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Doc number: 79 Abstract Background: Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Results: Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1 ), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1 ). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2 O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. Conclusions: P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components.

•A robust purification process for larger viruses such the Paramyxoviridae family, like measles.•The order of process steps influences measles virus purity and titer.•Process consists of restricted access chromatography and ultrafiltration.•Process is suited for operation in a functional closed system under aseptic conditions.•Scaleability to pilot scale level is proven. Purification of very large and complex, enveloped viruses, such as measles virus is very challenging, it must be performed in a closed system because the final product cannot be sterile filtered and often loss of virus titer and poor product purity has been observed. We developed a purification process where the clarified and endonuclease treated culture supernatant is loaded on a restricted access chromatography medium where small impurities are bound and the virus is collected in the flow-through, which is then concentrated, and buffer exchanged by ultra/diafiltration. Up to 98.5% of host cell proteins could be captured by direct loading of clarified and endonuclease treated cell culture supernatant. Reproducible process performance and scalability of the chromatography step were demonstrated from small to pilot scale, including loading volumes from 50 mL up to 9 L. A 10-fold virus concentration was achieved by the ultrafiltration using a 100 kDa flat-sheet membrane. The order of individual process steps had a large impact on the virus infectivity and total process yields. The developed process maintained virus infectivity and is twice as fast as the traditional process train, where concentration is performed before loading on the chromatography column. Capturing impurities by the restricted access medium makes it a platform purification process with a high flexibility, which can be easily and quickly adapted to other vectors based on the measles virus vector platform.

This chapter contains sections titled: Introduction Thin Wafer Processing ZoneBOND Room‐Temperature Debonding Conclusions

Wikidata is a community-maintained knowledge base that has been assembled from repositories in the fields of genomics, proteomics, genetic variants, pathways, chemical compounds, and diseases, and that adheres to the FAIR principles of findability, accessibility, interoperability and reusability. Here we describe the breadth and depth of the biomedical knowledge contained within Wikidata, and discuss the open-source tools we have built to add information to Wikidata and to synchronize it with source databases. We also demonstrate several use cases for Wikidata, including the crowdsourced curation of biomedical ontologies, phenotype-based diagnosis of disease, and drug repurposing.

Co -engineering of backside RDL, TSV and die stacking processes has changed the primary requirements for thin wafer processing. Temporarily bonding the device wafer to a carrier wafer enables wafer thinning and backside processing [2], After backside processing, the thin device wafer is separated from the carrier.

A typical process flow for temporary bonding first involves fully processing the device wafer on the front side. Subsequently, the carrier wafer and/ or the device wafer is coated with an adhesive. Both wafers are then transferred to a bond chamber, where they are carefully centered and vacuum bonded at elevated temperatures. The selection of the appropriate coating process and technology is influenced by the wafer topography that must be embedded by this material.

Targeted (nano-)drug delivery is essential for treating respiratory diseases, which are often confined to distinct lung regions. However, spatio-temporal profiling of drugs or nanoparticles (NPs) and their interactions with lung macrophages remains unresolved. Here, we present LungVis 1.0, an AI-powered imaging ecosystem that integrates light sheet fluorescence microscopy with deep learning-based image analysis pipelines to map NP deposition and dosage holistically and quantitatively across bronchial and alveolar (acinar) regions in murine lungs for widely-used bulk-liquid and aerosol-based delivery methods. We demonstrate that bulk-liquid delivery results in patchy NP distribution with elevated bronchial doses, whereas aerosols achieve uniform deposition reaching distal alveoli. Furthermore, we reveal that lung tissue-resident macrophages (TRMs) are dynamic, actively patrolling and redistributing NPs within alveoli, contesting the conventional paradigm of TRMs as static entities. LungVis 1.0 provides an advanced framework for exploring pulmonary delivery dynamics and deepening insights into TRM-mediated lung immunity. Targeted nanodrug delivery is crucial to treat respiratory diseases, but profiling the delivery and dynamics of nanoparticles (NPs) within the lung is a challenging task. Here, the authors present LungVis 1.0, an AI-driven 3D imaging ecosystem to profile inhaled substances in distinct lung regions while uncovering the role of tissue-resident macrophages in the fate of NPs.

Machine learning approaches for pattern recognition are increasingly popular. However, the underlying algorithms are often not open source, may require substantial data for model training, and are not geared toward specific tasks. We used open-source software to build a green toad breeding call detection algorithm that will aid in field data analysis. We provide instructions on how to reproduce our approach for other animal sounds and research questions. Our approach using 34 green toad call sequences and 166 audio files without green toad sounds had an accuracy of 0.99 when split into training (70%) and testing (30%) datasets. The final algorithm was applied to amphibian sounds newly collected by citizen scientists. Our function used three categories: “Green toad(s) detected”, “No green toad(s) detected”, and “Double check”. Ninety percent of files containing green toad calls were classified as “Green toad(s) detected”, and the remaining 10% as “Double check”. Eighty-nine percent of files not containing green toad calls were classified as “No green toad(s) detected”, and the remaining 11% as “Double check”. Hence, none of the files were classified in the wrong category. We conclude that it is feasible for researchers to build their own efficient pattern recognition algorithm.

Background Triple-negative breast cancer (TNBC) comprises a heterogeneous group of diseases which are generally associated with poor prognosis. Up to now, no targeted treatment beyond anti-VEGF therapy has been approved for TNBC and cytotoxic agents remain the mainstay of treatment. Ixazomib is a selective and reversible inhibitor of the proteasome, which has been mainly investigated in the treatment of multiple myeloma. In a preclinical study TNBC cells were treated with the first-generation proteasome inhibitor bortezomib in combination with cisplatin and synergistic efficacy was demonstrated. Clinical data are available for carboplatin plus bortezomib in metastatic ovarian and lung cancers showing remarkable antitumor activity and good tolerability (Mol Cancer 11:26 2012, J Thorac Oncol 4:87–92 2009, J Thorac Oncol 7:1032–1040, 2012). Based on this evidence, the phase I/II MBC-10 trial will evaluate the toxicity profile and efficacy of the second-generation proteasome inhibitor ixazomib in combination with carboplatin in patients with advanced TNBC. Methods Patients with metastatic TNBC pretreated with at least one prior line of chemotherapy for advanced disease with a confirmed disease progression and measurable disease according to RECIST criteria 1.1 are eligible for this study. Patients will receive ixazomib in combination with carboplatin on days 1, 8, and 15 in a 28-day cycle. The phase I part of this study utilizes an alternate dose escalation accelerated titration design. After establishing the maximum tolerated dose (MTD), the efficacy and safety of the combination will be further evaluated (phase II, including 41 evaluable patients). All patients will continue on study drugs until disease progression, unacceptable toxicity or discontinuation for any other reason. Primary endpoint of the phase II is overall response rate, secondary endpoints include progression-free survival, safety, and quality of life. This trial is open for patient enrollment since November 2016 in six Austrian cancer centers. Accrual is planned to be completed within 2 years. Discussion Based on preclinical and clinical findings an ixazomib and carboplatin combination is thought to be effective in metastatic TNBC patients. The MBC-10 trial is accompanied by a broad biomarker program investigating predictive biomarkers for treatment response and potential resistance mechanisms to the investigational drug combination. Trial registration EudraCT Number: 2016–001421-13 received on March 31, 2016, ClinicalTrials.gov Identifier: NCT02993094 first posted on December 15, 2016. This trial was registered prospectively.

The loss of suitable spawning habitats is a global threat to amphibians. Thus, establishing man-made ponds may serve as a successful conservation practice to counteract this threat. In this experiment, we used a citizen science (CS) approach to create mini-ponds in private gardens and asked participants to monitor amphibian activity. We provided 302 polyethylene mini-ponds (120 L × 90 W × 40 D cm) to citizen scientists across the range of the target species, the European green toad ( Bufotes viridis ), in Austria. In the first year of the experiment (2024), B. viridis and 12 other amphibian taxa were recorded at 38% of the monitored sites. Signs of amphibian reproduction were detected in 10% of the mini-ponds. Bayesian occupancy models showed detection probabilities consistent with other studies. Over time, we expect more amphibians to discover the mini-ponds and more individuals to use them for reproduction. Our experiment demonstrates that the creation of relatively small ponds is a promising method for supporting and monitoring amphibian populations.