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Wedel, Steffen Alexander ; Hudak, Lukasz ; Seibel, Jens-Michael ; Makarević, Jasmina ; Jüngel, Eva ; Tsaur, Igor ; Waaga, Ana Maria da Silva Rosa ; Haferkamp, Axel ; Blaheta, Roman A.
Background: Single drug use has not achieved satisfactory results in the treatment of prostate cancer, despite application of increasingly widespread targeted therapeutics. In the present study, the combined impact of the mammalian target of rapamycin (mTOR)-inhibitor RAD001, the dual EGFr and VGEFr tyrosine kinase inhibitor AEE788 and the histone deacetylase (HDAC)-inhibitor valproic acid (VPA) on prostate cancer growth and adhesion in vitro was investigated. Methods: PC-3, DU-145 and LNCaP cells were treated with RAD001, AEE788 or VPA or with a RAD-AEE-VPA combination. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT-assay, flow cytometry and western blotting, respectively. Furthermore, tumor cell adhesion to vascular endothelium or to immobilized extracellular matrix proteins as well as migratory properties of the cells was evaluated, and integrin alpha and beta subtypes were analyzed. Finally, effects of drug treatment on cell signaling pathways were determined. Results: All drugs, separately applied, reduced tumor cell adhesion, migration and growth. A much stronger anti-cancer effect was evoked by the triple drug combination. Particularly, cdk1, 2 and 4 and cyclin B were reduced, whereas p27 was elevated. In addition, simultaneous application of RAD001, AEE788 and VPA altered the membranous, cytoplasmic and gene expression pattern of various integrin alpha and beta subtypes, reduced integrin-linked kinase (ILK) and deactivated focal adhesion kinase (FAK). Signaling analysis revealed that EGFr and the downstream target Akt, as well as p70S6k was distinctly modified in the presence of the drug combination. Conclusions: Simultaneous targeting of several key proteins in prostate cancer cells provides an advantage over targeting a single pathway. Since strong anti-tumor properties became evident with respect to cell growth and adhesion dynamics, the triple drug combination might provide progress in the treatment of advanced prostate cancer.
The influence of visual tasks on short and long-term memory for visual features was investigated using a change-detection paradigm. Subjects completed 2 tasks: (a) describing objects in natural images, reporting a specific property of each object when a crosshair appeared above it, and (b) viewing a modified version of each scene, and detecting which of the previously described objects had changed. When tested over short delays (seconds), no task effects were found. Over longer delays (minutes) we found the describing task influenced what types of changes were detected in a variety of explicit and incidental memory experiments. Furthermore, we found surprisingly high performance in the incidental memory experiment, suggesting that simple tasks are sufficient to instill long-lasting visual memories. Keywords: visual working memory, natural scenes, natural tasks, change detection
Visual selective attention and visual working memory (WM) share the same capacity-limited resources. We investigated whether and how participants can cope with a task in which these 2 mechanisms interfere. The task required participants to scan an array of 9 objects in order to select the target locations and to encode the items presented at these locations into WM (1 to 5 shapes). Determination of the target locations required either few attentional resources (“popout condition”) or an attention-demanding serial search (“non pop-out condition”). Participants were able to achieve high memory performance in all stimulation conditions but, in the non popout conditions, this came at the cost of additional processing time. Both empirical evidence and subjective reports suggest that participants invested the additional time in memorizing the locations of all target objects prior to the encoding of their shapes into WM. Thus, they seemed to be unable to interleave the steps of search with those of encoding. We propose that the memory for target locations substitutes for perceptual pop-out and thus may be the key component that allows for flexible coping with the common processing limitations of visual WM and attention. The findings have implications for understanding how we cope with real-life situations in which the demands on visual attention and WM occur simultaneously. Keywords: attention, working memory, interference, encoding strategies
Wernicke, Caroline M. ; Grunewald, Thomas G. P. ; Juenger, Hendrik ; Kuçi, Selim ; Kuçi, Zyrafete ; Köhl, Ulrike ; Mueller, Ingo ; Döring, Michaela ; Peters, Christina ; Lawitschka, Anita ; Kolb, Hans-Jochem ; Bader, Peter ; Burdach, Stefan ; Teichert- von Lüttichau, Irene
Severe acute graft versus host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation. Human mesenchymal stromal cells (MSCs) play an important role in endogenous tissue repair and possess strong immune-modulatory properties making them a promising tool for the treatment of steroid-refractory GvHD. To date, a few reports exist on the use of MSCs in treatment of GvHD in children indicating that children tend to respond better than adults, albeit with heterogeneous results. We here present a review of the literature and the clinical course of two instructive pediatric patients with acute steroid-refractory GvHD after haploidentical stem cell transplantation, which exemplify the beneficial effects of third-party transplanted MSCs in treatment of acute steroid-refractory GvHD. Moreover, we provide a meta-analysis of clinical studies addressing the outcome of patients with steroid-refractory GvHD and treatment with MSCs in adults and in children (n = 183; 122 adults, 61 children). Our meta-analysis demonstrates that the overall response-rate is high (73.8%) and confirms, for the first time, that children indeed respond better to treatment of GvHD with MSCs than adults (complete response 57.4% vs. 45.1%, respectively). These data emphasize the significance of this therapeutic approach especially in children and indicate that future prospective studies are needed to assess the reasons for the observed differential response-rates in pediatric and adult patients. Additional file 1: MSCs expansion and release criteria.his file contains a detailed description of the MSCs expansion and release criteria for Case A and Case B.
Rainger, Joe ; Beusekom, Ellen van ; Ramsay, Jacqueline K. ; McKie, Lisa ; Al-Gazali, Lihadh ; Pallotta, Rosanna ; Saponari, Anita ; Branney, Peter ; Fisher, Malcolm ; Morrison, Harris ; Bicknell, Louise ; Gautier, Philippe ; Perry, Paul ; Sokhi, Kishan ; Sexton, David ; Bardakjian, Tanya M. ; Schneider, Adele S. ; Elcioglu, Nursel ; Ozkinay, Ferda ; Koenig, Rainer ; Mégarbané, Andre ; Semerci, C. Nur ; Khan, Ayesha ; Zafar, Saemah ; Hennekam, Raoul C. ; Sousa, Sérgio B. ; Ramos, Lina ; Garavelli, Livia ; Furga, Andrea Superti ; Wischmeijer, Anita ; Jackson, Ian J. ; Gillessen-Kaesbach, Gabriele Gertrud ; Brunner, Han G. ; Wieczorek, Dagmar ; Bokhoven, Hans van ; FitzPatrick, David R.
Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ~10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.
Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis
(2009)
Kozjak-Pavlovic, Vera ; Dian-Lothrop, Elke A. ; Meinecke, Michael ; Kepp, Oliver ; Ross, Katharina ; Rajalingam, Krishnaraj ; Harsman, Anke ; Hauf, Eva ; Brinkmann, Volker ; Günther, Dirk ; Herrmann, Ines ; Hurwitz, Robert ; Rassow, Joachim ; Wagner, Richard ; Rudel, Thomas
The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (delta psi m). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of delta psi m. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce delta psi m loss and apoptosis, demonstrating that dissipation of delta psi m is a requirement for cell death caused by neisserial infection.
The intensive foraging of insectivorous birds and bats is well known to reduce the density of arboreal herbivorous arthropods but quantification of collateral leaf damage remains limited for temperate forest canopies. We conducted exclusion experiments with nets in the crowns of young and mature oaks, Quercus robur, in south and central Germany to investigate the extent to which aerial vertebrates reduce herbivory through predation. We repeatedly estimated leaf damage throughout the vegetation period. Exclusion of birds and bats led to a distinct increase in arthropod herbivory, emphasizing the prominent role of vertebrate predators in controlling arthropods. Leaf damage (e.g., number of holes) differed strongly between sites and was 59% higher in south Germany, where species richness of vertebrate predators and relative oak density were lower compared with our other study site in central Germany. The effects of bird and bat exclusion on herbivory were 19% greater on young than on mature trees in south Germany. Our results support previous studies that have demonstrated clear effects of insectivorous vertebrates on leaf damage through the control of herbivorous arthropods. Moreover, our comparative approach on quantification of leaf damage highlights the importance of local attributes such as tree age, forest composition and species richness of vertebrate predators for control of arthropod herbivory.
Martin-Subero, Jose Ignacio ; Ammerpohl, Ole ; Bibikova, Marina ; Wickham-Garcia, Eliza ; Agirre, Xabier ; Alvarez, Sara ; Brüggemann, Monika ; Bug, Stefanie ; Calasanz, Maria J. ; Deckert, Martina ; Dreyling, Martin ; Du, Ming Q. ; Dürig, Jan ; Dyer, Martin J. S. ; Fan, Jian-Bing ; Gesk, Stefan ; Hansmann, Martin-Leo ; Harder, Lana ; Hartmann, Sylvia ; Klapper, Wolfram ; Küppers, Ralf ; Montesinos-Rongen, Manuel ; Nagel, Inga ; Pott, Christiane ; Richter, Julia ; Román-Gómez, José ; Seifert, Marc ; Stein, Harald ; Suela, Javier ; Trümper, Lorenz ; Vater, Inga ; Prosper, Felipe ; Haferlach, Claudia ; Cruz Cigudosa, Juan ; Siebert, Reiner
Background: Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings: Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance: We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.
Knauer, Shirley ; Fetz, Verena ; Rabenstein, Jens ; Friedl, Sandra ; Hofmann, Bettina ; Sabiani, Samaneh ; Schröder, Elisabeth ; Kunst, Lena ; Proschak, Ewgenij ; Thines, Eckhard ; Kindler, Thomas ; Schneider, Gisbert ; Marschalek, Rolf ; Stauber, Roland ; Bier, Carolin
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.
Eklöf, Johan S. ; Heide, Tjisse van der ; Donadi, Serena ; Zee, Els M. van der ; O'Hara, Robert B. ; Eriksson, Britas Klemens
Recovery of an ecosystem following disturbance can be severely hampered or even shift altogether when a point disturbance exceeds a certain spatial threshold. Such scale-dependent dynamics may be caused by preemptive competition, but may also result from diminished self-facilitation due to weakened ecosystem engineering. Moreover, disturbance can facilitate colonization by engineering species that alter abiotic conditions in ways that exacerbate stress on the original species. Consequently, establishment of such counteracting engineers might reduce the spatial threshold for the disturbance, by effectively slowing recovery and increasing the risk for ecosystem shifts to alternative states. We tested these predictions in an intertidal mudflat characterized by a two-state mosaic of hummocks (humps exposed during low tide) dominated by the sediment-stabilizing seagrass Zostera noltii) and hollows (low-tide waterlogged depressions dominated by the bioturbating lugworm Arenicola marina). In contrast to expectations, seagrass recolonized both natural and experimental clearings via lateral expansion and seemed unaffected by both clearing size and lugworm addition. Near the end of the growth season, however, an additional disturbance (most likely waterfowl grazing and/or strong hydrodynamics) selectively impacted recolonizing seagrass in the largest (1 m2) clearings (regardless of lugworm addition), and in those medium (0.25 m2) clearings where lugworms had been added nearly five months earlier. Further analyses showed that the risk for the disturbance increased with hollow size, with a threshold of 0.24 m2. Hollows of that size were caused by seagrass removal alone in the largest clearings, and by a weaker seagrass removal effect exacerbated by lugworm bioturbation in the medium clearings. Consequently, a sufficiently large disturbance increased the vulnerability of recolonizing seagrass to additional disturbance by weakening seagrass engineering effects (sediment stabilization). Meanwhile, the counteracting ecosystem engineering (lugworm bioturbation) reduced that threshold size. Therefore, scale-dependent interactions between habitat-mediated facilitation, competition and disturbance seem to maintain the spatial two-state mosaic in this ecosystem.