Rafael Andrade Buono

Post-translational modifications (PTMs) and genetic variants regulate protein function, signalling, and disease, but their interpretation requires integration of sequence annotations with structural, interaction, and biophysical context. Although resources such as Scop3P, UniProt, the Protein Data Bank, and AlphaFold provide extensive annotations and structural information, integrating these data into reproducible structure-aware analyses still requires custom scripting and manual coordination between multiple independent tools. To address this challenge, we developed Scop3P-Toolkit , an open-source executable analytical environment for interactive analysis of PTMs, mutations, and proteomics-derived peptides in their structural context. The toolkit integrates protein annotation retrieval with structural mapping, residue interaction network analysis, comparative structural analysis, and residue-level biophysical profiling within a unified framework. Experimentally supported phosphosites, phosphopeptides, and phosphoproteomics evidence are provided for human proteins through Scop3P, with optional integration of curated UniProt PTM annotations. UniProt-derived PTMs, sequence features, and genetic variants are available for proteins from any species, extending the framework beyond the human phosphoproteome. Scop3P-Toolkit supports structure-centric analyses including interpretation of PTMs and disease-associated variants, analysis of residue interaction networks and their rewiring across alternative conformations, structural localisation of peptides, and exploration of protein–protein, protein–ligand, and host–pathogen interfaces. Interactive visualisation links sequence annotations, three-dimensional structures, residue interaction networks, and biophysical profiles, enabling coordinated exploration across multiple molecular representations. The toolkit is distributed as Jupyter notebooks, browser-based Voilà applications, and a Galaxy interactive tool, providing transparent, accessible, and reproducible workflows for both computational and experimental researchers. By integrating biological annotation resources into executable, structure-aware workflows, Scop3P-Toolkit enables reproducible interpretation of PTMs, mutations, and proteomics data.

Artificial intelligence (AI) has seen transformative breakthroughs in the life sciences, expanding possibilities to interpret biological information at an unprecedented capacity. To maximize return on growing investments and accelerate progress, it is urgent to address long-standing research challenges arising from the rapid adoption of AI methods. We review the erosion of trust in AI outputs driven by poor reusability and reproducibility, and highlight their impact on environmental sustainability. Furthermore, we discuss the fragmented components of the AI ecosystem and lack of guiding pathways to support open and sustainable AI model development. In response, this Perspective introduces practical open and sustainable AI recommendations mapped to over 300 ecosystem components and provides guiding implementation pathways. Our work connects researchers with relevant AI resources, facilitating the implementation of sustainable, reusable and reproducible AI. Built upon community consensus and aligned to existing efforts, these outputs will aid future policy development and structured pathways for guiding AI implementation.

Programmed cell death (PCD) is a fundamental cellular process crucial to development, homeostasis, and immunity in multicellular eukaryotes. In contrast to our knowledge on the regulation of diverse animal cell death subroutines, information on execution of PCD in plants remains fragmentary. Here we make use of the accessibility of the Arabidopsis (Arabidopsis thaliana) root cap to visualize the execution process of developmentally controlled PCD. We identify a succession of selective decompartmentalization events and ion fluxes as part of the terminal differentiation program that is orchestrated by the NAC (NO APICAL MERISTEM, ARABIDOPSIS THALIANA ACTIVATING FACTOR, CUP-SHAPED COTYLEDON) transcription factor SOMBRERO. Surprisingly, breakdown of the large central vacuole is a relatively late and variable event, preceded by an increase of intracellular calcium levels and acidification, release of mitochondrial matrix proteins, leakage of nuclear and endoplasmic reticulum lumina, and release of fluorescent membrane reporters into the cytosol. In analogy to animal apoptosis, the plasma membrane remains impermeable for proteins during and after PCD execution. Elevated intracellular calcium levels and acidification are sufficient to trigger cell death execution specifically in terminally differentiated root cap cells, suggesting that these ion fluxes act as PCD-triggering signals. This detailed information on the cellular processes occurring during developmental PCD in plants is a pivotal prerequisite for future research into the molecular mechanisms of cell death execution.

The Horizon Europe project ISIDORe is dedicated to pandemic preparedness and responsiveness research. It brings together 17 research infrastructures (RIs) and networks to provide a broad range of services to infectious disease researchers. An efficient and structured treatment of data is central to ISIDORe’s aim to furnish seamless access to its multidisciplinary catalogue of services, and to ensure that users’ results are treated FAIRly. ISIDORe therefore requires a data management plan (DMP) covering both access management and research outputs, applicable over a broad range of disciplines, and compatible with the constraints and existing practices of its diverse partners. Here, we describe how, to achieve that aim, we undertook an iterative, step-by-step, process to build a community-approved living document, identifying good practices and processes, on the basis of use cases, presented as proof of concepts. International fora such as the RDA and EOSC, and primarily the BY-COVID project, furnished registries, tools and online data platforms, as well as standards, and the support of data scientists. Together, these elements provide a path for building an umbrella, FAIR-compliant DMP, aligned as fully as possible with FAIR principles, which could also be applied as a framework for data management harmonisation in other large-scale, challenge-driven projects. Finally, we discuss how data management and reuse can be further improved through the use of knowledge models when writing DMPs and, how, in the future, an inter-RI network of data stewards could contribute to the establishment of a community of practice, to be integrated subsequently into planned trans-RI competence centres.

Summary presentation reviewing all the components of the Elixir research data management ecosystem. Presented during the "ELIXIR FAIR & Research Data Management know-how ecosystem" workshop at the Elixir All Hands 2022.

CRISPR technology is an established tool for the generation of knockout plants (Zhang et al., 2019), yet limitations remain. First, the manipulation of individual genes may fail to produce phenotypes for groups of genes with redundant or synergistic functions. While this has been partially addressed by multiplexing guide RNAs (gRNAs), there is concern that as the number of targets increases, the chances of obtaining higher‐order knockouts diminish (Zhang et al., 2016). Second, knocking out fundamentally important genes can cause severe pleiotropic phenotypes or lethality. Tissue‐specific knockout of genes in somatic tissues can overcome this limitation (Decaestecker et al., 2019 ; Wang et al., 2020 ; Liang et al., 2019). However, the efficiency of simultaneously targeting more than three genes in a tissue‐specific context is unexplored. Here, by multiplexing gRNAs in Arabidopsis thaliana plants expressing Cas9 either ubiquitously (pPcUBI) or root cap‐specifically (pSMB), we show that six genes can be simultaneously mutated with high efficiency, generating higher‐order mutant phenotypes already in the first transgenic generation (T1). The mutation frequencies for all target genes were positively correlated and unaffected by the order of the gRNAs in the vector, showing that efficient higher‐order mutagenesis in specific plant tissues can be readily achieved.

Multiplex CRISPR approaches enable mutating multiple genes in plants, however it is unclear how feasible this is in tissue-specific mutagenesis. Here we simultaneously mutated six genes either ubiquitously or exclusively in the root cap of Arabidopsis. The mutation frequencies for all target genes were positively correlated and unaffected by the order of gRNAs in the vector, indicating that efficient higher-order mutagenesis in specific plant tissues can be readily achieved.

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

Proteases are among the key regulators of most forms of programmed cell death (PCD) in animals. Many PCD processes have also been associated with protease expression or activation in plants, However, functional evidence for the roles and actual modes of action of plant proteases in PCD remains surprisingly limited. In this review, we provide an update on protease involvement in the context of developmentally regulated plant PCD. To illustrate the diversity of protease functions, we focus on several prominent developmental PCD processes, including xylem and tapetum maturation, suspensor elimination, endosperm degradation, and seed coat formation, as well as plant senescence processes. Despite the substantial advances in the field, protease functions are often only correlatively linked to developmental PCD, and the specific molecular roles of proteases in many developmental PCD processes remain to be elucidated.