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The increasing importance of regenerative medicine has resulted in a growing need for advanced tissue replacement materials in head and neck surgery. Allo- and xenogenic graft processing is often time-consuming and can deteriorate the extracellular matrix (ECM). High hydrostatic pressure (HHP)-treatment could allow specific devitalization while retaining the essential properties of the ECM. Porcine connective tissue and cartilage were HHP-treated at 100-400 MPa for 10 min. Structural modifications following HHP-exposure were examined using electron microscopy, while devitalization was assessed through metabolism and cell death analyses. Furthermore, ECM alterations and decellularization were evaluated by histology, biomechanical testing, and DNA content analysis. Additionally, the inflammatory potential of HHP-treated tissue was evaluated in vivo using a dorsal skinfold chamber in a mouse model. The devitalization effects of HHP were dose-dependent, with a threshold identified at 200 MPa for fibroblasts and chondrocytes. At this pressure level, HHP induced structural alterations in cells, with a shift toward late-stage apoptosis. HHP-treatment preserved ECM structure and biomechanical properties, but did not remove cell debris from the tissue. This study observed a pressure-dependent increase of markers suggesting the occurrence of immunogenic cell death. In vivo investigations revealed an absence of inflammatory responses to HHP-treated tissue, indicating a favorable biological response to HHP. In conclusion, application of HHP devitalizes fibroblasts and chondrocytes at 200 MPa while retaining the essential properties of the ECM. Prospectively, HHP may simplify the preparation of allo- and xenogenic tissue replacement materials and increase the availability of grafts in head and neck surgery., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Understanding communication among microorganisms through the array of signal molecules and establishing controlled signal transfer between different species is a major goal of the future of biotechnology, and controlled multispecies bioreactor cultivations will open a wide range of applications. In this study, we used two quorum-sensing peptides from Bacillus subtilis - namely, the competence and sporulation factor (CSF) and regulator of the activity of phosphatase RapF (PhrF)-to establish a controlled interkingdom communication system between prokaryotes and eukaryotes. For this purpose, we engineered B. subtilis as a reporter capable of detecting the CSF and PhrF peptides heterologously produced by the yeast Saccharomyces cerevisiae . The reporter strain included the ComA-dependent srfAA promoter fused to the bioluminescence or fluorescence reporter gene(s) to monitor promoter activity measured in a multimode microplate reader. The first measurements of srfAA promoter activity showed a specific response of the reporter strain to the peptides CSF and PhrF. Based on this, systematic mutagenesis of genes that modulate the activity of ComA in the reporter strain resulted in increased activity of the promoter and, thereby, higher sensitivity to the heterologously produced CSF/PhrF. The robustness of the signal transfer was further confirmed in co-cultivation studies in both liquid and solid media. The reporter strain exhibited an up to 5-fold increase in promoter activity in the presence of quorum-sensing peptides-producing cells of S. cerevisiae . In summary, a quorum sensing peptide-driven interkingdom crosstalk between yeast and bacteria was successfully established, which might serve as a basis for controlled protein expression in co-cultivations, establishing biological sensor-actuator systems or study cell-cell interaction and metabolite exchange in bioreactors cultivations., Competing Interests: The authors declare and confirm that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vološen, Gutbier, Korn, Korp, Göttsche, Schuster, Pohl, Rau, Wolf and Ostermann.)

Inflammasome activation occurs in various diseases, including rare diseases that require multicenter studies for investigation. Flow cytometric analysis of ASC speck + cells in patient samples can be used to detect cell type-specific inflammasome activation. However, this requires standardized sample processing and the ability to compare data from different flow cytometers. To address this issue, we analyzed stimulated and unstimulated PBMCs from healthy donors using seven different flow cytometers. Additionally, human PBMCs were analyzed by fluorescence microscopy, imaging flow cytometry and high-content imaging (HCI). Flow cytometers differed significantly in their ability to detect ASC speck + cells. Aria III, Astrios EQ, and Canto II performed best in separating ASC speck + from diffuse ASC cells. Imaging flow cytometry and HCI provided additional insight into ASC speck formation based on image-based parameters. For optimal results, the ability to separate cells with diffuse ASC from ASC speck + cells is decisive. Image-based parameters can also differentiate cells with diffuse ASC from ASC speck + cells. For the first time, we analyzed ASC speck detection by HCI in PBMCs and demonstrated advantages of this technique, such as high-throughput, algorithm-driven image quantification and 3D-rendering. Thus, inflammasome activation by ASC speck formation can be detected by various technical methods. However, the results may vary depending on the device used., (© 2024. The Author(s).)

Humanity is often fascinated by structures and materials developed by Nature. While structural materials such as wood have been widely studied, the structural and mechanical properties of fungi are still largely unknown. One of the structurally interesting fungi is the polypore Fomes fomentarius. The present study deals with the investigation of the light but robust fruiting body of F. fomentarius. The four segments of the fruiting body (crust, trama, hymenium, and mycelial core) were examined. The comprehensive analysis included structural, chemical, and mechanical characterization with particular attention to cell wall composition, such as chitin/chitosan and glucan content, degree of deacetylation, and distribution of trace elements. The hymenium exhibited the best mechanical properties even though having the highest porosity. Our results suggest that this outstanding strength is due to the high proportion of skeletal hyphae and the highest chitin/chitosan content in the cell wall, next to its honeycomb structure. In addition, an increased calcium content was found in the hymenium and crust, and the presence of calcium oxalate crystals was confirmed by SEM-EDX. Interestingly, layers with different densities as well as layers of varying calcium and potassium depletion were found in the crust. Our results show the importance of considering the different structural and compositional characteristics of the segments when developing fungal-inspired materials and products. Moreover, the porous yet robust structure of hymenium is a promising blueprint for the development of advanced smart materials., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Klemm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Rivers are important transport pathways for microplastics into the ocean, but they can also be potential sinks due to microplastic deposition in the sediments of the river bed and adjacent floodplains. In particular, floods can (re)mobilise microplastics from sediments and floodplains, (re)deposit and relocate them depending on the floodplain topography. The knowledge about fluvial microplastic input to floodplains, their spatial distribution and their fate in floodplain soils is limited. To investigate this topic, we sampled soil at a depth of 5-20 cm along three transects in three different Rhine floodplains. We analysed the soil samples in tandem with pyrolysis GC/MS and ATR- & μ-FPA-FTIR for their microplastic abundance and mass concentrations. To study the influence of flood frequency on the microplastic abundance in the three floodplains, we fitted a hydrodynamic flood model (MIKE 21, DHI, Hørsholm, Denmark) and related the results to the respective spatial microplastic distribution. We found similar microplastic distribution patterns in each floodplain. The highest microplastic abundance (8516-70,124 microplastics kg -1 ) and mass concentration (46.2-141.6 mg kg -1 ) were consistently found in the farthest transects from the Rhine in a topographical depression. This microplastic distribution pattern is detectable with both, pyrolysis GC/MS and FTIR. The strongest correlation between the results of both methods was found for small, abundant microplastic particles. Our results suggest that the spatial distribution of microplastics in floodplains is related to the combination of flood frequency and local topography, that ought to be explicitly considered in future studies conducted in floodplains. Finally, our results indicate that pyrolysis GC/MS and FTIR data are comparable under certain conditions, which may help in the decision for the analytical method and sampling design in future studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A key player of excitable cells in the heart and brain is the L-type calcium channel Ca V 1.3. In the heart, it is required for voltage-dependent Ca 2+ -signaling, i.e., for controlling and modulating atrial cardiomyocyte excitation-contraction coupling. The clustering of Ca V 1.3 in functionally relevant channel multimers has not been addressed due to a lack of stoichiometric labeling combined with high-resolution imaging. Here, we developed a HaloTag-labeling strategy to visualize and quantify Ca V 1.3 clusters using STED nanoscopy to address the questions of cluster size and intra-cluster channel density. Channel clusters were identified in the plasma membrane of transfected live HEK293 cells as well as in giant plasma membrane vesicles derived from these cells that were spread on modified glass support to obtain supported plasma membrane bilayers (SPMBs). A small fraction of the channel clusters was colocalized with early and recycling endosomes at the membranes. STED nanoscopy in conjunction with live-cell and SPMB imaging enabled us to quantify Ca V 1.3 cluster sizes and their molecular density revealing significantly lower channel densities than expected for dense channel packing. Ca V 1.3 channel cluster size and molecular density were increased in SPMBs after treatment of the cells with the sympathomimetic compound isoprenaline, suggesting a regulated channel cluster condensation mechanism., (© 2024. The Author(s).)

Introduction: A distinct strain of Klebsiella pneumoniae ( K. pneumoniae ) referred to as hypervirulent (hvKp) is associated with invasive infections such as pyogenic liver abscess in young and healthy individuals. In South Africa, limited information about the prevalence and virulence of this hvKp strain is available. The aim of this study was to determine the prevalence of hvKp and virulence-associated factors in K. pneumoniae isolates from one of the largest tertiary hospitals in a South African province., Methods: A total of 74 K. pneumoniae isolates were received from Pelonomi Tertiary Hospital National Health Laboratory Service (NHLS), Bloemfontein. Virulence-associated genes ( rmpA , capsule serotype K1/K2, iroB and irp2 ) were screened using Polymerase Chain Reaction (PCR). The iutA (aerobactin transporter) gene was used as a primary biomarker of hvKp. The extracted DNAs were sequenced using the next-generation sequencing pipeline and the curated sequences were used for phylogeny analyses using appropriate bioinformatic tools. The virulence of hvKp vs. classical Klebsiella pneumoniae (cKp) was investigated using the Caenorhabditis elegans nematode model., Results: Nine (12.2%) isolates were identified as hvKp. Moreover, hvKp was significantly ( p < 0.05) more virulent in vivo in Caenorhabditis elegans relative to cKp. The virulence-associated genes [ rmpA, iroB , hypermucoviscous phenotype (hmv) phenotype and capsule K1/K2] were significantly ( p < 0.05) associated with hvKp. A homology search of the curated sequences revealed a high percentage of identity between 99.8 and 100% with other homologous iutA gene sequences of other hvKp in the GenBank., Conclusion: Findings from this study confirm the presence of hvKp in a large tertiary hospital in central South Africa. However, the low prevalence and mild to moderate clinical presentation of infected patients suggest a marginal threat to public health. Further studies in different settings are required to establish the true potential impact of hvKp in developing countries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dingiswayo, Adelabu, Arko-Cobbah, Pohl, Mokoena, Du Plessis and Musoke.)

Premise: Previous studies have suggested a trade-off between trichome density (D t ) and stomatal density (D s ) due to shared cell precursors. We clarified how, when, and why this developmental trade-off may be overcome across species., Methods: We derived equations to determine the developmental basis for D t and D s in trichome and stomatal indices (i t and i s ) and the sizes of epidermal pavement cells (e), trichome bases (t), and stomata (s) and quantified the importance of these determinants of D t and D s for 78 California species. We compiled 17 previous studies of D t -D s relationships to determine the commonness of D t -D s associations. We modeled the consequences of different D t -D s associations for plant carbon balance., Results: Our analyses showed that higher D t was determined by higher i t and lower e, and higher D s by higher i s and lower e. Across California species, positive D t -D s coordination arose due to i t -i s coordination and impacts of the variation in e. A D t -D s trade-off was found in only 30% of studies. Heuristic modeling showed that species sets would have the highest carbon balance with a positive or negative relationship or decoupling of D t and D s , depending on environmental conditions., Conclusions: Shared precursor cells of trichomes and stomata do not limit higher numbers of both cell types or drive a general D t -D s trade-off across species. This developmental flexibility across diverse species enables different D t -D s associations according to environmental pressures. Developmental trait analysis can clarify how contrasting trait associations would arise within and across species., (© 2024 The Authors. American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Cryosectioning is known as a common and well-established histological method, due to its easy accessibility, speed, and cost efficiency. However, the creation of bone cryosections is especially difficult. In this study, a cryosectioning protocol for trabecular bone that offers a relatively cheap and undemanding alternative to paraffin or resin embedded sectioning was developed. Sections are stainable with common histological dying methods while maintaining sufficient quality to answer a variety of scientific questions. Furthermore, this study introduces an automated protocol for analysing such sections, enabling users to rapidly access a wide range of different stainings. Therefore, an automated 'QuPath' neural network-based image analysis protocol for histochemical analysis of trabecular bone samples was established, and compared to other automated approaches as well as manual analysis regarding scattering, quality, and reliability. This highly automated protocol can handle enormous amounts of image data with no significant differences in its results when compared with a manual method. Even though this method was applied specifically for bone tissue, it works for a wide variety of different tissues and scientific questions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pohl et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO 2 emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material., Results: In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K) -1 which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites., Conclusion: This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards., (© 2023. The Author(s).)

The intake of omega-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA), is associated with health benefits due to its anti-inflammatory properties. This fatty acid also exhibits antifungal properties in vitro. In order to determine if this antifungal property is valid in vivo, we examined how EPA affects Candida albicans pathogenesis in the Caenorhabditis elegans infection model, an alternative to mammalian host models. The nematodes were supplemented with EPA prior to infection, and the influence of EPA on C. elegans lipid metabolism, survival and immune response was studied. In addition, the influence of EPA on hyphal formation in C. albicans was investigated. It was discovered that EPA supplementation changed the lipid composition, but not the unsaturation index of C. elegans by regulating genes involved in fatty acid and eicosanoid production. EPA supplementation also delayed killing of C. elegans by C. albicans due to the inhibition of hyphal formation in vivo, via the action of the eicosanoid metabolite of EPA, 17,18-epoxyeicosatetraenoic acid. Moreover, EPA supplementation also caused differential expression of biofilm-related gene expression in C. albicans and stimulated the immune response of C. elegans. This provides a link between EPA and host susceptibility to microbial infection in this model., (© 2023. The Author(s).)

Melanins are complex pigments with various biological functions and potential applications in space exploration and biomedicine due to their radioprotective properties. Aspergillus niger, a fungus known for its high radiation resistance, is widely used in biotechnology and a candidate for melanin production. In this study, we investigated the production of fungal pyomelanin (Pyo Fun ) in A. niger by inducing overproduction of the pigment using L-tyrosine in a recombinant Δ hmgA mutant strain (OS4.3). The Pyo Fun pigment was characterized using three spectroscopic methods, and its antioxidant properties were assessed using a DPPH-assay. Additionally, we evaluated the protective effect of Pyo Fun against non-ionizing radiation (monochromatic UV-C) and compared its efficacy to a synthetically produced control pyomelanin (Pyo Syn ). The results confirmed successful production of Pyo Fun in A. niger through inducible overproduction. Characterization using spectroscopic methods confirmed the presence of Pyo Fun , and the DPPH-assay demonstrated its strong antioxidant properties. Moreover, Pyo Fun exhibited a highly protective effect against radiation-induced stress, surpassing the protection provided by Pyo Syn . The findings of this study suggest that Pyo Fun has significant potential as a biological shield against harmful radiation. Notably, Pyo Fun is synthesized extracellularly, differing it from other fungal melanins (such as L-DOPA- or DHN-melanin) that require cell lysis for pigment purification. This characteristic makes Pyo Fun a valuable resource for biotechnology, biomedicine, and the space industry. However, further research is needed to evaluate its protective effect in a dried form and against ionizing radiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Koch, Freidank-Pohl, Siontas, Cortesao, Mota, Runzheimer, Jung, Rebrosova, Siler, Moeller and Meyer.)

Intraneuronal aggregates of the microtubule binding protein Tau are a hallmark of different neurodegenerative diseases including Alzheimer's disease (AD). In these aggregates, Tau is modified by posttranslational modifications such as phosphorylation as well as by proteolytic cleavage. Here we identify a novel Tau cleavage site at aspartate 65 (D65) that is specific for caspase-2. In addition, we show that the previously described cleavage site at D421 is also efficiently processed by caspase-2, and both sites are cleaved in human brain samples. Caspase-2-generated Tau fragments show increased aggregation potential in vitro, but do not accumulate in vivo after AAV-mediated overexpression in mouse hippocampus. Interestingly, we observe that steady-state protein levels of caspase-2 generated Tau fragments are low in our in vivo model despite strong RNA expression, suggesting efficient clearance. Consistent with this hypothesis, we find that caspase-2 cleavage significantly improves the recognition of Tau by the ubiquitin E3 ligase CHIP, leading to increased ubiquitination and faster degradation of Tau fragments. Taken together our data thus suggest that CHIP-induced ubiquitination is of particular importance for the clearance of caspase-2 generated Tau fragments in vitro and in vivo., Competing Interests: Declaration of Competing Interest JM, MS, JK, PR, LG, CP and CK are employees of AbbVie, LR, DCS and DEE joined AbbVie as employees during the study. JS was an employee of AbbVie at the time of the study. FM and MF are employees of the German Center for Neurodegenerative Diseases (DZNE), their contributions to the study were funded by BioMed X GmbH. PH, MS and IB are employees of EMBL, their contributions to the study were funded by BioMed X GmbH. EEH is an employee of BioMed X GmbH, MB and AB were employees of BioMed X GmbH at the time of the study (a contract research agreement was funded by AbbVie). The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.

Mental rotation is the ability to rotate objects in one's mind. Large age-related decreases in accuracy and processing time are often found in studies using paper-and-pencil or computerized mental rotation tests. For older participants, these tests are often too difficult. In the present study, real models consisting of cube figures were used to assess the mental rotation performance of middle and older aged adults. It should be investigated whether these tests were comparable to paper-and-pencil or chronometric tests and if very old participants were able to solve them. Eighty-four participants (49 females) between 40 and 90 years took part and were divided into middle (40-68 years) and older aged (69-90 years) and groups with higher (with college degree) and lower education (without college degree). For accuracy, main effects of gender and age group as well as interactions of age group and education were found. Younger participants outperformed older ones only in the group with lower education. For processing time, a main effect of age group as well as an interaction of age group and education was found. The age-related cognitive decline in the higher educated group was moderate, while a large effect appeared for the group without college degree. Age and gender effects of our new test with real objects were comparable to paper-pencil and computerized tests. Furthermore, a protective effect of education on the cognitive decline in mental rotation performance is discussed., (© 2022. The Author(s).)

Early adversity (e.g., family violence, parental depression, low income) places children at risk for maltreatment and negatively impacts developmental outcomes. Optimal parental reflective function (RF), defined as the parent's ability to think about and identify thoughts, feelings, and mental states in themselves and in their children, is linked to secure attachment and may protect against suboptimal outcomes. We present the results of Phase 2 randomized control trials (RCTs) and quasi-experimental studies (QES) of the Attachment and Child Health (ATTACH TM ) parental RF intervention for families with children at risk for maltreatment. Phase 2 parents experiencing adversity, along with their children aged 0-5 years ( n = 45), received the 10-12-week ATTACH TM intervention. Building on completed Phase 1 pilot data, Phase 2 examined outcomes of long-standing interest, including parental RF and child development, as well as new outcomes, including parental perceived social support and executive function, and children's behavior, sleep, and executive function. RCTs and QES revealed significant improvements in parents' RF, perception of social support, and executive function, children's development (i.e., communication, problem-solving, personal-social, and fine motor skills), and a decrease in children's sleep and behavioral problems (i.e., anxiety/depression, attention problems, aggressive behavior, and externalizing problems), post-intervention. ATTACH™ positively impacts parental RF to prevent negative impacts on children at risk of maltreatment., Competing Interests: The authors declare the following financial interests, which may be considered as potential competing interests: Drs. Nicole Letourneau and Martha Hart are co-owners of the not-for-profit company called ATTACH and Child Health Foundation and the for-profit company called ATTACH Ltd., both established to support community delivery of ATTACH&trade;. All other authors have no competing financial interests.

Across the world, societies have developed different customs, traditions, and practices to memorialize the death of a loved one. Commemorations vary greatly between cultures, and may incorporate different intensities of grieving and celebration. We have selected six unique cultural observances for the deceased which have found beauty and celebration in death., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Detection of de novo variants (DNVs) is critical for studies of disease-related variation and mutation rates. To accelerate DNV calling, we developed a graphics processing units-based workflow. We applied our workflow to whole-genome sequencing data from three parent-child sequenced cohorts including the Simons Simplex Collection (SSC), Simons Foundation Powering Autism Research (SPARK), and the 1000 Genomes Project (1000G) that were sequenced using DNA from blood, saliva, and lymphoblastoid cell lines (LCLs), respectively. The SSC and SPARK DNV callsets were within expectations for number of DNVs, percent at CpG sites, phasing to the paternal chromosome of origin, and average allele balance. However, the 1000G DNV callset was not within expectations and contained excessive DNVs that are likely cell line artifacts. Mutation signature analysis revealed 30% of 1000G DNV signatures matched B-cell lymphoma. Furthermore, we found variants in DNA repair genes and at Clinvar pathogenic or likely-pathogenic sites and significant excess of protein-coding DNVs in IGLL5; a gene known to be involved in B-cell lymphomas. Our study provides a new rapid DNV caller for the field and elucidates important implications of using sequencing data from LCLs for reference building and disease-related projects., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Background: After the World Health Organization declared COVID-19 a pandemic on March 11, 2020, public health restrictions were introduced to slow COVID-19 transmission and prevent health systems overload globally. Work-from-home requirements, online schooling, and social isolation measures required adaptations that may have exposed parents and children to family violence, including intimate partner violence and child abuse and neglect, especially in the early days of the pandemic. Thus, we sought to: (1) examine the occurrence of family violence; (2) identify factors associated with family violence; and (3) identify relevant recommendations, from COVID-19 literature published up to 1 year after the pandemic declaration., Methods: This review was registered on PROSPERO (CRD42021241622), employed rapid review methods, and extracted data from eligible papers in medical and health databases published between December 1, 2019 and March 11, 2021 in MEDLINE, PsycINFO, CINAHL, and Embase., Findings: 28 articles including 29 studies were included in the rapid review. While many studies of families/households revealed rises in family violence incidence, official justice, police, and emergency department records noted declines during the pandemic. Parental stress, burnout, mental distress (i.e. depression), difficulty managing COVID-19 measures, social isolation, and financial and occupational losses were related to increases in family violence. Health services should adopt approaches to prevent family violence, treat victims in the context of public health restrictions, and increase training for digital service usage by health and educational professionals., Interpretation: Globally, restrictions aimed to limit the spread of COVID-19 may have increased the risk factors and incidence of family violence in communities. Official records of family violence may be biased toward under-reporting in the context of pandemics and should be interpreted with caution., Funding: RESOLVE Alberta, Canada and the Emerging Leaders in the Americas Program (ELAP), Global Affairs Canada., Competing Interests: The authors report no competing interests., (© 2022 The Author(s).)

Mitophagy is essential to maintain mitochondrial function and prevent diseases. It activates upon mitochondria depolarization, which causes PINK1 stabilization on the mitochondrial outer membrane. Strikingly, a number of conditions, including mitochondrial protein misfolding, can induce mitophagy without a loss in membrane potential. The underlying molecular details remain unclear. Here, we report that a loss of mitochondrial protein import, mediated by the pre-sequence translocase-associated motor complex PAM, is sufficient to induce mitophagy in polarized mitochondria. A genome-wide CRISPR/Cas9 screen for mitophagy inducers identifies components of the PAM complex. Protein import defects are able to induce mitophagy without a need for depolarization. Upon mitochondrial protein misfolding, PAM dissociates from the import machinery resulting in decreased protein import and mitophagy induction. Our findings extend the current mitophagy model to explain mitophagy induction upon conditions that do not affect membrane polarization, such as mitochondrial protein misfolding., (© 2022. The Author(s).)

Rivers are major pathways for the transport of microplastics towards the oceans, and many studies focus on microplastic abundance in fluvial ecosystems. Although flooding strongly affects transport of microplastics, knowledge about the potential input via floodwaters, spatial distribution, and fate of microplastics in adjacent floodplains remains very limited. In this study, we suggest that local topography and flood frequency could influence the abundance of microplastics in floodplains. Based on this concept, we took soil samples in a Rhine River floodplain in two different depths (0-5 cm and 5-20 cm) along three transects with increasing distance to the river and analysed the abundance of microplastics via FTIR spectroscopy. Flood frequency of the transects was estimated by a combination of hydrodynamic modelling with MIKE 21 (DHI, Hørsholm Denmark) and analysis of time series of water levels. Microplastic abundance per kg dry soil varied between 25,502 to 51,119 particles in the top 5 cm and 25,616 to 84,824 particles in the deeper soil (5-20 cm). The results of our study indicate that local topography and resulting flooding patterns are responsible for the amount of microplastics found at the respective transect. Differences in soil properties, vegetation cover and signs of earthworm activity in the soil profile seem to be related to microplastic migration and accumulation in the deeper soil. The interdisciplinary approach we used in our work can be applied to other floodplains to elucidate the respective processes. This information is essentially important both for locating potential microplastic sinks for process-informed sampling designs and to identify areas of increased bioavailability of microplastics for proper ecological risk assessment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Self-assembly and fibril formation play important roles in protein behaviour. Amyloid fibril formation is well-studied due to its role in neurodegenerative diseases and characterized by refolding of the protein into predominantly β-sheet form. However, much less is known about the assembly of proteins into other types of supramolecular structures. Using cryo-electron microscopy at a resolution of 1.97 Å, we show that a triple-mutant of the anti-microbial peptide plectasin, PPI42, assembles into helical non-amyloid fibrils. The in vitro anti-microbial activity was determined and shown to be enhanced compared to the wildtype. Plectasin contains a cysteine-stabilised α-helix-β-sheet structure, which remains intact upon fibril formation. Two protofilaments form a right-handed protein fibril. The fibril formation is reversible and follows sigmoidal kinetics with a pH- and concentration dependent equilibrium between soluble monomer and protein fibril. This high-resolution structure reveals that α/β proteins can natively assemble into fibrils., (© 2022. The Author(s).)

Human noroviruses (hNoVs) are an important cause of acute gastroenteritis worldwide. However, the lack of a reproducible in vitro cell culture system has impaired research and the development of preventive measures, therapeutic drugs, and vaccines. The aim of this study was to analyze and optimize a suitable cell line for in vitro cultivation of hNoV. The Caco-2 cell line, which is of colorectal origin and differentiates spontaneously into intestinal enterocyte-like cells, was chosen as a model. It was found that differentiated cells were more susceptible to infection with hNoV, resulting in a higher virus yield. This was accompanied by an increase in H type 1 antigen in the cell membrane during differentiation, which functions as an attachment factor for hNoV. Induced overexpression of H type 1 antigen in undifferentiated Caco-2 cells resulted in an increase in viral output to a level similar to that in differentiated cells. However, the relatively low level of viral output, which contrasts with what is observed in vivo, shows that the viral replication cycle is restricted in this model. The results indicate that there is a block at the level of viral release., (© 2022. The Author(s).)

Background: Filamentous fungi of the phylum Basidiomycota are considered as an attractive source for the biotechnological production of composite materials. The ability of many basidiomycetes to accept residual lignocellulosic plant biomass from agriculture and forestry such as straw, shives and sawdust as substrates and to bind and glue together these otherwise loose but reinforcing substrate particles into their mycelial network, makes them ideal candidates to produce biological composites to replace petroleum-based synthetic plastics and foams in the near future., Results: Here, we describe for the first time the application potential of the tinder fungus Fomes fomentarius for lab-scale production of mycelium composites. We used fine, medium and coarse particle fractions of hemp shives and rapeseed straw to produce a set of diverse composite materials and show that the mechanical materials properties are dependent on the nature and particle size of the substrates. Compression tests and scanning electron microscopy were used to characterize composite material properties and to model their compression behaviour by numerical simulations. Their properties were compared amongst each other and with the benchmark expanded polystyrene (EPS), a petroleum-based foam used for thermal isolation in the construction industry. Our analyses uncovered that EPS shows an elastic modulus of 2.37 ± 0.17 MPa which is 4-times higher compared to the F. fomentarius composite materials whereas the compressive strength of 0.09 ± 0.003 MPa is in the range of the fungal composite material. However, when comparing the ability to take up compressive forces at higher strain values, the fungal composites performed better than EPS. Hemp-shive based composites were able to resist a compressive force of 0.2 MPa at 50% compression, rapeseed composites 0.3 MPa but EPS only 0.15 MPa., Conclusion: The data obtained in this study suggest that F. fomentarius constitutes a promising cell factory for the future production of fungal composite materials with similar mechanical behaviour as synthetic foams such as EPS. Future work will focus on designing materials characteristics through optimizing substrate properties, cultivation conditions and by modulating growth and cell wall composition of F. fomentarius, i.e. factors that contribute on the meso- and microscale level to the composite behaviour., (© 2022. The Author(s).)

High throughput screening for measuring the stability of industrially relevant proteins and their variants is necessary for quality assessment in the development process. Advances in automation, measurement time and sample consumption for many techniques allow rapid measurements with minimal amount of protein. However, many methods include automated data analysis, potentially neglecting important aspects of the protein's behavior in certain conditions. In this study we implement small angle X-ray scattering (SAXS), typically not used to assess protein behavior in industrial screening, in a high throughput screening workflow to address problems of contradicting results and reproducibility among different high throughput methods. As a case study we use the lipases of Thermomyces lanuginosus and Rhizomucor miehei, widely used industrial biocatalysts. We show that even the initial analysis of the SAXS data without performing any time-consuming modelling provide valuable information on interparticle interactions. We conclude that recent advances in automation and data processing, have enabled SAXS to be used more widely as a tool to gain in-depth knowledge highly useful for protein formulation development. This is especially relevant in light of increasing accessibility to SAXS due to the commercial availability of benchtop instruments., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The study investigates psychopathological symptoms and the Big Five personality traits as predictors of "problematic smartphone use" (PSU) in different generational groups. The generational groups were selected to analyze whether the different life stages and developmental tasks that need to be completed have an impact on PSU. The groups were divided into digital immigrants, digital natives, and Generation Y and Z as subgroups of digital natives. A total of 399 subjects participated (312 women, 86 men, 1 diverse; mean age = 25.9; range 14-67; 44 digital immigrants, 355 digital natives, 35 Generation Y, and 320 Generation Z). They completed the ICD-10 Symptom Rating (ISR), the Big Five Inventory-10 (BFI-10), and the Mobile Phone Problematic Use Scale (MPPUS). The results show differences between digital immigrants and digital natives in the expression of PSU, neuroticism, conscientiousness, depression, anxiety, and compulsivity. Concerning Generations Y and Z, differences were only found in the expression of the PSU. Regression revealed that symptoms for obsessive-compulsive disorders, depression, conscientiousness, and gender were significant predictors of PSU. Moderations have shown that being a digital immigrant/native moderates the impact of eating disorders on PSU. Belonging to Generation Y/Z moderates the influence of conscientiousness and depression on PSU. Thus, it shows that in different generations, different factors seem to play a role in the development and maintenance of PSU.

Lab-cultivated mycelia of Fomes fomentarius (FF), grown on a solid lignocellulose medium (FF-SM) and a liquid glucose medium (FF-LM), and naturally grown fruiting bodies (FF-FB) were studied as biosorbents for the removal of organic dyes methylene blue and Congo red (CR). Both the chemical and microstructural differences were revealed using X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, zeta potential analysis, and scanning electron microscopy, illuminating the superiority of FF-LM and FF-SM over FF-FB in dye adsorption. The adsorption process of CR on FF-LM and FF-SM is best described by the Redlich-Peterson model with β constants close to 1, that is, approaching the monolayer Langmuir model, which reach maximum adsorption capacities of 48.8 and 13.4 mg g -1 , respectively, in neutral solutions. Adsorption kinetics follow the pseudo-second-order model where chemisorption is the rate-controlling step. While the desorption efficiencies were low, adsorption performances were preserved and even enhanced under simulated dye effluent conditions. The results suggest that F. fomentarius can be considered an attractive biosorbent in industrial wastewater treatment and that its cultivation conditions can be specifically tailored to tune its cell wall composition and adsorption performance., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux., (© 2022. The Author(s).)

Stressors and mortality salience share considerable conceptual overlap. Thus, we examined the impact of a standard mortality salience and a standard stress manipulation on the activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis via endocrine measures of stress; a neutral control condition completed the design. The results revealed that stress elicits increased salivary α-amylase and salivary cortisol reactions; however, no endocrine reactions were found in the mortality salience and the control conditions. To the contrary, we did not find any differences regarding positive and negative affect between any conditions. Implications for social and health psychology are being discussed.

Experimental studies demonstrated antibodies against matrix and coating of polyester-based vascular prostheses. Thus, this study examined associations of these antibodies with serum cytokines (IL-2, IL-4, and IL-10) and local inflammatory reactions. Rats (n = 8/group) intramuscularly received prosthesis segments [PET-C, PET-G, and PET-A groups: polyethylene terephthalate (PET)-based prostheses coated with bovine collagen and gelatin or human serum albumin, respectively; uncoated polytetrafluoroethylene-based (PTFE) prosthesis], with sham-operated controls. Blood was drawn pre-operatively and weekly until day 22. Polymer-specific or coating-specific antibodies and cytokines were detected by enzyme immunoassays, inflammatory reactions were immunohistochemically evaluated on day 23. Polymer-specific antibodies were detected in all PET-groups using uncoated PET as antigenic target, but not for PTFE or controls, coating-specific antibodies only for PET-A. IL-10 was increased in all PET-groups and correlated with polymer-specific antibodies for PET-G and PET-A. IL-2 was increased for PET-A, but overall correlated with PET-specific antibodies. IL-4 remained unchanged in all groups. Intense local inflammatory reactions (ED1 + /ED2 + macrophages and T lymphocytes) were found within all PET-groups, but only minor for PTFE or controls. In conclusion, PET-specific antibodies were associated with increased IL-10 and along with concurrent coating-specific antibodies also with increased IL-2, indicating a specific T cell response. Thus, matrix and/or coating of polymeric vascular prostheses elicit distinct systemic immune reactions, probably influencing local inflammatory reactions., (© 2021 The Authors. Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Aggregation is a common phenomenon in the field of protein therapeutics and can lead to function loss or immunogenic patient responses. Two strategies are currently used to reduce aggregation: (1) finding a suitable formulation, which is labor-intensive and requires large protein quantities, or (2) engineering the protein, which requires extensive knowledge about the protein aggregation pathway. We present a biophysical characterization of the oligomerization and aggregation processes by Interferon alpha-2a (IFNα-2a), a protein drug with antiviral and immunomodulatory properties. This study combines experimental high throughput screening with detailed investigations by small-angle X-ray scattering and analytical ultracentrifugation. Metropolis Monte Carlo simulations are used to gain insight into the underlying intermolecular interactions. IFNα-2a forms soluble oligomers that are controlled by a fast pH and concentration-dependent equilibrium. Close to the isoelectric point of 6, IFNα-2a forms insoluble aggregates which can be prevented by adding salt. We show that monomer attraction is driven mainly by molecular anisotropic dipole-dipole interactions that increase with increasing pH. Repulsion is due to monopole-monopole interactions and depends on the charge of IFNα-2a. The study highlights how combining multiple methods helps to systematically dissect the molecular mechanisms driving oligomer formation and to design ultimately efficient strategies for preventing detrimental protein aggregation.

Background: Recent efforts in fungal biotechnology aim to develop new concepts and technologies that convert renewable plant biomass into innovative biomaterials. Hereby, plant substrates become metabolized by filamentous fungi to transform them into new fungal-based materials. Current research is thus focused on both understanding and optimizing the biology and genetics underlying filamentous fungal growth and on the development of new technologies to produce customized fungal-based materials., Results: This manuscript reports the production of stable pastes, composed of Fomes fomentarius mycelium, alginate and water with 71 wt.% mycelium in the solid content, for additive manufacturing of fungal-based composite materials. After printing complex shapes, such as hollow stars with up to 39 mm in height, a combination of freeze-drying and calcium-crosslinking processes allowed the printed shapes to remain stable even in the presence of water. The printed objects show low bulk densities of 0.12 ± 0.01 g/cm 3 with interconnected macropores., Conclusions: This work reports for the first time the application of mycelium obtained from the tinder fungus F. fomentarius for an extrusion-based additive manufacturing approach to fabricate customized light-weight 3D objects. The process holds great promise for developing light-weight, stable, and porous fungal-based materials that could replace expanded polystyrene produced from fossil resources., (© 2021. The Author(s).)

CD3-bispecific antibodies are a new class of immunotherapeutic drugs against cancer. The pharmacological activity of CD3-bispecifics is typically assessed through in vitro assays of cancer cell lines co-cultured with human peripheral blood mononuclear cells (PBMCs). Assay results depend on experimental conditions such as incubation time and the effector-to-target cell ratio, which can hinder robust quantification of pharmacological activity. In order to overcome these limitations, we developed a new, holistic approach for quantification of the in vitro dose-response relationship. Our experimental design integrates a time-independent analysis of the dose-response across different time points as an alternative to the static, "snap-shot" analysis based on a single time point commonly used in dose-response assays. We show that the potency values derived from static in vitro experiments depend on the incubation time, which leads to inconsistent results across multiple assays and compounds. We compared the potency values from the time-independent analysis with a model-based approach. We find comparably accurate potency estimates from the model-based and time-independent analyses and that the time-independent analysis provides a robust quantification of pharmacological activity. This approach may allow for an improved head-to-head comparison of different compounds and test systems and may prove useful for supporting first-in-human dose selection., (© 2021. The Author(s).)

The COVID-19 pandemic has posed a significant threat to US healthcare workers' mental and physical health. The US Food and Drug Administration approved the first mRNA COVID-19 vaccine for Emergency Use Authorization on December 11, 2020. High-risk healthcare workers were determined to be Phase 1a. Goal: Complete the two-dose vaccine series in all interested phase 1a staff immediately after the COVID-19 vaccine was available and distributed to our institution, December 14, 2020., Methods: A multidisciplinary team involving key stakeholders performed process mapping to develop four key drivers for vaccination success: rapid vaccine procurement, proper storage and handling, well-defined vaccine administration and follow-up plan, and system preparation. We tested interventions using plan-do-study-act cycles. We included employees and providers with direct patient care responsibilities, age 18 years or older, employed at the children's health system, or the affiliated academic medical center. We examined the total number of dose 1 and dose 2 vaccines administered for our primary outcome, and the balancing measure included the percent of wasted vaccines., Results: Three thousand nine-hundred twenty-one healthcare personnel completed the survey, and 73% reported intent to receive the COVID-19 vaccine immediately or at a later time. After 57 clinic days, we vaccinated 83% (n = 5,231) of healthcare personnel at our institution, and 99% completed the two-dose series. Due to surplus vaccines, we vaccinated an additional 1,258 community members with 99% completion of the two-dose series. Vaccine waste was minimal 0.1%., Conclusion: We describe the development and implementation of a successful COVID-19 employee and community vaccination program., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Filamentous fungi are highly productive cell factories, often used in industry for the production of enzymes and small bioactive compounds. Recent years have seen an increasing number of synthetic-biology-based applications in fungi, emphasizing the need for a synthetic biology toolkit for these organisms. Here we present a collection of 96 genetic parts, characterized in Penicillium or Aspergillus species, that are compatible and interchangeable with the Modular Cloning system. The toolkit contains natural and synthetic promoters (constitutive and inducible), terminators, fluorescent reporters, and selection markers. Furthermore, there are regulatory and DNA-binding domains of transcriptional regulators and components for implementing different CRISPR-based technologies. Genetic parts can be assembled into complex multipartite assemblies and delivered through genomic integration or expressed from an AMA1-sequence-based, fungal-replicating shuttle vector. With this toolkit, synthetic transcription units with established promoters, fusion proteins, or synthetic transcriptional regulation devices can be more rapidly assembled in a standardized and modular manner for novel fungal cell factories.

Carbon monoxide (CO) can occur in numerous situations and ambient conditions, such as fire smoke, indoor fireplaces, silos containing large quantities of wood pellets, engine exhaust fumes, and when using hookahs. Symptoms of CO poisoning are nonspecific and can range from dizziness, headache, and angina pectoris to unconsciousness and death. This guideline presents the current state of knowledge and national recommendations on the diagnosis and treatment of patients with CO poisoning. The diagnosis of CO poisoning is based on clinical symptoms and proven or probable exposure to CO. Negative carboxyhemoglobin (COHb) levels should not rule out CO poisoning if the history and symptoms are consistent with this phenomenon. Reduced oxygen-carrying capacity, impairment of the cellular respiratory chain, and immunomodulatory processes may result in myocardial and central nervous tissue damage even after a reduction in COHb. If CO poisoning is suspected, 100% oxygen breathing should be immediately initiated in the prehospital setting. Clinical symptoms do not correlate with COHb elimination from the blood; therefore, COHb monitoring alone is unsuitable for treatment management. Especially in the absence of improvement despite treatment, a reevaluation for other possible differential diagnoses ought to be performed. Evidence regarding the benefit of hyperbaric oxygen therapy (HBOT) is scant and the subject of controversy due to the heterogeneity of studies. If required, HBOT should be initiated within 6 h. All patients with CO poisoning should be informed about the risk of delayed neurological sequelae (DNS)., Competing Interests: See Attachment 2, (Copyright © 2021 Jüttner et al.)

Gender differences in mental-rotation tests with cube figures as rotational material are well examined and robust. Besides biological or socialization factors, task characteristics could partly be responsible for men's advantage in mental rotation. Therefore, we investigated in two studies the influence of different rotational materials on the gender differences in mental-rotation performance. In the first study, 134 undergraduate students (89 women, 45 men) participated using a mental-rotation test with either cube or pellet figures. Significant gender differences in favour of men but no interaction of gender and material were found. In the second study, 189 undergraduate students (110 women, 79 men) solved a mental-rotation test with either male or female-stereotyped objects. Significant gender differences appeared only when male-stereotyped objects were used as rotational material, but not for female-stereotyped material. A significant interaction of gender and material appeared. Hence, some rotational objects seem to have an influence on participants' mental-rotation performance and the gender differences in this task while others do not affect performances of women and men., (© 2020. Springer-Verlag GmbH Germany, part of Springer Nature.)

The aggregation of the human islet amyloid polypeptide (IAPP) is associated with diabetes type II. A quantitative understanding of this connection at the molecular level requires that the aggregation mechanism of IAPP is resolved in terms of the underlying microscopic steps. Here we have systematically studied recombinant IAPP, with amidated C-terminus in oxidised form with a disulphide bond between residues 3 and 7, using thioflavin T fluorescence to monitor the formation of amyloid fibrils as a function of time and IAPP concentration. We used global kinetic analyses to connect the macroscopic measurements of aggregation to the microscopic mechanisms, and show that the generation of new aggregates is dominated by the secondary nucleation of monomers on the fibril surface. We then exposed insulinoma cells to aliquots extracted from different time points of the aggregation process, finding the highest toxicity at the midpoint of the reaction, when the secondary nucleation rate reaches its maximum. These results identify IAPP oligomers as the most cytotoxic species generated during IAPP aggregation, and suggest that compounds that target secondary nucleation of IAPP could be most effective as therapeutic candidates for diabetes type II., Competing Interests: Authors DC, SKRC, JM, BM, JH and SC were employed by Wren Therapeutics Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rodriguez Camargo, Chia, Menzies, Mannini, Meisl, Lundqvist, Pohl, Bernfur, Lattanzi, Habchi, Cohen, Knowles, Vendruscolo and Linse.)

Fungal secondary metabolites are widely used as therapeutics and are vital components of drug discovery programs. A major challenge hindering discovery of novel secondary metabolites is that the underlying pathways involved in their biosynthesis are transcriptionally silent under typical laboratory growth conditions, making it difficult to identify the transcriptional networks that they are embedded in. Furthermore, while the genes participating in secondary metabolic pathways are typically found in contiguous clusters on the genome, known as biosynthetic gene clusters (BGCs), this is not always the case, especially for global and pathway-specific regulators of pathways' activities. To address these challenges, we used 283 genome-wide gene expression data sets of the ascomycete cell factory Aspergillus niger generated during growth under 155 different conditions to construct two gene coexpression networks based on Spearman's correlation coefficients (SCCs) and on mutual rank-transformed Pearson's correlation coefficients (MR-PCCs). By mining these networks, we predicted six transcription factors, named MjkA to MjkF, to regulate secondary metabolism in A. niger. Overexpression of each transcription factor using the Tet-On cassette modulated the production of multiple secondary metabolites. We found that the SCC and MR-PCC approaches complemented each other, enabling the delineation of putative global (SCC) and pathway-specific (MR-PCC) transcription factors. These results highlight the potential of coexpression network approaches to identify and activate fungal secondary metabolic pathways and their products. More broadly, we argue that drug discovery programs in fungi should move beyond the BGC paradigm and focus on understanding the global regulatory networks in which secondary metabolic pathways are embedded. IMPORTANCE There is an urgent need for novel bioactive molecules in both agriculture and medicine. The genomes of fungi are thought to contain vast numbers of metabolic pathways involved in the biosynthesis of secondary metabolites with diverse bioactivities. Because these metabolites are biosynthesized only under specific conditions, the vast majority of the fungal pharmacopeia awaits discovery. To discover the genetic networks that regulate the activity of secondary metabolites, we examined the genome-wide profiles of gene activity of the cell factory Aspergillus niger across hundreds of conditions. By constructing global networks that link genes with similar activities across conditions, we identified six putative global and pathway-specific regulators of secondary metabolite biosynthesis. Our study shows that elucidating the behavior of the genetic networks of fungi under diverse conditions harbors enormous promise for understanding fungal secondary metabolism, which ultimately may lead to novel drug candidates.

Selfish genetic elements that act as post-segregation distorters cause lethality in non-carrier individuals after fertilization. Two post-segregation distorters have been previously identified in Caenorhabditis elegans , the peel-1 / zeel-1 and the sup-35 / pha-1 elements. These elements seem to act as modification-rescue systems, also called toxin/antidote pairs. Here we show that the maternal-effect toxin/zygotic antidote pair sup-35 / pha-1 is required for proper expression of apical junction (AJ) components in epithelia and that sup-35 toxicity increases when pathways that establish and maintain basal epithelial characteristics, die-1 , elt-1 , lin-26 , and vab-10 , are compromised. We demonstrate that pha-1(e2123) embryos, which lack the antidote, are defective in epidermal morphogenesis and frequently fail to elongate. Moreover, seam cells are frequently misshaped and mispositioned and cell bond tension is reduced in pha-1(e2123) embryos, suggesting altered tissue material properties in the epidermis. Several aspects of this phenotype can also be induced in wild-type embryos by exerting mechanical stress through uniaxial loading. Seam cell shape, tissue mechanics, and elongation can be restored in pha-1(e2123) embryos if expression of the AJ molecule DLG-1/Discs large is reduced. Thus, our experiments suggest that maternal-effect toxicity disrupts proper development of the epidermis which involves distinct transcriptional regulators and AJ components., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lehmann and Pohl.)

CASK (Ca 2+ /calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.

Discoidin domain receptors 1 and 2 (DDR1/2) play a central role in fibrotic disorders, such as renal and pulmonary fibrosis, atherosclerosis, and various forms of cancer. Potent and selective inhibitors, so-called chemical probe compounds, have been developed to study DDR1/2 kinase signaling. However, these inhibitors showed undesired activity on other kinases such as the tyrosine protein kinase receptor TIE or tropomyosin receptor kinases, which are related to angiogenesis and neuronal toxicity. In this study, we optimized our recently published p38 mitogen-activated protein kinase inhibitor 7 toward a potent and cell-active dual DDR/p38 chemical probe and developed a structurally related negative control. The structure-guided design approach used provided insights into the P-loop folding process of p38 and how targeting of non-conserved amino acids modulates inhibitor selectivity. The developed and comprehensively characterized DDR/p38 probe, 30 (SR-302), is a valuable tool for studying the role of DDR kinase in normal physiology and in disease development.

Background: To assess the prognostic value of early echocardiographic indices of right ventricular function and vasoactive peptides for prediction of bronchopulmonary dysplasia (BPD) or death in very preterm infants., Methods: Prospective study involving 294 very preterm infants (median [IQR] gestational age 28.4 [26.4-30.4] weeks, birth weight 1065 [800-1380] g), of whom 57 developed BPD (oxygen supplementation at 36 weeks postmenstrual age) and 10 died. Tricuspid annular plane systolic excursion (TAPSE), right ventricular index of myocardial performance (RIMP), plasma concentrations of mid-regional pro-atrial natriuretic peptide (MR-proANP) and C-terminal pro-endothelin-1 (CT-proET1) were measured on day 7 of life., Results: RIMP was significantly increased (median [IQR] 0.3 [0.23-0.38] vs 0.22 [0.15-0.29]), TAPSE decreased (median [IQR] 5.0 [5.0-6.0] vs 6.0 [5.4-7.0] mm), MR-proANP increased (median [IQR] 784 [540-936] vs 353 [247-625] pmol/L), and CT-proET1 increased (median [IQR] 249 [190-345] vs 199 [158-284] pmol/L) in infants who developed BPD or died, as compared to controls. All variables showed significant but weak correlations with each other (rS -0.182 to 0.359) and predicted BPD/death with similar accuracy (areas under receiver operator characteristic curves 0.62 to 0.77). Multiple regression revealed only RIMP and birth weight as independent predictors of BPD or death., Conclusions: Vasoactive peptide concentrations and echocardiographic assessment employing standardized measures, notably RIMP, on day 7 of life are useful to identify preterm infants at increased risk for BPD or death., Competing Interests: The authors have declared that no competing interests exist.

Concrete exposure to high temperatures induces thermo-hygral phenomena, causing water phase changes, buildup of pore pressure and vulnerability to spalling. In order to predict these phenomena under various conditions, a three-phase transport model is proposed. The model is validated on X-ray CT data up to 320 °C, showing good agreement of the temperature profiles and moisture changes. A dehydration description, traditionally derived from thermogravimetric analysis, was replaced by a formulation based on data from neutron radiography. In addition, treating porosity and dehydration evolution as independent processes, previous approaches do not fulfil the solid mass balance. As a consequence, a new formulation is proposed that introduces the porosity as an independent variable, ensuring the latter condition.

The reconstruction of critical size bone defects is still clinically challenging. Even though the transplantation of autologous bone is used as gold standard, this therapy is accompanied by donor site morbidities as well as tissue limitations. The alternatively used allografts, which are devitalized due to thermal, chemical or physical processing, often lose their matrix integrity and have diminished biomechanical properties. High Hydrostatic Pressure (HHP) may represent a gentle alternative to already existing methods since HHP treated human osteoblasts undergo cell death and HHP treated bone cylinders maintain their mechanical properties. The aim of this study was to determine the biological effects caused by HHP treatment regarding protein/matrix integrity and type of cell death in trabecular bone cylinders. Therefore, different pressure protocols (250 and 300 MPa for 10, 20 and 30 min) and end point analysis such as quantification of DNA-fragmentation, gene expression, SDS-PAGE, FESEM analysis and histological staining were performed. While both protein and matrix integrity was preserved, molecular biological methods showed an apoptotic differentiation of cell death for lower pressures and shorter applications (250 MPa for 10 and 20 min) and necrotic differentiation for higher pressures and longer applications (300 MPa for 30 min). This study serves as a basis for further investigation as it shows that HHP successfully devitalizes trabecular bone cylinders., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Waletzko-Hellwig, Pohl, Riese, Schlosser, Dau, Engel, Springer and Bader.)

Orthopaedic implants and temporary osteosynthesis devices are commonly based on Titanium (Ti). For short-term devices, cell-material contact should be restricted for easy removal after bone healing. This could be achieved with anti-adhesive plasma-fluorocarbon-polymer (PFP) films created by low-temperature plasma processes. Two different PFP thin film deposition techniques, microwave (MW) and radiofrequency (RF) discharge plasma, were applied to receive smooth, hydrophobic surfaces with octafluoropropane (C 3 F 8 ) or hexafluorohexane (C 6 F 6 ) as precursors. This study aimed at examining the immunological local tissue reactions after simultaneous intramuscular implantation of four different Ti samples, designated as MW-C 3 F 8 , MW-C 6 F 6 , RF-C 3 F 8 and Ti-controls, in rats. A differentiated morphometric evaluation of the inflammatory reaction was conducted by immunohistochemical staining of CD68+ macrophages, CD163+ macrophages, MHC class II-positive cells, T lymphocytes, CD25+ regulatory T lymphocytes, NK cells and nestin-positive cells in cryosections of surrounding peri-implant tissue. Tissue samples were obtained on days 7, 14 and 56 for investigating the acute and chronical inflammation (n = 8 rats/group). Implants with a radiofrequency discharge plasma (RF-C 3 F 8 ) coating exhibited a favorable short- and long-term immune/inflammatory response comparable to Ti-controls. This was also demonstrated by the significant decrease in pro-inflammatory CD68+ macrophages, possibly downregulated by significantly increasing regulatory T lymphocytes.

The aim of this study was to assess the characteristics of asylum-seeking children with medical complexity visiting a tertiary care hospital in Switzerland, detailing their underlying medical conditions and management. Asylum-seeking patients with frequent visits between January 2016 and December 2017 were identified using administrative and electronic health records. Of 462 patients, 19 (4%) fulfilled the inclusion criteria with 811 (45%) visits. The age of the 19 patients ranged from 0 to 16.7 years (median of 7 years) with two main age groups identified: < 2 years and > 12 years. Nine (47%) patients originated from Syria. A total of 34/811(4%) visits were hospital admissions, 66/811 (8%) emergency department visits and 320/811(39%) outpatient department visits. In children < 2 years genetic diseases (5/8; 63%) and nutritional problems (6/8; 75%) were most common; in adolescents, orthopedic diseases (4/8; 50%) and mental health problems (4/8; 50%). Asylum-seeking children with medical complexity represent a small but important group of patients requiring frequent medical consultations. The high proportion of young patients with genetic diseases and severe nutritional problems suggests that new strategies are required in the management of this specific group of asylum-seeking children. This could be achieved by improved co-ordination between hospital and non-hospital care exploring options for integrated care.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification., (Copyright © 2021 Elsevier B.V. All rights reserved.)