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  • 1. Ahmed, Mona
    et al.
    Baumgartner, Roland
    Aldi, Silvia
    Dusart, Philip
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Hedin, Ulf
    Gustafsson, Bjorn
    Caidahl, Kenneth
    Human serum albumin-based probes for molecular targeting of macrophage scavenger receptors2019In: International Journal of Nanomedicine, ISSN 1176-9114, E-ISSN 1178-2013, Vol. 14, p. 3723-3741Article in journal (Refereed)
    Abstract [en]

    Background: Inflammation and accumulation of macrophages are key features of unstable atherosclerotic plaques. The ability of macrophages to take up molecular probes can be exploited in new clinical imaging methods for the detection of unstable atherosclerotic lesions. We investigated whether modifications of human serum albumin (HSA) could be used to target macrophages efficiently in vitro. Materials and methods: Maleylated and aconitylated HSA were compared with unmodified HSA. Fluorescent or radiolabeled (Zr-89) modified HSA was used in in vitro experiments to study cellular uptake by differentiated THP-1 cells and primary human macrophages. The time course of uptake was evaluated by flow cytometry, confocal microscopy, real-time microscopy and radioactivity measurements. The involvement of scavenger receptors (SR-Al, SR-B2, LOX-1) was assessed by knockdown experiments using RNA interference, by blocking experiments and by assays of competition by modified low-density lipoprotein. Results: Modified HSA was readily taken up by different macrophages. Uptake was mediated nonexclusively via the scavenger receptor SR-Al (encoded by the MSR1 gene). Knockdown of CD36 and ORL1 had no influence on the uptake. Modified HSA was preferentially taken up by human macrophages compared with other vascular cell types such as endothelial cells and smooth muscle cells. Conclusions: Modified Zr-89-labeled HSA probes were recognized by different subsets of polarized macrophages, and maleylated HSA may be a promising radiotracer for radio-nuclide imaging of macrophage-rich inflammatory vascular diseases.

  • 2.
    Ahmed, Mona
    et al.
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, S-17176 Stockholm, Sweden..
    Gustafsson, Björn
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, S-17176 Stockholm, Sweden..
    Aldi, Silvia
    Karolinska Inst, Sect Med Inflammat Res, Dept Med Biochem & Biophys, S-17177 Stockholm, Sweden..
    Dusart, Philip
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Egri, Gabriella
    Surflay Nanotec GmbH, Max Planck Str 3, D-12489 Berlin, Germany..
    Butler, Lynn M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Bone, Dianna
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, S-17176 Stockholm, Sweden..
    Dahne, Lars
    Surflay Nanotec GmbH, Max Planck Str 3, D-12489 Berlin, Germany..
    Hedin, Ulf
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, S-17176 Stockholm, Sweden..
    Caidahl, Kenneth
    Karolinska Inst, Dept Mol Med & Surg, Ctr Mol Med, S-17176 Stockholm, Sweden.;Univ Gothenburg, Sahlgrenska Acad, Inst Med, Dept Mol & Clin Med, S-41345 Gothenburg, Sweden..
    Molecular Imaging of a New Multimodal Microbubble for Adhesion Molecule Targeting2019In: Cellular and Molecular Bioengineering, ISSN 1865-5025, E-ISSN 1865-5033, Vol. 12, no 1, p. 15-32Article in journal (Refereed)
    Abstract [en]

    Introduction: Inflammation is an important risk-associated component of many diseases and can be diagnosed by molecular imaging of specific molecules. The aim of this study was to evaluate the possibility of targeting adhesion molecules on inflammation-activated endothelial cells and macrophages using an innovative multimodal polyvinyl alcohol-based microbubble (MB) contrast agent developed for diagnostic use in ultrasound, magnetic resonance, and nuclear imaging. Methods: We assessed the binding efficiency of antibody-conjugated multimodal contrast to inflamed murine or human endothelial cells (ECs), and to peritoneal macrophages isolated from rats with peritonitis, utilizing the fluorescence characteristics of the MBs. Single-photon emission tomography (SPECT) was used to illustrate 99m Tc-labeled MB targeting and distribution in an experimental in vivo model of inflammation. Results: Flow cytometry and confocal microscopy showed that binding of antibody-targeted MBs to the adhesion molecules ICAM-1, VCAM-1, or E-selectin, expressed on cytokine-stimulated ECs, was up to sixfold higher for human and 12-fold higher for mouse ECs, compared with that of non-targeted MBs. Under flow conditions, both VCAM-1- and E-selectin-targeted MBs adhered more firmly to stimulated human ECs than to untreated cells, while VCAM-1-targeted MBs adhered best to stimulated murine ECs. SPECT imaging showed an approximate doubling of signal intensity from the abdomen of rats with peritonitis, compared with healthy controls, after injection of anti-ICAM-1-MBs. Conclusions: This novel multilayer contrast agent can specifically target adhesion molecules expressed as a result of inflammatory stimuli in vitro, and has potential for use in disease-specific multimodal diagnostics in vivo using antibodies against targets of interest.

  • 3.
    Alm, Tove L.
    et al.
    KTH, School of Biotechnology (BIO).
    von Feilitzen, Kalle
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Uhlén, Mathias
    KTH, School of Biotechnology (BIO).
    Antibodypedia - The wiki of antibodies2015In: Molecular Biology of the Cell, ISSN 1059-1524, E-ISSN 1939-4586, Vol. 26Article in journal (Other academic)
  • 4. Altai, M.
    et al.
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Ding, Haozhong
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mitran, B.
    Edqvist, P. -H
    Tolmachev, V.
    Orlova, A.
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Affibody-derived drug conjugates: Potent cytotoxic molecules for treatment of HER2 over-expressing tumors2018In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 288, p. 84-95Article in journal (Refereed)
    Abstract [en]

    Patients with HER2-positive tumors often suffer resistance to therapy, warranting development of novel treatment modalities. Affibody molecules are small affinity proteins which can be engineered to bind to desired targets. They have in recent years been found to allow precise targeting of cancer specific molecular signatures such as the HER2 receptor. In this study, we have investigated the potential of an affibody molecule targeting HER2, ZHER2:2891, conjugated with the cytotoxic maytansine derivate MC-DM1, for targeted cancer therapy. ZHER2:2891 was expressed as a monomer (ZHER2:2891), dimer ((ZHER2:2891)2) and dimer with an albumin binding domain (ABD) for half-life extension ((ZHER2:2891)2-ABD). All proteins had a unique C-terminal cysteine that could be used for efficient and site-specific conjugation with MC-DM1. The resulting affibody drug conjugates were potent cytotoxic molecules for human cells over-expressing HER2, with sub-nanomolar IC50-values similar to trastuzumab emtansine, and did not affect cells with low HER2 expression. A biodistribution study of a radiolabeled version of (ZHER2:2891)2-ABD-MC-DM1, showed that it was taken up by the tumor. The major site of off-target uptake was the kidneys and to some extent the liver. (ZHER2:2891)2-ABD-MC-DM1 was found to have a half-life in circulation of 14 h. The compound was tolerated well by mice at 8.5 mg/kg and was shown to extend survival of mice bearing HER2 over-expressing tumors. The findings in this study show that affibody molecules are a promising class of engineered affinity proteins to specifically deliver small molecular drugs to cancer cells and that such conjugates are potential candidates for clinical evaluation on HER2-overexpressing cancers. 

  • 5.
    Altai, M.
    et al.
    Uppsala Univ, Imuunol Genet & Pathol, Uppsala, Sweden..
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Div Prot Technol, Stockholm, Sweden..
    Orlova, A.
    Div Mol Imaging, Dept Med Chem, Uppsala, Sweden..
    Tolmachev, V.
    Uppsala Univ, Imuunol Genet & Pathol, Uppsala, Sweden..
    Gräslund, Torbjörn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Div Prot Technol, Stockholm, Sweden..
    Improving of molecular design of a novel Affibody-fused HER2-recognising anticancer toxin using radionuclide-based techniques2016In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 43, p. S178-S178Article in journal (Other academic)
  • 6.
    Altai, M.
    et al.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    Vorobyeva, A.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    Westerlund, Kristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Mitran, B.
    Div Mol Imaging, Uppsala, Sweden..
    Orlova, A.
    Div Mol Imaging, Uppsala, Sweden..
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Tolmachev, V.
    Immunology, Genetics and Pathology, Uppsala, SWEDEN, .
    A novel method for conjugation of PNA to antibodies for radionuclide based pretargeting: proof of principal2018In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 45, p. S648-S648Article in journal (Other academic)
  • 7.
    Altai, Mohamed
    et al.
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden.;Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Leitao, Charles Dahlsson
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rinne, Sara S.
    Uppsala Univ, Dept Med Chem, S-75123 Uppsala, Sweden..
    Vorobyeva, Anzhelika
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Atterby, Christina
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    Ståhl, Stefan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Tolmachev, Vladimir
    Uppsala Univ, Dept Immunol Genet & Pathol, S-75185 Uppsala, Sweden..
    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-75237 Uppsala, Sweden..
    Influence of Molecular Design on the Targeting Properties of ABD-Fused Mono- and Bi-Valent Anti-HER3 Affibody Therapeutic Constructs2018In: CELLS, ISSN 2073-4409, Vol. 7, no 10, article id 164Article in journal (Refereed)
    Abstract [en]

    Overexpression of human epidermal growth factor receptor type 3 (HER3) is associated with tumour cell resistance to HER-targeted therapies. Monoclonal antibodies (mAbs) targeting HER3 are currently being investigated for treatment of various types of cancers. Cumulative evidence suggests that affibody molecules may be appropriate alternatives to mAbs. We previously reported a fusion construct (3A3) containing two HER3-targeting affibody molecules flanking an engineered albumin-binding domain (ABD 035) included for the extension of half-life in circulation. The 3A3 fusion protein (19.7 kDa) was shown to delay tumour growth in mice bearing HER3-expressing xenografts and was equipotent to the mAb seribantumab. Here, we have designed and explored a series of novel formats of anti-HER3 affibody molecules fused to the ABD in different orientations. All constructs inhibited heregulin-induced phosphorylation in HER3-expressing BxPC-3 and DU-145 cell lines. Biodistribution studies demonstrated extended the half-life of all ABD-fused constructs, although at different levels. The capacity of our ABD-fused proteins to accumulate in HER3-expressing tumours was demonstrated in nude mice bearing BxPC-3 xenografts. Formats where the ABD was located on the C-terminus of affibody binding domains (3A, 33A, and 3A3) provided the best tumour targeting properties in vivo. Further development of these promising candidates for treatment of HER3-overexpressing tumours is therefore justified.

  • 8. Altai, Mohamed
    et al.
    Liu, Hao
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Ding, Haozhong
    Mitran, Bogdan
    Edqvist, Per-Henrik
    Tolmachev, Vladimir
    Orlova, Anna
    Gräslund, Torbjorn
    Affibody-derived Drug Conjugates: Potent Cytotoxic Drugs ForTreatment Of HER2 Over-Expressing TumorsManuscript (preprint) (Other academic)
  • 9.
    Andersson, Annika
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Remnestål, Julia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nellgård, B.
    Vunk, Helian
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Kotol, David
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Edfors, Fredrik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Ilag, L. L.
    Zetterberg, H.
    Blennow, K.
    Månberg, Anna
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Fredolini, Claudia
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease2019In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 494, p. 79-93Article in journal (Refereed)
    Abstract [en]

    Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.

  • 10.
    Andersson, Ken G.
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Persson, Jonas
    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.
    Autotransporter-Mediated Display of a Naive Affibody Library on the Outer Membrane of Escherichia coli2019In: Biotechnology Journal, ISSN 1860-6768, E-ISSN 1860-7314, Vol. 14, no 4, article id 1800359Article in journal (Refereed)
    Abstract [en]

    Development of new affinity proteins using combinatorial protein engineering is today established for generation of monoclonal antibodies and also essential for discovery of binders that are based on non-immunoglobulin proteins. Phage display is most frequently used, but yeast display is becoming increasingly popular, partly due to the option of utilizing fluorescence-activated cell sorting (FACS) for isolation of new candidates. Escherichia coli has several valuable properties for library applications and in particular the high transformation efficiency. The use of various autotransporters and intimins for secretion and anchoring on the outer membrane have shown promising results and particularly for directed evolution of different enzymes. Here, the authors report on display of a large naive affibody library on the outer membrane of E. coli using the autotransporter Adhesin Involved in Diffuse Adherence (AIDA-I). The expression cassette is first engineered by removing non-essential sequences, followed by introduction of an affibody library, comprising more than 10(9) variants, into the new display vector. The quality of the library and general performance of the method is assessed by FACS against five different targets, which resulted in a panel of binders with down to nanomolar affinities, suggesting that the method has potential as a complement to phage display for generation of affibody molecules.

  • 11.
    Annelies, Nonneman
    et al.
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Nathan, Criem
    Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Cardiovasc Sci, Ctr Mol & Vasc Biol, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Human Genet, Herestr 49, B-3000 Leuven, Belgium..
    Lewandowski, Sebastian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Clin Neurosci, S-17177 Stockholm, Sweden..
    Rik, Nuyts
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Dietmar, Thal R.
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neuropathol, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    Frank, Pfrieger W.
    Univ Strasbourg, CNRS UPR 3212, Inst Cellular & Integrat Neurosci, F-67084 Strasbourg, France..
    John, Ravits
    Univ Calif San Diego, Dept Neurosci, 9500 Gilman Dr, San Diego, CA 92093 USA..
    Philip, Van Damme
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    An, Zwijsen
    Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Cardiovasc Sci, Ctr Mol & Vasc Biol, Herestr 49, B-3000 Leuven, Belgium.;KU Leuven Univ Leuven, Dept Human Genet, Herestr 49, B-3000 Leuven, Belgium..
    Ludo, Van Den Bosch
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium..
    Wim, Robberecht
    KU Leuven Univ Leuven, Dept Neurosci, Lab Neurobiol & Expt Neurol, Herestr 49, B-3000 Leuven, Belgium.;LBI, Herestr 49, B-3000 Leuven, Belgium.;Ctr Brain & Dis Res, VIB, Herestr 49, B-3000 Leuven, Belgium.;Univ Hosp Leuven, Dept Neurol, Herestr 49, B-3000 Leuven, Belgium..
    Astrocyte-derived Jagged-1 mitigates deleterious Notch signaling in amyotrophic lateral sclerosis2018In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 119, p. 26-40Article in journal (Refereed)
    Abstract [en]

    Amyotrophic lateral sclerosis (ALS) is a late-onset devastating degenerative disease mainly affecting motor neurons. Motor neuron degeneration is accompanied and aggravated by oligodendroglial pathology and the presence of reactive astrocytes and microglia. We studied the role of the Notch signaling pathway in ALS, as it is implicated in several processes that may contribute to this disease, including axonal retraction, microgliosis, astrocytosis, oligodendrocyte precursor cell proliferation and differentiation, and cell death. We observed abnormal activation of the Notch signaling pathway in the spinal cord of SOD1(G93A) mice, a well-established model for ALS, as well as in the spinal cord of patients with sporadic ALS (sALS). This increased activation was particularly evident in reactive GFAP-positive astrocytes. In addition, one of the main Notch ligands, Jagged-1, was ectopically expressed in reactive astrocytes in spinal cord from ALS mice and patients, but absent in resting astrocytes. Astrocyte-specific inactivation of Jagged-1 in presymptomatic SOD1(G93A) mice further exacerbated the activation of the Notch signaling pathway and aggravated the course of the disease in these animals without affecting disease onset. These data suggest that aberrant Notch signaling activation contributes to the pathogenesis of ALS, both in sALS patients and SOD1(G93A) mice, and that it is mitigated in part by the upregulation of astrocytic Jagged-1.

  • 12.
    Antypas, H.
    et al.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Veses-Garcia, M.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    Weibull, Emelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Svahn Andersson, Helene
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Richter-Dahlfors, A.
    Karolinska Inst, Dept Neurosci, Swedish Med Nanosci Ctr, Stockholm, Sweden..
    A universal platform for selection and high-resolution phenotypic screening of bacterial mutants using the nanowell slide2018In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 18, no 12, p. 1767-1777Article in journal (Refereed)
    Abstract [en]

    The Petri dish and microtiter plate are the golden standard for selection and screening of bacteria in microbiological research. To improve on the limited resolution and throughput of these methods, we developed a universal, user-friendly platform for selection and high-resolution phenotypic screening based on the nanowell slide. This miniaturized platform has an optimal ratio between throughput and assay complexity, holding 672 nanowells of 500 nl each. As monoclonality is essential in bacterial genetics, we used FACS to inoculate each nanowell with a single bacterium in 15 min. We further extended the protocol to select and sort only bacteria of interest from a mixed culture. We demonstrated this by isolating single transposon mutants generated by a custom-made transposon with dual selection for GFP fluorescence and kanamycin resistance. Optical compatibility of the nanowell slide enabled phenotypic screening of sorted mutants by spectrophotometric recording during incubation. By processing the absorbance data with our custom algorithm, a phenotypic screen for growth-associated mutations was performed. Alternatively, by processing fluorescence data, we detected metabolism-associated mutations, exemplified by a screen for -galactosidase activity. Besides spectrophotometry, optical compatibility enabled us to perform microscopic analysis directly in the nanowells to screen for mutants with altered morphologies. Despite the miniaturized format, easy transition from nano- to macroscale cultures allowed retrieval of bacterial mutants for downstream genetic analysis, demonstrated here by a cloning-free single-primer PCR protocol. Taken together, our FACS-linked nanowell slide replaces manual selection of mutants on agar plates, and enables combined selection and phenotypic screening in a one-step process. The versatility of the nanowell slide, and the modular workflow built on mainstream technologies, makes our universal platform widely applicable in microbiological research.

  • 13.
    Ayoglu, Burcu
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Multiplexed antigen bead arrays for the assessment of antibody selectivity and epitope mapping2018In: Epitope Mapping Protocols, Humana Press Inc. , 2018, p. 239-248Chapter in book (Refereed)
    Abstract [en]

    With the increasing number of binding reagents for affinity-based investigations of the human proteome, high-throughput tools for the characterization of the used reagents become essential. For the analysis of binding selectivity, bead-based antigen arrays offer a miniaturized and parallelized assay platform to meet such needs, as they enable two-dimensional multiplexing to analyze up to 384 samples against up to 500 analytes in a single round of analysis. In this chapter, we describe our protocols for the generation of multiplex bead arrays built on immobilized protein fragments, as well as biotinylated peptides. Combined together, these two versions of antigen arrays offer a versatile approach for multiplexed characterization of antibody binding selectivity, off-target interactions, as well as mapping for the amino acids of epitopes involved in antibody binding.

  • 14.
    Banerjee, Indradumna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Point of care microfluidic tool development for resource limited settings2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The development of point of care diagnostics using recent advances in microfluidics have the potential to transform health care in several ways, especially in resource limited settings with limited access to advanced health care infrastructure. However, translating a point of care device to reality is often a challenging task because of the complexities involved in integrating a number of diverse engineering concepts into an easy to use, accurate and portable device. This thesis focuses on miniaturization of crucial diagnostic laboratory tools, that can be used in a portable point of care format without compromising on the accuracy or performance. The first part of the thesis (Paper I-III) focuses on understanding and applying elasto-inertial microfluidics, which is a label-free and passive bio-particle sorting and separation method. A basic understanding of particle trajectories in both inertial (Paper I) and visco-elastic flows (Paper II) is established, followed by an investigation on the combined effects of inertia and elasticity (Paper III). The second part of the thesis (Paper IV-VI) focuses on developing integrated microfluidic platforms, each of which addresses different aspects of point of care diagnostic applications. The applications include neonatal diagnostics using a hand-driven Slipdisc technique (Paper IV), rapid nucleic acid quantification using a novel precipitate-based detection on a centrifugal microfluidics platform (Paper V), and hematocrit level measurement in blood using a portable lab-on- Disc platform operated by a mobile phone (Paper VI). The proof of concept microfluidic tools presented in the scope of this thesis have the potential to replace a number of functions of standard laboratory equipment, at a fraction of the price and without compromising performance. Hence, the different methods developed should contribute towards decentralization of medical testing laboratories, making healthcare accessible to one and all.

  • 15.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Aralaguppe, S. G.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Zhang, W.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Sönnerborg, A.
    Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Neogi, Ujjwal
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. Karolinska Inst, Dept Lab Med, Div Clin Microbiol, Stockholm, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Microfluidic centrifugation assisted precipitation based DNA quantification2019In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 9, p. 1657-1664Article in journal (Refereed)
    Abstract [en]

    Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often requires time consuming purification, washing or labeling steps. Here, we report a novel microfluidic centrifugation assisted precipitation (mu CAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, which can be quantified, when an intercalating dye (GelRed) is added to the DNA sample and centrifuged for a few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of the visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng mu l(-1). Furthermore, using an integrated lab-on-DVD platform we recently developed, the detection limit is lowered to 10 ng mu l(-1), which is comparable to those of current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the lab-on-DVD platform. The simple DNA quantification system could facilitate advanced point of care molecular diagnostics.

  • 16.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Aralaguppe, Shambhu Prasad
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Sönneborg, Anders
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Neogi, Ujjwal
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    MicroCAP: Microfluidic Centrifuge Assisted Precipitation for DNA Quantification on Lab-on-DVD2018Conference paper (Refereed)
    Abstract [en]

    We report for the first time the MicroCAP technique, for rapid DNA detection and quantification, that does not require any purification or fluorescent labelling of DNA. The invention is based on DNA interacting with a detection dye (Gelred) to form a complex, that forms a visible precipitate within seconds of centrifugation. MicroCAP can be used for DNA quantification, when combined with the Lab-on-DVD with inbuilt centrifugation and sub- micron imaging resolution. We quantify PCR and LAMP assay products using MicroCAP on the integrated Lab-on- DVD platform, and demonstrate a detection limit of 10 ng/!".

    KEYWORDS: MicroCAP, DNA detection, Centrifuge,Precipitate, LAMP, PCR.

    INTRODUCTION

    Detection of amplified DNA is often based on measurement of turbidity, fluorescence (after staining with a detec- tion dye) or absorbance. Commercially available instruments for DNA quantitation can be broadly divided into two categories: UV instruments based on absorbance (such as spectrophotometers, e.g. Nanodrop or Nanophotometer) and instruments based on measurement of a fluorescent dye (such as plate readers). One bottleneck in quantifying amplified DNA in a nucleic acid amplification test (NAAT) reaction, based on absorbance measurement technique, is the bias introduced due to the presence of the isothermal amplification buffer, dNTPs and other reagents. Each reagent or buffer may have an absorbance density at around 260 nm, elevating the apparent concentration measured by the device compared to the actual value. Hence, for most quantitation based NAATs, it is important to include an extra DNA purification step, which may result in non-negligible loss of the amplified product and increases the cost of the purification kit. Measurements based on fluorescence mostly use fluorescent dyes that are potentially hazardous for handling. In addition, fluorescence based quantitation methods require time consuming labelling and washing steps.

    In this report, we describe a new method, termed microfluidic centrifugation assisted precipitation (microCAP), involving quantification and detection of DNA based on precipitation of nucleic acids. The basis of the method is formation of a visible precipitate when GelRed, a nucleic acid intercalacting dye commonly used in gel electropho- resis, is mixed with DNA and centrifuged. A visible precipitate is formed after just a few seconds of centrifugation and enables rapid detection of the presence of DNA in a sample. To the best of our knowledge, the visible precipitate formed as a product of centrifuging GelRed mixed with DNA has not been reported before. We showed that the DNA GelRed complex is dense enough compared to water to precipitate upon centrifugation. Further, we extended the μCAP method to the Lab-on-DVD platform1 to quantify the DNA concentration from images generated using the optical DVD reader instrument. The modified DVD player was able to image the precipitate formed up to a detection limit of 10 ng/μl of DNA. For calibration of the images, known quantities of a purified PCR product were used to identify the relationship between the amounts of DNA and precipitate formed. We applied the method to quantify an unknown quantity of LAMP amplicons from a LAMP assay for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. A sensitivity limit of 10 ng/μl of DNA was achieved, comparable with that of a Nanophotometer.18 The results demonstrated that the method is able to quantitatively detect the presence of DNA in a sample in a few seconds without any purification step.

    EXPERIMENTAL

    The Lab-on-DVD system was employed for spinning and imaging the precipitate product using a modified DVD drive, as mentioned in our previous report.1 We began by dispensing the sample in the design chamber, adding GelRed dye (at a concentration of 4000X in water) and centrifuging the mixture at 1200 rpm. Figure 1a and 1b

    show schematics of the DNA sample precipitation process conducted in test tubes and the DVD platform, respec- tively. We used known amounts of a PCR product to calibrate the quantity of precipitate to the DNA concentration. We used a HIV genome amplified from 50 ng of plasmid pNL4.3 using the primers 0776F and 6231R.2 To evaluate the sensitivity of DNA detection of our system, we used the amplified products from a LAMP assay. The sensitivity of LAMP primers was tested on DNA from pNL4.3 (a HIV-1B genome containing plasmid). A 25X LAMP primer mix was prepared according to Curtis et al.,3 using the same template DNA sequence, set of primers and DNA polymerase. Eight concentrations (each being 5 μl volume) of the HIV-1B genome containing plasmid (pNL4.3) were tested, starting from 1 ng/!" serially diluted to 1 fg/!". Two negative controls were also prepared, one without DNA and primers and one without primers. The total reaction volume was increased to 30 μl (instead of 25 μl used in Curtis et al.3) by multiplying every component volume in the reaction by a factor of 1.2. Fabrication of the multi- layer microfluidic Disc followed the same procedure as described in our previous report.1 The Lab-on-DVD system was used to generate images of the precipitation zone. To quantify the amount of precipitate, an image processing script was written in MATLAB software (Mathworks, USA).

    RESULTS AND DISCUSSION

    MicroCAP was found to be suitable for determining the presence of DNA in a sample, We carried out the LAMP assay in Eppendorf tubes in an oven set at 65°C. After 45 minutes, 3 μl of 10,000X GelRed in water was added to two tubes of 30 μl volume each, one having an unknown concentration of LAMP amplified DNA and the other one with no DNA template as a control. After centrifugation for approximately 5 seconds, a visible precipitate was formed in the tube containing amplified DNA, whereas no precipitate was formed in the control tube (Fig. 2a). 10 μl volume of DNA was inserted into a U shaped channel of the DVD alongwith 1 μl of 10,000X GelRed in water, which was the same ratio of DNA sample to Gelred as used in the test tube. An imageable precipitate was observed in the Lab on DVD custom imaging software (fig.2b).

    A Matlab script was used for image analysis in which an original image(fig.3a) was transformed into a binary image (fig.3b) by defining a threshold pixel value, exploiting the difference in intensity of the precipitate from its background. The entire area to the left of the threshold line in the histogram (Fig. 3c), i.e. from value 0 to the threshold value (normally 90), was summed to estimate the total area of the precipitate.

    For DNA quantification, known concentrations of a PCR product was used for calibration. The initial concentration of purified PCR product was 129 ng/μl, measured with a Nanophotometer (in triplicates) after purification with a GeneJet PCR purification kit. The purified PCR product was subsequently diluted serially several times and each diluted concentration was measured again with the Nanophotometer (in triplicate). The measurements were then repeated with the Lab-on-DVD method. Fig. 4a shows four images recorded at four known concentrations together with their binary threshold images. Fig. 4b shows the precipitation area calculated from the images plotted against the known DNA concentrations, showing a linear relationship. 10 ng/μl was the lowest concentration detectable in the DVD images.

    For quantification of unknown quantities of nucleic acids, we carried out the LAMP assay on HIV-1B genome containing plasmid DNA using serial dilutions (10-fold dilutions from 1 ng/μl to 0.1 fg/μl) to evaluate the limit of detection (Fig.5). Two negative controls were also prepared, one comprising primers and no DNA template and second, no DNA template and no primers.

    Fig. 6 shows the precipitation area plotted against the starting concentration of DNA template. It shows that the amplification in the LAMP assay is not linear for all the starting concentrations of DNA template. The error bars in the figure show the standard deviation for a particular concentration. For a LAMP assay, which fluctuates somewhat in its yield of amplified prod- ucts, we believe that this error range is acceptable.

    The precipitation area was converted to an actual yield of DNA products for each of the concentrations. This conversion was based on the linear empirical equation generated from the calibration curve presented earlier in Fig. 4b, given by:

    y= 9.61x – 4.05 (1) Here, y denotes the precipitation area in arbitrary units while x denotes the DNA concentration.

    CONCLUSION

    We demonstrated an extremely fast visual DNA quantification method (μCAP) that can be made quantifiable on a Lab-on-DVD platform. The approach was based on DNA forming a precipitate upon centrifugation when in contact with the GelRed dye. Results using HIV-1B genome containing plasmid DNA revealed a detection limit of 0.01 pg/μl or total amount of 0.1 pg of starting DNA template, which is an acceptable standard for resource limited settings. The limit of detection of DNA with the Lab-on-DVD platform was found to be 10 ng/μl, which is almost comparable to the detection limits reported by commercially available instruments, such as the Nanophotometer. However, the μCAP method offers a distinct advantage over other state-of-the-art techniques as it does not require additional purification of the DNA. We believe the μCAP technique combined with the Lab-on-DVD platform provides a simple and low cost technology that can fulfil the need for a point-of-care device for DNA quantification.

    REFERENCES

    1. [1]  H. Ramachandraiah, M. Amasia, J. Cole, P. Sheard, S. Pickhaver, C. Walker, V. Wirta, P. Lexow, R. Lione and A. Russom, "Lab-on-DVD: standard DVD drives as a novel laser scanning microscope for image based point of care diagnostics."Lab. Chip, 2013, 13, 1578–1585.

    2. [2]  S. Grossmann, P. Nowak, and U. Neogi, “ Subtype-independent near full-length HIV-1 genome sequencing and assembly to be used in large molecular epidemiological studies and clinical man- agement.” Journal of the International AIDS Society, 2015,18(1), 20035.

    3. [3]  K. A. Curtis, D. L. Rudolph, I. Nejad, J. Singleton, A. Beddoe, B. Weigl, P. LaBarre and S. M. Owen, "Rapid detection of HIV-1 by reverse-transcription, loop-mediated isothermal amplification (RT- LAMP)." PLoS ONE, , DOI:10.1371/journal.pone.0031432.

    CONTACT

    *A. Russom; phone: +46-87909863; aman@kth.se

  • 17.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Ganeshappa Aralaguppe, Shambhu Prasad
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Lapins, Noa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Zhang, Wang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Kazemzadeh, Amin
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Sönnerborg, Anders
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Neogi, Ujjwal
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institute, Sweden..
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Microfluidic Centrifugation Assisted Precipitation based DNA QuantificationManuscript (preprint) (Other academic)
    Abstract [en]

    Nucleic acid amplification methods are increasingly being used to detect trace quantities of DNA in samples for various diagnostic applications. However, quantifying the amount of DNA from such methods often require time consuming purification, washing or labeling step. Here, we report a novel microfluidic centrifugation assisted precipitation (uCAP) method for single-step DNA quantification. The method is based on formation of a visible precipitate, that can be quantified, when an intercalating dye (GelRed) is added to DNA sample and centrifuged for few seconds. We describe the mechanism leading to the precipitation phenomenon. We utilize centrifugal microfluidics to precisely control the formation of visible and quantifiable mass. Using a standard CMOS sensor for imaging, we report a detection limit of 45 ng/ul. Furthermore, using an integrated Lab-on-DVD platform we recently developed, the detection limit was lowered to 10 ng/ul, which is comparable to current commercially available instruments for DNA quantification. As a proof of principle, we demonstrate the quantification of LAMP products for a HIV-1B type genome containing plasmid on the Lab-on-DVD platform. The simple DNA quantification system could facilitate advanced molecular diagnosis at point of care.

  • 18.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Rosti, Marco Edoardo
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Kumar, Tharagan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Brandt, Luca
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Analog particle position tuning in Elasto-inertial microfluidic flowsManuscript (preprint) (Other academic)
    Abstract [en]

    We observe for the first time an analog trend in particle focusing in a high throughput weakly viscoelastic regime, where it is possible to tune particles into multiple intermediate focusing positions that lie between the "Segre-Silberberg annulus" and the center of a circular microcapillary. The "Segre-Silberberg annulus" (0.6 times the pipe radius), that describes particle equilibrium in a predominantly inertial flow, shrinks consistently closer to the center for increasing elasticity in extremely dilute PEO concentrations (ranging from 0.001 wt% to 0.05wt%). The experimental observations are supported by direct numerical simulations, where an Immersed Boundary Method is used to account for the presence of particles and a FENE-P model is used to simulate the presence of polymers in a Non-Newtonian fluid. The numerical simulations study the dynamics and stability of finite size particles and are further used to analyze particle behavior at Reynolds number higher than what is allowed by the present experimental setup. In particular, we are able to report the entire migration trajectories of the particles as they reach their final equilibrium positions and extend our predictions to other geometries such as the square cross-section. We believe complex effects originate due to a combination of inertia and elasticity in a weakly viscoelastic regime, where neither inertia nor elasticity are able to mask each other's effect completely, thus leading to a number of intermediate focusing positions. The present study provides a new understanding into the mechanism of particle focusing in elasto-inertial flows and opens up new possibilities for exercising analog control in tuning the particle focusing positions.

  • 19.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Rosti, Marco Edoardo
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Kumar, Tharagan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH).
    Brandt, Luca
    KTH, School of Engineering Sciences (SCI), Mechanics.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Particle focusing dynamics in extended elasto inertial flow2018Conference paper (Refereed)
    Abstract [en]

    Elasto Inertial microfluidics has been exploited recently for a number of industrial and biological applications. Recently, we experimentally showed that it is possible to achieve single stream focusing of particles even at higher flow rates in the elasto inertial regime, relevant to flow cytometry applications, and , based on this concept, built a silica fibre based micro flow cytometer.1 However, the physics behind the focusing of particles is still poorly understood, specially for combinations of higher Reynolds (Re) and Weissenberg numbers(Wi).

    In the present study, for the first time, we seek to understand both experimentally and with numerical simulations, particle focusing across elasticity regimes. We vary the concentration of PEO (200 ppm to upto 10000 ppm) in PBS solution at sufficiently high flow rates of 100!l/min or above. We introduce a parameter, focusing bandwidth (F) to evaluate the extent of single stream focusing of 15 !m particles in a 75 !m diameter circular channel. Fig.1 shows the flow setup(fig.1a) along with images demonstrating the focused (fig.1b) and unfocused cases(fig.1c), as well as how F is calculated(fig.1d). We evaluate particle focusing by identifying the flow conditions for each concentration that leads to the minimum value of F. Fig.2 shows the variation of the focusing bandwidth(fig.2a) when changing PEO concentration, and the variation in Re along with Wi (fig.2b) and Elasticity number(El). The results show that for identical mass flow conditions across the different regimes the focusing bandwidth slowly shifts to a narrow single stream with increasing elasticity. We validated our experimental results as well as gained new insights into particle focusing with 3D numerical simulations based on a FENE P model. We studied the decoupled effects of Reynolds number and Weissenberg number on particle focusing, as well as the particle trajectories and migration dynamics as the particles reach equilibrium. Interestingly, enough we find a combination of high Re(Re=400) and sufficiently high Wi(Wi=3) for which the particles achieve a single stream focusing (fig.3a). The entire dynamics of particle migration in a circular cross section is also shown (in fig.3b) by changing Wi for a constant Re(Re=200). It can be seen that the particle goes through a longer amount of oscillations to reach its final equilibrium position as Wi is increased. Fig.4a shows the equilibrium position of the particle moving closer to the center with an increase in Wi at the same Re(Re=200). However, in the Non Newtonian cases, the particle has a slight oscillatory behaviour as it reaches its equilibrium position as compared to the Newtonian one. We introduced the particle at two different positions(at Re=200, We=0 and 1) and observed the same equilibrium positions in both cases (Fig.4b).

  • 20.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Lab-on-DVD: Optical Disk Drive-Based Platforms for Point-of-Care Diagnostics2018In: Frugal Innovation in Bioengineering for the Detection of Infectious Diseases / [ed] AK Chavali, R Ramji, Switzerland: Springer, 2018, 2, p. 23-38Chapter in book (Refereed)
    Abstract [en]

    There is a growing demand for simple, affordable, reliable and quality-assured point-of-care (POC) diagnostics for use in resource-limited settings. Among the top ten leading causes of death worldwide, three are infectious diseases, namely, respiratory infections, HIV/AIDS and diarrheal diseases (World Health Organization 2012). Although high-quality diagnostic tests are available, these are often not available to patients in developing countries. While recent development in microfluidics and “lab-on-a-chip” devices has the potential to spur the development of protocols and affordable instruments for diagnosis of infectious disease at POC, integration of complex sample preparation and detection into automated molecular and cellular systems remain a bottleneck for implementation of these systems at resource-limited settings. Towards this, we describe here how low-cost optical drives can, with minor modifications, be turned into POC diagnostic platforms. A DVD drive is essentially a highly advanced and low-cost optical laser-scanning microscope, with the capability to deliver high-resolution images for biological applications. Furthermore, the inherent centrifugal force on rotational discs is elegantly used for sample preparation and integration. Hence, the merging of low-cost optical disc drives with centrifugal microfluidics is feasible concept for POC diagnostics, specifically designed to meet the needs at resource-limited settings.

  • 21.
    Banerjee, Indradumna
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    MicroCAP2018Patent (Other (popular science, discussion, etc.))
    Abstract [en]

  • 22.
    Bedri, Sahl Khalid
    et al.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Nilsson, Ola B.
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Advice Foretagsassistans & Stockholm AB, TCER AB, Stockholm, Sweden..
    Fink, Katharina
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden.;Karolinska Univ Hosp, Dept Neurol, Stockholm, Sweden..
    Månberg, Anna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Affin Prote, SciLifeLab, Stockholm, Sweden..
    Hamsten, Carl
    Karolinska Inst, Dept Med, Immunol & Allergy Unit, Stockholm, Sweden..
    Ayoglu, Burcu
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Affin Prote, SciLifeLab, Stockholm, Sweden..
    Manouchehrinia, Ali
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Nilsson, Peter
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Affin Prote, SciLifeLab, Stockholm, Sweden..
    Olsson, Tomas
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Hillert, Jan
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Grönlund, Hans
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Glaser, Anna
    Karolinska Inst, Dept Clin Neurosci, Stockholm, Sweden.;Karolinska Inst, Ctr Mol Med, Stockholm, Sweden..
    Plasma protein profiling reveals candidate biomarkers for multiple sclerosis treatment2019In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 14, no 5, article id e0217208Article in journal (Refereed)
    Abstract [en]

    Multiple sclerosis (MS) treatment options have improved significantly over the past decades, but the consequences of MS can still be devastating and the needs for monitoring treatment surveillance are considerable. In the current study we used affinity proteomics technology to identify potential biomarkers which could ultimately be used to as facilitate treatment decisions. We profiled the intra-individual changes in the levels of 59 target proteins using an antibody suspension bead array in serial plasma samples from 44 MS patients during treatment with natalizumab followed by fingolimod. Nine proteins showed decreasing plasma levels during natalizumab treatment, with PEBP1 and RTN3 displaying the most significant changes. Protein levels remained stable during fingolimod treatment for both proteins. The decreasing PEBP1 levels during natalizumab treatment could be validated using ELISA and replicated in an independent cohort. These results support the use of this technology as a high throughput method of identifying potentially useful biomarkers of MS treatment.

  • 23.
    Benfeitas, Rui
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Institute of Technology, KTH.
    Bidkhori, Gholamreza
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Mukhopadhyay, Bani
    Klevstig, Martina
    Arif, Muhammad
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Zhang, Cheng
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lee, Sunjae
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Cinar, Resat
    Nielsen, Jens
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Boren, Jan
    Kunos, George
    Mardinoglu, Adil
    KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Characterization of heterogeneous redox responses in hepatocellular carcinoma patients using network analysis2019In: EBioMedicine, E-ISSN 2352-3964Article in journal (Refereed)
  • 24. Berger, Ashton C
    et al.
    Korkut, Anil
    Kanchi, Rupa S
    Hegde, Apurva M
    Lenoir, Walter
    Liu, Wenbin
    Liu, Yuexin
    Fan, Huihui
    Shen, Hui
    Ravikumar, Visweswaran
    Rao, Arvind
    Schultz, Andre
    Li, Xubin
    Sumazin, Pavel
    Williams, Cecilia
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics.
    Mestdagh, Pieter
    Gunaratne, Preethi H
    Yau, Christina
    Bowlby, Reanne
    Robertson, A Gordon
    Tiezzi, Daniel G
    Wang, Chen
    Cherniack, Andrew D
    Godwin, Andrew K
    Kuderer, Nicole M
    Rader, Janet S
    Zuna, Rosemary E
    Sood, Anil K
    Lazar, Alexander J
    Ojesina, Akinyemi I
    Adebamowo, Clement
    Adebamowo, Sally N
    Baggerly, Keith A
    Chen, Ting-Wen
    Chiu, Hua-Sheng
    Lefever, Steve
    Liu, Liang
    MacKenzie, Karen
    Orsulic, Sandra
    Roszik, Jason
    Shelley, Carl Simon
    Song, Qianqian
    Vellano, Christopher P
    Wentzensen, Nicolas
    Weinstein, John N
    Mills, Gordon B
    Levine, Douglas A
    Akbani, Rehan
    A Comprehensive Pan-Cancer Molecular Study of Gynecologic and Breast Cancers.2018In: Cancer Cell, ISSN 1535-6108, E-ISSN 1878-3686, Vol. 33, no 4, p. 690-705.e9, article id S1535-6108(18)30119-3Article in journal (Refereed)
    Abstract [en]

    We analyzed molecular data on 2,579 tumors from The Cancer Genome Atlas (TCGA) of four gynecological types plus breast. Our aims were to identify shared and unique molecular features, clinically significant subtypes, and potential therapeutic targets. We found 61 somatic copy-number alterations (SCNAs) and 46 significantly mutated genes (SMGs). Eleven SCNAs and 11 SMGs had not been identified in previous TCGA studies of the individual tumor types. We found functionally significant estrogen receptor-regulated long non-coding RNAs (lncRNAs) and gene/lncRNA interaction networks. Pathway analysis identified subtypes with high leukocyte infiltration, raising potential implications for immunotherapy. Using 16 key molecular features, we identified five prognostic subtypes and developed a decision tree that classified patients into the subtypes based on just six features that are assessable in clinical laboratories.

  • 25.
    Boutajangout, Allal
    et al.
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Psychiat, Langone Hlth, 550 1St Ave, New York, NY 10016 USA.;NYU, Langone Med Ctr, Dept Physiol & Neurosci, New York, NY USA..
    Lindberg, Hanna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Awwad, Abdulaziz
    King Abdulaziz Univ, Sch Med, Jeddah, Saudi Arabia..
    Paul, Arun
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Baitalmal, Rabaa
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Almokyad, Ismail
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA..
    Hoiden-Guthenberg, Ingmarie
    Affibody AB, Solna, Sweden..
    Gunneriusson, Elin
    Affibody AB, Solna, Sweden..
    Frejd, Fredrik Y.
    Affibody AB, Solna, Sweden..
    Hard, Torleif
    Swedish Univ Agr Sci SLU, Dept Chem & Biotechnol, Uppsala, Sweden..
    Löfblom, John
    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.
    Wisniewski, Thomas
    NYU, Ctr Cognit Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Neurol, Langone Hlth, New York, NY 10016 USA.;NYU, Dept Psychiat, Langone Hlth, 550 1St Ave, New York, NY 10016 USA.;NYU, Sch Med, Dept Pathol, New York, NY 10016 USA..
    Affibody-Mediated Sequestration of Amyloid beta Demonstrates Preventive Efficacy in a Transgenic Alzheimer's Disease Mouse Model2019In: Frontiers in Aging Neuroscience, ISSN 1663-4365, E-ISSN 1663-4365, Vol. 11, article id 64Article in journal (Refereed)
    Abstract [en]

    Different strategies for treatment and prevention of Alzheimer's disease (AD) are currently under investigation, including passive immunization with anti-amyloid beta (anti-A beta) monoclonal antibodies (mAbs). Here, we investigate the therapeutic potential of a novel type of A beta-targeting agent based on an affibody molecule with fundamentally different properties to mAbs. We generated a therapeutic candidate, denoted Z(SYM73)-albumin-binding domain (ABD; 16.8 kDa), by genetic linkage of the dimeric Z(SYM73) affibody for sequestering of monomeric A beta-peptides and an ABD for extension of its in vivo half-life. Amyloid precursor protein (APP)/PS1 transgenic AD mice were administered with Z(SYM73)-ABD, followed by behavioral examination and immunohistochemistry. Results demonstrated rescued cognitive functions and significantly lower amyloid burden in the treated animals compared to controls. No toxicological symptoms or immunology-related side-effects were observed. To our knowledge, this is the first reported in vivo investigation of a systemically delivered scaffold protein against monomeric A beta, demonstrating a therapeutic potential for prevention of AD.

  • 26.
    Bremer, Hanna D.
    et al.
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    Landegren, Nils
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden.;Uppsala Univ, Dept Med Sci, Sci Life Lab, Uppsala, Sweden..
    Sjöberg, Ronald
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hallgren, Asa
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden..
    Renneker, Stefanie
    Euroimmun AG, D-23560 Lubeck, Germany..
    Lattwein, Erik
    Euroimmun AG, D-23560 Lubeck, Germany..
    Leonard, Dag
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Eloranta, Maija-Leena
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Ronnblom, Lars
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Nordmark, Gunnel
    Uppsala Univ, Rheumatol & Sci Life Lab, Dept Med Sci, SE-75185 Uppsala, Sweden..
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Andersson, Goran
    Swedish Univ Agr Sci, Dept Anim Breeding & Genet, SE-75007 Uppsala, Sweden..
    Lilliehook, Inger
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    Lindblad-Toh, Kerstin
    Broad Inst Harvard & MIT, Cambridge, MA 02142 USA.;Uppsala Univ, Sci Life Lab, IMBIM, SE-75123 Uppsala, Sweden..
    Kampe, Olle
    Karolinska Inst, Karolinska Univ Hosp, Dept Med Solna, CMM, L8 01, SE-17176 Stockholm, Sweden.;Uppsala Univ, Dept Med Sci, Sci Life Lab, Uppsala, Sweden.;Univ Bergen, Dept Clin Sci, N-5021 Bergen, Norway.;Univ Bergen, KG Jebsen Ctr Autoimmune Disorders, N-5021 Bergen, Norway.;Haukeland Hosp, Dept Med, N-5021 Bergen, Norway..
    Hansson-Hamlin, Helene
    Swedish Univ Agr Sci, Dept Clin Sci, SE-75007 Uppsala, Sweden..
    ILF2 and ILF3 are autoantigens in canine systemic autoimmune disease2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4852Article in journal (Refereed)
    Abstract [en]

    Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjogren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

  • 27.
    Cavallaro, Sara
    et al.
    KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics.
    Horak, Josef
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Haag, Petra
    Karolinska Inst, Karolinska Univ Hosp, Dept Oncol Pathol, Theme Canc,Patient Area,Pelvis, Akad Straket 1, S-17164 Stockholm, Sweden..
    Gupta, Dhanu
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England..
    Stiller, Christiane
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Sahu, Siddharth S.
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Gorgens, Andre
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England.;Univ Duisburg Essen, Univ Hosp Essen, Inst Transfus Med, D-45122 Essen, Germany..
    Gatty, Hithesh K.
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Viktorsson, Kristina
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, Theme Canc,Patient Area,Head & Neck Lung & Skin, Akad Straket 1, S-17164 Solna, Sweden..
    El Andaloussi, Samir
    Karolinska Inst, Clin Res Ctr, Dept Lab Med, S-17177 Stockholm, Sweden.;Evox Therapeut Ltd, Oxford OX4 4HG, England..
    Lewensohn, Rolf
    Karolinska Inst, Dept Oncol Pathol, Karolinska Univ Hosp, Theme Canc,Patient Area,Head & Neck Lung & Skin, Akad Straket 1, S-17164 Solna, Sweden..
    Eriksson Karlström, Amelie
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering. KTH Royal Inst Technol, Sch Engn Sci Chem Biotechnol & Hlth, Dept Prot Sci, AlbalNova Univ Ctr, S-10691 Stockholm, Sweden..
    Linnros, Jan
    KTH, School of Engineering Sciences (SCI), Applied Physics, Photonics. KTH Royal Inst Technol, Sch Engn Sci, Dept Appl Phys, S-16440 Kista, Sweden..
    Dev, Apurba
    Uppsala Univ, Angstrom Lab, Dept Solid State Elect, Box 534, SE-75121 Uppsala, Sweden..
    Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor2019In: ACS SENSORS, ISSN 2379-3694, Vol. 4, no 5, p. 1399-1408Article in journal (Refereed)
    Abstract [en]

    Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

  • 28.
    Cengic, Ivana
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Synthetic biology approaches for improving production of fatty acid-derived compounds in cyanobacteria2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The environmental consequences associated with the use of fossil-sourced fuels and chemicals have brought with it a realization that future development must move in a more sustainable direction. Currently available biofuels or renewably produced chemical, such as bioethanol or biodiesel, are produced from microbial fermentation of sugar-rich crops or by chemical conversion of natural oils or fats. However, these strategies are not sustainable in the long run as fuel and chemical production competes with food supply and arable land usage. Instead of relying on photosynthetic feedstocks that require further conversion, one can engineer photosynthetic cyanobacteria to produce a product of interest directly from CO2 and sunlight. The first part of this thesis aimed to develop new synthetic biology tools for the model cyanobacteria Synechocystis sp. PCC 6803. The second part of the thesis focused on evaluating the regulation of fatty acid synthesis in cyanobacteria, and the production of fatty acid-derived chemicals in Synechocystis.

    In paper I, fusion of small affinity proteins (Affibodies) to the major type IV pili protein was shown to mediate successful surface display of the affibody. This surface display strategy was further shown to allow inter-species binding between Synechocystis and Escherichia coli or Staphylococcus carnosus displaying complementary polymerizing affibodies.

    In paper II, a CRISPR-interference tool was successfully implemented in Synechocystis for inducible gene repression. Further, its multiplexing ability was proven by simultaneous repression of up to four aldehyde reductase/dehydrogenase genes. In paper III, this established CRISPRi tool was used to target and repress native pathways competing with heterologous fatty alcohol production in Synechocystis. Repressing the gene encoding the PlsX phosphate acyltransferase allowed re-direction of carbon-flux from membrane lipids to fatty alcohol production, with a final best strain producing 10.4 mg g-1 DCW octadecanol and hexadecanol.

    In paper IV, the transcriptional response towards perturbations within the fatty acid synthesis pathway was evaluated for the two model cyanobacteria Synechocystis and Synechococcus elongatus PCC 7942. Preliminary results indicate that blocking fatty acid synthesis initiation/elongation causes a transcriptional response of the involved pathway genes only in S. elongatus PCC 7942, indicating differential transcriptional responses in these two strains.

    In paper V, metagenomically sourced aldehyde deformylating oxygenase (Ado) variants were evaluated for their alka(e)ne synthesizing ability. Several of these novel Ado enzymes outperformed the generally well-performing Ado from S. elongatus when relating alka(e)ne production to the soluble enzyme amount.

  • 29.
    Cengic, Ivana
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Asplund-Samuelsson, Johannes
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Hudson, Elton P.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Indications of differential transcriptional regulation of fatty acid synthesis in the model cyanobacteria Synechocystis sp. Strain PCC 6803 and Synechococcus elongatus PCC 7942Manuscript (preprint) (Other academic)
  • 30.
    Cengic, Ivana
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Sundh, John
    Ininbergs, Karolina
    Bergman, Birgitta
    Hudson, Elton Paul
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Evaluation of Baltic Sea metagenome-derived aldehyde deformylating oxygenases in Synechocystis sp. strain PCC 6803Manuscript (preprint) (Other academic)
  • 31.
    Charitou, Theodosia
    et al.
    South Australian Hlth & Med Res Inst, EMBL Australia Grp, Adelaide, SA 5000, Australia..
    Srihari, Sriganesh
    South Australian Hlth & Med Res Inst, EMBL Australia Grp, Adelaide, SA 5000, Australia..
    Lynn, Miriam A.
    South Australian Hlth & Med Res Inst, EMBL Australia Grp, Adelaide, SA 5000, Australia..
    Jarboui, Mohamed-Ali
    Univ Tubingen, Inst Ophthalm Res, Tubingen, Germany.;Univ Tubingen, Werner Siemens Imaging Ctr, Tubingen, Germany..
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Moldovan, Max
    South Australian Hlth & Med Res Inst, EMBL Australia Grp, Adelaide, SA 5000, Australia..
    Shirasawa, Senji
    Fukuoka Univ, Fac Med, Fukuoka, Fukuoka 8140133, Japan..
    Tsunoda, Toshiyuki
    Fukuoka Univ, Fac Med, Fukuoka, Fukuoka 8140133, Japan..
    Ueffing, Marius
    Univ Tubingen, Inst Ophthalm Res, Tubingen, Germany..
    Xie, Jianling
    South Australian Hlth & Med Res Inst, Nutr Diabet & Metab, Adelaide, SA 5000, Australia..
    Xin, Jin
    South Australian Hlth & Med Res Inst, Nutr Diabet & Metab, Adelaide, SA 5000, Australia..
    Wang, Xuemin
    South Australian Hlth & Med Res Inst, Nutr Diabet & Metab, Adelaide, SA 5000, Australia.;Univ Adelaide, Sch Biol Sci, Adelaide, SA 5000, Australia..
    Proud, Christopher G.
    South Australian Hlth & Med Res Inst, Nutr Diabet & Metab, Adelaide, SA 5000, Australia.;Univ Adelaide, Sch Biol Sci, Adelaide, SA 5000, Australia..
    Boldt, Karsten
    Univ Tubingen, Inst Ophthalm Res, Tubingen, Germany..
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Kolch, Walter
    Univ Coll Dublin, Syst Biol Ireland, Dublin, Ireland.;Univ Coll Dublin, Sch Med, Dublin, Ireland.;Univ Coll Dublin, Conway Inst, Dublin, Ireland..
    Lynn, David J.
    South Australian Hlth & Med Res Inst, EMBL Australia Grp, Adelaide, SA 5000, Australia.;Flinders Univ S Australia, Coll Med & Publ Hlth, Sch Med, Bedford Pk, SA 5042, Australia..
    Transcriptional and metabolic rewiring of colorectal cancer cells expressing the oncogenic KRAS(G13D) mutation2019In: British Journal of Cancer, ISSN 0007-0920, E-ISSN 1532-1827, Vol. 121, no 1, p. 37-50Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Activating mutations in KRAS frequently occur in colorectal cancer (CRC) patients, leading to resistance to EGFRtargeted therapies. METHODS: To better understand the cellular reprogramming which occurs in mutant KRAS cells, we have undertaken a systems-level analysis of four CRC cell lines which express either wild type (wt) KRAS or the oncogenic KRAS(G13D) allele (mtKRAS). RESULTS: RNAseq revealed that genes involved in ribosome biogenesis, mRNA translation and metabolism were significantly upregulated in mtKRAS cells. Consistent with the transcriptional data, protein synthesis and cell proliferation were significantly higher in the mtKRAS cells. Targeted metabolomics analysis also confirmed the metabolic reprogramming in mtKRAS cells. Interestingly, mtKRAS cells were highly transcriptionally responsive to EGFR activation by TGF alpha stimulation, which was associated with an unexpected downregulation of genes involved in a range of anabolic processes. While TGF alpha treatment strongly activated protein synthesis in wtKRAS cells, protein synthesis was not activated above basal levels in the TGF alpha-treated mtKRAS cells. This was likely due to the defective activation of the mTORC1 and other pathways by TGF alpha in mtKRAS cells, which was associated with impaired activation of PKB signalling and a transient induction of AMPK signalling. CONCLUSIONS: We have found that mtKRAS cells are substantially rewired at the transcriptional, translational and metabolic levels and that this rewiring may reveal new vulnerabilities in oncogenic KRAS CRC cells that could be exploited in future.

  • 32. Chen, Ziqing
    et al.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tao, Sheng-ce
    Current applications of antibody microarrays2018In: Clinical Proteomics, ISSN 1542-6416, E-ISSN 1559-0275, Vol. 15, article id 7Article, review/survey (Refereed)
    Abstract [en]

    The concept of antibody microarrays is one of the most versatile approaches within multiplexed immunoassay technologies. These types of arrays have increasingly become an attractive tool for the exploratory detection and study of protein abundance, function, pathways, and potential drug targets. Due to the properties of the antibody microarrays and their potential use in basic research and clinical analytics, various types of antibody microarrays have already been developed. In spite of the growing number of studies utilizing this technique, few reviews about antibody microarray technology have been presented to reflect the quality and future uses of the generated data. In this review, we provide a summary of the recent applications of antibody microarray techniques in basic biology and clinical studies, providing insights into the current trends and future of protein analysis.

  • 33.
    Chouhan, Dimple
    et al.
    Indian Inst Technol Guwahati, Dept Biosci & Bioengn, Biomat & Tissue Engn Lab, Gauhati 781039, Assam, India..
    Das, Piyali
    West Bengal Univ Anim & Fishery Sci, Dept Vet Surg & Radiol, Kolkata 700037, W Bengal, India..
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Nandi, Samit K.
    West Bengal Univ Anim & Fishery Sci, Dept Vet Surg & Radiol, Kolkata 700037, W Bengal, India..
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Mandal, Biman B.
    Indian Inst Technol Guwahati, Dept Biosci & Bioengn, Biomat & Tissue Engn Lab, Gauhati 781039, Assam, India..
    Silkworm Silk Matrices Coated with Functionalized Spider Silk Accelerate Healing of Diabetic Wounds2019In: ACS Biomaterials Science and Engineering, ISSN 2373-9878, Vol. 5, no 7, p. 3537-3548Article in journal (Refereed)
    Abstract [en]

    Complex cutaneous wounds like diabetic foot ulcers represent a critical clinical challenge and demand a large-scale and low-cost strategy for effective treatment. Herein, we use a rabbit animal model to investigate efficacy of bioactive wound dressings made up of silk biomaterials. Nanofibrous mats of Antheraea assama silkworm silk fibroin (AaSF) are coated with various recombinant spider silk fusion proteins through silk-silk interactions to fabricate multifunctional wound dressings. Two different types of spider silk coatings are used to compare their healing efficiency: FN-4RepCT (contains a cell binding motif derived from fibronectin) and Lac-4RepCT (contains a cationic antimicrobial peptide from lactoferricin). AaSF mats coated with spider silk show accelerated wound healing properties in comparison to the uncoated mats. Among the spider silk coated variants, dual coating of FN-4RepCT and Lac-4RepCT on top of AaSF mat demonstrated better wound healing efficiency, followed by FN-4RepCT and Lac-4RepCT single coated counterparts. The in vivo study also reveals excellent skin regeneration by the functionalized silk dressings in comparison to commercially used Duoderm dressing and untreated wounds. The spider silk coatings demonstrate early granulation tissue development, re-epithelialization, and efficient matrix remodelling of wounds. The results thus validate potential of bioactive silk matrices in faster repair of diabetic wounds.

  • 34. Chouhan, Dimple
    et al.
    Thatikonda, Naresh
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Nilebäck, Linnea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Engineering.
    Widhe, Mona
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Hedhammar, My
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Protein Technology.
    Mandal, Biman B.
    Recombinant Spider Silk Functionalized Silkworm Silk Matrices as Potential Bioactive Wound Dressings and Skin Grafts2018In: ACS Applied Materials and Interfaces, ISSN 1944-8244, E-ISSN 1944-8252, Vol. 10, no 28, p. 23560-23572Article in journal (Refereed)
    Abstract [en]

    Silk is considered to be a potential biomaterial for a wide number of biomedical applications. Silk fibroin (SF) can be retrieved in sufficient quantities from the cocoons produced by silkworms. While it is easy to formulate into scaffolds with favorable mechanical properties, the natural SF does not contain bioactive functions. Spider silk proteins, on the contrary, can be produced in fusion with bioactive protein domains, but the recombinant procedures are expensive, and large-scale production is challenging. We combine the two types of silk to fabricate affordable, functional tissue-engineered constructs for wound-healing applications. Nanofibrous mats and microporous scaffolds made of natural silkworm SF are used as a bulk material that are top-coated with the recombinant spider silk protein (4RepCT) in fusion with a cell-binding motif, antimicrobial peptides, and a growth factor. For this, the inherent silk properties are utilized to form interactions between the two silk types by self-assembly. The intended function, that is, improved cell adhesion, antimicrobial activity, and growth factor stimulation, could be demonstrated for the obtained functionalized silk mats. As a skin prototype, SF scaffolds coated with functionalized silk are cocultured with multiple cell types to demonstrate formation of a bilayered tissue construct with a keratinized epidermal layer under in vitro conditions. The encouraging results support this strategy of fabrication of an affordable bioactive SF-spider silk-based biomaterial for wound dressings and skin substitutes.

  • 35.
    Costeira-Paulo, Joana
    et al.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Gault, Joseph
    Univ Oxford, Dept Chem, Phys & Theoret Chem Lab, South Parks Rd, Oxford OX1 3QZ, England..
    Popova, Gergana
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Ladds, Marcus J. G. W.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    van Leeuwen, Ingeborg M. M.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden..
    Sarr, Medoune
    Karolinska Inst, Ctr Alzheimer Res, Dept Neurobiol Care Sci & Soc NVS, Div Neurogeriatr,Dept Neurobiol, S-14157 Huddinge, Sweden..
    Olsson, Anders
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Lane, David P.
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lain, Sonia
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Marklund, Erik G.
    Uppsala Univ, Dept Chem BMC, Box 576, S-75123 Uppsala, Sweden..
    Landreh, Michael
    Karolinska Inst, Dept Microbiol Tumour & Cell Biol, Nobels Vag 16, S-17177 Stockholm, Sweden.;Karolinska Inst, Sci Life Lab, Dept Microbiol Tumour & Cell Biol, Tomtebodavagen 23A, S-17165 Stockholm, Sweden..
    Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase2018In: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 25, no 3, p. 309-+Article in journal (Refereed)
    Abstract [en]

    The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among cofactor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

  • 36.
    Dahlsson Leitao, Charles
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Rinne, S. S.
    Mitran, B.
    Vorobyeva, A.
    Andersson, Ken Gösta
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Tolmachev, V.
    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, A.
    Molecular Design of HER3-Targeting Affibody Molecules: Influence of Chelator and Presence of HEHEHE-Tag on Biodistribution of 68 Ga-Labeled Tracers2019In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, no 5Article in journal (Refereed)
    Abstract [en]

    Affibody-based imaging of HER3 is a promising approach for patient stratification. We investigated the influence of a hydrophilic HEHEHE-tag ((HE)₃-tag) and two different gallium-68/chelator-complexes on the biodistribution of Z08698 with the aim to improve the tracer for PET imaging. Affibody molecules (HE)₃-Z08698-X and Z08698-X (X = NOTA, NODAGA) were produced and labeled with gallium-68. Binding specificity and cellular processing were studied in HER3-expressing human cancer cell lines BxPC-3 and DU145. Biodistribution was studied 3 h p.i. in Balb/c nu/nu mice bearing BxPC-3 xenografts. Mice were imaged 3 h p.i. using microPET/CT. Conjugates were stably labeled with gallium-68 and bound specifically to HER3 in vitro and in vivo. Association to cells was rapid but internalization was slow. Uptake in tissues, including tumors, was lower for (HE)₃-Z08698-X than for non-tagged variants. The neutral [68Ga]Ga-NODAGA complex reduced the hepatic uptake of Z08698 compared to positively charged [68Ga]Ga-NOTA-conjugated variants. The influence of the chelator was more pronounced in variants without (HE)3-tag. In conclusion, hydrophilic (HE)₃-tag and neutral charge of the [68Ga]Ga-NODAGA complex promoted blood clearance and lowered hepatic uptake of Z08698. [68Ga]Ga-(HE)₃-Z08698-NODAGA was considered most promising, providing the lowest blood and hepatic uptake and the best imaging contrast among the tested variants.

  • 37.
    Danielsson, Frida
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Cellular and Clinical Proteomics. Royal Inst Technol, Sci Life Lab, S-17165 Stockholm, Sweden..
    Peterson, McKenzie Kirsten
    Univ Utah, Sch Med, Dept Pathol, Salt Lake City, UT 84112 USA..
    Araujo, Helena Caldeira
    Univ Madeira, Ctr Quim, P-9020105 Funchal, Portugal..
    Lautenschlaeger, Franziska
    Saarland Univ, Leibniz Inst Neue Mat gGmbH INM & Expt Phys, NT Fac, Campus D2 2,E 2 6, D-66123 Saarbrucken, Germany..
    Britt Gad, Annica Karin
    Univ Madeira, Ctr Quim, P-9020105 Funchal, Portugal.;Uppsala Univ, Dept Med Biochem & Microbiol, S-75237 Uppsala, Sweden..
    Vimentin Diversity in Health and Disease2018In: CELLS, ISSN 2073-4409, Vol. 7, no 10, article id 147Article, review/survey (Refereed)
    Abstract [en]

    Vimentin is a protein that has been linked to a large variety of pathophysiological conditions, including cataracts, Crohn's disease, rheumatoid arthritis, HIV and cancer. Vimentin has also been shown to regulate a wide spectrum of basic cellular functions. In cells, vimentin assembles into a network of filaments that spans the cytoplasm. It can also be found in smaller, non-filamentous forms that can localise both within cells and within the extracellular microenvironment. The vimentin structure can be altered by subunit exchange, cleavage into different sizes, re-annealing, post-translational modifications and interacting proteins. Together with the observation that different domains of vimentin might have evolved under different selection pressures that defined distinct biological functions for different parts of the protein, the many diverse variants of vimentin might be the cause of its functional diversity. A number of review articles have focussed on the biology and medical aspects of intermediate filament proteins without particular commitment to vimentin, and other reviews have focussed on intermediate filaments in an in vitro context. In contrast, the present review focusses almost exclusively on vimentin, and covers both ex vivo and in vivo data from tissue culture and from living organisms, including a summary of the many phenotypes of vimentin knockout animals. Our aim is to provide a comprehensive overview of the current understanding of the many diverse aspects of vimentin, from biochemical, mechanical, cellular, systems biology and medical perspectives.

  • 38. Deyev, S.
    et al.
    Vorobyeva, A.
    Schulga, A.
    Proshkina, G.
    Güler, Rezan
    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.
    Mitran, B.
    Garousi, J.
    Altai, M.
    Buijs, J.
    Chernov, V.
    Orlova, A.
    Tolmachev, V.
    Comparative Evaluation of Two DARPin Variants: Effect of Affinity, Size, and Label on Tumor Targeting Properties2019In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 16, no 3, p. 995-1008Article in journal (Refereed)
    Abstract [en]

    Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins that can be selected for binding to desirable molecular targets. High affinity and small size of DARPins render them promising probes for radionuclide molecular imaging. However, detailed knowledge on many factors influencing their imaging properties is still lacking. We have evaluated two human epidermal growth factor 2 (HER2)-specific DARPins with different size and binding properties. DARPins 9-29-H 6 and G3-H 6 were radiolabeled with iodine-125 and tricarbonyl technetium-99m and evaluated in vitro. A side-by-side comparison of biodistribution and tumor targeting was performed. HER2-specific tumor accumulation of G3-H 6 was demonstrated. A combination of smaller size and higher affinity resulted in a higher tumor uptake of G3-H 6 in comparison to 9-29-H 6 . Technetium-99m labeled G3-H 6 demonstrated a better biodistribution profile than 9-29-H 6 , with several-fold lower uptake in liver. Radioiodinated G3-H 6 showed the best tumor-to-organ ratios. The combined effect of affinity, molecular weight, scaffold composition, and nonresidualizing properties of iodine label provided radioiodinated G3-H 6 with high clinical potential for imaging of HER2.

  • 39. Djureinovic, D.
    et al.
    Dodig-Crnkovic, Tea
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hellström, Cecilia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Holgersson, G.
    Bergqvist, M.
    Mattsson, J. S. M.
    Pontén, F.
    Ståhle, E.
    Schwenk, Jochen M.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Micke, P.
    Detection of autoantibodies against cancer-testis antigens in non-small cell lung cancer2018In: Lung Cancer, ISSN 0169-5002, E-ISSN 1872-8332, Vol. 125, p. 157-163Article in journal (Refereed)
    Abstract [en]

    Objectives: Cancer-testis antigens (CTAs) are defined as proteins that are specifically expressed in testis or placenta and their expression is frequently activated in cancer. Due to their ability to induce an immune response, CTAs may serve as suitable targets for immunotherapy. The aim of this study was to evaluate if there is reactivity against CTAs in the plasma of non-small cell lung cancer (NSCLC) patients through the detection of circulating antibodies. Materials and methods: To comprehensively analyze autoantibodies against CTAs the multiplexing capacities of suspension bead array technology was used. Bead arrays were created with 120 protein fragments, representing 112 CTAs. Reactivity profiles were measured in plasma samples from 133 NSCLC patients and 57 cases with benign lung diseases. Results: Altogether reactivity against 69 antigens, representing 81 CTAs, was demonstrated in at least one of the analyzed samples. Twenty-nine of the antigens (45 CTAs) demonstrated exclusive reactivity in NSCLC samples. Reactivity against cancer-testis antigen family 47; member A (CT47A) genes, P antigen family member 3 (PAGE3), variable charge X-linked (VCX), melanoma antigen family B1 (MAGEB1), lin-28 homolog B (LIN28B) and chromosome 12 open reading frame 54 (C12orf54) were only found in NSCLC patients at a frequency of 1%–4%. The presence of autoantibodies towards these six antigens was confirmed in an independent group of 34 NSCLC patients. Conclusion: We identified autoantibodies against CTAs in the plasma of lung cancer patients. The reactivity pattern of autoantibodies was higher in cancer patients compared to the benign group, stable over time, but low in frequency of occurrence. The findings suggest that some CTAs are immunogenic and that these properties can be utilized as immune targets. 

  • 40.
    Drobin, Kimi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Antibody-based bead arrays for high-throughput protein profiling in human plasma and serum2018Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    Affinity-based proteomics utilizes affinity binders to detect target proteins in a large-scale manner. This thesis describes a high-throughput method, which enables the search for biomarker candidates in human plasma and serum. A highly multiplexed antibody-based suspension bead array is created by coupling antibodies generated in the Human Protein Atlas project to color-coded beads. The beads are combined for parallel analysis of up to 384 analytes in patient and control samples. This provides data to compare protein levels from the different groups.

    In paper I osteoporosis patients are compared to healthy individuals to find disease-linked proteins. An untargeted discovery screening was conducted using 4608 antibodies in 16 cases and 6 controls. This revealed 72 unique proteins, which appeared differentially abundant. A validation screening of 91 cases and 89 controls confirmed that the protein autocrine motility factor receptor (AMFR) is decreased in the osteoporosis patients.

    Paper II investigates the risk proteome of inflammatory bowel disease (IBD). Antibodies targeting 209 proteins corresponding to 163 IBD genetic risk loci were selected. To find proteins related to IBD or its subgroups, sera from 49 patients with Crohn’s disease, 51 with ulcerative colitis and 50 matched controls were analyzed. From these targeted assays, the known inflammation-related marker serum amyloid protein A (SAA) was shown to be elevated in the IBD cases. In addition, the protein laccase (multi-copper oxidoreductase) domain containing 1 (LACC1) was found to be decreased in the IBD subjects.

    In conclusion, assays using affinity-based bead arrays were developed and applied to screen human plasma and serum samples in two disease contexts. Untargeted and targeted screening strategies were applied to discover disease-associated proteins. Upon further validation, these potential biomarker candidates could be valuable in future disease studies.

  • 41.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics.
    Assadi, Ghazaleh
    Hong, Mun-Gwan
    Andersson, Eni
    Fredolini, Claudia
    Forsström, Björn
    Reznichenko, Anna
    Akhter, Tahmina
    Ek, Weronica
    Bonfiglio, Ferdinando
    Berner Hansen, Mark
    Sandberg, Kristian
    Greco, Dario
    Repsilber, Dirk
    Schwenk, Jochen
    D'Amato, Mauro
    Halfvarson, Jonas
    Targeted analysis of serum proteins encoded at known inflammatory bowel disease risk lociManuscript (preprint) (Other academic)
  • 42.
    Drobin, Kimi
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Assadi, Ghazaleh
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Hong, Mun-Gwan
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Anggraeni Andersson, Margaretha
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab. Royal Inst Technol, KTH, Sch Biotechnol, Affin Prote,SciLifeLab, Stockholm, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Reznichenko, Anna
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Akhter, Tahmina
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden..
    Ek, Weronica E.
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;Uppsala Univ, Sci Life Lab, Dept Immunol Genet & Pathol, Uppsala, Sweden..
    Bonfiglio, Ferdinando
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;Biodonostia Hlth Res Inst, Dept Gastrointestinal & Liver Dis, San Sebastian, Spain..
    Hansen, Mark Berner
    AstraZeneca R&D, Innovat & Global Med, Molndal, Sweden.;Univ Copenhagen, Bispebjerg Hosp, Ctr Digest Dis, Copenhagen, Denmark..
    Sandberg, Kristian
    Uppsala Univ, Sci Life Lab, Drug Discovery & Dev Platform, Uppsala, Sweden.;Uppsala Univ, Uppsala Biomed Ctr, Dept Med Chem, Organ Pharmaceut Chem, Uppsala, Sweden.;Karolinska Inst, Dept Physiol & Pharmacol, Stockholm, Sweden..
    Greco, Dario
    Univ Helsinki, Inst Biotechnol, Helsinki, Finland..
    Repsilber, Dirk
    Orebro Univ, Sch Med Sci, Orebro, Sweden..
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    D'Amato, Mauro
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;BioDonostia Hlth Res Inst, San Sebastian, Spain.;Ikerbasque, Basque Fdn Sci, Bilbao, Spain..
    Halfvarson, Jonas
    Orebro Univ, Fac Med & Hlth, Dept Gastroenterol, SE-70182 Orebro, Sweden..
    Targeted Analysis of Serum Proteins Encoded at Known Inflammatory Bowel Disease Risk Loci2019In: Inflammatory Bowel Diseases, ISSN 1078-0998, E-ISSN 1536-4844, Vol. 25, no 2, p. 306-316Article in journal (Refereed)
    Abstract [en]

    Few studies have investigated the blood proteome of inflammatory bowel disease (IBD). We characterized the serum abundance of proteins encoded at 163 known IBD risk loci and tested these proteins for their biomarker discovery potential. Based on the Human Protein Atlas (HPA) antibody availability, 218 proteins from genes mapping at 163 IBD risk loci were selected. Targeted serum protein profiles from 49 Crohns disease (CD) patients, 51 ulcerative colitis (UC) patients, and 50 sex- and age-matched healthy individuals were obtained using multiplexed antibody suspension bead array assays. Differences in relative serum abundance levels between disease groups and controls were examined. Replication was attempted for CD-UC comparisons (including disease subtypes) by including 64 additional patients (33 CD and 31 UC). Antibodies targeting a potentially novel risk protein were validated by paired antibodies, Western blot, immuno-capture mass spectrometry, and epitope mapping. By univariate analysis, 13 proteins mostly related to neutrophil, T-cell, and B-cell activation and function were differentially expressed in IBD patients vs healthy controls, 3 in CD patients vs healthy controls and 2 in UC patients vs healthy controls (q < 0.01). Multivariate analyses further differentiated disease groups from healthy controls and CD subtypes from UC (P < 0.05). Extended characterization of an antibody targeting a novel, discriminative serum marker, the laccase (multicopper oxidoreductase) domain containing 1 (LACC1) protein, provided evidence for antibody on-target specificity. Using affinity proteomics, we identified a set of IBD-associated serum proteins encoded at IBD risk loci. These candidate proteins hold the potential to be exploited as diagnostic biomarkers of IBD.

  • 43.
    Edfors, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Vunk, Helian
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Kotol, David
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fredolini, Claudia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svensson, Anne-Sophie
    KTH.
    Boström, Tove
    KTH.
    Tegel, Hanna
    KTH.
    Nilsson, Peter
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Schwenk, Jochen M.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Affinity Proteomics. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology. KTH, Centres, Science for Life Laboratory, SciLifeLab. Karolinska Inst, Dept Neurosci, SE-17165 Solna, Sweden.;Tech Univ Denmark, Novo Nordisk Fdn Ctr Biosustainabil, DK-2970 Horsholm, Denmark..
    Screening a Resource of Recombinant Protein Fragments for Targeted Proteomics2019In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 7, p. 2706-2718Article in journal (Refereed)
    Abstract [en]

    The availability of proteomics resources hosting protein and peptide standards, as well as the data describing their analytical performances, will continue to enhance our current capabilities to develop targeted proteomics methods for quantitative biology. This study describes the analysis of a resource of 26,840 individually purified recombinant protein fragments corresponding to more than 16,000 human protein-coding genes. The resource was screened to identify proteotypic peptides suitable for targeted proteomics efforts, and we report LC-MS/MS assay coordinates for more than 25,000 proteotypic peptides, corresponding to more than 10,000 unique proteins. Additionally, peptide formation and digestion kinetics were, for a subset of the standards, monitored using a time-course protocol involving parallel digestion of isotope-labeled recombinant protein standards and endogenous human plasma proteins. We show that the strategy by adding isotope-labeled recombinant proteins before trypsin digestion enables short digestion protocols (<= 60 min) with robust quantitative precision. In a proof-of-concept study, we quantified 23 proteins in human plasma using assay parameters defined in our study and used the standards to describe distinct clusters of individuals linked to different levels of LPA, APOE, SERPINAS, and TFRC. In summary, we describe the use and utility of a resource of recombinant proteins to identify proteotypic peptides useful for targeted proteomics assay development.

  • 44.
    Edfors, Fredrik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Andreas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Linderbäck, Klas
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Maddalo, Gianluca
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Azimi, Alireza
    Karolinska Inst, Karolinska Univ Hosp, Dept Oncol Pathol, SE-17177 Stockholm, Sweden..
    Sivertsson, Åsa
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Tegel, Hanna
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Hober, Sophia
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Fagerberg, Linn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    von Feilitzen, Kalle
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Oksvold, Per
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Lindskog, Cecilia
    Uppsala Univ, Dept Immunol Genet & Pathol, SE-75185 Uppsala, Sweden..
    Forsström, Björn
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science. KTH, Centres, Science for Life Laboratory, SciLifeLab. Biosustainabil, DK-2970 Horsholm, Denmark..
    Enhanced validation of antibodies for research applications2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 4130Article in journal (Refereed)
    Abstract [en]

    There is a need for standardized validation methods for antibody specificity and selectivity. Recently, five alternative validation pillars were proposed to explore the specificity of research antibodies using methods with no need for prior knowledge about the protein target. Here, we show that these principles can be used in a streamlined manner for enhanced validation of research antibodies in Western blot applications. More than 6,000 antibodies were validated with at least one of these strategies involving orthogonal methods, genetic knockdown, recombinant expression, independent antibodies, and capture mass spectrometry analysis. The results show a path forward for efforts to validate antibodies in an application-specific manner suitable for both providers and users.

  • 45.
    Faridi, Muhammad Asim
    et al.
    KTH.
    Shahzad, Adnan Faqui
    KTH.
    Russom, Aman
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Nano Biotechnology.
    Wiklund, Martin
    KTH, School of Engineering Sciences (SCI), Applied Physics, Biomedical and X-ray Physics.
    Milliliter scale acoustophoresis based bioparticle processing platform2018In: ASME 2018 16th International Conference on Nanochannels, Microchannels, and Minichannels, ICNMM 2018, ASME Press, 2018Conference paper (Refereed)
    Abstract [en]

    Bioparticles such as mammalian cells and bacteria can be manipulated directly or indirectly for multiple applications such as sample preparation for diagnostic applications mainly up-concentration, enrichment & separation as well as immunoassay development. There are various active and passive microfluidic particle manipulation techniques where Acoustophoresis is a powerful technique showing high cell viability. The use of disposable glass capillaries for acoustophoresis, instead of cleanroom fabricated glass-silicon chip can potentially bring down the cost factor substantially, aiding the realization of this technique for real-world diagnostic devices. Unlike available chips and capillary-based microfluidic devices, we report milliliter-scale platform able to accommodate 1ml of a sample for acoustophoresis based processing on a market available glass capillary. Although it is presented as a generic platform but as a demonstration we have shown that polystyrene suspending medium sample can be processed with trapping efficiency of 87% and the up-concentration factor of 10 times in a flow through manner i.e., at 35µl/min. For stationary volume accommodation, this platform practically offers 50 times more sample handling capacity than most of the microfluidic setups. Furthermore, we have also shown that with diluted blood (0.6%) in a flow-through manner, 82% of the white blood cells (WBCs) per ml could be kept trapped. This milliliter platform could potentially be utilized for assisting in sample preparation, plasma separation as well as a flow-through immunoassay assay development for clinical diagnostic applications.

  • 46.
    Fasterius, Erik
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    seqCAT: a Bioconductor R-package for variant analysis of high throughput sequencing dataManuscript (preprint) (Other academic)
  • 47.
    Fasterius, Erik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Al-Khalili Szigyarto, Cristina
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Analysis of public RNA-sequencing data reveals biological consequences of genetic heterogeneity in cell line populations2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 11226Article in journal (Refereed)
    Abstract [en]

    Meta-analysis of datasets available in public repositories are used to gather and summarise experiments performed across laboratories, as well as to explore consistency of scientific findings. As data quality and biological equivalency across samples may obscure such analyses and consequently their conclusions, we investigated the comparability of 85 public RNA-seq cell line datasets. Thousands of pairwise comparisons of single nucleotide variants in 139 samples revealed variable genetic heterogeneity of the eight cell line populations analysed as well as variable data quality. The H9 and HCT116 cell lines were found to be remarkably stable across laboratories (with median concordances of 99.2% and 98.5%, respectively), in contrast to the highly variable HeLa cells (89.3%). We show that the genetic heterogeneity encountered greatly affects gene expression between same-cell comparisons, highlighting the importance of interrogating the biological equivalency of samples when comparing experimental datasets. Both the number of differentially expressed genes and the expression levels negatively correlate with the genetic heterogeneity. Finally, we demonstrate how comparing genetically heterogeneous datasets affect gene expression analyses and that high dissimilarity between same-cell datasets alters the expression of more than 300 cancer-related genes, which are often the focus of studies using cell lines.

  • 48.
    Fasterius, Erik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Uhlén, Mathias
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Single cell RNA-seq variant analysis for exploration of inter- and intra-tumour genetic heterogeneityManuscript (preprint) (Other academic)
  • 49.
    Fasterius, Erik
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science, Systems Biology.
    Uhlén, Mathias
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Centres, Albanova VinnExcellence Center for Protein Technology, ProNova. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
    Al-Khalili Szigyarto, Cristina
    KTH, Superseded Departments (pre-2005), Biotechnology.
    Single-cell RNA-seq variant analysis for exploration of genetic heterogeneity in cancer2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 9524Article in journal (Refereed)
    Abstract [en]

    Inter-and intra-tumour heterogeneity is caused by genetic and non-genetic factors, leading to severe clinical implications. High-throughput sequencing technologies provide unprecedented tools to analyse DNA and RNA in single cells and explore both genetic heterogeneity and phenotypic variation between cells in tissues and tumours. Simultaneous analysis of both DNA and RNA in the same cell is, however, still in its infancy. We have thus developed a method to extract and analyse information regarding genetic heterogeneity that affects cellular biology from single-cell RNA-seq data. The method enables both comparisons and clustering of cells based on genetic variation in single nucleotide variants, revealing cellular subpopulations corroborated by gene expression-based methods. Furthermore, the results show that lymph node metastases have lower levels of genetic heterogeneity compared to their original tumours with respect to variants affecting protein function. The analysis also revealed three previously unknown variants common across cancer cells in glioblastoma patients. These results demonstrate the power and versatility of scRNA-seq variant analysis and highlight it as a useful complement to already existing methods, enabling simultaneous investigations of both gene expression and genetic variation.

  • 50. Fornell, Anna
    et al.
    Nilsson, Johan
    Jonsson, Linus
    Periyannan Rajeswari, Prem Kumar
    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.
    Tenje, Maria
    Particle enrichment in droplet acoustofluidics2016In: Micronano System Workshop (MSW 2016), Lund, Sweden, May 17-18 2016, 2016Conference paper (Refereed)
1234 1 - 50 of 183
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