Folding and self-assembly of the TatA translocation pore based on a charge zipper mechanism.

We propose a concept for the folding and self-assembly of the pore-forming TatA complex from the Twin-arginine translocase and of other membrane proteins based on electrostatic "charge zippers." Each subunit of TatA consists of a transmembrane segment, an amphiphilic helix (APH), and a C-terminal densely charged region (DCR). The sequence of charges in the DCR is complementary to the charge pattern on the APH, suggesting that the protein can be "zipped up" by a ladder of seven salt bridges. The length of the resulting hairpin matches the lipid bilayer thickness, hence a transmembrane pore could self-assemble via intra- and intermolecular salt bridges. The steric feasibility was rationalized by molecular dynamics simulations, and experimental evidence was obtained by monitoring the monomer-oligomer equilibrium of specific charge mutants. Similar "charge zippers" are proposed for other membrane-associated proteins, e.g., the biofilm-inducing peptide TisB, the human antimicrobial peptide dermcidin, and the pestiviral E(RNS) protein.

Capturing spatially resolved kinetic data and coking of Ga-Pt supported catalytically active liquid metal solutions during propane dehydrogenation in situ.

Supported liquid phase catalysis has great potential to unify the advantages from both homogeneous and heterogeneous catalysis. Recently, we reported supported catalytically active liquid metal solutions (SCALMS) as a new class of liquid phase catalysts. SCALMS enable high temperature application due to the high thermal stability of liquid metals when compared to supported molten salts or ionic liquids. The highly dynamic liquid metal/gas interface of SCALMS allows for catalysis over single atoms of an active metal atom within a matrix of liquid gallium. In the present study, kinetic data is acquired along the catalyst bed in a compact profile reactor during propane dehydrogenation (PDH) over gallium-platinum SCALMS. The reactor design allows for the analysis of the temperature and gas phase composition along the catalyst bed with a high spatial resolution using a sampling capillary inside the reactor. The concentration profiles suggest enhanced deactivation of the catalyst at the end of the bed with a deactivation front moving from the end to the beginning of the catalyst bed over time on stream. Only minor amounts of side products, formed via cracking of propane, were identified, supporting previously reported high selectivity of SCALMS during alkane dehydrogenation. The acquired data is supported by in situ high-resolution thermogravimetry coupled with mass spectrometry to monitor the activity and coking behaviour of SCALMS during PDH. The results strongly suggest an enhanced formation of coke over Al2O3-supported SCALMS when compared to using SiO2 as the support material.

Process-wide control and automation of an integrated continuous manufacturing platform for antibodies.

Integrated continuous manufacturing is entering the biopharmaceutical industry. The main drivers range from improved economics, manufacturing flexibility, and more consistent product quality. However, studies on fully integrated production platforms have been limited due to the higher degree of system complexity, limited process information, disturbance, and drift sensitivity, as well as difficulties in digital process integration. In this study, we present an automated end-to-end integrated process consisting of a perfusion bioreactor, CaptureSMB, virus inactivation (VI), and two polishing steps to produce an antibody from an instable cell line. A supervisory control and data acquisition (SCADA) system was developed, which digitally integrates unit operations and analyzers, collects and centrally stores all process data, and allows process-wide monitoring and control. The integrated system consisting of bioreactor and capture step was operated initially for 4 days, after which the full end-to-end integrated run with no interruption lasted for 10 days. In response to decreasing cell-specific productivity, the supervisory control adjusted the loading duration of the capture step to obtain high capacity utilization without yield loss and constant antibody quantity for subsequent operations. Moreover, the SCADA system coordinated VI neutralization and discharge to enable constant loading conditions on the polishing unit. Lastly, the polishing was sufficiently robust to cope with significantly increased aggregate levels induced on purpose during virus inactivation. It is demonstrated that despite significant process disturbances and drifts, a robust process design and the supervisory control enabled constant (optimum) process performance and consistent product quality.

Process design and development of a mammalian cell perfusion culture in shake-tube and benchtop bioreactors.

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCDmax ), minimum cell-specific perfusion rate (CSPRmin ), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell-1 ·day-1 ) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 106 cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures.

Development of a shake tube-based scale-down model for perfusion cultures.

The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption.

Size dependent stability of cobalt nanoparticles on silica under high conversion Fischer-Tropsch environment.

Highly monodisperse cobalt crystallites, supported on Stöber silica spheres, as model catalysts for the Fischer-Tropsch synthesis were exposed to simulated high conversion environments in the presence and absence of CO utilising an in house developed in situ magnetometer. The catalyst comprising the smallest crystallites in the metallic state (average diameter of 3.2 nm) experienced pronounced oxidation whilst the ratio of H2O to H2 was increased stepwise to simulate CO conversions from 26% up to complete conversion. Direct exposure of this freshly reduced catalyst to a high conversion Fischer-Tropsch environment resulted in almost spontaneous oxidation of 40% of the metallic cobalt. In contrast, a model catalyst with cobalt crystallites of 5.3 nm only oxidised to a small extent even when exposed to a simulated conversion of over 99%. The largest cobalt crystallites were rather stable and only experienced measurable oxidation when subjected to H2O in the absence of H2. This size dependency of the stability is in qualitative accordance with reported thermodynamic calculations. However, the cobalt crystallites showed an unexpected low susceptibility to oxidation, i.e. only relatively high ratios of H2O to H2 partial pressure caused oxidation. Similar experiments in the presence of CO revealed the significance of the actual Fischer-Tropsch synthesis on the metallic surface as the dissociation of CO, an elementary step in the Fischer-Tropsch mechanism, was shown to be a prerequisite for oxidation. Direct oxidation of cobalt to CoO by H2O seems to be kinetically hindered. Thus, H2O may only be capable of indirect oxidation, i.e. high concentrations prevent the removal of adsorbed oxygen species on the cobalt surface leading to oxidation. However, a spontaneous direct oxidation of cobalt at the interface between the support and the crystallites by H2O forming presumably cobalt silicate type species was observed in the presence and absence of CO. The formation of these metal-support compounds is in accordance with conducted thermodynamic predictions. None of the extreme Fischer-Tropsch conditions initiated hydrothermal sintering. Seemingly, the formation of metal-support compounds stabilised the metallic crystallites and/or higher partial pressures of CO are required to increase the concentration of mobile, cobalt oxide-type species on the metallic surface.

Structure of the membrane anchor of pestivirus glycoprotein E(rns), a long tilted amphipathic helix.

E(rns) is an essential virion glycoprotein with RNase activity that suppresses host cellular innate immune responses upon being partially secreted from the infected cells. Its unusual C-terminus plays multiple roles, as the amphiphilic helix acts as a membrane anchor, as a signal peptidase cleavage site, and as a retention/secretion signal. We analyzed the structure and membrane binding properties of this sequence to gain a better understanding of the underlying mechanisms. CD spectroscopy in different setups, as well as Monte Carlo and molecular dynamics simulations confirmed the helical folding and showed that the helix is accommodated in the amphiphilic region of the lipid bilayer with a slight tilt rather than lying parallel to the surface. This model was confirmed by NMR analyses that also identified a central stretch of 15 residues within the helix that is fully shielded from the aqueous layer, which is C-terminally followed by a putative hairpin structure. These findings explain the strong membrane binding of the protein and provide clues to establishing the E(rns) membrane contact, processing and secretion.

Experimental Validation of a Real-Time Adaptive 4D-Optimized Particle Radiotherapy Approach to Treat Irregularly Moving Tumors.

PURPOSE: Treatment of locally advanced lung cancer is limited by toxicity and insufficient local control. Particle therapy could enable more conformal treatment than intensity modulated photon therapy but is challenged by irregular tumor motion, associated range changes, and tumor deformations. We propose a new strategy for robust, online adaptive particle therapy, synergizing 4-dimensional optimization with real-time adaptive beam tracking. The strategy was tested and the required motion monitoring precision was determined. METHODS AND MATERIALS: In multiphase 4-dimensional dose delivery (MP4D), a dedicated quasistatic treatment plan is delivered to each motion phase of periodic 4-dimensional computed tomography (4DCT). In the new extension, "MP4D with residual tracking" (MP4DRT), lateral beam tracking compensates for the displacement of the tumor center-of-mass relative to the current phase in the planning 4DCT. We implemented this method in the dose delivery system of a clinical carbon facility and tested it experimentally for a lung cancer plan based on a periodic subset of a virtual lung 4DCT (planned motion amplitude 20 mm). Treatments were delivered in a quality assurance-like setting to a moving ionization chamber array. We considered variable motion amplitudes and baseline drifts. The required motion monitoring precision was evaluated by adding noise to the motion signal. Log-file-based dose reconstructions were performed in silico on the entire 4DCT phantom data set capable of simulating nonperiodic motion. MP4DRT was compared with MP4D, rescanned beam tracking, and internal target volume plans. Treatment quality was assessed in terms of target coverage (D95), dose homogeneity (D5-D95), conformity number, and dose to heart and lung. RESULTS: For all considered motion scenarios and metrics, MP4DRT produced the most favorable metrics among the tested motion mitigation strategies and delivered high-quality treatments. The conformity was similar to static treatments. The motion monitoring precision required for D95 >95% was 1.9 mm. CONCLUSIONS: With clinically feasible motion monitoring, MP4DRT can deliver highly conformal dose distributions to irregularly moving targets.

Dry reforming of methane over gallium-based supported catalytically active liquid metal solutions.

Gallium-rich supported catalytically active liquid metal solutions (SCALMS) were recently introduced as a new way towards heterogeneous single atom catalysis. SCALMS were demonstrated to exhibit a certain resistance against coking during the dehydrogenation of alkanes using Ga-rich alloys of noble metals. Here, the conceptual catalytic application of SCALMS in dry reforming of methane (DRM) is tested with non-noble metal (Co, Cu, Fe, Ni) atoms in the gallium-rich liquid alloy. This study introduces SCALMS to high-temperature applications and an oxidative reaction environment. Most catalysts were shown to undergo severe oxidation during DRM, while Ga-Ni SCALMS retained a certain level of activity. This observation is explained by a kinetically controlled redox process, namely oxidation to gallium oxide species and re-reduction via H2 activation over Ni. Consequentially, this redox process can be shifted to the metallic side when using increasing concentrations of Ni in Ga, which strongly suppresses coke formation. Density-functional theory (DFT) based ab initio molecular dynamics (AIMD) simulations were performed to confirm the increased availability of Ni at the liquid alloy-gas interface. However, leaching of gallium via the formation of volatile oxidic species during the hypothesised redox cycles was identified indicating a critical instability of Ga-Ni SCALMS for prolonged test durations.

Thermodynamic assessment of the stability of bulk and nanoparticulate cobalt and nickel during dry and steam reforming of methane.

The high reaction temperatures during steam and dry reforming of methane inevitably entail catalyst deactivation. Evaluation of the feasibility or potentially relevant mechanisms at play is of utmost importance to develop highly active and stable catalysts. Herein, various oxidation reactions of bulk-sized nickel and cobalt to the corresponding metal oxide or in the presence of a metal oxide carrier are evaluated thermodynamically and linked to approximated conditions during methane reforming. In particular cobalt aluminate, as well as cobalt or nickel titanates are likely to form. As oxidation to bulk-sized metal oxide is unlikely, a thermodynamic analysis of metallic nanoparticles was performed to calculate the size dependent stability against oxidation to nickel oxide or cobalt oxide in water and carbon dioxide-rich environments. The calculations indicate that nickel nanoparticles >3 nm and cobalt nanoparticles >10 nm are expected to withstand oxidation during steam and dry reforming of methane with stoichiometric feed compositions and methane conversion levels >10% at temperatures up to 1100 and 900 °C, respectively. Lastly, the reduced thermal stability of nanoparticles due to melting point suppression was assessed, leading to similar recommendations concerning minimum particle sizes.

Dosimetric Validation of a System to Treat Moving Tumors Using Scanned Ion Beams That Are Synchronized With Anatomical Motion.

PURPOSE: The purpose of this study was to validate the dosimetric performance of scanned ion beam deliveries with motion-synchronization to heterogenous targets. METHODS: A 4D library of treatment plans, comprised of up to 10 3D sub-plans, was created with robust and conventional 4D optimization methods. Each sub-plan corresponded to one phase of periodic target motion. The plan libraries were delivered to a test phantom, comprising plastic slabs, dosimeters, and heterogenous phantoms. This phantom emulated range changes that occur when treating moving tumors. Similar treatment plans, but without motion synchronization, were also delivered to a test phantom with a stationary target and to a moving target; these were used to assess how the target motion degrades the quality of dose distributions and the extent to which motion synchronization can improve dosimetric quality. The accuracy of calculated dose distributions was verified by comparison with corresponding measurements. Comparisons utilized the gamma index analysis method. Plan quality was assessed based on conformity, dose coverage, overdose, and homogeneity values, each extracted from calculated dose distributions. RESULTS: High pass rates for the gamma index analysis confirmed that the methods used to calculate and reconstruct dose distributions were sufficiently accurate for the purposes of this study. Calculated and reconstructed dose distributions revealed that the motion-synchronized and static deliveries exhibited similar quality in terms of dose coverage, overdose, and homogeneity for all deliveries considered. Motion-synchronization substantially improved conformity in deliveries with moving targets. Importantly, measurements at multiple locations within the target also confirmed that the motion-synchronized delivery system satisfactorily compensated for changes in beam range caused by the phantom motion. Specifically, the overall planning and delivery approach achieved the desired dose distribution by avoiding range undershoots and overshoots caused by tumor motion. CONCLUSIONS: We validated a dose delivery system that synchronizes the movement of the ion beam to that of a moving target in a test phantom. Measured and calculated dose distributions revealed that this system satisfactorily compensated for target motion in the presence of beam range changes due to target motion. The implication of this finding is that the prototype system is suitable for additional preclinical research studies, such as irregular anatomic motion.

Preliminary tests of dosimetric quality and projected therapeutic outcomes of multi-phase 4D radiotherapy with proton and carbon ion beams.

Objective. The purpose of this study was to perform preliminary pre-clinical tests to compare the dosimetric quality of two approaches to treating moving tumors with ion beams: synchronously delivering the beam with the motion of a moving planning target volume (PTV) using the recently developed multi-phase 4D dose delivery (MP4D) approach, and asynchronously delivering the ion beam to a motion-encompassing internal tumor volume (ITV) combined with rescanning.Approach. We created 4D optimized treatment plans with proton and carbon ion beams for two patients who had previously received treatment for non-small cell lung cancer. For each patient, we created several treatment plans, using approaches with and without motion mitigation: MP4D, ITV with rescanning, static deliveries to a stationary PTV, and deliveries to a moving tumor without motion compensation. Two sets of plans were optimized with margins or robust uncertainty scenarios. Each treatment plan was delivered using a recently-developed motion-synchronized dose delivery system (M-DDS); dose distributions in water were compared to measurements using gamma index analysis to confirm the accuracy of the calculations. Reconstructed dose distributions on the patient CT were analyzed to assess the dosimetric quality of the deliveries (conformity, uniformity, tumor coverage, and extent of hotspots).Main results. Gamma index analysis pass rates confirmed the accuracy of dose calculations. Dose coverage was >95% for all static and MP4D treatments. The best conformity and the lowest lung doses were achieved with MP4D deliveries. Robust optimization led to higher lung doses compared to conventional optimization for ITV deliveries, but not for MP4D deliveries.Significance. We compared dosimetric quality for two approaches to treating moving tumors with ion beams. Our findings suggest that the MP4D approach, using an M-DDS, provides conformal motion mitigation, with full target coverage and lower OAR doses.

A Modular System for Treating Moving Anatomical Targets With Scanned Ion Beams at Multiple Facilities: Pre-Clinical Testing for Quality and Safety of Beam Delivery.

BACKGROUND: Quality management and safety are integral to modern radiotherapy. New radiotherapy technologies require new consensus guidelines on quality and safety. Established analysis strategies, such as the failure modes and effects analysis (FMEA) and incident learning systems have been developed as tools to assess the safety of several types of radiation therapies. An extensive literature documents the widespread application of risk analysis methods to photon radiation therapy. Relatively little attention has been paid to performing risk analyses of nascent radiation therapy systems to treat moving tumors with scanned heavy ion beams. The purpose of this study was to apply a comprehensive safety analysis strategy to a motion-synchronized dose delivery system (M-DDS) for ion therapy. METHODS: We applied a risk analysis method to new treatment planning and treatment delivery processes with scanned heavy ion beams. The processes utilize a prototype, modular dose delivery system, currently undergoing preclinical testing, that provides new capabilities for treating moving anatomy. Each step in the treatment process was listed in a process map, potential errors for each step were identified and scored using the risk probability number in an FMEA, and the possible causes of each error were described in a fault tree analysis. Solutions were identified to mitigate the risk of these errors, including permanent corrective actions, periodic quality assurance (QA) tests, and patient specific QA (PSQA) tests. Each solution was tested experimentally. RESULTS: The analysis revealed 58 potential errors that could compromise beam delivery quality or safety. Each of the 14 binary (pass-or-fail) tests passed. Each of the nine QA and four PSQA tests were within anticipated clinical specifications. The modular M-DDS was modified accordingly, and was found to function at two centers. CONCLUSION: We have applied a comprehensive risk analysis strategy to the M-DDS and shown that it is a clinically viable motion mitigation strategy. The described strategy can be utilized at any ion therapy center that operates with the modular M-DDS. The approach can also be adapted for use at other facilities and can be combined with existing safety analysis systems.

GaPt Supported Catalytically Active Liquid Metal Solution Catalysis for Propane Dehydrogenation-Support Influence and Coking Studies.

Supported catalytically active liquid metal solutions (SCALMS) of Pt in Ga (2 at.-% Pt) were studied in the temperature range of 500 to 600 °C for propane dehydrogenation. A facile synthesis procedure using ultrasonication was implemented and compared to a previously reported organo-chemical route for gallium deposition. The procedure was applied to synthesize GaPt-SCALMS catalyst on silica (SiO2), alumina (Al2O3), and silicon carbide (SiC) to investigate the effect of the support material on the catalytic performance. The SiC-based SCALMS catalyst showed the highest activity, while SiO2-based SCALMS showed the highest stability and lowest cracking tendency at higher temperatures. The selectivity toward propene for the SiO2-based catalyst remained above 93% at 600 °C. The catalysts were analyzed for coke content after use by temperature-programmed oxidation (TPO) and Raman spectroscopy. While the SiC- and SiO2-supported SCALMS systems showed hardly any coke formation, the Al2O3-supported systems suffered from pronounced coking. SEM-EDX analyses of the catalysts before and after reaction indicated that no perceivable morphological changes occur during reaction. The SCALMS catalysts under investigation are compared with supported Pt and supported GaPt solid-phase catalyst, and possible deactivation pathways are discussed.