Standard SARS-CoV-2 testing protocols using nasopharyngeal/throat (NP/T) swabs are invasive and require trained medical staff for reliable sampling. In addition, it has been shown that PCR is more sensitive as compared to antigen-based tests. Here we describe the analytical and clinical evaluation of our in-house RNA extraction-free saliva-based molecular assay for the detection of SARS-CoV-2. Analytical sensitivity of the test was equal to the sensitivity obtained in other Dutch diagnostic laboratories that process NP/T swabs. In this study, 955 individuals participated and provided NP/T swabs for routine molecular analysis (with RNA extraction) and saliva for comparison. Our RT-qPCR resulted in a sensitivity of 82,86% and a specificity of 98,94% compared to the gold standard. A false-negative ratio of 1,9% was found. The SARS-CoV-2 detection workflow described here enables easy, economical, and reliable saliva processing, useful for repeated testing of individuals.
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Summary: Xpaths is a collection of algorithms that allow for the prediction of compound-induced molecular mechanisms of action by integrating phenotypic endpoints of different species; and proposes follow-up tests for model organisms to validate these pathway predictions. The Xpaths algorithms are applied to predict developmental and reproductive toxicity (DART) and implemented into an in silico platform, called DARTpaths.
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Despite tremendous efforts, the exact structure of SARS-CoV-2 and related betacoronaviruses remains elusive. SARS-CoV-2 envelope is a key structural component of the virion that encapsulates viral RNA. It is composed of three structural proteins, spike, membrane (M), and envelope, which interact with each other and with the lipids acquired from the host membranes. Here, we developed and applied an integrative multi-scale computational approach to model the envelope structure of SARS-CoV-2 with near atomistic detail, focusing on studying the dynamic nature and molecular interactions of its most abundant, but largely understudied, M protein. The molecular dynamics simulations allowed us to test the envelope stability under different configurations and revealed that the M dimers agglomerated into large, filament-like, macromolecular assemblies with distinct molecular patterns. These results are in good agreement with current experimental data, demonstrating a generic and versatile approach to model the structure of a virus de novo.
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Routine neuropathology diagnostic methods are limited to histological staining techniques or directed PCR for pathogen detection and microbial cultures of brain abscesses are negative in one-third of the cases. Fortunately, due to improvements in technology, metagenomic sequencing of a conserved bacterial gene could provide an alternative diagnostic method. For histopathological work up, formalin-fixed paraffin-embedded (FFPE) tissue with highly degraded nucleic acids is the only material being available. Innovative amplicon-specific next-generation sequencing (NGS) technology has the capability to identify pathogens based on the degraded DNA within a few hours. This approach significantly accelerates diagnostics and is particularly valuable to identify challenging pathogens. This ensures optimal treatment for the patient, minimizing unnecessary health damage. Within this project, highly conserved primers in a universal PCR will be used, followed by determining the nucleotide sequence. Based on the obtained data, it is then precisely determined which microorganism(s) is/are responsible for the infection, even in cases of co-infection with multiple pathogens. This project will focus to answer the following research question; how can a new form of rapid molecular diagnostics contribute to the identification of microbial pathogens in CNS infections? The SME partner Molecular Biology Systems B.V. (MBS) develops and sells equipment for extremely rapid execution of the commonly used PCR. In this project, the lectorate Analysis Techniques in the Life Sciences (Avans) will, in collaboration with MBS, Westerdijk Institute (WI-KNAW) and the Institute of Neuropathology (Münster, DE) establish a new molecular approach for fast diagnosis within CNS infections using this MBS technology. This enables the monitoring of infectious diseases in a fast and user-friendly manner, resulting in an improved treatment plan.
Bijna alle klinische samples bevatten genetisch materiaal van het infectieus agentia, waardoor sequencing een aantrekkelijke benadering is voor de detectie en identificatie van pathogenen. Deze nieuwe vorm van moleculaire diagnostiek omzeilt het lange proces van traditionele, op cultuur gebaseerde analyses. Innovatieve amplicon specifieke next generation sequencing (NGS) technologie heeft de mogelijkheid de ziekteverwekkers op basis van DNA binnen enkele uren te identificeren. Deze aanpak versnelt daarmee de diagnostiek enorm en is met name een waardevolle aanvulling bij niet- of moeilijk kweekbare pathogenen. Dit zorgt ervoor dat de patiënt optimaal behandeld kan worden en er geen onnodige gezondheidsschade optreedt. Binnen dit project zal gebruik worden gemaakt van sterk geconserveerde primers in een universele PCR, gevolgd door het bepalen van de nucleotiden volgorde. Aan de hand van de verkregen data wordt vervolgens exact bepaald welk(e) micro-organisme(n) verantwoordelijk is/zijn voor de infectie, zelfs wanneer er co-infectie van meerdere pathogenen is opgetreden. MKB partner Molecular Biology Systems B.V. (MBS) ontwikkelen en verkopen apparatuur waarmee de veelgebruikte PCR extreem snel kan worden uitgevoerd. In samenwerking met MBS, MUMC+ en het lectoraat Analyse Technieken in de Life Sciences wordt binnen dit project een nieuwe moleculaire aanpak opgezet middels de MBS technologie, waarbij op een snelle en gebruiksvriendelijke manier infectieziekten gemonitord kunnen worden wat resulteert in een verbeterd behandelplan. Alle partners kunnen direct profiteren van de resultaten van dit onderzoek. Zo is er directe vertaling van resultaten naar een nieuwe werkwijze voor MUMC+, zal Avans de resultaten direct integreren in haar onderwijs en zal MBS deze snelle innovatieve werkmethode verder door ontwikkelen in de medische diagnostiek.
Structural Biology plays a crucial role in understanding the Chemistry of Life by providing detailed information about the three-dimensional structures of biological macromolecules such as proteins, DNA, RNA and complexes thereof. This knowledge allows researchers to understand how these molecules function and interact with each other, which forms the basis for a molecular understanding of disease and the development of targeted therapies. For decades, X-ray crystallography has been the dominant technique to determine these 3D structures. Only a decade ago, advances in technology and data processing resulted in a dramatic improvement of the resolution at which structures of biomolecular assemblies can be determined using another technique: cryo-electron microscopy (cryo-EM). This has been referred to as “the resolution revolution”. Since then, an ever increasing group of structural biologists are using cryo-EM. They employ a technique named Single Particle Analysis (SPA), in which thousands of individual macromolecules are imaged. These images are then computationally iteratively aligned and averaged to generate a three-dimensional reconstruction of the macromolecule. SPA works best if a very pure and concentrated macromolecule of interest can be captured in random orientations within a thin layer (10-50nm) of vitreous ice. Maastricht University has been the inventor of the machine that is found in most labs worldwide used for this: the VitroBot. We have been the inventor of succeeding technologies that allow for much better control of this process: the VitroJet. In here, we will develop a novel chemical way to expand our arsenal for preparing SPA samples of defined thickness. We will design, produce and test chemical spacers to allow for a controlled sample thickness. If successful, this will provide an easy, affordable solution for the ~1000 laboratories worldwide using SPA, and help them with their in vitro studies necessary for an improved molecular understanding of the Chemistry of Life.
