Directed evolution of Rhodotorula gracilisd-amino acid oxidase using single-cell hydrogel encapsulation and ultrahigh-throughput screening

Engineering catalytic and biophysical properties of enzymes is an essential step en route to advanced biomedical and industrial applications. Here, we developed a high-throughput screening and directed evolution strategy relying on single-cell hydrogel encapsulation to enhance the performance of D -Amino acid oxidase from Rhodotorula gracilis ( Rg DAAOx), a candidate enzyme for cancer therapy. We used a cascade reaction between Rg DAAOx variants surface displayed on yeast and horseradish peroxidase (HRP) in the bulk media to trigger enzyme-mediated crosslinking of phenol-bearing fluorescent alginate macromonomers, resulting in hydrogel formation around single yeast cells. The fluorescent hydrogel capsules served as an artificial phenotype and basis for pooled library screening by fluorescence activated cell sorting (FACS). We screened a Rg DAAOx variant library containing ∼10 6 clones while lowering the D -Ala substrate concentration over three sorting rounds in order to isolate variants with low K m . After three rounds of FACS sorting and regrowth, we isolated and fully characterized four variants displayed on the yeast surface. We identified variants with a more than 5-fold lower K m than the parent sequence, with an apparent increase in substrate binding affinity. The mutations we identified were scattered across the Rg DAAOx structure, demonstrating the difficulty in rationally predicting allosteric sites and highlighting the advantages of scalable library screening technologies for evolving catalytic enzymes

AUTOMATIC MAPPING FROM ULTRA-LIGHT UAV IMAGERY

This paper presents an affordable, fully automated and accurate mapping solutions based on ultra-light UAV imagery, which is commercialized by Pix4D. We show interesting application in the field of UAV mapping, analyse the accuracy of the automated processing on several datasets. The accuracy highly depends on the ground resolution (flying height) of the input imagery. When chosen appropriately this mapping solution can compete with traditional mapping solutions that capture fewer high-resolution images from airplanes and that rely on highly accurate orientation and positioning sensors on board. Due to the careful integration with recent computer vision techniques, the result is robust and fully automatic and can deal with inaccurate position and orientation information which are typically problematic with traditional techniques

Insights Into Today’s Real Estate Market - A Focus on Switzerland

Real estate is at the confluence of several major transformations: higher inflation, higher interest rates, and increasing ESG risks. In this Roundup, experts from academia, industry, and regulation discuss how investors and households navigate new difficult tradeoffs in the Swiss context. Will macroeconomic uncertainties affect prices and impact the financial sector? What is the proper place of real estate in an investor's portfolio? Should homeowners rethink their financing options? And will increasingly strict energy regulations impact the market

Gonadal Malformations in Whitefish from Lake Thun: Defining the Case and Evaluating the Role of EDCs

The objectives of this project were to evaluate i) whether the gonad alterations of whitefish (Coregonus lavaretus spp.) in Lake Thun represent abnormal morphological variations specific to this lake, and, if so, ii) whether the malformations are related to chemical exposure, in particular to exposure to endocrine-disrupting compounds (EDCs). Large-scale monitoring data revealed that, although whitefish in other lakes display some background variation of gonad morphology, the situation in Lake Thun, is unique because of the significantly higher prevalence of gonad malformations. The abnormal variations of whitefish gonad morphology include aplasias, compartmentations, fusions, and intersex. In the search for the factor(s) causing the gonad malformations, coregonids were exposed from fertilization up to maturity to Lake Thun water and plankton or to contaminants possibly being present in the lake, including trinitrotoluenes, and naphtalene sulfonates. Since these experiments are still ongoing, a conclusive answer cannot be given yet, but initial observations point to a role of the lake plankton. The possible presence of EDCs in Lake Thun was assessed using bioanalytics and biomarkers. The bioanalytical studies found estrogenic activities in concentrated plankton extracts of Lake Thun, however, estrogenic activities occurred also in plankton extracts of reference lakes. Bioassay-directed fractionation of the plankton samples points to degradation products of natural substances as a cause of the estrogenic activity. Examination of Lake Thun whitefish for EDC biomarkers such as vitellogenin, sex steroid levels or intersex frequency yielded no indications of exposure to EDCs, neither in fish with normal nor in fish with abnormal gonad morphology. Long-term laboratory exposure of developing coregonids to the prototype estrogenic compound, 17?-estradiol, resulted in an increased frequency of intersex gonads, but did not induce the other gonad malformations typical for Lake Thun coregonids. In summing up, the currently available evidence does not support an EDC or chemical etiology of the gonad malformations, however, this preliminary conclusion needs to be substantiated in the ongoing investigations. The project also highlights the need for more detailed knowledge of natural variation in wildlife populations to be able to recognize anthropogenically caused variation

Simplified Building Models Extraction From Ultra-Light UAV Imagery

Generating detailed simplified building models such as the ones present on Google Earth is often a difficult and lengthy manual task, requiring advanced CAD software and a combination of ground imagery, LIDAR data and blueprints. Nowadays, UAVs such as the Falcon 8 have reached the maturity to offer an affordable, fast and easy way to capture large amounts of oblique images covering all part of a building. In this paper we present a state-of-the-art photogrammetry and visual reconstruction pipeline provided by Pix4D applied to medium resolution imagery acquired by such UAVs. The key element of simplified building models extraction is the seamless integration of the outputs of such a pipeline for a final manual refinement step in order to minimize the amount of manual wor

Swiss general internal medicine board examination: quantitative effects of publicly available and unavailable questions on question difficulty and test performance.

BACKGROUND Formerly, a substantial number of the 120 multiple-choice questions of the Swiss Society of General Internal Medicine (SSGIM) board examination were derived from publicly available MKSAP questions (Medical Knowledge Self-Assessment Program®). The possibility to memorise publicly available questions may unduly influence the candidates' examination performance. Therefore, the examination board raised concerns that the examination did not meet the objective of evaluating the application of knowledge. The society decided to develop new, "Helvetic" questions to improve the examination. The aim of the present study was to quantitatively assess the degree of difficulty of the Helvetic questions (HQ) compared with publicly available and unavailable MKSAP questions and to investigate whether the degree of difficulty of MKSAP questions changed over time as their status changed from publicly available to unavailable. METHODS The November 2019 examination consisted of 40 Helvetic questions, 40 publicly available questions from MKSAP edition 17 (MKSAP-17) and 40 questions from MKSAP-15/16, which were no longer publicly available at the time of the examination. An one factorial univariate analysis of variance (ANOVA) examined question difficulty (lower values mean higher difficulty) between these three question sets. A repeated ANOVA compared the difficulty of MKSAP-15/16 questions in the November 2019 examination with the difficulty of the exact same questions from former examinations, when these questions belonged to the publicly available MKSAP edition. The publicly available MKSAP-17 and the publicly unavailable Helvetic questions served as control. RESULTS The analysis of the November 2019 exam showed a significant difference in average item difficulty between Helvetic and MKSAP-17 questions (71% vs 86%, p <0.001) and between MKSAP-15/16 and MKSAP-17 questions (70% vs 86%, p <0.001). There was no significant difference in item difficulty between Helvetic and MKSAP-15/16 questions (71% vs 70%, p = 0.993). The repeated measures ANOVA on question use and the three question categories showed a significant interaction (p <0.001, partial eta-squared = 0.422). The change in the availability of MKSAP-15/16 questions had a strong effect on difficulty. Questions became on average 21.9% more difficult when they were no longer publicly available. In contrast, the difficulty of the MKSAP-17 and Helvetic questions did not change significantly across administrations. DISCUSSION This study provides the quantitative evidence that the public availability of questions has a decisive influence on question difficulty and thus on SSGIM board examination performance. Reducing the number of publicly available questions in the examination by introducing confidential, high-quality Helvetic questions contributes to the validity of the board examination by addressing higher order cognitive skills and making rote-learning strategies less effective

SAKK 24/09: safety and tolerability of bevacizumab plus paclitaxel vs. bevacizumab plus metronomic cyclophosphamide and capecitabine as first-line therapy in patients with HER2-negative advanced stage breast cancer - a multicenter, randomized phase III trial.

BACKGROUND: Adding bevacizumab to chemotherapy improves response rates and progression-free survival (PFS) in metastatic breast cancer (mBC). We aimed to demonstrate decreased toxicity with metronomic chemotherapy/bevacizumab compared with paclitaxel/bevacizumab. METHODS: This multicenter, randomized phase III trial compared bevacizumab with either paclitaxel (arm A) or daily oral capecitabine-cyclophosphamide (arm B) as first-line treatment in patients with HER2-negative advanced breast cancer. The primary endpoint was the incidence of selected grade 3-5 adverse events (AE) including: febrile neutropenia, infection, sensory/motor neuropathy, and mucositis. Secondary endpoints included objective response rate, disease control rate, PFS, overall survival (OS), quality of life (QoL), and pharmacoeconomics. The study was registered prospectively with ClinicalTrials.gov, number NCT01131195 on May 25, 2010. RESULTS: Between September 2010 and December 2012, 147 patients were included at 22 centers. The incidence of primary endpoint-defining AEs was similar in arm A (25 % [18/71]; 95 % CI 15-35 %) and arm B (24 % [16/68]; 95 % CI 13-34 %; P = 0.96). Objective response rates were 58 % (42/73; 95 % CI 0.46-0.69) and 50 % (37/74; 95 % CI 0.39-0.61) in arms A and B, respectively (P = 0.45). Median PFS was 10.3 months (95 % CI 8.7-11.3) in arm A and 8.5 months (95 % CI 6.5-11.9) in arm B (P = 0.90). Other secondary efficacy endpoints were not significantly different between study arms. The only statistically significant differences in QoL were less hair loss and less numbness in arm B. Treatment costs between the two arms were equivalent. CONCLUSION: This trial failed to meet its primary endpoint of a reduced rate of prespecified grade 3-5 AEs with metronomic bevacizumab, cyclophosphamide and capecitabine

Zooplankton Feeding Induces Macroscopical Gonad Malformations in Whitefish (Coregonus ssp.) from Lake Thun, Switzerland

Alterations in gonad morphology are widespread in wild fish populations. Whitefish (Coregonus spp.) from Lake Thun, Switzerland, display a high prevalence of macroscopical gonad malformations including fusions to the musculature, segmented gonads and intersex condition. The aim of the present study was to evaluate whether the gonad morphological changes in Lake Thun whitefish are caused by genetic factors, environmental factors (water, diet) or &ldquo;gene &times; environment&rdquo; interaction. We performed two independent experiments of a three-year duration in which we reared whitefish from fertilization until adulthood and tested the possible causative factors using a matrix design: (i) genetics&mdash;comparing the prevalence of gonad malformations in whitefish of different genetic origin reared under identical environmental conditions (same diet, same water, same experimental facility); (ii) environment&mdash;comparing the prevalence of gonad malformations in whitefish of the same genetic origin reared in different water sources and/or fed with different diets; and (iii) gene-environment interaction&mdash;comparing the prevalence of gonad morphological alterations in relation to the combinations of genetics and environmental factors. Two diets were used for the rearing experiments: either zooplankton collected in Lake Thun which represents the natural diet of whitefish, or an artificial dry food which was used as control. The key finding of this study is that the inducing factor of the gonad malformations is contained in the zooplankton of Lake Thun. Fish fed with this diet developed a significantly higher prevalence of malformations than fish from any other treatment, irrespective of the genetic origin and/or the water source. This result could be repeated in the two independent experiments. Importantly, the prevalence values observed in the experimental fish fed with Lake Thun zooplankton were similar to the prevalence values recorded in free-ranging whitefish in the lake. The findings of this study advance the understanding of the causes of gonad morphological alterations in wild fish populations

Fused Filament Fabrication Based on Polyhydroxy Ether (Phenoxy) Polymers and Related Properties

This paper describes the first-time application of polyhydroxy ether polymers, so-called phenoxy, to fused filament fabrication (FFF). Phenoxy is an amorphous thermoplastic polymer that is based on the same building blocks as epoxide resins. This similarity creates some unique properties such as dissolution to epoxide systems, which is why phenoxy is used as an additive for toughening. In this study, the processing parameters were characterized, a filament was extruded and applied to FFF printing, and the final mechanical characteristics were determined. The study concludes with a comparison with other standard FFF materials

Predictive Simulation of Process Windows for Powder Bed Fusion Additive Manufacturing: Influence of the Powder Bulk Density

The resulting properties of parts fabricated by powder bed fusion additive manufacturing processes are determined by their porosity, local composition, and microstructure. The objective of this work is to examine the influence of the stochastic powder bed on the process window for dense parts by means of numerical simulation. The investigations demonstrate the unique capability of simulating macroscopic domains in the range of millimeters with a mesoscopic approach, which resolves the powder bed and the hydrodynamics of the melt pool. A simulated process window reveals the influence of the stochastic powder layer. The numerical results are verified with an experimental process window for selective electron beam-melted Ti-6Al-4V. Furthermore, the influence of the powder bulk density is investigated numerically. The simulations predict an increase in porosity and surface roughness for samples produced with lower powder bulk densities. Due to its higher probability for unfavorable powder arrangements, the process stability is also decreased. This shrinks the actual parameter range in a process window for producing dense parts