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  • 51.
    Jönsson, Håkan
    et al.
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Nano Biotechnology (closed 20130101).
    Tröpfchen-Mikrofluidik für die Einzelzellanalyse2012In: Angewandte Chemie, ISSN 0044-8249, E-ISSN 1521-3757, Angewandte Chemie, Vol. 124, no 49, p. 12342-12359Article in journal (Refereed)
    Abstract [de]

    Die tröpfchenbasierte Mikrofluidik dient der Isolierung und Manipulation von einzelnen Zellen und Reagentien innerhalb von monodispersen, pikolitergroßen Flüssigkapseln bei einem Umsatz von tausenden Tröpfchen pro Sekunde. Diese Qualitäten machen die Tröpfchen‐Mikrofluidik geeignet für viele Anforderungen der Einzelzellanalyse. Durch die Monodispersität lässt sich die Konzentration in den Tröpfchen quantitativ einstellen. Die Tröpfchen bieten der Zelle und ihrer unmittelbaren Umgebung ein isoliertes Kompartiment, und bei einem Durchsatz von tausenden Tröpfchen pro Sekunde ist es möglich, zehntausende bis millionen verkapselte Zellen zu prozessieren. Heterogene Zellpopulationen lassen sich somit exakt beschreiben oder seltene Zellarten identifizieren. Das kleine Volumen der Tröpfchen macht auch sehr große Screenings ökonomisch machbar. Dieser Aufsatz gibt einen Überblick über den aktuellen Stand der Einzelzellanalyse durch die Tröpfchen‐Mikrofluidik und nennt Beispiele, bei denen sie biologische Vorgänge besser verstehen hilft.

  • 52.
    Khorshidi, Mohammad Ali
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Live Single Cell Imaging and Analysis Using Microfluidic Devices2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Today many cell biological techniques study large cell populations where an average estimate of individual cells’ behavior is observed. On the other hand, single cell analysis is required for studying functional heterogeneities between cells within populations. This thesis presents work that combines the use of microfluidic devices, optical microscopy and automated image analysis to design various cell biological assays with single cell resolution including cell proliferation, clonal expansion, cell migration, cell-cell interaction and cell viability tracking. In fact, automated high throughput single cell techniques enable new studies in cell biology which are not possible with conventional techniques.

    In order to automatically track dynamic behavior of single cells, we developed a microwell based device as well as a droplet microfluidic platform. These high throughput microfluidic assays allow automated time-lapse imaging of encapsulated single cells in micro droplets or confined cells inside microwells. Algorithms for automatic quantification of cells in individual microwells and micro droplets are developed and used for the analysis of cell viability and clonal expansion. The automatic counting protocols include several image analysis steps, e.g. segmentation, feature extraction and classification. The automatic quantification results were evaluated by comparing with manual counting and revealed a high success rate. In combination these automatic cell counting protocols and our microfluidic platforms can provide statistical information to better understand behavior of cells at the individual level under various conditions or treatments in vitro exemplified by the analysis of function and regulation of immune cells. Thus, together these tools can be used for developing new cellular imaging assays with resolution at the single cell level.

    To automatically characterize transient migration behavior of natural killer (NK) cells compartmentalized in microwells, we developed a method for single cell tracking. Time-lapse imaging showed that the NK cells often exhibited periods of high motility, interrupted with periods of slow migration or complete arrest. These transient migration arrest periods (TMAPs) often overlapped with periods of conjugations between NK cells and target cells. Such conjugation periods sometimes led to cell-mediated killing of target cells. Analysis of cytotoxic response of NK cells revealed that a small sub-class of NK cells called serial killers was able to kill several target cells. In order to determine a starting time point for cell-cell interaction, a novel technique based on ultrasound was developed to aggregate NK and target cells into the center of the microwells. Therefore, these assays can be used to automatically and rapidly assess functional and migration behavior of cells to detect differences between health and disease or the influence of drugs.

    The work presented in this thesis gives good examples of how microfluidic devices combined with automated imaging and image analysis can be helpful to address cell biological questions where single cell resolution is necessary. 

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  • 53.
    Koeck, P. J. B.
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Karshikoff, A.
    Limitations of the linear and the projection approximations in three-dimensional transmission electron microscopy of fully hydrated proteins2015In: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, no 3, p. 197-209Article in journal (Refereed)
    Abstract [en]

    We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors. Non-Technical Abstract Cryo transmission electron microscopy and three-dimensional reconstruction can be used to determine a three-dimensional model of a protein molecule. In the mathematical methods used for three-dimensional reconstruction assumptions are made about a linear relationship between the images recorded in the electron microscope and the objects being imaged. In this paper we investigate with computer simulations at what specimen thickness these assumptions start breaking down and what sort of errors can be expected in the three-dimensional reconstructions when the assumptions are not valid anymore.

  • 54.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hjelm, Barbara
    KTH, School of Biotechnology (BIO), Proteomics.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies2010In: Recent Patents on Biotechnology, ISSN 1872-2083, Vol. 4, no 3, p. 171-182Article in journal (Refereed)
    Abstract [en]

    The field of combinatorial protein engineering for generation of new affinity proteins started in the mid 80s by the development of phage display. Although phage display is a prime example of a simple yet highly efficient method, manifested by still being the standard technique 25 years later, new alternative technologies are available today. One of the more successful new display technologies is cell display. Here we review the field of cell display for directed evolution purposes, with focus on a recently developed method employing Gram-positive staphylococci as display host. Patents on the most commonly used cell display systems and on different modifications as well as specific applications of these systems are also included. General strategies for selection of new affinity proteins from cell-displayed libraries are discussed, with detailed examples mainly from studies on the staphylococcal display system. In addition, strategies for characterization of recombinant proteins on the staphylococcal cell surface, with an emphasis on an approach for epitope mapping of antibodies, are included.

  • 55.
    Kronqvist, Nina
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Rockberg, Johan
    KTH, School of Biotechnology (BIO), Proteomics.
    Hjelm, Barbara
    KTH, School of Biotechnology (BIO), Proteomics.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    New Ways for Discovery of Biopharmaceuticals: Emerging Techniques using Surface Display on Gram-positive Bacteria for Combinatorial Protein Engineering and Characterization2009In: Bioforum Europe, ISSN 1611-597X, Vol. 13, no 6-7, p. 022-Article in journal (Refereed)
  • 56. Kuktaite, Ramune
    et al.
    Plivelic, Tomas S.
    Cerenius, Yngve
    Hedenqvist, Mikael S.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Gallstedt, Mikael
    Marttila, Salla
    Ignell, Rickard
    Popineau, Yves
    Tranquet, Oliver
    Shewry, Peter R.
    Johansson, Eva
    Structure and Morphology of Wheat Gluten Films: From Polymeric Protein Aggregates toward Superstructure Arrangements2011In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 12, no 5, p. 1438-1448Article in journal (Refereed)
    Abstract [en]

    Evaluation of structure and morphology of extruded wheat gluten (WG) films showed WG protein assemblies elucidated on a range of length scales from nano (4.4 angstrom and 9 to 10 angstrom, up to 70 angstrom) to micro (10 mu m). The presence of NaOH in WG films induced a tetragonal structure with unit cell parameters, a = 51.85 angstrom and c = 40.65 angstrom, whereas NH4OH resulted in a bidimensional hexagonal close-packed (HCP) structure with a lattice parameter of 70 angstrom. In the WG films with NH4OH, a highly polymerized protein pattern with intimately mixed glutenins and gliadins bounded through SH/SS interchange reactions was found. A large content of beta-sheet structures was also found in these films, and the film structure was oriented in the extrusion direction. In conclusion, this study highlights complexities of the supramolecular structures and conformations of wheat gluten polymeric proteins in biofilms not previously reported for biobased materials.

  • 57.
    Langer, Krzysztof
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Jönsson, Håkan
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019Manuscript (preprint) (Other academic)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 58.
    Langer, Krzysztof
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Jönsson, Håkan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rapid production and recovery of cell spheroids by automated droplet microfluidics2019In: bioRxivArticle in journal (Refereed)
    Abstract [en]

    Droplet microfluidics enables high throughput cell processing, analysis and screening by miniaturizing the reaction vessels to nano- or pico-liter water-in oil droplets, but like many other microfluidic formats, droplet microfluidics have not been interfaced with or automated by laboratory robotics. Here we demonstrate automation of droplet microfluidics based on an inexpensive liquid handling robot for the automated production of human scaffold-free cell spheroids, using pipette actuation and interfacing the pipetting tip with a droplet generating microfluidic chip. In this chip we produce highly mono-disperse 290μm droplets with diameter CV of 1.7%. By encapsulating cells in these droplets, we produce cell spheroids in droplets and recover them to standard formats at a throughput of 85000 spheroids per microfluidic circuit per hour. The viability of the cells in spheroids remains high after recovery only decreased by 4% starting from 96% after 16 hours incubation in nanoliter droplets. Scaffold-free cell spheroids and 3D tissue constructs recapitulate many aspects of functional human tissue more accurately than 2D or single cell cultures, but assembly methods for spheroids, e.g. hanging drop micro-plates, has had limited throughput. The increased throughput and decreased cost of our method enables spheroid production at the scale needed for lead discovery drug screening and approaches the cost where these micro tissues could be used as building blocks for organ scale regenerative medicine.

  • 59.
    Larsbrink, Johan
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Thompson, Andrew J.
    Lundqvist, Magnus
    KTH, School of Biotechnology (BIO), Glycoscience.
    Gardner, Jeffrey G.
    Davies, Gideon J.
    Brumer, Harry
    KTH, School of Biotechnology (BIO), Glycoscience. Univ British Columbia, Canada; .
    A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 94, no 2, p. 418-433Article in journal (Refereed)
    Abstract [en]

    The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an -xylosidase, a -galactosidase, and an -l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities - and notable differences - to the complex polysaccharide utilization loci of the Bacteroidetes.

  • 60.
    Lindberg, Hanna
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Härd, Torleif
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    A truncated and dimeric format of an Affibody library on bacteria enables FACS-mediated isolation of amyloid-beta aggregation inhibitors with subnanomolar affinity2015In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 10, no 11, p. 1707-1718Article in journal (Refereed)
    Abstract [en]

    The amyloid hypothesis suggests that accumulation of amyloid β (Aβ) peptides in the brain is involved in development of Alzheimer's disease. We previously generated a small dimeric affinity protein that inhibited Aβ aggregation by sequestering the aggregation prone parts of the peptide. The affinity protein is originally based on the Affibody scaffold, but is evolved to a distinct interaction mechanism involving complex structural rearrangement in both the Aβ peptide and the affinity proteins upon binding. The aim of this study was to decrease the size of the dimeric affinity protein and significantly improve its affinity for the Aβ peptide to increase its potential as a future therapeutic agent. We combined a rational design approach with combinatorial protein engineering to generate two different affinity maturation libraries. The libraries were displayed on staphylococcal cells and high-affinity Aβ-binding molecules were isolated using flow-cytometric sorting. The best performing candidate binds Aβ with a KD value of around 300 pM, corresponding to a 50-fold improvement in affinity relative to the first-generation binder. The new dimeric Affibody molecule was shown to capture Aβ1-42 peptides from spiked E. coli lysate. Altogether, our results demonstrate successful engineering of this complex binder for increased affinity to the Aβ peptide.

  • 61.
    Lindbo, Sarah
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Garousi, Javad
    Uppsala university.
    Mitran, Bogdan
    Uppsala university.
    Vorobyeva, Anzhelika
    Uppsala university.
    Oroujeni, Maryam
    Uppsala university.
    Orlova, Anna
    Uppsala university.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). KTH, School of Biotechnology (BIO), Centres, Centre for Bioprocess Technology, CBioPT. KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova.
    Tolmachev, Vladimir
    Uppsala university.
    Optimized molecular design of ADAPT-based HER2-imaging probes labelled with 111In and 68Ga2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 7, p. 2674-2683Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging is a promising tool for visualization of cancer associated molecular abnormalities in vivo and stratification of patients for specific therapies. ADAPT is a new type of small engineered proteins based on the scaffold of an albumin binding domain of protein G. ADAPTs have been utilized to select and develop high affinity binders to different proteinaceous targets. ADAPT6 binds to human epidermal growth factor 2 (HER2) with low nanomolar affinity and can be used for its in vivo visualization. Molecular design of 111In-labeled anti-HER2 ADAPT has been optimized in several earlier studies. In this study, we made a direct comparison of two of the most promising variants, having either a DEAVDANS or a (HE)3DANS sequence at the N-terminus, conjugated with a maleimido derivative of DOTA to a GSSC amino acids sequence at the C-terminus. The variants (designated DOTA-C59-DEAVDANS-ADAPT6-GSSC and DOTA-C61-(HE)3DANS-ADAPT6-GSSC) were stably labeled with 111In for SPECT and 68Ga for PET. Biodistribution of labeled ADAPT variants was evaluated in nude mice bearing human tumor xenografts with different levels of HER2 expression. Both variants enabled clear discrimination between tumors with high and low levels of HER2 expression. 111In-labeled ADAPT6 derivatives provided higher tumor-to-organ ratios compared to 68Ga-labeled counterparts. The best performing variant was DOTA-C61-(HE)3DANS-ADAPT6-GSSC, which provided tumor-to-blood ratios of 208 ± 36 and 109 ± 17 at 3 h for 111In and 68Ga labels, respectively.

  • 62. Liotta, L. A.
    et al.
    Davis, J. B.
    Couch, R. D.
    Fredolini, Claudia
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Zhou, W.
    Petricoin, E.
    Espina, V.
    Clinical proteomics and molecular pathology2017In: Molecular Pathology: The Molecular Basis of Human Disease, Elsevier Inc. , 2017, p. 183-203Chapter in book (Other academic)
    Abstract [en]

    Genomic and proteomic research is launching the next era of cancer molecular medicine. Molecular expression profiles can uncover clues to functionally important molecules in the development of human disease and generate information to subclassify human tumors and tailor a treatment to the individual patient. The next revolution is the synthesis of proteomic information into functional pathways and circuits in cells and tissues. Such synthesis must take into account the dynamic state of protein post-translational modifications; protein-protein or protein-DNA/RNA interactions; cross-talk between signal pathways; and feedback regulation within cells, between cells, and between tissues. This full set of information may be required before we can fully dissect the specific dysregulated pathways driving tumorigenesis. This higher level of functional understanding will be the basis for true rational therapeutic design that specifically targets the molecular lesions underlying human disease. 

  • 63.
    Liu, Hao
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. ltai, Mohamed; Garousi, Javad; Tolmachev, Vladimir.
    Lindbo, Sarah
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Ding, Haozhong
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Altai, Mohamed
    Garousi, Javad
    Orlova, Anna
    Tolmachev, Vladimir
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A2019In: International Journal of Oncology, ISSN 1019-6439, Vol. 55, no 1, p. 309-319Article in journal (Refereed)
    Abstract [en]

    Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain-derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA-derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half-life extension, can be used for specific killing of HER2-expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity. All constructs had strong affinity for HER2 (K-D 10 to 26 nM) and no tendency for aggregation could be detected. The fusion toxins including the ABD showed strong interaction with human and mouse serum albumin [equilibrium dissociation constant (K-D) 1 to 3 nM and 2 to 10 nM, respectively]. The in vitro investigation of the cytotoxic potential revealed IC50-values in the picomolar range for cells expressing high levels of HER2. The specificity was also demonstrated, by showing that free HER2 receptors on the target cells are required for fusion toxin activity. In mice, the fusion toxins containing the ABD exhibited an appreciably longer time in circulation. The uptake was highest in liver and kidney. Fusion with PE25 was associated with the highest hepatic uptake. Collectively, the results suggest that fusion toxins consisting of ADAPTs and ETA-derivatives are promising agents for targeted cancer therapy.

  • 64. Liu, Xiao
    et al.
    Spicarova, Zuzana
    Rydholm, Susanna
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Li, Juan
    Brismar, Hjalmar
    KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
    Aperia, Anita
    Ankyrin B Modulates the Function of Na,K-ATPase/Inositol 1,4,5-Trisphosphate Receptor Signaling Microdomain2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 17, p. 11461-11468Article in journal (Refereed)
    Abstract [en]

    Na, K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappa B activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na, K-ATPase/ IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na, K-ATPase and IP3R. We identify the N terminus tail of the Na, K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na, K-ATPase and IP3R, reduced the number of cells responding to pM doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappa B. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na, K-ATPase and no effect on the distribution and apparent mobility of Na, K-ATPase in the plasma membrane.

  • 65.
    Lopez Navarro, Indira Patricia
    KTH, School of Biotechnology (BIO).
    Utveckling av affinitets-baserade analys av muskel dialys prover från patienter med facioscapulohumeral muskel dystrofi2016Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    Interstitial Fluid is a complex sample, highly abundant in the human body that can give information regardingtissue secretion, intracellular signaling and tissue health status. The composition of the interstitial fluid can giveinformation regarding the processes occurring in muscles and alterations due to pathological changes occurringduring disease progression. Currently this sample has not yet been characterized within rare diseases like musculardystrophies. Facioscapulohumeral Muscular Dytrophy is an inherited progressive myopathy, characterized by thedegeneration and progressive muscular fiber necrosis of muscles from the face, upper arms and lower limbs. It canbe diagnosed; but in an advanced stage where weakness in the muscles have already occur. Meanwhile there is nocurrent understanding of the mechanisms happening in the muscle. In this project an immunoassay protocol wasdeveloped using suspension bead array technology to create an optimal method to analyze the protein content ofthese samples. The technological platform allows antibody-based capturing and detection of protein targets frombiotinylated biological samples. By modifying an existing protocol for analysis of serum and plasma samplesabundance of 63 protein targets was measured in muscle interstitial fluid from healthy individuals and patientsaffected by facioscapulohumeral dystrophy (FSHD), The optimized steps were the sample pre-treatment, the assaybuffer dilution ratio and the incubation time for capturing the protein targets. The findings of this project indicatethat using 1 μl of muscle interstitial fluid sample with minimized dilution factor and 60-fold molar excess biotinrelative to sample protein concentration enables detection of Interstitial fluid protein components. The proteinsdetected are ret finger protein-like 4B (RFPL4B) and albumin in from affected muscle and histone cluster(HIST1H3A) and albumin in non affected muscle.

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  • 66.
    Lundqvist, Magnus
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Methods for cell line and protein engineering2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Therapeutic proteins are becoming increasingly important. They are desirable, as they typically possess low adverse effects and higher specificity compared to the traditional, small molecule drugs. But they are also more complex and involve different intricate and expensive development and production processes. Through new technologies in protein and cell line development, more efficient and safer drugs can be readily available and at a lower cost. This thesis gives an overview of how protein therapeutics are developed and produced. It explores strategies to improve the efficacy and safety of protein drugs and how to improve production yields. In the present investigation, two papers present new methods for high-throughput cloning and site-directed mutagenesis using solid-phase immobilization of DNA fragments. These methods were designed to generate new drug candidates with swiftness and ease. Three papers show the development of a new cell line screening system that combines droplet microfluidics and the split-GFP reporter system. This combination allows for relative quantification of secreted recombinant proteins between individual cells and provides a tool for the selection of the best-producing clones for final production from a heterologous cell pool. The final paper explores the possibility to produce proteins at a higher cell density by examining how the metabolome and proteome of a perfusion bioreactor evolve as the cell density reaches exceptionally high levels. The consistent goal of all of these studies is to expedite the development and improve the production of therapeutic proteins, to assist the discovery of new drugs and to bring down production and development costs. Engineered proteins can be used to cure previously incurable diseases or give current medications a higher efficacy. Lower production and development costs can make the treatments available to more people.

    Download full text (pdf)
    fulltext
  • 67.
    Lundqvist, Magnus
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Thalén, Niklas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Volk, Anna-Luisa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hansen, Henning Gram
    Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, Lyngby, Denmark..
    von Otter, Eric
    Nanyang Technol Univ, Sch Biol Sci, Singapore 637551, Singapore..
    Nygren, Per-Åke
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
    Rockberg, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 310Article in journal (Refereed)
    Abstract [en]

    Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.

  • 68.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Affibody molecules binding to the ErbB3 receptorPatent (Other (popular science, discussion, etc.))
  • 69.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Bacterial display in directed evolution for generation of new biopharmaceuticals2011In: Biotech International, ISSN 2032-2887, Vol. 23, no June, p. 26-29Article in journal (Other academic)
    Abstract [en]

    The successful use of monoclonal antibodies and antibody derivatives for therapeutic and in vivo diagnostic applications has resulted in a rapid progression in the fields surrounding biopharmaceutical drug discovery and combinatorial protein engineering. Consequently, investigations on new and improved technologies for both generation and characterisation of specific antibodies and alternative protein scaffolds are continuously being reported in the literature. This review summarises the recent efforts in development and application of bacterial display platforms for such purposes.

  • 70.
    Löfblom, John
    et al.
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Frejd, Fredrik Y.
    Uppsala University.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology.
    Non-immunoglobulin based protein scaffolds2011In: Current Opinion in Biotechnology, ISSN 0958-1669, E-ISSN 1879-0429, Vol. 22, no 6, p. 843-848Article, review/survey (Refereed)
    Abstract [en]

    Non-immunoglobulin based protein scaffolds have been reported as promising alternatives to traditional monoclonal antibodies for over a decade and are often mentioned as part of the next-generation immunotherapeutics. Today, this class of biologics is beginning to demonstrate its potential for therapeutic applications and several are currently in preclinical or clinical development. A common denominator for most of these new scaffolds is the attractive properties that differentiate them from monoclonal antibodies including small size, cysteine-free sequence, flexible pharmacokinetic properties, and ease of generating multispecific molecules. In addition to therapeutic applications, substantial evidence point to superior performance of several of these scaffolds in molecular imaging compared to full-length antibodies. Here we review the most recent progress using alternative protein scaffolds for therapy and medical imaging.

  • 71.
    Magnusson, Anders
    KTH, School of Biotechnology (BIO).
    Rational redesign of Candida antarctica lipase B2005Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis describes the use of rational redesign to modify the properties of the enzyme Candida antarctica lipase B. Through carefully selected single-point mutations, we were able to introduce substrate-assisted catalysis and to alter the reaction specificity. Other single-point mutations afforded variants with greatly changed substrate selectivity and enantioselectivity.

    Mutation of the catalytic serine changed the hydrolase activity into an aldolase activity. The mutation decreased the activation energy for aldol addition by 4 kJ×mol-1, while the activation energy increased so much for hydrolysis that no hydrolysis activity could be detected. This mutant can catalyze aldol additions that no natural aldolases can catalyze.

    Mutation of the threonine in the oxyanion hole proved the great importance of its hydroxyl group in the transition-state stabilization. The lost transition-state stabilization was partly replaced through substrate-assisted catalysis with substrates carrying a hydroxyl group. The poor selectivity of the wild-type lipase for ethyl 2-hydroxypropanoate (E=1.6) was greatly improved in the mutant (E=22), since only one enantiomer could perform substrate-assisted catalysis.

    The redesign of the size of the stereospecificity pocket was very successful. Mutation of the tryptophan at the bottom of this pocket removed steric interactions with secondary alcohols that have to position a substituent larger than an ethyl in this pocket. This mutation increased the activity 5 500 times towards 5-nonanol and 130 000 times towards (S)-1-phenylethanol. The acceptance of such large substituents (butyl and phenyl) in the redesigned stereospecificity pocket increases the utility of lipases in biocatalysis. The improved activity with (S)-1-phenylethanol strongly contributed to the 8 300 000 times change in enantioselectivity towards 1-phenylethanol; example of such a large change was not found in the literature. The S-selectivity of the mutant is unique for lipases. Its enantioselectivity increases strongly with temperature reaching a useful S-selectivity (E=44) at 69 °C.

    Thermodynamics analysis of the enantioselectivity showed that the mutation in the stereospecificity pocket mainly changed the entropic term, while the enthalpic term was only slightly affected. This pinpoints the importance of entropy in enzyme catalysis and entropy should not be neglected in rational redesign.

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    FULLTEXT01
  • 72.
    Magnusson, Anders O.
    et al.
    KTH, School of Biotechnology (BIO).
    Rotticci Mulder, Johanna C.
    KTH, School of Biotechnology (BIO).
    Santagostino, Alberto
    KTH, School of Biotechnology (BIO).
    Hult, Karl
    KTH, School of Biotechnology (BIO).
    Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B2005In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 6, no 6, p. 1051-1056Article in journal (Refereed)
    Abstract [en]

    The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser-His-Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast-reacting enantiomer of secondary alcohols places its medium-sized substituent in the stereospecificity pocket and its large substituent towards the active-site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild-type lipase increased 270 times towards heptan-4-ol and 5500 times towards nonan-5-ol; this resulted in the high specificity constants 1100 and 830 s(-1)m(-1), respectively. The substrate selectivity changed over 400000 times for nonan-5-ol over propan-2-ol with both Trp104Ala and the Trp104Gln mutations.

  • 73.
    Malm, Magdalena
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology.
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Protein Technology.
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Protein Technology.
    Gudmundsdotter, Lindvi
    Affibody AB, Stockholm, Sweden.
    Bass, Tarek
    KTH, School of Biotechnology (BIO), Protein Technology.
    Frejd, Fredrik Y.
    Höidén-Guthenberg, Ingmarie
    Affibody AB, Stockholm, Sweden.
    Varasteh, Zohreh
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Orlova, Anna
    Departmaent of Medical Chemistry, Preclinical PET Platform, Uppasala University, Uppsala, Sweden.
    Tolmachev, Vladimir
    Unit of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Inhibiting HER3-Mediated Tumor Cell Growth with Affibody Molecules Engineered to Low Picomolar Affinity by Position-Directed Error-Prone PCR-Like Diversification2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 5, p. e62791-Article in journal (Refereed)
    Abstract [en]

    The HER3 receptor is implicated in the progression of various cancers as well as in resistance to several currently used drugs, and is hence a potential target for development of new therapies. We have previously generated Affibody molecules that inhibit heregulin-induced signaling of the HER3 pathways. The aim of this study was to improve the affinity of the binders to hopefully increase receptor inhibition efficacy and enable a high receptor-mediated uptake in tumors. We explored a novel strategy for affinity maturation of Affibody molecules that is based on alanine scanning followed by design of library diversification to mimic the result from an error-prone PCR reaction, but with full control over mutated positions and thus less biases. Using bacterial surface display and flow-cytometric sorting of the maturation library, the affinity for HER3 was improved more than 30-fold down to 21 PM. The affinity is among the higher that has been reported for Affibody molecules and we believe that the maturation strategy should be generally applicable for improvement of affinity proteins. The new binders also demonstrated an improved thermal stability as well as complete refolding after denaturation. Moreover, inhibition of ligand-induced proliferation of HER3-positive breast cancer cells was improved more than two orders of magnitude compared to the previously best-performing clone. Radiolabeled Affibody molecules showed specific targeting of a number of HER3-positive cell lines in vitro as well as targeting of HER3 in in vivo mouse models and represent promising candidates for future development of targeted therapies and diagnostics.

  • 74.
    Mesmar, Fahmi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Univ Houston, Ctr Nucl Receptors & Cell Signaling, Dept Biol & Biochem, Houston, TX USA.;Indiana Univ, Sch Informat Comp & Engn, Dept Intelligent Syst Engn, Bloomington, IN USA..
    Dai, Bingbing
    Univ Texas MD Anderson Canc Ctr, Dept Surg Oncol, Houston, TX 77030 USA..
    Ibrahim, Ahmed
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Hases, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Jafferali, Mohammed Hakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Augustine, Jithesh Jose
    Univ Texas MD Anderson Canc Ctr, Dept Surg Oncol, Houston, TX 77030 USA..
    DiLorenzo, Sebastian
    Uppsala Univ, Dept Med Sci, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Natl Bioinformat Infrastruct Sweden, Uppsala, Sweden..
    Kang, Ya'an
    Univ Texas MD Anderson Canc Ctr, Dept Surg Oncol, Houston, TX 77030 USA..
    Zhao, Yang
    Univ Texas MD Anderson Canc Ctr, Dept Bioinformat & Comp Sci, Houston, TX 77030 USA..
    Wang, Jing
    Univ Texas MD Anderson Canc Ctr, Dept Bioinformat & Comp Sci, Houston, TX 77030 USA..
    Kim, Michael
    Univ Texas MD Anderson Canc Ctr, Dept Surg Oncol, Houston, TX 77030 USA..
    Lin, Chin-Yo
    Univ Houston, Ctr Nucl Receptors & Cell Signaling, Dept Biol & Biochem, Houston, TX USA..
    Berkenstam, Anders
    Axcentua Pharmaceut AB, Stockholm, Sweden.;ABK, Stockholm, Sweden..
    Fleming, Jason
    Univ Texas MD Anderson Canc Ctr, Dept Surg Oncol, Houston, TX 77030 USA.;H Lee Moffitt Canc Ctr & Res Inst, Dept Gastrointestinal Oncol, Tampa, FL USA..
    Williams, Cecilia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Clinical candidate and genistein analogue AXP107-11 has chemoenhancing functions in pancreatic adenocarcinoma through G protein-coupled estrogen receptor signaling2019In: Cancer Medicine, ISSN 2045-7634, E-ISSN 2045-7634, Vol. 8, no 18, p. 7705-7719Article in journal (Refereed)
    Abstract [en]

    Despite advances in cancer therapeutics, pancreatic cancer remains difficult to treat and often develops resistance to chemotherapies. We have evaluated a bioavailable genistein analogue, AXP107-11 which has completed phase Ib clinical trial, as an approach to sensitize tumor cells to chemotherapy. Using organotypic cultures of 14 patient-derived xenografts (PDX) of pancreatic ductal adenocarcinoma, we found that addition of AXP107-11 indeed sensitized 57% of cases to gemcitabine treatment. Results were validated using PDX models in vivo. Further, RNA-Seq from responsive and unresponsive tumors proposed a 41-gene treatment-predictive signature. Functional and molecular assays were performed in cell lines and demonstrated that the effect was synergistic. Transcriptome analysis indicated activation of G-protein-coupled estrogen receptor (GPER1) as the main underlying mechanism of action, which was corroborated using GPER1-selective agonists and antagonists. GPER1 expression in pancreatic tumors was indicative of survival, and our study proposes that activation of GPER1 may constitute a new avenue for pancreatic cancer therapeutics.

  • 75.
    Neiman, Maja
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Bead based protein profiling in blood2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is about protein profiling in blood-derived samples using suspension bead ar- rays built with protein affinity reagents, and the evaluation of binding characteristics and potential disease relation of such profiles.

    A central aim of the presented work was to discover and verify disease associated protein profiles in blood-derived samples such as serum or plasma. This was based on immobiliz- ing antigens or antibodies on color-coded beads for a multiplexed analysis. This concept generally allow for a dual multiplexing because hundreds of samples can be screened for hundreds of proteins in a miniaturized and parallelized fashion. At first, protein antigens were used to study humoral immune responses in cattle suffering from a mycoplasma infec- tion (Paper I). Here, the most immunogenic of the applied antigens were identified based on reactivity profiles from the infected cattle, and were combined into an antigen cocktail to serve as a diagnostic assay in a standard ELISA set-up. Next, antibodies and their em- ployment in assays with directly labeled human samples was initiated. This procedure was applied in a study of kidney disorders where screening of plasma resulted in the discovery of a biomarker candidate, fibulin-1 (Paper II). In parallel to the disease related applica- tions, systematic evaluations of the protein profiles were conducted. Protein profiles from 2,300 antibodies were classified on the bases of binding properties in relation to sample heating and stringent washing (Paper III). With a particular focus on heat dependent de- tectability, a method was developed to visualize those proteins that were captured to the beads in an immunoassay by using Western blotting (Paper IV). In conclusion, this thesis presents examples of the possibilities of comparative plasma profiling enabled by protein bead arrays. 

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    Neiman PhD Thesis
  • 76.
    Neiman, Maja
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nygren, Per-Åke
    KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Nilsson, Peter
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101). KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Validating the selectivity of antibodies used in multiplexed serum profiling via parallel immunocapture analysisManuscript (preprint) (Other academic)
    Abstract [en]

    The increasing availability of antibodies towards human proteins drives the generation of new and exploratory data, which can be generated by profiling e.g. plasma samples using multiplexed arrays. However, antibody assays can lead to erroneous results due to cross-­‐reactivity to off-­‐targets. Here, we describe an approach in which an antibody-­‐based suspension bead array is combined with subsequent validation of on-­‐ target binding using a coupled affinity purification assay. Based on differential heat treatment of samples, antibody performance was investigated and the results for antibodies directed towards several complement factors provide insights on the detection of proteins in sera from patients with complement deficiency. In conclusion, the combined parallel flow and blot based immunocapture analysis serves an important, first line tool for resolving differently detected serum or plasma protein profiles proposed by antibody arrays. 

  • 77. Nguyen, T. N.
    et al.
    Libon, C.
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Subunit vaccine candidates engineered from the central conserved region of the RSV G protein aimed for parenteral or mucosal delivery2013In: Molecular Vaccines: From Prophylaxis to Therapy-Volume 1, Springer, 2013, p. 103-118Chapter in book (Other academic)
    Abstract [en]

    Respiratory syncytial virus (RSV) is a major pathogen causing severe upper and lower respiratory disease in infants and in elderly worldwide. ccording to WHO, an RSV vaccine is urgently needed. Here, we describe the design of various types of subunit vaccine concepts based on molecular engineering aimed to deliver RSV antigens. Gene segment encoding parts of the conserved central region of the RSV G protein (G2Na) were prepared for various expression and delivery formats: (1) prokaryotically expressed and purifi ed G2Na alone or fused to different carrier proteins, one of them, namely, BBG2Na (Alum), has reached clinical trials in the elderly; (2) G protein-derived antigens surface displayed on lived vectors (non pathogenic bacteria) and (3) nucleic acid vectors. These subunit vaccines were administered with or without adjuvant in rodents and in non-human primates by different routes (parenteral or mucosal). We summarise and compare immunogenicity, protective effi cacy and safety conferred by each immunisation format in RSV experimental animal models. Among these, G2Na proved to be the most promising component for an RSV subunit vaccine.

  • 78. Niklasson, Mia
    et al.
    Maddalo, Gianluca
    Sramkova, Zuzana
    Mutlu, Ercan
    Wee, Shimei
    Sekyrova, Petra
    Schmidt, Linnea
    Fritz, Nicolas
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Dehnisch, Ivar
    Kyriatzis, Gregorios
    Krafcikova, Michaela
    Carson, Brittany B.
    Feenstra, Jennifer M.
    Marinescu, Voichita D.
    Segerman, Anna
    Haraldsson, Martin
    Gustavsson, Anna-Lena
    Hammarstrom, Lars G. J.
    Jensen, Annika Jenmalm
    Uhrbom, Lene
    Altelaar, A. F. Maarten
    Linnarsson, Sten
    Uhlen, Per
    Trantirek, Lukas
    Vincent, C. Theresa
    Nelander, Sven
    Enger, Per Oyvind
    Andang, Michael
    Membrane-Depolarizing Channel Blockers Induce Selective Glioma Cell Death by Impairing Nutrient Transport and Unfolded Protein/Amino Acid Responses2017In: Cancer Research, ISSN 0008-5472, E-ISSN 1538-7445, Vol. 77, no 7, p. 1741-1752Article in journal (Refereed)
    Abstract [en]

    Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca2+ and KCa channels can effectively induce selective cell death of GIC and increase host survival in an orthotopic mouse model of human glioma. At present, the precise cellular pathways affected by the drugs affecting these channels are unknown. However, using cell-based assays and integrated proteomics, phosphoproteomics, and transcriptomics analyses, we identified the downstreamsignaling events these drugs affect. Changes in plasma membrane depolarization and elevated intracellular Na+, which compromised Na+-dependent nutrient transport, were documented. Deficits in nutrient deficit acted in turn to trigger the unfolded protein response and the amino acid response, leading ultimately to nutrient starvation and GIC cell death. Our results suggest new therapeutic targets to attack aggressive gliomas.

  • 79. Nilsson, D.
    et al.
    Pettersson, M.
    Gustavsson, P.
    Förster, A.
    Hofmeister, W.
    Wincent, J.
    Zachariadis, V.
    Anderlid, B. -M
    Nordgren, A.
    Mäkitie, O.
    Wirta, Valtteri
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Käller, Max
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vezzi, F.
    Lupski, J. R.
    Nordenskjöld, M.
    Syk Lundberg, E.
    Carvalho, C. M. B.
    Lindstrand, A.
    Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation2017In: Human Mutation, ISSN 1059-7794, E-ISSN 1098-1004, Vol. 38, no 2, p. 180-192Article in journal (Refereed)
    Abstract [en]

    Most balanced translocations are thought to result mechanistically from nonhomologous end joining or, in rare cases of recurrent events, by nonallelic homologous recombination. Here, we use low-coverage mate pair whole-genome sequencing to fine map rearrangement breakpoint junctions in both phenotypically normal and affected translocation carriers. In total, 46 junctions from 22 carriers of balanced translocations were characterized. Genes were disrupted in 48% of the breakpoints; recessive genes in four normal carriers and known dominant intellectual disability genes in three affected carriers. Finally, seven candidate disease genes were disrupted in five carriers with neurocognitive disabilities (SVOPL, SUSD1, TOX, NCALD, SLC4A10) and one XX-male carrier with Tourette syndrome (LYPD6, GPC5). Breakpoint junction analyses revealed microhomology and small templated insertions in a substantive fraction of the analyzed translocations (17.4%; n = 4); an observation that was substantiated by reanalysis of 37 previously published translocation junctions. Microhomology associated with templated insertions is a characteristic seen in the breakpoint junctions of rearrangements mediated by error-prone replication-based repair mechanisms. Our data implicate that a mechanism involving template switching might contribute to the formation of at least 15% of the interchromosomal translocation events.

  • 80. Nogueira, Eugenia
    et al.
    Mangialavori, Irene C.
    Loureiro, Ana
    Azoia, Nuno G.
    Sarria, Marisa P.
    Nogueira, Patricia
    Freitas, Jaime
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Inst, Sch Technol & Hlth.
    Shimanovich, Ulyana
    Rollett, Alexandra
    Lacroix, Ghislaine
    Bernardes, Goncalo J. L.
    Guebitz, Georg
    Hebert, Hans
    Moreira, Alexandra
    Carmo, Alexandre M.
    Rossi, Juan Pablo F. C.
    Gomes, Andreia C.
    Preto, Ana
    Cavaco-Paulo, Artur
    Peptide Anchor for Folate-Targeted Liposomal Delivery2015In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 16, no 9, p. 2904-2910Article in journal (Refereed)
    Abstract [en]

    Specific folate receptors are abundantly overexpressed in chronically activated macrophages and in most cancer cells. Directed folate receptor targeting using liposomes is usually achieved using folate linked to a phospholipid or cholesterol anchor. This link is formed using a large spacer like polyethylene glycol. Here, we report an innovative strategy for targeted liposome delivery that uses a hydrophobic fragment of surfactant protein D linked to folate. Our proposed spacer is a small 4 amino acid residue linker. The peptide conjugate inserts deeply into the lipid bilayer without affecting liposomal integrity, with high stability and specificity. To compare the drug delivery potential of both liposomal targeting systems, we encapsulated the nuclear dye Hoechst 34580. The eventual increase in blue fluorescence would only be detectable upon liposome disruption, leading to specific binding of this dye to DNA. Our delivery system was proven to be more efficient (2-fold) in Caco-2 cells than classic systems where the folate moiety is linked to liposomes by polyethylene glycol.

  • 81.
    Ohlsson, Anna B.
    et al.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Djerbi, Soraya
    KTH, School of Biotechnology (BIO), Glycoscience.
    Winzell, Anders
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bessueille, Laurence
    Ståldal, Veronika
    Li, Xinguo
    KTH, School of Biotechnology (BIO), Glycoscience.
    Blomqvist, Kristina
    KTH, School of Biotechnology (BIO), Glycoscience.
    Bulone, Vincent
    Teeri, Tuula T.
    KTH, School of Biotechnology (BIO), Glycoscience.
    Berglund, Torkel
    KTH, School of Biotechnology (BIO), Glycoscience.
    Cell suspension cultures of Populus tremula x P. tremuloides exhibit a high level of cellulose synthase gene expression that coincides with increased in vitro cellulose synthase activity.2006In: Protoplasma, ISSN 0033-183X, E-ISSN 1615-6102, Vol. 228, no 4, p. 221-9Article in journal (Refereed)
    Abstract [en]

    Compared to wood, cell suspension cultures provide convenient model systems to study many different cellular processes in plants. Here we have established cell suspension cultures of Populus tremula L. x P. tremuloides Michx. and characterized them by determining the enzymatic activities and/or mRNA expression levels of selected cell wall-specific proteins at the different stages of growth. While enzymes and proteins typically associated with primary cell wall synthesis and expansion were detected in the exponential growth phase of the cultures, the late stationary phase showed high expression of the secondary-cell-wall-associated cellulose synthase genes. Interestingly, detergent extracts of membranes from aging cell suspension cultures exhibited high levels of in vitro cellulose synthesis. The estimated ratio of cellulose to callose was as high as 50 : 50, as opposed to the ratio of 30 : 70 so far achieved with membrane preparations extracted from other systems. The increased cellulose synthase activity was also evidenced by higher levels of Calcofluor white binding in the cell material from the stationary-phase cultures. The ease of handling cell suspension cultures and the improved capacity for in vitro cellulose synthesis suggest that these cultures offer a new basis for studying the mechanism of cellulose biosynthesis.

  • 82.
    Olofsson, Karl
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Carannante, V.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden..
    Ohlin, Mathias
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Frisk, Thomas
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Kushiro, K.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Takai, M.
    Univ Tokyo, Dept Bioengn, Tokyo, Japan..
    Lundqvist, A.
    Karolinska Inst, Dept Oncol Pathol, Stockholm, Sweden..
    Önfelt, Björn
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Acoustic formation of multicellular tumor spheroids enabling on-chip functional and structural imaging2018In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 16, p. 2466-2476Article in journal (Refereed)
    Abstract [en]

    Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound-coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

  • 83. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Lindberg, Hanna
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Varasteh, Zohreh
    Rosestedt, Maria
    Tolmachev, Vadimir
    Kronqvist, Nina
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Feasibility of radionuclide imaging of HER3-expressing tumors using affibody molecules2013In: Journal of labelled compounds & radiopharmaceuticals, ISSN 0362-4803, E-ISSN 1099-1344, Vol. 56, p. S11-S11Article in journal (Other academic)
  • 84. Orlova, Anna
    et al.
    Malm, Magdalena
    KTH, School of Biotechnology (BIO), Protein Technology.
    Rosestedt, Maria
    Varasteh, Zohreh
    Andersson, Ken
    KTH, School of Biotechnology (BIO), Protein Technology.
    Selvaraju, Ram Kumar
    Altai, Mohamed
    Honarvar, Hadis
    Strand, Joanna
    Stahl, Stefan
    KTH, School of Biotechnology (BIO), Protein Technology.
    Tolmachev, Vladimir
    Löfblom, John
    KTH, School of Biotechnology (BIO), Protein Technology.
    Imaging of HER3-expressing xenografts in mice using a Tc-99m(CO)(3)-HEHEHE-Z(HER3:08699) affibody molecule2014In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 41, no 7, p. 1450-1459Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is a transmembrane receptor tyrosine kinase belonging to the HER (ErbB) receptor family. Membranous expression of HER3 is associated with trastuzumab resistance in breast cancer and the transition to androgen independence in prostate cancer. Imaging of HER3 expression in malignant tumors may provide important diagnostic information that can influence patient management. Affibody molecules with low picomolar affinity to HER3 were recently selected. The aim of this study was to investigate the feasibility of HER3 imaging using radiolabeled Affibody molecules. A HER3-binding Affibody molecule, Z(08699), with a HEHEHE-tag on N-terminus was labeled with Tc-99m(CO)(3) using an IsoLink kit. In vitro and in vivo binding specificity and the cellular processing of the labeled binder were evaluated. Biodistribution of Tc-99m(CO)(3)-HEHEHE-Z(08699) was studied over time in mice bearing HER3-expressing xenografts. HEHEHE-Z(08699) was labeled with Tc-99m(CO)(3) with an isolated yield of > 80 % and a purity of > 99 %. Binding of Tc-99m(CO)(3)-HEHEHE-Z(08699) was specific to BT474 and MCF7 (breast cancer), and LS174T (colon cancer) cells. Cellular processing showed rapid binding and relatively quick internalization of the receptor/Affibody molecule complex (70 % of cell-associated radioactivity was internalized after 24 h). The tumor targeting was receptor mediated and the excretion was predominantly renal. Receptor-mediated uptake was also found in the liver, lung, stomach, intestine, and salivary glands. At 4 h pi, tumor-to-blood ratios were 7 +/- 3 for BT474, and 6 +/- 2 for LS174T xenografts. LS174T tumors were visualized by microSPECT 4 h pi. The results of this study suggest the feasibility of HER3-imaging in malignant tumors using Affibody molecules.

  • 85.
    Pathak, Anuj
    et al.
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Bergstrand, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Sender, Vicky
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Spelmink, Laura
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Aschtgen, Marie-Stephanie
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Muschiol, Sandra
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden..
    Widengren, Jerker
    KTH, School of Engineering Sciences (SCI), Applied Physics, Quantum and Biophotonics.
    Henriques-Normark, Birgitta
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, SE-17177 Stockholm, Sweden.;Nanyang Technol Univ, Lee Kong Chian Sch Med LKC, Singapore 639798, Singapore.;Nanyang Technol Univ, Singapore Ctr Environm Life Sci Engn SCELSE, Singapore 639798, Singapore.;Karolinska Univ Hosp, Dept Clin Microbiol, SE-17176 Stockholm, Sweden..
    Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3398Article in journal (Refereed)
    Abstract [en]

    Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

  • 86.
    Perez-Zabaleta, Mariel
    KTH, School of Biotechnology (BIO), Industrial Biotechnology.
    (R)-3-Hydroxybutyrate production in a metabolically engineered Escherichia coli2016In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 33, p. S117-S117Article in journal (Refereed)
  • 87.
    Persson, Helena
    et al.
    KTH, School of Biotechnology (BIO), Protein Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Preger, C.
    Marcon, E.
    Lengqvist, J.
    Gräslund, S.
    Antibody validation by immunoprecipitation followed by mass spectrometry analysis2017In: Methods in Molecular Biology, Humana Press Inc. , 2017, p. 175-187Conference paper (Refereed)
    Abstract [en]

    We describe a mass spectrometry-based approach for validation of antibody specificity. This method allows validation of antibodies or antibody fragments, against their endogenous targets. It can assess if the antibody is able to bind to its native antigen in cell lysates among thousands of other proteins, DNA, RNA, and other cellular components. In addition, it identifies other proteins the antibody is able to immunoprecipitate allowing for the assessment of antibody specificity and selectivity. This method is easily scalable, adaptable to different cell lines and conditions and has been shown to be reproducible between multiple laboratories.

  • 88.
    Petrou, Georgia
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Jansson, Ronnie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Hogqvist, Mark
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Erlandsson, Johan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Wågberg, Lars
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Fibre- and Polymer Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Crouzier, Thomas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Chemistry, Glycoscience.
    Genetically Engineered Mucoadhesive Spider Silk2018In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 19, no 8, p. 3268-3279Article in journal (Refereed)
    Abstract [en]

    Mucoadhesion is defined as the adhesion of a material to the mucus gel covering the mucous membranes. The mechanisms controlling mucoadhesion include nonspecific electrostatic interactions and specific interactions between the materials and the mucins, the heavily glycosylated proteins that form the mucus gel. Mucoadhesive materials can be used to develop mucosal wound dressings and noninvasive transmucosal drug delivery systems. Spider silk, which is strong, biocompatible, biodegradable, nontoxic, and lightweight would serve as an excellent base for the development of such materials. Here, we investigated two variants of the partial spider silk protein 4RepCT genetically engineered in order to functionalize them with mucoadhesive properties. The pLys-4RepCT variant was functionalized with six cationically charged lysines, aiming to provide nonspecific adhesion from electrostatic interactions with the anionically charged mucins, while the hGal3-4RepCT variant was genetically fused with the Human Galectin-3 Carbohydrate Recognition Domain which specifically binds the mucin glycans Gal beta 1-3GlcNAc and Gal beta 1-4GlcNAc. First, we demonstrated that coatings, fibers, meshes, and foams can be readily made from both silk variants. Measured by the adsorption of both bovine submaxillary mucin and pig gastric mucin, the newly produced silk materials showed enhanced mucin binding properties compared with materials of wild-type (4RepCT) silk. Moreover, we showed that pLys-4RepCT silk coatings bind mucins through electrostatic interactions, while hGal3-4RepCT silk coatings bind mucins through specific glycan-protein interactions. We envision that the two new mucoadhesive silk variants pLys-4RepCT and hGal3-4RepCT, alone or combined with other biofunctional silk proteins, constitute useful new building blocks for a range of silk protein-based materials for mucosal treatments.

  • 89.
    Polyutov, Sergey
    KTH, School of Biotechnology (BIO).
    Electron-nuclear Dynamics in Nonlinear Optics and X-ray spectroscopy2007Doctoral thesis, comprehensive summary (Other scientific)
    Abstract [en]

    This thesis is devoted to theoretical studies of the role of nuclear vibrations on nonlinear and linear absorption, pulse propagation, and resonant scattering of light. The molecular parameters needed for the simulations are obtained through suitable quantum chemical calculations, which are compared with available experimental data.

    The first part of the thesis addresses to modeling of ampli ed spontaneous emission (ASE) in organic chromophores recently studied in a series of experiments. To explain the threshold behavior of the ASE spectra we invoke the idea of competition between di erent ASE channels and non-radiative quenching of the lasing levels. We show that the ASE spectrum changes drastically when the pump intensity approaches the threshold level, namely, when the ASE rate approaches the rate of vibrational relaxation or the rate of solute-solvent relaxation in the rst excited state. According to our simulations the ASE intensity experiences oscillations. Temporal self-pulsations of forward and backward propagating ASE pulses occur due to two reasons: i) the interaction of co- and counter-propagating ASE, and ii) the competition between the ampli ed spontaneous emission and o -resonant absorption.

    In the second part of the thesis we explore two-photon absorption taking into account nuclear vibrational degrees of freedom. The theory, applied to the N101 molecule [p-nitro-p'- diphenylamine stilbene], shows that two-step absorption is red shifted relative to one-photon absorption spectrum in agreement with the measurements. The reason for this e ect is the one-photon absorption from the first excited state. Simulations show that two mechanisms are responsible for the population of this state, two-photon absorption and offresonant one-photon absorption by the wing of the spectral line.

    In the third part of the thesis we study multi-photon dynamics of photobleaching by a periodical sequence of short laser pulses. It is found that the photobleaching as well as the uorescence follow double-exponential dynamics.

    The fourth part of the thesis is devoted to the role of the nuclear dynamics in x-ray spectroscopy. Our studies show that the vibronic coupling of close lying core excited states strongly a ects the resonant x-ray Raman scattering from ethylene and benzene molecules. We demonstrate that the manifestation of the non-adiabatic e ects depends strongly on the detuning of photon energy from the top of photoabsorption. The electronic selection rules are shown to break down when the excitation energy is tuned in resonance with the symmetry breaking vibrational modes. Selection rules are then restored for large detuning. We obtained good agreement with experiment. Finally, our multi-mode theory is applied to simulations of the resonant Auger and x-ray absorption spectra of the ethyne molecule.

    Download full text (pdf)
    FULLTEXT01
  • 90.
    Poux, Candice
    et al.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Dondalska, Aleksandra
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Bergenstråhle, Joseph
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Palsson, Sandra
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Contreras, Vanessa
    CEA, Inst Biol Francois Jacob, UMR1184, IDMIT Dept,DRF, Fontenay Aux Roses, France..
    Arasa, Claudia
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Jarver, Peter
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    Albert, Jan
    Karolinska Inst, Dept Microbiol Tumor & Cell Biol, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Clin Microbiol, Stockholm, Sweden..
    Busse, David C.
    Imperial Coll London, Dept Infect Dis, London, England..
    LeGrand, Roger
    CEA, Inst Biol Francois Jacob, UMR1184, IDMIT Dept,DRF, Fontenay Aux Roses, France..
    Lundeberg, Joakim
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Tregoning, John S.
    Imperial Coll London, Dept Infect Dis, London, England..
    Spetz, Anna-Lena
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, Stockholm, Sweden..
    A Single-Stranded Oligonucleotide Inhibits Toll-Like Receptor 3 Activation and Reduces Influenza A (H1N1) Infection2019In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 10, article id 2161Article in journal (Refereed)
    Abstract [en]

    The initiation of an immune response is dependent on the activation and maturation of dendritic cells after sensing pathogen associated molecular patterns by pattern recognition receptors. However, the response needs to be balanced as excessive pro-inflammatory cytokine production in response to viral or stress-induced pattern recognition receptor signaling has been associated with severe influenza A virus (IAV) infection. Here, we use an inhibitor of Toll-like receptor (TLR)3, a single-stranded oligonucleotide (ssON) with the capacity to inhibit certain endocytic routes, or a TLR3 agonist (synthetic double-stranded RNA Polyl:C), to evaluate modulation of innate responses during H1N1 IAV infection. Since IAV utilizes cellular endocytic machinery for viral entry, we also assessed ssON's capacity to affect IAV infection. We first show that IAV infected human monocyte-derived dendritic cells (MoDC) were unable to up-regulate the co-stimulatory molecules CD80 and CD86 required for T cell activation. Exogenous TLR3 stimulation did not overcome the IAV-mediated inhibition of co-stimulatory molecule expression in MoDC. However, TLR3 stimulation using Polyl:C led to an augmented pro-inflammatory cytokine response. We reveal that ssON effectively inhibited Polyl:C-mediated pro-inflammatory cytokine production in MoDC, notably, ssON treatment maintained an interferon response induced by IAV infection. Accordingly, RNAseq analyses revealed robust up-regulation of interferon-stimulated genes in IAV cultures treated with ssON. We next measured reduced IAV production in MoDC treated with ssON and found a length requirement for its anti-viral activity, which overlapped with its capacity to inhibit uptake of Polyl:C. Hence, in cases wherein an overreacting TLR3 activation contributes to IAV pathogenesis, ssON can reduce this signaling pathway. Furthermore, concomitant treatment with ssON and IAV infection in mice resulted in maintained weight and reduced viral load in the lungs. Therefore, extracellular ssON provides a mechanism for immune regulation of TLR3-mediated responses and suppression of IAV infection in vitro and in vivo in mice.

  • 91. Quince, Christopher
    et al.
    Delmont, Tom O.
    Raguideau, Sebastien
    Alneberg, Johannes
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Darling, Aaron E.
    Collins, Gavin
    Eren, A. Murat
    DESMAN: a new tool for de novo extraction of strains from metagenomes2017In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 18, article id 181Article in journal (Refereed)
    Abstract [en]

    We introduce DESMAN for De novo Extraction of Strains from Metagenomes. Large multi-sample metagenomes are being generated but strain variation results in fragmentary co-assemblies. Current algorithms can bin contigs into metagenome-assembled genomes but are unable to resolve strain-level variation. DESMAN identifies variants in core genes and uses co-occurrence across samples to link variants into haplotypes and abundance profiles. These are then searched for against non-core genes to determine the accessory genome of each strain. We validated DESMAN on a complex 50-species 210-genome 96-sample synthetic mock data set and then applied it to the Tara Oceans microbiome.

  • 92.
    Qundos, Ulrika
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Antibody based plasma protein profiling2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided.

    Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V).

    As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected.

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    Antibody based plasma protein profiling
  • 93.
    Redin, David
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Phasing single DNA molecules with barcode linked sequencing2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Elucidation of our genetic constituents has in the past decade predominately taken the form of short-read DNA sequencing. Revolutionary technology developments have enabled vast amounts of biological information to be obtained, but from a medical standpoint it has yet to live up to the promise of associating individual genotypes to phenotypic states of wide-spread clinical relevance. The mechanisms by which complex phenotypes arise have been difficult to ascertain and the value of short-read sequencing platforms have been limited in this regard. It has become evident that resolving the full spectrum of genetic heterogeneity requires accurate long range information of individual haplotypes to be distinguished. Long-range haplotyping information can be obtained experimentally by long-read sequencing platforms or through linkage of short sequencing reads by means of a common barcode. This thesis explores these solutions, primarily through the development of novel technologies to phase short sequences of single molecules using DNA barcoding. A new method for high-throughput phasing of single DNA molecules, achieved by the production and utilization of uniquely barcoded beads in emulsion droplets, is described in Paper I. The results confirm that complex libraries of beads featuring mutually exclusive barcodes can be generated through clonal PCR amplification, and that these beads can be used to phase variations of the 16s rRNA gene which reduces the ambiguity of classifying bacterial species for metagenomics. Paper II describes a second methodology (‘Droplet Barcode Sequencing’) which simplifies the concept of barcoding DNA fragments by omitting the need for beads and instead relying on clonal amplification of single barcoding oligonucleotides. This study also increases the amount of information that can be linked, which is showcased by phasing all exons of the HLA-A gene and successfully resolving all the alleles present in a sample pool of eight individuals. Paper III expands on this work and explores the use of a single molecule sequencing platform to provide full-length sequencing coverage of six genes of the HLA family. The results show that while genes shorter than 10 kb can be resolved with a high degree of accuracy, compensating for a relatively high error rate by means of increased coverage can be challenging for larger genomic loci. Finally, Paper IV introduces the use of barcode-linked reads on an unprecedented scale, with a new assay that enables low-cost haplotyping of whole genomes without the need for predetermined capture sequences. This technology is utilized to generate a haplotype-resolved human genome, call large-scale structural variants and perform reference-free assembly of bacterial and human genomes. At a cost of only $19 USD per sample, this technology makes the benefits of long-range haplotyping available to the vast majority of laboratories which currently rely solely on short-read sequencing platforms.

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    Redin_Thesis
  • 94.
    Redin, David
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Frick, Tobias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Aghelpasand, Hooman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Theland, Jennifer
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Käller, Max
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Borgström, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Olsen, Remi-Andre
    Stockholms Universitet.
    Ahmadian, Afshin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Gene Technology.
    Efficient whole genome haplotyping and single molecule phasing with barcode-linked readsManuscript (preprint) (Other academic)
    Abstract [en]

    The future of human genomics is one that seeks to resolve the entirety of genetic variation through sequencing. The prospect of utilizing genomics for medical purposes require cost-efficient and accurate base calling, long-range haplotyping capability, and reliable calling of structural variants. Short-read sequencing has lead the development towards such a future but has struggled to meet the latter two of these needs. To address this limitation, we developed a technology that preserves the molecular origin of short sequencing reads, with an insignificant increase to sequencing costs. We demonstrate a library preparation method which enables whole genome haplotyping, long-range phasing of single DNA molecules, and de novo genome assembly through barcode-linked reads (BLR). Millions of random barcodes are used to reconstruct megabase-scale phase blocks and call structural variants. We also highlight the versatility of our technology by generating libraries from different organisms using picograms to nanograms of input material.

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    BLR.incl.SI
  • 95.
    Rinne, Sara S.
    et al.
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mitran, Bogdan
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden..
    Bass, Tarek
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Andersson, Ken G.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, Uppsala, Sweden..
    Optimization of HER3 expression imaging using affibody molecules: Influence of chelator for labeling with indium-1112019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 655Article in journal (Refereed)
    Abstract [en]

    Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N ''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid), and DOTAGA (1,4,7,10-tetraazacyclododececane, 1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z(08698) and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged In-111-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.

  • 96.
    Rinne, Sara S.
    et al.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Xu, Tianqi
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Löfblom, John
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Orlova, Anna
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden.;Uppsala Univ, Sci Life Lab, S-75123 Uppsala, Sweden.;Natl Res Tomsk Polytech Univ, Ctr Oncotheranost, Tomsk 634050, Russia..
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden.;Natl Res Tomsk Polytech Univ, Ctr Oncotheranost, Tomsk 634050, Russia..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden.;Natl Res Tomsk Polytech Univ, Ctr Oncotheranost, Tomsk 634050, Russia..
    Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules2020In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 21, no 4, article id 1312Article in journal (Refereed)
    Abstract [en]

    Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)(3)-Z(HER3:08698)-DOTAGA affibody molecule with non-residualizing [I-125]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA was compared side-by-side with [In-111]In-(HE)(3)-Z(HER3:08698)-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24h pi showed faster clearance of the [I-125]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [I-125]I-PIB-(HE)(3)-Z(HER3:08698)-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 +/- 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [I-125]I-PIB label. In conclusion, the use of non-residualizing [I-125]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.

  • 97.
    Robinson, Jonathan L.
    et al.
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden..
    Feizi, Amir
    Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden.;Novo Nordisk Res Ctr Oxford, Old Campus Rd, Oxford, England..
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nielsen, Jens
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Chalmers Univ Technol, Dept Biol & Biol Engn, Kemivagen 10, Gothenburg, Sweden ; Chalmers Univ Technol, Wallenberg Ctr Prot Res, Kemivagen 10, Gothenburg, Sweden ; Tech Univ Denmark, Novo Nordisk Fdn, Ctr Biosustainabil, DK-2800 Lyngby, Denmark.
    A Systematic Investigation of the Malignant Functions and Diagnostic Potential of the Cancer Secretome2019In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 26, no 10, p. 2622-+Article in journal (Refereed)
    Abstract [en]

    The collection of proteins secreted from a cell-the secretome-is of particular interest in cancer pathophysiology due to its diagnostic potential and role in tumorigenesis. However, cancer secretome studies are often limited to one tissue or cancer type or focus on biomarker prediction without exploring the associated functions. We therefore conducted a pan-cancer analysis of secretome gene expression changes to identify candidate diagnostic biomarkers and to investigate the underlying biological function of these changes. Using transcriptomic data spanning 32 cancer types and 30 healthy tissues, we quantified the relative diagnostic potential of secretome proteins for each cancer. Furthermore, we offer a potential mechanism by which cancer cells relieve secretory pathway stress by decreasing the expression of tissue-specific genes, thereby facilitating the secretion of proteins promoting invasion and proliferation. These results provide a more systematic understanding of the cancer secretome, facilitating its use in diagnostics and its targeting for therapeutic development.

  • 98.
    Rockberg, Johan
    et al.
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Löfblom, John
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Hjelm, Barbara
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Ståhl, Stefan
    KTH, School of Biotechnology (BIO), Molecular Biotechnology (closed 20130101).
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO), Proteomics (closed 20130101).
    Epitope mapping using gram-positive surface display2010In: Current Protocols in Immunology, ISSN 1934-3671, no SUPPL. 90, p. 9.9.1-9.9.17Article, review/survey (Refereed)
    Abstract [en]

    Antibodies have proven to be invaluable tools for a vast number of applications during the last decades, including protein purification and characterization, medical diagnosis and imaging, and treatment using therapeutic antibiotics. No matter what the aims of the application are, the antibodys binding characteristics will still be the main features determining the assays reliability. Here, we describe a protocol for determination of antibody-binding epitopes using an antigen-focused, library-based approach where library members are generated by fragmentation of antigen DNA and presented as cloned peptides on the cell surface of the Gram-positive bacterium Staphylococcus carnosus. The rigid cell structure of this organism allows for multivalent expression and permits rapid library analysis and sorting of antibody-binding cells using flow-sorting devices. Epitopes are determined by DNA sequencing of the sorted cells and alignment back to the antigen sequence. The protocol described here has been shown useful for mapping of both monoclonal and polyclonal binders with varying epitope lengths.

  • 99.
    Ronaghi, Martin
    et al.
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Uhlén, Mathias
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    Nyrén, Pål
    KTH, Superseded Departments (pre-2005), Biochemistry and Biotechnology.
    A sequencing method based on real-time pyrophosphate1998In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 281, no 5375, p. 363-365Article in journal (Refereed)
  • 100.
    Rozkov, Aleksei D.
    et al.
    KTH, Superseded Departments, Biotechnology.
    Enfors, Sven-Olof
    KTH, Superseded Departments, Biotechnology.
    Analysis and control of proteolysis of recombinant proteins in Escherichia coli.2004In: Advances in biochemical engineering/biotechnology, ISSN 0724-6145, Vol. 89, p. 163-195Article in journal (Refereed)
    Abstract [en]

    Proteolysis is one of the reasons for poor production of recombinant proteins in Escherichia coli. Important properties of E. coli proteases, which are relevant for the production of recombinant proteins, are reviewed. Furthermore, various strategies to control the proteolysis of the recombinant proteins are presented. These strategies for control of proteolysis can be applied on various stages of the process: design of more stable protein, a modification of the host cell in respect to proteolytic activity, optimisation of cultivation and downstream processing. However, before implementing these measures the proteolysis rate should be measured in order to calculate a potential benefit of reduced proteolysis rate.

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