We developed a lesson where students construct a qualitative representation to learn how clock genes are regulated. Qualitative representations provide a non-numerical description of system behavior, focusing on causal relation-ships and system states. They align with human reasoning about system dy-namics and serve as valuable learning tools for understanding both domain-specific systems and developing broader systems thinking skills.The lesson, designed for upper secondary and higher education, is imple-mented in the DynaLearn software at Level 4, where students can model feedback loops. Students construct the representation step by step, guided by a structured workbook and built-in support functions within the software. At each step, they run simulations to examine system behavior and reflect on the results through workbook questions. To ensure scientific accuracy, the representation and workbook were evaluated by domain experts.The lesson begins with modeling how increasing BMAL:CLOCK activity enhances the transcription of PER and CRY genes through binding to the E-box. Next, students explore how mRNA production and degradation—two opposing processes—regulate mRNA levels. This is followed by modeling translation at the ribosomes, where PER and CRY proteins are synthesized and subsequently degraded, again illustrating competing regulatory process-es. Students then model how PER and CRY proteins form a complex that translocates to the nucleus, inhibiting CLOCK:BMAL binding and establish-ing a negative feedback loop. Finally, they extend their understanding by ex-ploring how CLOCK:BMAL also regulates the AVP gene, linking clock genes to broader physiological processes.
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The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.
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The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00–2.49 log CFU/g of manured soil, ranging between 10–5 and 10–4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.
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Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.
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Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome‐wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site‐directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101.
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Hyperhomocysteinemia is a risk factor for cardiovascular disease, neurological disorders, and bone abnormalities. The key enzyme in homocysteine metabolism, cystathionine-β-synthase (CBS) is recognized as a target for new homocysteine-lowering therapies including enzyme replacement and gene therapy. Currently, there are no pharmacotherapies available that enhance CBS activity through its allosteric mechanism. The only known allosteric activator of CBS is S-adenosyl-L-methionine (SAM), which is available as a food supplement, but its effectiveness is limited by low membrane permeability and universal involvement in methylation reactions as a substrate. The discovery of CBS activators in high-throughput screening is challenging due to a lack of dedicated assays. Available HTS-compatible activity assays for CBS rely on measuring the product hydrogen sulfide or methanethiol where the signal increases with increased CBS activity. In the case of fluorescence-based assays, it is challenging to discern activators from autofluorescent compounds. In this study, we introduce a homocysteine consumption assay for isolated human CBS (HconCBS) based on the absorbance of Ellman's reagent. This assay leverages a decrease in signal upon CBS activation, with performance parameters exceeding the requirements for high-throughput screening. In a commercial library of 3010 compounds, the HconCBS assay identified 10 hit compounds as more active than SAM, whereas a fluorescence-based assay using 7-azido-4-methylcoumarin (AzMC) identified 141 hits. HconCBS identified 101 compounds with autoabsorbance which did not include hit compounds, while the fluorescence-based assay identified 383 autofluorescent compounds which included all hit compounds. While 4 out of 10 HconCBS hits were confirmed when purchased from a new source, the compounds affected homocysteine rather than CBS. Nevertheless, HconCBS consistently detected the CBS activator seleno-adenosyl-L-methionine (SeAM) added to 4 library plates and re-discovered the same library hits in 3 out of 4 re-screened plates. Taken together, HconCBS was designed to enable the discovery of allosteric CBS activators with greater reliability than fluorescence-based methods. Despite identifying some compounds that acted on homocysteine rather than CBS, the assay consistently identified the CBS activators SAM and SeAM and demonstrated reproducibility across two screening rounds. These findings establish HconCBS as a valuable tool for identifying potential therapeutic candidates for hyperhomocysteinemia, addressing a key gap in the development of CBS-targeted pharmacotherapies.
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We tested the hypothesis that in human ageing a decreased intramuscular acylcarnitine status is associated with (pre-)frailty, reduced physical performance and altered mitochondrial function. Results showed that intramuscular total carnitine levels and acetylcarnitine levels were lower in (pre-)frail old females compared to fit old females and young females, whereas no differences were observed in males. The low intramuscular acetylcarnitine levels in females correlated with low physical performance, even after correction for muscle mass (%), and were accompanied with lowered expression of genes involved in mitochondrial energy production and functionality. We concluded that in (pre-)frail old females, intramuscular total carnitine levels and acetylcarnitine levels are decreased, and this decrease is associated with reduced physical performance and low expression of a wide range of genes critical for mitochondrial function. The results stress the importance of taking sex differences into account in ageing research.
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Most endogenous viruses, an important proportion of eukaryote genomes, are doomed to slowly decay. Little is known, however, on how they evolve when they confer a benefit to their host. Bracoviruses are essential for the parasitism success of parasitoid wasps, whose genomes they integrated ~103 million years ago. Here we show, from the assembly of a parasitoid wasp genome, for the first time at a chromosomal scale, that symbiotic bracovirus genes spread to and colonized all the chromosomes. Moreover, large viral clusters are stably maintained suggesting strong evolutionary constraints. Genomic comparison with another wasps revealed that this organization was already established ~53 mya. Transcriptomic analyses highlight temporal synchronization of viral gene expression, leading to particle production. Immune genes are not induced, however, indicating the virus is not perceived as foreign by the wasp. This recognition suggests that no conflicts remain between symbiotic partners when benefits to them converge.
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Om inzicht te krijgen in spierveroudering is genexpressie gemeten in vastus lateralis biopten van jonge en oude mannen en vrouwen. We vonden dat tijdens het ouder worden bij beide geslachten dezelfde categorieën genen in spieren worden aan- en uitgeschakeld (“gereguleerd”); de mate van deze zogenaamde differentiële expressie was echter geslachtsspecifiek. Bij mannen was oxidatieve fosforylering het meest in het oog springende proces, en bij vrouwen was dit celgroei gemedieerd door AKT-signalering. De conclusie is dat dezelfde processen zijn geassocieerd met skeletspierveroudering bij mannen en vrouwen, maar dat de differentiële expressie van die processen geslachtsspecifiek is.
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