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121.
122.
Dag Heinemann Markus Schomaker Stefan Kalies Maximilian Schieck Regina Carlson Hugo Murua Escobar Tammo Ripken Heiko Meyer Alexander Heisterkamp 《PloS one》2013,8(3)
Laser based transfection methods have proven to be an efficient and gentle alternative to established molecule delivery methods like lipofection or electroporation. Among the laser based methods, gold nanoparticle mediated laser transfection bears the major advantage of high throughput and easy usability. This approach uses plasmon resonances on gold nanoparticles unspecifically attached to the cell membrane to evoke transient and spatially defined cell membrane permeabilization. In this study, we explore the parameter regime for gold nanoparticle mediated laser transfection for the delivery of molecules into cell lines and prove its suitability for siRNA mediated gene knock down. The developed setup allows easy usage and safe laser operation in a normal lab environment. We applied a 532 nm Nd:YAG microchip laser emitting 850 ps pulses at a repetition rate of 20.25 kHz. Scanning velocities of the laser spot over the sample of up to 200 mm/s were tested without a decline in perforation efficiency. This velocity leads to a process speed of ∼8 s per well of a 96 well plate. The optimal particle density was determined to be ∼6 particles per cell using environmental scanning electron microscopy. Applying the optimized parameters transfection efficiencies of 88% were achieved in canine pleomorphic adenoma ZMTH3 cells using a fluorescent labeled siRNA while maintaining a high cell viability of >90%. Gene knock down of d2-EGFP was demonstrated and validated by fluorescence repression and western blot analysis. On basis of our findings and established mathematical models we suppose a mixed transfection mechanism consisting of thermal and multiphoton near field effects. Our findings emphasize that gold nanoparticle mediated laser transfection provides an excellent tool for molecular delivery for both, high throughput purposes and the transfection of sensitive cells types. 相似文献
123.
P. Sivaramakrishna Rachakonda Andrea S. Bauer Huaping Xie Daniele Campa Cosmeri Rizzato Federico Canzian Stefania Beghelli William Greenhalf Eithne Costello Michaela Schanne Anette Heller Aldo Scarpa John P. Neoptolemos Jens Werner Markus Büchler J?rg D. Hoheisel Kari Hemminki Nathalia Giese Rajiv Kumar 《PloS one》2013,8(4)
KRAS mutations are major factors involved in initiation and maintenance of pancreatic tumors. The impact of different mutations on patient survival has not been clearly defined. We screened tumors from 171 pancreatic cancer patients for mutations in KRAS and CDKN2A genes. Mutations in KRAS were detected in 134 tumors, with 131 in codon 12 and only 3 in codon 61. The GGT>GAT (G12D) was the most frequent mutation and was present in 60% (80/134). Deletions and mutations in CDKN2A were detected in 43 tumors. Analysis showed that KRAS mutations were associated with reduced patient survival in both malignant exocrine and ductal adenocarcinomas (PDAC). Patients with PDACs that had KRAS mutations showed a median survival of 17 months compared to 30 months for those without mutations (log-rank P = 0.07) with a multivariate hazard ratio (HR) of 2.19 (95%CI 1.09–4.42). The patients with G12D mutation showed a median survival of 16 months (log-rank-test P = 0.03) and an associated multivariate HR 2.42 (95%CI 1.14–2.67). Although, the association of survival in PDAC patients with CDKN2A aberrations in tumors was not statistically significant, the sub-group of patients with concomitant KRAS mutations and CDKN2A alterations in tumors were associated with a median survival of 13.5 months compared to 22 months without mutation (log-rank-test P = 0.02) and a corresponding HR of 3.07 (95%CI 1.33–7.10). Our results are indicative of an association between mutational status and survival in PDAC patients, which if confirmed in subsequent studies can have potential clinical application. 相似文献
124.
Julian Zimmermann Marius Krauthausen Markus J. Hofer Michael T. Heneka Iain L. Campbell Marcus Müller 《PloS one》2013,8(2)
Interleukin-17A (IL-17A) is a key cytokine modulating the course of inflammatory diseases. Whereas effector functions of IL-17A like induction of antimicrobial peptides and leukocyte infiltration could clearly be demonstrated for peripheral organs, CNS specific effects are not well defined and appear controversial. To further clarify the functional significance of IL-17A in the CNS, we generated a transgenic mouse line with astrocyte-restricted expression of the IL-17A gene. GFAP/IL-17A transgenic mice develop normally and do not show any signs of neurological dysfunction. However, histological characterization revealed astrocytosis and activation of microglia. Demyelination, neurodegeneration or prominent tissue damage was not observed but a vascular pathology mimicking microangiopathic features was evident. Histological and flow cytometric analysis demonstrated the absence of parenchymal infiltration of immune cells into the CNS of GFAP/IL-17A transgenic mice. In GFAP/IL-17A mice, LPS-induced endotoxemia led to a more pronounced microglial activation with expansion of a distinct CD45high/CD11b+ population and increased induction of proinflammatory cytokines compared with controls. Our data argues against a direct role of IL-17A in mediating tissue damage during neuroinflammation. More likely IL-17A acts as a modulating factor in the network of induced cytokines. This novel mouse model will be a very useful tool to further characterize the role of IL-17A in neuroinflammatory disease models. 相似文献
125.
126.
Vera Jankowski Markus T?lle Thi Nguyet Anh Tran Markus van der Giet Mirjam Schuchardt Kerstin Lehmann Doreen Janke Burkhard Flick Alberto Arduan Ortiz Ni?o Maria Dolores Sanchez Martin Tepel Walter Zidek Joachim Jankowski 《PloS one》2013,8(7)
The secretion of angiogenic factors by vascular endothelial cells is one of the key mechanisms of angiogenesis. Here we report on the isolation of a new potent angiogenic factor, diuridine tetraphosphate (Up4U) from the secretome of human endothelial cells. The angiogenic effect of the endothelial secretome was partially reduced after incubation with alkaline phosphatase and abolished in the presence of suramin. In one fraction, purified to homogeneity by reversed phase and affinity chromatography, Up4U was identified by MALDI-LIFT-fragment-mass-spectrometry, enzymatic cleavage analysis and retention-time comparison. Beside a strong angiogenic effect on the yolk sac membrane and the developing rat embryo itself, Up4U increased the proliferation rate of endothelial cells and, in the presence of PDGF, of vascular smooth muscle cells. Up4U stimulated the migration rate of endothelial cells via P2Y2-receptors, increased the ability of endothelial cells to form capillary-like tubes and acts as a potent inducer of sprouting angiogenesis originating from gel-embedded EC spheroids. Endothelial cells released Up4U after stimulation with shear stress. Mean total plasma Up4U concentrations of healthy subjects (N = 6) were sufficient to induce angiogenic and proliferative effects (1.34±0.26 nmol L-1). In conclusion, Up4U is a novel strong human endothelium-derived angiogenic factor. 相似文献
127.
Leiz M. C. Véras Vanessa R. R. Cunha Filipe C. D. A. Lima Maria A. Guimar?es Marianne M. Vieira Yuri D. M. Campelo Vanessa Y. Sakai David F. Lima Paulo S. Carvalho Jr Javier A. Ellena Paulo R. P. Silva Luciene C. Vasconcelos Markus Godejohann Helena M. Petrilli Vera R. L. Constantino Yvonne P. Mascarenhas José Roberto de Souza de Almeida Leite 《PloS one》2013,8(6)
This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), 1H and 13C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution. 相似文献
128.
Markus Burgmer Maimu Alissa Rehbein Marco Wrenger Judith Kandil Gereon Heuft Christian Steinberg Bettina Pfleiderer Markus Jungh?fer 《PloS one》2013,8(8)
Background
In chronic PTSD, a preattentive neural alarm system responds rapidly to emotional information, leading to increased prefrontal cortex (PFC) activation at early processing stages (<100 ms). Enhanced PFC responses are followed by a reduction in occipito-temporal activity during later processing stages. However, it remains unknown if this neuronal pattern is a result of a long lasting mental disorder or if it represents changes in brain function as direct consequences of severe trauma.Methodology
The present study investigates early fear network activity in acutely traumatized patients with PTSD. It focuses on the question whether dysfunctions previously observed in chronic PTSD patients are already present shortly after trauma exposure. We recorded neuromagnetic activity towards emotional pictures in seven acutely traumatized PTSD patients between one and seven weeks after trauma exposure and compared brain responses to a balanced healthy control sample. Inverse modelling served for mapping sources of differential activation in the brain.Principal Findings
Compared to the control group, acutely traumatized PTSD patients showed an enhanced PFC response to high-arousing pictures between 60 to 80 ms. This rapid prefrontal hypervigilance towards arousing pictorial stimuli was sustained during 120–300 ms, where it was accompanied by a reduced affective modulation of occipito-temporal neural processing.Conclusions
Our findings indicate that the hypervigilance-avoidance pattern seen in chronic PTSD is not necessarily a product of an endured mental disorder, but arises as an almost immediate result of severe traumatisation. Thus, traumatic experiences can influence emotion processing strongly, leading to long-lasting changes in trauma network activation and expediting a chronic manifestation of maladaptive cognitive and behavioral symptoms. 相似文献129.
130.
Christian Ehrnthaller Markus Huber-Lang Per Nilsson Ronny Bindl Simon Redeker Stefan Recknagel Anna Rapp Tom Mollnes Michael Amling Florian Gebhard Anita Ignatius 《PloS one》2013,8(11)
There is increasing evidence that complement may play a role in bone development. Our previous studies demonstrated that the key complement receptor C5aR was strongly expressed in the fracture callus not only by immune cells but also by bone cells and chondroblasts, indicating a function in bone repair. To further elucidate the role of complement in bone healing, this study investigated fracture healing in mice in the absence of the key complement molecules C3 and C5. C3-/- and C5-/- as well as the corresponding wildtype mice received a standardized femur osteotomy, which was stabilized using an external fixator. Fracture healing was investigated after 7 and 21 days using histological, micro-computed tomography and biomechanical measurements. In the early phase of fracture healing, reduced callus area (C3-/-: -25%, p=0.02; C5-/-: -20% p=0.052) and newly formed bone (C3-/-: -38%, p=0.01; C5-/-: -52%, p=0.009) was found in both C3- and C5-deficient mice. After 21 days, healing was successful in the absence of C3, whereas in C5-deficient mice fracture repair was significantly reduced, which was confirmed by a reduced bending stiffness (-45%; p=0.029) and a smaller callus volume (-17%; p=0.039). We further demonstrated that C5a was activated in C3-/- mice, suggesting cleavage via extrinsic pathways. Our results suggest that the activation of the terminal complement cascade in particular may be crucial for successful fracture healing. 相似文献