This article gives information on an international ring trial of the epidermal-equivalent (EE) sensitizer potency assay.
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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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To accelerate differentiation between Staphylococcus aureus and Coagulase Negative Staphylococci (CNS), this study aimed to compare six different DNA extraction methods from 2 commonly used blood culture materials, i.e. BACTEC and Bact/ALERT. Furthermore, we analyzed the effect of reduced blood culture times for detection of Staphylococci directly from blood culture material. A real-time PCR duplex assay was used to compare 6 different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with MRSA. Bacterial DNA was isolated with: automated extractor EasyMAG (3 protocols), automated extractor MagNA Pure LC (LC Microbiology Kit MGrade), a manual kit MolYsis Plus, and a combination between MolYsis Plus and the EasyMAG. The most optimal isolation method was used to evaluate reduced bacterial culture times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the EasyMAG resulted in the most sensitive detection of S.aureus, with a detection limit of 10 CFU/ml, in Bact/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S.aureus load of 1 CFU/ml blood can be detected after 5 hours of culture in Bact/ALERT3D by combining the sensitive isolation method and the tuf LightCycler assay.
DOCUMENT
Structural colour (SC) is created by light interacting with regular nanostructures in angle-dependent ways resulting in vivid hues. This form of intense colouration offers commercial and industrial benefits over dyes and other pigments. Advantages include durability, efficient use of light, anti-fade properties and the potential to be created from low cost materials (e.g. cellulose fibres). SC is widely found in nature, examples include butterflies, squid, beetles, plants and even bacteria. Flavobacterium IR1 is a Gram-negative, gliding bacterium isolated from Rotterdam harbour. IR1 is able to rapidly self-assemble into a 2D photonic crystal (a form of SC) on hydrated surfaces. Colonies of IR1 are able to display intense, angle-dependent colours when illuminated with white light. The process of assembly from a disordered structure to intense hues, that reflect the ordering of the cells, is possible within 10-20 minutes. This bacterium can be stored long-term by freeze drying and then rapidly activated by hydration. We see these properties as suiting a cellular reporter system quite distinct from those on the market, SC is intended to be “the new Green Fluorescent Protein”. The ability to understand the genomics and genetics of SC is the unique selling point to be exploited in product development. We propose exploiting SC in IR1 to create microbial biosensors to detect, in the first instance, volatile compounds that are damaging to health and the environment over the long term. Examples include petroleum or plastic derivatives that cause cancer, birth defects and allergies, indicate explosives or other insidious hazards. Hoekmine, working with staff and students within the Hogeschool Utrecht and iLab, has developed the tools to do these tasks. We intend to create a freeze-dried disposable product (disposables) that, when rehydrated, allow IR1 strains to sense and report multiple hazardous vapours alerting industries and individuals to threats. The data, visible as brightly coloured patches of bacteria, will be captured and quantified by mobile phone creating a system that can be used in any location by any user without prior training. Access to advice, assay results and other information will be via a custom designed APP. This work will be performed in parallel with the creation of a business plan and market/IP investigation to prepare the ground for seed investment. The vision is to make a widely usable series of tests to allow robust environmental monitoring for all to improve the quality of life. In the future, this technology will be applied to other areas of diagnostics.
Organ-on-a-chip technology holds great promise to revolutionize pharmaceutical drug discovery and development which nowadays is a tremendously expensive and inefficient process. It will enable faster, cheaper, physiologically relevant, and more reliable (standardized) assays for biomedical science and drug testing. In particular, it is anticipated that organ-on-a-chip technology can substantially replace animal drug testing with using the by far better models of true human cells. Despite this great potential and progress in the field, the technology still lacks standardized protocols and robust chip devices, which are absolutely needed for this technology to bring the abovementioned potential to fruition. Of particular interest is heart-on-a-chip for drug and cardiotoxicity screening. There is presently no preclinical test system predicting the most important features of cardiac safety accurately and cost-effectively. The main goal of this project is to fabricate standardized, robust generic heart-on-a-chip demonstrator devices that will be validated and further optimized to generate new physiologically relevant models to study cardiotoxicity in vitro. To achieve this goal various aspects will be considered, including (i) the search for alternative chip materials to replace PDMS, (ii) inner chip surface modification and treatment (chemistry and topology), (iii) achieving 2D/3D cardiomyocyte (long term) cell culture and cellular alignment within the chip device, (iv) the possibility of integrating in-line sensors in the devices and, finally, (v) the overall chip design. The achieved standardized heart-on-a-chip technology will be adopted by pharmaceutical industry. This proposed project offers a unique opportunity for the Netherlands, and Twente in particular, which has relevant expertise, potential, and future perspective in this field as it hosts world-leading companies pioneering various core aspects of the technology that are relevant for organs-on-chips, combined with two world-leading research institutes within the University of Twente.
