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Kreuger, Johan
Publications (10 of 46) Show all publications
Wistrand-Yuen, P., Malmberg, C., Fatsis-Kavalopoulos, N., Lübke, M., Tängdén, T. & Kreuger, J. (2020). A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing. mBio, 11, Article ID e03109-19.
Open this publication in new window or tab >>A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing
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2020 (English)In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 11, article id e03109-19Article in journal (Refereed) Published
Abstract [en]

Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases.

Place, publisher, year, edition, pages
AMER SOC MICROBIOLOGY, 2020
Keywords
antibiotic susceptibility testing, clinical isolates, fluidic chip, microfluidics, multiplex
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-408914 (URN)10.1128/mBio.03109-19 (DOI)000518763400114 ()32098819 (PubMedID)
Funder
Vinnova, 2016-02286EU, Horizon 2020, 642866
Available from: 2020-04-17 Created: 2020-04-17 Last updated: 2020-04-17Bibliographically approved
Fatsis-Kavalopoulos, N., O'Callaghan, P., Xie, B., Hernández Vera, R., Idevall Hagren, O. & Kreuger, J. (2019). Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution. Lab on a Chip, 19(6), 1071-1081
Open this publication in new window or tab >>Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
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2019 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed) Published
Abstract [en]

The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-381586 (URN)10.1039/c8lc01153b (DOI)000462666200012 ()30783638 (PubMedID)
Funder
Swedish Cancer Society, CAN 2017/703EU, Horizon 2020, 642866Swedish Research Council, MH2015-03087Göran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2019-04-12 Created: 2019-04-12 Last updated: 2019-04-24Bibliographically approved
Hernández Vera, R., O'Callaghan, P., Fatsis-Kavalopoulos, N. & Kreuger, J. (2019). Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures. Scientific Reports, 9, Article ID 11321.
Open this publication in new window or tab >>Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures
2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11321Article in journal (Refereed) Published
Abstract [en]

Microfluidic systems are very useful for in vitro studies of interactions between blood cells and vascular endothelial cells under flow, and several commercial solutions exist. However, the availability of customizable, user-designed devices is largely restricted to researchers with expertise in photolithography and access to clean room facilities. Here we describe a strategy for producing tailor-made modular microfluidic systems, cast in PDMS from 3D-printed molds, to facilitate studies of leukocyte adherence to endothelial cells. A dual-chamber barrier module was optimized for culturing two endothelial cell populations, separated by a 250 μm wide dividing wall, on a glass slide. In proof-of-principle experiments one endothelial population was activated by TNFα, while the other served as an internal control. The barrier module was thereafter replaced with a microfluidic flow module, enclosing both endothelial populations in a common channel. A suspension of fluorescently-labeled leukocytes was then perfused through the flow module and leukocyte interactions with control and tnfα-treated endothelial populations were monitored in the same field of view. Time-lapse microscopy analysis confirmed the preferential attachment of leukocytes to the TNFα-activated endothelial cells. We conclude that the functionality of these modular microfluidic systems makes it possible to seed and differentially activate adherent cell types, and conduct controlled side-by-side analysis of their capacity to interact with cells in suspension under flow. Furthermore, we outline a number of practical considerations and solutions associated with connecting and switching between the microfluidic modules, and the advantages of simultaneously and symmetrically analyzing control and experimental conditions in such a microfluidic system.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-382276 (URN)10.1038/s41598-019-47475-z (DOI)000478743700033 ()
Funder
Swedish Cancer Society, CAN 2017/703EU, Horizon 2020, 642866
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-09-27Bibliographically approved
Ahl, D., Eriksson, O., Sedin, J., Seignez, C., Schwan, E., Kreuger, J., . . . Phillipson, M. (2019). Turning Up the Heat: Local Temperature Control During in vivo Imaging of Immune Cells. Frontiers in Immunology, 10, Article ID 2036.
Open this publication in new window or tab >>Turning Up the Heat: Local Temperature Control During in vivo Imaging of Immune Cells
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2019 (English)In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 10, article id 2036Article in journal (Refereed) Published
Abstract [en]

Intravital imaging is an invaluable tool for studying the expanding range of immune cell functions. Only in vivo can the complex and dynamic behavior of leukocytes and their interactions with their natural microenvironment be observed and quantified. While the capabilities of high-speed, high-resolution confocal and multiphoton microscopes are well-documented and steadily improving, other crucial hardware required for intravital imaging is often developed in-house and less commonly published in detail. In this report, we describe a low-cost, multipurpose, and tissue-stabilizing in vivo imaging platform that enables sensing and regulation of local tissue temperature. The effect of tissue temperature on local blood flow and leukocyte migration is demonstrated in muscle and skin. Two different models of vacuum windows are described in this report, however, the design of the vacuum window can easily be adapted to fit different organs and tissues.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2019
Keywords
intravital microscopy, skin, blood flow, leukocytes, vacuum window, confocal microscopy
National Category
Immunology Immunology in the medical area
Identifiers
urn:nbn:se:uu:diva-398848 (URN)10.3389/fimmu.2019.02036 (DOI)000482819900001 ()31507619 (PubMedID)
Funder
Swedish Research Council, 2018-02552Knut and Alice Wallenberg FoundationRagnar Söderbergs stiftelseSwedish Society for Medical Research (SSMF)Göran Gustafsson Foundation for promotion of scientific research at Uppala University and Royal Institute of TechnologyNovo Nordisk
Available from: 2019-12-18 Created: 2019-12-18 Last updated: 2019-12-18Bibliographically approved
O'Callaghan, P., Zarb, Y., Noborn, F. & Kreuger, J. (2018). Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec. PLoS ONE, 13(8), Article ID e0201557.
Open this publication in new window or tab >>Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec
2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 8, article id e0201557Article in journal (Refereed) Published
Abstract [en]

The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.

Place, publisher, year, edition, pages
San Francisco, California, US: , 2018
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-360991 (URN)10.1371/journal.pone.0201557 (DOI)000441129300022 ()30086153 (PubMedID)
Funder
The Dementia Association - The National Association for the Rights of the DementedSwedish Cancer Society, CAN 2014/820
Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-11-28Bibliographically approved
Heldin, J., O'Callaghan, P., Hernández Vera, R., Fredlund Fuchs, P., Gerwins, P. & Kreuger, J. (2017). FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation. Cellular Signalling, 40, 125-132
Open this publication in new window or tab >>FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation
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2017 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 40, p. 125-132Article in journal (Refereed) Published
Abstract [en]

The complete repertoire of endothelial functions elicited by FGD5, a guanine nucleotide exchange factor activating the Rho GTPase Cdc42, has yet to be elucidated. Here we explore FGD5's importance during vascular endothelial growth factor A (VEGFA) signaling via VEGF receptor 2 (VEGFR2) in human endothelial cells. In microvascular endothelial cells, FGD5 is located at the inner surface of the cell membrane as well as at the outer surface of EEAl-positive endosomes carrying VEGFR2. The latter finding prompted us to explore if FGD5 regulates VEGFR2 dynamics. We found that depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. Our results thus suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation.

Keywords
Angiogenesis, Cdc42, FGD5, Vascular biology, VEGFR2, Degradation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341984 (URN)10.1016/j.cellsig.2017.09.009 (DOI)000414620900013 ()28927665 (PubMedID)
Available from: 2018-02-19 Created: 2018-02-19 Last updated: 2018-02-19Bibliographically approved
Blom, M., Reis, K., Heldin, J., Kreuger, J. & Aspenström, P. (2017). The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration. Experimental Cell Research, 352(2), 255-264
Open this publication in new window or tab >>The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration
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2017 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 352, no 2, p. 255-264Article in journal (Refereed) Published
Abstract [en]

RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.

Keywords
RhoD, Rho GTPase, Actin, Stress fiber, Cell migration
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-319045 (URN)10.1016/j.yexcr.2017.02.013 (DOI)000396964600010 ()28196728 (PubMedID)
Funder
The Karolinska Institutet's Research FoundationSwedish Cancer Society, JK-2014/820 PA-2014/0644
Available from: 2017-03-30 Created: 2017-03-30 Last updated: 2018-01-13Bibliographically approved
Christoffersson, G., Lomei, J., O'Callaghan, P., Kreuger, J., Engblom, S. & Phillipson, M. (2017). Vascular sprouts induce local attraction of proangiogenic neutrophils. Journal of Leukocyte Biology, 102, 741-751
Open this publication in new window or tab >>Vascular sprouts induce local attraction of proangiogenic neutrophils
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2017 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 102, p. 741-751Article in journal (Refereed) Published
National Category
Physiology Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-196483 (URN)10.1189/jlb.1MA0117-018R (DOI)000413395700019 ()
Projects
eSSENCE
Available from: 2017-06-05 Created: 2013-03-10 Last updated: 2018-11-12Bibliographically approved
Hernández Vera, R., Schwan, E., Fatsis-Kavalopoulos, N. & Kreuger, J. (2016). A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D Printed Parts, a Smartphone, and Off-The-Shelf Electronics. PLoS ONE, 11(12), Article ID e0167583.
Open this publication in new window or tab >>A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D Printed Parts, a Smartphone, and Off-The-Shelf Electronics
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167583Article in journal (Refereed) Published
Abstract [en]

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

National Category
Biomedical Laboratory Science/Technology
Identifiers
urn:nbn:se:uu:diva-316428 (URN)10.1371/journal.pone.0167583 (DOI)000392853100013 ()28002463 (PubMedID)
Funder
Swedish Cancer Society, CAN 2014/820EU, European Research Council, 642866
Available from: 2017-03-01 Created: 2017-03-01 Last updated: 2019-04-24Bibliographically approved
Kreuger, J. & O'Callaghan, P. (2016). Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence. PLoS ONE, 11(10), Article ID e0165012.
Open this publication in new window or tab >>Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0165012Article in journal (Refereed) Published
Abstract [en]

Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2(tm1a( KOMP)Wtsi) embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2(tm1a(KOMP)Wtsi) construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models. As PCR products amplified from KO1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity of individual mice from a single PCR. In parallel we performed PCR with a primer specifically targeting the synthetic loxP sequence. Unexpectedly, while the latter strategy detected the synthetic loxP region and correctly genotyped KO1st chimeric mice, the same individuals were genotyped as wild-type when using the primers that flanked the synthetic loxP region. We discuss the possibility that secondary DNA structures, formed due to the palindromic nature of the synthetic loxP region, may have caused the KO1st template to elude the PCR when using primers that flanked this region. This brief report aims to raise awareness regarding this potential source of false-negative genotype results, particularly for those who are devising genotyping strategies for similarly engineered animal models.

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-308918 (URN)10.1371/journal.pone.0165012 (DOI)000386205400044 ()27768725 (PubMedID)
Funder
Swedish Cancer Society, CAN 2014/820Swedish Research Council, 2010-3968NIH (National Institute of Health), U01HG004085
Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2017-11-29Bibliographically approved
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