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Sulcius, S., Simoliunas, E., Alzbutas, G., Gasiunas, G., Jauniskis, V., Kuznecova, J., . . . Holmfeldt, K. (2019). Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions. Applied and Environmental Microbiology, 85(1), Article ID UNSP e01311-18.
Open this publication in new window or tab >>Genomic Characterization of Cyanophage vB_AphaS-CL131 Infecting Filamentous Diazotrophic Cyanobacterium Aphanizomenon flos-aquae Reveals Novel Insights into Virus-Bacterium Interactions
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2019 (English)In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 85, no 1, article id UNSP e01311-18Article in journal (Refereed) Published
Abstract [en]

While filamentous cyanobacteria play a crucial role in food web dynamics and biogeochemical cycling of many aquatic ecosystems around the globe, the knowledge regarding the phages infecting them is limited. Here, we describe the complete genome of the virulent cyanophage vB_AphaS-CL131 (here, CL 131), a Siphoviridae phage that infects the filamentous diazotrophic bloom-forming cyanobacterium Aphanizomenon flos-aquae in the brackish Baltic Sea. CL 131 features a 112,793-bp double-stranded DNA (dsDNA) genome encompassing 149 putative open reading frames (ORFs), of which the majority (86%) lack sequence homology to genes with known functions in other bacteriophages or bacteria. Phylogenetic analysis revealed that CL 131 possibly represents a new evolutionary lineage within the group of cyanophages infecting filamentous cyanobacteria, which form a separate cluster from phages infecting unicellular cyanobacteria. CL 131 encodes a putative type V-U2 CRISPR-Cas system with one spacer (out of 10) targeting a DNA primase pseudogene in a cyanobacterium and a putative type II toxin-antitoxin system, consisting of a GNAT family N-acetyltransferase and a protein of unknown function containing the PRK09726 domain (characteristic of HipB antitoxins). Comparison of CL 131 proteins to reads from Baltic Sea and other available fresh- and brackish-water metagenomes and analysis of CRISPR-Cas arrays in publicly available A. flos-aquae genomes demonstrated that phages similar to CL 131 are present and dynamic in the Baltic Sea and share a common history with their hosts dating back at least several decades. In addition, different CRISPR-Cas systems within individual A. flos-aquae genomes targeted several sequences in the CL 131 genome, including genes related to virion structure and morphogenesis. Altogether, these findings revealed new genomic information for exploring viral diversity and provide a model system for investigation of virus-host interactions in filamentous cyanobacteria. IMPORTANCE The genomic characterization of novel cyanophage vB_AphaS-CL131 and the analysis of its genomic features in the context of other viruses, metagenomic data, and host CRISPR-Cas systems contribute toward a better understanding of aquatic viral diversity and distribution in general and of brackish-water cyanophages infecting filamentous diazotrophic cyanobacteria in the Baltic Sea in particular. The results of this study revealed previously undescribed features of cyanophage genomes (e.g., self-excising intein-containing putative dCTP deaminase and putative cyanophage-encoded CRISPR-Cas and toxin-antitoxin systems) and can therefore be used to predict potential interactions between bloom-forming cyanobacteria and their cyanophages.

Place, publisher, year, edition, pages
American Society of Microbiology, 2019
Keywords
Baltic Sea, brackish environment, phage-encoded CRISPR-Cas, Siphoviridae, TA system
National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-79743 (URN)10.1128/AEM.01311-18 (DOI)000452985900002 ()30367000 (PubMedID)
Available from: 2019-01-24 Created: 2019-01-24 Last updated: 2019-01-24Bibliographically approved
Sulcius, S., Mazur-Marzec, H., Vitonyte, I., Kvederaviciute, K., Kuznecova, J., Simoliunas, E. & Holmfeldt, K. (2018). Insights into cyanophage-mediated dynamics of nodularin and other non-ribosomal peptides in Nodularia spumigena. Harmful Algae, 78, 69-74
Open this publication in new window or tab >>Insights into cyanophage-mediated dynamics of nodularin and other non-ribosomal peptides in Nodularia spumigena
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2018 (English)In: Harmful Algae, ISSN 1568-9883, E-ISSN 1878-1470, Vol. 78, p. 69-74Article in journal (Refereed) Published
Abstract [en]

The effect of cyanophage infection and lysis on the dynamics of the hepatotoxin nodularin (NOD) and other nonribosomal peptides (NRPs) produced by cyanobacteria is poorly understood. In this study, changes in concentration of NOD and other NRPs during cyanophage infection of the filamentous cyanobacteria Nodularia spumigena were assessed using incubation experiments. Viral infection and lysis were associated with a significant reduction (93% at the 96 h post infection) of N. spumigena cell density. While no correlation between N. spumigena abundance and total concentration of NOD (ng mL(-1)) within the infected cells was observed, cellular NOD quota (ng cell(-1)) gradually increased in the remaining cyanophage resistant N. spumigena subpopulation. Lysis of N. spurnigena cells resulted in a substantial increase (>57 times) of dissolved NOD concentration in the culture medium. The relative concentration of other cyclic (anabaenopeptins) and linear (aeruginosins, spumigins) NRPs produced by N. spumigena also increased in response to cyanophage addition. This study highlights the importance of cyanophage infection on the population toxicity of filamentous cyanobacteria and demonstrates a significant contribution of virus-mediated cell lysis on the conversion of NOD from the particulate to dissolved phase.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Aquatic viruses, Baltic Sea, Cyanotoxins, Filamentous cyanobacteria, virus-bacterium interactions
National Category
Ecology
Research subject
Ecology, Aquatic Ecology
Identifiers
urn:nbn:se:lnu:diva-78620 (URN)10.1016/j.hal.2018.07.004 (DOI)000447112000008 ()30196926 (PubMedID)
Available from: 2018-11-01 Created: 2018-11-01 Last updated: 2018-11-01Bibliographically approved
Lopez-Fernandez, M., Simone, D., Wu, X., Soler, L., Nilsson, E., Holmfeldt, K., . . . Dopson, M. (2018). Metatranscriptomes Reveal That All Three Domains of Life Are Active but Are Dominated by Bacteria in the Fennoscandian Crystalline Granitic Continental Deep Biosphere. mBio, 9(6), Article ID e01792-18.
Open this publication in new window or tab >>Metatranscriptomes Reveal That All Three Domains of Life Are Active but Are Dominated by Bacteria in the Fennoscandian Crystalline Granitic Continental Deep Biosphere
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2018 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 9, no 6, article id e01792-18Article in journal (Refereed) Published
Abstract [en]

The continental subsurface is suggested to contain a significant part of the earth's total biomass. However, due to the difficulty of sampling, the deep subsurface is still one of the least understood ecosystems. Therefore, microorganisms inhabiting this environment might profoundly influence the global nutrient and energy cycles. In this study, in situ fixed RNA transcripts from two deep continental groundwaters from the Aspo Hard Rock Laboratory (a Baltic Sea-influenced water with a residence time of <20 years, defined as "modern marine," and an "old saline" groundwater with a residence time of thousands of years) were subjected to metatranscriptome sequencing. Although small subunit (SSU) rRNA gene and mRNA transcripts aligned to all three domains of life, supporting activity within these community subsets, the data also suggested that the groundwaters were dominated by bacteria. Many of the SSU rRNA transcripts grouped within newly described candidate phyla or could not be mapped to known branches on the tree of life, suggesting that a large portion of the active biota in the deep biosphere remains unexplored. Despite the extremely oligotrophic conditions, mRNA transcripts revealed a diverse range of metabolic strategies that were carried out by multiple taxa in the modern marine water that is fed by organic carbon from the surface. In contrast, the carbon dioxide- and hydrogen-fed old saline water with a residence time of thousands of years predominantly showed the potential to carry out translation. This suggested these cells were active, but waiting until an energy source episodically becomes available. IMPORTANCE A newly designed sampling apparatus was used to fix RNA under in situ conditions in the deep continental biosphere and benchmarks a strategy for deep biosphere metatranscriptomic sequencing. This apparatus enabled the identification of active community members and the processes they carry out in this extremely oligotrophic environment. This work presents for the first time evidence of eukaryotic, archaeal, and bacterial activity in two deep subsurface crystalline rock groundwaters from the Aspo Hard Rock Laboratory with different depths and geochemical characteristics. The findings highlight differences between organic carbonfed shallow communities and carbon dioxide- and hydrogen-fed old saline waters. In addition, the data reveal a large portion of uncharacterized microorganisms, as well as the important role of candidate phyla in the deep biosphere, but also the disparity in microbial diversity when using standard microbial 165 rRNA gene amplification versus the large unknown portion of the community identified with unbiased metatranscriptomes.

Place, publisher, year, edition, pages
American Society of Microbiology, 2018
Keywords
metatranscriptomes, mRNA, rRNA, deep biosphere, groundwaters
National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-79757 (URN)10.1128/mBio.01792-18 (DOI)000454730100031 ()30459191 (PubMedID)
Available from: 2019-01-23 Created: 2019-01-23 Last updated: 2019-02-27Bibliographically approved
Howard-Varona, C., Roux, S., Dore, H., Solonenko, N. E., Holmfeldt, K., Markillie, L. M., . . . Sullivan, M. B. (2017). Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus.. The ISME Journal, 11(1), 284-295
Open this publication in new window or tab >>Regulation of infection efficiency in a globally abundant marine Bacteriodetes virus.
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2017 (English)In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 11, no 1, p. 284-295Article in journal (Refereed) Published
Abstract [en]

Bacteria impact humans, industry and nature, but do so under viral constraints. Problematically, knowledge of viral infection efficiencies and outcomes derives from few model systems that over-represent efficient lytic infections and under-represent virus-host natural diversity. Here we sought to understand infection efficiency regulation in an emerging environmental Bacteroidetes-virus model system with markedly different outcomes on two genetically and physiologically nearly identical host strains. For this, we quantified bacterial virus (phage) and host DNA, transcripts and phage particles throughout both infections. While phage transcriptomes were similar, transcriptional differences between hosts suggested host-derived regulation of infection efficiency. Specifically, the alternative host overexpressed DNA degradation genes and underexpressed translation genes, which seemingly targeted phage DNA particle production, as experiments revealed they were both significantly delayed (by >30 min) and reduced (by >50%) in the inefficient infection. This suggests phage failure to repress early alternative host expression and stress response allowed the host to respond against infection by delaying phage DNA replication and protein translation. Given that this phage type is ubiquitous and abundant in the global oceans and that variable viral infection efficiencies are central to dynamic ecosystems, these data provide a critically needed foundation for understanding and modeling viral infections in nature.

National Category
Microbiology Ecology
Research subject
Natural Science, Ecology
Identifiers
urn:nbn:se:lnu:diva-61477 (URN)10.1038/ismej.2016.81 (DOI)000394537700025 ()27187794 (PubMedID)
Available from: 2017-03-21 Created: 2017-03-21 Last updated: 2018-05-31Bibliographically approved
Holmfeldt, K., Solonenko, N., Howard-Varona, C., Moreno, M., Malmstrom, R. R., Blow, M. J. & Sullivan, M. B. (2016). Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems. Environmental Microbiology, 18(11), 3949-3961
Open this publication in new window or tab >>Large-scale maps of variable infection efficiencies in aquatic Bacteroidetes phage-host model systems
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2016 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 18, no 11, p. 3949-3961Article in journal (Refereed) Published
Abstract [en]

Microbes drive ecosystem functioning and their viruses modulate these impacts through mortality, gene transfer and metabolic reprogramming. Despite the importance of virus-host interactions and likely variable infection efficiencies of individual phages across hosts, such variability is seldom quantified. Here, we quantify infection efficiencies of 38 phages against 19 host strains in aquatic Cellulophaga (Bacteroidetes) phage-host model systems. Binary data revealed that some phages infected only one strain while others infected 17, whereas quantitative data revealed that efficiency of infection could vary 10 orders of magnitude, even among phages within one population. This provides a baseline for understanding and modeling intrapopulation host range variation. Genera specific host ranges were also informative. For example, the Cellulophaga Microviridae, showed a markedly broader intra-species host range than previously observed in Escherichia coli systems. Further, one phage genus, Cba41, was examined to investigate nonheritable changes in plating efficiency and burst size that depended on which host strain it most recently infected. While consistent with host modification of phage DNA, no differences in nucleotide sequence or DNA modifications were detected, leaving the observation repeatable, but the mechanism unresolved. Overall, this study highlights the importance of quantitatively considering replication variations in studies of phage-host interactions.

National Category
Ecology Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-59469 (URN)10.1111/1462-2920.13392 (DOI)000388614800026 ()27235779 (PubMedID)2-s2.0-84994151527 (Scopus ID)
Available from: 2016-12-23 Created: 2016-12-23 Last updated: 2018-10-24Bibliographically approved
Wu, X., Holmfeldt, K., Hubalek, V., Lundin, D., Åström, M. E., Bertilsson, S. & Dopson, M. (2016). Microbial metagenomes from three aquifers in the Fennoscandian shield terrestrial deep biosphere reveal metabolic partitioning among populations. The ISME Journal, 10(5), 1192-1203
Open this publication in new window or tab >>Microbial metagenomes from three aquifers in the Fennoscandian shield terrestrial deep biosphere reveal metabolic partitioning among populations
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2016 (English)In: The ISME Journal, ISSN 1751-7362, E-ISSN 1751-7370, Vol. 10, no 5, p. 1192-1203Article in journal (Refereed) Published
Abstract [en]

Microorganisms in the terrestrial deep biosphere host up to 20% of the earth's biomass and are suggested to be sustained by the gases hydrogen and carbon dioxide. A metagenome analysis of three deep subsurface water types of contrasting age (from <20 to several thousand years) and depth (171 to 448 m) revealed phylogenetically distinct microbial community subsets that either passed or were retained by a 0.22 μm filter. Such cells of <0.22 μm would have been overlooked in previous studies relying on membrane capture. Metagenomes from the three water types were used for reconstruction of 69 distinct microbial genomes, each with >86% coverage. The populations were dominated by Proteobacteria, Candidate divisions, unclassified archaea and unclassified bacteria. The estimated genome sizes of the <0.22 μm populations were generally smaller than their phylogenetically closest relatives, suggesting that small dimensions along with a reduced genome size may be adaptations to oligotrophy. Shallow 'modern marine' water showed community members with a predominantly heterotrophic lifestyle. In contrast, the deeper, 'old saline' water adhered more closely to the current paradigm of a hydrogen-driven deep biosphere. The data were finally used to create a combined metabolic model of the deep terrestrial biosphere microbial community.

National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-49416 (URN)10.1038/ismej.2015.185 (DOI)000374377200016 ()26484735 (PubMedID)2-s2.0-84944910110 (Scopus ID)
Available from: 2016-01-29 Created: 2016-01-29 Last updated: 2018-10-24Bibliographically approved
Bunse, C., Lundin, D., Karlsson, C. M. G., Akram, N., Vila-Costa, M., Palovaara, J., . . . Pinhassi, J. (2016). Response of marine bacterioplankton pH homeostasis gene expression to elevated CO2. Nature Climate Change, 6(5), 483-487
Open this publication in new window or tab >>Response of marine bacterioplankton pH homeostasis gene expression to elevated CO2
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2016 (English)In: Nature Climate Change, ISSN 1758-678X, E-ISSN 1758-6798, Vol. 6, no 5, p. 483-487Article in journal (Refereed) Published
Abstract [en]

Human-induced ocean acidification impacts marine life. Marine bacteria are major drivers of biogeochemical nutrient cycles and energy fluxes1; hence, understanding their performance under projected climate change scenarios is crucial for assessing ecosystem functioning. Whereas genetic and physiological responses of phytoplankton to ocean acidification are being disentangled2, 3, 4, corresponding functional responses of bacterioplankton to pH reduction from elevated CO2 are essentially unknown. Here we show, from metatranscriptome analyses of a phytoplankton bloom mesocosm experiment, that marine bacteria responded to lowered pH by enhancing the expression of genes encoding proton pumps, such as respiration complexes, proteorhodopsin and membrane transporters. Moreover, taxonomic transcript analysis showed that distinct bacterial groups expressed different pH homeostasis genes in response to elevated CO2. These responses were substantial for numerous pH homeostasis genes under low-chlorophyll conditions (chlorophyll a <2.5 μg l−1); however, the changes in gene expression under high-chlorophyll conditions (chlorophyll a >20 μg l−1) were low. Given that proton expulsion through pH homeostasis mechanisms is energetically costly, these findings suggest that bacterioplankton adaptation to ocean acidification could have long-term effects on the economy of ocean ecosystems.

National Category
Microbiology Ecology Climate Research
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-49969 (URN)10.1038/nclimate2914 (DOI)000375125200015 ()2-s2.0-84964949342 (Scopus ID)
Projects
EcoChange
Available from: 2016-02-29 Created: 2016-02-29 Last updated: 2018-10-24Bibliographically approved
Šulčius, S. & Holmfeldt, K. (2016). Viruses of microorganisms in the Baltic Sea: current state of research and perspectives. Marine Biology Research, 12(2), 115-124
Open this publication in new window or tab >>Viruses of microorganisms in the Baltic Sea: current state of research and perspectives
2016 (English)In: Marine Biology Research, ISSN 1745-1000, E-ISSN 1745-1019, Vol. 12, no 2, p. 115-124Article in journal (Refereed) Published
Abstract [en]

This paper reviews the current state of research into viruses of microorganisms in the Baltic Sea. Field and experimental studies reveal the great phenotypic and genotypic variety of the Baltic Sea viruses and highlights the importance of phages in the Baltic Sea food-web dynamics. In total, 93 virus isolates from the Baltic Sea have been described, the genome sequence being available for 39 of these. However, these isolates were derived from only eight host species and, therefore, underrepresent the overall viral diversity in the Baltic Sea. The most studied group of viruses is bacteriophages that infect Bacteroidetes, whereas, by contrast, phages of Cyanobacteria, Proteobacteria and Actinobacteria, as well as algal viruses, have been investigated to a lesser extent. No reports on viruses infecting Baltic Sea fungi and archaea isolates have been reported to date. This paper also identifies gaps in our knowledge of the aquatic virology of the Baltic Sea and emphasizes the need for more comprehensive analysis of those viruses that occur in one of the world’s largest brackish water ecosystems.

National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-50174 (URN)10.1080/17451000.2015.1118514 (DOI)000372363500001 ()2-s2.0-84960392590 (Scopus ID)
Available from: 2016-03-04 Created: 2016-03-04 Last updated: 2018-11-15Bibliographically approved
Lara, E., Holmfeldt, K., Solonenko, N., Sà, E. L., Ignacio-Espinoza, J. C., Cornejo-Castillo, F. M., . . . Acinas, S. G. (2015). Life-style and genome structure of marine Pseudoalteromonas siphovirus B8b isolated from the northwestern Mediterranean Sea.. PLoS ONE, 10(1), Article ID e0114829.
Open this publication in new window or tab >>Life-style and genome structure of marine Pseudoalteromonas siphovirus B8b isolated from the northwestern Mediterranean Sea.
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 1, article id e0114829Article in journal (Refereed) Published
Abstract [en]

Marine viruses (phages) alter bacterial diversity and evolution with impacts on marine biogeochemical cycles, and yet few well-developed model systems limit opportunities for hypothesis testing. Here we isolate phage B8b from the Mediterranean Sea using Pseudoalteromonas sp. QC-44 as a host and characterize it using myriad techniques. Morphologically, phage B8b was classified as a member of the Siphoviridae family. One-step growth analyses showed that this siphovirus had a latent period of 70 min and released 172 new viral particles per cell. Host range analysis against 89 bacterial host strains revealed that phage B8b infected 3 Pseudoalteromonas strains (52 tested, >99.9% 16S rRNA gene nucleotide identity) and 1 non-Pseudoaltermonas strain belonging to Alteromonas sp. (37 strains from 6 genera tested), which helps bound the phylogenetic distance possible in a phage-mediated horizontal gene transfer event. The Pseudoalteromonas phage B8b genome size was 42.7 kb, with clear structural and replication modules where the former were delineated leveraging identification of 16 structural genes by virion structural proteomics, only 4 of which had any similarity to known structural proteins. In nature, this phage was common in coastal marine environments in both photic and aphotic layers (found in 26.5% of available viral metagenomes), but not abundant in any sample (average per sample abundance was 0.65% of the reads). Together these data improve our understanding of siphoviruses in nature, and provide foundational information for a new 'rare virosphere' phage-host model system.

National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-49392 (URN)10.1371/journal.pone.0114829 (DOI)000347928300004 ()25587991 (PubMedID)
Available from: 2016-01-29 Created: 2016-01-28 Last updated: 2017-11-30Bibliographically approved
Holmfeldt, K., Howard-Varona, C., Solonenko, N. & Sullivan, M. B. (2014). Contrasting genomic patterns and infection strategies of two co-existing Bacteroidetes podovirus genera. Environmental Microbiology, 16(8), 2501-2513
Open this publication in new window or tab >>Contrasting genomic patterns and infection strategies of two co-existing Bacteroidetes podovirus genera
2014 (English)In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 16, no 8, p. 2501-2513Article in journal (Refereed) Published
Abstract [en]

Bacterial viruses (phages) are abundant, ecologically important biological entities. However, our understanding of their impact is limited by model systems that are primarily not well represented in nature, e.g. Enterophages and their hosts. Here, we investigate genomic characteristics and infection strategies among six aquatic Bacteroidetes phages that represent two genera of exceptionally large (∼70-75 kb genome) podoviruses, which were isolated from the same seawater sample using Cellulophaga baltica as host. Quantitative host range studies reveal that these genera have contrasting narrow (specialist) and broad (generalist) host ranges, with one-step growth curves revealing reduced burst sizes for the generalist phages. Genomic comparisons suggest candidate genes in each genus that might explain this host range variation, as well as provide hypotheses about receptors in the hosts. One generalist phage, φ38:1, was more deeply characterized, as its infection strategy switched from lytic on its original host to either inefficient lytic or lysogenic on an alternative host. If lysogenic, this phage was maintained extrachromosomally in the alternative host and could not be induced by mitomycin C. This work provides fundamental knowledge regarding phage-host ranges and their genomic drivers while also exploring the 'host environment' as a driver for switching phage replication mode.

National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-38860 (URN)10.1111/1462-2920.12391 (DOI)24428166 (PubMedID)
Funder
Swedish Research Council, 623-2012-1395
Available from: 2015-01-09 Created: 2015-01-09 Last updated: 2017-12-05Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6887-6661

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