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111.
Roberta Bianchi Alvaro Teijeira Steven T. Proulx Ailsa J. Christiansen Catharina D. Seidel Thomas Rülicke Taija M?kinen René H?gerling Cornelia Halin Michael Detmar 《PloS one》2015,10(4)
The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research. 相似文献
112.
Cornelia Blume Riccardo Reale Marie Held Timothy M. Millar Jane E. Collins Donna E. Davies Hywel Morgan Emily J. Swindle 《PloS one》2015,10(10)
The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms. 相似文献
113.
Cornelia Braicu Valentina Pileczki Laura Pop Roxana Cojocneanu Petric Sergiu Chira Eve Pointiere Patriciu Achimas-Cadariu Ioana Berindan-Neagoe 《PloS one》2015,10(4)
Triple-negative breast cancer (TNBC) is a highly aggressive phenotype that is resistant to standard therapy. Thus, the development of alternative therapeutic strategies for TNBC is essential. The purpose of our in vitro study was to evaluate the impact of p53 gene silencing in conjunction with the administration of a natural compound, epigallocatechingallate (EGCG). RT2Profiler PCR Array technology was used to evaluate the impact of dual treatment on the main genes involved in apoptosis in the Hs578T cell culture model of TNBC. Gene expression analysis revealed 28 genes were significantly altered (16 upregulated and 12 downregulated) in response to combined p53 siRNA and EGCG treatment. Further analysis revealed that p53 siRNA and EGCG dual therapy leads to the activation of pro-apoptotic genes and the inhibition of pro-survival genes, autophagy, and cell network formation. These results indicate that this dual therapy targets both the apoptotic and angiogenic pathways, which may improve treatment effectiveness for tumors resistant to conventional treatment. 相似文献
114.
Mao H Graziano JJ Chase TM Bentley CA Bazirgan OA Reddy NP Song BD Smider VV 《Nature biotechnology》2010,28(11):1195-1202
Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ~10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens. 相似文献
115.
High-speed, biplanar X-ray motion analysis, X-ray reconstruction of moving morphology (XROMM) and morphological studies have led to the identification of those traits which are considered to be crucial for the evolution of arboreal locomotion in chameleons. The loss of the extensive lateral undulation typical of reptiles needs to be compensated by high mobility in the shoulder girdle and a clear functional regionalization of the trunk. Large limb excursion angles provide a compliant gait and are made possible by a functional parasagittalization of fore- and hind limbs, at least temporarily. All these evolutionary novelties parallel very similar modifications in the evolution of the locomotor apparatus in therian mammals. We propose that the convergent “invention” of dynamic stability and a compliant gait seem to be responsible for the locomotor similarities between chameleons and mammals. 相似文献
116.
Gene duplication and divergence is widely considered to be a fundamental mechanism for generating evolutionary novelties. The Bone Morphogenetic Proteins (BMPs) are a diverse family of signalling molecules found in all metazoan genomes that have evolved by duplication and divergence from a small number of ancestral types. In the fruit fly Drosophila, there are three BMPs: Decapentaplegic (Dpp) and Glass bottom boat (Gbb), which are the orthologues of vertebrate BMP2/4 and BMP5/6/7/8, respectively, and Screw (Scw), which, at the sequence level, is equally divergent from Dpp and Gbb. It has recently been shown that Scw has arisen from a duplication of Gbb in the lineage leading to higher Diptera. We show that since this duplication event, Gbb has maintained the ancestral BMP5/6/7/8 functionality while Scw has rapidly diverged. The evolution of Scw was accompanied by duplication and divergence of a suite of extracellular regulators that continue to diverge together in the higher Diptera. In addition, Scw has become restricted in its receptor specificity: Gbb proteins can signal through the Type I receptors Thick veins (Tkv) and Saxophone (Sax), while Scw signals through Sax. Thus, in a relatively short span of evolutionary time, the duplication event that gave rise to Scw produced not only a novel ligand but also a novel signalling mode that is functionally distinct from the ancestral Gbb mode. Our results demonstrate the plasticity of the BMP pathway not only in evolving new family members and new functions but also new signalling modes by redeploying key regulators in the pathway. 相似文献
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119.
Alexander Weng Kristina Jenett-Siems Peter Schmieder Diana Bachran Christopher Bachran Cornelia Görick Mayank Thakur Hendrik Fuchs Matthias F. Melzig 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(7-8):713-718
Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy. 相似文献
120.
Carsten Schmidt Margret Schilli-Westermann Reinhard Klinger Cornelia Kirsch 《The protein journal》2010,29(2):127-135
Phosphoinositide 3-kinase γ is a multifunctional enzyme with lipid and protein kinase activities that also acts as a scaffold
protein in many diverse signalling processes. The enzyme contains five different domains, but their individual contributions
to membrane binding are not fully understood. Here, using in vitro liposome binding assays of individual domains and deletion
constructs of human phosphoinositide 3-kinase γ, we show that each domain is capable of binding anionic phospholipids to varying
degrees, depending on the charge of the anionic substrate. Moreover, with the exception of the C2-domain, deletion of any
single protein domain results in a complete loss of kinase activity toward both lipids and proteins. 相似文献