding proteins (+TIPs), bind and regulate the proper functioning of microtubules. However, the molecular mechanism of plus-end association by +TIPs and their ability to track the growing end are not well understood. Here we report the in vitro reconstitution of a minimal plus-end tracking system consisting of the three fission yeast proteins Mal3, Tip1 and the kinesin Tea2. Using time-lapse total internal reflection fluorescence microscopy, we show that the EB1 homologue Mal3 has an enhanced affinity for growing microtubule end structures as opposed to the microtubule lattice. This allows it to track growing microtubule ends autonomously by an end recognition mechanism. In addition, Mal3 acts as a factor that mediates loading of the processive motor Tea2 and its cargo, the Clip170 homologue Tip1, onto the microtubule lattice. The interaction of all three proteins is required for the selective tracking of growing microtubule plus ends by both Tea2 and Tip1. Our results dissect the collective interactions of the constituents of this plus-end tracking system and show how these interactions lead to the emergence of its dynamic behaviour. We expect that such in vitro reconstitutions will also be essential for the mechanistic dissection of other plus-end tracking systems.

  • 23.
    Björnham, Oscar
    et al.
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Nilsson, Håkan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Schedin, Staffan
    Umeå University, Faculty of Science and Technology, Department of Applied Physics and Electronics.
    Physical properties of the specific PapG–galabiose binding in E. coli P pili-mediated adhesion2009In: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 38, no 2, p. 245-254Article in journal (Refereed)
    Abstract [en]

    Detailed analyses of the mechanisms thatmediate binding of the uropathogenic Escherichia coli tohost cells are essential, as attachment is a prerequisite forthe subsequent infection process. We explore, by means offorce measuring optical tweezers, the interaction betweenthe galabiose receptor and the adhesin PapG expressed byP pili on single bacterial cells. Two variants of dynamicforce spectroscopy were applied based on constant andnon-linear loading force. The specific PapG–galabiosebinding showed typical slip-bond behaviour in the forceinterval (30–100 pN) set by the pilus intrinsic biomechanicalproperties. Moreover, it was found that the bondhas a thermodynamic off-rate and a bond length of2.6×10-3 s-1 and 5.0 Å , respectively. Consequently, thePapG–galabiose complex is significantly stronger thanthe internal bonds in the P pilus structure that stabilizes thehelical chain-like macromolecule. This finding suggeststhat the specific binding is strong enough to enable the Ppili rod to unfold when subjected to strong shear forces inthe urinary tract. The unfolding process of the P pili rodpromotes the formation of strong multipili interaction,which is important for the bacterium to maintain attachmentto the host cells.

  • 24.
    Blanco, Nicolas E.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Centro de Estudios Fotosintéticos y Bioquímicos, Universidad Nacional de Rosario (CEFOBI-CONICET/UNR), Rosario, Argentina.
    Liebsch, Daniela
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Rosario, Argentina.
    Guinea Diaz, Manuel
    Strand, Åsa
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Whelan, James
    Dual and dynamic intracellular localization of Arabidopsis thaliana SnRK1.12019In: Journal of Experimental Botany, ISSN 0022-0957, E-ISSN 1460-2431, Vol. 70, no 8, p. 2325-2338Article in journal (Refereed)
    Abstract [en]

    Sucrose non-fermenting 1 (SNF1)-related protein kinase 1.1 (SnRK1.1; also known as KIN10 or SnRK1 alpha) has been identified as the catalytic subunit of the complex SnRK1, the Arabidopsis thaliana homologue of a central integrator of energy and stress signalling in eukaryotes dubbed AMPK/Snf1/SnRK1. A nuclear localization of SnRK1.1 has been previously described and is in line with its function as an integrator of energy and stress signals. Here, using two biological models (Nicotiana benthamiana and Arabidopsis thaliana), native regulatory sequences, different microscopy techniques, and manipulations of cellular energy status, it was found that SnRK1.1 is localized dynamically between the nucleus and endoplasmic reticulum (ER). This distribution was confirmed at a spatial and temporal level by co-localization studies with two different fluorescent ER markers, one of them being the SnRK1.1 phosphorylation target HMGR. The ER and nuclear localization displayed a dynamic behaviour in response to perturbations of the plastidic electron transport chain. These results suggest that an ER-associated SnRK1.1 fraction might be sensing the cellular energy status, being a point of crosstalk with other ER stress regulatory pathways.

  • 25. Blaschek, Leonard
    et al.
    Champagne, Antoine
    Dimotakis, Charilaos
    Nuoendagula,
    Decou, Raphaël
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hishiyama, Shojiro
    Kratzer, Susanne
    Kajita, Shinya
    Pesquet, Edouard
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, Stockholm, Sweden.
    Cellular and Genetic Regulation of Coniferaldehyde Incorporation in Lignin of Herbaceous and Woody Plants by Quantitative Wiesner Staining2020In: Frontiers in Plant Science, E-ISSN 1664-462X, Vol. 11, article id 109Article in journal (Refereed)
    Abstract [en]

    Lignin accumulates in the cell walls of specialized cell types to enable plants to stand upright and conduct water and minerals, withstand abiotic stresses, and defend themselves against pathogens. These functions depend on specific lignin concentrations and subunit composition in different cell types and cell wall layers. However, the mechanisms controlling the accumulation of specific lignin subunits, such as coniferaldehyde, during the development of these different cell types are still poorly understood. We herein validated the Wiesner test (phloroglucinol/HCl) for the restrictive quantitative in situ analysis of coniferaldehyde incorporation in lignin. Using this optimized tool, we investigated the genetic control of coniferaldehyde incorporation in the different cell types of genetically-engineered herbaceous and woody plants with modified lignin content and/or composition. Our results demonstrate that the incorporation of coniferaldehyde in lignified cells is controlled by (a) autonomous biosynthetic routes for each cell type, combined with (b) distinct cell-to-cell cooperation between specific cell types, and (c) cell wall layer-specific accumulation capacity. This process tightly regulates coniferaldehyde residue accumulation in specific cell types to adapt their property and/or function to developmental and/or environmental changes.

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  • 26.
    Blaschek, Leonard
    et al.
    Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, Stockholm, Sweden.
    Murozuka, Emiko
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, Stockholm, Sweden.
    Serk, Henrik
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Menard, Delphine
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, Stockholm, Sweden.
    Pesquet, Edouard
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Arrhenius Laboratories, Department of Ecology, Environment and Plant Sciences (DEEP), Stockholm University, Stockholm, Sweden; Bolin Centre for Climate Research, Stockholm University, Stockholm, Sweden.
    Different combinations of laccase paralogs nonredundantly control the amount and composition of lignin in specific cell types and cell wall layers in Arabidopsis2023In: The Plant Cell, ISSN 1040-4651, E-ISSN 1532-298X, Vol. 35, no 2, p. 889-909Article in journal (Refereed)
    Abstract [en]

    Vascular plants reinforce the cell walls of the different xylem cell types with lignin phenolic polymers. Distinct lignin chemistries differ between each cell wall layer and each cell type to support their specific functions. Yet the mechanisms controlling the tight spatial localization of specific lignin chemistries remain unclear. Current hypotheses focus on control by monomer biosynthesis and/or export, while cell wall polymerization is viewed as random and nonlimiting. Here, we show that combinations of multiple individual laccases (LACs) are nonredundantly and specifically required to set the lignin chemistry in different cell types and their distinct cell wall layers. We dissected the roles of Arabidopsis thaliana LAC4, 5, 10, 12, and 17 by generating quadruple and quintuple loss-of-function mutants. Loss of these LACs in different combinations led to specific changes in lignin chemistry affecting both residue ring structures and/or aliphatic tails in specific cell types and cell wall layers. Moreover, we showed that LAC-mediated lignification has distinct functions in specific cell types, waterproofing fibers, and strengthening vessels. Altogether, we propose that the spatial control of lignin chemistry depends on different combinations of LACs with nonredundant activities immobilized in specific cell types and cell wall layers.

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  • 27. Blazkova, Hana
    et al.
    Krejcikova, Katerina
    Moudry, Pavel
    Frisan, Teresa
    Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
    Hodny, Zdenek
    Bartek, Jiri
    Bacterial intoxication evokes cellular senescence with persistent DNA damage and cytokine signalling2010In: Journal of Cellular and Molecular Medicine (Print), ISSN 1582-1838, E-ISSN 1582-4934, Vol. 14, no 1-2, p. 357-367Article in journal (Refereed)
    Abstract [en]

    Cytolethal distending toxins (CDTs) are proteins produced and secreted by facultative pathogenic strains of Gram‐negative bacteria with potentially genotoxic effects. Mammalian cells exposed to CDTs undergo cell type‐dependent cell‐cycle arrest or apoptosis; however, the cell fate responses to such intoxication are mechanistically incompletely understood. Here we show that both normal and cancer cells (BJ, IMR‐90 and WI‐38 fibroblasts, HeLa and U2‐OS cell lines) that survive the acute phase of intoxication by Haemophilus ducreyi CDT possess the hallmarks of cellular senescence. This characteristic phenotype included persistently activated DNA damage signalling (detected as 53BP1/γH2AX+ foci), enhanced senescence‐associated β‐galactosidase activity, expansion of promyelocytic leukaemia nuclear compartments and induced expression of several cytokines (especially interleukins IL‐6, IL‐8 and IL‐24), overall features shared by cells undergoing replicative or premature cellular senescence. We conclude that analogous to oncogenic, oxidative and replicative stresses, bacterial intoxication represents another pathophysiological stimulus that induces premature senescence, an intrinsic cellular response that may mechanistically underlie the ‘distended’ morphology evoked by CDTs. Finally, the activation of the two anticancer barriers, apoptosis and cellular senescence, together with evidence of chromosomal aberrations (micronucleation) reported here, support the emerging genotoxic and potentially oncogenic effects of this group of bacterial toxins, and warrant further investigation of their role(s) in human disease.

  • 28. Blokhina, Olga
    et al.
    Laitinen, Teresa
    Hatakeyama, Yuto
    Delhomme, Nicolas
    Paasela, Tanja
    Zhao, Lei
    Street, Nathaniel
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Wada, Hiroshi
    Karkonen, Anna
    Fagerstedt, Kurt
    Ray Parenchymal Cells Contribute to Lignification of Tracheids in Developing Xylem of Norway Spruce1[OPEN]2019In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 181, no 4, p. 1552-1572Article in journal (Refereed)
    Abstract [en]

    A comparative transcriptomic study and a single-cell metabolome analysis were combined to determine whether parenchymal ray cells contribute to the biosynthesis of monolignols in the lignifying xylem of Norway spruce (Picea abies). Ray parenchymal cells may function in the lignification of upright tracheids by supplying monolignols. To test this hypothesis, parenchymal ray cells and upright tracheids were dissected with laser-capture microdissection from tangential cryosections of developing xylem of spruce trees. The transcriptome analysis revealed that among the genes involved in processes typical for vascular tissues, genes encoding cell wall biogenesis-related enzymes were highly expressed in both developing tracheids and ray cells. Interestingly, most of the shikimate and monolignol biosynthesis pathway-related genes were equally expressed in both cell types. Nonetheless, 1,073 differentially expressed genes were detected between developing ray cells and tracheids, among which a set of genes expressed only in ray cells was identified. In situ single cell metabolomics of semi-intact plants by picoliter pressure probe-electrospray ionization-mass spectrometry detected monolignols and their glycoconjugates in both cell types, indicating that the biosynthetic route for monolignols is active in both upright tracheids and parenchymal ray cells. The data strongly support the hypothesis that in developing xylem, ray cells produce monolignols that contribute to lignification of tracheid cell walls. Transcriptomics combined with single-cell metabolomics give new information on the role of rays in lignification of developing xylem in Norway spruce.

  • 29. Boija, Ann
    et al.
    Mahat, Dig Bijay
    Zare, Aman
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holmqvist, Per-Henrik
    Philip, Philge
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Meyers, David J
    Cole, Philip A
    Lis, John T
    Stenberg, Per
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Mannervik, Mattias
    CBP Regulates Recruitment and Release of Promoter-Proximal RNA Polymerase II2017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 3, p. 491-503.e5Article in journal (Refereed)
    Abstract [en]

    Transcription activation involves RNA polymerase II (Pol II) recruitment and release from the promoter into productive elongation, but how specific chromatin regulators control these steps is unclear. Here, we identify a novel activity of the histone acetyltransferase p300/CREB-binding protein (CBP) in regulating promoter-proximal paused Pol II. We find that Drosophila CBP inhibition results in "dribbling" of Pol II from the pause site to positions further downstream but impedes transcription through the +1 nucleosome genome-wide. Promoters strongly occupied by CBP and GAGA factor have high levels of paused Pol II, a unique chromatin signature, and are highly expressed regardless of cell type. Interestingly, CBP activity is rate limiting for Pol II recruitment to these highly paused promoters through an interaction with TFIIB but for transit into elongation by histone acetylation at other genes. Thus, CBP directly stimulates both Pol II recruitment and the ability to traverse the first nucleosome, thereby promoting transcription of most genes.

  • 30.
    Boussardon, Clément
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Cell Type–Specific Isolation of Mitochondria in Arabidopsis2022In: Plant Mitochondria: Methods and Protocols / [ed] Olivier Van Aken; Allan G. Rasmusson, New York: Humana Press, 2022, 1, , p. 11p. 13-23Chapter in book (Refereed)
    Abstract [en]

    Membrane-bound organelles are unique features of eukaryotic cell structures. Among them, mitochondria host key metabolic functions and pathways, including the aerobic respiration. In plants, several procedures are available to isolate mitochondria from the other cell compartments, as high-quality purified extracts are often necessary for accurate molecular biology or biochemistry investigations. Protocols based on differential centrifugations and subsequent density gradients are an effective way to extract rather pure and intact mitochondria within a few hours. However, while mitochondria from seedlings, large leaves or tubers are relatively easy to extract, tissue-specific isolation of organelles had remained a challenge. This has recently been circumvented, only in transformable plants though, by the use of affinity-tagged mitochondria and their isolation with magnetic beads. We hereby describe a step-by-step protocol for the rapid and tissue-specific isolation of Arabidopsis thaliana mitochondria, a method named IMTACT (Isolation of Mitochondria TAgged in specific Cell Types). Cell-specific biotinylated mitochondria are isolated with streptavidin magnetic beads in less than 30 min from sampling to final extract. Key steps, enrichment, bead size comparison, and mitochondrial depletion in the sample are also reported in order to facilitate the experimental setup of the user.

  • 31.
    Boussardon, Clément
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Przybyla-Toscano, Jonathan
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Carrie, Chris
    Keech, Olivier
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Tissue-specific isolation of Arabidopsis/plant mitochondria - IMTACT (isolation of mitochondria tagged in specific cell types)2020In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 103, no 1, p. 459-473Article in journal (Refereed)
    Abstract [en]

    Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed inArabidopsis thaliana(Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of theOM64(Outer Membrane 64) gene combined withBLRPand theBirAbiotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g.CAB3andSUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild-type and mutant Arabidopsis plants we generated a set of OM64-BLRP one-shot constructs with different selection markers and tissue-specific promoters.

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  • 32. Brauner, Annelie
    et al.
    Brandt, Lena
    Frisan, Teresa
    Department of Clinical Microbiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.
    Thelestam, Monica
    Ekbom, Anders
    Is there a risk of cancer development after Campylobacter infection?2010In: Scandinavian Journal of Gastroenterology, ISSN 0036-5521, E-ISSN 1502-7708, Vol. 45, no 7-8, p. 893-897Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: All Campylobacter jejuni species produce a genotoxin, which induce DNA double strand breaks, could lead to an increased risk of cancer especially in the gastro-intestinal tract.

    MATERIAL AND METHODS: All individuals in Stockholm County who tested positive with C. jejuni between 1989 and 2006 were included. The cohort was followed-up until December 31, 2007 for the occurrence of cancer, overall and site specific. Standard incidence ratios (SIR) with 95% confidence intervals (CI) were calculated by comparisons with the background population.

    RESULTS: There were 16,276 individuals who tested positive for C. jejuni generating 124,387 person years. Excluding the first year of follow-up the overall risk for cancer did neither differ from that expected SIR = 0.95 (95% CI 0.82-1.09) nor after 10 years or more of follow-up; SIR = 0.91 (95% CI 0.71-1.16). There was no increased risk for cancer in the gastro-intestinal tract, but there were significantly increased risks for melanomas SIR = 1.84 (95% CI 1.27-2.57) and squamous cell skin cancer SIR = 1.52 (95% CI 1.01-2.19) while a significantly decreased risk of respiratory cancers among males SIR = 0.32 (95% CI 0.12-0.70) was observed.

    CONCLUSIONS: Our results indicate no excess risks of malignancies following an infection by C. jejuni at least during the first decade. Furthermore, the finding of a decreased risk of respiratory cancers could be of interest, if the results are reproduced in future studies in other populations.

  • 33. Bykova, Natalia V.
    et al.
    Møller, Ian M.
    Gardeström, Per
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Igamberdiev, Abir U.
    The function of glycine decarboxylase complex is optimized to maintain high photorespiratory flux via buffering of its reaction products2014In: Mitochondrion (Amsterdam. Print), ISSN 1567-7249, E-ISSN 1872-8278, Vol. 19, p. 357-364Article in journal (Refereed)
    Abstract [en]

    Oxidation of glycine in photorespiratory pathway is the major flux through mitochondria of C3 plants in the light. It sustains increased intramitochondrial concentrations of NADH and NADPH, which are required to engage the internal rotenone-insensitive NAD(P)H dehydrogenases and the alternative oxidase. We discuss here possible mechanisms of high photorespiratory flux maintenance in mitochondria and suggest that it is fulfilled under conditions where the concentrations of glycine decarboxylase reaction products NADH and CO2 achieve an equilibrium provided by malate dehydrogenase and carbonic anhydrase, respectively. This results in the removal of these products from the glycine decarboxylase multienzyme active sites and in the maintenance of their concentrations at levels sufficiently low to prevent substrate inhibition of the reaction. 

  • 34. Campos, Manuel
    et al.
    Nilges, Michaël
    Cisneros, David A.
    Institut Pasteur, Unité de Génétique Moléculaire, Département de Microbiologie, F-75015 Paris, France.
    Francetic, Olivera
    Detailed structural and assembly model of the type II secretion pilus from sparse data2010In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, no 29, p. 13081-13086Article in journal (Refereed)
    Abstract [en]

    Many gram-negative bacteria secrete specific proteins via the type II secretion systems (T2SS). These complex machineries share with the related archaeal flagella and type IV pilus (T4P) biogenesis systems the ability to assemble thin, flexible filaments composed of small, initially inner membrane-localized proteins called "pilins." In the T2SS from Klebsiella oxytoca, periplasmic pseudopili that are essential for pullulanase (PulA) secretion extend beyond the bacterial surface and form pili when the major pilin PulG is overproduced. Here, we describe the detailed, experimentally validated structure of the PulG pilus generated from crystallographic and electron microscopy data by a molecular modeling approach. Two intermolecular salt bridges crucial for function were demonstrated using single and complementary charge inversions. Double-cysteine substitutions in the transmembrane segment of PulG led to position-specific cross-linking of protomers in assembled pili. These biochemical data provided information on residue distances in the filament that were used to derive a refined model of the T2SS pilus at pseudoatomic resolution. PulG is organized as a right-handed helix of subunits, consistent with protomer organization in gonococcal T4P. The conserved character of residues involved in key hydrophobic and electrostatic interactions within the major pseudopilin family supports the general relevance of this model for T2SS pseudopilus structure.

  • 35. Cerqua, Cristina
    et al.
    Morbidoni, Valeria
    Desbats, Maria Andrea
    Doimo, Mara
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Clinical Genetics Unit, Department of Women and Children's Health, University of Padova, Padova, Italy.
    Frasson, Chiara
    Sacconi, Sabrina
    Baldoin, Maria Cristina
    Sartori, Geppo
    Basso, Giuseppe
    Salviati, Leonardo
    Trevisson, Eva
    COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX22018In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1859, no 4, p. 244-252Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits.

    We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant.

    Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

  • 36. Cha, Shih-Ting
    et al.
    Chen, Pai-Sheng
    Johansson, Gunnar
    1Laboratory of Molecular and Cellular Toxicology, Institute of Toxicology, College of Medicine, National Taiwan University.
    Chu, Chia-Yu
    Wang, Ming-Yang
    Jeng, Yung-Ming
    Yu, Sung-Liang
    Chen, Jin-Shing
    Chang, King-Jen
    Jee, Shiou-Hwa
    Tan, Ching-Ting
    Lin, Ming-Tsan
    Kuo, Min-Liang
    MicroRNA-519c suppresses hypoxia-inducible factor-1alpha expression and tumor angiogenesis.2010In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 7, p. 2675-2685Article in journal (Refereed)
    Abstract [en]

    Hypoxia-inducible factor-1alpha (HIF-1alpha) is widely considered to be one of the key regulators of tumor angiogenesis. The upstream regulation is complex and involves several growth factors, cytokines, and hypoxia. Herein, we have identified miR-519c as a hypoxia-independent regulator of HIF-1alpha, acting through direct binding to the HIF-1alpha 3' untranslated region and leading to reduced tumor angiogenesis. Overexpression of miR-519c resulted in a significant decrease of HIF-1alpha protein levels and reduced the tube formation of human umbilical vein endothelial cells; similarly, antagomir inhibition of miR-519c increased the level of HIF-1alpha protein and enhanced angiogenic activity, suggesting an important role of miR-519c in HIF-1alpha-mediated angiogenesis. Consistent with the overexpression of miR-519c in cancer patients with better prognosis, mice injected with miR-519c-overexpressing cells exhibited dramatically reduced HIF-1alpha levels, followed by suppressed tumor angiogenesis, growth, and metastasis. In addition, we found that hepatocyte growth factor (HGF), a known HIF-1alpha inducer, reduced the miR-519c levels through an Akt-dependent pathway. This regulation was posttranscriptional and may be mediated by suppression of miR-519c maturation. Taken together, our findings provide the first evidence that miR-519c is a pivotal regulator of tumor angiogenesis and that microenvironmental HGF contributes to regulating miR-519c biogenesis in cancer cells.

  • 37.
    Chang, Yanhai
    et al.
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Wang, Xiao
    Department of Galactophore, Shaanxi Provincial Cancer Hospital, Xi’an, PR China.
    Sun, Zhengming
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Jin, Zhankui
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Chen, Ming
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Wang, Xiaoqing
    Department of Orthopaedics, The Third Affiliated Hospital (Shaanxi Provincial People's Hospital), Health Science Center of Xi’an Jiaotong University, Xi’an, PR China.
    Lammi, Mikko
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Guo, Xiong
    School of Public Health, Xi’an Jiaotong University Health Science Center, Key Laboratory of Trace Elements and Endemic Diseases, Ministry of Health, Xi’an, China.
    Inflammatory cytokine of IL-1β is involved in T-2 toxin-triggered chondrocyte injury and metabolism imbalance by the activation of Wnt/β-catenin signaling2017In: Molecular Immunology, ISSN 0161-5890, E-ISSN 1872-9142, Vol. 91, p. 195-201Article in journal (Refereed)
    Abstract [en]

    Mycotoxin T-2 exerts a causative role in Kashin-Beck disease (KBD) suffering chondrocyte apoptosis and cartilage matrix homeostasis disruption. Recent research corroborated the aberrant levels of pro-inflammatory cytokine IL-1ß in KBD patients and mycotoxin environment. In the present study, we investigated the relevance of IL-1ß in T-2 toxin-evoked chondrocyte cytotoxic injury and aberrant catabolism. High levels of IL-1ß were detected in serum and cartilages from KBD patients and in T-2-stimulated chondrocytes. Moreover, knockdown of IL-1ß antagonized the adverse effects of T-2 on cytotoxic injury by enhancing cell viability and inhibiting apoptosis. However, exogenous supplementation of IL-1β further aggravated cell damage in response to T-2. Additionally, cessation of IL-1β rescued T-2-elicited tilt of matrix homeostasis toward catabolism by elevating the transcription of collagen II and aggrecan, promoting release of sulphated glycosaminoglycans (sGAG) and TIMP1, and suppressing matrix metalloproteinases production including MMP-1, MMP-3 and MMP-13. Conversely, IL-1β stimulation deteriorated T-2-induced disruption of matrix metabolism balance toward catabolism. Mechanistic analysis found the high activation of Wnt/β-catenin in KBD patients and chondrocytes upon T-2. Furthermore, this activation was mitigated after IL-1β inhibition, but further enhanced following IL-1β precondition. Importantly, blocking this pathway by transfection with β-catenin alleviated the adverse roles of IL-1β on cytotoxic injury and metabolism disorders under T-2 conditioning. Together, this study elucidates a new insight into how T-2 deteriorates the pathological progression of KBD by regulating inflammation-related pathways, indicating a promising anti-inflammation strategy for KBD therapy.

  • 38. Chen, Chi-Kuan
    et al.
    Yang, Ching-Yao
    Hua, Kuo-Tai
    Hua, Kuo-Ti
    Ho, Ming-Chih
    Johansson, Gunnar
    Department of Neurology, National Taiwan University Hospital, and National Taiwan University College of Medicine, Taipei, Taiwan.
    Jeng, Yung-Ming
    Chen, Chiung-Nien
    Chen, Min-Wei
    Lee, Wei-Jiunn
    Su, Jen-Liang
    Lai, Tsung-Ching
    Chou, Chi-Chi
    Ho, Bing-Ching
    Chang, Chuan-Fa
    Lee, Po-Huang
    Chang, King-Jen
    Hsiao, Michael
    Lin, Ming-Tsan
    Kuo, Min-Liang
    Leukocyte cell-derived chemotaxin 2 antagonizes MET receptor activation to suppress hepatocellular carcinoma vascular invasion by protein tyrosine phosphatase 1B recruitment2014In: Hepatology, ISSN 0270-9139, E-ISSN 1527-3350, Vol. 59, no 3, p. 974-985Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Leukocyte cell-derived chemotoxin 2 (LECT2) has been shown to act as a tumor suppressor in hepatocellular carcinoma (HCC). However, the underlying mechanism has not yet been completely defined. Here, we employ a LECT2-affinity column plus liquid chromatography coupled with tandem mass spectrometry to identify LECT2-binding proteins and found that MET receptor strongly interacted with LECT2 protein. Despite the presence of hepatocyte growth factor, the LECT2 binding causes an antagonistic effect to MET receptor activation through recruitment of protein tyrosine phosphatase 1B. The antagonistic effect of LECT2 on MET activation also mainly contributes to the blockage of vascular invasion and metastasis of HCC. Furthermore, serial deletions and mutations of LECT2 showed that the HxGxD motif is primarily responsible for MET receptor binding and its antagonistic effects.

    CONCLUSION: These findings reveal a novel, specific inhibitory function of LECT2 in HCC by the direct binding and inactivation of MET, opening a potential avenue for treating MET-related liver cancer.

  • 39. Chen, Min-Wei
    et al.
    Hua, Kuo-Tai
    Kao, Hsin-Jung
    Chi, Chia-Chun
    Wei, Lin-Hung
    Johansson, Gunnar
    Graduate Institute of Toxicology, National Taiwan University College of Medicine.
    Shiah, Shine-Gwo
    Chen, Pai-Sheng
    Jeng, Yung-Ming
    Cheng, Tsu-Yao
    Lai, Tsung-Ching
    Chang, Jeng-Shou
    Jan, Yi-Hua
    Chien, Ming-Hsien
    Yang, Chih-Jen
    Huang, Ming-Shyan
    Hsiao, Michael
    Kuo, Min-Liang
    H3K9 histone methyltransferase G9a promotes lung cancer invasion and metastasis by silencing the cell adhesion molecule Ep-CAM2010In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 70, no 20, p. 7830-7840Article in journal (Refereed)
    Abstract [en]

    G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Emerging evidence suggests that G9a is required to maintain the malignant phenotype, but the role of G9a function in mediating tumor metastasis has not been explored. Here, we show that G9a is expressed in aggressive lung cancer cells, and its elevated expression correlates with poor prognosis. RNAi-mediated knockdown of G9a in highly invasive lung cancer cells inhibited cell migration and invasion in vitro and metastasis in vivo. Conversely, ectopic G9a expression in weakly invasive lung cancer cells increased motility and metastasis. Mechanistic investigations suggested that repression of the cell adhesion molecule Ep-CAM mediated the effects of G9a. First, RNAi-mediated knockdown of Ep-CAM partially relieved metastasis suppression imposed by G9a suppression. Second, an inverse correlation between G9a and Ep-CAM expression existed in primary lung cancer. Third, Ep-CAM repression was associated with promoter methylation and an enrichment for dimethylated histone H3K9. G9a knockdown reduced the levels of H3K9 dimethylation and decreased the recruitment of the transcriptional cofactors HP1, DNMT1, and HDAC1 to the Ep-CAM promoter. Our findings establish a functional contribution of G9a overexpression with concomitant dysregulation of epigenetic pathways in lung cancer progression.

  • 40. Chen, Xi
    et al.
    Venkatachalapathy, Muthukumaran
    Kamps, Dominic
    Weigel, Simone
    Kumar, Ravi
    Orlich, Michael
    Garrecht, Ruben
    Hirtz, Michael
    Niemeyer, Christof M.
    Wu, Yao-Wen
    Chemical Genomics Centre of the Max-Planck Society, Dortmund, Germany.
    Dehmelt, Leif
    “Molecular Activity Painting”: Switch-like, light-controlled perturbations inside living cells2017In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 56, no 21, p. 5916-5920Article in journal (Refereed)
    Abstract [en]

    Acute subcellular protein targeting is a powerful tool to study biological networks. However, signaling at the plasma membrane is highly dynamic, making it difficult to study in space and time. In particular, sustained local control of molecular function is challenging due to lateral diffusion of plasma membrane targeted molecules. Here we present “Molecular Activity Painting” (MAP), a novel technology which combines photoactivatable chemically induced dimerization (pCID) with immobilized artificial receptors. The immobilization of artificial receptors by surface-immobilized antibodies blocks lateral diffusion, enabling rapid and stable “painting” of signaling molecules and their activity at the plasma membrane with micrometer precision. Using this method, we show that painting of the RhoA-myosin activator GEF-H1 induces patterned acto-myosin contraction inside living cells.

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  • 41. Chien, Ming-Hsien
    et al.
    Ku, Chia-Chi
    Johansson, Gunnar
    Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Umeå University.
    Chen, Min-Wei
    Hsiao, Michael
    Su, Jen-Liang
    Inoue, Hiroyasu
    Hua, Kuo-Tai
    Wei, Lin-Hung
    Kuo, Min-Liang
    Vascular endothelial growth factor-C (VEGF-C) promotes angiogenesis by induction of COX-2 in leukemic cells via the VEGF-R3/JNK/AP-1 pathway.2009In: Carcinogenesis, ISSN 0143-3334, E-ISSN 1460-2180, Vol. 30, no 12, p. 2005-13Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor (VEGF)-C is recognized as a tumor lymphangiogenic factor based on the effects of activated VEGF-R3 on lymphatic endothelial cells. Many tumor cells express VEGF-R3 but the function of this receptor in tumor cells is largely unknown. It has been reported that the VEGF-C/VEGF-R3 axis is activated in subsets of leukemia patients. Herein, we have shown that VEGF-C induces angiogenic activity in the tube formation assay invitro and Matrigel plug assay in vivo by upregulating an angiogenic factor, cyclooxygenase-2 (COX-2), through VEGF-R3 in the human acute myeloid leukemia (AML) cell line, THP-1. COX-2 induction by VEGF-C was also observed in other VEGF-R3(+) human AML cell lines (U937 and HL60). Moreover, immunohistochemical analysis of bone marrow specimens of 37 patients diagnosed with AML revealed that VEGF-C expression in specimens was associated with the expression of COX-2 (P < 0.001). The manner by which signaling pathways transduced by VEGF-C is responsible for COX-2 upregulation was further investigated. Blocking the p42/44 mitogen-activated protein kinase (MAPK) pathway with the MAPK kinase inhibitor, PD 98059, failed to inhibit VEGF-C-mediated COX-2 expression. However, VEGF-C-induced COX-2 upregulation was effectively abolished by overexpression of dominant-negative c-Jun N-terminal kinase (JNK) or treatment with the JNK inhibitor, SP 600125. VEGF-C induced JNK-dependent nuclear translocation of c-Jun. Furthermore, chromatin immunoprecipitation and reporter assays revealed that VEGF-C enhanced c-Jun binding to the cyclic adenosine 3',5'-monophosphate-response element of the COX-2 promoter and induced COX-2 expression. In sum, the data herein highlight the pathogenic role of VEGF-C in leukemia via regulation of angiogenesis through upregulation of COX-2.

  • 42.
    Chowdhury, Jamil
    et al.
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Ferdous, Jannatul
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Lihavainen, Jenna
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Albrectsen, Benedicte Riber
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Lundberg-Felten, Judith
    Umeå Plant Science Center, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Fluorogenic properties of 4-dimethylaminocinnamaldehyde (DMACA) enable high resolution imaging of cell-wall-bound proanthocyanidins in plant root tissues2023In: Frontiers in Plant Science, E-ISSN 1664-462X, Vol. 13, article id 1060804Article in journal (Refereed)
    Abstract [en]

    Proanthocyanidins (PAs) are polymeric phenolic compounds found in plants and used in many industrial applications. Despite strong evidence of herbivore and pathogen resistance-related properties of PAs, their in planta function is not fully understood. Determining the location and dynamics of PAs in plant tissues and cellular compartments is crucial to understand their mode of action. Such an approach requires microscopic localization with fluorescent dyes that specifically bind to PAs. Such dyes have hitherto been lacking. Here, we show that 4-dimethylaminocinnamaldehyde (DMACA) can be used as a PA-specific fluorescent dye that allows localization of PAs at high resolution in cell walls and inside cells using confocal microscopy, revealing features of previously unreported wall-bound PAs. We demonstrate several novel usages of DMACA as a fluorophore by taking advantage of its double staining compatibility with other fluorescent dyes. We illustrate the use of the dye alone and its co-localization with cell wall polymers in different Populus root tissues. The easy-to-use fluorescent staining method, together with its high photostability and compatibility with other fluorogenic dyes, makes DMACA a valuable tool for uncovering the biological function of PAs at a cellular level in plant tissues. DMACA can also be used in other plant tissues than roots, however care needs to be taken when tissues contain compounds that autofluoresce in the red spectral region which can be confounded with the PA-specific DMACA signal.

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  • 43.
    Colomé, Núria
    et al.
    ProteoRed-ISCIII, Vall d'Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain.
    Abian, Joaquín
    ProteoRed-ISCIII, Instituto de Investigaciones Biomédicas de Barcelona, IIBB-CSIC/IDIBAPS, 08036 Barcelona, Spain.
    Aloria, Kerman
    ProteoRed-ISCIII, Proteomics Core Facility-SGIKER, University of the Basque Country (UPV/EHU), Leioa, Spain.
    Arizmendi, Jesús M.
    Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain.
    Barceló-Batllori, Silvia
    ProteoRed-ISCIII, Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
    Braga-Lagache, Sophie
    Department for BioMedical Research (DBMR), Proteomics and Mass Spectrometry Core Facility, University of Bern, CH-3010 Bern, Switzerland.
    Burlet-Schiltz, Odile
    Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, UPS, CNRS, Toulouse, France.
    Carrascal, Montse
    ProteoRed-ISCIII, Instituto de Investigaciones Biomédicas de Barcelona, IIBB-CSIC/IDIBAPS, 08036 Barcelona, Spain.
    Casal, J. Ignacio
    ProteoRed-ISCIII, Centro de Investigaciones Biológicas-CSIC, Madrid 28040, Spain.
    Chicano-Gálvez, Eduard
    ProteoRed-ISCIII, Proteomics Unit, IMIBIC/UCO/HURS, IMIBIC Building Fl.3, 14004 Córdoba, Spain.
    Chiva, Cristina
    Proteomics Unit, Center for Genomics Regulation, Barcelona Institute of Science and Technology (BIST), Barcelona, Spain.
    Clemente, Luis Felipe
    ProteoRed-ISCIII, Proteomics Unit, Complutense University, 28040 Madrid, Spain.
    Elortza, Felix
    ProteoRed-ISCIII, CIC bioGUNE, Proteomics Platform, Basque Research & Technology Alliance (BRTA), CIBERehd,Bizkaia Science and Technology Park, 48160 Derio, Spain.
    Estanyol, Josep M.
    ProteoRed-ISCIII, Scientific and Technological Centers (CCiTUB), University of Barcelona, 08036 Barcelona, Spain.
    Fernandez-Irigoyen, Joaquín
    Proteored-ISCIII. Proteomics Unit, Clinical Neuroproteomics Group, Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Pública de Navarra (UPNA), IdiSNA, 31008 Pamplona, Spain.
    Fernández-Puente, Patricia
    Grupo de Investigación de Reumatología (GIR), Agrupación CICA-INIBIC, Universidad de A Coruña, A Coruña, Spain.
    Fidalgo, María José
    ProteoRed-ISCIII, Scientific and Technological Centers (CCiTUB), University of Barcelona, 08036 Barcelona, Spain.
    Froment, Carine
    Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, UPS, CNRS, Toulouse, France.
    Fuentes, Manuel
    Department of Medicine and General Cytometry Service-Nucleus, Proteomics Unit, CIBERONC, Cancer Research Center (IBMCC/CSIC/USAL/IBSAL), Universidad de Salamanca, Spain.
    Fuentes-Almagro, Carlos
    Proteomics Unit, SCAI, University of Córdoba, Ramón y Cajal Building, Rabanales Campus, 14071, Córdoba, Spain.
    Gay, Marina
    ProteoRed-ISCIII, Institute for Research in Biomedicine (IRB Barcelona), BIST (The Barcelona Institute of Science and Technology), Baldiri i Reixac 10, 08028 Barcelona, Spain.
    Hainard, Alexandre
    Proteomics Core Facility, CMU, University of Geneva, Switzerland.
    Heller, Manfred
    Department for BioMedical Research (DBMR), Proteomics and Mass Spectrometry Core Facility, University of Bern, CH-3010 Bern, Switzerland.
    Hernández, María Luisa
    ProteoRed-ISCIII, Proteomics Unit, Complutense University, 28040 Madrid, Spain.
    Ibarrola, Nieves
    ProteoRed-ISCIII, Proteomics Unit. Cancer Research Center (IBMCC/CSIC/USAL/IBSAL), Universidad de Salamanca-CSIC, Salamanca, Spain.
    Iloro, Ibon
    ProteoRed-ISCIII, CIC bioGUNE, Proteomics Platform, Basque Research & Technology Alliance (BRTA), CIBERehd,Bizkaia Science and Technology Park, 48160 Derio, Spain.
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lario, Antonio
    ProteoRed-ISCIII, IPBLN -CSIC, 18016 Granada, Spain.
    Locard-Paulet, Marie
    Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Université de Toulouse, UPS, CNRS, Toulouse, France.
    Marina-Ramírez, Anabel
    ProteoRed-ISCIII, CBM Severo Ochoa (CSIC-UAM), Madrid 28049, Spain.
    Martín, Luna
    ProteoRed-ISCIII, Vall d'Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain.
    Morato-López, Esperanza
    ProteoRed-ISCIII, CBM Severo Ochoa (CSIC-UAM), Madrid 28049, Spain.
    Muñoz, Javier
    ProteoRed-ISCIII, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain.
    Navajas, Rosana
    ProteoRed-ISCIII, Centro Nacional de Biotecnologia (CSIC), 28049, Madrid, Spain.
    Odena, M. Antonia
    ProteoRed-ISCIII, Proteomics Platform, Barcelona Science Park, 08028, Barcelona, Spain.
    Odriozola, Leticia
    ProteoRed-ISCIII, CIMA, University of Navarra, 31008, Pamplona, Spain.
    de Oliveira, Eliandre
    ProteoRed-ISCIII, Proteomics Platform, Barcelona Science Park, 08028, Barcelona, Spain.
    Paradela, Alberto
    ProteoRed-ISCIII, Centro Nacional de Biotecnologia (CSIC), 28049, Madrid, Spain.
    Pasquarello, Carla
    Proteomics Core Facility, CMU, University of Geneva, Switzerland.
    de los Rios, Vivian
    ProteoRed-ISCIII, Centro de Investigaciones Biológicas-CSIC, Madrid 28040, Spain.
    Ruiz-Romero, Cristina
    Grupo de Investigación de Reumatología (GIR) - ProteoRed-ISCIII, Unidad de Proteómica, INIBIC–Complejo Hospitalario Universitario de A Coruña, SERGAS, A Coruña, Spain.
    Sabidó, Eduard
    ProteoRed ISCIII, Proteomics Unit, Universitat Pompeu Fabra, Barcelona, Spain.
    Sánchez del Pino, Manuel
    Biotechnology and Biomedicine Interdisciplinary Research Unit (ERI BIOTECMED), University of Valencia, 46100 Burjassot, Spain.
    Sancho, Jaime
    ProteoRed-ISCIII, IPBLN -CSIC, 18016 Granada, Spain.
    Santamaría, Enrique
    Proteored-ISCIII. Proteomics Unit, Clinical Neuroproteomics Group, Navarrabiomed, Complejo Hospitalario de Navarra (CHN), Universidad Pública de Navarra (UPNA), IdiSNA, 31008 Pamplona, Spain.
    Schaeffer-Reiss, Christine
    Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000, Strasbourg, France.
    Schneider, Justine
    Laboratoire de Spectrométrie de Masse BioOrganique, Université de Strasbourg, CNRS, IPHC UMR 7178, 67000, Strasbourg, France.
    de la Torre, Carolina
    ProteoRed-ISCIII, Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain.
    Valero, M. Luz
    ProteoRed-ISCIII, Proteomics Unit, Central Service for Experimental Research (SCSIE), University of Valencia, 46100, Burjassot, Spain.
    Vilaseca, Marta
    ProteoRed-ISCIII, Institute for Research in Biomedicine (IRB Barcelona), BIST (The Barcelona Institute of Science and Technology), Baldiri i Reixac 10, 08028 Barcelona, Spain.
    Wu, Shuai
    ProteoRed-ISCIII, Vall d’Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain; Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Universit´e de Toulouse, UPS, CNRS, Toulouse, France .
    Wu, Linfeg
    ProteoRed-ISCIII, Vall d’Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain; Proteomics and Mass Spectrometry of Biomolecules, Proteomics Infrastructure of Toulouse, Proteomics French Infrastructure, ProFI. Institut de Pharmacologie et Biologie Structurale (IPBS), Universit´e de Toulouse, UPS, CNRS, Toulouse, France .
    de Embún, Pilar Ximénez
    ProteoRed-ISCIII, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain.
    Canals, Francesc
    ProteoRed-ISCIII, Vall d’Hebron Institute of Oncology (VHIO), Barcelona 08035, Spain .
    Corrales, Fernando J.
    ProteoRed-ISCIII, Centro Nacional de Biotecnologia (CSIC), 28049, Madrid, Spain; ProteoRed-ISCIII, CIMA, University of Navarra, 31008, Pamplona, Spain.
    Multi-laboratory experiment PME11 for the standardization of phosphoproteome analysis2022In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 251, article id 104409Article in journal (Refereed)
    Abstract [en]

    Global analysis of protein phosphorylation by mass spectrometry proteomic techniques has emerged in the last decades as a powerful tool in biological and biomedical research. However, there are several factors that make the global study of the phosphoproteome more challenging than measuring non-modified proteins. The low stoichiometry of the phosphorylated species and the need to retrieve residue specific information require particular attention on sample preparation, data acquisition and processing to ensure reproducibility, qualitative and quantitative robustness and ample phosphoproteome coverage in phosphoproteomic workflows. Aiming to investigate the effect of different variables in the performance of proteome wide phosphoprotein analysis protocols, ProteoRed-ISCIII and EuPA launched the Proteomics Multicentric Experiment 11 (PME11). A reference sample consisting of a yeast protein extract spiked in with different amounts of a phosphomix standard (Sigma/Merck) was distributed to 31 laboratories around the globe. Thirty-six datasets from 23 laboratories were analyzed. Our results indicate the suitability of the PME11 reference sample to benchmark and optimize phosphoproteomics strategies, weighing the influence of different factors, as well as to rank intra and inter laboratory performance.

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  • 44. Concepcion Cruz-Santos, Maria
    et al.
    Aragon-Raygoza, Alejandro
    Espinal-Centeno, Annie
    Arteaga-Vazquez, Mario
    Cruz-Hernandez, Andres
    Bako, Laszlo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology.
    Cruz-Ramirez, Alfredo
    The Role of microRNAs in Animal Cell Reprogramming2016In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 25, no 14, p. 1035-1049Article, review/survey (Refereed)
    Abstract [en]

    Our concept of cell reprogramming and cell plasticity has evolved since John Gurdon transferred the nucleus of a completely differentiated cell into an enucleated Xenopus laevis egg, thereby generating embryos that developed into tadpoles. More recently, induced expression of transcription factors, oct4, sox2, klf4, and c-myc has evidenced the plasticity of the genome to change the expression program and cell phenotype by driving differentiated cells to the pluripotent state. Beyond these milestone achievements, research in artificial cell reprogramming has been focused on other molecules that are different than transcription factors. Among the candidate molecules, microRNAs (miRNAs) stand out due to their potential to control the levels of proteins that are involved in cellular processes such as self-renewal, proliferation, and differentiation. Here, we review the role of miRNAs in the maintenance and differentiation of mesenchymal stem cells, epimorphic regeneration, and somatic cell reprogramming to induced pluripotent stem cells.

  • 45.
    Corkery, Dale P.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wu, Yao-Wen
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Eating while intoxicated: characterizing the molecular mechanism behind V. cholerae toxin MakA-regulated autophagy2023In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 19, no 6, p. 1885-1886Article in journal (Refereed)
    Abstract [en]

    Extracellular pathogens utilize secreted virulence factors to regulate host cell function. Recently we characterized the molecular mechanism behind host macroautophagy/autophagy regulation by the Vibrio cholerae toxin MakA. Cholesterol binding at the plasma membrane induces MakA endocytosis and pH-dependent pore assembly. Membrane perforation of late endosomal membranes induces cellular membrane repair pathways and V-ATPase-dependent unconventional LC3 lipidation on damaged membranes.

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  • 46. Cruz-Ramírez, Alfredo
    et al.
    Díaz-Triviño, Sara
    Blilou, Ikram
    Grieneisen, Verônica A.
    Sozzani, Rosangela
    Zamioudis, Christos
    Miskolczi, Pál
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Nieuwland, Jeroen
    Benjamins, René
    Dhonukshe, Pankaj
    Caballero-Pérez, Juan
    Horvath, Beatrix
    Long, Yuchen
    Mähönen, Ari Pekka
    Zhang, Hongtao
    Xu, Jian
    Murray, James A. H.
    Benfey, Philip N.
    Bako, Laszlo
    Umeå University, Faculty of Science and Technology, Department of Plant Physiology. Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Department of Forest Genetics and Plant Physiology, Umeå Plant Science Center, Swedish University of Agricultural Sciences, Umeå, Sweden.
    Marée, Athanasius F. M.
    Scheres, Ben
    A Bistable Circuit Involving SCARECROW-RETINOBLASTOMA Integrates Cues to Inform Asymmetric Stem Cell Division2012In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 150, no 5, p. 1002-1015Article in journal (Refereed)
    Abstract [en]

    In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOM-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a "flip flop" that constrains asymmetric cell division to the stem cell region.

  • 47. Dal Molin, Federica
    et al.
    Zornetta, Irene
    Puhar, Andrea
    Dipartimento di Scienze Biomediche and Istituto C.N.R. Neuroscienze, Università di Padova, Viale G. Colombo n. 3, 35121 Padova, Italy.
    Tonello, Fiorella
    Zaccolo, Manuela
    Montecucco, Cesare
    cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin2008In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 376, no 2, p. 429-433Article in journal (Refereed)
    Abstract [en]

    The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.

  • 48.
    Das, Biswajit
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Mishra, Pradeep
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Pandey, Praveen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Sharma, Sushma
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Chabes, Andrei
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    dNTP concentrations do not increase in mammalian cells in response to DNA damage2022In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 34, no 12, p. 1895-1896Article in journal (Refereed)
  • 49. Daste, Frederic
    et al.
    Walrant, Astrid
    Holst, Mikkel R.
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Gadsby, Jonathan R.
    Mason, Julia
    Lee, Ji-Eun
    Brook, Daniel
    Mettlen, Marcel
    Larsson, Elin
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Lee, Steven F.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Gallop, Jennifer L.
    Control of actin polymerization via the coincidence of phosphoinositides and high membrane curvature2017In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 216, no 11, p. 3745-3765Article in journal (Refereed)
    Abstract [en]

    The conditional use of actin during clathrin-mediated endocytosis in mammalian cells suggests that the cell controls whether and how actin is used. Using a combination of biochemical reconstitution and mammalian cell culture, we elucidate a mechanism by which the coincidence of PI(4,5)P-2 and PI(3)P in a curved vesicle triggers actin polymerization. At clathrin-coated pits, PI(3) P is produced by the INPP4A hydrolysis of PI(3,4)P-2, and this is necessary for actin-driven endocytosis. Both Cdc42.guanosine triphosphate and SNX9 activate N-WASP-WIP-and Arp2/3-mediated actin nucleation. Membrane curvature, PI(4,5)P-2, and PI(3) P signals are needed for SNX9 assembly via its PX-BAR domain, whereas signaling through Cdc42 is activated by PI(4,5)P-2 alone. INPP4A activity is stimulated by high membrane curvature and synergizes with SNX9 BAR domain binding in a process we call curvature cascade amplification. We show that the SNX9-driven actin comets that arise on human disease-associated oculocerebrorenal syndrome of Lowe (OCRL) deficiencies are reduced by inhibiting PI(3) P production, suggesting PI(3) P kinase inhibitors as a therapeutic strategy in Lowe syndrome.

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  • 50.
    Debar, Louis
    et al.
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    Ishak, Layal
    Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
    Moretton, Amandine
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    Anoosheh, Saber
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morel, Frederic
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    Jenninger, Louise
    Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden.
    Garreau-Balandier, Isabelle
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    Vernet, Patrick
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    Hofer, Anders
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    van den Wildenberg, Siet
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France; Université Clermont Auvergne, CNRS, IRD, Université Jean Monnet Saint Etienne, LMV, Clermont-Ferrand, France.
    Farge, Geraldine
    Université Clermont Auvergne, CNRS, Laboratoire de Physique de Clermont, Clermont-Ferrand, France.
    NUDT6 and NUDT9, two mitochondrial members of the NUDIX family, have distinct hydrolysis activities2023In: Mitochondrion (Amsterdam. Print), ISSN 1567-7249, E-ISSN 1872-8278, Vol. 71, p. 93-103Article in journal (Refereed)
    Abstract [en]

    The 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and in the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mitochondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6- and NUDT9-deficiency and found that the depletion of either of the two proteins results in an increased activity of the respiratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD+(H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR.

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