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NOVELTY - Calibrating a microfluidic system involves providing bead having a structure based on DNA origami, where structure comprises a predetermined number of fluorophores, measuring the fluorescence of bead, and calibrating microfluidic system on basis of fluorescence measurement, and obtaining a calibration curve of the microfluidic system.USE - Method for calibrating a microfluidic system.ADVANTAGE - The method enables to calibrate a microfluidic system, which allows quantification of antigens present in a sample at a very low level, and allows fluorescence quantification in microfluidics.DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for the following:(1) a method for determining level of an antigen in a sample by a microfluidics system, which involves calibrating a microfluidics system, obtaining a calibration curve; providing a sample comprising an antigen, where antigen is attached to a cell or to a vesicle or to a virus-like particle or a viral particle or an extracellular vesicle; contacting sample with ligand binding molecule coupled to a fluorescent label, where ligand binds the antigen; measuring fluorescence of fluorescence label; scoring fluorescence of fluorescence label based on calibration curve, and determining level of antigen in sample;(2) a method for determining presence or state of a hematological disease in an individual by a microfluidics system, which involves calibrating a microfluidics system, obtaining a calibration curve; providing a fluid sample from individual comprising blood cell; contacting sample with ligand binding molecule coupled to a fluorescent label, where ligand is associated with presence or state of hematological disease; measuring fluorescence of fluorescent label; scoring fluorescence of fluorescent label based on calibration curve; and determining level and/or concentration of antigen associated with presence or state of hematological disease; and(3) a kit for calibration of microfluidic instrument, which comprises a bead having a structure based on DNA origami where structure comprises a predetermined number of fluorophores.

In the current study, we describe a novel DNA sensor system for specific and quantitative detection of mycobacteria, which is the causative agent of tuberculosis. Detection is achieved by using the enzymatic activity of the mycobacterial encoded enzyme topoisomerase IA (TOP1A) as a biomarker. The presented work is the first to describe how the catalytic activities of a member of the type IA family of topoisomerases can be exploited for specific detection of a bacteria. The principle for detection relies on a solid support anchored DNA substrate with dual functions namely; 1) the ability to isolate mycobacterial TOP1A from crude sample, and 2) the ability to be converted into a closed DNA circle upon reaction with the isolated enzyme. The DNA circle can act as a template for rolling circle amplification generating a tandem repeat product that can be visualized at the single molecule level by fluorescent labelling. This reaction scheme ensures specific, sensitive, and quantitative detection of the mycobacteria TOP1A biomarker as demonstrated by the use of purified mycobacterial TOP1A and extracts from an array of non-mycobacteria and mycobacteria species. When combined with mycobacteriophage induced lysis as a novel way of effective yet gentle extraction of the cellular content from the model Mycobacterium smegmatis, the DNA sensor system allowed detection of mycobacteria in small volumes of cell suspensions. Moreover, it was possible to detect M. smegmatis added to human saliva. Depending on the composition of the sample, we were able to detect 0.6 or 0.9 million colony forming units (CFU)/mL of mycobacteria, which is within the range of clinically relevant infection numbers. We, therefore, believe that the presented assay, which relies on techniques that can be adapted to limited resource settings, may be the first step towards the development of a new point-of-care diagnostic test for tuberculosis.

Rainbow trout is a major worldwide aquaculture species and viral disease has a high cost to fish farmers every year why effective treatment and a deeper understanding of immune components involved in the coexistence between fish and virus is of big concern to our field. We present here a study of microRNA regulation in rainbow trout during infection with the fish pathogenic rhabdovirus viral haemorrhagic septicaemia virus (VHSV). Infected fish as well as infected and immune stimulated cell cultures have been tested for microRNA regulation by microarray using a ´all species´ approach followed by qPCR. Two regulated rainbow trout microRNAs have been cloned, sequenced and upstream promoter areas characterized and tested for functionality upon immune stimulation.

Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells.

Udgivelsesdato: Sept. 27