Application of Quantum Cascade Laser-Infrared Spectroscopy and Chemometrics for In-Line Discrimination of Coeluting Proteins from Preparative Size Exclusion Chromatography

An external-cavity quantum cascade laser (EC-QCL)-based flow-through mid-infrared (IR) spectrometer was placed in line with a preparative size exclusion chromatography system to demonstrate real-time analysis of protein elutions with strongly overlapping chromatographic peaks. Two different case studies involving three and four model proteins were performed under typical lab-scale purification conditions. The large optical path length (25 μm), high signal-to-noise ratios, and wide spectral coverage (1350 to 1750 cm-1) of the QCL-IR spectrometer allow for robust spectra acquisition across both the amide I and II bands. Chemometric analysis by self-modeling mixture analysis and multivariate curve resolution enabled accurate quantitation and structural fingerprinting across the protein elution transient. The acquired concentration profiles were found to be in excellent agreement with the off-line high-performance liquid chromatography reference analytics performed on the collected effluent fractions. These results demonstrate that QCL-IR detectors can be used effectively for in-line, real-time analysis of protein elutions, providing critical quality attribute data that are typically only accessible through time-consuming and resource-intensive off-line methods.Fil: Akhgar, Christopher K.. Vienna University of Technology; AustriaFil: Ebner, Julian. Vienna University of Technology; AustriaFil: Alcaraz, Mirta R.. Institute Of Chemical Technologies And Analytics; AustriaFil: Kopp, Julian. Vienna University of Technology; AustriaFil: Goicoechea, Hector Casimiro. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Spadiut, Oliver. Vienna University of Technology; AustriaFil: Schwaighofer, Andreas. Vienna University of Technology; AustriaFil: Lendl, Bernhard. Vienna University of Technology; Austri

Timing and Grounding in Motor Skill Coaching Interaction: Consequences for the Information State

Hough J, de Kok I, Schlangen D, Kopp S. Timing and Grounding in Motor Skill Coaching Interaction: Consequences for the Information State. In: Proceedings of the 19th SemDial Workshop on the Semantics and Pragmatics of Dialogue (goDIAL). 2015: 86-94

Deictic gestures in coaching interactions

de Kok I, Hough J, Schlangen D, Kopp S. Deictic gestures in coaching interactions. In: Proceedings of the Workshop on Multimodal Analyses enabling Artificial Agents in Human-Machine Interaction - MA3HMI '16. New York, NY: Association for Computing Machinery (ACM); 2016: 1

State-of-the-art and novel approaches to mild solubilization of inclusion bodies

Throughout the twenty-first century, the view on inclusion bodies (IBs) has shifted from undesired by-products towards a targeted production strategy for recombinant proteins. Inclusion bodies can easily be separated from the crude extract after cell lysis and contain the product in high purity. However, additional solubilization and refolding steps are required in the processing of IBs to recover the native protein. These unit operations remain a highly empirical field of research in which processes are developed on a case-by-case basis using elaborate screening strategies. It has been shown that a reduction in denaturant concentration during protein solubilization can increase the subsequent refolding yield due to the preservation of correctly folded protein structures. Therefore, many novel solubilization techniques have been developed in the pursuit of mild solubilization conditions that avoid total protein denaturation. In this respect, ionic liquids have been investigated as promising agents, being able to solubilize amyloid-like aggregates and stabilize correctly folded protein structures at the same time. This review briefly summarizes the state-of-the-art of mild solubilization of IBs and highlights some challenges that prevent these novel techniques from being yet adopted in industry. We suggest mechanistic models based on the thermodynamics of protein unfolding with the aid of molecular dynamics simulations as a possible approach to solve these challenges in the future

Dialogue Structure of Coaching Sessions

de Kok I, Hough J, Frank C, Schlangen D, Kopp S. Dialogue Structure of Coaching Sessions. In: Proceedings of the 18th SemDial Workshop on the Semantics and Pragmatics of Dialogue (DialWatt), Posters. Herriot-Watt University; 2014: 167-169

A Multimodal System for Real-Time Action Instruction in Motor Skill Learning

de Kok I, Hough J, Hülsmann F, Botsch M, Schlangen D, Kopp S. A Multimodal System for Real-Time Action Instruction in Motor Skill Learning. In: Proceedings of ACM ICMI 17th ACM International Conference on Multimodal Interaction. 2015: 355-362

Intestinal PTGS2 mRNA Levels, PTGS2 Gene Polymorphisms, and Colorectal Carcinogenesis

<div><p>Background & Aims</p><p>Inflammation is a major risk factor for development of colorectal cancer (CRC). Prostaglandin synthase cyclooxygenase-2 (COX-2) encoded by the <i>PTGS2</i> gene is the rate limiting enzyme in prostaglandin synthesis and therefore plays a distinct role as regulator of inflammation.</p><p>Methods</p><p><i>PTGS2</i> mRNA levels were determined in intestinal tissues from 85 intestinal adenoma cases, 115 CRC cases, and 17 healthy controls. The functional <i>PTGS2</i> polymorphisms A-1195G (rs689466), G-765C (rs20417), T8473C (rs5275) were assessed in 200 CRC cases, 991 adenoma cases and 399 controls from the Norwegian KAM cohort.</p><p>Results</p><p><i>PTGS2</i> mRNA levels were higher in mild/moderate adenoma tissue compared to morphologically normal tissue from the same individual (P<0.0001) and (P<0.035) and compared to mucosa from healthy individuals (P<0.0039) and (P<0.0027), respectively. In CRC patients, <i>PTGS2</i> mRNA levels were 8–9 times higher both in morphologically normal tissue and in cancer tissue, compared to healthy individuals (P<0.0001). <i>PTGS2</i> A-1195G variant allele carriers were at reduced risk of CRC (odds ratio (OR) = 0.52, 95% confidence interval (95% CI): 0.28–0.99, P = 0.047). Homozygous carriers of the haplotype encompassing the A-1195G and G-765C wild type alleles and the T8473C variant allele <i>(PTGS2</i> AGC) were at increased risk of CRC as compared to homozygous carriers of the <i>PTGS2</i> AGT (<u>A</u>-1195G, <u>G</u>-765C, <u>T</u>8473C) haplotype (OR = 5.37, 95% CI: 1.40–20.5, P = 0.014). No association between the investigated polymorphisms and <i>PTGS2</i> mRNA levels could be detected.</p><p>Conclusion</p><p>High intestinal <i>PTGS2</i> mRNA level is an early event in colorectal cancer development as it occurs already in mild/moderate dysplasia. <i>PTGS2</i> polymorphisms that have been associated with altered <i>PTGS2</i> mRNA levels/COX-2 activity in some studies, although not the present study, were associated with colorectal cancer risk. Thus, both <i>PTGS2</i> polymorphisms and <i>PTGS2</i> mRNA levels may provide information regarding CRC risk.</p></div

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing.

peer reviewedContinuous cultivation with Escherichia coli has several benefits compared to classical fed-batch cultivation. The economic benefits would be a stable process, which leads to time independent quality of the product, and hence ease the downstream process. However, continuous biomanufacturing with E. coli is known to exhibit a drop of productivity after about 4-5 days of cultivation depending on dilution rate. These cultivations are generally performed on glucose, being the favorite carbon source for E. coli and used in combination with isopropyl β-D-1 thiogalactopyranoside (IPTG) for induction. In recent works, harsh induction with IPTG was changed to softer induction using lactose for T7-based plasmids, with the result of reducing the metabolic stress and tunability of productivity. These mixed feed systems based on glucose and lactose result in high amounts of correctly folded protein. In this study we used different mixed feed systems with glucose/lactose and glycerol/lactose to investigate productivity of E. coli based chemostats. We tested different strains producing three model proteins, with the final aim of a stable long-time protein expression. While glucose fed chemostats showed the well-known drop in productivity after a certain process time, glycerol fed cultivations recovered productivity after about 150 h of induction, which corresponds to around 30 generation times. We want to further highlight that the cellular response upon galactose utilization in E. coli BL21(DE3), might be causing fluctuating productivity, as galactose is referred to be a weak inducer. This "Lazarus" phenomenon has not been described in literature before and may enable a stabilization of continuous cultivation with E. coli using different carbon sources