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Maronde, Erik ; Schilling, Arndt ; Seitz, Sebastian ; Schinke, Thorsten ; Schmutz, Isabelle ; Horst, Gijsbertus van der ; Amling, Michael ; Albrecht, Urs
Background: Clock genes and their protein products regulate circadian rhythms in mammals but have also been implicated in various physiological processes, including bone formation. Osteoblasts build new mineralized bone whereas osteoclasts degrade it thereby balancing bone formation. To evaluate the contribution of clock components in this process, we investigated mice mutant in clock genes for a bone volume phenotype. Methodology/Principal Findings: We found that Per2Brdm1 mutant mice as well as mice lacking Cry2-/- displayed significantly increased bone volume at 12 weeks of age, when bone turnover is high. Per2Brdm1 mutant mice showed alterations in parameters specific for osteoblasts whereas mice lacking Cry2-/- displayed changes in osteoclast specific parameters. Interestingly, inactivation of both Per2 and Cry2 genes leads to normal bone volume as observed in wild type animals. Importantly, osteoclast parameters affected due to the lack of Cry2, remained at the level seen in the Cry2-/- mutants despite the simultaneous inactivation of Per2. Conclusions/Significance: This indicates that Cry2 and Per2 affect distinct pathways in the regulation of bone volume with Cry2 influencing mostly the osteoclastic cellular component of bone and Per2 acting on osteoblast parameters.
Haarmann, Axel ; Deiß, Annika ; Prochaska, Jürgen ; Förch, Christian ; Weksler, Babette ; Romero, Ignacio ; Couraud, Pierre-Olivier ; Stoll, Guido ; Rieckmann, Peter ; Buttmann, Mathias
Background: An inducible release of soluble junctional adhesion molecule-A (sJAM-A) under pro-inflammatory conditions was described in cultured non-CNS endothelial cells (EC) and increased sJAM-A serum levels were found to indicate inflammation in non-CNS vascular beds. Here we studied the regulation of JAM-A expression in cultured brain EC and evaluated sJAM-A as a serum biomarker of blood-brain barrier (BBB) function. Methodology/Principal Findings: As previously reported in non-CNS EC types, pro-inflammatory stimulation of primary or immortalized (hCMEC/D3) human brain microvascular EC (HBMEC) induced a redistribution of cell-bound JAM-A on the cell surface away from tight junctions, along with a dissociation from the cytoskeleton. This was paralleled by reduced immunocytochemical staining of occludin and zonula occludens-1 as well as by increased paracellular permeability for dextran 3000. Both a self-developed ELISA test and Western blot analysis detected a constitutive sJAM-A release by HBMEC into culture supernatants, which importantly was unaffected by pro-inflammatory or hypoxia/reoxygenation challenge. Accordingly, serum levels of sJAM-A were unaltered in 14 patients with clinically active multiple sclerosis compared to 45 stable patients and remained unchanged in 13 patients with acute ischemic non-small vessel stroke over time. Conclusion: Soluble JAM-A was not suited as a biomarker of BBB breakdown in our hands. The unexpected non-inducibility of sJAM-A release at the human BBB might contribute to a particular resistance of brain EC to inflammatory stimuli, protecting the CNS compartment.
Respiratory chain complexes in dynamic mitochondria display a patchy distribution in life cells
(2010)
Muster, Britta ; Kohl, Wladislaw ; Wittig, Ilka ; Strecker, Valentina ; Joos, Friederike ; Haase, Winfried ; Bereiter-Hahn, Jürgen ; Busch, Karin
Background: Mitochondria, the main suppliers of cellular energy, are dynamic organelles that fuse and divide frequently. Constraining these processes impairs mitochondrial is closely linked to certain neurodegenerative diseases. It is proposed that functional mitochondrial dynamics allows the exchange of compounds thereby providing a rescue mechanism. Methodology/Principal Findings: The question discussed in this paper is whether fusion and fission of mitochondria in different cell lines result in re-localization of respiratory chain (RC) complexes and of the ATP synthase. This was addressed by fusing cells containing mitochondria with respiratory complexes labelled with different fluorescent proteins and resolving their time dependent re-localization in living cells. We found a complete reshuffling of RC complexes throughout the entire chondriome in single HeLa cells within 2–3 h by organelle fusion and fission. Polykaryons of fused cells completely re-mixed their RC complexes in 10–24 h in a progressive way. In contrast to the recently described homogeneous mixing of matrix-targeted proteins or outer membrane proteins, the distribution of RC complexes and ATP synthase in fused hybrid mitochondria, however, was not homogeneous but patterned. Thus, complete equilibration of respiratory chain complexes as integral inner mitochondrial membrane complexes is a slow process compared with matrix proteins probably limited by complete fusion. In co-expressing cells, complex II is more homogenously distributed than complex I and V, resp. Indeed, this result argues for higher mobility and less integration in supercomplexes. Conclusion/Significance: Our results clearly demonstrate that mitochondrial fusion and fission dynamics favours the re-mixing of all RC complexes within the chondriome. This permanent mixing avoids a static situation with a fixed composition of RC complexes per mitochondrion.
Grosskinsky, Sonja ; Schott, Melanie ; Brenner, Christiane ; Cutler, Sally J. ; Kraiczy, Peter ; Zipfel, Peter F. ; Simon, Markus M. ; Wallich, Reinhard
Borrelia recurrentis, the etiologic agent of louse-borne relapsing fever in humans, has evolved strategies, including antigenic variation, to evade immune defence, thereby causing severe diseases with high mortality rates. Here we identify for the first time a multifunctional surface lipoprotein of B. recurrentis, termed HcpA, and demonstrate that it binds human complement regulators, Factor H, CFHR-1, and simultaneously, the host protease plasminogen. Cell surface bound factor H was found to retain its activity and to confer resistance to complement attack. Moreover, ectopic expression of HcpA in a B. burgdorferi B313 strain, deficient in Factor H binding proteins, protected the transformed spirochetes from complement-mediated killing. Furthermore, HcpA-bound plasminogen/plasmin endows B. recurrentis with the potential to resist opsonization and to degrade extracellular matrix components. Together, the present study underscores the high virulence potential of B. recurrentis. The elucidation of the molecular basis underlying the versatile strategies of B. recurrentis to escape innate immunity and to persist in human tissues, including the brain, may help to understand the pathological processes underlying louse-borne relapsing fever.
Scheckhuber, Christian Quintus ; Houthoofd, Koen ; Weil, Andrea C. ; Werner, Alexandra ; De Vreese, Annemie ; Vanfleteren, Jacques R. ; Osiewacz, Heinz D.
The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms.
This paper discusses the sustainability impact (contribution to sustainability, reduction of adverse environmental impacts) of online second-hand trading. A survey of eBay users shows that a relationship between the trading of used goods and the protection of natural resources is hardly realized. Secondly, the environmental motivation and the willingness to act in a sustainable manner differ widely between groups of consumers. Given these results from a user perspective, the paper tries to find some objective hints of online second-hand trading’s environmental impact. The greenhouse gas emissions resulting from the energy used for the trading transactions seem to be considerably lower than the emissions due to the (avoided) production of new goods. The paper concludes with a set of recommendations for second-hand trade and consumer policy. Information about the sustainability benefits of purchasing second-hand goods should be included in general consumer information, and arguments for changes in behavior should be targeted to different groups of consumers. Keywords: online marketplaces; online auctions; consumer; electronic commerce; used products; second-hand market; sustainable consumption
O'Dwyer, Laurence ; Lamberton, Franck ; Bokde, Arun L. W. ; Ewers, Michael ; Faluyi, Yetunde O. ; Tanner, Colby ; Mazoyer, Bernard M. ; O'Neill, Des ; Bartley, Máiréad ; Collins, D. Rónán ; Coughlan, Tara ; Prvulovic, David ; Hampel, Harald
The study of multiple indices of diffusion, including axial (DA), radial (DR) and mean diffusion (MD), as well as fractional anisotropy (FA), enables WM damage in Alzheimer's disease (AD) to be assessed in detail. Here, tract-based spatial statistics (TBSS) were performed on scans of 40 healthy elders, 19 non-amnestic MCI (MCIna) subjects, 14 amnestic MCI (MCIa) subjects and 9 AD patients. Significantly higher DA was found in MCIna subjects compared to healthy elders in the right posterior cingulum/precuneus. Significantly higher DA was also found in MCIa subjects compared to healthy elders in the left prefrontal cortex, particularly in the forceps minor and uncinate fasciculus. In the MCIa versus MCIna comparison, significantly higher DA was found in large areas of the left prefrontal cortex. For AD patients, the overlap of FA and DR changes and the overlap of FA and MD changes were seen in temporal, parietal and frontal lobes, as well as the corpus callosum and fornix. Analysis of differences between the AD versus MCIna, and AD versus MCIa contrasts, highlighted regions that are increasingly compromised in more severe disease stages. Microstructural damage independent of gross tissue loss was widespread in later disease stages. Our findings suggest a scheme where WM damage begins in the core memory network of the temporal lobe, cingulum and prefrontal regions, and spreads beyond these regions in later stages. DA and MD indices were most sensitive at detecting early changes in MCIa.
Krenn, Brigitte M. ; Egorov, Andrej ; Romanovskaya-Romanko, Ekaterina ; Wolschek, Markus ; Nakowitsch, Sabine ; Ruthsatz, Tanja ; Kiefmann, Bettina ; Morokutti, Alexander ; Humer, Johannes ; Geiler, Janina ; Cinatl, Jindrich ; Michaelis, Martin ; Wressnigg, Nina ; Sturlan, Sanda ; Ferko, Boris ; Batishchev, Oleg V. ; Indenbom, Andrey V. ; Zhu, Rong ; Kastner, Markus ; Hinterdorfer, Peter ; Kiselev, Oleg A. ; Muster, Thomas ; Romanova, Julia
Background: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. Methodology/Principal Findings: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH <= 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K -> I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K -> I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. Conclusion/Significance: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.
Maurer, Gabriele D. ; Brucker, Daniel P. ; Bähr, Roy Oliver ; Harter, Patrick Nikolaus ; Hattingen, Elke ; Walenta, Stefan ; Müller-Klieser, Wolfgang ; Steinbach, Joachim Peter ; Rieger, Johannes
Background: Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells. Methods: To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model. Results: The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1alpha (HIF-1alpha) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival. Conclusion: In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.
Rickerts, Volker ; Khot, Prasanna D. ; Myerson, David ; Ko, Daisy L. ; Lambrecht, Evelyn ; Fredricks, David N.
Background: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. Methods: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broadrange fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. Results: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. Conclusions: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.
Background: The apoptosis-inducing serine protease granzyme B (GrB) is an important factor contributing to lysis of target cells by cytotoxic lymphocytes. Expression of enzymatically active GrB in recombinant form is a prerequisite for functional analysis and application of GrB for therapeutic purposes. Methods and Findings: We investigated the influence of bacterial maltose-binding protein (MBP) fused to GrB via a synthetic furin recognition motif on the expression of the MBP fusion protein also containing an N-terminal alpha-factor signal peptide in the yeast Pichia pastoris. MBP markedly enhanced the amount of GrB secreted into culture supernatant, which was not the case when GrB was fused to GST. MBP-GrB fusion protein was cleaved during secretion by an endogenous furin-like proteolytic activity in vivo, liberating enzymatically active GrB without the need of subsequent in vitro processing. Similar results were obtained upon expression of a recombinant fragment of the ErbB2/HER2 receptor protein or GST as MBP fusions. Conclusions: Our results demonstrate that combination of MBP as a solubility enhancer with specific in vivo cleavage augments secretion of processed and functionally active proteins from yeast. This strategy may be generally applicable to improve folding and increase yields of recombinant proteins.
Background: Fragmented distribution ranges of species with little active dispersal capacity raise the question about their place of origin and the processes and timing of either range fragmentation or dispersal. The peculiar distribution of the land snail Tudorella sulcata s. str. in Southern France, Sardinia and Algeria is such a challenging case. Methodology: Statistical phylogeographic analyses with mitochondrial COI and nuclear hsp70 haplotypes were used to answer the questions of the species' origin, sequence and timing of dispersal. The origin of the species was on Sardinia. Starting from there, a first expansion to Algeria and then to France took place. Abiotic and zoochorous dispersal could be excluded by considering the species' life style, leaving only anthropogenic translocation as parsimonious explanation. The geographic expansion could be dated to approximately 8,000 years before present with a 95% confidence interval of 10,000 to 3,000 years before present. Conclusions: This period coincides with the Neolithic expansion in the Western Mediterranean, suggesting a role of these settlers as vectors. Our findings thus propose that non-domesticated animals and plants may give hints on the direction and timing of early human expansion routes.
Leuner, Kristina ; Kraus, Margarethe ; Wölfle, Ute ; Beschmann, Heike A. ; Harteneck, Christian ; Boehncke, Wolf-Henning ; Schempp, Christoph Mathis ; Müller, Walter E.
Psoriasis is a characteristic inflammatory and scaly skin condition with typical histopathological features including increased proliferation and hampered differentiation of keratinocytes. The activation of innate and adaptive inflammatory cellular immune responses is considered to be the main trigger factor of the epidermal changes in psoriatic skin. However, the molecular players that are involved in enhanced proliferation and impaired differentiation of psoriatic keratinocytes are only partly understood. One important factor that regulates differentiation on the cellular level is Ca2+. In normal epidermis, a Ca2+ gradient exists that is disturbed in psoriatic plaques, favoring impaired keratinocyte proliferation. Several TRPC channels such as TRPC1, TRPC4, or TRPC6 are key proteins in the regulation of high [Ca2+]ex induced differentiation. Here, we investigated if TRPC channel function is impaired in psoriasis using calcium imaging, RT-PCR, western blot analysis and immunohistochemical staining of skin biopsies. We demonstrated substantial defects in Ca2+ influx in psoriatic keratinocytes in response to high extracellular Ca2+ levels, associated with a downregulation of all TRPC channels investigated, including TRPC6 channels. As TRPC6 channel activation can partially overcome this Ca2+ entry defect, specific TRPC channel activators may be potential new drug candidates for the topical treatment of psoriasis.
Michaelis, Martin ; Geiler, Janina ; Naczk, Patrizia ; Sithisarn, Patchima ; Leutz, Anke ; Doerr, Hans Wilhelm ; Cinatl, Jindrich
Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFKappaB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.
Background: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFKappaB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). Methodology/Principal Findings: Overexpression of a dominant negative AMPKalpha2 mutant in tumor necrosis factor-alpha-stimulated human endothelial cells resulted in increased NFKappaB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKalpha2-/- mice the interleukin (IL)-1beta induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFKappaB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKalpha2-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IKappaB and p65, indicating a link between AMPK and the IKappaB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKalpha2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKalpha2+/+ versus AMPKalpha2-/- mice. Conclusions: These results demonstrate that the IKK is a direct substrate of AMPKalpha2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFKappaB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.
Steinert, Gunnar ; Oancea, Claudia ; Roos, Jessica ; Hagemeyer, Heike ; Maier, Thorsten Jürgen ; Ruthardt, Martin ; Puccetti, Elena
Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARalpha, PLZF/RARalpha, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARalpha and PLZF/RARalpha or AML-1/ETO activate Wnt signaling by upregulating gamma-catenin and beta-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARalpha-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both beta-catenin and gamma-catenin in X-RARalpha-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARalpha-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARalpha, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.
Background: Decoding of frequency-modulated (FM) sounds is essential for phoneme identification. This study investigates selectivity to FM direction in the human auditory system. Methodology/Principal Findings: Magnetoencephalography was recorded in 10 adults during a two-tone adaptation paradigm with a 200-ms interstimulus-interval. Stimuli were pairs of either same or different frequency modulation direction. To control that FM repetition effects cannot be accounted for by their on- and offset properties, we additionally assessed responses to pairs of unmodulated tones with either same or different frequency composition. For the FM sweeps, N1m event-related magnetic field components were found at 103 and 130 ms after onset of the first (S1) and second stimulus (S2), respectively. This was followed by a sustained component starting at about 200 ms after S2. The sustained response was significantly stronger for stimulation with the same compared to different FM direction. This effect was not observed for the non-modulated control stimuli. Conclusions/Significance: Low-level processing of FM sounds was characterized by repetition enhancement to stimulus pairs with same versus different FM directions. This effect was FM-specific; it did not occur for unmodulated tones. The present findings may reflect specific interactions between frequency separation and temporal distance in the processing of consecutive FM sweeps.
Huszthy, Peter C. ; Giroglou, Tsanan ; Tsinkalovsky, Oleg ; Euskirchen, Philipp ; Skaftnesmo, Kai Ove ; Bjerkvig, Rolf ; Laer, Dorothee von ; Miletic, Hrvoje
Background: Glioblastoma is the most frequent and most malignant primary brain tumor with a poor prognosis. The translation of therapeutic strategies for glioblastoma from the experimental phase into the clinic has been limited by insufficient animal models, which lack important features of human tumors. Lentiviral gene therapy is an attractive therapeutic option for human glioblastoma, which we validated in a clinically relevant animal model. Methodology/Principal Findings: We used a rodent xenograft model that recapitulates the invasive and angiogenic features of human glioblastoma to analyze the transduction pattern and therapeutic efficacy of lentiviral pseudotyped vectors. Both, lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors very efficiently transduced human glioblastoma cells in vitro and in vivo. In contrast, pseudotyped gammaretroviral vectors, similar to those evaluated for clinical therapy of glioblastoma, showed inefficient gene transfer in vitro and in vivo. Both pseudotyped lentiviral vectors transduced cancer stem-like cells characterized by their CD133-, nestin- and SOX2-expression, the ability to form spheroids in neural stem cell medium and to express astrocytic and neuronal differentiation markers under serum conditions. In a therapeutic approach using the suicide gene herpes simplex virus thymidine kinase (HSV-1-tk) fused to eGFP, both lentiviral vectors mediated a complete remission of solid tumors as seen on MRI resulting in a highly significant survival benefit (p<0.001) compared to control groups. In all recurrent tumors, surviving eGFP-positive tumor cells were found, advocating prodrug application for several cycles to even enhance and prolong the therapeutic effect. Conclusions/Significance: In conclusion, lentiviral pseudotyped vectors are promising candidates for gene therapy of glioma in patients. The inefficient gene delivery by gammaretroviral vectors is in line with the results obtained in clinical therapy for GBM and thus confirms the high reproducibility of the invasive glioma animal model for translational research.
Köhler, Barbara ; Anguissola, Sergio ; Concannon, Caoimhin G. ; Rehm, Markus ; Kögel, Donat ; Prehn, Jochen H. M.
Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases. Methodology/Principal Findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations. Conclusions/Significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.
Madsen, Karina M. ; Udatha, Gupta D. B. R. K. ; Semba, Saori ; Otero, Jose M. ; Kötter, Peter ; Nielsen, Jens ; Ebizuka, Yutaka ; Kushiro, Tetsuo ; Panagiotou, Gianni
Background: Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. Methodology/Principal Findings: Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a beta-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. Conclusions: The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and beta-amyrin production. In the case of beta-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers.