TermineR: Extracting information on endogenous proteolytic processing from shotgun proteomics data.

State-of-the-art mass spectrometers combined with modern bioinformatics algorithms for peptide-to-spectrum matching (PSM) with robust statistical scoring allow for more variable features (i.e., post-translational modifications) being reliably identified from (tandem-) mass spectrometry data, often without the need for biochemical enrichment. Semi-specific proteome searches, that enforce a theoretical enzymatic digestion to solely the N- or C-terminal end, allow to identify of native protein termini or those arising from endogenous proteolytic activity (also referred to as "neo-N-termini" analysis or "N-terminomics"). Nevertheless, deriving biological meaning from these search outputs can be challenging in terms of data mining and analysis. Thus, we introduce TermineR, a data analysis approach for the (1) annotation of peptides according to their enzymatic cleavage specificity and known protein processing features, (2) differential abundance and enrichment analysis of N-terminal sequence patterns, and (3) visualization of neo-N-termini location. We illustrate the use of TermineR by applying it to tandem mass tag (TMT)-based proteomics data of a mouse model of polycystic kidney disease, and assess the semi-specific searches for biological interpretation of cleavage events and the variable contribution of proteolytic products to general protein abundance. The TermineR approach and example data are available as an R package at https://github.com/MiguelCos/TermineR.

Immunopeptidomics mapping of Listeria monocytogenes T cell epitopes in mice.

Listeria monocytogenes is a foodborne intracellular bacterial model pathogen. Protective immunity against Listeria depends on an effective CD8+ T cell response, but very few T cell epitopes are known in mice as a common animal infection model for listeriosis. To identify epitopes we screened for Listeria immunopeptides presented in the spleen of infected mice by mass spectrometry-based immunopeptidomics. We mapped more than 6,000 mouse self-peptides presented on MHC Class I molecules, including 12 high confident Listeria peptides from 12 different bacterial proteins. Bacterial immunopeptides with confirmed fragmentation spectra were further tested for their potential to activate CD8+ T cells, revealing VTYNYINI from the putative cell wall surface anchor family protein LMON_0576 as a novel bona fide peptide epitope. The epitope showed high biological potency in a prime boost model and can be used as a research tool to probe CD8+ T cell responses in mouse models of Listeria infection. Together, our results demonstrate the power of immunopeptidomics for bacterial antigen identification.

Assessing the performance of blue-green solutions through a fine-scale water balance model for an urban area.

Blue and Green Infrastructures (BGIs) are natural or semi-natural systems that are considered an efficient solution to enhance stormwater management. To assess the performance of BGIs in mitigating floods and droughts in an urban area, a water balance model was developed in this study to simulate runoff formation and propagation. The developed model features fine spatial and temporal resolutions and flexibly integrates BGIs. Combining the conceptual single reservoir approach and the empirical continuous Soil Conservation Service Curve Number (SCS-CN) method, the model achieves computational efficiency, enabling long-term simulations that capture both short-term extreme events and long-term water balance. Its high transferability allows for easy incorporation of local datasets, making it adaptable to various urban contexts. Applied on a university campus located in Belgium, the model was used to simulate the water balance components of feasible BGIs for the study area, which were green roofs, permeable surfaces and rainwater tanks. Scenario analysis of both single BGI and combined BGI implementations was conducted, and all BGI scenarios were evaluated based on peak flow and runoff volume reduction and water balance analysis. Results demonstrate that the implementation of a combination of several BGIs with different functions is an effective solution for both flood control and drought mitigation, as these solutions can significantly reduce runoff flows, increase infiltration and provide considerable rainwater reuse.

Biomechanics of Human Locomotion in the Wind.

In laboratory settings, human locomotion encounters minimal opposition from air resistance. However, moving in nature often requires overcoming airflow. Here, the drag force exerted on the body by different headwind or tailwind speeds (between 0-15 m s-1) was measured during walking at 1.5 m s-1 and running at 4 m s- 1. To our knowledge, the biomechanical effect of drag in human locomotion has only been evaluated by simulations. Data were collected on eight male subjects using an instrumented treadmill placed in a wind tunnel. From the ground reaction forces, the drag and external work done to overcome wind resistance and to sustain the motion of the center of mass of the body were measured. Drag increased with wind speed: a 15 m s-1 headwind exerted a drag of ~60 N in walking and ~50 N in running. The same tailwind exerted -55 N of drag in both gaits. At this wind speed, the work done to overcome the airflow represented ~80% of the external work in walking and ~50% in running. Furthermore, in the presence of fast wind speeds, subjects altered their drag area (CdA) by adapting their posture to limit the increase in air friction. Moving in the wind modified the ratio between positive and negative external work performed. The modifications observed when moving with a head- or tailwind have been compared with moving uphill or downhill. The present findings may have implications for optimizing aerodynamic performance in competitive running, whether in sprints or marathons.

The Plant PTM Viewer 2.0: in-depth exploration of plant protein modification landscapes.

Post-translational modifications (PTMs) greatly increase protein diversity and functionality. To help the plant research community interpret the ever-increasing number of reported PTMs, the Plant PTM Viewer (https://www.psb.ugent.be/PlantPTMViewer) provides an intuitive overview of plant protein PTMs and the tools to assess it. This update includes 62 novel PTM profiling studies, adding a total of 112 000 modified peptides reporting plant PTMs, including 14 additional PTM types and three species (moss, tomato, and soybean). Furthermore, an open modification re-analysis of a large-scale Arabidopsis thaliana mass spectrometry tissue atlas identified previously uncharted landscapes of lysine acylations predominant in seed and flower tissues and 3-phosphoglycerylation on glycolytic enzymes in plants. An extra 'Protein list analysis' tool was developed for retrieval and assessing the enrichment of PTMs in a protein list of interest. We conducted a protein list analysis on nuclear proteins, revealing a substantial number of redox modifications in the nucleus, confirming previous assumptions regarding the redox regulation of transcription. We encourage the plant research community to use PTM Viewer 2.0 for hypothesis testing and new target discovery, and also to submit new data to expand the coverage of conditions, plant species, and PTM types, thereby enriching our understanding of plant biology.

Analysing spatial variability in drought sensitivity of rivers using explainable artificial intelligence.

Hydrological drought can have a severe negative impact on aquatic ecosystems and human living standards. Therefore, being able to predict and gain more insights in the spatial variability in drought sensitivity of rivers is of relevance for water managers. The drought sensitivity of a river is in this study represented by four drought metrics, of which three are relative towards the ecological minimal flow. Statistical and machine learning methods were evaluated to predict these metrics for rivers in the Flanders region of Belgium based on catchment characteristics and data on human interferences. XGBoost had the best performance, with an explained variance of 80 % to 90 %. After applying explaining AI on these models, insights were obtained in the spatial variability of the drought metrics. Irrigation is the most important variable, a high percentage of irrigation leads to a higher drought sensitivity. If there are a lot of human interferences, there is a higher drought sensitivity. Many of the observed dependencies can be explained by the differences in soil infiltration capacity and transferability of water for sandy versus clay soils. No clear dependence with the amount of forest or agriculture was observed, implying that the impact of forest and agriculture on the drought sensitivity of a river is complex.

Kinetics and mechanical work done to move the body centre of mass along a curve.

When running on a curve, the lower limbs interact with the ground to redirect the trajectory of the centre of mass of the body (CoM). The goal of this paper is to understand how the trajectory of the CoM and the work done to maintain its movements relative to the surroundings (Wcom) are modified as a function of running speed and radius of curvature. Eleven participants ran at different speeds on a straight line and on circular curves with a 6 m and 18 m curvature. The trajectory of the CoM and Wcom were calculated using force-platforms measuring the ground reaction forces and infrared cameras recording the movements of the pelvis. To follow a circular path, runners overcompensate the rotation of their trajectory during contact phases. The deviation from the circular path increases when the radius of curvature decreases and speed increases. Interestingly, an asymmetry between the inner and outer lower limbs emerges as speed increases. The method to evaluate Wcom on a straight-line was adapted using a referential that rotates at heel strike and remains fixed during the whole step cycle. In an 18 m radius curve and at low speeds on a 6 m radius, Wcom changes little compared to a straight-line run. Whereas at 6 m s-1 on a 6 m radius, Wcom increases by ~25%, due to an augmentation in the work to move the CoM laterally. Understanding these adaptations provides valuable insight for sports sciences, aiding in optimizing training and performance in sports with multidirectional movements.

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry.

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

Interactome of Arabidopsis ATG5 Suggests Functions beyond Autophagy.

Autophagy is a catabolic pathway capable of degrading cellular components ranging from individual molecules to organelles. Autophagy helps cells cope with stress by removing superfluous or hazardous material. In a previous work, we demonstrated that transcriptional upregulation of two autophagy-related genes, ATG5 and ATG7, in Arabidopsis thaliana positively affected agronomically important traits: biomass, seed yield, tolerance to pathogens and oxidative stress. Although the occurrence of these traits correlated with enhanced autophagic activity, it is possible that autophagy-independent roles of ATG5 and ATG7 also contributed to the phenotypes. In this study, we employed affinity purification and LC-MS/MS to identify the interactome of wild-type ATG5 and its autophagy-inactive substitution mutant, ATG5K128R Here we present the first interactome of plant ATG5, encompassing not only known autophagy regulators but also stress-response factors, components of the ubiquitin-proteasome system, proteins involved in endomembrane trafficking, and potential partners of the nuclear fraction of ATG5. Furthermore, we discovered post-translational modifications, such as phosphorylation and acetylation present on ATG5 complex components that are likely to play regulatory functions. These results strongly indicate that plant ATG5 complex proteins have roles beyond autophagy itself, opening avenues for further investigations on the complex roles of autophagy in plant growth and stress responses.

Shift in vacuolar to cytosolic regime of infecting Salmonella from a dual proteome perspective.

By applying dual proteome profiling to Salmonella enterica serovar Typhimurium (S. Typhimurium) encounters with its epithelial host (here, S. Typhimurium infected human HeLa cells), a detailed interdependent and holistic proteomic perspective on host-pathogen interactions over the time course of infection was obtained. Data-independent acquisition (DIA)-based proteomics was found to outperform data-dependent acquisition (DDA) workflows, especially in identifying the downregulated bacterial proteome response during infection progression by permitting quantification of low abundant bacterial proteins at early times of infection when bacterial infection load is low. S. Typhimurium invasion and replication specific proteomic signatures in epithelial cells revealed interdependent host/pathogen specific responses besides pointing to putative novel infection markers and signalling responses, including regulated host proteins associated with Salmonella-modified membranes.

Estimation of the impact of combined sewer overflows on surface water quality in a sparsely monitored area.

Combined sewer overflows (CSOs) can have a severe negative, local impact on surface water systems. To assure good ecological surface water quality and drinking water production that meets the demands, the impact of sewer system overflows on the surrounding water bodies for current and future climate conditions needs to be assessed. Typically, integrated, detailed hydrological and hydrodynamic water quantity and quality models are used for this purpose, but often data and computational resource requirements limit their applicability. Therefore, an alternative computationally efficient, integrated water quantity and quality model of sewer systems and their receiving surface waters is proposed to assess the impact of CSOs on surface water quality in a sparsely observed area. A conceptual model approach to estimate CSO discharges is combined with an empirical model for estimating CSO pollutant concentrations based on waste water treatment plant influent observations. Both methods are compared with observations and independent results of established reference methods as to evaluate their performance. The methodology is demonstrated by modelling the current impact of CSOs on the water abstraction area of a major drinking water production centre in Flanders, Belgium. It is concluded that the proposed conceptual models achieve similar results for daily WWTP effluent and CSO frequency, whereby the accumulated CSO volume is similar to more detailed full hydrodynamic models. Further, the estimated pollutant concentrations correspond with another dataset based on high resolution sampled overflows. As a result, the proposed computational efficient method can give insights in the impact of CSOs on the water quality at a catchment level and can be used for planning monitoring campaigns or performing analyses of e.g. the current and future water availability for a data scarce areas.

Mutation of Arabidopsis SME1 and Sm core assembly improves oxidative stress resilience.

Alternative splicing is a key posttranscriptional gene regulatory process, acting in diverse adaptive and basal plant processes. Splicing of precursor-messenger RNA (pre-mRNA) is catalyzed by a dynamic ribonucleoprotein complex, designated the spliceosome. In a suppressor screen, we identified a nonsense mutation in the Smith (Sm) antigen protein SME1 to alleviate photorespiratory H2O2-dependent cell death in catalase deficient plants. Similar attenuation of cell death was observed upon chemical inhibition of the spliceosome, suggesting pre-mRNA splicing inhibition to be responsible for the observed cell death alleviation. Furthermore, the sme1-2 mutants showed increased tolerance to the reactive oxygen species inducing herbicide methyl viologen. Both an mRNA-seq and shotgun proteomic analysis in sme1-2 mutants displayed a constitutive molecular stress response, together with extensive alterations in pre-mRNA splicing of transcripts encoding metabolic enzymes and RNA binding proteins, even under unstressed conditions. Using SME1 as a bait to identify protein interactors, we provide experimental evidence for almost 50 homologs of the mammalian spliceosome-associated protein to reside in the Arabidopsis thaliana spliceosome complexes and propose roles in pre-mRNA splicing for four uncharacterized plant proteins. Furthermore, as for sme1-2, a mutant in the Sm core assembly protein ICLN resulted in a decreased sensitivity to methyl viologen. Taken together, these data show that both a perturbed Sm core composition and assembly results in the activation of a defense response and in enhanced resilience to oxidative stress.

Modification of the locomotor pattern when deviating from the characteristic heel-to-toe rolling pattern during walking.

PURPOSE: Humans are amongst few animals that step first on the heel, and then roll on the ball of the foot and toes. While this heel-to-toe rolling pattern has been shown to render an energetic advantage during walking, the effect of different foot contact strategies, on the neuromuscular control of adult walking gaits has received less attention. We hypothesised that deviating from heel-to-toe rolling pattern affects the energy transduction and weight acceptance and re-propulsive phases in gait along with the modification of spinal motor activity. METHODS: Ten subjects walked on a treadmill normally, then placed their feet flat on the ground at each step and finally walked on the balls of the feet. RESULTS: Our results show that when participants deviate from heel-to-toe rolling pattern strategy, the mechanical work increases on average 85% higher (F = 15.5; p < 0.001), mainly linked to a lack of propulsion at late stance. This modification of the mechanical power is related to a differential involvement of lumbar and sacral segment activation. Particularly, the delay between the major bursts of activation is on average 65% smaller, as compared to normal walking (F = 43.2; p < 0.001). CONCLUSION: Similar results are observable in walking plantigrade animals, but also at the onset of independent stepping in toddlers, where the heel-to-toe rolling pattern is not yet established. These indications seem to bring arguments to the fact that the rolling of the foot during human locomotion has evolved to optimise gait, following selective pressures from the evolution of bipedal posture.

Functionally annotating cysteine disulfides and metal binding sites in the plant kingdom using AlphaFold2 predicted structures.

Deep learning algorithms such as AlphaFold2 predict three-dimensional protein structure with high confidence. The recent release of more than 200 million structural models provides an unprecedented resource for functional protein annotation. Here, we used AlphaFold2 predicted structures of fifteen plant proteomes to functionally and evolutionary analyze cysteine residues in the plant kingdom. In addition to identification of metal ligands coordinated by cysteine residues, we systematically analyzed cysteine disulfides present in these structural predictions. Our analysis demonstrates most of these predicted disulfides are trustworthy due their high agreement (∼96%) with those present in X-ray and NMR protein structures, their characteristic disulfide stereochemistry, the biased subcellular distribution of their proteins and a higher degree of oxidation of their respective cysteines as measured by proteomics. Adopting an evolutionary perspective, zinc binding sites are increasingly present at the expense of iron-sulfur clusters in plants. Interestingly, disulfide formation is increased in secreted proteins of land plants, likely promoting sequence evolution to adapt to changing environments encountered by plants. In summary, Alphafold2 predicted structural models are a rich source of information for studying the role of cysteines residues in proteins of interest and for protein redox biology in general.

Cysteine thiol-based post-translational modification: What do we know about transcription factors?

Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression.

International collaboration to improve physiotherapists' training, Viet Nam.

Problem: Like most low- and middle-income countries, Viet Nam has a scarcity of rehabilitation professionals and lacks training programmes that meet international standards. Approach: In 2018, four Vietnamese medical universities, the Université Catholique de Louvain, the Université Libre de Bruxelles, the Humanity & Inclusion charity and World Physiotherapy agreed to collaborate on strengthening pre-service education for physiotherapists in the country. Local setting: Viet Nam has a favourable environment for nurturing rehabilitation services and education: development funds have been available; government investment is increasing; and rehabilitation education has existed for many decades. Relevant changes: The collaboration resulted in the establishment of: (i) a 4-year, competency-based, entry-level curriculum for physiotherapists (bachelor's degree); (ii) opportunities for continuing professional development; (iii) a 2-year master's programme for physiotherapy lecturers and clinical supervisors; and (iv) a national physiotherapy association. In addition, four students were supported in studying for PhD degrees. Strong collaboration and comprehensive and complementary interventions have laid the foundations for sustainable, high-quality, educational programmes for physiotherapists, which will improve access to, and the standard of, rehabilitation services in Viet Nam, thereby leading to better patient outcomes. Lessons learnt: Curricula for entry-level physiotherapy programmes should be competency-based, be actively managed by national educators and meet international standards while being responsive to local priorities. To strengthen the rehabilitation workforce, educators involved in teaching and supervising training programmes should have the skills and knowledge required. A national professional physiotherapy association should be established to provide continuing professional development for physiotherapists and to take part in international collaborations.

Further clinical and genetic evidence of ASC-1 complex dysfunction in congenital neuromuscular disease.

Transcriptional coregulators modulate the efficiency of transcription factors. Bi-allelic variants in TRIP4 and ASCC1, two genes that encode members of the tetrameric coregulator ASC-1, have recently been associated with congenital bone fractures, hypotonia, and muscular dystrophy in a total of 22 unrelated families. Upon exome sequencing and data repository mining, we identified six new patients with pathogenic homozygous variants in either TRIP4 (n = 4, two novel variants) or ASCC1 (n = 2, one novel variant). The associated clinical findings confirm and extend previous descriptions. Considering all patients reported to date, we provide supporting evidence suggesting that ASCC1-related disease has a more severe phenotype compared to TRIP4-related disorder regarding higher incidence of perinatal bone fractures and shorter survival.

Analysis of ROS-Triggered Changes in the Transcriptome.

RNA sequencing is routinely used for determining transcriptome-wide expression changes during various conditions, including oxidative stress conditions. In this chapter, a basic workflow to determine differentially expressed genes between two conditions of interest is provided. After providing brief guidelines for experimental design, we provide step-by-step instructions for genome alignment of reads and differential expression analysis.

Exploring Posttranslational Modifications with the Plant PTM Viewer.

An increasing numbed and diversity of protein posttranslational modifications are mapped by proteomics. Knowledge of modified protein sites can be of direct relevance to steer downstream functional studies, but such information can be difficult to distill from the numerous individual studies that often report modified sites in supplementary data files. In this chapter, we provide basic instructions and use cases to browse and collect plant protein modifications from over 100 studies in a user-friendly manner with the Plant PTM Viewer. The Plant PTM Viewer ( https://www.psb.ugent.be/PlantPTMViewer ) is a central resource compiling currently 24 modifications types for ten thousands of plant proteins. Next to viewing modifications for a protein of interest, we demonstrate how evolutionary conserved modifications can be retrieved using PTM Blast. In addition, modifications themselves located in a motif or sequence context of interest can be retrieved by PTM search. Altogether, these operations can be performed, are intuitive, and open for non-bioinformatic experts.