Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

The determination of cell viability is essential to many areas of life sciences and biotechnology. Typically, cell viability measurements are based on the optical analysis of stained cells, which requires additional labeling steps and is hard to implement online. Frequency-dependent impedance flow cytometry (IFC) provides a label-free, fast, and reliable alternative to determine cell viability at the single cell level based on the Coulter principle. Here, we describe the application of IFC to eukaryotic cell cultures and compare the results to commonly used staining methods. Yeast cell parameters were assessed in normal and heat-inactivated cells as well as in alcoholic fermentation and long-term batch cultures providing a precise and fast determination of the cell viability and further quantitative measures of the cell culture status. As an important new application, we have investigated recombinant protein production in the widely used baculovirus insect cell expression system. The IFC analysis revealed the presence of a subpopulation of cells, which correlates with the protein expression yield, but it is not detectable with conventional optical cell counters. We tentatively identify this subpopulation as cells in the late phase of infection. Their detection can serve as a predictor for the optimal time point of harvest. The IFC technique should be generally applicable to many eukaryotic cell cultures in suspension, possibly also implemented online

Ambipolar Blends of Cu-Phthalocyanine and Fullerene: Charge Carrier Mobility, Electronic Structure and their Implications for Solar Cell Applications

Summary: Ambipolar transport has been realised in blends of the molecular hole conductor Cu-phthalocyanine (CuPc) and the electron conducting fullerene C 60 . Charge carrier mobilities and the occupied electronic levels have been analyzed as a function of the mixing ratio using field-effect transistor measurements and photoelectron spectroscopy. These results are discussed in the context of photovoltaic cells based on these materials

Multistep crystallization pathways in the ambient-temperature synthesis of a new alkali-activated binder

Concrete is the most prevalent manufactured material that has shaped the built environment, but the high-temperature production of cement, the main component of concrete, has a massive carbon footprint. It is shown that CO2 emissions during clinker production of cement can be circumvented by a metathesis reaction at room temperature in ball-mills, where the cement clinker is replaced by non-calcined limestone and alkali-activated binders/geopolymers. An amorphous intermediate (aNaSiCC) containing a random mixture of the ionic constituents in “molecular” dispersion is formed by mechanochemical activation of CaCO3 and Na2SiO3. This allows molecular transport during crystallization and low activated reactions, as precipitation of solids from liquids (nucleation limited and kinetically controlled) and solid-state transformations (diffusion-limited and thermodynamically controlled) have equal weight. Several steps of the hydration reaction could be resolved. Activating the amorphous aNaSiCC precursor with NaOH leads to a CSH-like phase with a C/S ratio of ≈1 containing some sodium. The carbonate components pass through a multistep crystallization from aNaSiCC via pirssonite and gaylussite to monohydrocalcite. The findings help unravel the interplay between thermodynamics and kinetics in complex reactions of alkali-activated binders and for CaCO3 crystallization in industrial and geochemical settings, where dissolved silicate is always involved

Automated Liquid Handling Extraction and Rapid Quantification of Underivatized Amino Acids and Tryptophan Metabolites from Human Serum and Plasma Using Dual-Column U(H)PLC-MRM-MS and Its Application to Prostate Cancer Study

Amino acids (AAs) and their metabolites are important building blocks, energy sources, and signaling molecules associated with various pathological phenotypes. The quantification of AA and tryptophan (TRP) metabolites in human serum and plasma is therefore of great diagnostic interest. Therefore, robust, reproducible sample extraction and processing workflows as well as rapid, sensitive absolute quantification are required to identify candidate biomarkers and to improve screening methods. We developed a validated semi-automated robotic liquid extraction and processing workflow and a rapid method for absolute quantification of 20 free, underivatized AAs and six TRP metabolites using dual-column U(H)PLC-MRM-MS. The extraction and sample preparation workflow in a 96-well plate was optimized for robust, reproducible high sample throughput allowing for transfer of samples to the U(H)PLC autosampler directly without additional cleanup steps. The U(H)PLC-MRM-MS method, using a mixed-mode reversed-phase anion exchange column with formic acid and a high-strength silica reversed-phase column with difluoro-acetic acid as mobile phase additive, provided absolute quantification with nanomolar lower limits of quantification within 7.9 min. The semi-automated extraction workflow and dual-column U(H)PLC-MRM-MS method was applied to a human prostate cancer study and was shown to discriminate between treatment regimens and to identify metabolites responsible for discriminating between healthy controls and patients on active surveillance.</p

Prospective Life Cycle Assessment of Recycled Carbon Fibres from a Solvolysis Process and their Potential Applications in the Transport and Building Sector

Due to the high energy input and consumption of non-renewable resources for the production of today’s virgin carbon fibres (vCF) based on Polyacrylonitrile (PAN), their multiple use in a potential circular economy seems desirable from economic and ecological point of view. The EDISON-rCF project demonstrates the potentials of a recycling process chain for carbon fibre reinforced polymers (CFRP). One- and two-dimensional semi-finished products for subsequential use in the transport and construction sectors were developed and tested based on recycled carbon fibres (rCF) obtained from an improved Solvolysis process. In a Life Cycle Assessment (LCA) for CFRP based lightweight systems the modelling and interpretation of a future cascading product system is a challenge because of Life Cycle Inventory (LCI) data quality and allocation methods. The availability of reliable data for the virgin carbon fibre production is limited and literature data shows considerable scattering of energy consumption and incompleteness regarding the covered phases of the production. Particularly in the recycling of high-performance materials such as carbon fibres, attention must be paid to potential performance losses (downcycling) and corresponding influence on their applicability. An important factor for the establishment of recycling routes is the creation of incentives for the recycling including a useful application of such materials. The allocation of potential environmental impacts based on different EoL Formulas plays a crucial role here. The presentation sheds a light on the preliminary results for the use of rCF in two potential use-cases, a bicycle transport box and rebars for carbon concrete. For this, the whole process chain rom a cradle-to-gate perspective was modeled using foreground and background data with different level of completeness. Therefore, the uncertainty of this prospective LCA is high and shows the potential shortcomings of incomplete data based on missing information and low technology readiness level

Comparative Assessment of Quantification Methods for Tumor Tissue Phosphoproteomics

With increasing sensitivity and accuracy in mass spectrometry, the tumor phosphoproteome is getting into reach. However, the selection of quantitation techniques best-suited to the biomedical question and diagnostic requirements remains a trial and error decision as no study has directly compared their performance for tumor tissue phosphoproteomics. We compared label-free quantification (LFQ), spike-in-SILAC (stable isotope labeling by amino acids in cell culture), and tandem mass tag (TMT) isobaric tandem mass tags technology for quantitative phosphosite profiling in tumor tissue. Compared to the classic SILAC method, spike-in-SILAC is not limited to cell culture analysis, making it suitable for quantitative analysis of tumor tissue samples. TMT offered the lowest accuracy and the highest precision and robustness toward different phosphosite abundances and matrices. Spike-in-SILAC offered the best compromise between these features but suffered from a low phosphosite coverage. LFQ offered the lowest precision but the highest number of identifications. Both spike-in-SILAC and LFQ presented susceptibility to matrix effects. Match between run (MBR)-based analysis enhanced the phosphosite coverage across technical replicates in LFQ and spike-in-SILAC but further reduced the precision and robustness of quantification. The choice of quantitative methodology is critical for both study design such as sample size in sample groups and quantified phosphosites and comparison of published cancer phosphoproteomes. Using ovarian cancer tissue as an example, our study builds a resource for the design and analysis of quantitative phosphoproteomic studies in cancer research and diagnostics

Pacific Northwest National Laboratory Site Environmental Report for Calendar Year 2011

The PNNL Site Environmental Report for Calendar Year 2011 was prepared pursuant to the requirements of Department of Energy (DOE) Order 231.1B, "Environment, Safety and Health Reporting" to provide a synopsis of calendar year 2011 information related to environmental management performance and compliance efforts. It summarizes site compliance with federal, state, and local environmental laws, regulations, policies, directives, permits, and orders and environmental management performance

On the Role of Summary Content Units in Text Summarization Evaluation

At the heart of the Pyramid evaluation method for text summarization lie human written summary content units (SCUs). These SCUs are concise sentences that decompose a summary into small facts. Such SCUs can be used to judge the quality of a candidate summary, possibly partially automated via natural language inference (NLI) systems. Interestingly, with the aim to fully automate the Pyramid evaluation, Zhang and Bansal (2021) show that SCUs can be approximated by automatically generated semantic role triplets (STUs). However, several questions currently lack answers, in particular: i) Are there other ways of approximating SCUs that can offer advantages? ii) Under which conditions are SCUs (or their approximations) offering the most value? In this work, we examine two novel strategies to approximate SCUs: generating SCU approximations from AMR meaning representations (SMUs) and from large language models (SGUs), respectively. We find that while STUs and SMUs are competitive, the best approximation quality is achieved by SGUs. We also show through a simple sentence-decomposition baseline (SSUs) that SCUs (and their approximations) offer the most value when ranking short summaries, but may not help as much when ranking systems or longer summaries.Comment: 10 Pages, 3 Figures, 3 Tables, camera ready version accepted at NAACL 202

Real-World Federated Learning in Radiology: Hurdles to overcome and Benefits to gain

Objective: Federated Learning (FL) enables collaborative model training while keeping data locally. Currently, most FL studies in radiology are conducted in simulated environments due to numerous hurdles impeding its translation into practice. The few existing real-world FL initiatives rarely communicate specific measures taken to overcome these hurdles, leaving behind a significant knowledge gap. Minding efforts to implement real-world FL, there is a notable lack of comprehensive assessment comparing FL to less complex alternatives. Materials & Methods: We extensively reviewed FL literature, categorizing insights along with our findings according to their nature and phase while establishing a FL initiative, summarized to a comprehensive guide. We developed our own FL infrastructure within the German Radiological Cooperative Network (RACOON) and demonstrated its functionality by training FL models on lung pathology segmentation tasks across six university hospitals. We extensively evaluated FL against less complex alternatives in three distinct evaluation scenarios. Results: The proposed guide outlines essential steps, identified hurdles, and proposed solutions for establishing successful FL initiatives conducting real-world experiments. Our experimental results show that FL outperforms less complex alternatives in all evaluation scenarios, justifying the effort required to translate FL into real-world applications. Discussion & Conclusion: Our proposed guide aims to aid future FL researchers in circumventing pitfalls and accelerating translation of FL into radiological applications. Our results underscore the value of efforts needed to translate FL into real-world applications by demonstrating advantageous performance over alternatives, and emphasize the importance of strategic organization, robust management of distributed data and infrastructure in real-world settings

Multidisciplinary Approach to Unravelling the Relative Contribution of Different Oxylipins in Indirect Defense of Arabidopsis thaliana

The oxylipin pathway is commonly involved in induced plant defenses, and is the main signal-transduction pathway induced by insect folivory. Herbivory induces the production of several oxylipins, and consequently alters the so-called ‘oxylipin signature’ in the plant. Jasmonic acid (JA), as well as pathway intermediates are known to induce plant defenses. Indirect defense against herbivorous insects comprises the production of herbivore-induced plant volatiles (HIPVs). To unravel the precise oxylipin signal-transduction underlying the production of HIPVs in Arabidopsis thaliana and the resulting attraction of parasitoid wasps, we used a multidisciplinary approach that includes molecular genetics, metabolite analysis, and behavioral analysis. Mutant plants affected in the jasmonate pathway (18:0 and/or 16:0 -oxylipin routes; mutants dde2-2, fad5, opr3) were studied to assess the effects of JA and its oxylipin intermediates 12-oxo-phytodienoate (OPDA) and dinor-OPDA (dnOPDA) on HIPV emission and parasitoid (Diadegma semiclausum) attraction. Interference with the production of the oxylipins JA and OPDA altered the emission of HIPVs, in particular terpenoids and the phenylpropanoid methyl salicylate, which affected parasitoid attraction. Our data show that the herbivore-induced attraction of parasitoid wasps to Arabidopsis plants depends on HIPVs that are induced through the 18:0 oxylipin-derivative JA. Furthermore, our study shows that the 16:0-oxylipin route towards dnOPDA does not play a role in HIPV induction, and that the role of 18:0 derived oxylipin-intermediates, such as OPDA, is either absent or limited