Articles
Cite This: ACS Chem. Biol. 2018, 13, 1890−1896
Unique Fluorescent Imaging Probe for Bacterial Surface Localization
and Resistant Enzyme Imaging
Hui Ling Chan,† Linna Lyu,† Junxin Aw,† Wenmin Zhang,†,‡ Juan Li,‡ Huang-Hao Yang,‡
Hirohito Hayashi,† Shunsuke Chiba,† and Bengang Xing*,†,‡
†
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Division of Chemistry and Biological Chemistry, School of Physical & Mathematical Sciences, Nanyang Technological University,
Singapore, 637371, Singapore
‡
College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China
S Supporting Information
*
ABSTRACT: Emergence of antibiotic bacterial resistance has caused serious
clinical issues worldwide due to increasingly difficult treatment. Development of a
specific approach for selective visualization of resistant bacteria will be highly
significant for clinical investigations to promote timely diagnosis and treatment of
bacterial infections. In this article, we present an effective method that not only is
able to selectively recognize drug resistant AmpC β-lactamases enzyme but, more
importantly, is able to interact with bacterial cell wall components, resulting in a
desired localization effect on the bacterial surface. A unique and specific enzymeresponsive cephalosporin probe (DFD-1) has been developed for the selective
recognition of resistance bacteria AmpC β-lactamase, by employing fluorescence
resonance energy transfer with an “off−on” bioimaging. To achieve the desired
localization, a lipid−azide conjugate (LA-12) was utilized to facilitate its penetration
into the bacterial surface, followed by copper-free click chemistry. This enables the
probe DFD-1 to be anchored onto the cell surface. In the presence of AmpC
enzymes, the cephalosporin β-lactam ring on DFD-1 will be hydrolyzed, leading to the quencher release, thus generating
fluorescence for real-time resistant bacterial screening. More importantly, the bulky dibenzocyclooctyne group in DFD-1 allowed
selective recognition toward the AmpC bacterial enzyme instead of its counterpart (e.g., TEM-1 β-lactamase). Both live cell
imaging and cell cytometry assays showed the great selectivity of DFD-1 to drug resistant bacterial pathogens containing the
AmpC enzyme with significant fluorescence enhancement (∼67-fold). This probe presented promising capability to selectively
localize and screen for AmpC resistance bacteria, providing great promise for clinical microbiological applications.
W
lactam drugs by resistance bacteria transmitting in human
communities.11,12 So far, extensive investigations specific to
class A Blas have been widely conducted.13−17 Although a
similar serine hydroxyl group and geometries of active sites are
observed in both class A and C Blas enzymes, their detailed
secondary structures indicated a significant difference in the
pocket size and the amino acid residue arrangement.18−20
Critically, recent outbreaks of resistance in bacterial species
previously not expressing class C Blas have emerged due to
conjugative transmission of a bacterial plasmid, which led to a
heightened clinical risk of antibiotic resistance in hospitals
globally.21−24 Therefore, a simple and unique approach that is
capable of selectively identifying class C Blas enzyme activities
will be clinically important in combatting microbial resistance.
Over the years, various effective strategies have been
developed to facilitate Blas drug resistant enzyme investiga-
idespread antibiotic resistance among pathogenic
bacteria is a critical problem currently faced by both
hospitals and community health worldwide, thus placing
individuals’ health at stake on both an international and
national scale.1−3 One of the primary defense mechanisms that
antibiotic resistance bacteria possess is the production of drug
resistant enzymes, e.g., β-lactamases (Blas), a family of bacterial
enzymes that specifically break down β-lactam antibiotic
structure.4,5 Generally, such resistant Blas enzymes can hinder
the antibiotic ability to inhibit penicillin-binding proteins for
cell wall biosynthesis, hence rendering these drugs ineffective
for antibacterial treatment.6,7 Therefore, extensive investigation
is still required. Additionally, the development of specific
approaches for selective recognition of resistance bacteria to
gain a better understanding of the biochemical processes
conferring bacterial drug resistance will be highly desirable.
In terms of distinct expression patterns and related catalytic
mechanisms, Blas enzymes can be categorized into four
different Ambler classes: A, B, C, and D, each with its unique
enzyme sequence conferring intrinsic antibiotic bacterial
resistance.8−10 Among these enzyme classes, class A and C
Blas are considered the most significant in hydrolysis of β© 2018 American Chemical Society
Special Issue: Sensors
Received: February 20, 2018
Accepted: March 29, 2018
Published: March 29, 2018
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tions.25−29 Among which, fluorescence optical imaging has
been widely employed due to its promising possibilities in
offering accurate and reliable results in real-time monitoring of
biochemical functions and resolving drug-resistant bacterial
pathogens in vitro and in vivo.30,31 Despite great dependability,
most of the established methods through chemical-based probe
molecules may have concerns for real-time monitoring of the
dynamics of enzyme functions, or limited precision for the
accurate detection of bacterially resistant processes within a
targeted and localized region which is mainly attributed to the
multidimensional, heterogeneous, and spatial complexity of the
cellular environment. Moreover, the inevitable diffusion of the
chemical probe during the imaging process may present a
potential issue of specificity or limited spatial resolution for
real-time imaging analysis in living systems. Although several
localization strategies based on covalent labeling or enzyme
triggered probe molecule aggregation have been previously
proposed to selectively report different resistant enzyme
expression in bacterial pathogens;32−35 these methods may
potentially induce perturbation toward bacterial structure and
functions in living settings. Therefore, the development of a
simple and efficient method that allows effective probe
localization on the bacterial surface, especially for the analysis
of drug resistant bacteria, remains a technical challenge in the
field, and extensive studies are required.
Fatty acid lipid molecules are naturally expressed cell surface
components that are well recognized as integral membrane
structures with a close relation to cell wall stiffness and
susceptibility to various types of cell functions.36,37 So far,
incorporation of fatty acid lipidated groups with therapeutic
moieties and contrast agents have been proposed to achieve an
enhanced pharmacokinetic profile, improved treatment efficacy,
as well as cellular structure-localized imaging to track real-time
enzymatic dynamics and subcellular organelle position.38−47
Inspired by the promising capability of such unique cell surface
components, in this study, we present an effective method that
can selectively recognize class C drug resistant Blas and, more
importantly, interacts with bacterial cell components, allowing
the desired localization effect to be obtained (Scheme 1).
Effective immobilization of the probe onto the bacterial surface
was achieved through insertion of the lipidated fatty acid chain,
which is a part of the cell membrane component’s favorable
structure. Typically, a FRET pair, fluorescein isothiocyanate
(FITC) fluorophore as a donor and 4-(4,2-dimethylaminophe-
nylazo) benzoic acid (DABCYL) as a quencher, can be utilized
to amplify enzyme activity. Under the presence of class C Blas
(e.g., AmpC), the cephalosporin β-lactam ring in DFD-1 was
cleaved, releasing the 3′-position conjugated DABCYL. Such
enzyme triggered probe release led to fluorescence enhancement and allowed real-time visualization of resistance bacteria.
To attain AmpC enzyme selectivity, a bulky dibenzocyclooctyne (DBCO) group was attached to the cephalosporin βlactam ring 7′-position to introduce steric hindrance, which
may enable selective recognition toward AmpC Blas. More
importantly, through fatty acid chain exploitation, insertion of
the lipid into the bacterial surface can allow the observation of a
desired localization. To achieve such a localization property, we
employed a lipid-azide conjugate for its penetration into the
bacterial cell surface, followed by copper-free click chemistry,
which immobilized the fluorescence signal onto the bacterial
surface. Such a unique strategy can promote efficient
localization of the fluorescence probe DFD-1 and can thus
greatly reduce the active diffusion of the probe molecules in
bacterial structures, therefore providing great promise for
performing precise and reliable screening of bacterial resistance
in clinical practice.
■
RESULTS AND DISCUSSION
Synthetic Strategy. The synthetic route of probe DFD-1 is
depicted in Scheme 2. Cephalosporin β-lactam 1 was first
linked with Mtt-Lys 3 at the 7′-position to yield Mtt-Lys-Lac 4
(step a). Next, at the 3′-position, cephalosporin was coupled
with Thio-DABCYL 8 (prepared from trityl-protected 4aminothiophenol with DABCYL) to afford 9 (step b).
Subsequently, at the 7′-position of 9, FITC was conjugated
under basic reaction conditions, which was then followed by
installation of the DBCO moiety through amide coupling with
dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCONHS ester) to give the desired product DFD-1. The reaction
mixture was purified by high performance liquid chromatography (HPLC) and characterized by mass spectroscopy analysis
(MS).
Enzymatic Properties. To study the enzyme activity of the
reporter molecule DFD-1, the fluorescence emission was
recorded upon the addition of AmpC Blas in phosphate
buffered saline (PBS) solution (0.1 M, pH = 7.4). As shown in
Figure 1A, under the absence of the Blas enzyme, little
fluorescence was observed with DFD-1 alone due to the
efficient FRET quenching. Notably, with the treatment of the
AmpC enzyme in PBS, significant enhancement in the
fluorescence intensity at 516 nm was detected (∼67-folds)
before and after the enzyme reaction. This indicates the ability
of the AmpC Blas enzyme to efficiently cleave the β-lactam ring
and release the quencher from the cephalosporin structure,
thereby resulting in the observed intensity increment. Moreover, such enzyme cleavage is further supported by the LC-MS
spectral analysis of the fragments corresponding to the
hydrolyzed cephalosporin ring and quencher Dabcyl moieties
at 1062.73 and 377.23, respectively (Supporting Information
Figure S1). In addition, a low fluorescence enhancement was
found in the presence of a typical AmpC inhibitor, aztreonam
(AZT; Figure 1B). Such effective suppression of the AmpC
enzymatic activity resulted in a substantial reduction in the
fluorescence readout, which clearly demonstrates the specificity
of DFD-1 to the AmpC enzyme instead of the spontaneous
degradation of the probe or nonspecific interactions. As a
control, a similar enzymatic analysis was carried out with the
Scheme 1. Bacterial Surface Localization and Enzymatic
Responsive Fluorescence Changes upon the Reaction of
Probe DFD-1 and AmpC Blas
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Scheme 2. Synthesis of the Enzyme Responsive Probe DFD-1a
Reagents and conditions: (a) Mtt-Lys 3, EDC·HCl, HOBt, TEA, CH3CN/dioxane (1:1), 0−23 °C, 12 h; (b) Thio-DABCYL 8, 2,6-lutidine, NaI,
DMF 23 °C, 13 h; (c) (i) 2% v/v TFA, CH2Cl2, 23 °C, 1 h; (ii) FITC, TEA, DMF, 23 °C, 10 h; (d) (i) 12.5% v/v TFA, 2.5% v/v TIPS, CH2Cl2, 23
°C, 4 h; (ii) EDC·HCl, 4-DMAP, DBCO-NHS, DMF, 23 °C, 10 h.
a
microscopy with the excitation at 488 nm (Figure 2A).
Furthermore, to examine whether the lipid moiety chain length
Figure 1. Emission spectra of (A) probe DFD-1 (10 μM) before and
after incubation with AmpC and TEM-1 Blas, respectively (30 nM).
(B) Fluorescence enhancement of DFD-1 incubated with AmpC,
TEM-1 enzymes, inhibitor AZT, and CA (100 μM) in 0.1 M PBS (pH
= 7.4).
use of TEM-1 enzyme, a typical class A Blas, to investigate the
reaction selectivity. As shown in Figure 1A and B, treatment
with the TEM-1 enzyme showed weak fluorescence activity
after enzyme reaction. Further analysis of enzyme kinetics was
also conducted to determine the activity of both AmpC and
TEM-1 Blas (Supporting Information Figure S2). The probe
DFD-1 cleavage was identified with the Michaelis constant, Km
= 7.4 μM and 10.0 μM, and the catalytic constant, kcat = 142.9
min−1 and 47.6 min−1, for AmpC and TEM-1, respectively.
Hence, these results clearly demonstrated that the synthesized
DFD-1 exhibited a greater specificity and selectivity recognition
toward AmpC Blas rather than to the TEM-1 counterpart,
mostly attributed to the additional bulky DBCO moiety within
the DFD-1 cephalosporin 7′-position that could accommodate
well into the larger binding pocket located in the AmpC Blas
enzyme structure.18−20
Ability of Lipids to Anchor onto Bacterial Surface. To
investigate the feasibility of the enzyme responsive probe
molecule to be efficiently anchored onto the bacterial surface
for facilitated cell structure immobilization, we first employed a
fatty acid molecule, an intrinsic lipid component of the bacterial
surface as the targeting moiety modified with an azide group,
for incubation with the bacterial pathogens. As a proof of
concept in this study, two commonly used bacterial strains, the
Gram-negative Pseudomonas aeruginosa PAO1 (P. aeruginosa
PAO1) and Gram-positive Enterococcus faecium (E. faecium)
were chosen. Both are well-known as typical pathogens for
bacterial resistance studies.48−51 Upon bacterial incubation with
the lipid, a simple fluorescence molecule, DBCO-FITC (DF),
was subsequently conjugated through a copper-free click
reaction. The fluorescence images were recorded to investigate
the effective bacterial surface insertion by using confocal
Figure 2. (A) Scheme of bacterial insertion with lipid of LA-18, LA12, and LA-6, followed by click reaction with DF. (B) Confocal
imaging of lipid screening in P. aeruginosa PAO1 and E. faecium
bacterial strains upon treatment of lipid moieties (LA-18, LA-12, and
LA-6) (2 μM) and DF (2 μM) in 0.1 M PBS, pH = 7.4. Scale bar: 5
μm.
may affect bacterial insertion, the azido-coupled fatty acids with
different carbons (N-(2-azidoethyl) stearamide, N-(2-azidoethyl) dodecanamide, and N-(2-azidoethyl) hexanamide abbreviated as LA-18, LA-12, and LA-6, respectively; Supporting
Information Scheme S2) were also synthesized, and their
subsequent bacterial incubations were carried out for imaging
analysis.
As shown in Figure 2B, the P. aeruginosa PAO1 strain, there
was a weak fluorescence observed in fatty acid moiety LA-18
incubated bacteria when compared to the pathogens treated
with LA-6 and LA-12 structures. Although both bacteria treated
with LA-6 and LA-12 moieties led to an increment in
fluorescence signal, LA-12 exhibited a stronger fluorescence
intensity than that of LA-6. Similarly, for the E. faecium bacteria,
among three fatty acids moieties, LA-12 demonstrated the
strongest fluorescence for lipid facilitated bacterial imaging
(Figure 2B and Supporting Information Figure S3). The results
indicated that the increased length of the carbon chain could
likely enhance the immobilization of the probe onto the
bacterial surface. The shorter hydrophobic chain in LA-6 would
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Figure 3. Fluorescence imaging of selective enzyme reaction and bacterial surface localization in living bacteria: antibiotic susceptible strains S. aureus
and P. putida OUS82, TEM-1 Blas producing MRSA BAA-44 and E. faecium, as well as AmpC Blas producing P. aeruginosa PAO1 and E. cloacae,
with subsequent treatment of LA-12 (50 μM) and DFD-1 (10 μM) in 0.1 M PBS, pH = 7.4. Scale bar: 5 μm.
producing bacterial strains in P. aeruginosa PAO1 and E. cloacae.
On the other hand, similar bacterial treatment with LA-12 and
DFD-1 in the TEM-1 Blas enzyme expressing MRSA BAA-44
and E. faecium strains only resulted in weak fluorescence signals.
Importantly, the pretreatment of targeted strains producing the
AmpC enzyme (e.g., P. aeruginosa PAO1) with a typical class C
Blas AZT inhibitor greatly reduced the fluorescence intensity in
bacterial imaging, while similar incubation of P. aeruginosa
PAO1 bacteria with class A TEM-1 enzyme inhibitor CA
demonstrated little effect on the fluorescence signal (Supporting Information Figure S5). In the negative control, there was
almost no fluorescence detected in antibiotic susceptible S.
aureus and P. putida OUS82 bacteria under treatment with the
probe molecule (Figure 3). Importantly, there was minimum
effect observed in inhibition of bacterial growth during the
bacterial imaging by using the DFD-1 probe molecule
(Supporting Information Table S1). These results clearly
enforced the capability of the designed enzymatic substrate
DFD-1 as a safe probe molecule for the selective recognition of
AmpC enzyme activities in live bacterial strains. Moreover,
compared to the imaging of bacteria strains treated with the
LA-12 lipid moiety and DF, the bright fluorescence signals
observed in P. aeruginosa PAO1 and E. cloacae strains further
confirmed the localization ability of LA-12 that facilitated
immobilization of the enzyme responsive probe onto the
bacterial surface (Figure 3 and Supporting Information Figure
S6).
Enzyme Activity and Click Reaction Contribution
toward Imaging. In order to investigate the fluorescent
staining effects of enzyme hydrolysis and click chemistry
triggered bacterial localization contributed to live bacterial
imaging, we compared these individual performances in the
drug resistance bacteria (e.g., P. aeruginosa PAO1) with AmpC
Blas expression. In this typical study, the bacterial strain P.
aeruginosa PAO1 was first incubated with the LA-12 lipid
moiety for 1 h at 37 °C, followed by subsequent addition of
DF. The bacterial surface immobilization triggered by the
copper-free click chemistry reaction was determined by
measuring the fluorescence change at different time durations.
Alternatively, the enzyme cleavage activity was also carried out
in a similar manner, through incubation of LA-12 lipid-labeled
lead to less staining, which could be easily washed away, giving
decreased fluorescence intensity. Although LA-18 could also
show an enhanced tracking ability on the bacterial surface, this
azido-coupled lipid moiety indicated limited solubility in
aqueous solutions, which may potentially affect cell activity
and reduce the effective concentration for bacterial imaging.
These fluorescent staining studies in both P. aeruginosa PAO1
and E. faecium bacteria clearly showed the lipid moiety LA-12
as the ideal tracer to specifically immobilize DF onto the
bacterial cell surfaces, which may thus greatly facilitate dynamic
visualization of drug-resistant enzymes in living bacteria.
Additionally, staining of LA-12 treated bacterial strains with
propidium iodide (PI) showed minimum bacterial perturbation
(Supporting Information Figure S4). These studies unequivocally indicated the great possibilities of LA-12 as a promising
localizing agent for bacterial imaging under living conditions.
Imaging Resistant Bacterial with LA-12 and DFD-1.
Encouraged by the specific enzymatic hydrolysis of DFD-1 as
well as the promising bacterial immobilization ability of the LA12 lipid moiety, we investigated the possibility for localization
of the reporter molecular DFD-1 onto the bacterial surface for
real-time imaging of drug resistant enzymes in living bacterial
pathogens (Figure 3). In this typical study, two antibiotic
resistant bacterial strains, Enterobacter cloacae (E. cloacae,
ATCC 13047) and P. aeruginosa PAO1 (ATCC 15692), were
selected as our main targets primarily due to their native
capability to produce class C AmpC Blas enzyme. Another two
drug resistant bacterial pathogens E. faecium (ATCC 51559)
and methicillin-resistant Staphylococcus aureus (MRSA BAA-44,
ATCC BAA44) were utilized as control strains owing to their
high expression levels of class A TEM-1 Blas. In addition, two
antibiotic susceptible strains Pseudomonas putida OUS82 (P.
putida OUS82) and Staphylococcus aureus (S. aureus, ATCC
29213) without Blas expression were selected as the negative
control. Typically, these bacterial strains were separately treated
with lipid LA-12 for 1 h at 37 °C. After washing and
subsequent incubation with the DFD-1 probe for another 30
min, the bacterial samples were subjected to confocal
microscopy for fluorescence imaging analysis. As shown in
Figure 3, strong fluorescence emissions were observed upon the
incubation of the DFD-1 probe with the AmpC Blas enzyme
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P. aeruginosa PAO1 bacteria with the probe DFD-1; the
fluorescence change was recorded to evaluate surface localized
enzyme activities. As shown in Figure 4B, under the same
Figure 5. Flow cytometry analysis of resistant bacterial strains
incubated with (A) lipid LA-12 (50 μM) and probe DFD-1 (10
μM) or (B) lipid LA-12 (50 μM) and inhibitor AZT (100 μM),
followed by probe DFD-1 (10 μM) staining in 0.1 M PBS, pH = 7.4.
Concentration of bacterial was approximately 108 cells mL−1.
could efficiently be inserted into the bacterial surface and thus
greatly facilitated the localization of the enzyme triggered
fluorescent signal onto the bacterial surface. By taking
advantage of the bulky DBCO group at the 7′-position of
cephalosporin structure, the fluorescent probe DFD-1 could
selectively recognize the AmpC Blas enzyme. The significant
fluorescence enhancement toward selective detection of the
AmpC Blas enzyme indicated a promising strategy for direct
observation of resistant bacterial infections under living
conditions. More importantly, such lipid facilitated surface
localization of fluorescence labeling could also provide the
promising capability for dynamic monitoring of bacterial
development and assessing effective antibacterial therapeutics
in vitro and in vivo.
Figure 4. (A) Scheme of the different staining effects from the enzyme
hydrolysis and click reaction toward fluorescence imaging in live
bacteria. (B) Comparison of fluorescence intensity between the
enzyme hydrolysis and click chemistry in live bacteria. LA-12 lipidated
(50 μM) P. aeruginosa PAO1 strains were separately treated with DF
and DFD-1 (10 μM), under different time intervals in 0.1 M PBS, pH
= 7.4.
reaction conditions, DF labeling triggered by click reaction
exhibited a relatively higher fluorescence intensity than the
fluorescence generated from the enzyme reaction. The
fluorescence profiles implied that both enzyme hydrolysis and
click chemistry triggered surface insertion would contribute to
the bacterial imaging, and the click coupling would facilitate the
imaging probe staining on the bacterial surface for effective
imaging studies. (Figure 4).
Flow Cytometry Analysis. Additionally, we studied the
possibility to quantify the specific labeling of AmpC Blas
expressing resistant bacteria (e.g., P. aeruginosa PAO1) using
flow cytometer analysis (FCM). The antibiotic susceptible S.
aureus and resistant MRSA BAA-44 strains with expression of
TEM-1 Blas were used as the negative control (Figure 5). In
this study, the different strains were first incubated with the LA12 moiety and subsequently treated with DFD-1 for flow
cytometry analysis. Fluorescence signals were collected at 525
nm. As shown in Figure 5A, a stronger intensity of fluorescence
was observed in the AmpC expressing P. aeruginosa PAO1
strain compared to the negative control S. aureus bacteria that
do not produce β-lactamase. Meanwhile, a lower fluorescence
change was detected for the TEM-1 producing MRSA BAA-44
strain. Moreover, a similar flow cytometry analysis with the
treatment of AmpC enzyme inhibitor AZT showed a significant
decrease in the fluorescence intensity (Figure 5B). Therefore,
these data clearly demonstrate the specificity of DFD-1 as a
reliable reporter molecule for quantification of AmpC activities
in antibiotic resistant bacteria.
In conclusion, this work presented a specific and selective
approach toward efficient bacterial surface localization and realtime imaging of drug resistant bacteria with AmpC enzyme
expression. In this study, the optimized lipid moiety (LA-12)
■
■
METHODS
See the Supporting Information for a detailed description of the
experimental methods.
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acschembio.8b00172.
Supplemental experimental procedures, figures, data, and
spectra for compounds (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +65-6316 8758. Fax: + 65-6316 6984. E-mail:
bengang@ntu.edu.sg.
ORCID
Juan Li: 0000-0001-7175-2320
Huang-Hao Yang: 0000-0001-5894-0909
Shunsuke Chiba: 0000-0003-2039-023X
Bengang Xing: 0000-0002-8391-1234
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was partially supported by NTU-AIT-MUV NAM/
16001, Tier 1 RG110/16 (S), RG 11/13, and RG 35/15
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awarded in Nanyang Technological University, Singapore and
National Natural Science Foundation of China (NSFC; No.
51628201). We thank L. Yang at the Singapore Centre for
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