Plant cell packs: a scalable platform for recombinant protein production and metabolic engineering

Industrial plant biotechnology applications include the production of sustainable fuels, complex metabolites and recombinant proteins, but process development can be impaired by a lack of reliable and scalable screening methods. Here, we describe a rapid and versatile expression system which involves the infusion of Agrobacterium tumefaciens into three‐dimensional, porous plant cell aggregates deprived of cultivation medium, which we have termed plant cell packs (PCPs). This approach is compatible with different plant species such as Nicotiana tabacum BY2, Nicotiana benthamiana or Daucus carota and 10‐times more effective than transient expression in liquid plant cell culture. We found that the expression of several proteins was similar in PCPs and intact plants, for example, 47 and 55 mg/kg for antibody 2G12 expressed in BY2 PCPs and N. tabacum plants respectively. Additionally, the expression of specific enzymes can either increase the content of natural plant metabolites or be used to synthesize novel small molecules in the PCPs. The PCP method is currently scalable from a microtiter plate format suitable for high‐throughput screening to 150‐mL columns suitable for initial product preparation. It therefore combined the speed of transient expression in plants with the throughput of microbial screening systems. Plant cell packs therefore provide a convenient new platform for synthetic biology approaches, metabolic engineering and conventional recombinant protein expression techniques that require the multiplex analysis of several dozen up to hundreds of constructs for efficient product and process development

The transmembrane protein fibrocystin/polyductin regulates cell mechanics and cell motility

Polycystic kidney disease is a disorder that leads to fluid filled cysts that replace normal renal tubes. During the process of cellular development and in the progression of the diseases, fibrocystin can lead to impaired organ formation and even cause organ defects. Besides cellular polarity, mechanical properties play major roles in providing the optimal apical-basal or anterior–posterior symmetry within epithelial cells. A breakdown of the cell symmetry that is usually associated with mechanical property changes and it is known to be essential in many biological processes such as cell migration, polarity and pattern formation especially during development and diseases such as the autosomal recessive cystic kidney disease. Since the breakdown of the cell symmetry can be evoked by several proteins including fibrocystin, we hypothesized that cell mechanics are altered by fibrocystin. However, the effect of fibrocystin on cell migration and cellular mechanical properties is still unclear. In order to explore the function of fibrocystin on cell migration and mechanics, we analyzed fibrocystin knockdown epithelial cells in comparison to fibrocystin control cells. We found that invasiveness of fibrocystin knockdown cells into dense 3D matrices was increased and more efficient compared to control cells. Using optical cell stretching and atomic force microscopy, fibrocystin knockdown cells were more deformable and exhibited weaker cell–matrix as well as cell–cell adhesion forces, respectively. In summary, these findings show that fibrocystin knockdown cells displayed increased 3D matrix invasion through providing increased cellular deformability, decreased cell–matrix and reduced cell–cell adhesion force

Effect of Nuclear Stiffness on Cell Mechanics and Migration of Human Breast Cancer Cells

The migration and invasion of cancer cells through 3D confined extracellular matrices is coupled to cell mechanics and the mechanics of the extracellular matrix. Cell mechanics is mainly determined by both the mechanics of the largest organelle in the cell, the nucleus, and the cytoskeletal architecture of the cell. Hence, cytoskeletal and nuclear mechanics are the major contributors to cell mechanics. Among other factors, steric hindrances of the extracellular matrix confinement are supposed to affect nuclear mechanics and thus also influence cell mechanics. Therefore, we propose that the percentage of invasive cells and their invasion depths into loose and dense 3D extracellular matrices is regulated by both nuclear and cytoskeletal mechanics. In order to investigate the effect of both nuclear and cytoskeletal mechanics on the overall cell mechanics, we firstly altered nuclear mechanics by the chromatin de-condensing reagent Trichostatin A (TSA) and secondly altered cytoskeletal mechanics by addition of actin polymerization inhibitor Latrunculin A and the myosin inhibitor Blebbistatin. In fact, we found that TSA-treated MDA-MB-231 human breast cancer cells increased their invasion depth in dense 3D extracellular matrices, whereas the invasion depths in loose matrices were decreased. Similarly, the invasion depths of TSA-treated MCF- 7 human breast cancer cells in dense matrices were significantly increased compared to loose matrices, where the invasion depths were decreased. These results are also valid in the presence of a matrix-metalloproteinase inhibitor GM6001. Using atomic force microscopy (AFM), we found that the nuclear stiffnesses of both MDA-MB- 231 and MCF-7 breast cancer cells were pronouncedly higher than their cytoskeletal stiffness, whereas the stiffness of the nucleus of human mammary epithelial cells was decreased compared to their cytoskeleton. TSA treatment reduced cytoskeletal and nuclear stiffness of MCF-7 cells, as expected. However, a softening of the nucleus by TSA treatment may induce a stiffening of the cytoskeleton of MDA-MB-231 cells and subsequently an apparent stiffening of the nucleus. Inhibiting actin polymerization using Latrunculin A revealed a softer nucleus of MDA-MB-231 cells under TSA treatment. This indicates that the actin-dependent cytoskeletal stiffness seems to be influenced by the TSA-induced nuclear stiffness changes. Finally, the combined treatment with TSA and Latrunculin A further justifies the hypothesis of apparent nuclear stiffening, indicating that cytoskeletal mechanics seem to be regulated by nuclear mechanics

Inhomogeneities in 3D Collagen Matrices Impact Matrix Mechanics and Cancer Cell Migration

Cell motility under physiological and pathological conditions including malignant progression of cancer and subsequent metastasis are founded on environmental confinements. During the last two decades, three-dimensional cell migration has been studied mostly by utilizing biomimetic extracellular matrix models. In the majority of these studies, the in vitro collagen scaffolds are usually assumed to be homogenous, as they consist commonly of one specific type of collagen, such as collagen type I, isolated from one species. These collagen matrices should resemble in vivo extracellular matrix scaffolds physiologically, however, mechanical phenotype and functional reliability have been addressed poorly due to certain limitations based on the assumption of homogeneity. How local variations of extracellular matrix structure impact matrix mechanics and cell migration is largely unknown. Here, we hypothesize that local inhomogeneities alter cell movement due to alterations in matrix mechanics, as they frequently occur in in vivo tissue scaffolds and were even changed in diseased tissues. To analyze the effect of structural inhomogeneities on cell migration, we used a mixture of rat tail and bovine dermal collagen type I as well as pure rat and pure bovine collagens at four different concentrations to assess three-dimensional scaffold inhomogeneities. Collagen type I from rat self-assembled to elongated fibrils, whereas bovine collagen tended to build node-shaped inhomogeneous scaffolds. We have shown that the elastic modulus determined with atomic force microscopy in combination with pore size analysis using confocal laser scanning microscopy revealed distinct inhomogeneities within collagen matrices. We hypothesized that elastic modulus and pore size govern cancer cell invasion in three-dimensional collagen matrices. In fact, invasiveness of three breast cancer cell types is altered due to matrix-type and concentration indicating that these two factors are crucial for cellular invasiveness. Our findings revealed that local matrix scaffold inhomogeneity is another crucial parameter to explain differences in cell migration, which not solely depended on pore size and stiffness of the collagen matrices. With these three distinct biophysical parameters, characterizing structure and mechanics of the studied collagen matrices, we were able to explain differences in the invasion behavior of the studied cancer cell lines in dependence of the used collagen model

Von der Gegenkunst zur Gegenwelt.Die gestalteten Orte Niki de Saint Phalles im Kontext postmoderner Subjektkulturen.

Im Zuge des Paradigmenwechsels in der Kunst der 1960er Jahre, entwickelte Niki de Saint Phalle in Gemeinschaftsausstellungen mit französischen und amerikanischen Künstlern ein Kunstverständnis, das auf der Partizipation des Betrachters gründet und durch die Stimulation verschiedener Sinne ein Kunsterlebnis erzeugen will, dessen Ziel es ist, vergnüglich zu sein. 1966 übertrug sie dieses Paradigma gemeinsam mit Jean Tinguely in der temporären Installation Hon - einer begehbaren Riesin mit sinnlichen Erlebnisräumen im Inneren - in die Monumentalität. Die dort erprobten Strategien zur Betrachteraktivierung und-partizipation wurden in ihren späteren Skulpturengärten verfeinert. Landschaft, Skulptur und Architektur werden dort zu nicht alltäglichen Erlebnisorten zusammengeführt, deren Wirkung durch den Einsatz von grotesken Körperkonzepten und aufwendigen Materialien gesteigert wird. Die Dissertation geht über die bislang dominante individual-psychologische Deutung des Werkes Niki de Saint Phalles hinaus. Die bisher nur unzureichend untersuchten Skulpturengärten Il Giardino dei Tarocchi (1978-1998) in der südlichen Toskana und Queen Califa's Magical Circle (1999-2003) in Südkalifornien werden monographisch erfasst und in die Geschichte der Kunst des 20. Jahrhunderts und der Gartenkunst mit Exkursen zu Vergnügungsparks eingeordnet. Inhaltlich wird die Untersuchung entlang der De- und Refigurierung von Weiblichkeitsbildern durchgeführt

Funktionelle Magnetresonanztomographie (MRT) des Beckenbodens

Harninkontinenz und Deszensus genitalis sind bei Frauen häufig auftretende Probleme, deren Ursache in einer Insuffizienz des Beckenbodenverschlußsystems liegen. Als eine der Hauptursachen für die Schädigung von Muskulatur und Innervation des Beckenbodens gilt die vaginale Geburt. In der vorliegenden Arbeit wurden die Auswirkungen der vaginalen Entbindung anhand funktioneller MRT-Studien an Erstgebärenden nach spontanvaginaler Geburt untersucht im Vergleich zu Probandinnen, die noch nicht geboren hatten. Die MRT bietet sich aufgrund der fehlenden Strahlen- und Kontrastmittelbelastung besonders zur Untersuchung junger Frauen an. Sie ermöglicht dabei eine exzellente Darstellung aller am Beckenbodenaufbau beteiligter Strukturen und erlaubt in den funktionellen Sequenzen die Beobachtung des Zusammenspiels aller drei Beckenbodenkompartimente beim Zwick- und Pressmanöver. Es zeigten sich signifikante Unterschiede beim Vergleich der beiden Kollektive. Die Primiparae wiesen beim Pressen im Durchschnitt ein signifikant ausgeprägteres Tiefertreten von Harnblase, hinterem Scheidengewölbe und anorektalem Übergang auf, die Weite des Levatortores sowie die Rektozelentiefe waren im Vergleich mit den Nulliparae ebenfalls deutlich vergrößert. Eine Häufung von pathologischen MRT-Befunden bei Frauen mit geburtshilflichen Risikofaktoren wie großem Kindsgewicht und Kopfumfang sowie langer Austreibungsperiode fand sich am untersuchten Primiparae-Kollektiv jedoch nicht. In Hinblick auf klinisch vorliegende Harn- und Stuhlinkontinenz ließ sich bis auf eine erhöhte Rate an Rektumdeszensus und erweitertem Hiatus genitalis in der Gruppe der mittel- bis höhergradig stuhlinkontinenten Patientinnen ebenfalls keine Korrelation finden zwischen klinischem Kontinenzbefund und MRT-Bildgebung. Bei diesen Beobachtungen ist jedoch die sehr kleinen Anzahl an untersuchten harn- bzw. stuhlinkontinenten Frauen zu berücksichtigen, so daß hier für eine verlässliche Beurteilung Studien an größeren Kollektiven unerlässlich sind. Zusammenfassend lässt sich feststellen, daß die funktionelle MRT eine sehr gute und umfassende Darstellung der postpartalen Beckenbodenveränderungen ermöglicht. Sie trägt neben Kontinenzanamnese, gynäkologischer Untersuchung, Sonographie und urodynamischer Diagnostik erheblich zu einer differenzierten Beurteilung der postpartalen Beckenbodensituation bei. Weiterführende Studien unter Berücksichtigung der verschiedenen Entbindungsmodalitäten (vaginal-operativ, Sectio) sind bereits in Arbeit und werden das Verständnis der geburtsbedingten Veränderungen am Beckenboden sicherlich noch weiter vertiefen können

Implementation of the UN Convention on the Rights of Persons with Disabilities: A Comparison of Four European Countries with Regards to Assistive Technologies

The United Nations Convention on the Rights of Persons with Disabilities (CRPD) is an international treaty that aims to promote, protect and ensure the rights of persons with disabilities so that they can fully participate in society and enjoy the same freedoms and opportunities as others. It provides an important framework for the inclusion of persons with disabilities with the help of Assistive Technologies (AT). This paper assesses and compares the implementation of the CRPD with regards to the availability of AT in four countries (Germany, Hungary, Portugal and Sweden), which to some extent represent different European regions. The paper is based on a review of relevant academic literature, the DOTCOM database and regulatory documents as well as on five validation interviews with national experts. In the countries studied, anti-discriminatory and other legislation is included at the highest level of the legal framework and contains detailed rules on definitions, remedies and legal procedures. There are specific prohibitions in several fields, such as employment, housing, and healthcare. Nonetheless, there are still cases of non-compliance with the CRPD and of laws and regulations which discriminate against persons with a disability. Additionally, there are great variations between countries. As very positive examples of favourable regulatory frameworks for furthering inclusion with the help of ATs do exist, there may be benefit in raising awareness of such examples to support other countries in developing their own measures