scholarly journals Phagocytic Receptors Dictate Phagosomal Escape and Intracellular Proliferation of Francisella tularensis

2011 ◽  
Vol 79 (6) ◽  
pp. 2204-2214 ◽  
Author(s):  
Henriette Geier ◽  
Jean Celli
Keyword(s):  
Francisella Tularensis ◽  
Scavenger Receptor ◽  
Mannose Receptor ◽  
Dependent Manner ◽  
Content Type ◽  
Murine Bone Marrow ◽  
Delayed Maturation ◽  
Schu S4 ◽  
Intracellular Proliferation

ABSTRACTFrancisella tularensis, the causative agent of tularemia, survives and proliferates within macrophages of the infected host as part of its pathogenic strategy, through an intracellular life cycle that includes phagosomal escape and extensive proliferation within the macrophage cytosol. Variousin vitromodels ofFrancisella-macrophage interactions have been developed, using either opsonic or nonopsonic phagocytosis, and have generated discrepant results on the timing and extent ofFrancisellaphagosomal escape. Here we have investigated whether either complement or antibody opsonization of the virulent prototypical type A strainFrancisella tularensissubsp.tularensisSchu S4 affects its intracellular cycle within primary murine bone marrow-derived macrophages. Opsonization of Schu S4 with either human serum or purified IgG enhanced phagocytosis but restricted phagosomal escape and intracellular proliferation. Opsonization of Schu S4 with either fresh serum or purified antibodies redirected bacteria from the mannose receptor (MR) to the complement receptor CR3, the scavenger receptor A (SRA), and the Fcγ receptor (FcγR), respectively. CR3-mediated uptake delayed maturation of the earlyFrancisella-containing phagosome (FCP) and restricted phagosomal escape, while FcγR-dependent phagocytosis was associated with superoxide production in the early FCP and restricted phagosomal escape and intracellular growth in an NADPH oxidase-dependent manner. Taken together, these results demonstrate that opsonophagocytic receptors alter the intracellular fate ofFrancisellaby delivering bacteria through phagocytic pathways that restrict phagosomal escape and intracellular proliferation.

scholarly journals Restricted cytosolic growth of Francisella tularensis subsp. tularensis by IFN-γ activation of macrophages

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 327-339 ◽  
Author(s):  
Jessica A. Edwards ◽  
Dedeke Rockx-Brouwer ◽  
Vinod Nair ◽  
Jean Celli
Keyword(s):  
Francisella Tularensis ◽  
Murine Bone Marrow ◽  
Bacterial Proliferation ◽  
Phagocyte Nadph Oxidase ◽  
Human Blood Monocyte ◽  
Oxide Synthase ◽  
Schu S4 ◽  
Intracellular Proliferation

The intracellular bacterium Francisella tularensis ensures its survival and proliferation within phagocytes of the infected host through phagosomal escape and cytosolic replication, to cause the disease tularemia. The cytokine interferon-γ (IFN-γ) is important in controlling primary infections in vivo, and in vitro intracellular proliferation of Francisella in macrophages, but its actual effects on the intracellular cycle of the bacterium are ambiguous. Here, we have performed an extensive analysis of the intracellular fate of the virulent F. tularensis subsp. tularensis strain Schu S4 in primary IFN-γ-activated murine and human macrophages to understand how this cytokine controls Francisella proliferation. In both murine bone marrow-derived macrophages (muBMMs) and human blood monocyte-derived macrophages (MDMs), IFN-γ controlled bacterial proliferation. Schu S4 growth inhibition was not due to a defect in phagosomal escape, since bacteria disrupted their phagosomes with indistinguishable kinetics in both muBMMs and MDMs, regardless of their activation state. Rather, IFN-γ activation restricted cytosolic replication of Schu S4 in a manner independent of reactive oxygen or nitrogen species. Hence, IFN-γ induces phagocyte NADPH oxidase Phox- and inducible nitric oxide synthase (iNOS)-independent cytosolic effector mechanisms that restrict growth of virulent Francisella in macrophages.

scholarly journals Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Philip M. Ireland ◽  
Helen L. Bullifent ◽  
Nicola J. Senior ◽  
Stephanie J. Southern ◽  
Zheng Rong Yang ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Insertion Site ◽  
Therapeutic Drug ◽  
Content Type ◽  
Fischer 344 ◽  
A Genome ◽  
Fischer 344 Rat ◽  
Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

scholarly journals A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

2011 ◽  
Vol 79 (9) ◽  
pp. 3665-3676 ◽  
Author(s):  
Brian C. Russo ◽  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Deanna M. Schmitt ◽  
Matthew J. Brown ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Febrile Illness ◽  
Host Cells ◽  
High Morbidity ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Schu S4

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agentFrancisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by whichF. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions ofF. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis ofF. tularensissubsp.holarcticalive vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 inF. tularensissubsp.tularensisSchu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophagesin vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuatedin vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as anF. tularensisgene important for virulence and evasion of the host immune response.

scholarly journals Kdo Hydrolase Is Required for Francisella tularensis Virulence and Evasion of TLR2-Mediated Innate Immunity

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Nihal A. Okan ◽  
Sabina Chalabaev ◽  
Tae-Hyun Kim ◽  
Avner Fink ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immunity ◽  
Innate Immune Response ◽  
Francisella Tularensis ◽  
Innate Immune ◽  
Content Type ◽  
Tnf Α ◽  
Schu S4 ◽  
Highly Virulent

ABSTRACT The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification—Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal—has recently been reported in F. tularensis. We found that the ∆kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

scholarly journals Gallium Potentiates the Antibacterial Effect of Gentamicin against Francisella tularensis

2015 ◽  
Vol 60 (1) ◽  
pp. 288-295 ◽  
Author(s):  
Helena Lindgren ◽  
Anders Sjöstedt
Keyword(s):  
Francisella Tularensis ◽  
Iron Uptake ◽  
Combined Treatment ◽  
Bactericidal Effect ◽  
Intracellular Growth ◽  
Content Type ◽  
Intimate Link ◽  
Schu S4

ABSTRACTThe reasons why aminoglycosides are bactericidal have not been not fully elucidated, and evidence indicates that the cidal effects are at least partly dependent on iron. We demonstrate that availability of iron markedly affects the susceptibility of the facultative intracellular bacteriumFrancisella tularensisstrain SCHU S4 to the aminoglycoside gentamicin. Specifically, the intracellular depots of iron were inversely correlated to gentamicin susceptibility, whereas the extracellular iron concentrations were directly correlated to the susceptibility. Further proof of the intimate link between iron availability and antibiotic susceptibility were the findings that a ΔfslAmutant, which is defective for siderophore-dependent uptake of ferric iron, showed enhanced gentamicin susceptibility and that a ΔfeoBmutant, which is defective for uptake of ferrous iron, displayed complete growth arrest in the presence of gentamicin. Based on the aforementioned findings, it was hypothesized that gallium could potentiate the effect of gentamicin, since gallium is sequestered by iron uptake systems. The ferrozine assay demonstrated that the presence of gallium inhibited >70% of the iron uptake. Addition of gentamicin and/or gallium to infected bone marrow-derived macrophages showed that both 100 μM gallium and 10 μg/ml of gentamicin inhibited intracellular growth of SCHU S4 and that the combined treatment acted synergistically. Moreover, treatment ofF. tularensis-infected mice with gentamicin and gallium showed an additive effect. Collectively, the data demonstrate that SCHU S4 is dependent on iron to minimize the effects of gentamicin and that gallium, by inhibiting the iron uptake, potentiates the bactericidal effect of gentamicinin vitroandin vivo.

scholarly journals FTT0831c/FTL_0325 Contributes to Francisella tularensis Cell Division, Maintenance of Cell Shape, and Structural Integrity

2014 ◽  
Vol 82 (7) ◽  
pp. 2935-2948 ◽  
Author(s):  
Gregory T. Robertson ◽  
Elizabeth Di Russo Case ◽  
Nicole Dobbs ◽  
Christine Ingle ◽  
Murat Balaban ◽  
...  
Keyword(s):  
Cell Wall ◽  
Francisella Tularensis ◽  
Structural Integrity ◽  
Content Type ◽  
Mueller Hinton ◽  
Host Innate Immune Response ◽  
Altered Surface ◽  
Schu S4

ABSTRACTTheFrancisellaFTT0831c/FTL_0325 gene encodes amino acid motifs to suggest it is a lipoprotein and that it may interact with the bacterial cell wall as a member of the OmpA-like protein family. Previous studies have suggested that FTT0831c is surface exposed and required for virulence ofFrancisella tularensisby subverting the host innate immune response (M. Mahawar et al., J. Biol. Chem. 287:25216–25229, 2012). We also found that FTT0831c is required for murine pathogenesis and intramacrophage growth of Schu S4, but we propose a different model to account for the proinflammatory nature of the resultant mutants. First, inactivation of FTL_0325 from live vaccine strain (LVS) or FTT0831c from Schu S4 resulted in temperature-dependent defects in cell viability and morphology. Loss of FTT0831c was also associated with an unusual defect in lipopolysaccharide O-antigen synthesis, but loss of FTL_0325 was not. Full restoration of these properties was observed in complemented strains expressing FTT0831cin trans, but not in strains lacking the OmpA motif, suggesting that cell wall contact is required. Finally, growth of the LVS FTL_0325 mutant in Mueller-Hinton broth at 37°C resulted in the appearance of membrane blebs at the poles and midpoint, prior to the formation of enlarged round cells that showed evidence of compromised cellular membranes. Taken together, these data are more consistent with the known structural role of OmpA-like proteins in linking the OM to the cell wall and, as such, maintenance of structural integrity preventing altered surface exposure or release of Toll-like receptor 2 agonists during rapid growth ofFrancisellain vitroandin vivo.

scholarly journals The Fluorocycline TP-271 Is Efficacious in Models of Aerosolized Francisella tularensis SCHU S4 Infection in BALB/c Mice and Cynomolgus Macaques

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Trudy H. Grossman ◽  
Michael S. Anderson ◽  
David Christ ◽  
Melanie Gooldy ◽  
Lisa N. Henning ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Respiratory Infections ◽  
Vehicle Control ◽  
Primate Model ◽  
Once Daily ◽  
Content Type ◽  
Aerosol Exposure ◽  
Cynomolgus Macaques ◽  
Schu S4

ABSTRACT TP-271 is a novel, fully synthetic fluorocycline in development for complicated bacterial respiratory infections. TP-271 was active in vitro against a panel of 29 Francisella tularensis isolates, showing MICs against 50% and 90% of isolates of 0.25 and 0.5 μg/ml, respectively. In a mouse model of inhalational tularemia, animals were exposed by aerosol to 91 to 283 50% lethal doses (LD50)/mouse of F. tularensis SCHU S4. Following 21 days of once-daily intraperitoneal dosing with TP-271 at 3, 6, 12, and 18 mg/kg of body weight/day, initiating at 24 h postchallenge, survival was 80%, 100%, 100%, and 100%, respectively. When treatment was initiated at 72 h postchallenge, survival was 89%, 100%, 100%, and 100% in the 3-, 6-, 12-, and 18-mg/kg/day TP-271 groups, respectively. No mice treated with the vehicle control survived. Surviving mice treated with TP-271 showed little to no relapse during 14 days posttreatment. In a nonhuman primate model of inhalational tularemia, cynomolgus macaques received an average aerosol exposure of 1,144 CFU of F. tularensis SCHU S4. Once-daily intravenous infusion with 1 or 3 mg/kg TP-271, or vehicle control, for 21 days was initiated within 6 h of confirmed fever. All animals treated with TP-271 survived to the end of the study, with no relapse during 14 days after the last treatment, whereas no vehicle control-treated animals survived. The protection and low relapse afforded by TP-271 treatment in these studies support continued investigation of TP-271 for use in the event of aerosolized exposure to F. tularensis.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

Cerebrovascular characterization of clazosentan, the first nonpeptide endothelin receptor antagonist clinically effective for the treatment of cerebral vasospasm. Part I: Inhibitory effect on endothelinA receptor—mediated contraction

2005 ◽  
Vol 102 (6) ◽  
pp. 1101-1107 ◽  
Author(s):  
Hartmut Vatter ◽  
Michael Zimmermann ◽  
Veronika Tesanovic ◽  
Andreas Raabe ◽  
Lothar Schilling ◽  
...  
Keyword(s):  
Receptor Antagonist ◽  
Cerebral Vasospasm ◽  
Isometric Force ◽  
Inhibitory Effect ◽  
Affinity Constant ◽  
Dependent Manner ◽  
Content Type ◽  
The Right ◽  
Fine Print

Object. The central role of endothelin (ET)—1 in the development of cerebral vasospasm after subarachnoid hemorrhage is indicated by the successful treatment of this vasospasm in several animal models by using selective ETA receptor antagonists. Clazosentan is a selective ETA receptor antagonist that provides for the first time clinical proof that ET-1 is involved in the pathogenesis of cerebral vasospasm. The aim of the present investigation was, therefore, to define the pharmacological properties of clazosentan that affect ETA receptor—mediated contraction in the cerebrovasculature. Methods. Isometric force measurements were performed in rat basilar artery (BA) ring segments with (E+) and without (E−) endothelial function. Concentration effect curves (CECs) were constructed by cumulative application of ET-1 or big ET-1 in the absence or presence of clazosentan (10−9, 10−8, and 10−7 M). The inhibitory potency of clazosentan was determined by the value of the affinity constant (pA2). The CECs for contraction induced by ET-1 and big ET-1 were shifted to the right in the presence of clazosentan in a parallel dose-dependent manner, which indicates competitive antagonism. The pA2 values for ET-1 were 7.8 (E+) and 8.6 (E−) and the corresponding values for big ET-1 were 8.6 (E+) and 8.3 (E−). Conclusions. The present data characterize clazosentan as a potent competitive antagonist of ETA receptor—mediated constriction of the cerebrovasculature by ET-1 and its precursor big ET-1. These functional data may also be used to define an in vitro profile of an ET receptor antagonist with a high probability of clinical efficacy.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

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