Magnetic Resonance Im

Genetically controlled MRI contrast mechanisms and their prospects in

systems neuroscience research

Gil G. Westmeyera, Alan Jasanoff b,c,d,4aMcGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

bDepartment of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USAcDepartment of Nuclear Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

dBiological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

Received 27 October 2006; accepted 3 November 2006

Abstract

Application of MRI contrast agents to neural systems research is complicated by the need to deliver agents past the blood–brain barrier or

into cells, and the difficulty of targeting agents to specific brain structures or cell types. In the future, these barriers may be wholly or partially

overcome using genetic methods for producing and directing MRI contrast. Here we review MRI contrast mechanisms that have used gene

expression to manipulate MRI signal in cultured cells or in living animals. We discuss both fully genetic systems involving endogenous

biosynthesis of contrast agents, and semi-genetic systems in which expressed proteins influence the localization or activity of exogenous

contrast agents. We close by considering which contrast-generating mechanisms might be most suitable for applications in neuroscience, and

we ask how genetic control machinery could be productively combined with existing molecular agents to enable next-generation

neuroimaging experiments.

D 2007 Elsevier Inc. All rights reserved.

Keywords:Magnetic resonance imaging; fMRI;MRI contrast agent; Nanoparticles; Reporter genes; Ferritin; Hemoglobin; Transferrin receptor;h-Galactosidase; LacZ

1. Introduction

Hemodynamic functional magnetic resonance imaging

(fMRI) is a powerful tool for mapping brain activity patterns

in humans and animals, but its utility for analysis of neural

mechanisms is limited by the complex properties of neuro-

vascular coupling [1]. More informative indicators of neural

function could be produced using MRI contrast agents with

specificity for molecular or cellular signaling events.

Calcium-sensitive contrast agents [2,3] may be suitable for

bmolecular fMRIQ because of the close relationship between

neuronal signaling and intracellular calcium concentration,

but many other approaches are conceivable [4]. Experiments

using molecular neuroimaging agents would combine the

specificity of cellular neuroscience measurement techniques

0730-725X/$ – see front matter D 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.mri.2006.11.027

Abbreviations: fMRI, functional magnetic resonance imaging; Ft,

ferritin; SPIO, superparamagnetic iron oxide; Tf, transferrin; TfR,

transferrin receptor; Hb, hemoglobin; Mb, myoglobin; GFP, green

fluorescent protein; h-gal, h-galactosidase.4 Corresponding author. Tel.: +1 617 452 2538; fax: +1 617 253 0760.

E-mail address: jasanoff@mit.edu (A. Jasanoff).

with the noninvasiveness, whole-brain coverage and rela-

tively high resolution of MRI.

Contrast agent-based imaging strategies have limitations

of their own, however: many agents are laborious or

expensive to synthesize and modify, compounds must be

delivered to their site of action in vivo, and cell- or

compartment-specific targeting (which could significantly

increase the specificity of consequent measurements) is

difficult. The hurdles may be particularly severe for cytosolic

neuroimaging agents, like the synthetic calcium sensors,

which must cross both the blood–brain barrier and cell

membranes to be effective. In such cases, invasive injection

procedures or the use of special vehicles is required [5].

Some of the most promising strategies for addressing

the challenges of delivery and targeting in the context of

optical imaging have involved using genetically encoded

molecules (fluorescent proteins) or marker proteins that

interact with synthetic chromophores, fluorophores or

luminescence-generating substrates. Notable examples in-

clude molecular probes derived from green fluorescent

protein, in vivo protein labeling reagents such as FlAsH

and streptavidin-coated quantum dots [6], and luciferase-

aging 25 (2007) 1004–1010

Fig. 1. Mechanisms for genetic control of MRI contrast. (A) MRI contrast

can be genetically controlled using expression of protein contrast agents (or

in principle RNA contrast agents), without the need for synthesis or

delivery of exogenous agents. In this example, a gene (bottom left) directs

intracellular production of a metalloprotein (red) that becomes a contrast

agent in complex with endogenous metal ions (green). (B) In a

representative semi-genetic system, gene expression leads to synthesis of

a cell surface protein (red) that acts as a receptor for an exogenous contrast

G.G. Westmeyer, A. Jasanoff / Magnetic Resonance Imaging 25 (2007) 1004–1010 1005

based bioluminescent systems [7]. Many optical imaging

agents have been generated by facile protein engineering

techniques that illustrate the advantage of genetic encoding

for design and synthesis. Once produced, genes encoding

the agents have been delivered using transfection methods

or incorporation into transgenic animals. Spatial control of

image contrast in the brain is possible by using targeted

injection of transfection vectors or nongenetic contrast-

related factors. Cell-specific targeted expression can be

accomplished with promoter-based methods [8], and

subcellular targeting of protein agents is possible using

appropriate localization sequences.

In the past several years, a number of genetic or bsemi-

geneticQ techniques have also been demonstrated to create

MRI contrast. Additional MRI contrast mechanisms have

been designed around endogenously expressed proteins that

could be adapted to act as genetically encoded reporters. In

this article, we review and categorize systems reported to

produce MRI contrast using genetically encoded molecules.

Our discussion emphasizes bcontrast mechanismsQ as

opposed to contrast agents, because many of the approaches

to genetic control involve molecules that interact with

contrast agents, but do not themselves produce contrast. We

also consider possible applications of genetic MRI contrast

control mechanisms to problems in neuroscience and

speculate about how genetically controlled indicators of

neural function could be constructed using approaches

derived from the current literature.

agent (green) and subsequently promotes internalization of the agent into

vesicles (blue oval). The contrast agent accumulates only in cells expressing

the receptor and influences MRI contrast selectively in their vicinity. This

example is modeled after the TfR-based mechanism of Ref. [19]. (C) A

responsive genetically controlled MRI contrast mechanism can be designed

around protein contrast agents that change their properties in the presence

of a physiological target or environmental parameter. In this hypothetical

example, a contrast agent formed as in (A) from expressed polypeptides and

endogenous metal ions binds reversibly to a target molecule (blue triangle).

Target binding influences the structure of the metalloprotein and changes its

relaxivity (yellow halo). (D) Even relatively low-level gene expression can

influence MRI contrast if the expressed gene encodes an enzyme that

activates (or deactivates) contrast agents; one enzyme molecule can process

many contrast agent molecules, so signal amplification is achieved. In this

example (derived from Ref. [21]), an expressed enzyme (red) cleaves an

exogenous caged contrast agent (black/green) and increases its relaxivity

(yellow halo).

2. Classification of genetically controlled

contrast mechanisms

Genetically controlled MRI contrast mechanisms can

be grouped broadly into those that require exogenous

contrast agents to be supplied and those that only involve

endogenous or ectopically expressed components (Fig. 1A

and B). Subsequent sections of this review classify

contrast mechanisms based on this distinction (see also

Table 1). Scenarios requiring exogenous contrast agents

are somewhat analogous to semi-genetic protein labeling

methods developed for fluorescence microscopy, or to

techniques like lacZ staining that require a synthetic

substrate for an expressed enzyme. These systems do not

forgo problems of synthesis and delivery associated with

whatever compounds must be administered. On the other

hand, they may incorporate particularly robust contrast

agents or contrast amplification strategies that are impos-

sible to construct from endogenous and ectopically

expressed building blocks.

The MRI contrast mechanisms we consider are also

distinguished by other salient qualities (Table 1). A

distinction is to be made between contrast mechanisms

that can respond to environmental variables like calcium,

oxygen and specific molecular targets (Fig. 1C), and those

that do not. Responsive contrast mechanisms may have

particular significance for neuroscientists interested in

detecting correlates of transient brain activity, but insen-

sitive contrast mechanisms may be more useful for

examining spatiotemporal aspects of genetic regulation

itself. Another important difference is between mechanisms

that involve expression of enzymes (Fig. 1D) and those

that involve expression of protein contrast agents or

contrast agent receptors. While expression of protein

contrast agents and receptors might lead more directly to

MRI contrast, expression of enzymes that transport or

activate contrast agents may lead to significantly amplified

contrast, even if protein expression levels are relatively

low — this is because each expressed enzyme molecule

Table 1

Genetically controlled MRI contrast mechanisms

Fully genetic/endogenous

Gene/protein Contrast mechanism Observed change Test system Ref.

Ferritin Sequesters iron from labile

intracellular iron pool and acts as

an intracellular SPIO analog

T2 changes from 45 to 20 ms with

30 Ag Ft expressed per mg of total

protein (14 T)

Cell culture; viral-mediated

transfection in mouse brain

[9]

Tyrosine hydroxylase Catalyzes initial steps in the

synthesis of melanin, which forms

intracellular aggregates in

complex with iron

37% increase in T1-weighted MRI

signal at the highest expression

level used (1.5 T)

Transfected mouse fibroblasts

and HEK cells grown in culture

[13]

Myoglobin Heme protein and T2-based

oxygen sensor naturally expressed

at high levels in myocytes

No statistically significant contrast

with ectopic expression (4.7 T)

Transgenic mouse [10]

Hemoglobin Heme protein that acts as an innate

T2-based oxygen sensor in

erythrocytes

BOLD contrast: T2 changes from

50 to 4 ms with 100% modulation

of blood oxygenation at 7 T

Rat (and others) [26]

Protein amide 1H Amide proton exchange rates vary

depending on pH; aggregate signal

from endogenous protein amide

protons is observed

pH-dependent 50–70% signal

change using saturation

transfer-weighted MRI at 4.7 T

Ischemic rat brain and

gliosarcoma rat model

[28]

Semi-genetic/semi-endogenous

Gene/protein Contrast mechanism Observed change Test system Ref.

Transferrin receptor Transferrin-conjugated SPIO

particles bind and are internalized

by ectopically expressed TfR on

the surface of transfected cells

50% change in T2-weighted MRI

signal at highest TfR expression

levels (3 mg iron injected per

mouse, 7.1 T)

Mice implanted with

TfR-expressing gliosarcoma cells

[19]

Truncated progesterone receptor Gd3+-conjugated RU-486 binds

and activates intracellular

expressed PR

100% greater uptake of 50 AMcontrast agent by PR-expressing

cells; little contrast change

Cultured T47D cells [20]

h-Galactosidase Expressed h-gal cleaves a caged

synthetic Gd3+ compound,

increasing its relaxivity

60% T1-weighted signal increase

with injection of 3.2 nmol of

contrast agent injected per frog

embryo (12 T)

Xenopus laevis embryos

transfected with lacZ

[21]

G.G. Westmeyer, A. Jasanoff / Magnetic Resonance Imaging 25 (2007) 1004–10101006

can process superstoichiometric amounts of the actual

contrast agent.

3. MRI contrast without exogenous agents

Iron is by far the most abundant paramagnetic ion found

in biological systems, and a number of genetically

controlled MRI contrast mechanisms have been designed

to harness endogenous iron. The simplest approach has been

to express iron-containing proteins. Endogenous expression

levels of the iron storage protein ferritin (Ft) have been

known for some time to influence MRI contrast in the brain,

and more recently Genove et al. [9] have shown that ectopic

expression of Ft can be used to manipulate contrast

artificially in vivo. These authors used viral transfection to

introduce Ft genes into mouse brain (Fig. 2A and B). Ft-

expressing cells took up more iron than control cells,

without apparent toxic side-effects. Iron was thought to be

incorporated into partially superparamagnetic Ft cores,

which are similar to synthetic superparamagnetic iron oxide

(SPIO) contrast agents, but have lower relaxivity. The heme

iron protein myoglobin (Mb) has also been overexpressed in

transgenic animals [10]. Mb acts as an oxygen-sensitive

contrast agent in vitro, and oxygen-sensitive imaging using

endogenous Mb contrast in muscle has been performed [11].

In the transgenic mouse study, however, little MRI Mb-

related contrast was observed, probably because expression

levels and relaxivity were not high enough. Oxygen

measurements would therefore not be feasible without

enhancements to the system.

Two approaches have generated contrast using enzymatic

systems and fully endogenous contrast sources. Weissleder

et al. [12,13] investigated whether the accumulation of

melanin, a biopolymer pigment shown to influence MRI

contrast by scavenging paramagnetic ions, could be placed

under genetic control. Melanin is generated in a pathway

initiated by the enzyme tyrosine hydroxylase, which

catalyzes the rate-limiting transformation from tyrosine to

Fig. 2. Image contrast enhanced in vivo by genetic and semi-genetic

methods. Ectopic expression of iron storage protein Ft influences MRI

contrast without the need for exogenous agents [9]: (A) T2-weighted MRI

scan from a mouse (coronal section) 5 days after inoculation with viral

vectors harboring the genes for Ft H and L chains. Signal decrease in the

neighborhood of the injection site is apparent (arrowhead), due to increased

iron localization in cells expressing Ft holomers. (B) Immunohistochem-

istry in sectioned brains was used to visualize the pattern of Ft protein

expression after scanning; MRI signal decreases in (A) appear to correlate

with protein localization (arrowhead) observed in this magnified coronal

section. In a test of a semi-genetic MRI contrast mechanism [19], mice were

implanted with control tumors and tumors expressing human TfR as a

genetically encoded receptor for Tf-conjugated SPIO contrast agents. (C)

Whole-body MRI scan of a mouse with control and test tumors implanted

into the left and right flanks (arrowheads). (D) SPIO contrast agents

injected systemically produced differential T2-weighted MRI signal in the

control and test tumors. The TfR-expressing tumor (left side) appears

darker because of enhanced binding or uptake of the Tf-functionalized

contrast agent.

G.G. Westmeyer, A. Jasanoff / Magnetic Resonance Imaging 25 (2007) 1004–1010 1007

the melanin precursor dopaquinone. These authors showed

that overexpression of tyrosine hydroxylase in cultured

human cells noticeably increased metal uptake and pro-

duced MRI contrast with respect to controls. The second

approach to enzymatic generation of MRI contrast has

involved the overexpression of components from the

magnetotaxis system of Magnetospirillum magneticum.

These bacteria produce intracellular biosynthetic SPIOs

called magnetosomes, using a set of specialized enzymes

and binding proteins. Although results in this area are still

preliminary, it appears from the work of several groups

[14–16] that gene products from the magnetosome synthesis

pathway may be able to generate MRI contrast when

overexpressed ectopically in eukaryotic cells.

4. Genetic control mechanisms involving synthetic

contrast agents

In the bfully geneticQ MRI contrast mechanisms de-

scribed above, ectopic expression of a single gene is

intended to produce MRI contrast changes in transfected

cells or animals. More complex bsemi-geneticQ or bsemi-

endogenousQ systems involve genetic expression of a

protein that indirectly influences MRI contrast through its

interactions with exogenous agents. In these systems, the

exogenous component is typically a contrast agent that

produces more dramatic effects (e.g., has higher relaxivity),

or that can be used in higher concentration, than endogenous

agents. The most straightforward semi-genetic systems

involve expression of a receptor specific for a contrast

agent; the contrast agent binds to the receptor and thus

localizes preferentially at sites where the receptor is

expressed. An early example of this approach was the

use of the transferrin receptor (TfR) by Weissleder et al.

[17–19]. This group showed that expression of TfR on the

surfaces of cells led to uptake of SPIOs conjugated to the

protein transferrin (Tf). The authors implanted mice with

tumor cells transfected with the gene for an engineered form

of TfR. Tf-conjugated SPIOs were injected systemically and

found to create T2 MRI contrast at TfR-transfected tumor

sites, compared with controls (Fig. 2C and D). Another

ectopically expressed receptor imaging strategy was recent-

ly introduced by Lee et al. [20]. These authors generated a

Gd3+-containing analog of the steroid RU-486 and found

that it exhibited enhanced uptake in cultured cells express-

ing a receptor for RU-486. Genetically controlled MRI

contrast mechanisms based on expression of a marker

enzyme have also been developed; these systems allow the

expressed protein to be detected with greater sensitivity than

receptor-based approaches. A paradigmatic example was

described by Louie et al. [21], who generated a caged

contrast agent substrate for the popular marker enzyme

h-galactosidase (h-gal), which is encoded by the lacZ gene.

Cleavage of the caged contrast agent by h-gal increased its

T1 relaxivity significantly. The caged agent was injected

into early stage Xenopus laevis embryos and allowed cells

transfected with lacZ to be distinguished from untransfected

cells based on T1-weighted MRI. Additional lacZ detection

reagents have been reported by Mason et al. [22–24], who

synthesized several fluorinated galactopyranosides designed

to be detected by 19F-based MRI methods. The fluorine

resonances produced by these reagents undergo a chemical

shift change after cleavage by h-gal. Fluorine MRI

techniques required for imaging with these agents produce

much lower signal-to-noise levels than proton MRI used

with relaxation contrast agents, but the fluorine methods

allow the total amount of agent to be measured, in addition

to its cleavage state. This potentially allows more quanti-

tative measures of enzyme activity to be obtained.

Among the semi-genetic MRI contrast systems described

in this section, the TfR system has an advantage in terms of

delivery, because the Tf-conjugated contrast agent binds to

expressed TfR at the cell surface and therefore need not be

G.G. Westmeyer, A. Jasanoff / Magnetic Resonance Imaging 25 (2007) 1004–10101008

cell permeable. In principle, h-gal could also be expressed atthe cell surface (using protein targeting methods); this might

lead to a loss of specificity of the resulting contrast,

however, because the h-gal substrate, once cleaved, would

be free to diffuse throughout the extracellular space.

Himmelreich et al. [25] have proposed an innovative

solution to this problem, by suggesting that lipophilic

contrast agents be used in combination with marker

enzymes that cleave the agents into a trapped soluble form

with greater relaxivity. This strategy remains to be fully

developed, however.

5. Additional systems suitable for genetic manipulation

of contrast

Various proteins have been targeted by contrast agents or

shown to influence MRI contrast without being used

explicitly as markers under genetic control. In neuroscience,

the most famous such protein is hemoglobin (Hb), a

genetically encoded tetramer present at millimolar concen-

trations in red blood cells. Oxygen-sensitive T2 contrast due

to Hb is the basis of the blood oxygenation level dependent

(BOLD) effect in fMRI [26]. Hb has also been used as an

exogenous oxygen-sensitive contrast agent in flies, but it

has not been applied in ectopic expression studies [27]. In

principle, however, cell-specific Hb expression could be

used to sense oxygen tension in the PO2 range from roughly

20 to 40 mm Hg (higher than Mb), assuming that the protein

could be produced without deleterious side-effects. Another

intrinsic protein-related signal has been used for MRI

imaging dependent on tissue pH. Zhou et al. [28] found

that the aggregate proton resonance due to protein amide

backbone protons gives rise to saturation transfer-based

contrast. Because this contrast is not ascribable to any

particular protein, and because of the large amide proton

concentration (2 mM) required for noticeable effects, it

would probably be difficult to adapt this approach to explicit

genetic control.

There are now numerous examples in the literature of

MRI contrast agents targeted towards cell surface receptors

or enzymes. Most of the targeted molecules have been

chosen because of their association with cancers, and some

of these, because they are expressed in healthy tissue at low

abundance, may be suitable marker proteins for genetically

controlled contrast generation. Several receptors would be

candidates for selective imaging strategies analogous to the

TfR-based system described above, though they would not

necessarily lead to cellular uptake of the contrast agent.

These include the Her2/neu receptor [29], LHRH receptor

[30], LDL receptor [31], secretin receptor [32] and EGF

receptor [33]. The Gal80/Gal80BP interaction demonstrated

by De Leon-Rodriguez et al. [34] to affect T1 contrast could

also potentially be used to direct contrast agent binding in a

semi-genetic system. Finally, it might be possible to use a

biotin–streptavidin-based approach [35] to direct labeling of

genetically expressed BirA consensus sequence-tagged

proteins by a contrast agent, in analogy to quantum dot

labeling approaches used in fluorescence imaging [36]. This

kind of system could also be used to genetically target

hyperpolarized imaging agents in the manner pioneered by

Lowery et al. [37].

Various enzymes have been used to manipulate the

aggregation state of SPIO contrast agents and therefore to

influence T2 contrast in vitro. For the most part, this work

has been performed without speculation that the enzymatic

mechanisms could be placed under genetic control. Of the

enzymes used, bacterial restriction endonucleases [38] and

tumor-specific proteases [39] might be the best suited to use

as marker proteins because of their low background activity

in mammals. Chen et al. [40] reported activation of a

gadolinium-based T1 contrast agent (5-hydroxytryptamine-

DTPA-Gd3+) by myeloperoxidase, an enzyme secreted by

neutrophils, monocytes and macrophages during inflamma-

tion. Myeloperoxidase activity triggers the generation of

free radicals in the presence of H2O2 and the authors

showed relaxation rate changes due to localized cross-

linking and immobilization of the contrast agent in the

radical-rich environment. In another example, an enzyme

called TAFI was used to cleave off a masking group from a

human serum albumin (HSA)-binding gadolinium conjugate

[41]. In the presence of HSA and the enzyme, the conjugate

was activated and bound to HSA, leading to an increase in

rotational correlation time and T1 relaxation rate. These

enzymes could be used in analogy to the h-gal-basedamplification systems, though their effects could turn out to

be complicated by unwanted interactions or side effects in

actual experiments.

6. Genetically controlled MRI contrast mechanisms in

neural systems research?

Gene expression is of immense interest in systems

neuroscience research as it is in every other facet of

biology. Many genes are known to be regulated during

neural activity; modulated expression of various genes

(NMDAR, CPGs, etc.) is also closely associated with

learning and memory, as well as developmental plasticity.

The ability to monitor regulation of these genes in three

dimensions, throughout behavioral or developmental time

courses, could be quite important for understanding the

relationship between specific stimuli, tasks and interven-

tions, and the neurophysiological events they influence. A

clear significance of genetically controlled MRI contrast

mechanisms would therefore be to complement, and to

some extent supplant, existing histological methods for

gene expression mapping in neurobiological experiments.

Environmentally insensitive contrast mechanisms incorpo-

rating amplification mechanisms (e.g., tyrosine hydroxy-

lase) may be best suited to this context, because sensitivity

is critical and contrast should ideally reflect only the

expression level of the marker protein(s).

G.G. Westmeyer, A. Jasanoff / Magnetic Resonance Imaging 25 (2007) 1004–1010 1009

Genetically controlled MRI contrast mechanisms were

introduced at the beginning of this article as a possible

solution to the problems of synthesis, delivery and

targeting encountered with synthetic MRI contrast agents.

In systems neuroscience, the contrast agents of paramount

interest are those that detect activity correlates like

voltage fluctuations, synaptic transmission and intracellu-

lar calcium. Genetic methods could be combined with

existing calcium sensors to improve their potential for

neuroimaging applications in two respects: First, ectopi-

cally expressed uncaging enzymes (cf. h-gal) could be

used to activate caged analogs of calcium sensors

selectively in genetically targeted cells. Of course, this

approach would not circumvent the need to deliver the

agents and would require that a new generation of caged

agents be designed and synthesized. Second, in the case

of part protein agents like the SPIO-based contrast

mechanisms [3,38,39], the required protein components

could be selectively expressed only in targeted cells,

while the remaining synthetic component is delivered

systemically to the brain. This approach could simplify

the delivery problem somewhat because a single bone-size-fits-allQ SPIO (or equivalent) could be used to form

multiple different types of sensor; exogenous SPIOs and

endogenous protein domains could be connected using

streptavidin/Strep-tag [42], peptide Velcro [43] or other

specific protein–protein interaction domains. A third

approach would be to modify bfully geneticQ contrast

agents like Ft or Mb to act as responsive molecules —

this would involve potentially difficult protein design

efforts, but would solve synthesis, delivery and targeting

problems more or less in full if successful.

A set of caveats associated with every one of the

genetically controlled contrast generation approaches de-

scribed in this paper is that none is in general use, none has

been demonstrated to work in more than a handful of

contexts, and none has been extensively characterized in

vivo to determine stability, ultrastructural localization,

expressed quantity and kinetics of contrast generation. A

further limitation, inherent to virtually all genetic

approaches, is the incompatibility of these MRI methods

with human subjects. In some sense, this is a particular

drawback because of the otherwise uniquely advantageous

capabilities MRI offers for research involving humans. For

the foreseeable future, genetically controlled contrast

mechanisms will be used only in animals, but it is in

animals that the most advanced imaging and targeting

methods can best be used alongside existing neural

recording techniques to help build mechanistic explanations

of brain function.

Acknowledgments

The authors wish to acknowledge generous support from

the Raymond & Beverly Sackler Foundation and from NIH

grant EB5723 (to AJ).

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