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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This study aimed to evaluate technological (acidification, proteolysis, lipolysis, resistance to low pH, NaCl, and bile salts) and biopreservation (antimicrobial activity against foodborne pathogens) features of 1002 LAB by high throughput screening (HTS) methods. The LAB was isolated from 11 types of Brazilian artisanal cheeses (BAC) marketed in the main 5 producing regions. Remarkable intra-species variability in acidification rates have been found, which was most pronounced between isolates from Mina's artisanal cheeses, Caipira and Coalho cheeses. Lacticaseibacillus paracasei and Levilactobacillus brevis showed the fastest acidification rate; however, all isolates showed slower acidification rates than a lactococcal control strain (4.3 × lower). When testing inhibitory effects, > 75% of LAB isolates could inhibit the growth of Staphylococcus aureus ATCC 19095 and Listeria monocytogenes ATCC 7644. Two of these isolates, identified as Lactiplantibacillus plantarum and Lentilactobacillus buchneri, the sterile and neutral supernatants alone, were sufficient to inhibit L. monocytogenes growth. Principal component analysis (PCA) allowed the identification of functional groups based on proteolytic and lipolytic activity, osmotic stress resistance, and inhibition of L. monocytogenes. The type of cheese the isolates were recovered from influenced properties such as anti-listerial compounds and lipolytic enzyme production. The use of HTS and multivariate statistics allowed insights into a diverse set of LAB technological and biopreservation properties. These findings allow a profound knowledge of the heterogeneity of a large set of isolates, which can be further used to design starter cultures with varied and combined properties, such as biopreservation and technological features. Besides that, HTS makes it possible to analyze a vast panel of LAB strains, reducing costs and time within laboratory analysis, while avoiding the loss of information once all LAB are tested at the same time (differently from the traditional labor-intensive approach, in which a few numbers of strains is tested per time).
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