A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression

Pointing Enhancement for an Optical Laser Downlink Using Automated Image Processing

The small satellite Flying Laptop, launched in July 2017, was developed and built by graduate and undergraduate students at the Institute of Space Systems of the University of Stuttgart with support by space industry and research institutions. The mission goals are technology demonstration, earth observation, and serving as an educational satellite. At a mass of 110 kg, it features three-axis stabilized attitude control and several payloads, including an AIS receiver, a multi spectral camera system, a wide angle camera, and an optical communication terminal. The pointing requirement for the optical communication is an accuracy of less than 150 arcseconds during a target overflight. To fulfill this requirement, several measures are needed. A major part of them is the characterization of the attitude control system (ACS). Since there is no optical receiver onboard, it is not possible to perform closed loop tracking of the satellite attitude. Therefore, the absolute performance and the characteristic noise levels of the attitude control system, can only be determined with other payloads. In this case the multi-spectral camera system was used, providing a ground resolution of 25 m. To use the images from the satellite to improve the ACS, three steps have to be taken. As a first action, the images have to be georeferenced to know the position of each pixel in the WGS84 coordinate system. With this information, the deviation of the image center from the desired target is measured. This second step includes the calculation of the deviation matrix. To avoid a corruption of the attitude control of the satellite, the matrix is checked for unrealistic values in a third and final step. These three actions can be repeated as needed without human interaction. By updating the ACS model onboard the satellite, the results of the image processing are used to correct the off-pointing. This deviation is time invariant and is caused by an insufficient alignment of the satellite axes and the cameras on ground. In contrast to that, characterizing noise as a time variant factor, the ACS is tested over a long period of time. This is achieved by analyzing images from one, as well as from multiple target overflights. This conquers the issue of a very low image rate while observing high frequency attitude changes. Using this mechanism, the proposed process can be used to continuously monitor the pointing quality. As a first approach the described processing is done manually by comparing the target position on Earth with the center of the taken image. The method successfully showed an improvement of the pointing in the pictures, paving the way for their automation. This paper gives an overview of the needed image processing and tools to automatically use cameras on board the satellite to validate and improve the ACS periodically. First results of the long term characteristics and pointing improvements are shown

Cultured Peribulbar Dermal Sheath Cells Can Induce Hair Follicle Development and Contribute to the Dermal Sheath and Dermal Papilla

Green fluorescent protein (GFP)-expressing wild-type, and nontransgenic mouse vibrissa follicle cells were cultured and implanted to mouse ears and footpads. Dermal papiller (DP)-derived cells and cells from the peribulbar dermal sheath “cup” (DSC) induced new hair follicles in both implanted ears and footpads, while nonbulbar dermal sheath cells did not. Confocal microscopy revealed that GFP-expressing DP and DSC cells induced hair growth associated with the formation of DP exclusively comprised of fluorescent cells. In mouse ears, but not footpads, fluorescent DP and DSC cells could also be identified in DP along with nonfluorescent cells. DSC cells were characterized in vivo and in vitro by low alkaline phosphatase activity in contrast to high alkaline phosphatase in DP cells. The results indicate transplanted DP and DSC cells were equally capable of DP formation and hair follicle induction. This suggests the DP and peribulbar DSC may be functionally similar. In addition to observing papillae exclusively composed of GFP-expressing cells, DP and DSC cells may also have combined with resident cells to form papillae composed of implanted GFP-expressing cells and host-derived non-GFP-expressing cells. Alkaline phosphatase expression may be utilized as a simple marker to identify hair follicle mesenchyme derived cells with hair follicle inductive abilities

Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system

The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion

Verbesserte Transparenz im Naturkostmarkt: Datenerhebung Naturkostfacheinzelhandel unter Berücksichtigung der Ausbildungsangebote

Der spezialisierte Naturkosthandel (NKH) stellt in Deutschland traditionell einen wichtigen Absatzkanal für ökologisch erzeugte Lebensmittel dar. Dieses Marktsegment ist dominiert von Klein- und Mittleren Unternehmen (KMU) und damit gekennzeichnet von einer relativ hohen Marktintransparenz. Da alle Marktbeteiligten ebenso wie die Politik aktuelle und belastbare Daten benötigen, wurden im BÖLN-Projekt 08OE123 die Strukturdaten des Naturkostfachhandels erstmals für das Jahr 2009 ff erhoben (siehe: https://orgprints.org/20521/1/20521-08OE123-bnn-roeder-kuhnert-2011-strukturdaten_naturkostfachhandel.pdf). Inzwischen ist aufgrund der dynamischen Marktentwicklung eine neue, grundlegende Erhebung der aktuellen Strukturdaten erforderlich. Durch diese fundierte Vollerhebung wird sichergestellt, dass die strukturelle Basis aufgrund von Annahmen und Schätzungen möglichst wenig anfällig für Ungenauigkeiten ist. Die wichtigsten Ergebnisse der Strukturdatenerhebung lassen sich wie folgt zusammenfassen: Anhand der Erhebung konnte die Existenz von 2.516 stationären Verkaufsstellen des Naturkostfachhandels verifiziert werden. Dies entspricht einem Zuwachs von sieben Prozent seit der letzten Erhebung im Jahr 2010. Die Läden sind im Durchschnitt größer als 2010, vor allem die Anzahl der Bio-Supermärkte ab 400 m² hat stark zugenommen. Allerdings stellen die Bio-Fachgeschäfte bis 399 m² mit einem Anteil von 60 Prozent immer noch den größten Teil der Verkaufsstellen dar. Nach wie vor konzentrieren sich die Naturkost-Verkaufsstellen in städtischen Ballungsräumen sowie in Süd- und Westdeutschland. In fast allen Bundesländern ist ein Zuwachs bezüglich Anzahl und Größe der Verkaufsstellen zu verzeichnen. Viele stationäre Verkaufsstellen erweitern ihr Angebot mittlerweile mit Zusatzangeboten wie Café, Bistro, Lieferservice oder Online-Shop. Der Grad der Diversifizierung hat zugenommen. Für 38 Prozent steht nach eigener Einschätzung die Nachfolgeplanung noch an. Je größer die Fläche einer Verkaufsstelle ist, desto größer ist deren Umsatz, desto mehr Mitarbeiter werden in dieser beschäftigt, desto eher werden Azubis ausgebildet und desto aktiver sind diese hinsichtlich der Nutzung von Weiterbildungsangeboten. Es sind 58 Prozent der Antwortenden nach eigener Einschätzung im Bereich Aus- und Weiterbildung aktiv, der weit überwiegende Teil aus dieser Gruppe ist der Kategorie Bio-Supermarkt zuzurechnen. Während 31 Prozent der Antwortenden angeben keine Zeit für Aus- und Weiterbildung zu haben. Diese Gruppe wird weit überwiegend aus der Kategorie der Bio-Fachgeschäfte und der Hofläden gespeist. Praxisbezug, Aktualität und Nähe sind die wesentlichen Kriterien für die Teilnahme an einer Weiterbildung

Operations System vs. Operating System: Towards a Ground System Supporting Satellite Application Programming

The term operating system refers to a software component, which traditionally controls the resources and the processes of a computer, and by providing the appropriate interfaces allows for the implementation of custom user applications. This is a common definition, working very well for ordinary computer systems. Yet, what if the operating system and a corresponding application are physically separated, because the computer is within a satellite in space, while the user program is executed on ground? Then, capabilities must be created to connect both, which is of course complicated by the natural boundaries in satellite communication, for example the limited satellite contact times. Over the past decades, several systems have been developed, which are capable of managing satellite resources and the mission schedule from ground. Although this covers quite well the purpose of an operating system, other terms have evolved in this domain: operations system, ground system, mission control system, ground data handling, etc. The problem though is, those systems primarily focus on the exchange of data and satellite TM/TC, rather than the actual control process. This creates an artificial barrier between ground and space, which harms the development capabilities for ground based satellite applications. This paper introduces a novel approach for an operations system architecture, which can be considered as a ground extension of the satellite’s operating system. This approach shall not break with the existing conventions and definitions, especially in terms of operating systems, but shall introduce a new view on satellite operations. In a layered, functional software architecture, the operating system is the lowest layer between the hardware and the application. Through the definition of the appropriate interfaces in the ground system, a software architecture can be created that actively supports outsourcing parts of the satellite control process to ground. The proposed approach has great potential for various applications in satellite operations. It supports the implementation of automatic system control processes, the implementation of custom payload applications, and the integration of respective activities into the satellite schedule. As applications and operators interact with a verified schedule, and operations is thus no longer limited to low-level commanding, the approach further reduces the risk of the mission being jeopardized by human mistake

Evidence for a Pathophysiological Role of Keratinocyte-Derived Type III Interferon (IFNλ) in Cutaneous Lupus Erythematosus

Type I IFNs (IFNα/β) have been shown to have a central role in the pathophysiology of lupus erythematosus (LE). The recently discovered type III IFNs (IFNλ1/IL29, IFNλ2/IL28a, IFNλ3/IL28b) share several functional similarities with type I IFNs, particularly in antiviral immunity. As IFNλs act primarily on epithelial cells, we investigated whether type III IFNs might also have a role in the pathogenesis of cutaneous LE (CLE). Our investigations demonstrate that IFNλ and the IFNλ receptor were strongly expressed in the epidermis of CLE skin lesions and related autoimmune diseases (lichen planus and dermatomyositis). Significantly enhanced IFNλ1 could be measured in the serum of CLE patients with active skin lesions. Functional analyses revealed that human keratinocytes are able to produce high levels of IFNλ1 but only low amounts of IFNα/β/γ in response to immunostimulatory nuclear acids, suggesting that IFNλ is a major IFN produced by these cells. Exposure of human keratinocytes to IFNλ1 induced the expression of several proinflammatory cytokines, including CXCL9 (CXC-motiv ligand 9), which drive the recruitment of immune cells and are associated with the formation of CLE skin lesions. Our results provide evidence for a role of type III IFNs in not only antiviral immunity but also autoimmune diseases of the skin

Automated cell cultivation and analysis: an approach for a user interface design

While automation is standard in most industrial and research fields, cell cultivation is still dominated “handling”, manual processes. To improve the situation, our group focusses on the development of a bio-centred, automated cell cultivation tool. Modular design may help to realize a variety of experiments. Cultivation processes are observed and controlled via a graphical user interface. This article describes the systematic approach, the overall speci-fication (with process analyses) and the preliminary results for the prototype of a graphical user interface

Tree species traits but not diversity mitigate stem breakage in a subtropical forest following a rare and extreme ice storm

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