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  • 1.
    Fallqvist, Björn
    et al.
    KTH Royal Institute of Technology, Sweden.
    Fielden, Matthew L.
    KTH Royal Institute of Technology, Sweden.
    Pettersson, Torbjörn
    KTH Royal Institute of Technology, Sweden.
    Niklas, Nordgren
    RISE, SP – Sveriges Tekniska Forskningsinstitut, SP Kemi Material och Ytor, Material och ytteknik.
    Kroon, Martin
    KTH Royal Institute of Technology, Sweden.
    Gad, Annica K. B.
    Karolinska Institute, Sweden.
    Experimental and computational assessment of F-actin influence in regulating cellular stiffness and relaxation behaviour of fibroblasts2016In: Journal of The Mechanical Behavior of Biomedical Materials, ISSN 1751-6161, E-ISSN 1878-0180, Vol. 59, p. 168-184Article in journal (Refereed)
    Abstract [en]

    In biomechanics, a complete understanding of the structures and mechanisms that regulate cellular stiffness at a molecular level remain elusive. In this paper, we have elucidated the role of filamentous actin (F-actin) in regulating elastic and viscous properties of the cytoplasm and the nucleus. Specifically, we performed colloidal-probe atomic force microscopy (AFM) on BjhTERT fibroblast cells incubated with Latrunculin B (LatB), which results in depolymerisation of F-actin, or DMSO control. We found that the treatment with LatB not only reduced cellular stiffness, but also greatly increased the relaxation rate for the cytoplasm in the peripheral region and in the vicinity of the nucleus. We thus conclude that F-actin is a major determinant in not only providing elastic stiffness to the cell, but also in regulating its viscous behaviour. To further investigate the interdependence of different cytoskeletal networks and cell shape, we provided a computational model in a finite element framework. The computational model is based on a split strain energy function of separate cellular constituents, here assumed to be cytoskeletal components, for which a composite strain energy function was defined. We found a significant influence of cell geometry on the predicted mechanical response. Importantly, the relaxation behaviour of the cell can be characterised by a material model with two time constants that have previously been found to predict mechanical behaviour of actin and intermediate filament networks. By merely tuning two effective stiffness parameters, the model predicts experimental results in cells with a partly depolymerised actin cytoskeleton as well as in untreated control. This indicates that actin and intermediate filament networks are instrumental in providing elastic stiffness in response to applied forces, as well as governing the relaxation behaviour over shorter and longer time-scales, respectively.

  • 2.
    Sidstedt, Maja
    et al.
    National Forensic Centre, Sweden.
    Gynnå, Arvid H.
    National Forensic Centre, Sweden.
    Kiesler, Kevin M.
    NIST, USA.
    Jansson, Linda
    National Forensic Centre, Sweden; Lund University, Sweden.
    Steffen, Carolyn R.
    NIST, USA.
    Håkansson, Joakim
    RISE Research Institutes of Sweden, Materials and Production, Product Realisation Methodology. University of Gothenburg, Sweden.
    Johansson, Gustav
    SIMSEN Diagnostics, Sweden.
    Österlund, Tobias
    University of Gothenburg, Sweden; Sahlgrenska University Hospital, Sweden.
    Bogestål, Yalda
    RISE Research Institutes of Sweden, Materials and Production, Product Realisation Methodology.
    Tillmar, Andreas
    National Board of Forensic Medicine, Sweden.
    Rådström, Peter
    Lund University, Sweden.
    Ståhlberg, Anders
    University of Gothenburg, Sweden;Sahlgrenska University Hospital, Sweden.
    Vallone, Peter M.
    NIST, USA.
    Hedman, Johannes
    National Forensic Centre, Sweden; Lund University, Sweden.
    Ultrasensitive sequencing of STR markers utilizing unique molecular identifiers and the SiMSen-Seq method2024In: Forensic Science International: Genetics, ISSN 1872-4973, E-ISSN 1878-0326, Vol. 71, article id 103047Article in journal (Refereed)
    Abstract [en]

    Massively parallel sequencing (MPS) is increasingly applied in forensic short tandem repeat (STR) analysis. The presence of stutter artefacts and other PCR or sequencing errors in the MPS-STR data partly limits the detection of low DNA amounts, e.g., in complex mixtures. Unique molecular identifiers (UMIs) have been applied in several scientific fields to reduce noise in sequencing. UMIs consist of a stretch of random nucleotides, a unique barcode for each starting DNA molecule, that is incorporated in the DNA template using either ligation or PCR. The barcode is used to generate consensus reads, thus removing errors. The SiMSen-Seq (Simple, multiplexed, PCR-based barcoding of DNA for sensitive mutation detection using sequencing) method relies on PCR-based introduction of UMIs and includes a sophisticated hairpin design to reduce unspecific primer binding as well as PCR protocol adjustments to further optimize the reaction. In this study, SiMSen-Seq is applied to develop a proof-of-concept seven STR multiplex for MPS library preparation and an associated bioinformatics pipeline. Additionally, machine learning (ML) models were evaluated to further improve UMI allele calling. Overall, the seven STR multiplex resulted in complete detection and concordant alleles for 47 single-source samples at 1 ng input DNA as well as for low-template samples at 62.5 pg input DNA. For twelve challenging mixtures with minor contributions of 10 pg to 150 pg and ratios of 1–15% relative to the major donor, 99.2% of the expected alleles were detected by applying the UMIs in combination with an ML filter. The main impact of UMIs was a substantially lowered number of artefacts as well as reduced stutter ratios, which were generally below 5% of the parental allele. In conclusion, UMI-based STR sequencing opens new means for improved analysis of challenging crime scene samples including complex mixtures. © 2024 The Authors

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