TY - JOUR A1 - Szabó, Áron A1 - Papin, Christian A1 - Zorn, Daniela A1 - Ponien, Prishila A1 - Weber, Frank A1 - Raabe, Thomas A1 - Rouyer, François T1 - The CK2 Kinase Stabilizes CLOCK and Represses Its Activity in the Drosophila Circadian Oscillator JF - PLoS Biology N2 - Phosphorylation is a pivotal regulatory mechanism for protein stability and activity in circadian clocks regardless of their evolutionary origin. It determines the speed and strength of molecular oscillations by acting on transcriptional activators and their repressors, which form negative feedback loops. In Drosophila, the CK2 kinase phosphorylates and destabilizes the PERIOD (PER) and TIMELESS (TIM) proteins, which inhibit CLOCK (CLK) transcriptional activity. Here we show that CK2 also targets the CLK activator directly. Downregulating the activity of the catalytic alpha subunit of CK2 induces CLK degradation, even in the absence of PER and TIM. Unexpectedly, the regulatory beta subunit of the CK2 holoenzyme is not required for the regulation of CLK stability. In addition, downregulation of \(CK2\alpha\) activity decreases CLK phosphorylation and increases per and tim transcription. These results indicate that CK2 inhibits CLK degradation while reducing its activity. Since the CK1 kinase promotes CLK degradation, we suggest that CLK stability and transcriptional activity result from counteracting effects of CK1 and CK2. KW - negative feedback loop KW - PER-TIM complex KW - posttranslational regulation KW - transcription factor KW - in-vivo KW - behavioral rhythms KW - proteins period KW - beta-subunit KW - phosphorylation KW - gene KW - CT, circadian time KW - LD, light:dark KW - DD, constant darkness Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127234 SN - 1545-7885 VL - 11 IS - 8 ER - TY - JOUR A1 - Staab, Wieland A1 - Hottowitz, Ralf A1 - Sohns, Christian A1 - Sohns, Jan Martin A1 - Gilbert, Fabian A1 - Menke, Jan A1 - Niklas, Andree A1 - Lotz, Joachim T1 - Accelerometer and Gyroscope Based Gait Analysis Using Spectral Analysis of Patients with Osteoarthritis of the Knee JF - Journal of Physical Therapy Science N2 - [Purpose] A wide variety of accelerometer tools are used to estimate human movement, but there are no adequate data relating to gait symmetry parameters in the context of knee osteoarthritis. This study's purpose was to evaluate a 3D-kinematic system using body-mounted sensors (gyroscopes and accelerometers) on the trunk and limbs. This is the first study to use spectral analysis for data post processing. [Subjects] Twelve patients with unilateral knee osteoarthritis (OA) (10 male) and seven age-matched controls (6 male) were studied. [Methods] Measurements with 3-D accelerometers and gyroscopes were compared to video analysis with marker positions tracked by a six-camera optoelectronic system (VICON 460, Oxford Metrics). Data were recorded using the 3D-kinematic system. [Results] The results of both gait analysis systems were significantly correlated. Five parameters were significantly different between the knee OA and control groups. To overcome time spent in expensive post-processing routines, spectral analysis was performed for fast differentiation between normal gait and pathological gait signals using the 3D-kinematic system. [Conclusions] The 3D-kinematic system is objective, inexpensive, accurate and portable, and allows long-term recordings in clinical, sport as well as ergonomic or functional capacity evaluation (FCE) settings. For fast post-processing, spectral analysis of the recorded data is recommended. KW - gait KW - accelerometer KW - gyroscope KW - HIP osteoarthritis KW - kinematic analysis KW - human movement KW - in-vivo KW - artifact KW - systems KW - people Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115907 VL - 26 IS - 7 ER - TY - JOUR A1 - Schlereth, Katharina A1 - Heyl, Charlotte A1 - Krampitz, Anna-Maria A1 - Mernberger, Marco A1 - Finkernagel, Florian A1 - Scharfe, Maren A1 - Jarek, Michael A1 - Leich, Ellen A1 - Rosenwald, Andreas A1 - Stiewe, Thorsten T1 - Characterization of the p53 Cistrome - DNA Binding Cooperativity Dissects p53's Tumor Suppressor Functions JF - PLOS Genetics N2 - p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context-and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53's apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing. KW - cell-cycle arrest KW - gene expression KW - breast cancer KW - human genome KW - transcriptional repression KW - consensus DNA KW - in-vivo KW - apoptosis KW - network KW - damage Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-127579 SN - 1553-7404 VL - 9 IS - 8 ER - TY - JOUR A1 - Scheer, Sebastian A1 - Krempl, Christine A1 - Kallfass, Carsten A1 - Frey, Stefanie A1 - Jakob, Thilo A1 - Mouahid, Gabriel A1 - Mone, Helene A1 - Schmitt-Graeff, Anette A1 - Staeheli, Peter A1 - Lamers, Marinus C. T1 - S-mansoni Bolsters Anti-Viral Immunity in the Murine Respiratory Tract JF - PLOS ONE N2 - The human intestinal parasite Schistosoma mansoni causes a chronic disease, schistosomiasis or bilharzia. According to the current literature, the parasite induces vigorous immune responses that are controlled by Th2 helper cells at the expense of Th1 helper cells. The latter cell type is, however, indispensable for anti-viral immune responses. Remarkably, there is no reliable literature among 230 million patients worldwide describing defective anti-viral immune responses in the upper respiratory tract, for instance against influenza A virus or against respiratory syncitial virus (RSV). We therefore re-examined the immune response to a human isolate of S. mansoni and challenged mice in the chronic phase of schistosomiasis with influenza A virus, or with pneumonia virus of mice (PVM), a mouse virus to model RSV infections. We found that mice with chronic schistosomiasis had significant, systemic immune responses induced by Th1, Th2, and Th17 helper cells. High serum levels of TNF-alpha, IFN-gamma, IL-5, IL-13, IL-2, IL-17, and GM-CSF were found after mating and oviposition. The lungs of diseased mice showed low-grade inflammation, with goblet cell hyperplasia and excessive mucus secretion, which was alleviated by treatment with an anti-TNF-alpha agent (Etanercept). Mice with chronic schistosomiasis were to a relative, but significant extent protected from a secondary viral respiratory challenge. The protection correlated with the onset of oviposition and TNF-alpha-mediated goblet cell hyperplasia and mucus secretion, suggesting that these mechanisms are involved in enhanced immune protection to respiratory viruses during chronic murine schistosomiasis. Indeed, also in a model of allergic airway inflammation mice were protected from a viral respiratory challenge with PVM. KW - innate lymphoid cells KW - necrosis-factor-alpha KW - CD4(+) T-cells KW - helminth infection KW - pneumona virus KW - dendritic cells KW - TNF-alpha KW - in-vivo KW - cytokine responses Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114723 SN - 1932-6203 VL - 9 IS - 11 ER - TY - JOUR A1 - Panayotova-Dimitrova, Diana A1 - Feoktistova, Maria A1 - Ploesser, Michaela A1 - Kellert, Beate A1 - Hupe, Mike A1 - Horn, Sebastian A1 - Makarov, Roman A1 - Jensen, Federico A1 - Porubsky, Stefan A1 - Schmieder, Astrid A1 - Zenclussen, Ana Claudia A1 - Marx, Alexander A1 - Kerstan, Andreas A1 - Geserick, Peter A1 - He, You-Wen A1 - Leverkus, Martin T1 - cFLIP Regulates Skin Homeostasis and Protects against TNF-Induced Keratinocyte Apoptosis JF - Cell Reports N2 - FADD, caspase-8, and cFLIP regulate the outcome of cell death signaling. Mice that constitutively lack these molecules die at an early embryonic age, whereas tissue-specific constitutive deletion of FADD or caspase-8 results in inflammatory skin disease caused by increased necroptosis. The function of cFLIP in the skin in vivo is unknown. In contrast to tissue-specific caspase-8 knockout, we show that mice constitutively lacking cFLIP in the epidermis die around embryonic days 10 and 11. When cFLIP expression was abrogated in adult skin of cFLIP(fl/fl)-K14CreER(tam) mice, severe inflammation of the skin with concomitant caspase activation and apoptotic, but not necroptotic, cell death developed. Apoptosis was dependent of autocrine tumor necrosis factor production triggered by loss of cFLIP. In addition, epidermal cFLIP protein was lost in patients with severe drug reactions associated with epidermal apoptosis. Our data demonstrate the importance of cFLIP for the integrity of the epidermis and for silencing of spontaneous skin inflammation. KW - eczematous dermatitis KW - programmed necrosis KW - gene induction KW - in-vivo KW - activation KW - mediated apoptosis KW - c-flip KW - cell-death KW - Stevens-Johnson syndrome KW - toxic epidermal necrolysis Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122155 VL - 5 ER - TY - JOUR A1 - Nanguneri, Siddharth A1 - Flottmann, Benjamin A1 - Horstmann, Heinz A1 - Heilemann, Mike A1 - Kuner, Thomas T1 - Three-Dimensional, Tomographic Super-Resolution Fluorescence Imaging of Serially Sectioned Thick Samples JF - PLoS One N2 - Three-dimensional fluorescence imaging of thick tissue samples with near-molecular resolution remains a fundamental challenge in the life sciences. To tackle this, we developed tomoSTORM, an approach combining single-molecule localization-based super-resolution microscopy with array tomography of structurally intact brain tissue. Consecutive sections organized in a ribbon were serially imaged with a lateral resolution of 28 nm and an axial resolution of 40 nm in tissue volumes of up to 50 \(\mu\)mx50\(\mu\)mx2.5\(\mu\)m. Using targeted expression of membrane bound (m)GFP and immunohistochemistry at the calyx of Held, a model synapse for central glutamatergic neurotransmission, we delineated the course of the membrane and fine-structure of mitochondria. This method allows multiplexed super-resolution imaging in large tissue volumes with a resolution three orders of magnitude better than confocal microscopy. KW - architecture KW - rat calyx KW - in-vivo KW - microscopy KW - resolution KW - proteins KW - transmission KW - ultrastructure KW - reconstruction KW - localization Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134434 VL - 7 IS - 5 ER - TY - JOUR A1 - Maudet, Claire A1 - Sourisce, Adèle A1 - Dragin, Loïc A1 - Lahouassa, Hichem A1 - Rain, Jean-Christopher A1 - Bouaziz, Serge A1 - Ramirez, Bertha Cécilia A1 - Margottin-Goguet, Florence T1 - HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP JF - PLOS ONE N2 - The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered. KW - immunodeficiency-virus type-1 KW - MI-2/NURD complex KW - cell-cycle arrest KW - CUL4-DDB1 ubiquitin ligase KW - viral protein-R KW - NF-KAPPA-B KW - macrophage infection KW - enzyme APOBEC3G KW - in-vivo KW - transcription Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-128316 SN - 1932-6203 VL - 8 IS - 10 ER - TY - JOUR A1 - Kühlhorn, Franziska A1 - Rath, Matthias A1 - Schmoeckel, Katrin A1 - Cziupka, Katharina A1 - Nguyen, Huu Hung A1 - Hildebrandt, Petra A1 - Hünig, Thomas A1 - Sparwasser, Tim A1 - Huehn, Jochen A1 - Pötschke, Christian A1 - Bröker, Barbara M. T1 - \(Foxp3^+\) Regulatory T Cells Are Required for Recovery from Severe Sepsis JF - PLoS ONE N2 - The role of regulatory T cells (Tregs) in bacterial sepsis remains controversial because antibody-mediated depletion experiments gave conflicting results. We employed DEREG mice (DEpletion of REGulatory T cells) and a caecal ligation and puncture model to elucidate the role of \(CD4^+Foxp3^+\) Tregs in sepsis. In DEREG mice natural Tregs can be visualized easily and selectively depleted by diphtheria toxin because the animals express the diphtheria toxin receptor and enhanced green fluorescent protein as a fusion protein under the control of the foxp3 locus. We confirmed rapid Treg-activation and an increased ratio of Tregs to Teffs in sepsis. Nevertheless, 24 h after sepsis induction, Treg-depleted and control mice showed equally strong inflammation, immune cell immigration into the peritoneum and bacterial dissemination. During the first 36 h of disease survival was not influenced by Treg-depletion. Later, however, only Treg-competent animals recovered from the insult. We conclude that the suppressive capacity of Tregs is not sufficient to control overwhelming inflammation and early mortality, but is a prerequisite for the recovery from severe sepsis. KW - CD4(+)CD25(+) KW - toll-like receptors KW - TGF-BETA KW - mediated suppression KW - polymicrobial sepsis KW - improves survival KW - adoptive transfer KW - infected mice KW - in-vivo KW - CD4(+) Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130940 VL - 8 IS - 5 ER - TY - JOUR A1 - Hopp, Sarah A1 - Albert-Weissenberger, Christiane A1 - Mencl, Stine A1 - Bieber, Michael A1 - Schuhmann, Michael K. A1 - Stetter, Christian A1 - Nieswandt, Bernhard A1 - Schmidt, Peter M. A1 - Monoranu, Camelia-Maria A1 - Alafuzoff, Irina A1 - Marklund, Niklas A1 - Nolte, Marc W. A1 - Sirén, Anna-Leena A1 - Kleinschnitz, Christoph T1 - Targeting coagulation factor XII as a novel therapeutic option in brain trauma JF - Annals of Neurology N2 - Objective: Traumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism-based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury. Methods: We investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin-fused infestin-4 (rHA-Infestin-4) on trauma-induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury. Results: Our study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma-induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII-deficient mice fully restored injury-induced microvascular thrombus formation and brain damage. Interpretation: The robust protective effect of rHA-Infestin-4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. KW - Molecular-weight heparin KW - Thrombus formation KW - Cerebral-ischemia KW - in-vivo KW - Intravascular coagulation KW - Hemodynamic depression KW - Head-injury KW - Rats KW - Model KW - Mice Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188800 VL - 79 IS - 6 ER - TY - JOUR A1 - Hoffmann, Linda S A1 - Schmidt, Peter M A1 - Keim, Yvonne A1 - Hoffmann, Carsten A1 - Schmidt, Harald H H W A1 - Stasch, Johannes-Peter T1 - Fluorescence Dequenching Makes Haem-Free Soluble Guanylate Cyclase Detectable in Living Cells JF - PLOS ONE N2 - In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e. g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions. KW - spontaneously hypersensitive-rats KW - nitric-oxide KW - down-regulation KW - energy-transfer KW - cyclic-gmp KW - protein KW - activation KW - identification KW - in-vivo KW - no Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139631 VL - 6 IS - 8 ER -