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Syvänen, Stina
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Publications (10 of 41) Show all publications
Gustafsson, S., Lindström, V., Ingelsson, M., Hammarlund-Udenaes, M. & Syvänen, S. (2018). Intact blood-brain barrier transport of small molecular drugs in animal models of amyloid beta and alpha-synuclein pathology. Neuropharmacology, 128, 482-491
Open this publication in new window or tab >>Intact blood-brain barrier transport of small molecular drugs in animal models of amyloid beta and alpha-synuclein pathology
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2018 (English)In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 128, p. 482-491Article in journal (Refereed) Published
Abstract [en]

Pathophysiological impairment of the neurovascular unit, including the integrity and dynamics of the blood-brain barrier (BBB), has been denoted both a cause and consequence of neurodegenerative diseases. Pathological impact on BBB drug delivery has also been debated. The aim of the present study was to investigate BBB drug transport, by determining the unbound brain-to-plasma concentration ratio (K-p,K-uu,K-brain), in aged A beta PP-transgenic mice, alpha-synuclein transgenic mice, and wild type mice. Mice were dosed with a cassette of five compounds, including digoxin, levofloxacin (1 mg/kg, s.c.), paliperidone, oxycodone, and diazepam (0.25 mg/kg, s.c.). Brain and blood were collected at 0.5,1, or 3 h after dosage. Drug concentrations were measured using LC-MS/MS. The total brain-to-plasma concentration ratio was calculated and equilibrium dialysis was used to determine the fraction of unbound drug in brain and plasma for all compounds. Together, these three measures were used to determine the Kp,uu,brain value. Despite A beta or alpha-synuclein pathology in the current animal models, no difference was observed in the extent of drug transport across the BBB compared to wild type animals for any of the compounds investigated. Hence, the present study shows that the concept of a leaking barrier within neurodegenerative conditions has to be interpreted with caution when estimating drug transport into the brain. The capability of the highly dynamic BBB to regulate brain drug exposure still seems to be intact despite the presence of pathology. (C) 2017 The Authors. Published by Elsevier Ltd.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Blood-brain barrier, Pharmacokinetics, Drug transport, Disease, Amyloid beta, Alpha-synuclein
National Category
Neurology Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-340458 (URN)10.1016/j.neuropharm.2017.08.002 (DOI)000418977200043 ()28797721 (PubMedID)
Available from: 2018-02-15 Created: 2018-02-15 Last updated: 2018-03-27Bibliographically approved
Syvänen, S., Fang, X. T., Hultqvist, G., Meier, S. R., Lannfelt, L. & Sehlin, D. (2017). A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging. NeuroImage, 148, 55-63
Open this publication in new window or tab >>A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging
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2017 (English)In: NeuroImage, ISSN 1053-8119, E-ISSN 1095-9572, Vol. 148, p. 55-63Article in journal (Refereed) Published
Abstract [en]

Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the Tribody(TM) format, for PET imaging of soluble amyloid-beta (All) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the A beta protofibril selective antibody mAb158. Five Tribody proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and All protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human All. The systemic pharmacokinetics were similar with half-lives in blood of around 9 h for all three ligands. Brain concentrations at 2 h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72 h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for Al, A2 and A3 respectively), except in the cerebellum, an area largely devoid of A beta pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with A beta pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017
Keywords
PET, Amyloid-beta, Antibody, Transferrin receptor, Tribody
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-320289 (URN)10.1016/j.neuroimage.2017.01.004 (DOI)000396803100006 ()28069541 (PubMedID)
Funder
Swedish Research Council, 2012-1593The Swedish Brain FoundationTorsten Söderbergs stiftelse
Available from: 2017-04-25 Created: 2017-04-25 Last updated: 2017-04-25Bibliographically approved
Fang, X., Hultqvist, G., Meier, S., Sehlin, D. & Syvänen, S. (2017). A small bispecific antibody-based construct based on bapineuzumab as a PET tracer for amyloid beta pathology in brain. Paper presented at 28th International Symposium on Cerebral Blood Flow, Metabolism and Function / 13th International Conference on Quantification of Brain Function with PET, APR 01-04, 2017, Int Soc Cerebral Blood Flow & Metab, Berlin, GERMANY. Meeting abstract
Open this publication in new window or tab >>A small bispecific antibody-based construct based on bapineuzumab as a PET tracer for amyloid beta pathology in brain
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2017 (English)In: Meeting abstractArticle in journal, Meeting abstract (Other academic) Published
National Category
Endocrinology and Diabetes Hematology Neurology
Identifiers
urn:nbn:se:uu:diva-331029 (URN)000400157400102 ()
Conference
28th International Symposium on Cerebral Blood Flow, Metabolism and Function / 13th International Conference on Quantification of Brain Function with PET, APR 01-04, 2017, Int Soc Cerebral Blood Flow & Metab, Berlin, GERMANY
Note

Supplement: 1, Meeting Abstract: BPS01-1

Available from: 2017-10-11 Created: 2017-10-11 Last updated: 2017-11-08
Syvänen, S., Fang, X. T., Hultqvist, G., Falting, J., Antoni, G., Lannfelt, L. & Sehlin, D. (2017). Antibody-based PET radioligands for imaging of amyloid-beta protofibrils. Paper presented at 28th International Symposium on Cerebral Blood Flow, Metabolism and Function / 13th International Conference on Quantification of Brain Function with PET, APR 01-04, 2017, Int Soc Cerebral Blood Flow & Metab, Berlin, GERMANY. Journal of Cerebral Blood Flow and Metabolism, 37, 84-84
Open this publication in new window or tab >>Antibody-based PET radioligands for imaging of amyloid-beta protofibrils
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2017 (English)In: Journal of Cerebral Blood Flow and Metabolism, ISSN 0271-678X, E-ISSN 1559-7016, Vol. 37, p. 84-84Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
Sage Publications, 2017
National Category
Endocrinology and Diabetes Hematology Neurology
Identifiers
urn:nbn:se:uu:diva-331033 (URN)000400157400120 ()
Conference
28th International Symposium on Cerebral Blood Flow, Metabolism and Function / 13th International Conference on Quantification of Brain Function with PET, APR 01-04, 2017, Int Soc Cerebral Blood Flow & Metab, Berlin, GERMANY
Note

Supplement: 1, Meeting Abstract: BPS04-1.

Available from: 2017-10-11 Created: 2017-10-11 Last updated: 2017-10-11
Hultqvist, G., Syvänen, S., Fang, X. T., Lannfelt, L. & Sehlin, D. (2017). Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor. Theranostics, 7(2), 308-318
Open this publication in new window or tab >>Bivalent Brain Shuttle Increases Antibody Uptake by Monovalent Binding to the Transferrin Receptor
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2017 (English)In: Theranostics, ISSN 1838-7640, E-ISSN 1838-7640, Vol. 7, no 2, p. 308-318Article in journal (Refereed) Published
Abstract [en]

The blood-brain barrier (BBB) is an obstacle for antibody passage into the brain, impeding the development of immunotherapy and antibody-based diagnostics for brain disorders. In the present study, we have developed a brain shuttle for active transport of antibodies across the BBB by receptor-mediated transcytosis. We have thus recombinantly fused two single-chain variable fragments (scFv) of the transferrin receptor (TfR) antibody 8D3 to the light chains of mAb158, an antibody selectively binding to A beta protofibrils, which are involved in the pathogenesis of Alzheimer's disease (AD). Despite the two TfR binders, a monovalent interaction with TfR was achieved due to the short linkers that sterically hinder bivalent binding to the TfR dimer. The design enabled efficient receptor-mediated brain uptake of the fusion protein. Two hours after administration, brain concentrations were 2-3% of the injected dose per gram brain, comparable to small molecular drugs and 80-fold higher than unmodified mAb158. After three days, fusion protein concentrations in AD transgenic mouse brains were 9-fold higher than in wild type mice, demonstrating high in vivo specificity. Thus, our innovative recombinant design markedly increases mAb158 brain uptake, which makes it a strong candidate for improved Aa immunotherapy and as a PET radioligand for early diagnosis and evaluation of treatment effect in AD. Moreover, this approach could be applied to any target within the brain.

Place, publisher, year, edition, pages
IVYSPRING INT PUBL, 2017
Keywords
BBB shuttle, TfR, antibodies, Alzheimer's disease, immunotherapy, PET
National Category
Geriatrics
Identifiers
urn:nbn:se:uu:diva-319779 (URN)10.7150/thno.17155 (DOI)000396555900006 ()28042336 (PubMedID)
Funder
Swedish Research Council, 2012-1593 2012-2172The Swedish Brain FoundationStiftelsen Gamla TjänarinnorMagnus Bergvall Foundation
Available from: 2017-04-12 Created: 2017-04-12 Last updated: 2017-11-29Bibliographically approved
Fang, X. T., Eriksson, J., Antoni, G., Yngve, U., Cato, L., Lannfelt, L., . . . Syvänen, S. (2017). Brain mGluR5 in mice with amyloid beta pathology studied with in vivo [(11)C]ABP688 PET imaging and ex vivo immunoblotting. Neuropharmacology, 113(Pt A), 293-300, Article ID S0028-3908(16)30459-2.
Open this publication in new window or tab >>Brain mGluR5 in mice with amyloid beta pathology studied with in vivo [(11)C]ABP688 PET imaging and ex vivo immunoblotting
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2017 (English)In: Neuropharmacology, ISSN 0028-3908, E-ISSN 1873-7064, Vol. 113, no Pt A, p. 293-300, article id S0028-3908(16)30459-2Article in journal (Refereed) Published
Abstract [en]

Alzheimer's disease (AD) is characterized by aggregation of amyloid beta (Aβ) into insoluble plaques. Intermediates, Aβ oligomers (Aβo), appear to be the mechanistic cause of disease. The de facto PET AD ligand, [(11)C]PIB, binds and visualizes Aβ plaque load, which does not correlate well with disease severity. Therefore, finding a dynamic target that changes with pathology progression in AD is of great interest. Aβo alter synaptic plasticity, inhibit long-term potentiation, and facilitate long-term depression; key mechanisms involved in memory and learning. In order to convey these neurotoxic effects, Aβo requires interaction with the metabotropic glutamate 5 receptor (mGluR5). The aim was to investigate in vivo mGluR5 changes in an Aβ pathology model using PET. Wild type C57/BL6 (wt) and AβPP transgenic mice (tg-ArcSwe), 4, 8, and 16 months old, were PET scanned with [(11)C]ABP688, which is highly specific to mGluR5, to investigate changes in mGluR5. Mouse brains were extracted postscan and mGluR5 and Aβ protofibril levels were assessed with immunoblotting and ELISA respectively. Receptor-dense brain regions (hippocampus, thalamus, and striatum) displayed higher [(11)C]ABP688 concentrations corresponding to mGluR5 expression pattern. Mice had similar uptake levels of [(11)C]ABP688 regardless of genotype or age. Immunoblotting revealed general decline in mGluR5 expression and elevated levels of mGluR5 in 16 months old tg-ArcSwe compared with wt mice. [(11)C]ABP688 could visualize mGluR5 in the mouse brain. In conclusion, mGluR5 levels were found to decrease with age and tended to be higher in tg-ArcSwe compared with wt mice, however these changes could not be quantified with PET.

Keywords
Alzheimer's disease, PET, [(11)C]ABP688, mGluR5
National Category
Geriatrics Radiology, Nuclear Medicine and Medical Imaging Neurology Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-317721 (URN)10.1016/j.neuropharm.2016.10.009 (DOI)000390502200028 ()27743932 (PubMedID)
Available from: 2017-03-17 Created: 2017-03-17 Last updated: 2018-01-13Bibliographically approved
Syvänen, S., Edén, D. & Sehlin, D. (2017). Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment. Biochemical and Biophysical Research Communications - BBRC, 493(1), 120-125
Open this publication in new window or tab >>Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment
2017 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 493, no 1, p. 120-125Article in journal (Refereed) Published
Abstract [en]

Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to different extents and the specific and unspecific binding was studied in vitro. Next, cationized F(ab')2-h158 was labelled with iodine-125 and its brain distribution and pharmacokinetics was studied in mice. Cationization did not alter the in vitro affinity to A beta protofibrils, but increased the unspecific binding somewhat. Ex vivo experiments revealed a doubling of brain concentrations compared with unmodified F(ab')2-h158 and in vivo imaging with single photon emission computed tomography (SPECT) showed that the cationized F(ab')2-h158, but not the unmodified F(ab')2-h158 could be visualized in the brain. To conclude, cationization is a means to increase brain concentrations of therapeutic antibodies or fragments and may facilitate the use of antibodies/fragments as imaging biomarkers in the brain.

Keywords
Cationization, Adsorptive-mediated transcytosis, Alzheimer's disease, Amyloid-beta protofibrils, Molecular imaging, Blood-brain barrier
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-339734 (URN)10.1016/j.bbrc.2017.09.065 (DOI)000413134200019 ()28919420 (PubMedID)
Funder
Swedish Research Council, 2012-1593
Available from: 2018-01-26 Created: 2018-01-26 Last updated: 2018-01-26Bibliographically approved
Gustafsson, S., Eriksson, J., Syvänen, S., Eriksson, O., Hammarlund-Udenaes, M. & Antoni, G. (2017). Combined PET and microdialysis for in vivo estimation of drug blood-brain barrier transport and brain unbound concentrations. NeuroImage, 155, 177-186
Open this publication in new window or tab >>Combined PET and microdialysis for in vivo estimation of drug blood-brain barrier transport and brain unbound concentrations
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2017 (English)In: NeuroImage, ISSN 1053-8119, E-ISSN 1095-9572, Vol. 155, p. 177-186Article in journal (Refereed) Published
Abstract [en]

Methods to investigate blood-brain barrier transport and pharmacologically active drug concentrations in the human brain are limited and data translation between species is challenging. Hence, there is a need to further develop the read-out of techniques like positron emission tomography ( PET) for studying neuropharmacokinetics. PET has a high translational applicability from rodents to man and measures total drug concentrations in vivo. The aim of the present study was to investigate the possibility of translating total drug concentrations, acquired through PET, to unbound concentrations, resembling those measured in the interstitial fluid by microdialysis sampling. Simultaneous PET scanning and brain microdialysis sampling were performed in rats throughout a 60 min infusion of [N-methyl-C-11] oxycodone in combination with a therapeutic dose of oxycodone and during a 60 min follow up period after the end of infusion. The oxycodone concentrations acquired with PET were converted into unbound concentrations by compensating for brain tissue binding and brain intracellular distribution, using the unbound volume of distribution in brain (Vu, brain), and were compared to microdialysis measurements of unbound concentrations. A good congruence between the methods was observed throughout the infusion. However, an accumulating divergence in the acquired PET and microdialysis data was apparent and became more pronounced during the elimination phase, most likely due to the passage of radioactive metabolites into the brain. In conclusion, the study showed that PET can be used to translate non-invasively measured total drug concentrations into unbound concentrations as long as the contribution of radiolabelled metabolites is minor or can be compensated for.

Keywords
Blood-brain barrier, Unbound concentration, Positron emission tomography, Microdialysis, Pharmacokinetics, Oxycodone
National Category
Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:uu:diva-332421 (URN)10.1016/j.neuroimage.2017.04.068 (DOI)000405460900015 ()28467891 (PubMedID)
Available from: 2017-11-02 Created: 2017-11-02 Last updated: 2018-03-27Bibliographically approved
Kaya, I., Brinet, D., Michno, W., Syvänen, S., Sehlin, D., Zetterberg, H., . . . Hanrieder, J. (2017). Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer's Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry. ACS Chemical Neuroscience, 8(2), 347-355
Open this publication in new window or tab >>Delineating Amyloid Plaque Associated Neuronal Sphingolipids in Transgenic Alzheimer's Disease Mice (tgArcSwe) Using MALDI Imaging Mass Spectrometry
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2017 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 8, no 2, p. 347-355Article in journal (Refereed) Published
Abstract [en]

The major pathological hallmarks of Alzheimer's disease (AD) are the progressive aggregation and accumulation of beta-amyloid (A beta) and hyperphosphorylated tau protein into neurotoxic deposits. A beta aggregation has been suggested as the critical early inducer, driving the disease progression. However, the factors that promote neurotoxic A beta aggregation remain elusive. Imaging mass spectrometry (IMS) is a powerful technique to comprehensively elucidate the spatial distribution patterns of lipids, peptides, and proteins in biological tissue sections. In the present study, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)-based imaging was used on transgenic Alzheimer's disease mouse (tgArcSwe) brain tissue to investigate the sphingolipid microenvironment of individual A beta plaques and elucidate plaque-associated sphingolipid alterations. Multivariate data analysis was used to interrogate the IMS data for identifying pathologically relevant, anatomical features based on their lipid chemical profile. This approach revealed sphingolipid species that distinctly located to cortical and hippocampal deposits, whose A beta identity was further verified using fluorescent amyloid staining and immunohistochemistry. Subsequent multivariate statistical analysis of the spectral data revealed significant localization of gangliosides and ceramides species to A beta positive plaques, which was accompanied by distinct local reduction of sulfatides. These plaque-associated changes in sphingolipid levels implicate a functional role of sphingolipid metabolism in A beta plaque pathology and AD pathogenesis. Taken together, the presented data highlight the potential of imaging mass spectrometry as a powerful approach for probing A beta plaque-associated lipid changes underlying AD pathology.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
Alzheimer's disease, amyloid-beta plaque pathology, MALDI imaging mass spectrometry, sphingolipids, tgArcSwe
National Category
Geriatrics Neurology
Identifiers
urn:nbn:se:uu:diva-320510 (URN)10.1021/acschemneuro.6b00391 (DOI)000394483300017 ()27984697 (PubMedID)
Funder
Swedish Research Council, 2014-6447 2012-1593 2013-2546 2013-14002EU, European Research Council, 681712The Dementia Association - The National Association for the Rights of the DementedThe Swedish Brain FoundationStiftelsen Gamla Tjänarinnor
Available from: 2017-04-20 Created: 2017-04-20 Last updated: 2017-04-20Bibliographically approved
Fang, X. T., Sehlin, D., Lannfelt, L., Syvänen, S. & Hultqvist, G. (2017). Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells. Biological Procedures Online, 19, Article ID 11.
Open this publication in new window or tab >>Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells
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2017 (English)In: Biological Procedures Online, ISSN 1480-9222, E-ISSN 1480-9222, Vol. 19, article id 11Article in journal (Refereed) Published
Abstract [en]

Background: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.

Methods: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost).

Results: The protocol has been used to generate multiple variants of tetra-and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (A beta) aggregates in vivo.

Conclusions: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

Keywords
Bispecific antibodies, Expi293, HEK293, PEI, Transient protein expression
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-335186 (URN)10.1186/s12575-017-0060-7 (DOI)000410951400001 ()000410951400001 (PubMedID)
Available from: 2017-12-08 Created: 2017-12-08 Last updated: 2017-12-08Bibliographically approved
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