Prädiktoren bei der schubförmigen Multiplen Sklerose im Hinblick auf die Entwicklung einer sekundären Progression

Hintergrund: Im klinischen Verlauf der MS unterscheidet man einen akuten Schub im frühen Stadium von einer chronischen Neurodegeneration in der späten pro-gressiven Phase. Einem Schub liegt eine akute Entzündungsreaktion zugrunde. Mit zunehmender Krankheitsdauer nimmt die Neurodegeneration und eine damit verbundene klinische Behinderung der Betroffenen zu. Im natürlichen Verlauf geht die schubförmige MS (RR-MS) bei 2/3 der Patienten nach ca. 10–19 Jahren in eine sekundär progrediente Verlaufsform (SP-MS) über. Eine immunmodulatorische Behandlung der RR-MS mit den Basistherapeutika der 1. Wahl, ß-Interferone (IFNß) und Copaxone (CPX), wird früh begonnen, um die Entzündung zu hemmen und die Schubrate zu reduzieren sowie die entzündungsbedingte Krankheitspro-gression und Entwicklung der SP-MS zu verzögern. In großen kontrollierten Stu-dien wurden die Therapieeffekte von IFNß und CPX im frühen Krankheitsverlauf verglichen (CHAMPS-, BEYOND-, REGARD-, BECOME-Studie). Ziel dieser retro-spektiven Verlaufsstudie war es, die langfristigen Wirkungen von IFNß und CPX auf die Entwicklung der SP-MS in der Überlebensanalyse nach Kaplan-Meier zu vergleichen. Methoden: Insgesamt 777 MS-Patienten wurden nach folgenden Einschlußkriterien gefiltert: (1) klinisch gesicherte RR-MS nach McDonald, (2) IFN oder CPX als Erst- und Monotherapie für mindestens 9 Monate oder Therapiewechsel innerhalb von 9 Monaten wegen Nebenwirkungen, (3) Diagnosestellung bei Therapiebeginn  5 Jahre, (4) EDSS  5. Ausschlußkriterien waren: (1) Entwicklung einer SP-MS vor und innerhalb von 6 Monaten nach Therapiebeginn, (2) Therapiewechsel wegen Unwirksamkeit. Als primäre Effektivitätsparameter wurden verglichen: %-Anteil der MS-Patienten, die nach 6 Jahren Beobachtungszeit keine SP-MS zeigten oder ei-ne SP-MS entwickelten. Eine SP-MS wurde als Krankheitsprogression im Sinne einer Verschlechterung des EDSS um > 0,5 Punkte innerhalb von 6 Monaten definiert. Als statistische Verfahren kamen die Überlebenszeitanalyse nach Kaplan-Meier, die Cox-Regressionsanalyse und der Box-Plot zur Anwendung. Die Auswertung erfolgte mit dem SPSS-Programm 15,0 (SPSS inc., Chicago, Illinois). Signifikanzbe-rechnungen wurden mittels des Log-Rank-Tests durchgeführt und Ergebnisse mit P  0,05 als statistisch signifikant betrachtet. Ergebnisse: Insgesamt 388 Patienten mit RR-MS wurden in die Studie einge-schlossen. Davon erhielten 264 Patienten über einen 6-Jahreszeitraum IFNß und 124 Patienten alternativ CPX. Es gab zum Therapiebeginn keine deutlichen Unterschiede in den Basis-Patientendaten zwischen den Gruppen (Frauenanteil, Alter und EDSS bei Diagnosestellung und Erkrankungsdauer). In einem 6-Jahreszeitraum nach Therapiebeginn waren die prozentualen Anteile der Pati-enten ohne SP-MS für CPX und IFNß nicht signifikant unterschiedlich (CPX: 104/124 oder 83,9% vs. IFNß: 212/264 oder 80,3%; p=0,39). Gleichzeitig entwi-ckelten 20/124 oder 16,1% der CPX-Patienten und 52/264 oder 19,7 % der IFN-Patienten eine SP-MS. Mittels Hazard-Funktion wurde kein signifikanter Unter-schied zwischen beiden Medikamenten nachgewiesen (p=0,39). Mit diesen Da-ten und Werten für Power, Signifikanzniveau und klinisch relevantem Unter-schied (0,80; 0,05 und 4/5 bzw. 5/4) liess sich für eine prospektive Studie eine Fallzahl von 3000 – 4000 Patienten für beide Gruppen berechnen. Schlußfolgerungen: Insgesamt waren die langfristigen Therapieeffekte von IFNß und CPX auf die Entwicklung der SP-MS vergleichbar. Es ergaben sich keine klinisch relevanten Unterschiede zwischen den Wirkstoffen (p=0,39). Mögliche klinische Unterschiede zwischen den Medikamenten können in einer prospekti-ven Studie mit Fallzahlkalkulation und einer Power von 80 % nachgewiesen werden

A Cost-Effective Pichia pastoris Cell-Free System Driven by Glycolytic Intermediates Enables the Production of Complex Eukaryotic Proteins

Cell-free systems are particularly attractive for screening applications and the production of difficult-to-express proteins. However, the production of cell lysates is difficult to implement on a larger scale due to large time requirements, cultivation costs, and the supplementation of cell-free reactions with energy regeneration systems. Consequently, the methylotrophic yeast Pichia pastoris, which is widely used in recombinant protein production, was utilized in the present study to realize cell-free synthesis in a cost-effective manner. Sensitive disruption conditions were evaluated, and appropriate signal sequences for translocation into ER vesicles were identified. An alternative energy regeneration system based on fructose-1,6-bisphosphate was developed and a ~2-fold increase in protein production was observed. Using a statistical experiment design, the optimal composition of the cell-free reaction milieu was determined. Moreover, functional ion channels could be produced, and a G-protein-coupled receptor was site-specifically modified using the novel cell-free system. Finally, the established P. pastoris cell-free protein production system can economically produce complex proteins for biotechnological applications in a short time

Enhancing the performance of a mutant pyrrolysyl-tRNA synthetase to create a highly versatile eukaryotic cell-free protein synthesis tool

Modification of proteins with a broad range of chemical functionalities enables the investigation of protein structure and activity by manipulating polypeptides at single amino acid resolution. Indeed, various functional groups including bulky non-canonical amino acids like strained cyclooctenes could be introduced by the unique features of the binding pocket of the double mutant pyrrolysyl-tRNA synthetase (Y306A, Y384F), but the instable nature of the enzyme limits its application in vivo. Here, we constructed a cell-free protein production system, which increased the overall enzyme stability by combining different reaction compartments. Moreover, a co-expression approach in a one-pot reaction allowed straightforward site-specific fluorescent labeling of the functional complex membrane protein cystic fibrosis transmembrane conductance regulator. Our work provides a versatile platform for introducing various non-canonical amino acids into difficult-to-express proteins for structural and fluorescence based investigation of proteins activity

Promoting the production of challenging proteins via induced expression in CHO cells and modified cell-free lysates harboring T7 RNA polymerase and mutant eIF2α

Chinese hamster ovary (CHO) cells are crucial in biopharmaceutical production due to their scalability and capacity for human-like post-translational modifications. However, toxic proteins and membrane proteins are often difficult-to-express in living cells. Alternatively, cell-free protein synthesis can be employed. This study explores innovative strategies for enhancing the production of challenging proteins through the modification of CHO cells by investigating both, cell-based and cell-free approaches. A major result in our study involves the integration of a mutant eIF2 translation initiation factor and T7 RNA polymerase into CHO cell lysates for cell-free protein synthesis. This resulted in elevated yields, while eliminating the necessity for exogenous additions during cell-free production, thereby substantially enhancing efficiency. Additionally, we explore the potential of the Rosa26 genomic site for the integration of T7 RNA polymerase and cell-based tetracycline-controlled protein expression. These findings provide promising advancements in bioproduction technologies, offering flexibility to switch between cell-free and cell-based protein production as needed

One to one comparison of cell-free synthesized erythropoietin conjugates modified with linear polyglycerol and polyethylene glycol

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation

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The existence of spacetime singularities is irrelevant for the irreversible appearance of black holes. However, confirmation of the latter's unitary dynamics would require the preparation of a coherent superposition of a tremendous number of appropriate ``Everett worlds''.Comment: 10 pages, 1 figure, Latex - Invited paper for a special Einstein issue of Physics Letters

Erweiterung der kennfeldbasierten Verdichtermodellierung für sequentielle Aufladesysteme

The overall goal of this thesis is to extend the map-based compressor modeling, for example for the layout of sequential boosting systems using 1D engine process simulation. The need for research is initially demonstrated with a mixed-sequential boosting concept. Due to serial arranged compressors, low pressure ratios occur in both compressors during stationary full load operation. The conventional methods for stationary compressor measurements on a hot gas test bench do not cover the operating range at low compressor pressure ratios. With new measuring methods the examined operating range is extended to zero speed and pressure ratios of less than one (ΠC < 1). The evaluation of the operating ranges in which power input or power output, i.e. turbine operation, occurs provides a fundamental understanding of the processes in the compressor. The findings of the extended measurements show that the usual map-based compressor modelling approach based on isentropic efficiencies is not sufficient to describe the system behavior at ΠC < 1. An extended modelling approach, as well as a new compressor model based on the newly introduced corrected compressor torque, allows a predictive 1D engine process simulation. Extended measurements are not always available. Different methods from literature to extrapolate the compressor map to low pressure ratios are investigated. However, these show weaknesses in the prediction of choking mass flow rates. A detailed analysis by means of 3D-CFD compressor simulations allows the localization of the choking cross-sections of the compressor in engine-relevant operating areas. At high circumferential speeds, choking occurs at the impeller inlet, whereas at medium circumferential speeds choking can be observed at the impeller outlet. At the lowest circumferential speeds choking occurs in the junction of the compressor volute. Based on these findings, a 0D/1D-modelling approach for extrapolation of the compressor map is presented. The model allows the calculation of flow conditions along the flow path in the compressor based on a few geometric dimensions as well as empirical loss models. The parameters of the loss models are adjusted so that the model matching the measured data in the conventionally measured operating range. The model shows a good prediction accuracy of the choking mass flow rate in relation to the extended measured data. Finally, the extended compressor modelling is validated with measurements on the engine test bench. Compared to state of the art compressor modelling, the new modelling approach shows clear advantages in predicting pressure losses for engine operation points at low compressor pressure ratios

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase