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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Background: Profiling the plant root architecture is vital for selecting resilient crops that can efficiently take up water and nutrients. The high-performance imaging tools available to study root-growth dynamics with the optimal resolution are costly and stationary. In addition, performing nondestructive high-throughput phenotyping to extract the structural and morphological features of roots remains challenging. Results: We developed the MultipleXLab: a modular, mobile, and cost-effective setup to tackle these limitations. The system can continuously monitor thousands of seeds from germination to root development based on a conventional camera attached to a motorized multiaxis-rotational stage and custom-built 3D-printed plate holder with integrated light-emitting diode lighting. We also developed an image segmentation model based on deep learning that allows the users to analyze the data automatically. We tested the MultipleXLab to monitor seed germination and root growth of Arabidopsis developmental, cell cycle, and auxin transport mutants non-invasively at high-throughput and showed that the system provides robust data and allows precise evaluation of germination index and hourly growth rate between mutants. Conclusion: MultipleXLab provides a flexible and user-friendly root phenotyping platform that is an attractive mobile alternative to high-end imaging platforms and stationary growth chambers. It can be used in numerous applications by plant biologists, the seed industry, crop scientists, and breeding companies.
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Many lithographically created optical components, such as photonic crystals, require the creation of periodically repeated structures [1]. The optical properties depend critically on the consistency of the shape and periodicity of the repeated structure. At the same time, the structure and its period may be similar to, or substantially below that of the optical diffraction limit, making inspection with optical microscopy difficult. Inspection tools must be able to scan an entire wafer (300 mm diameter), and identify wafers that fail to meet specifications rapidly. However, high resolution, and high throughput are often difficult to achieve simultaneously, and a compromise must be made. TeraNova is developing an optical inspection tool that can rapidly image features on wafers. Their product relies on (a) knowledge of what the features should be, and (b) a detailed and accurate model of light diffraction from the wafer surface. This combination allows deviations from features to be identified by modifying the model of the surface features until the calculated diffraction pattern matches the observed pattern. This form of microscopy—known as Fourier microscopy—has the potential to be very rapid and highly accurate. However, the solver, which calculates the wafer features from the diffraction pattern, must be very rapid and precise. To achieve this, a hardware solver will be implemented. The hardware solver must be combined with mechatronic tracking of the absolute wafer position, requiring the automatic identification of fiduciary markers. Finally, the problem of computer obsolescence in instrumentation (resulting in security weaknesses) will also be addressed by combining the digital hardware and software into a system-on-a-chip (SoC) to provide a powerful, yet secure operating environment for the microscope software.
DISCO aims at fast-tracking upscaling to new generation of urban logistics and smart planning unblocking the transition to decarbonised and digital cities, delivering innovative frameworks and tools, Physical Internet (PI) inspired. To this scope, DISCO will deploy and demonstrate innovative and inclusive urban logistics and planning solutions for dynamic space re-allocation integrating urban freight at local level, within efficiently operated network-of-networks (PI) where the nodes and infrastructure are fixed and mobile based on throughput demands. Solutions are co-designed with the urban logistics community – e.g., cities, logistics service providers, retailers, real estate/public and private infrastructure owners, fleet owners, transport operators, research community, civil society - all together moving a paradigm change from sprawl to data driven, zero-emission and nearby-delivery-based models.
Aanleiding Nieuwe stoffen en producten van de farmaceutische sector en de (agro)chemie moeten uitgebreid getest worden voordat ze op de markt kunnen verschijnen. Voor die testen is nu nog een groot aantal proefdieren nodig. Dit stuit echter op een aantal bezwaren: de uitkomsten van deze studies zijn niet altijd goed vertaalbaar naar effecten bij de mens, proefdierstudies zijn duur en de ethische kant van dierproeven staat steeds vaker ter discussie. Bedrijven zijn naarstig op zoek naar alternatieve testsystemen die ervoor kunnen zorgen dat proefdierstudies met zoogdieren worden verminderd, verfijnd en vervangen (de drie V's). Doelstelling In twee eerdere RAAK-projecten is ontdekt dat nematode C. elegans een kansrijk alternatief voor dierproeven is. Het is nu aan het multidisciplinaire team van Elegant! om de potentie van deze rondworm uit te bouwen en te ontwikkelen in een gevalideerd onderzoeksmodel voor de chemische, agrochemische en farmaceutische sector. Zij gaan C. elegans inzetten als alternatief testmodel om complexe responsen te meten. De vragen die zij willen beantwoorden met het onderzoek zijn: " Hoe effectief is het gebruik van C. elegans als alternatief testsysteem in het voorspellen van mogelijke toxische effecten en farmaceutische activiteiten? " In hoeverre kan C. elegans een meerwaarde hebben met betrekking tot het bestuderen en begrijpen van het onderliggende werkingsmechanisme? Beoogde resultaten Het resultaat van het project is kennis over de effectiviteit van C. elegans als alternatief systeem voor het screenen van stoffen op veiligheid en activiteit. Tijdens het onderzoek wordt er ook nieuwe technologie ontwikkeld, waaronder: " een productieproces voor de continue aanlevering van wormen; " lab-on-chipmodule voor high-throughput microscopie; " zelfregulerende mappingtool voor verzameling en interpretatie van data.
