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Golovliov, Igor
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Publications (10 of 20) Show all publications
Alam, A., Golovliov, I., Javed, E. & Sjöstedt, A. (2018). ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication. Scientific Reports, 8, Article ID 11324.
Open this publication in new window or tab >>ClpB mutants of Francisella tularensis subspecies holarctica and tularensis are defective for type VI secretion and intracellular replication
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 11324Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for the virulence of the bacterium. Recent data suggest that the HSP100 family member, ClpB, is involved in T6SS disassembly in the subspecies Francisella novicida. Here, we investigated the role of ClpB for the function of the T6SS and for phenotypic characteristics of the human pathogenic subspecies holarctica and tularensis. The Delta clpB mutants of the human live vaccine strain, LVS, belonging to subspecies holarctica, and the highly virulent SCHU S4 strain, belonging to subspecies tularensis, both showed extreme susceptibility to heat shock and low pH, severely impaired type VI secretion (T6S), and significant, but impaired intracellular replication compared to the wild-type strains. Moreover, they showed essentially intact phagosomal escape. Infection of mice demonstrated that both Delta clpB mutants were highly attenuated, but the SCHU S4 mutant showed more effective replication than the LVS strain. Collectively, our data demonstrate that ClpB performs multiple functions in the F. tularensis subspecies holarctica and tularensis and its function is important for T6S, intracellular replication, and virulence.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-150672 (URN)10.1038/s41598-018-29745-4 (DOI)000439965200017 ()30054549 (PubMedID)2-s2.0-85050803246 (Scopus ID)
Funder
Swedish Research Council, 2013-4581; 2013-8621
Available from: 2018-08-16 Created: 2018-08-16 Last updated: 2018-08-21Bibliographically approved
Eneslätt, K., Golovliov, I., Rydén, P. & Sjöstedt, A. (2018). Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system. Frontiers in Cellular and Infection Microbiology, 8, Article ID 27.
Open this publication in new window or tab >>Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system
2018 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, article id 27Article in journal (Refereed) Published
Abstract [en]

Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
Keywords
F. tularensis, in vitro model, human immune response, IFN-gamma, TNF, MIP-1 beta, correlates of immunity
National Category
Microbiology in the medical area Immunology
Identifiers
urn:nbn:se:umu:diva-144645 (URN)10.3389/fcimb.2018.00027 (DOI)000424355900001 ()29468144 (PubMedID)
Available from: 2018-02-08 Created: 2018-02-08 Last updated: 2018-08-20Bibliographically approved
Wallet, P., Benaoudia, S., Mosnier, A., Lagrange, B., Martin, A., Lindgren, H., . . . Henry, T. (2017). IFN-gamma extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection. PLoS Pathogens, 13(10), Article ID e1006630.
Open this publication in new window or tab >>IFN-gamma extends the immune functions of Guanylate Binding Proteins to inflammasome-independent antibacterial activities during Francisella novicida infection
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2017 (English)In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 10, article id e1006630Article in journal (Refereed) Published
Abstract [en]

Guanylate binding proteins (GBPs) are interferon-inducible proteins involved in the cellintrinsic immunity against numerous intracellular pathogens. The molecular mechanisms underlying the potent antibacterial activity of GBPs are still unclear. GBPs have been functionally linked to the NLRP3, the AIM2 and the caspase-11 inflammasomes. Two opposing models are currently proposed to explain the GBPs-inflammasome link: i) GBPs would target intracellular bacteria or bacteria-containing vacuoles to increase cytosolic PAMPs release ii) GBPs would directly facilitate inflammasome complex assembly. Using Francisella novicida infection, we investigated the functional interactions between GBPs and the inflammasome. GBPs, induced in a type I IFN-dependent manner, are required for the F. novicida-mediated AIM2-inflammasome pathway. Here, we demonstrate that GBPs action is not restricted to the AIM2 inflammasome, but controls in a hierarchical manner the activation of different inflammasomes complexes and apoptotic caspases. IFN-gamma induces a quantitative switch in GBPs levels and redirects pyroptotic and apoptotic pathways under the control of GBPs. Furthermore, upon IFN-gamma priming, F. novicida-infected macrophages restrict cytosolic bacterial replication in a GBP-dependent and inflammasome-independent manner. Finally, in a mouse model of tularemia, we demonstrate that the inflammasome and the GBPs are two key immune pathways functioning largely independently to control F. novicida infection. Altogether, our results indicate that GBPs are the master effectors of IFN-gamma-mediated responses against F. novicida to control antibacterial immune responses in inflammasome-dependent and independent manners.

Place, publisher, year, edition, pages
Public Library Science, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-141986 (URN)10.1371/journal.ppat.1006630 (DOI)000414163300008 ()
Available from: 2017-12-06 Created: 2017-12-06 Last updated: 2018-06-09Bibliographically approved
Golovliov, I., Lindgren, H., Eneslätt, K., Conlan, W., Mosnier, A., Henry, T. & Sjöstedt, A. (2016). An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy. Frontiers in Cellular and Infection Microbiology, 6, Article ID 152.
Open this publication in new window or tab >>An In Vitro Co-culture Mouse Model Demonstrates Efficient Vaccine-Mediated Control of Francisella tularensis SCHU S4 and Identifies Nitric Oxide as a Predictor of Efficacy
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2016 (English)In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 152Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a highly virulent intracellular bacterium and cell-mediated immunity is critical for protection, but mechanisms of protection against highly virulent variants, such as the prototypic strain F. tularensis strain SCHU S4, are poorly understood. To this end, we established a co-culture system, based on splenocytes from naive, or immunized mice and in vitro infected bone marrow-derived macrophages that allowed assessment of mechanisms controlling infection with F. tularensis. We utilized the system to understand why the clpB gene deletion mutant, Delta clpB, of SCHU S4 shows superior efficacy as a vaccine in the mouse model as compared to the existing human vaccine, the live vaccine strain (LVS). Compared to naive splenocytes, Delta clpB-, or LVS-immune splenocytes conferred very significant control of a SCHU S4 infection and the Delta clpB-immune splenocytes were superior to the LVS-immune splenocytes. Cultures with the Delta clpB-immune splenocytes also contained higher levels of IFN-gamma, IL-17, and GM-CSF and nitric oxide, and T cells expressing combinations of IFN-gamma, TNF-alpha, and IL-17, than did cultures with LVS-immune splenocytes. There was strong inverse correlation between bacterial replication and levels of nitrite, an end product of nitric oxide, and essentially no control was observed when BMDM from iNOS(-/-) mice were infected. Collectively, the co-culture model identified a critical role of nitric oxide for protection against a highly virulent strain of F. tularensis.

Keywords
F. tularensis SCHU S4, in vitro co-culturemodel, mouse immune response, correlates of protection
National Category
Immunology Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-129814 (URN)10.3389/fcimb.2016.00152 (DOI)000388557800001 ()27933275 (PubMedID)
Available from: 2017-01-10 Created: 2017-01-09 Last updated: 2018-06-09Bibliographically approved
Lindgren, H., Lindgren, L., Golovliov, I. & Sjöstedt, A. (2015). Mechanisms of heme utilization by Francisella tularensis. PLoS ONE, 10(3), Article ID e0119143.
Open this publication in new window or tab >>Mechanisms of heme utilization by Francisella tularensis
2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 3, article id e0119143Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a highly virulent facultative intracellular pathogen causing the severe disease tularemia in mammals. As for other bacteria, iron is essential for its growth but very few mechanisms for iron acquisition have been identified. Here, we analyzed if and how F. tularensis can utilize heme, a major source of iron in vivo. This is by no means obvious since the bacterium lacks components of traditional heme-uptake systems. We show that SCHU S4, the prototypic strain of subspecies tularensis, grew in vitro with heme as the sole iron source. By screening a SCHU S4 transposon insertion library, 16 genes were identified as important to efficiently utilize heme, two of which were required to avoid heme toxicity. None of the identified genes appeared to encode components of a potential heme-uptake apparatus. Analysis of SCHU S4 deletion mutants revealed that each of the components FeoB, the siderophore system, and FupA, contributed to the heme-dependent growth. In the case of the former two systems, iron acquisition was impaired, whereas the absence of FupA did not affect iron uptake but led to abnormally high binding of iron to macromolecules. Overall, the present study demonstrates that heme supports growth of F. tularensis and that the requirements for the utilization are highly complex and to some extent novel.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-102224 (URN)10.1371/journal.pone.0119143 (DOI)000351275700039 ()25756756 (PubMedID)
Available from: 2015-05-05 Created: 2015-04-22 Last updated: 2018-06-07Bibliographically approved
Lindgren, M., Tancred, L., Golovliov, I., Conlan, W., Twine, S. M. & Sjöstedt, A. (2014). Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4. Infection and Immunity, 82(9), 3622-3635
Open this publication in new window or tab >>Identification of Mechanisms for Attenuation of the FSC043 Mutant of Francisella tularensis SCHU S4
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2014 (English)In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 9, p. 3622-3635Article in journal (Refereed) Published
Abstract [en]

Previously, we identified a spontaneous, essentially avirulent mutant, FSC043, of the highly virulent strain SCHU S4 of Francisella tularensis subsp. tularensis. We have now characterized the phenotype of the mutant and the mechanisms of its attenuation in more detail. Genetic and proteomic analyses revealed that the pdpE gene and most of the pdpC gene were very markedly downregulated and, as previously demonstrated, that the strain expressed partially deleted and fused fupA and fupB genes. FSC043 showed minimal intracellular replication and induced no cell cytotoxicity. The mutant showed delayed phagosomal escape; at 18 h, colocalization with LAMP-1 was 80%, indicating phagosomal localization, whereas the corresponding percentages for SCHU S4 and the Delta fupA mutant were < 10%. However, a small subset of the FSC043-infected cells contained up to 100 bacteria with LAMP-1 colocalization of around 30%. The unusual intracellular phenotype was similar to that of the Delta pdpC and Delta pdpC Delta pdpE mutants. Complementation of FSC043 with the intact fupA and fupB genes did not affect the phenotype, whereas complementation with the pdpC and pdpE genes restored intracellular replication and led to marked virulence. Even higher virulence was observed after complementation with both double-gene constructs. After immunization with the FSC043 strain, moderate protection against respiratory challenge with the SCHU S4 strain was observed. In summary, FSC043 showed a highly unusual intracellular phenotype, and based on our findings, we hypothesize that the mutation in the pdpC gene makes an essential contribution to the phenotype.

Place, publisher, year, edition, pages
American Society for Microbiology, 2014
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-94934 (URN)10.1128/IAI.01406-13 (DOI)000341932700011 ()
Available from: 2014-11-10 Created: 2014-10-20 Last updated: 2018-06-07Bibliographically approved
Golovlev, I., Twine, S. M., Shen, H., Sjöstedt, A. & Conlan, W. (2013). A Delta clpB Mutant of Francisella tularensis Subspecies holarctica Strain, FSC200, Is a More Effective Live Vaccine than F. tularensis LVS in a Mouse Respiratory Challenge Model of Tularemia. PLoS ONE, 8(11), e78671
Open this publication in new window or tab >>A Delta clpB Mutant of Francisella tularensis Subspecies holarctica Strain, FSC200, Is a More Effective Live Vaccine than F. tularensis LVS in a Mouse Respiratory Challenge Model of Tularemia
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. e78671-Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis subsp. tularensis is a highly virulent pathogen for humans especially if inhaled. Consequently, it is considered to be a potential biothreat agent. An experimental vaccine, F. tularensis live vaccine strain, derived from the less virulent subsp. holarctica, was developed more than 50 years ago, but remains unlicensed. Previously, we developed a novel live vaccine strain, by deleting the chaperonin clpB gene from F. tularensis subsp. tularensis strain, SCHU S4. SCHU S4 Delta clpB was less virulent for mice than LVS and a more effective vaccine against respiratory challenge with wild type SCHU S4. In the current study, we were interested to determine whether a similar mutant on the less virulent subsp. holarctica background would also outperform LVS in terms of safety and efficacy. To this end, clpB was deleted from clinical holarctica strain, FSC200. FSC200 Delta clpB had a significantly higher intranasal LD50 than LVS for BALB/c mice, but replicated to higher numbers at foci of infection after dermal inoculation. Moreover, FSC200 Delta clpB killed SCID mice more rapidly than LVS. However, dermal vaccination of BALB/c mice with the former versus the latter induced greater protection against respiratory challenge with SCHU S4. This increased efficacy was associated with enhanced production of pulmonary IL-17 after SCHU S4 challenge.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-85560 (URN)10.1371/journal.pone.0078671 (DOI)000327254700029 ()24236032 (PubMedID)
Available from: 2014-02-07 Created: 2014-02-06 Last updated: 2018-06-08Bibliographically approved
Lindgren, M., Bröms, J. E., Meyer, L., Golovliov, I. & Sjöstedt, A. (2013). The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection. BMC Microbiology, 13, Article ID 20.
Open this publication in new window or tab >>The Francisella tularensis LVS ΔpdpC mutant exhibits a unique phenotype during intracellular infection
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2013 (English)In: BMC Microbiology, ISSN 1471-2180, E-ISSN 1471-2180, Vol. 13, article id 20Article in journal (Refereed) Published
Abstract [en]

Background: A prerequisite for the virulence of the facultative intracellular bacterium Francisella tularensis is effective intramacrophage proliferation, which is preceded by phagosomal escape into the cytosol, and ultimately leads to host cell death. Many components essential for the intracellular life cycle are encoded by a gene cluster, the Francisella pathogenicity island (FPI), constituting a type VI secretion system.

Results: We characterized the FPI mutant ΔpdpC of the live vaccine strain (LVS) of F. tularensis and found that it exhibited lack of intracellular replication, incomplete phagosomal escape, and marked attenuation in the mouse model, however, unlike a phagosomally contained FPI mutant, it triggered secretion of IL-1β, albeit lower than LVS, and markedly induced LDH release.

Conclusions: The phenotype of the ΔpdpC mutant appears to be unique compared to previously described F. tularensis FPI mutants.

Place, publisher, year, edition, pages
BioMed Central, 2013
Keywords
Francisella tularensis, type VI secretion, cytopathogenicity, intracellular replication, PdpC
National Category
Microbiology Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-66129 (URN)10.1186/1471-2180-13-20 (DOI)000314827600002 ()23356941 (PubMedID)
Note

The Francisella tularensis LVS Delta pdpC mutant exhibits a unique phenotype during intracellular infection

Available from: 2013-02-15 Created: 2013-02-15 Last updated: 2018-06-08Bibliographically approved
Tancred, L., Telepnev, M., Golovlev, I., Andersson, B., Andersson, H., Lindgren, H. & Sjöstedt, A. (2011). Administration of a donor of nitric oxide inhibits mglA expression of intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion. Journal of Medical Microbiology, 30(11), 1570-1583
Open this publication in new window or tab >>Administration of a donor of nitric oxide inhibits mglA expression of intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion
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2011 (English)In: Journal of Medical Microbiology, ISSN 0022-2615, E-ISSN 1473-5644, Vol. 30, no 11, p. 1570-1583Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide or 3-morpholinosydnonimine hydrochloride (SIN-1), which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50- to 100-fold. F. tularensis-infected J774 cells are incapable of secreting TNF-alpha in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-alpha secretion of J774 cells. Strong staining for nitrotyrosine was observed in SNAP-treated bacteria and mass spectrometry identified nitration of two ribosomal 50S proteins, a CBS domain pair protein, and bacterioferritin. The results demonstrate that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in nitration of several F. tularensis proteins.

Keywords
Francisella tularensis, macrophage activation, TNF-α, Igl proteins, nitric oxide
National Category
Microbiology in the medical area
Research subject
Microbiology
Identifiers
urn:nbn:se:umu:diva-45864 (URN)10.1099/jmm.0.032870-0 (DOI)000296547900002 ()21700740 (PubMedID)
Available from: 2011-08-19 Created: 2011-08-19 Last updated: 2018-06-08Bibliographically approved
Conlan, J. W., Shen, H., Golovliov, I., Zingmark, C., Oyston, P. C., Chen, W., . . . Sjöstedt, A. (2010). Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization. Vaccine, 28(7), 1824-1831
Open this publication in new window or tab >>Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization
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2010 (English)In: Vaccine, ISSN 0264-410X, E-ISSN 1873-2518, Vol. 28, no 7, p. 1824-1831Article in journal (Refereed) Published
Abstract [en]

Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4DeltaclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4DeltaclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Delta0918DeltacapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4DeltaclpB, or SCHU S4Delta0918DeltacapB provided no obvious correlate to their relative efficacies.

Keywords
Francisella tularensis; Mutants; Vaccines; Aerosol
National Category
Immunology in the medical area
Identifiers
urn:nbn:se:umu:diva-42613 (URN)10.1016/j.vaccine.2009.12.001 (DOI)000275158500024 ()20018266 (PubMedID)
Available from: 2011-04-11 Created: 2011-04-11 Last updated: 2018-06-08Bibliographically approved
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