Process Optimization for Recombinant Protein Expression in Insect Cells

Insect cells can be used for the efficient production of heterologous proteins. The baculovirus expression vector system (BEVS) in Spodoptera frugiperda cells and the stable transformation of Drosophila melanogaster S2 cells are widely used for this purpose. Whereas BEVS is a transient expression system for rapid protein production, stable D. melanogaster cell lines are compatible with more complex processes modes. This chapter describes the setup of both systems, including steps for the generation of expression vectors and comprehensive optimization approaches. The genetic elements available in each system are described, as well as the use of different cloning and transfection methods and advanced process monitoring to achieve robust protein expression in larger-scale bioreactors

The species status of the Otiorhynchus clavipes (Bonsdorff, 1785) species group (Coleoptera: Curculionidae) : an integrative approach using molecular, morphological, ecological, and biogeographical data

The species of the Otiorhynchus clavipes (Bonsdorff, 1785) group (Coleoptera: Curculionidae) treated here, are characterized by their unusually high phenotypical variation, which often caused taxonomic problems and controversies. Molecular markers COI and EF1-$\alpha$, karyological analysis, as well as morphological, biogeographical and ecological data are used to study weevils collected in the Alps, Carpathians, Sudetes and different areas of Germany. In the investigated populations of the flightless species O. fagi Gyllenhal, 1834 and O. clavipes, we detected an interspecific genetic distance of 11.3-15.8% (COI) and 3.1-3.7% (EF1-$\alpha$) depending on geographical distance. The phylogenetic trees indicate that both species are monophyletic and that they were correctly delimited from each other. Both species have also separate geographical ranges in Central Europe. Male specimens differ in the morphology of the aedeagus and the last abdominal sternite. Our study supports the legitimacy of species delimitation of O. fagi and O. clavipes as separate species, which can be treated as stable taxonomic hypotheses. The determination of the species status required the re-examination of spe cies ranges and allowed together with data on biology and altitudinal preferences a better biogeographical and ecological characterization of the species

Steric exclusion chromatography for the purification of recombinant baculovirus

Steric exclusion chromatography (SXC) has already proven to be a valuable tool in the purification of proteins and virus particles. An important benefit of the method is the fast and simple procedure at mild chromatography conditions as no harsh binding and elution buffers are needed. The sample is initially mixed with a polyethylene glycol (PEG) containing buffer of choice. The steric exclusion of a macromolecule from the polyethylene glycol and the stationary phase allows a selective retention of the product, depending, among others, mainly on its size as well as on the molecular weight and concentration of the PEG. Here, SXC was set up in order that smaller process contaminants, i.e. host cell proteins and DNA, did not bind to the stationary phase, in contrast to the targeted larger virus particles. These were subsequently eluted reducing the PEG concentration in the mobile phase. Regenerated cellulose was used as stationary phase to purify VSV-G pseudotyped AcMNPV baculoviruses derived from Spodoptera frugiperda cells (Sf9 cells) by SXC. The purified virus particles are used as gene transfer tools for human mesenchymal stroma cells. For this purpose, the baculovirus was clarified prior to the SXC by sequential centrifugation (4700 gmax). The SXC conditions were optimized in terms of yield and purity by a design of experiment approach considering the PEG molecular weight, its concentration and the ionic strength of the elution buffer as critical process parameters. Within the design space virus recovery was ≥70%. Without further nuclease treatment the depletion of double-stranded DNA was \u3e90% and the amount of host cell proteins were reduced \u3e90% in the virus fraction. In conclusion, SXC can drastically reduce the process development in terms of time and equipment requirements for the purification of recombinant baculoviruses, as well as for the achieved purity which is superior over classical methods

Penestragania apicalis (Osborn & Ball, 1898), another invasive Nearctic leafhopper found in Europe: (Hemiptera: Cicadellidae, Iassinae)

Penestragania apicalis (Osborn & Ball, 1898), eine weitere invasive nearktische Zikade in Europa gefunden (Hemiptera: Cicadellidae, Iassinae). – Im Jahr 2010 wurde die nearktische Zikadenart Penestragania apicalis (Osb. & Ball) erstmalig in Europa gefunden. Insgesamt sind derzeit 16 Fundorte in Frankreich, der Schweiz, Deutschland und Österreich bekannt; daher ist davon auszugehen, dass die Art bereits seit längerer Zeit fest etabliert und in Europa und vielleicht weltweit weiter verbreitet ist. Wie in Nordamerika lebt sie an Gleditschie (Gleditsia triacanthos L.), überwintert im Eistadium und hat eine oder zwei Generation pro Jahr, mit adulten Tieren von mindestens Ende Juni bis Anfang Oktober. Ob in Europa wirtschaftlich relevante Schäden verursacht werden, ist noch unklar.In 2010 the Nearctic leafhopper Penestragania apicalis (Osb. & Ball) was found for the first time in Europe. Altogether there are now 16 known localities in France, Switzerland, Germany and Austria indicating that the species is well es‐ tablished for a rather long period and more widespread in Europe and perhaps worldwide. As in North America it lives on honeylocust (Gleditsia triacanthos L.), overwinters in the egg stage and probably has one or two generations a year, with adults at least from late June until early October. It is yet unclear if it causes relevant damage to the host plant in Europe

Developmental Biology and Identification of a Garden Pest, Otiorhynchus (Podoropelmus) smreczynskii Cmoluch, 1968 (Coleoptera, Curculionidae, Entiminae), with Comments on Its Origin and Distribution

The mature larva and pupa of Otiorhynchus smreczynskii are described and illustrated with complete chaetotaxy for the first time. Five larval instars and the factors for larval growth are determined, and the larval development of this species is comprehensively described. In order to confirm species affiliation, selected larvae were subjected to genetic analysis (mtCOI). Host plants and unique feeding signs of some Entiminae species are presented, and all available data on development are documented and interpreted. Additionally, the morphometry of 78 specimens (48 of O. smreczynskii and 30 of O. rotundus) was examined in order to verify the usefulness of morphological features in distinguishing both species. The female genitalia of both species are illustrated, described and compared with each other for the first time. Finally, the updated distribution of O. smreczynskii is given, and a possible origin of O. smreczynskii and O. rotundus is proposed

Noble gas solubility in silicate melts:a review of experimentation and theory, and implications regarding magma degassing processes

Noble gas solubility in silicate melts and glasses has gained a crucial role in Earth Sciences investigations and in the studies of non-crystalline materials on a micro to a macro-scale. Due to their special geochemical features, noble gases are in fact ideal tracers of magma degassing. Their inert nature also allows them to be used to probe the structure of silicate melts. Owing to the development of modern high pressure and temperature technologies, a large number of experimental investigations have been performed on this subject in recent times. This paper reviews the related literature, and tries to define our present state of knowledge, the problems encountered in the experimental procedures and the theoretical questions which remain unresolved. Throughout the manuscript I will also try to show how the thermodynamic and structural interpretations of the growing experimental dataset are greatly improving our understanding of the dissolution mechanisms, although there are still several points under discussion. Our improved capability of predicting noble gas solubilities in conditions closer to those found in magma has allowed scientists to develop quantitative models of magma degassing, which provide constraints on a number of questions of geological impact. Despite these recent improvements, noble gas solubility in more complex systems involving the main volatiles in magmas, is poorly known and a lot of work must be done. Expertise from other fields would be extremely valuable to upcoming research, thus focus should be placed on the structural aspects and the practical and commercial interests of the study of noble gas solubility

A national training program for simulation educators and technicians: evaluation strategy and outcomes

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.BACKGROUND: Simulation-based education (SBE) has seen a dramatic uptake in health professions education over the last decade. SBE offers learning opportunities that are difficult to access by other methods. Competent faculty is seen as key to high quality SBE. In 2011, in response to a significant national healthcare issue--the need to enhance the quality and scale of SBE--a group of Australian universities was commissioned to develop a national training program--Australian Simulation Educator and Technician Training (AusSETT) Program. This paper reports the evaluation of this large-scale initiative. METHODS: The AusSETT Program adopted a train-the-trainer model, which offered up to three days of workshops and between four and eight hours of e-learning. The Program was offered across all professions in all states and territories. Three hundred and three participants attended workshops with 230 also completing e-learning modules. Topics included: foundational learning theory; orientation to diverse simulation modalities; briefing; and debriefing. A layered objectives-oriented evaluation strategy was adopted with multiple stakeholders (participants, external experts), methods of data collection (end of module evaluations, workshop observer reports and individual interviews) and at multiple data points (immediate and two months later). Descriptive statistics were used to analyse numerical data while textual data (written comments and transcripts of interviews) underwent content or thematic analysis. RESULTS: For each module, between 45 and 254 participants completed evaluations. The content and educational methods were rated highly with items exceeding the pre-established standard. In written evaluations, participants identified strengths (e.g. high quality facilitation, breadth and depth of content) and areas for development (e.g. electronic portfolio, learning management system) of the Program. Interviews with participants suggested the Program had positively impacted their educational practices. Observers reported a high quality educational experience for participants with alignment of content and methods with perceived participant needs. CONCLUSIONS: The AusSETT Program is a significant and enduring learning resource. The development of a national training program to support a competent simulation workforce is feasible. The Program objectives were largely met. Although there are limitations with the study design (e.g. self-report), there are strengths such as exploring the impact two months later. The evaluation of the Program informs the next phase of the national strategy for simulation educators and technicians with respect to content and processes, strengths and areas for development

Penestragania apicalis (Osborn & Ball,1898),another invasive Nearctic leafhopper found in Europe

In 2010 the Nearctic leafhopper Penestragania apicalis (Osb.& Ball) was found for the first time in Europe. Altogether there are now 16 known localities in France, Switzerland, Germany and Austria indicating that the species is well established for a rather long period and more widespread in Europe and perhaps worldwide. As in North America it lives on honeylocust (Gleditsia triacanthos L.), overwinters in the egg stage and probably has one or two generations a year, with adults at least from late June until early October. It is yet unclear if it causes relevant damage to the host plant in Europe

Photocatalytic proton reduction by a computationally identified, molecular hydrogen-bonded framework

We show that a hydrogen-bonded framework, TBAP-α, with extended π-stacked pyrene columns has a sacrificial photocatalytic hydrogen production rate of up to 3108 μmol g-1 h-1. This is the highest activity reported for a molecular organic crystal. By comparison, a chemically-identical but amorphous sample of TBAP was 20-200 times less active, depending on the reaction conditions, showing unambiguously that crystal packing in molecular crystals can dictate photocatalytic activity. Crystal structure prediction (CSP) was used to predict the solid-state structure of TBAP and other functionalised, conformationally-flexible pyrene derivatives. Specifically, we show that energy-structure-function (ESF) maps can be used to identify molecules such as TBAP that are likely to form extended π-stacked columns in the solid state. This opens up a methodology for the a priori computational design of molecular organic photocatalysts and other energy-relevant materials, such as organic electronics

Protocol of the IntenSify-Trial:An open-label phase I trial of the CYP3A inhibitor cobicistat and the cytostatics gemcitabine and nab-paclitaxel in patients with advanced stage or metastatic pancreatic ductal adenocarcinoma to evaluate the combination's pharmacokinetics, safety, and efficacy

Expression of CYP3A5 protein is a basal and acquired resistance mechanism of pancreatic ductal adenocarcinoma cells conferring protection against the CYP3A and CYP2C8 substrate paclitaxel through metabolic degradation. Inhibition of CYP3A isozymes restores the cells sensitivity to paclitaxel. The combination of gemcitabine and nab-paclitaxel is an established regimen for the treatment of metastasized or locally advanced inoperable pancreatic cancer. Cobicistat is a CYP3A inhibitor developed for the pharmacoenhancement of protease inhibitors. The addition of cobicistat to gemcitabine and nab-paclitaxel may increase the antitumor effect. We will conduct a phase I dose escalation trial with a classical 3 + 3 design to investigate the safety, tolerability, and pharmacokinetics (PKs) of gemcitabine, nab-paclitaxel, and cobicistat. Although the doses of gemcitabine (1000 mg/m2) and cobicistat (150 mg) are fixed, three dose levels of nab-paclitaxel (75, 100, and 125 mg/m2) will be explored to account for a potential PK drug interaction. After the dose escalation phase, we will set the recommended dose for expansion (RDE) and treat up to nine patients in an expansion part of the trial. The trial is registered under the following identifiers EudraCT-Nr. 2019-001439-29, drks.de: DRKS00029409, and ct.gov: NCT05494866. Overcoming resistance to paclitaxel by CYP3A5 inhibition may lead to an increased efficacy of the gemcitabine and nab-paclitaxel regimen. Safety, efficacy, PK, and RDE data need to be acquired before investigating this combination in a large-scale clinical study.</p