Optimization of insect cell based protein production processes - online monitoring, expression systems, scale-up

Due to the increasing use of insect cell based expression systems in research and industrial recombinant protein production, the development of efficient and reproducible production processes remains a challenging task. In this context, the application of online monitoring techniques is intended to ensure high and reproducible product qualities already during the early phases of process development. In the following chapter, the most common transient and stable insect cell based expression systems are briefly introduced. Novel applications of insect cell based expression systems for the production of insect derived antimicrobial peptides/proteins (AMPs) are discussed using the example of G. mellonella derived gloverin. Suitable in situ sensor techniques for insect cell culture monitoring in disposable and common bioreactor systems are outlined with respect to optical and capacitive sensor concepts. Since scale-up of production processes is one of the most critical steps in process development, a conclusive overview is given about scale up aspects for industrial insect cell culture processes

Increased adipose tissue oxygen tension in obese compared with lean men is accompanied by insulin resistance, impaired adipose tissue capillarization, and inflammation

Background- Adipose tissue (AT) dysfunction in obesity contributes to chronic, low-grade inflammation that predisposes to type 2 diabetes mellitus and cardiovascular disease. Recent in vitro studies suggest that AT hypoxia may induce inflammation. We hypothesized that adipose tissue blood flow (ATBF) regulates AT oxygen partial pressure (AT Po(2)), thereby affecting AT inflammation and insulin sensitivity. Methods and Results- We developed an optochemical measurement system for continuous monitoring of AT Po(2) using microdialysis. The effect of alterations in ATBF on AT Po(2) was investigated in lean and obese subjects with both pharmacological and physiological approaches to manipulate ATBF. Local administration of angiotensin II (vasoconstrictor) in abdominal subcutaneous AT decreased ATBF and AT Po(2), whereas infusion of isoprenaline (vasodilator) evoked opposite effects. Ingestion of a glucose drink increased ATBF and AT Po(2) in lean subjects, but these responses were blunted in obese individuals. However, AT Po(2) was higher (hyperoxia) in obese subjects despite lower ATBF, which appears to be explained by lower AT oxygen consumption. This was accompanied by insulin resistance, lower AT capillarization, lower AT expression of genes encoding proteins involved in mitochondrial biogenesis and function, and higher AT gene expression of macrophage infiltration and inflammatory markers. Conclusions- Our findings establish ATBF as an important regulator of AT Po(2). Nevertheless, obese individuals exhibit AT hyperoxia despite lower ATBF, which seems to be explained by lower AT oxygen consumption. This is accompanied by insulin resistance, impaired AT capillarization, and higher AT gene expression of inflammatory cell markers. Clinical Trial Registration- URL: http://www.trialregister.nl. Unique identifier: NTR2451

Diet-induced weight loss decreases adipose tissue oxygen tension with parallel changes in adipose tissue phenotype and insulin sensitivity in overweight humans

Background/objectives: Although adipose tissue (AT) hypoxia is present in rodent models of obesity, evidence for this in humans is limited. Here, we investigated the effects of diet-induced weight loss (WL) on abdominal subcutaneous AT oxygen tension (pO 2), AT blood flow (ATBF), AT capillary density, AT morphology and transcriptome, systemic inflammatory markers and insulin sensitivity in humans. Subjects/methods: Fifteen overweight and obese individuals underwent a dietary intervention (DI), consisting of a 5-week very-low-calorie diet (VLCD, 500 kcal day -1; WL), and a subsequent 4-week weight stable diet (WS). Body composition, AT pO 2 (optochemical monitoring), ATBF (133 Xe washout), and whole-body insulin sensitivity were determined, and AT biopsies were collected at baseline, end of WL (week 5) and end of WS (week 9). Result: Body weight, body fat percentage and adipocyte size decreased significantly during the DI period. The DI markedly decreased AT pO 2 and improved insulin sensitivity, but did not alter ATBF. Finally, the DI increased AT gene expression of pathways related to mitochondrial biogenesis and non-mitochondrial oxygen consumption. Conclusions: VLCD-induced WL markedly decreases abdominal subcutaneous AT pO 2, which is paralleled by a reduction in adipocyte size, increased AT gene expression of mitochondrial biogenesis markers and non-mitochondrial oxygen consumption pathways, and improved whole-body insulin sensitivity in humans.</p

A Metal–Organic Framework-Based Material for Electrochemical Sensing of Carbon Dioxide

The free primary hydroxyl groups in the metal-organic framework of CDMOF-2, an extended cubic structure containing units of six Î-cyclodextrin tori linked together in cube-like fashion by rubidium ions, has been shown to react with gaseous CO2 to form alkyl carbonate functions. The dynamic covalent carbon-oxygen bond, associated with this chemisorption process, releases CO2 at low activation energies. As a result of this dynamic covalent chemistry going on inside a metal-organic framework, CO2 can be detected selectively in the atmosphere by electrochemical impedance spectroscopy. The as-synthesized CDMOF-2 which exhibits high proton conductivity in pore-filling methanolic media, displays a ∼550-fold decrease in its ionic conductivity on binding CO2. This fundamental property has been exploited to create a sensor capable of measuring CO2 concentrations quantitatively even in the presence of ambient oxygen. © 2014 American Chemical Society.1