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Tükenmez, H., Edström, I., Kalsum, S., Braian, C., Ummanni, R., Lindberg, S., . . . Larsson, C. (2019). Corticosteroids protect infected cells against mycobacterial killing in vitro. Biochemical and Biophysical Research Communications - BBRC, 511(1), 117-121
Open this publication in new window or tab >>Corticosteroids protect infected cells against mycobacterial killing in vitro
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2019 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 511, no 1, p. 117-121Article in journal (Refereed) Published
Abstract [en]

The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 mu M. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling. (C) 2019 The Authors. Published by Elsevier Inc.

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2019
Keywords
Mycobacterium, Tuberculosis, Corticosteroids, Cell death, Drug discovery
National Category
Immunology
Identifiers
urn:nbn:se:umu:diva-157508 (URN)10.1016/j.bbrc.2019.02.044 (DOI)000460849800019 ()30773257 (PubMedID)
Available from: 2019-04-05 Created: 2019-04-05 Last updated: 2019-04-05Bibliographically approved
Massai, F., Saleeb, M., Doruk, T., Elofsson, M. & Forsberg, Å. (2019). Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa. Frontiers in Cellular and Infection Microbiology, 9, Article ID 250.
Open this publication in new window or tab >>Development, Optimization, and Validation of a High Throughput Screening Assay for Identification of Tat and Type II Secretion Inhibitors of Pseudomonas aeruginosa
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2019 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 9, article id 250Article in journal (Refereed) Published
Abstract [en]

Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z' of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
Pseudomonas aeruginosa, high-throughput screening, twin arginine translocase, type II secretion, virulence inhibitors, phospholipase C
National Category
Infectious Medicine
Identifiers
urn:nbn:se:umu:diva-161822 (URN)10.3389/fcimb.2019.00250 (DOI)000474778200001 ()31355152 (PubMedID)
Available from: 2019-08-13 Created: 2019-08-13 Last updated: 2019-08-13Bibliographically approved
Beck, C., Rodriguez-Vargas, J. M., Boehler, C., Robert, I., Heyer, V., Hanini, N., . . . Dantzer, F. (2019). PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers. Cell Death and Differentiation, 26(9), 1615-1630
Open this publication in new window or tab >>PARP3, a new therapeutic target to alter Rictor/mTORC2 signaling and tumor progression in BRCA1-associated cancers
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2019 (English)In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 26, no 9, p. 1615-1630Article in journal (Refereed) Published
Abstract [en]

PARP3 has been shown to be a key driver of TGF beta-induced epithelial-to-mesenchymal transition (EMT) and sternness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3(-/- )BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9(D10A) gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3 beta, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

Place, publisher, year, edition, pages
Nature Publishing Group, 2019
National Category
Biochemistry and Molecular Biology Cell Biology
Identifiers
urn:nbn:se:umu:diva-162838 (URN)10.1038/s41418-018-0233-1 (DOI)000480648400005 ()30442946 (PubMedID)
Available from: 2019-09-13 Created: 2019-09-13 Last updated: 2019-09-13Bibliographically approved
Chandra, N., Frängsmyr, L., Imhof, S., Caraballo, R., Elofsson, M. & Arnberg, N. (2019). Sialic Acid-Containing Glycans as Cellular Receptors for Ocular Human Adenoviruses: Implications for Tropism and Treatment. Viruses, 11(5), Article ID 395.
Open this publication in new window or tab >>Sialic Acid-Containing Glycans as Cellular Receptors for Ocular Human Adenoviruses: Implications for Tropism and Treatment
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2019 (English)In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 11, no 5, article id 395Article in journal (Refereed) Published
Abstract [en]

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.

Place, publisher, year, edition, pages
MDPI, 2019
Keywords
adenovirus, cellular receptor, epidemic keratoconjunctivitis, pharyngoconjunctival fever, sialic acid, tropism
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-159268 (URN)10.3390/v11050395 (DOI)000472676600006 ()31035532 (PubMedID)2-s2.0-85065483937 (Scopus ID)
Funder
Knut and Alice Wallenberg Foundation, 2013.0019
Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-08-28Bibliographically approved
Puigvert, M., Sole, M., Lopez-Garcia, B., Coll, N. S., Beattie, K. D., Davis, R. A., . . . Valls, M. (2019). Type III secretion inhibitors for the management of bacterial plant diseases. Molecular plant pathology, 20(1), 20-32
Open this publication in new window or tab >>Type III secretion inhibitors for the management of bacterial plant diseases
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2019 (English)In: Molecular plant pathology, ISSN 1464-6722, E-ISSN 1364-3703, Vol. 20, no 1, p. 20-32Article in journal (Refereed) Published
Abstract [en]

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds—including molecules effective against human pathogens—for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down‐regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases.

Place, publisher, year, edition, pages
John Wiley & Sons, 2019
Keywords
bacterial speck, bacterial wilt, disease control, Pseudomonas syringae, Ralstonia solanacearum, type III secretion system, virulence inhibitors
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-155091 (URN)10.1111/mpp.12736 (DOI)000453710700003 ()30062690 (PubMedID)
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Karlberg, T., Hornyak, P., Pinto, A. F., Milanova, S., Ebrahimi, M., Lindberg, M. J., . . . Schüler, H. (2018). 14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface. Nature Communications, 9, Article ID 3785.
Open this publication in new window or tab >>14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface
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2018 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3785Article in journal (Refereed) Published
Abstract [en]

Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3 beta: ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Cell and Molecular Biology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-152249 (URN)10.1038/s41467-018-06194-1 (DOI)000444757900003 ()30224724 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SB12-0022Swedish Research Council, 2012-2802Swedish Research Council, 2015-4200Swedish Research Council, 2015-4603Wenner-Gren Foundations
Available from: 2018-10-04 Created: 2018-10-04 Last updated: 2018-10-04Bibliographically approved
Islam, M. K. K., Strand, M., Saleeb, M., Svensson, R., Baranczewski, P., Artursson, P., . . . Evander, M. (2018). Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound. Scientific Reports, 8, Article ID 1925.
Open this publication in new window or tab >>Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1925Article in journal (Refereed) Published
Abstract [en]

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-144950 (URN)10.1038/s41598-018-20362-9 (DOI)000423663100004 ()29386590 (PubMedID)
Available from: 2018-02-22 Created: 2018-02-22 Last updated: 2018-09-14Bibliographically approved
Kumar, A., Saleeb, M., Werz, D. & Elofsson, M. (2018). Cyclopropylmethyl Protection of Phenols: Total Synthesis of the Resveratrol Dimers Anigopreissin A and Resveratrol-Piceatannol Hybrid. ChemistryOpen, 7(12), 953-956
Open this publication in new window or tab >>Cyclopropylmethyl Protection of Phenols: Total Synthesis of the Resveratrol Dimers Anigopreissin A and Resveratrol-Piceatannol Hybrid
2018 (English)In: ChemistryOpen, ISSN 2191-1363, Vol. 7, no 12, p. 953-956Article in journal (Refereed) Published
Abstract [en]

We demonstrate the versatile use of the cyclopropylmethyl group to protect phenols through the total synthesis of two benzofuran-based natural products, that is, anigopreissin A and the resveratrol-piceatannol hybrid. This protecting group is a good alternative to the conventional methyl group, owing to the feasibility of introduction, stability under a variety of conditions, and its relative ease of removal under different acidic conditions.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2018
Keywords
anigopreissin A, cyclopropylmethyl protecting group, deprotection, resveratrol-piceatannol hybrid, total synthesis
National Category
Organic Chemistry
Identifiers
urn:nbn:se:umu:diva-155651 (URN)10.1002/open.201800214 (DOI)000454525600002 ()30524921 (PubMedID)
Funder
Swedish Research Council, 621-2014-4670
Available from: 2019-01-25 Created: 2019-01-25 Last updated: 2019-01-25Bibliographically approved
Ekblad, T., Verheugd, P., Lindgren, A. E., Nyman, T., Elofsson, M. & Schüler, H. (2018). Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol. SLAS DISCOVERY, 23(4), 353-362
Open this publication in new window or tab >>Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol
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2018 (English)In: SLAS DISCOVERY, ISSN 2472-5552, Vol. 23, no 4, p. 353-362Article in journal (Refereed) Published
Abstract [en]

Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a similar to 1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

Place, publisher, year, edition, pages
Sage Publications, 2018
Keywords
ADP-ribosylation, macrodomain inhibitor, PARP, protein-protein interaction, AlphaScreen
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-147337 (URN)10.1177/2472555217750870 (DOI)000429940100005 ()29316839 (PubMedID)
Available from: 2018-05-14 Created: 2018-05-14 Last updated: 2018-06-09Bibliographically approved
Saleeb, M., Mojica, S., Eriksson, A. U., Andersson, C. D., Gylfe, Å. & Elofsson, M. (2018). Natural product inspired library synthesis – Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis. European Journal of Medicinal Chemistry, 143, 1077-1089
Open this publication in new window or tab >>Natural product inspired library synthesis – Identification of 2,3-diarylbenzofuran and 2,3-dihydrobenzofuran based inhibitors of Chlamydia trachomatis
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 143, p. 1077-1089Article in journal (Refereed) Published
Abstract [en]

A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial activity in a panel of test systems including phenotypic, biochemical and image-based screening assays. We identified several potent inhibitors that block intracellular replication of pathogenic Chlamydia trachomatis with IC50 ≤ 3 μM. These new C. trachomatis inhibitors can serve as starting points for the development of specific treatments that reduces the global burden of C. trachomatis infections.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
2, 3-diaryl-2, 3-dihydrobenzofuran, 2, 3-diaryl-benzofuran, Antibacterial, Benzofuran, Chlamydia
National Category
Organic Chemistry Biological Sciences
Identifiers
urn:nbn:se:umu:diva-143062 (URN)10.1016/j.ejmech.2017.11.099 (DOI)000428216700089 ()29232584 (PubMedID)
Funder
Swedish Foundation for Strategic Research , SB12-0022Swedish Research Council, 621-2014-4670
Available from: 2017-12-18 Created: 2017-12-18 Last updated: 2018-08-24Bibliographically approved
Projects
Chemical attenuation of bacterial virulence: Inhibitors of type III secretion [2009-02938_VR]; Umeå UniversityUsing Small Molecules to Study Big Questions in Microbiology [2010-04746_VR]; Umeå UniversityChemical attenuation of bacterial virulence - Inhibitors of type III secretion [2012-02802_VR]; Umeå UniversityNya anti-infektiva terapier mot tuberkolos (Beviljad ansökan överförd från Vinnova med dnr 2013-02030) [2013-08775_VR]; Umeå UniversitySmall molecules answer big questions in microbiology [2014-04670_VR]; Umeå UniversityDevelopment of orally bioavailable antibacterials to treat Chlamydia infections [2018-05886_VR]; Umeå University
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-3219-4669

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