Synthesis of mono Cytochrome P450 in a modified CHO-CPR cell-free protein production platform

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup

Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha

The ability to catalyze diverse reactions with relevance for chemical and pharmaceutical research and industry has led to an increasing interest in fungal enzymes. There is still an enormous potential considering the sheer amount of new enzymes from the huge diversity of fungi. Most of these fungal enzymes have not been characterized yet due to the lack of high throughput synthesis and analysis methods. This bottleneck could be overcome by means of cell-free protein synthesis. In this study, cell-free protein synthesis based on eukaryotic cell lysates was utilized to produce a functional glycoside hydrolase (GH78) from the soft-rot fungus Xylaria polymorpha (Ascomycota). The enzyme was successfully synthesized under different reaction conditions. We characterized its enzymatic activities and immobilized the protein via FLAG-Tag interaction. Alteration of several conditions including reaction temperature, template design and lysate supplementation had an influence on the activity of cell-free synthesized GH78. Consequently this led to a production of purified GH78 with a specific activity of 15.4 U mg− 1. The results of this study may be foundational for future high throughput fungal enzyme screenings, including substrate spectra analysis and mutant screenings

The FLASHForward Facility at DESY

The FLASHForward project at DESY is a pioneering plasma-wakefield acceleration experiment that aims to produce, in a few centimetres of ionised hydrogen, beams with energy of order GeV that are of quality sufficient to be used in a free-electron laser. The plasma wave will be driven by high-current density electron beams from the FLASH linear accelerator and will explore both external and internal witness-beam injection techniques. The plasma is created by ionising a gas in a gas cell with a multi-TW laser system, which can also be used to provide optical diagnostics of the plasma and electron beams due to the <30 fs synchronisation between the laser and the driving electron beam. The operation parameters of the experiment are discussed, as well as the scientific program.Comment: 19 pages, 9 figure

Dilatação dos confins: caminhos, vilas e cidades na formação da Capitania de São Paulo (1532-1822)

Este ensaio analisa a formação da rede urbana das capitanias de São Vicente e Santo Amaro, depois unidas na Capitania de São Paulo. Discute o processo de apropriação do sertão, a pulsação e dilatação dos confins ao sabor dos deslocamentos humanos e de interesses políticos. Interpreta o papel de capelas, freguesias, vilas e cidades no controle e produção de territórios metropolitanos em solos ultramarinos.This essay analyzes the development of urban networks in the Captaincies of São Vicente and Santo Amaro, later merged into the Captaincy of São Paulo. It discusses the process of appropriation of the sertão (backcountry), the commotion and expansion beyond the confines to the tune of population movements and political interests. The paper also interprets the role of chapels, parishes, villages and towns in initiatives to create and control metropolitan areas on overseas soil

A História da Alimentação: balizas historiográficas

Os M. pretenderam traçar um quadro da História da Alimentação, não como um novo ramo epistemológico da disciplina, mas como um campo em desenvolvimento de práticas e atividades especializadas, incluindo pesquisa, formação, publicações, associações, encontros acadêmicos, etc. Um breve relato das condições em que tal campo se assentou faz-se preceder de um panorama dos estudos de alimentação e temas correia tos, em geral, segundo cinco abardagens Ia biológica, a econômica, a social, a cultural e a filosófica!, assim como da identificação das contribuições mais relevantes da Antropologia, Arqueologia, Sociologia e Geografia. A fim de comentar a multiforme e volumosa bibliografia histórica, foi ela organizada segundo critérios morfológicos. A seguir, alguns tópicos importantes mereceram tratamento à parte: a fome, o alimento e o domínio religioso, as descobertas européias e a difusão mundial de alimentos, gosto e gastronomia. O artigo se encerra com um rápido balanço crítico da historiografia brasileira sobre o tema

“Light” versus “classic” laser treatment for clinically significant diabetic macular oedema

Aim: To compare the effectiveness of “light” versus “classic” laser photocoagulation in diabetic patients with clinically significant macular oedema (CSMO). Methods: A prospective randomised pilot clinical trial in which 29 eyes of 24 diabetic patients with mild to moderate non-proliferative diabetic retinopathy (NPDR) and CSMO were randomised to either “classic” or “light” Nd:YAG 532 nm (frequency doubled) green laser. “Light” laser treatment differed from conventional (“classic”) photocoagulation in that the energy employed was the lowest capable to produce barely visible burns at the level of the retinal pigment epithelium. Primary outcome measure was the change in foveal retinal thickness as measured by optical coherence tomography (OCT); secondary outcomes were the reduction/elimination of macular oedema on contact lens biomicroscopy and fluorescein angiography, change in visual acuity, contrast sensitivity, and mean deviation in the central 10° visual field. Examiners were masked to patients’ treatment. Results: 14 eyes were assigned to “classic” and 15 were assigned to “light” laser treatment. At 12 months, seven (50%) of 14 eyes treated with “classic” and six (43%) of 14 eyes treated with “light” laser had a decrease of foveal retinal thickness on OCT (p = 0.79). A comparison of reduction/elimination of oedema, visual improvement, visual loss, change in contrast sensitivity, and mean deviation in the central 10° showed no statistical difference between the groups at 12 months (p>0.05 for all groups). Conclusions: This study suggests that “light” photocoagulation for CSMO may be as effective as “classic” laser treatment, thus supporting the rationale for a larger equivalence trial

Eukaryotic cell-free systems: A novel platform technology for production and functional characterization of pore-forming toxins

Question: Bacterial infections still cause tremendous health risks worldwide. A variety of pathogenic bacteria in particular causes virulence by pore-forming toxins that insert themselves into membranes and cause cell lysis. Since highly resistant bacterial strains have evolved over the last decades, new methods for the characterization of bacterial toxins are needed in order to develop new and specific diagnostic tools as well as to improve present-day treatment regimens. Methods: In vivo production of toxins is rather difficult as toxins harm the cells" viability. Cell-free protein synthesis has emerged as a fast and cost-efficient method to synthesize and characterize a variety of toxins while using a cell lysate rather than viable cells. Here, we describe the cell-free synthesis and functional characterization of enterotoxins from Bacillus cereus as well as Staphylococcus aureus, Cytolysin from Vibrio vulnificus and Aerolysin from Aeromonas hydrophila.Cell-based toxicity assays, blood agar plates and electrophysiological measurements on planar lipid bilayers were used to determine the toxins" functionality. Results: In our studies, we showed the successful synthesis and characterization of various pore-forming toxins and the ability to use the cell-free system in versatile ways. Conclusion: This platform technology enables a fast and efficient characterization of a variety of toxins as a prerequisite to develop diagnostic methods and treatments