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To make modern data management and analysis that is committed to FAIR principles and improved reproducibility a reality, Flanders BioImaging in a team effort with the Flemish supercomputing center (Vlaams Supercomputer Centrum, VSC) and KU Leuven’s central IT infrastructure, is exploring new models for data management, metadata handling, and image analysis., Grant FWO: Flanders BioImaging Leading Imaging Application Integrated Service & Enablement (FBI-LIAISE) 2023-2026

Recently, LifeSciTrainers.org held two community calls to discuss the potential of using ChatGPT, a large-scale AI language model, as a tool for training and teaching bioinformatics. The calls brought together experts, educators, and practitioners in the field to share their thoughts and experiences on the benefits and challenges of integrating ChatGPT into their teaching methods. This blog post summarizes the major points from these discussions.

The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)(.) In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutieres syndrome (AGS). In mice, complete loss of ADAR1 activity is embryonically lethal, and mutations similar to those found in patients with AGS cause autoinflammation. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Z alpha domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Z alpha domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.

Many trainers and organizations are passionate about sharing their training material. Sharing training material has several benefits, such as providing a record of recognition as an author, offering inspiration to other trainers, enabling researchers to discover training resources for their personal learning path, and improving the training resource landscape using data-driven gap analysis from the bioinformatics community. In this article, we present a series of protocols for using the ELIXIR online training registry Training eSupport System (TeSS). TeSS provides a one-stop shop for trainers and trainees to discover online information and content, including training materials, events, and interactive tutorials. For trainees, we provide protocols for registering and logging in and for searching and filtering content. For trainers and organizations, we also show how to manually or automatically register training events and materials. Following these protocols will contribute to promoting training events and add to a growing catalog of materials. This will concomitantly increase the FAIRness of training materials and events. Training registries like TeSS use a scraping mechanism to aggregate training resources from many providers when they have been annotated using Bioschemas specifications. Finally, we describe how to enrich training resources to allow for more efficient sharing of the structured metadata, such as prerequisites, target audience, and learning outcomes using Bioschemas specification. As increasing training events and material are aggregated in TeSS, searching the registry for specific events and materials becomes crucial.

PLAZA is a platform for comparative, evolutionary, and functional plant genomics. It makes a broad set of genomes, data types and analysis tools available to researchers through a user-friendly website, an API, and bulk downloads. In this latest release of the PLAZA platform, we are integrating a record number of 134 high-quality plant genomes, split up over two instances: PLAZA Dicots 5.0 and PLAZA Monocots 5.0. This number of genomes corresponds with a massive expansion in the number of available species when compared to PLAZA 4.0, which offered access to 71 species, a 89% overall increase. The PLAZA 5.0 release contains information for 5 882 730 genes, and offers pre-computed gene families and phylogenetic trees for 5 274 684 protein-coding genes. This latest release also comes with a set of new and updated features: a new BED import functionality for the workbench, improved interactive visualizations for functional enrichments and genome-wide mapping of gene sets, and a fully redesigned and extended API. Taken together, this new version offers extended support for plant biologists working on different families within the green plant lineage and provides an efficient and versatile toolbox for plant genomics. All PLAZA releases are accessible from the portal website: https://bioinformatics.psb.ugent.be/plaza/.

Summary report about a workshop on research data management and data deposition for biological images. This workshop took place on 23/06/2022 and was organized by VIB and Flanders Bioimaging consortium in collaboration with Elixir Belgium and Flemish Supercomputer Center. https://training.vib.be/all-trainings/workshop-data-management-and-data-deposition-biological-images, {"references":["Woller, Tatiana, Botzki, Alexander and Munck, Sebastian (2022, October), Workshop on data management and data deposition for biological images"]}

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures.[Image: see text]

Summary The Unipept Visualizations library is a JavaScript package to generate interactive visualizations of both hierarchical and non-hierarchical quantitative data. It provides four different visualizations: a sunburst, a treemap, a treeview and a heatmap. Every visualization is fully configurable, supports TypeScript and uses the excellent D3.js library. Availability and implementation The Unipept Visualizations library is available for download on NPM: https://npmjs.com/unipept-visualizations. All source code is freely available from GitHub under the MIT license: https://github.com/unipept/unipept-visualizations

Summary: Loss of function of adenosine deaminase acting on double-stranded RNA (dsRNA)-1 (ADAR1) causes the severe autoinflammatory disease Aicardi-Goutières syndrome (AGS). ADAR1 converts adenosines into inosines within dsRNA. This process called A-to-I editing masks self-dsRNA from detection by the antiviral dsRNA sensor MDA5. ADAR1 binds to dsRNA in both the canonical A-form and the poorly defined Z conformation (Z-RNA). Mutations in the Z-RNA-binding Zα domain of ADAR1 are common in patients with AGS. How loss of ADAR1/Z-RNA interaction contributes to disease development is unknown. We demonstrate that abrogated binding of ADAR1 to Z-RNA leads to reduced A-to-I editing of dsRNA structures formed by base pairing of inversely oriented short interspersed nuclear elements. Preventing ADAR1 binding to Z-RNA triggers an MDA5/MAVS-mediated type I interferon response and leads to the development of lethal autoinflammation in mice. This shows that the interaction between ADAR1 and Z-RNA restricts sensing of self-dsRNA and prevents AGS development.

Post-translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370 000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows the submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

Scop3D is a tool that automatically annotates protein structure with sequence conservation starting from a set of protein sequence variants. We present a complete upgrade and rewrite of Scop3D. We have included a DNA module that allows the analysis of single nucleotide polymorphisms in relation to the structural context of the protein. Scop3D therefore forms a bridge between genomics and protein structure. Moreover, Scop3D is now also available through an intuitive web-interface that makes the tool highly user-friendly.

iRefWeb is a resource that provides web interface to a large collection of protein-protein interactions aggregated from major primary databases. The underlying data-consolidation process, called iRefIndex, implements a rigorous methodology of identifying redundant protein sequences and integrating disparate data records that reference the same peptide sequences, despite many potential differences in data identifiers across various source databases. iRefWeb offers a unified user interface to all interaction records and associated information collected by iRefIndex, in addition to a number of data filters and visual features that present the supporting evidence. Users of iRefWeb can explore the consolidated landscape of protein-protein interactions, establish the provenance and reliability of each data record, and compare annotations performed by different data curator teams. The iRefWeb portal is freely available at http://wodaklab.org/iRefWeb .

Presentation at the ELIXIR-NL Session about Data Analysis and Stewardship Training at the BioSB Conference 2020 (https://www.aanmelder.nl/biosb2020).

ELIXIR is a pan-European intergovernmental organisation for life science that aims to coordinate bioinformatics resources in a single infrastructure across Europe; bioinformatics training is central to its strategy, which aims to develop a training community that spans all ELIXIR member states. In an evidence-based approach for strengthening bioinformatics training programmes across Europe, the ELIXIR Training Platform, led by the ELIXIR EXCELERATE Quality and Impact Assessment Subtask in collaboration with the ELIXIR Training Coordinators Group, has implemented an assessment strategy to measure quality and impact of its entire training portfolio. Here, we present ELIXIR’s framework for assessing training quality and impact, which includes the following: specifying assessment aims, determining what data to collect in order to address these aims, and our strategy for centralised data collection to allow for ELIXIR-wide analyses. In addition, we present an overview of the ELIXIR training data collected over the past 4 years. We highlight the importance of a coordinated and consistent data collection approach and the relevance of defining specific metrics and answer scales for consortium-wide analyses as well as for comparison of data across iterations of the same course. ELIXIR-EXCELERATE is funded by the European Commission within the Research Infrastructures programme of Horizon 2020, grant agreement number 676559 (https://ec.europa.eu/programmes/horizon2020/en/area/researchinfrastructures). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Compensating in classical flow cytometry or unmixing in spectral systems is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. In both cases, spillover coefficients are estimated for each fluorophore using single-color controls. This approach has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, a novel approach for calculating spillover coefficients or spectral signatures of fluorophores. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software, but it differs in two key aspects from current methods: (1) it is much less demanding in the preparation of controls, as it does not require the presence of well-defined positive and negative populations, and (2) it does not require manual tuning of the spillover matrix, as the algorithm iteratively computes the tuning, producing an optimal compensation matrix. Another algorithm, AutoSpread, complements this approach, providing a robust estimate of the Spillover Spreading Matrix (SSM), while avoiding the need for well-defined positive and negative populations. Together, AutoSpill and AutoSpread provide a superior solution to the problem of fluorophore spillover, allowing simpler and more robust workflows in high-parameter flow cytometry.

Advances in high-throughput sequencing have resulted in a massive increase of RNA-Seq transcriptome data. However, the promise of rapid gene expression profiling in a specific tissue, condition, unicellular organism, or microbial community comes with new computational challenges. Owing to the limited availability of well-resolved reference genomes, de novo assembled (meta)transcriptomes have emerged as popular tools for investigating the gene repertoire of previously uncharacterized organisms. Yet, despite their potential, these datasets often contain fragmented or contaminant sequences, and their analysis remains difficult. To alleviate some of these challenges, we developed TRAPID 2.0, a web application for the fast and efficient processing of assembled transcriptome data. The initial processing phase performs a global characterization of the input data, providing each transcript with several layers of annotation, comprising structural, functional, and taxonomic information. The exploratory phase enables downstream analyses from the web application. Available analyses include the assessment of gene space completeness, the functional analysis and comparison of transcript subsets, and the study of transcripts in an evolutionary context. A comparison with similar tools highlights TRAPID’s unique features. Finally, analyses performed within TRAPID 2.0 are complemented by interactive data visualizations, facilitating the extraction of new biological insights, as demonstrated with diatom community metatranscriptomes.

Transcription factors are important gene regulators with distinctive roles in development, cell signaling and cell cycling, and they have been associated with many diseases. The ConTra v3 web server allows easy visualization and exploration of predicted transcription factor binding sites (TFBSs) in any genomic region surrounding coding or non-coding genes. In this updated version, with a completely re-implemented user interface using latest web technologies, users can choose from nine reference organisms ranging from human to yeast. ConTra v3 can analyze promoter regions, 5΄-UTRs, 3΄-UTRs and introns or any other genomic region of interest. Thousands of position weight matrices are available to choose from for detecting specific binding sites. Besides this visualization option, additional new exploration functionality is added to the tool that will automatically detect TFBSs having at the same time the highest regulatory potential, the highest conservation scores of the genomic regions covered by the predicted TFBSs and strongest co-localizations with genomic regions exhibiting regulatory activity. The ConTra v3 web server is freely available at http://bioit2.irc.ugent.be/contra/v3.

Motivation In vivo protein folding is governed by molecular chaperones, that escort proteins from their translational birth to their proteolytic degradation. In E.coli the main classes of chaperones that interact with the nascent chain are trigger factor, DnaK/J and GroEL/ES and several authors have performed whole-genome experiments to construct exhaustive client lists for each of these. Results We constructed a database collecting all publicly available data of experimental chaperone-interaction and -dependency data for the E.coli proteome, and enriched it with an extensive set of protein-specific as well as cell context-dependent proteostatic parameters. We made this publicly accessible via a web interface that allows to search for proteins or chaperone client lists, but also to profile user-specified datasets against all the collected parameters. We hope this will accelerate research in this field by quickly identifying differentiating features in datasets. Availability and implementation The Protein Homeostasis Database is freely available without any registration requirement at http://PHDB.switchlab.org/.

SUMMARYPosttranslational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased number and types of mapped protein modifications, a database is necessary that simultaneouly integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 200,000 PTM sites for 17 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools facilitate to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository that allows submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

Triterpenes constitute a large and important class of plant natural products with diverse structures and functions. Their biological roles range from membrane structural components over plant hormones to specialized plant defence compounds. Furthermore, triterpenes have great potential for a variety of commercial applications such as vaccine adjuvants, anti-cancer drugs, food supplements and agronomic agents. Their biosynthesis is carried out through complicated, branched pathways by multiple enzyme types that include oxidosqualene cyclases, cytochrome P450s, and UDP-glycosyltransferases. Given that the number of characterized triterpene biosynthesis enzymes has been growing fast recently, the need for a database specifically focusing on triterpene enzymology became eminent. Here, we present the TriForC database (http://bioinformatics.psb.ugent.be/triforc/), encompassing a comprehensive catalogue of triterpene biosynthesis enzymes. This highly interlinked database serves as a user-friendly access point to versatile data sets of enzyme and compound features, enabling the scanning of a complete catalogue of experimentally validated triterpene enzymes, their substrates and products, as well as the pathways they constitute in various plant species. The database can be accessed by direct browsing or through convenient search tools including keyword, BLAST, plant species and substructure options. This database will facilitate gene mining and creating genetic toolboxes for triterpene synthetic biology.

Motivation Comparative and evolutionary studies utilize phylogenetic trees to analyze and visualize biological data. Recently, several web-based tools for the display, manipulation and annotation of phylogenetic trees, such as iTOL and Evolview, have released updates to be compatible with the latest web technologies. While those web tools operate an open server access model with a multitude of registered users, a feature-rich open source solution using current web technologies is not available. Results Here, we present an extension of the widely used PhyloXML standard with several new options to accommodate functional genomics or annotation datasets for advanced visualization. Furthermore, PhyD3 has been developed as a lightweight tool using the JavaScript library D3.js to achieve a state-of-the-art phylogenetic tree visualization in the web browser, with support for advanced annotations. The current implementation is open source, easily adaptable and easy to implement in third parties’ web sites. Availability and implementation More information about PhyD3 itself, installation procedures and implementation links are available at http://phyd3.bits.vib.be and at http://github.com/vibbits/phyd3/. Supplementary information Supplementary data are available at Bioinformatics online.

The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties.

Hyaluronidases depolymerize hyaluronic acid, an important component of the extracellular matrix. Bacterial hyaluronan lyases (EC 4.2.2.1) contribute to the spreading of microorganisms in tissues. Potent and selective inhibitors are not known up to now, but would be interesting pharmacological tools and potential drugs for the treatment of bacterial infections. Starting from two crystal structures of streptococcal hyaluronan lyases, a homology model of the S. agalactiae strain 4755 enzyme hylB4755 was generated and applied to structure-based inhibitor design with the de novo design program LUDI. The databases LeadQuest® Vol. 1&2, Accelrys and ChemACX containing 228000 compounds were screened resulting in 1275 hits. Based on high LUDI scores, synthetic feasibility and commercial availability, 19 of these hits were investigated for inhibition of hylB4755. 13 compounds were active in the milli- and submillimolar range. 1,3-diacetylbenzimidazole-2-thione with IC50 values of 5 M and 160 M at physiological and optimum pH, respectively, is one of the two most potent inhibitors of hyaluronan lyases known to date. Depending on the LUDI sphere radius, it was docked in two different poses with similar scores. To analyze whether the hits represent (physico)chemical properties typical for drugability and optimal pharmacokinetics, the distributions of six descriptors within all hits from LeadQuest® and ChemACX and within the original databases were compared. Generally, the hit distributions represent the type of the original ones, but are narrower with slightly different medians. More than 80% of the hits have molecular weights between 150 and 350, XLOGP values between 1 and 4, one H-donor, 1-3 H-acceptors, 0-3 rotatable bonds and 1-3 rings. In conclusion, the application of LUDI to both databases has led to the accumulation of relatively rigid, drug-like molecules.

Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.

The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal α-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527–534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants Δ528–534 and Δ527–533, whereas the C-terminal β-strand and/or α-helix is displaced in the nonresponsive mutant Δ527–534.

Bacterial hyaluronan lyases (Hyal) degrade hyaluronan, an important component of the extracellular matrix, and are involved in microbial spread. Hyal inhibitors may serve as tools to study the role of the enzyme, its substrates and products in the course of bacterial infections. Moreover, such enzyme inhibitors are potential candidates for antibacterial combination therapy. Based on crystal structures of Streptococcus pneumoniae Hyal in complex with a hexasaccharide substrate and with different inhibitors, 1-acylated benzimidazole-2-thiones and benzoxazole-2-thiones were derived as new leads for the inhibition of Streptococcus agalactiae strain 4755 Hyal. Structure-based optimization led to N-(3-phenylpropionyl)benzoxazole-2-thione, one of the most potent compounds known to date (IC50 values: 24 μM at pH 7.4, 15 μM at pH 5). Among the 27 new derivatives, other N-acylated benzimidazoles and benzoxazoles are just as active at pH 7.4, but not at pH 5. The results support a binding mode characterized by interactions with residues in the catalytic site and with a hydrophobic patch.

Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH.

The Toll/interleukin 1 (IL-1) receptor family plays an important role in both innate and adaptive immunity. These receptors are characterized by a C-terminal homology motif called the Toll/IL-1 receptor (TIR) domain. A principal function of the TIR domain is mediating homotypic protein-protein interactions in the signal transduction pathway. To suggest interaction sites of TIR domains in the IL-1 receptor complex, we modeled the putative three-dimensional structure of the TIR domain within the co-receptor chain, IL-1 receptor accessory protein. The model was based on homology with the crystal structures of human TLR1 and TLR2. The final structure of the IL-1 receptor accessory protein TIR domain suggests the conserved regions box 1 and 2, including Pro-446, as well as box 3 within the C-terminal alpha-helix as possible protein-protein interaction sites due to their exposure and their electrostatic potential. Pro-446, corresponding to the Pro/His mutation in dominant negative TLR4, is located in the third loop at the outmost edge of the TIR domain and does not play any structural role. Inhibition of IL-1 responsiveness seen after substitution of Pro-446 by charged amino acids is due to the loss of an interaction site for other TIR domains. Amino acids 527-534 as part of the loop close to the conserved box 3 are critical for recruitment of myeloid differentiation factor 88 and to a lesser extent for IL-1 responsiveness. Modeling suggests that native folding of the TIR domain may be approached by the responsive deletion mutants delta528-534 and delta527-533, whereas the C-terminal beta-strand and/or alpha-helix is displaced in the nonresponsive mutant delta527-534.

The novel lead bis(1H-2-indolyl)methanone inhibits autophosphorylation of platelet-derived growth factor (PDGF) receptor tyrosine kinase in intact cells. Various substituents in the 5- or 6-position of one indole ring increase or preserve potency, whereas most modifications of the ring structures and of the methanone group as well as substitution at both indoles result in weak or no activity. An ATP binding site model, derived by homology from the FGFR-1 tyrosine kinase crystal structure suggesting hydrogen bonds of one indole NH and the methanone oxygen with the backbone carbonyl and amide, respectively, of Cys684, explains why only one indole moiety is open for substitution and locates groups in the 5- or 6-position outside the pocket. The hitherto most active derivatives, 39, 53 and 67, inhibit both isoforms of the PDGF receptor kinase in intact cells, with IC(50) of 0.1-0.3 microM, and purified PDGFbeta-receptor in vitro, with IC(50) of 0.09, 0.1, or 0.02 microM, respectively. PDGF-stimulated DNA synthesis is inhibited by these derivatives with IC(50) values of 1-3 microM. Kinetic analysis of 53 showed an ATP-competitive mode of inhibition. The compounds are inactive or weakly active toward a number of other tyrosine kinases, including the FGF receptor 1, EGF receptor, and c-Src kinase, as well as toward serine-threonine kinases, including different PKC isoforms and GRK2, and appear therefore selective for PDGF receptor inhibition.

PLAZA (https://bioinformatics.psb.ugent.be/plaza) is a plant-oriented online resource for comparative, evolutionary and functional genomics. The PLAZA platform consists of multiple independent instances focusing on different plant clades, while also providing access to a consistent set of reference species. Each PLAZA instance contains structural and functional gene annotations, gene family data and phylogenetic trees and detailed gene colinearity information. A user-friendly web interface makes the necessary tools and visualizations accessible, specific for each data type. Here we present PLAZA 4.0, the latest iteration of the PLAZA framework. This version consists of two new instances (Dicots 4.0 and Monocots 4.0) providing a large increase in newly available species, and offers access to updated and newly implemented tools and visualizations, helping users with the ever-increasing demands for complex and in-depth analyzes. The total number of species across both instances nearly doubles from 37 species in PLAZA 3.0 to 71 species in PLAZA 4.0, with a much broader coverage of crop species (e.g. wheat, palm oil) and species of evolutionary interest (e.g. spruce, Marchantia). The new PLAZA instances can also be accessed by a programming interface through a RESTful web service, thus allowing bioinformaticians to optimally leverage the power of the PLAZA platform.

Compensating in flow cytometry is an unavoidable challenge in the data analysis of fluorescence-based flow cytometry. Even the advent of spectral cytometry cannot circumvent the spillover problem, with spectral unmixing an intrinsic part of such systems. The calculation of spillover coefficients from single-color controls has remained essentially unchanged since its inception, and is increasingly limited in its ability to deal with high-parameter flow cytometry. Here, we present AutoSpill, an alternative method for calculating spillover coefficients. The approach combines automated gating of cells, calculation of an initial spillover matrix based on robust linear regression, and iterative refinement to reduce error. Moreover, autofluorescence can be compensated out, by processing it as an endogenous dye in an unstained control. AutoSpill uses single-color controls and is compatible with common flow cytometry software. AutoSpill allows simpler and more robust workflows, while reducing the magnitude of compensation errors in high-parameter flow cytometry., Flow cytometry allows the simultaneous quantification of many markers in and on a cell, but the analysis of such data is complicated. Here, the authors propose AutoSpill, a framework that facilitates the analysis of such data by automating parts of the analysis and requiring fewer controls.