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  • 51.
    Maïno, Nicolas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Lunaphore Technologies SA, Switzerland.
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). University College London, United Kingdom.
    Cappi, G.
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Dupouy, D. G.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    A microfluidic platform towards automated multiplexed in situ sequencing2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 3542Article in journal (Refereed)
    Abstract [en]

    Advancements in multiplexed in situ RNA profiling techniques have given unprecedented insight into spatial organization of tissues by enabling single-molecule quantification and sub-micron localization of dozens to thousands of RNA species simultaneously in cells and entire tissue sections. However, the lack of automation of the associated complex experimental procedures represents a potential hurdle towards their routine use in laboratories. Here, we demonstrate an approach towards automated generation and sequencing of barcoded mRNA amplicons in situ, directly in fixed cells. This is achieved through adaptation of a microfluidic tool compatible with standard microscope slides and cover glasses. The adapted tool combines a programmable reagent delivery system with temperature controller and flow cell to perform established in situ sequencing protocols, comprising hybridization and ligation of gene-specific padlock probes, rolling circle amplification of the probes yielding barcoded amplicons and identification of amplicons through barcode sequencing. By adapting assay parameters (e.g. enzyme concentration and temperature), we achieve a near-identical performance in identifying mouse beta-actin transcripts, in comparison with the conventional manual protocol. The technically adapted assay features i) higher detection efficiency, ii) shorter protocol time, iii) lower consumption of oligonucleotide reagents but slightly more enzyme. Such an automated microfluidic tissue processor for in situ sequencing studies would greatly enhance its research potentials especially for cancer diagnostics, thus paving way to rapid and effective therapies.

  • 52. McGinn, Steven
    et al.
    Bauer, David
    Brefort, Thomas
    Dong, Liqin
    EI-Sagheer, Afaf
    Elsharawy, Abdou
    Evans, Geraint
    Falk-Sorqvist, Elin
    Forster, Michael
    Fredriksson, Simon
    Freeman, Peter
    Freitag, Camilla
    Fritzsche, Joachim
    Gibson, Spencer
    Gullberg, Mats
    Gut, Marta
    Heath, Simon
    Heath-Brun, Isabelle
    Heron, Andrew J.
    Hohlbein, Johannes
    Ke, Rongqin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Lancaster, Owen
    Le Reste, Ludovic
    Maglia, Giovanni
    Marie, Rodolphe
    Mauger, Florence
    Mertes, Florian
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Moens, Lotte
    Oostmeijer, Jelle
    Out, Ruud
    Nyvold Pedersen, Jonas
    Persson, Fredrik
    Picaud, Vincent
    Rotem, Dvir
    Schracke, Nadine
    Sengenes, Jennifer
    Stähler, Peer F.
    Stade, Björn
    Stoddart, David
    Teng, Xia
    Veal, Colin D.
    Zahra, Nathalie
    Bayley, Hagan
    Beier, Markus
    Brown, Tom
    Dekker, Cees
    Ekström, Björn
    Flyvbjerg, Henrik
    Franke, Andre
    Guenther, Simone
    Kapanidis, Achillefs N.
    Kaye, Jane
    Kristensen, Anders
    Lehrach, Hans
    Mangion, Jonathan
    Sauer, Sascha
    Schyns, Emile
    Tost, Jörg
    van Helvoort, Joop M. L. M.
    van der Zaag, Pieter J.
    Tegenfeldt, Jonas O.
    Brookes, Anthony J.
    Mir, Kalim
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Willcocks, James P.
    Gut, Ivo G.
    New technologies for DNA analysis - a review of the READNA Project2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, no 3, p. 311-330Article, review/survey (Refereed)
    Abstract [en]

    The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3rd and 4th generation of sequencing methods with nanopores and in situ sequencing, respectively.

  • 53.
    Mezger, Anja
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Allen, Stephanie
    Cavelier, Lucia
    Hulten, Maj
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elimination of maternal aneuploidy DNA for accurate non-invasive prenatal diagnosis: a pilot studyManuscript (preprint) (Other academic)
  • 54.
    Mezger, Anja
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fock, Jeppe
    Antunes, Paula
    Østerberg, Frederik W.
    Boisen, Anja
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hansen, Mikkel F.
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Donolato, Marco
    Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples2015In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 9, no 7, p. 7374-7382Article in journal (Refereed)
    Abstract [en]

    We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.

  • 55.
    Mezger, Anja
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Gullberg, Erik
    Göransson, Jenny
    Zorzet, Anna
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Tano, Eva
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Andersson, Dan I.
    A General Method for Rapid Determination of Antibiotic Susceptibility and Species in Bacterial Infections2015In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 53, no 2, p. 425-432Article in journal (Refereed)
    Abstract [en]

    To ensure correct antibiotic treatment and reduce the unnecessary use of antibiotics, there is an urgent need for new rapid methods for species identification and determination of antibiotic susceptibility in infectious pathogenic bacteria. We have developed a general method for the rapid identification of the bacterial species causing an infection and the determination of their antibiotic susceptibility profiles. An initial short cultivation step in the absence and presence of different antibiotics was combined with sensitive species-specific padlock probe detection of the bacterial target DNA to allow a determination of growth (i.e., resistance) and no growth (i.e., susceptibility). A proof-of-concept was established for urinary tract infections in which we applied the method to determine the antibiotic susceptibility profiles of Escherichia coli for two drugs with 100% accuracy in 3.5 h. The short assay time from sample to readout enables fast appropriate treatment with effective drugs and minimizes the need to prescribe broad-spectrum antibiotics due to unknown resistance profiles of the treated infection.

  • 56.
    Mezger, Anja
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Kuhnemund, Malte
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Highly specific DNA detection employing ligation on suspension bead array readout2015In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 32, no 5, p. 504-510Article in journal (Refereed)
    Abstract [en]

    We show for the first time that monomerized rolling circle amplification (RCA) products can be directly detected with the Luminex suspension bead array readout without the need of PCR amplification. Furthermore, using monomerized RCA products to guide ligation of the detection oligonucleotide (DO) to barcode sequences on the magnetic Luminex beads, combined with efficient washing and increased measurement temperature, yields a higher signal to noise ratio. As a proof-of-principle, we demonstrate detection of pathogenic DNA sequences with high reproducibility, sensitivity and a dynamic range over four orders of magnitude. Using padlock probes in combination with bead suspension arrays opens up the possibility for highly multiplexed DNA targeting and readout.

  • 57.
    Mezger, Anja
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Öhrmalm, Christina
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Blomberg, Jonas
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Detection of Rotavirus Using Padlock Probes and Rolling Circle Amplification2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 11, p. e111874-Article in journal (Refereed)
    Abstract [en]

    Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

  • 58.
    Mignardi, Marco
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mezger, Anja
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Qian, Xiaoyan
    La Fleur, Linnea
    Botling, Johan
    Larsson, Chatarina
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oligonucleotide gap-fill ligation for mutation detection and sequencing in situManuscript (preprint) (Other academic)
  • 59.
    Mignardi, Marco
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mezger, Anja
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Qian, Xiaoyan
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    La Fleur, Linnea
    Botling, Johan
    Larsson, Chatarina
    Nilsson, Mats
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ2015In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 22, article id e151Article in journal (Refereed)
    Abstract [en]

    In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) pre-serving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.

  • 60.
    Mignardi, Marco
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fourth-generation sequencing in the cell and the clinic2014In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 6, p. 31-31Article in journal (Other academic)
    Abstract [en]

    Nearly 40 years ago, DNA was sequenced for the first time. Since then, DNA sequencing has undergone continuous development, passing through three generations of sequencing technology. We are now entering the beginning of a new phase of genomic analysis in which massively parallel sequencing is performed directly in the cell. Two methods have recently been described for in situ RNA sequencing, one targeted and one untargeted, that rely on ligation chemistry. This fourth generation of sequencing technology opens up prospects for transcriptomic analysis, biomarker validation, diagnosis and patient stratification for cancer treatment.

  • 61. Moens, Lotte N.
    et al.
    Falk-Sorqvist, Elin
    Asplund, A. Charlotta
    Bernatowska, Ewa
    Smith, C. I. Edvard
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Diagnostics of Primary Immunodeficiency Diseases: A Sequencing Capture Approach2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 12, p. e114901-Article in journal (Refereed)
    Abstract [en]

    Primary Immunodeficiencies (PID) are genetically inherited disorders characterized by defects of the immune system, leading to increased susceptibility to infection. Due to the variety of clinical symptoms and the complexity of current diagnostic procedures, accurate diagnosis of PID is often difficult in daily clinical practice. Thanks to the advent of next generation'' sequencing technologies and target enrichment methods, the development of multiplex diagnostic assays is now possible. In this study, we applied a selector-based target enrichment assay to detect disease-causing mutations in 179 known PID genes. The usefulness of this assay for molecular diagnosis of PID was investigated by sequencing DNA from 33 patients, 18 of which had at least one known causal mutation at the onset of the experiment. We were able to identify the disease causing mutations in 60% of the investigated patients, indicating that the majority of PID cases could be resolved using a targeted sequencing approach. Causal mutations identified in the unknown patient samples were located in STAT3, IGLL1, RNF168 and PGM3. Based on our results, we propose a stepwise approach for PID diagnostics, involving targeted resequencing, followed by whole transcriptome and/or whole genome sequencing if causative variants are not found in the targeted exons.

  • 62. Moens, Lotte N. J.
    et al.
    Falk-Sörqvist, Elin
    Ljungström, Viktor
    Mattsson, Johanna
    Sundström, Magnus
    La Fleur, Linnéa
    Mathot, Lucy
    Micke, Patrick
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Botling, Johan
    HaloPlex Targeted Resequencing for Mutation Detection in Clinical Formalin-Fixed, Paraffin-Embedded Tumor Samples2015In: Journal of Molecular Diagnostics, ISSN 1525-1578, E-ISSN 1943-7811, Vol. 17, no 6, p. 729-739Article in journal (Refereed)
    Abstract [en]

    In recent years, the advent of massively parallel next-generation sequencing technologies has enabled substantial advances in the study of human diseases. Combined with targeted DNA enrichment methods, high sequence coverage can be obtained for different genes simultaneously at a reduced cost per sample, creating unique opportunities for clinical cancer diagnostics. However, the formalin-fixed, paraffin-embedded (FFPE) process of tissue samples, routinely used in pathology departments, results in DNA fragmentation and nucleotide modifications that introduce a number of technical challenges for downstream biomotecular analyses. We evaluated the HaloPlex target enrichment system for somatic mutation detection in 80 tissue fractions derived from 20 clinical cancer cases with paired tumor and normal tissue available in both FFPE and fresh-frozen format. Several modifications to the standard method were introduced, including a reduced target fragment Length and two strand capturing. We found that FFPE material can be used for HaloPlex-based target enrichment and next-generation sequencing, even when starting from small amounts of DNA. By specifically capturing both strands for each target fragment, we were able to reduce the number of false-positive errors caused by FFPE-induced artifacts and Lower the detection limit for somatic mutations. We believe that the HaloPlex method presented here will be broadly applicable as a tool for somatic mutation detection in clinical cancer settings.

  • 63.
    Neumann, Felix
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Grabbe, Malin
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Albert, Jan
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Padlock Probe Assay for Detection and Subtyping of Seasonal Influenza2018In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 64, no 12, p. 1704-1712Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Influenza remains a constant threat worldwide, and WHO estimates that it affects 5% to 15% of the global population each season, with an associated 3 to 5 million severe cases and up to 500000 deaths. To limit the morbidity and the economic burden of influenza, improved diagnostic assays are needed. METHODS: We developed a multiplexed assay for the detection and subtyping of seasonal influenza based on padlock probes and rolling circle amplification. The assay simultaneously targets all 8 genome segments of the 4 circulating influenza variants-A(H1N1), A(H3N2), B/Yamagata, and B/Victoria-and was combined with a prototype cartridge for inexpensive digital quantification. Characterized virus isolates and patient nasopharyngeal swabs were used for assay design and analytical validation. The diagnostic performance was assessed by blinded testing of 50 clinical samples analyzed in parallel with a commercial influenza assay, Simplexa (TM) Flu A/B & RSV Direct. RESULTS: The assay had a detection limit of 18 viral RNA copies and achieved 100% analytical and clinical specificity for differential detection and subtyping of seasonal circulating influenza variants. The diagnostic sensitivity on the 50 clinical samples was 77.5% for detecting influenza and up to 73% for subtyping seasonal variants. CONCLUSIONS: We have presented a proof-of-concept padlock probe assay combined with an inexpensive digital read-out for the detection and subtyping of seasonal influenza strains A and B. The demonstrated high specificity and multiplexing capability, together with the digital quantification, established the assay as a promising diagnostic tool for seasonal influenza.

  • 64.
    Neumann, Felix
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Salas, Jeanpierre
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Devyser AB, Sweden.
    Mecea, Vasile
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    QCM mass underestimation in molecular biotechnology: Proximity ligation assay for norovirus detection as a case study2018In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 273, p. 742-750Article in journal (Refereed)
    Abstract [en]

    The development of piezoelectric mass-sensitive devices is based on the shift in resonance frequency that is proportional to the deposited mass. However, this holds true only for small, rigid masses, while it can result in mass underestimation for heavy, non-rigid masses. In this work, we demonstrate this 'missing mass' phenomenon by measurement of high molecular weight biomolecules on a Quartz Crystal Microbalance (QCM) platform. For this, we present a model bioassay consisting of a sandwich-type proximity ligation assay for the detection of norovirus-like particles, and its real-time build-up on QCM as an experimental evidence. Upon combination with a localized QCM platform, we explain the pronounced slipping effect in multilayer biological systems resulting in energy dissipation and subsequent mass underestimation. This helps in pointing out the limitations of mega-gravity field sensors for molecular diagnostics where absolute quantification of pathogen load becomes indispensable towards biosensing applications.

  • 65.
    Pavankumar, Asalapuram R.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Engström, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Liu, Jie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Herthnek, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Proficient Detection of Multi-Drug-Resistant Mycobacterium tuberculosis by Padlock Probes and Lateral Flow Nucleic Acid Biosensors2016In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 88, no 8, p. 4277-4284Article in journal (Refereed)
    Abstract [en]

    Tuberculosis is a major communicable disease. Its causative agent, Mycobacterium tuberculosis, becomes resistant to antibiotics by acquisition of point mutations in the chromosome. Multi-drug-resistant tuberculosis (MDR-TB) is an increasing public health threat, and prompt detection of such strains is of critical importance. As rolling circle amplification of padlock probes can be used to robustly distinguish single-nucleotide variants, we combined this technique with a sensitive lateral flow nucleic acid biosensor to develop a rapid molecular diagnostic test for MDR-TB, A proof-of-concept test was established for detection of the most common mutations [rpoB 531 (TCG/TTG) and katG 315 (AGC/ACC)] causing MDR-TB and verification of loss of the respective wild type. The molecular diagnostic test produces visual signals corresponding to the respective genotypes on lateral flow strips in approximately 75 min. By detecting only two mutations, the test can detect about 60% of all MDR-TB cases. The padlock probe-lateral flow (PLP-LF) test is the first of its kind and can ideally be performed at resource-limited clinical laboratories. Rapid information about the drug-susceptibility pattern can assist clinicians to choose suitable treatment regimens and take appropriate infection control actions rather than prescribing empirical treatment, thereby helping to control the spread of MDR-TB in the community.

  • 66.
    Qian, Xiaoyan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Harris, Kenneth D.
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). University College London, UK.
    Nicoloutsopoulos, Dimitris
    Muñoz-Manchado, Ana B.
    Skene, Nathan
    Hjerling-Leffler, Jens
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Probabilistic cell typing enables fine mapping of closely related cell types in situ2020In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 17, no 1, p. 101-106Article in journal (Refereed)
    Abstract [en]

    Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related neuronal types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages prior scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method by mapping the inhibitory neurons of mouse hippocampal area CA1, for which ground truth is available from extensive prior work identifying their laminar organization. Our method identified these neuronal classes in a spatial arrangement matching ground truth, and further identified multiple classes of isocortical pyramidal cell in a pattern matching their known organization. This method will allow identifying the spatial organization of closely related cell types across the brain and other tissues.

  • 67.
    Qian, Xiaoyan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wu, Di
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wu, Chenglin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Target sequence design of padlock probes based on experimentally determined in situ synthesized cDNA fragmentsManuscript (preprint) (Other academic)
    Abstract [en]

    Padlock probes are widely used to target a short fragment of DNA. For example, in in situ sequencing (ISS), an image-based technology for highly multiplexed spatial gene expression analysis, cDNA target detection is mediated by padlock probes. Transcript counts from ISS generally has good correlation with next-generation sequencing read counts, but bias between different genes are also observed. Therefore, we developed a new method to isolate and sequence in situ synthesized cDNA and sought to use the read coverage information from it to guide padlock probe design. The results show limited correlation between cDNA library sequencing and ISS counts, but it can still help the probe design process by eliminating target sequences that are very unlikely to be detected. In addition, the method provides a way to systematically characterize in situ reverse transcription.

  • 68. Russell, Camilla
    et al.
    Roy, Subhadeep
    Ganguly, Saheli
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Caruthers, Marvin H.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Formation of Silver Nanostructures by Rolling Circle Amplification Using Boranephosphonate-Modified Nucleotides2015In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 87, no 13, p. 6660-6666Article in journal (Refereed)
    Abstract [en]

    We investigate the efficiency of incorporation of boranephosphonate-modified nucleotides by phi29 DNA poly, merase and present a simple method for forming large defined silver nanostructures by rolling circle amplification (RCA) using boranephosphonate internudeotide linkages. RCA is a linear DNA amplification technique that can use specifically circularized DNA probes for detection of target nucleic acids and proteins. The resulting product is a collapsed single-stranded DNA molecule with tandem repeats of the DNA probe. By substituting each of the natural nucleotides with the corresponding 5'-(alpha-P-borano)-deoxynudeosidetriphosphate, only a small reduction in amplification rate is observed. Also, by substituting all four natural nucleotides, it is possible to enzymatically synthesize a micrometensized, single-stranded DNA molecule with only boranephosphonate internucleotide linkages. Well-defined silver particles are then readily formed along the rolling circle product.

  • 69. Russell, Camilla
    et al.
    Welch, Ken
    Jarvius, Jonas
    Cai, Yixiao
    Brucas, Rimantas
    Nikolajeff, Fredrik
    Svedlindh, Peter
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Gold Nanowire Based Electrical DNA Detection Using Rolling Circle Amplification2014In: ACS Nano, ISSN 1936-0851, E-ISSN 1936-086X, Vol. 8, no 2, p. 1147-1153Article in journal (Refereed)
    Abstract [en]

    We present an electrical sensor that uses rolling circle amplification (RCA) of DNA to stretch across the gap between two electrodes, interact with metal nanoparticle seeds to generate an electrically conductive nanowire, and produce electrical signals upon detection of specific target DNA sequences. RCA is a highly specific molecular detection mechanism based on DNA probe circularization. With this technique, long single-stranded DNA with simple repetitive sequences are produced. Here we show that stretched RCA products can be metalized using silver or gold solutions to form metal wires. Upon metallization, the resistance drops from T Omega to k Omega for silver and to Omega for gold. Metallization is seeded by gold nanoparticles aligned along the single-stranded DNA product through hybridization of functionalized oligonucleotides. We show that combining RCA with electrical DNA detection produces results in readout with very high signal-to-noise ratio, an essential feature for sensitive and specific detection assays. Finally, we demonstrate detection of 10 ng of Escherichia coli genomic DNA using the sensor concept.

  • 70. Salamon, John
    et al.
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Lynn, David John
    Network Visualization and Analysis of Spatially Aware Gene Expression Data with InsituNet2018In: Cell Systems, ISSN 2405-4712, Vol. 6, no 5, p. 626-630Article in journal (Refereed)
    Abstract [en]

    In situ sequencing methods generate spatially resolved RNA localization and expression data at an almost single-cell resolution. Few methods, however, currently exist to analyze and visualize the complex data that is produced, which can encode the localization and expression of a million or more individual transcripts in a tissue section. Here, we present InsituNet, an application that converts in situ sequencing data into interactive network-based visualizations, where each unique transcript is a node in the network and edges represent the spatial co-expression relationships between transcripts. InsituNet is available as an app for the Cytoscape platform at http://apps.cytoscape.org/apps/insitunet. InsituNet enables the analysis of the relationships that exist between these transcripts and can uncover how spatial co-expression profiles change in different regions of the tissue or across different tissue sections.

  • 71.
    Salih, Tagrid
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ahlford, Annika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Plichta, Zdenek
    Horak, Daniel
    Streptavidin-modified monodispersed magnetic poly(2-hydroxyethyl methacrylate) microspheres as solid support in DNA-based molecular protocols2016In: Materials science & engineering. C, biomimetic materials, sensors and systems, ISSN 0928-4931, E-ISSN 1873-0191, Vol. 61, p. 362-367Article in journal (Refereed)
    Abstract [en]

    Molecular diagnostics may provide tailored and cost efficient treatment for infectious disease and cancer. Rolling circle amplification (RCA) of padlock probes guarantees high specificity to identify nucleic acid targets down to single nucleotide resolution in a multiplex fashion. This makes the assay suitable for molecular analysis of various diseases, and interesting to integrate into automated devices for point-of-care analysis. A critical prerequisite for many molecular assays is (i) target-specific isolation from complex clinical samples and (ii) removal of reagents, inhibitors and contaminants between reaction steps. Efficient solid supports are therefore essential to enable multi-step, multi-analyte protocols. Superparamagnetic micro- and nanoparticles, with large surface area and rapid liquid-phase kinetics, are attractive for multi-step protocols. Recently, streptavidin-modified magnetic monodispersed poly(2-hydroxyethyl methacrylate) (STV-mag.PHEMA) microspheres were developed by multiple swelling polymerization. They are easily separated by a magnet and exhibit low non-specific protein sorption. In this study, the performance and the binding efficiency of STV-mag.PHEMA was addressed by circle-to-circle amplification (C2CA). A lower number of RCA products were detected as compared to the gold standard Dynabeads. Nevertheless, this study was the first to successfully adapt STV-mag.PHEMA microspheres as solid support in a DNA-based protocol, which is an important finding. The STV-mag.PHEMA microspheres were larger with about 16 times less surface area as compared to the Dynabeads, which might partly explain the lower rolling circle product (RCP) count obtained. Further research is currently ongoing comparing particles of similar sizes and optimizing reaction conditions to establish their full utility in the field. Ultimately, low cost and versatile particles are a great resource to facilitate future clinical molecular diagnostics.

  • 72. Schütte, Moritz
    et al.
    Risch, Thomas
    Abdavi-Azar, Nilofar
    Boehnke, Karsten
    Schumacher, Dirk
    Keil, Marlen
    Yildirimman, Reha
    Jandrasits, Christine
    Borodina, Tatiana
    Amstislavskiy, Vyacheslav
    Worth, Catherine L.
    Schweiger, Caroline
    Liebs, Sandra
    Lange, Martin
    Warnatz, Hans-Jörg
    Butcher, Lee M.
    Barrett, James E.
    Sultan, Marc
    Wierling, Christoph
    Golob-Schwarzl, Nicole
    Lax, Sigurd
    Uranitsch, Stefan
    Becker, Michael
    Welte, Yvonne
    Regan, Joseph Lewis
    Silvestrov, Maxine
    Kehler, Inge
    Fusi, Alberto
    Kessler, Thomas
    Herwig, Ralf
    Landegren, Ulf
    Wienke, Dirk
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Velasco, Juan A.
    Garin-Chesa, Pilar
    Reinhard, Christoph
    Beck, Stephan
    Schäfer, Reinhold
    Regenbrecht, Christian R. A.
    Henderson, David
    Lange, Bodo
    Haybaeck, Johannes
    Keilholz, Ulrich
    Hoffmann, Jens
    Lehrach, Hans
    Yaspo, Marie-Laure
    Molecular dissection of colorectal cancer in pre-clinical models identifies biomarkers predicting sensitivity to EGFR inhibitors2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 14262Article in journal (Refereed)
    Abstract [en]

    Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling 44,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

  • 73. Sepehri, Sobhan
    et al.
    Agnarsson, Björn
    de la Torre, Teresa Zardán Gómez
    Schneiderman, Justin E.
    Blomgren, Jakob
    Jesorka, Aldo
    Johansson, Christer
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Albert, Jan
    Strømme, Maria
    Winkler, Dag
    Kalaboukhov, Alexei
    Characterization of Binding of Magnetic Nanoparticles to Rolling Circle Amplification Products by Turn-On Magnetic Assay2019In: Biosensors, ISSN 2079-6374, Vol. 9, no 3, article id 109Article in journal (Refereed)
    Abstract [en]

    The specific binding of oligonucleotide-tagged 100 nm magnetic nanoparticles (MNPs) to rolling circle products (RCPs) is investigated using our newly developed differential homogenous magnetic assay (DHMA). The DHMA measures ac magnetic susceptibility from a test and a control samples simultaneously and eliminates magnetic background signal. Therefore, the DHMA can reveal details of binding kinetics of magnetic nanoparticles at very low concentrations of RCPs. From the analysis of the imaginary part of the DHMA signal, we find that smaller MNPs in the particle ensemble bind first to the RCPs. When the RCP concentration increases, we observe the formation of agglomerates, which leads to lower number of MNPs per RCP at higher concentrations of RCPs. The results thus indicate that a full frequency range of ac susceptibility observation is necessary to detect low concentrations of target RCPs and a long amplification time is not required as it does not significantly increase the number of MNPs per RCP. The findings are critical for understanding the underlying microscopic binding process for improving the assay performance. They furthermore suggest DHMA is a powerful technique for dynamically characterizing the binding interactions between MNPs and biomolecules in fluid volumes.

  • 74. Sepehri, Sobhan
    et al.
    de la Torre, Teresa Zardán Gomez
    Schneiderman, Justin F.
    Blomgren, Jakob
    Jesorka, Aldo
    Johansson, Christer
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Albert, Jan
    Strømme, Maria
    Winkler, Dag
    Kalaboukhovf, Alexei
    Homogeneous Differential Magnetic Assay2019In: Acs Sensors, ISSN 2379-3694, Vol. 4, no 9, p. 2381-2388Article in journal (Refereed)
    Abstract [en]

    Assays are widely used for detection of various targets, including pathogens, drugs, and toxins. Homogeneous assays are promising for the realization of point-of-care diagnostics as they do not require separation, immobilization, or washing steps. For low concentrations of target molecules, the speed and sensitivity of homogeneous assays have hitherto been limited by slow binding kinetics, time-consuming amplification steps, and the presence of a high background signal. Here, we present a homogeneous differential magnetic assay that utilizes a differential magnetic readout that eliminates previous limitations of homogeneous assays. The assay uses a gradiometer sensor configuration combined with precise microfluidic sample handling. This enables simultaneous differential measurement sample containing a synthesized Vibrio cholerae target and a negative control sample, which reduces the background signal and increases the readout speed. Very low concentrations of targets down to femtomolar levels are thus detectable without any additional amplification of the number of targets. Our homogeneous differential magnetic assay method opens new possibilities for rapid and highly sensitive diagnostics at the point of care.

  • 75. Soares, Ruben R. G.
    et al.
    Neumann, Felix
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Caneira, Catarina R. F.
    Madaboosi, Narayanan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ciftci, Sibel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pinto, Ines F.
    Santos, Denis R.
    Chu, Virginia
    Russom, Aman
    Conde, Joao P.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Silica bead-based microfluidic device with integrated photodiodes for the rapid capture and detection of rolling circle amplification products in the femtomolar range2019In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 128, p. 68-75Article in journal (Refereed)
    Abstract [en]

    The rapid and sensitive detection of specific nucleic acid sequences at the point-of-care (PoC) is becoming increasingly in demand for a variety of emergent biomedical applications ranging from infectious disease diagnostics to the screening of antimicrobial resistance. To meet such demand, considerable efforts have been invested towards the development of portable and integrated analytical devices combining microfluidics with miniaturized signal transducers. Here, we demonstrate the combination of rolling circle amplification (RCA)-based nucleic acid amplification with an on-chip size-selective trapping of amplicons on silica beads (similar to 8 nL capture chamber) coupled with a thin-film photodiode (200 x 200 mu m area) fluorescence readout. Parameters such as the flow rate of the amplicon solution and trapping time were optimized as well as the photodiode measurement settings, providing minimum detection limits below 0.5 fM of targeted nucleic acids and requiring only 5 mu L of pre-amplified sample. Finally, we evaluated the analytical performance of our approach by benchmarking it against a commercial instrument for RCA product (RCP) quantification and further investigated the effect of the number of RCA cycles and elongation times (ranging from 10 to 120 min). Moreover, we provide a demonstration of the application for diagnostic purposes by detecting RNA from influenza and Ebola viruses, thus highlighting its suitability for integrated PoC systems.

  • 76. Soldatov, Ruslan
    et al.
    Kaucka, Marketa
    Kastriti, Maria Eleni
    Petersen, Julian
    Chontorotzea, Tatiana
    Englmaier, Lukas
    Akkuratova, Natalia
    Yang, Yunshi
    Häring, Martin
    Dyachuk, Viacheslav
    Bock, Christoph
    Farlik, Matthias
    Piacentino, Michael L.
    Boismoreau, Franck
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Cartana AB, Sweden.
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Bronner, Marianne E.
    Croci, Laura
    Hsiao, Wen-Yu
    Guertin, David A.
    Brunet, Jean-Francois
    Consalez, Gian Giacomo
    Ernfors, Patrik
    Fried, Kaj
    Kharchenko, Peter V.
    Adameyko, Igor
    Spatiotemporal structure of cell fate decisions in murine neural crest2019In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 364, no 6444, article id eaas9536Article in journal (Refereed)
    Abstract [en]

    Neural crest cells are embryonic progenitors that generate numerous cell types in vertebrates. With single-cell analysis, we show that mouse trunk neural crest cells become biased toward neuronal lineages when they delaminate from the neural tube, whereas cranial neural crest cells acquire ectomesenchyme potential dependent on activation of the transcription factor Twist1. The choices that neural crest cells make to become sensory, glial, autonomic, or mesenchymal cells can be formalized as a series of sequential binary decisions. Each branch of the decision tree involves initial coactivation of bipotential properties followed by gradual shifts toward commitment. Competing fate programs are coactivated before cells acquire fate-specific phenotypic traits. Determination of a specific fate is achieved by increased synchronization of relevant programs and concurrent repression of competing fate programs.

  • 77.
    Strell, Carina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Laxman, Navya
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Svedlund, Jessica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wu, Chenglin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Placing RNA in context and space - methods for spatially resolved transcriptomics2019In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 286, no 8, p. 1468-1481Article, review/survey (Refereed)
    Abstract [en]

    Single-cell transcriptomics provides us with completely new insights into the molecular diversity of different cell types and the different states they can adopt. The technique generates inventories of cells that constitute the building blocks of multicellular organisms. However, since the method requires isolation of discrete cells, information about the original location within tissue is lost. Therefore, it is not possible to draw detailed cellular maps of tissue architecture and their positioning in relation to other cells. In order to better understand the cellular and tissue function of multicellular organisms, we need to map the cells within their physiological, morphological, and anatomical context and space. In this review, we will summarize and compare the different methods of in situ RNA analysis and the most recent developments leading to more comprehensive and highly multiplexed spatially resolved transcriptomic approaches. We will discuss their highlights and advantages as well as their limitations and challenges and give an outlook on promising future applications and directions both within basic research as well as clinical integration.

  • 78.
    Strell, Carina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Paulsson, Janna
    Jin, Shao-Bo
    Tobin, Nicholas P.
    Mezheyeuski, Artur
    Roswall, Pernilla
    Mutgan, Ceren
    Mitsios, Nicholas
    Johansson, Hemming
    Wickberg, Sarah Marie
    Svedlund, Jessica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hall, Per
    Mulder, Jan
    Radisky, Derek C.
    Pietras, Kristian
    Bergh, Jonas
    Lendahl, Urban
    Warnberg, Fredrik
    Ostman, Arne
    Impact of Epithelial-Stromal Interactions on Peritumoral Fibroblasts in Ductal Carcinoma in Situ2019In: Journal of the National Cancer Institute, ISSN 0027-8874, E-ISSN 1460-2105, Vol. 111, no 9, p. 983-995, article id djy234Article in journal (Refereed)
    Abstract [en]

    Background: A better definition of biomarkers and biological processes related to local recurrence and disease progression is highly warranted for ductal breast carcinoma in situ (DCIS). Stromal-epithelial interactions are likely of major importance for the biological, clinical, and pathological distinctions between high- and low-risk DCIS cases.

    Methods: Stromal platelet derived growth factor receptor (PDGFR) was immunohistochemically assessed in two DCIS patient cohorts (n = 458 and n = 80). Cox proportional hazards models were used to calculate the hazard ratios of recurrence. The molecular mechanisms regulating stromal PDGFR expression were investigated in experimental in vitro co-culture systems of DCIS cells and fibroblasts and analyzed using immunoblot and quantitative real-time PCR. Knock-out of JAG1 in DCIS cells and NOTCH2 in fibroblasts was obtained through CRISPR/Cas9. Experimental data were validated by mammary fat pad injection of DCIS and DCIS-JAG1 knock-out cells (10 mice per group). All statistical tests were two-sided.

    Results: PDGFR alpha((low))/PDGFR beta((high)) fibroblasts were associated with increased risk for recurrence in DCIS (univariate hazard ratio = 1.59, 95% confidence interval [CI] = 1.02 to 2.46; P = .04 Wald test; multivariable hazard ratio = 1.78, 95% CI = 1.07 to 2.97; P = .03). Tissue culture and mouse model studies indicated that this fibroblast phenotype is induced by DCIS cells in a cell contact-dependent manner. Epithelial Jagged1 and fibroblast Notch2 were identified through loss-of-function studies as key juxtacrine signaling components driving the formation of the poor prognosis-associated fibroblast phenotype.

    Conclusions: A PDGFR alpha((low))/PDGFR beta((high)) fibroblast subset was identified as a marker for high-risk DCIS. The Jagged-1/Notch2/PDGFR stroma-epithelial pathway was described as a novel signaling mechanism regulating this poor prognosis-associated fibroblast subset. In general terms, the study highlights epithelial-stromal crosstalk in DCIS and contributes to ongoing efforts to define clinically relevant fibroblast subsets and their etiology.

  • 79. Strömberg, Mattias
    et al.
    Gómez de la Torre, Teresa Zardán
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Svedlindh, Peter
    Strømme, Maria
    A magnetic nanobead-based bioassay provides sensitive detection of single- and biplex bacterial DNA using a portable AC susceptometer2014In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 9, no 1, p. 137-145Article in journal (Refereed)
    Abstract [en]

    Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin-biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM-NDA further towards implementation in point-of-care and outpatient settings.

  • 80.
    Svedlund, Jessica
    et al.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Strell, Carina
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Qian, Xiaoyan
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zilkens, Kilian J. C.
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tobin, Nicholas P.
    Bergh, Jonas
    Sieuwerts, Anieta M.
    Nilsson, Mats
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Generation of in situ sequencing based OncoMaps to spatially resolve gene expression profiles of diagnostic and prognostic markers in breast cancer2019In: EBioMedicine, E-ISSN 2352-3964, Vol. 48, p. 212-223Article in journal (Refereed)
    Abstract [en]

    Background: Gene expression analysis of breast cancer largely relies on homogenized tissue samples. Due to the high degree of cellular and molecular heterogeneity of tumor tissues, bulk tissue-based analytical approaches can only provide very limited system-level information about different signaling mechanisms and cellular interactions within the complex tissue context. Methods: We describe an analytical approach using in situ sequencing (ISS), enabling highly multiplexed, spatially and morphologically resolved gene expression profiling. Ninety-one genes including prognostic and predictive marker profiles, as well as genes involved in specific cellular pathways were mapped within whole breast cancer tissue sections, covering luminal A/B-like, HER2-positive and triple negative tumors. Finally, all these features were combined and assembled into a molecular-morphological OncoMap for each tumor tissue. Findings: Our in situ approach spatially revealed intratumoral heterogeneity with regard to tumor subtype as well as to the OncotypeDX recurrence score and even uncovered areas of minor cellular subpopulations. Since ISS-resolved molecular profiles are linked to their histological context, a deeper analysis of the core and periphery of tumor foci enabled identification of specific gene expression patterns associated with these morphologically relevant regions. Interpretation :15S generated OncoMaps represent useful tools to extend our general understanding of the biological processes behind tumor progression and can further support the identification of novel therapeutical targets as well as refine tumor diagnostics.

  • 81. Tiklova, Katarina
    et al.
    Björklund, Åsa K.
    Lahti, Laura
    Fiorenzano, Alessandro
    Nolbrant, Sara
    Gillberg, Linda
    Volakakis, Nikolaos
    Yokota, Chika
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hilscher, Markus M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Hauling, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Holmstrom, Fredrik
    Joodmardi, Eliza
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Parmar, Malin
    Perlmann, Thomas
    Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 581Article in journal (Refereed)
    Abstract [en]

    Midbrain dopamine (mDA) neurons constitute a heterogenous group of cells that have been intensely studied, not least because their degeneration causes major symptoms in Parkinson's disease. Understanding the diversity of mDA neurons - previously well characterized anatomically - requires a systematic molecular classification at the genome-wide gene expression level. Here, we use single cell RNA sequencing of isolated mouse neurons expressing the transcription factor Pitx3, a marker for mDA neurons. Analyses include cells isolated during development up until adulthood and the results are validated by histological characterization of newly identified markers. This identifies seven neuron subgroups divided in two major branches of developing Pitx3-expressing neurons. Five of them express dopaminergic markers, while two express glutamatergic and GABAergic markers, respectively. Analysis also indicate evolutionary conservation of diversity in humans. This comprehensive molecular characterization will provide a valuable resource for elucidating mDA neuron subgroup development and function in the mammalian brain.

  • 82. Weibrecht, Irene
    et al.
    Lundin, Elin
    Kiflemariam, Sara
    Mignardi, Marco
    Grundberg, Ida
    Larsson, Chatarina
    Koos, Björn
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala universitet.
    Söderberg, Ola
    In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 2, p. 355-372Article in journal (Refereed)
    Abstract [en]

    Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.

  • 83.
    Wu, Chenglin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Simonetti, Michele
    Rossell, Carla
    Mignardi, Marco
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mirzazadeh, Reza
    Annaratone, Laura
    Marchiò, Caterina
    Sapino, Anna
    Bienko, Magda
    Crosetto, Nicola
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples2018In: Communications biology, E-ISSN 2399-3642, Vol. 1, article id 209Article in journal (Refereed)
    Abstract [en]

    Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 x) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of similar to 90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.

  • 84.
    Wu, Di
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Yan, Junhong
    Shen, Xia
    Sun, Yu
    Thulin, Måns
    Cai, Yanling
    Wik, Lotta
    Shen, Qiujin
    Oelrich, Johan
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Dubois, K. Louise
    Ronquist, K. Göran
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Landegren, Ulf
    Kamali-Moghaddam, Masood
    Profiling surface proteins on individual exosomes using a proximity barcoding assay2019In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, article id 3854Article in journal (Refereed)
    Abstract [en]

    Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease.

  • 85. Yasui, Takao
    et al.
    Ogawa, Kensuke
    Kaji, Noritada
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ajiri, Taiga
    Tokeshi, Manabu
    Horiike, Yasuhiro
    Baba, Yoshinobu
    Label-free detection of real-time DNA amplification using a nanofluidic diffraction grating2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 31642Article in journal (Refereed)
    Abstract [en]

    Quantitative DNA amplification using fluorescence labeling has played an important role in the recent, rapid progress of basic medical and molecular biological research. Here we report a label-free detection of real-time DNA amplification using a nanofluidic diffraction grating. Our detection system observed intensity changes during DNA amplification of diffracted light derived from the passage of a laser beam through nanochannels embedded in a microchannel. Numerical simulations revealed that the diffracted light intensity change in the nanofluidic diffraction grating was attributed to the change of refractive index. We showed the first case reported to date for label-free detection of real-time DNA amplification, such as specific DNA sequences from tubercle bacilli (TB) and human papillomavirus (HPV). Since our developed system allows quantification of the initial concentration of amplified DNA molecules ranging from 1 fM to 1 pM, we expect that it will offer a new strategy for developing fundamental techniques of medical applications.

  • 86. Zaghlool, Ammar
    et al.
    Ameur, Adam
    Wu, Chenglin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Orzechowski Westholm, Jakub
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Niazi, Adnan
    Manivannan, Manimozhi
    Bramlett, Kelli
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Uppsala University, Sweden.
    Feuk, Lars
    Expression profiling and in situ screening of circular RNAs in human tissues2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 16953Article in journal (Refereed)
    Abstract [en]

    Circular RNAs (circRNAs) were recently discovered as a class of widely expressed noncoding RNA and have been implicated in regulation of gene expression. However, the function of the majority of circRNAs remains unknown. Studies of circRNAs have been hampered by a lack of essential approaches for detection, quantification and visualization. We therefore developed a target-enrichment sequencing method suitable for screening of circRNAs and their linear counterparts in large number of samples. We also applied padlock probes and in situ sequencing to visualize and determine circRNA localization in human brain tissue at subcellular levels. We measured circRNA abundance across different human samples and tissues. Our results highlight the potential of this RNA class to act as a specific diagnostic marker in blood and serum, by detection of circRNAs from genes exclusively expressed in the brain. The powerful and scalable tools we present will enable studies of circRNA function and facilitate screening of circRNA as diagnostic biomarkers.

  • 87. Österberg, Frederik W.
    et al.
    Rizzi, Giovanni
    Donolato, Marco
    Bejhed, Rebecca S.
    Mezger, Anja
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Strömberg, Mattias
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Strømme, Maria
    Svedlindh, Peter
    Hansen, Mikkel F.
    On-Chip Detection of Rolling Circle Amplified DNA Molecules from Bacillus Globigii Spores and Vibrio Cholerae2014In: Small, ISSN 1613-6810, E-ISSN 1613-6829, Vol. 10, no 14, p. 2877-2882Article in journal (Refereed)
    Abstract [en]

    For the first time DNA coils formed by rolling circle amplification are quantified on-chip by Brownian relaxation measurements on magnetic nanobeads using a magnetoresistive sensor. No external magnetic fields are required besides the magnetic field arising from the current through the sensor, which makes the setup very compact. Limits of detection down to 500 Bacillus globigii spores and 2 pM of Vibrio cholerae are demonstrated, which are on the same order of magnitude or lower than those achieved previously using a commercial macro-scale AC susceptometer. The chip-based readout is an important step towards the realization of field tests based on rolling circle amplification molecular analyses.

12 51 - 87 of 87
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