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author:("raji, Hideo")
1.  Feasibility of Amylin Imaging in Pancreatic Islets with β-Amyloid Imaging Probes 
Scientific Reports  2014;4:6155.
Islet amyloid deposition composed of amylin aggregates is regarded as one of the hallmarks of type 2 diabetes mellitus (T2DM). For the diagnosis of T2DM, several nuclear medical imaging probes have been developed. However, there have been no reports regarding the development of imaging probes targeting amylin. In this report, we investigated the feasibility of amylin imaging using [125I]IPBF as one of the model compounds of β-amyloid (Aβ) imaging probes. In in vitro experiments, [125I]IPBF exhibited high binding affinity for amylin aggregates (Kd = 8.31 nM). Moreover, autoradiographic images showed that [125I]IPBF specifically bound to islet amyloid composed of amylin. These results suggest the potential application of Aβ imaging probes to amylin imaging. In addition, [125I]IPBF is one of the promising lead compounds for amylin imaging, and further structural optimization based on [125I]IPBF may lead to useful tracers for the in vivo imaging of islet amyloids in the pancreas.
PMCID: PMC4139942  PMID: 25142178
2.  Radiolabeled Probes Targeting Hypoxia-Inducible Factor-1-Active Tumor Microenvironments 
The Scientific World Journal  2014;2014:165461.
Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α), which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of 18F-FDG or 18F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.
PMCID: PMC4151590  PMID: 25215311
3.  Investigation of cyanine dyes for in vivo optical imaging of altered mitochondrial membrane potential in tumors 
Cancer Medicine  2014;3(4):775-786.
Mitochondrial membrane potential (Δψm) alteration is an important target for cancer diagnosis. In this study, we designed a series of near-infrared fluorescent cationic cyanine dyes with varying alkyl chain lengths (IC7-1 derivatives) to provide diverse lipophilicities and serum albumin-binding rates, and we evaluated the usefulness of these derivatives for in vivo Δψm imaging. IC7-1 derivatives with side chains from methyl to hexyl (IC7-1-Me to IC7-1-He) were synthesized, and their optical properties were measured. Cellular uptake and intracellular distribution were investigated with depolarized HeLa cells from carbonyl cyanine m-chlorophenylhydrazone (CCCP) treatment using a spectrofluorometer and a fluorescence microscope. Serum albumin-binding rates were evaluated using albumin-binding inhibitors. In vivo optical imaging was performed with HeLa cell xenograft mice following intravenous administration of IC7-1 derivatives with or without warfarin and CCCP as in vivo blocking agents. IC7-1 derivatives showing maximum excitation and emission wavelengths at 823 nm and ∼845 nm, respectively, were synthesized. IC7-1-Me to -Bu showed fluorescence in mitochondria that decreased with CCCP treatment in a concentration-dependent manner, which showed that IC7-1-Me to -Bu successfully indicated Δψm. Tumors were clearly visualized after IC7-1-Bu administration. Treatment with warfarin or CCCP significantly decreased IC7-1-Bu fluorescence in the tumor region. In summary, IC7-1-Bu exhibited fluorescence localized to mitochondria dependent on Δψm, which enabled clear in vivo tumor imaging via serum albumin as a drug carrier for effective tumor targeting. Our data suggest that IC7-1-Bu is a promising NIR probe for in vivo imaging of the altered Δψm of tumor cells.
PMCID: PMC4303146  PMID: 24737784
Albumin binding; cancer diagnosis; mitochondrial membrane potential; near infrared; optical imaging
4.  Structure–Activity Relationships and in Vivo Evaluation of Quinoxaline Derivatives for PET Imaging of β-Amyloid Plaques 
ACS Medicinal Chemistry Letters  2013;4(7):596-600.
This letter describes the synthesis, structure–activity relationships, and in vivo evaluation of a new series of 2-phenylquinoxaline (PQ) derivatives for imaging β-amyloid (Aβ) plaques in Alzheimer’s disease (AD). In experiments in vitro, the affinity of the derivatives for Aβ aggregates varied, with Ki values of 0.895 to 1180 nM. In brain sections from AD patients, derivatives with a Ki of less than 111 nM intensely labeled Aβ plaques, while those with values over 242 nM showed no marked labeling. In biodistribution experiments using normal mice, the derivatives showed good uptake into (4.69–7.59 %ID/g at 2 or 10 min postinjection) and subsequent washout from (1.48–3.08 %ID/g at 60 min postinjection) the brain. In addition, [18F]PQ-6 labeled Aβ plaques in vivo in APP transgenic mice, while it showed nonspecific binding in the white matter. Further structural optimization based on [18F]PQ-6 may lead to more useful PET probes for imaging Aβ plaques.
PMCID: PMC4027474  PMID: 24900717
Alzheimer’s disease (AD); β-amyloid (Aβ); PET; quinoxaline; structure−activity relationships
5.  Investigation of a MMP-2 Activity-Dependent Anchoring Probe for Nuclear Imaging of Cancer 
PLoS ONE  2014;9(7):e102180.
Since matrix metalloproteinase-2 (MMP-2) is an important marker of tumor malignancy, we developed an original drug design strategy, MMP-2 activity dependent anchoring probes (MDAP), for use in MMP-2 activity imaging, and evaluated the usefulness of this probe in in vitro and in vivo experiments.
We designed and synthesized MDAP1000, MDAP3000, and MDAP5000, which consist of 4 independent moieties: RI unit (111In hydrophilic chelate), MMP-2 substrate unit (short peptide), anchoring unit (alkyl chain), and anchoring inhibition unit (polyethylene glycol (PEGn; where n represents the approximate molecular weight, n = 1000, 3000, and 5000). Probe cleavage was evaluated by chromatography after MMP-2 treatment. Cellular uptake of the probes was then measured. Radioactivity accumulation in tumor xenografts was evaluated after intravenous injection of the probes, and probe cleavage was evaluated in tumor homogenates.
MDAP1000, MDAP3000, and MDAP5000 were cleaved by MMP-2 in a concentration-dependent manner. MDAP3000 pretreated with MMP-2 showed higher accumulation in tumor cells, and was completely blocked by additional treatment with an MMP inhibitor. MDAP3000 exhibited rapid blood clearance and a high tumor accumulation after intravenous injection in a rodent model. Furthermore, pharmacokinetic analysis revealed that MDAP3000 exhibited a considerably slow washout rate from tumors to blood. A certain fraction of cleaved MDAP3000 existed in tumor xenografts in vivo.
The results indicate the possible usefulness of our MDAP strategy for tumor imaging.
PMCID: PMC4092090  PMID: 25010662
7.  Development of a Radioiodinated Triazolopyrimidine Probe for Nuclear Medical Imaging of Fatty Acid Binding Protein 4 
PLoS ONE  2014;9(4):e94668.
Fatty acid binding protein 4 (FABP4) is the most well-characterized FABP isoform. FABP4 regulates inflammatory pathways in adipocytes and macrophages and is involved in both inflammatory diseases and tumor formation. FABP4 expression was recently reported for glioblastoma, where it may participate in disease malignancy. While FABP4 is a potential molecular imaging target, with the exception of a tritium labeled probe there are no reports of other nuclear imaging probes that target this protein. Here we designed and synthesized a nuclear imaging probe, [123I]TAP1, and evaluated its potential as a FABP4 targeting probe in in vitro and in vivo assays. We focused on the unique structure of a triazolopyrimidine scaffold that lacks a carboxylic acid to design the TAP1 probe that can undergo facilitated delivery across cell membranes. The affinity of synthesized TAP1 was measured using FABP4 and 8-anilino-1-naphthalene sulfonic acid. [125I]TAP1 was synthesized by iododestannylation of a precursor, followed by affinity and selectivity measurements using immobilized FABPs. Biodistributions in normal and C6 glioblastoma-bearing mice were evaluated, and excised tumors were subjected to autoradiography and immunohistochemistry. TAP1 and [125I]TAP1 showed high affinity for FABP4 (Ki = 44.5±9.8 nM, Kd = 69.1±12.3 nM). The FABP4 binding affinity of [125I]TAP1 was 11.5- and 35.5-fold higher than for FABP3 and FABP5, respectively. In an in vivo study [125I]TAP1 displayed high stability against deiodination and degradation, and moderate radioactivity accumulation in C6 tumors (1.37±0.24% dose/g 3 hr after injection). The radioactivity distribution profile in tumors partially corresponded to the FABP4 positive area and was also affected by perfusion. The results indicate that [125I]TAP1 could detect FABP4 in vitro and partly in vivo. As such, [125I]TAP1 is a promising lead compound for further refinement for use in in vivo FABP4 imaging.
PMCID: PMC3986099  PMID: 24732569
8.  Quantification of Small Molecule Drugs in Biological Tissue Sections by Imaging Mass Spectrometry Using Surrogate Tissue-Based Calibration Standards 
Mass Spectrometry  2014;3(1):A0025.
Quantitative analysis of administered drugs in biological tissues is essential for understanding the mechanisms underlying their efficacy or toxicity. Imaging mass spectrometry (IMS) may allow the quantification of targeted drugs in tissue sections along with the visualization of their spatial distribution. In this study, surrogate tissue-based calibration standards were prepared to quantify a small molecule drug (S-777469 or raclopride) in tissue sections of mice administered with the drug, followed by analysis with a linear ion trap mass spectrometer equipped with a matrix-assisted laser desorption/ionization (MALDI) source. The distribution of the drugs in the dissected organs was clearly visualized by MALDI-IMS. The drug concentration determined using the calibration standards prepared for MALDI-IMS analysis was highly consistent with that determined by liquid chromatography-tandem mass spectrometry, and the quantification in multiple organs was enabled. The results of this study show that MALDI-IMS can be used to quantify small molecule drugs in biological tissue sections using surrogate tissue-based calibration standards.
PMCID: PMC3968287  PMID: 24738041
imaging mass spectrometry; matrix-assisted laser desorption/ionization; quantification
9.  Development of Novel 123I-Labeled Pyridyl Benzofuran Derivatives for SPECT Imaging of β-Amyloid Plaques in Alzheimer’s Disease 
PLoS ONE  2013;8(9):e74104.
Imaging of β-amyloid (Aβ) plaques in the brain may facilitate the diagnosis of cerebral β-amyloidosis, risk prediction of Alzheimer’s disease (AD), and effectiveness of anti-amyloid therapies. The purpose of this study was to evaluate novel 123I-labeled pyridyl benzofuran derivatives as SPECT probes for Aβ imaging. The formation of a pyridyl benzofuran backbone was accomplished by Suzuki coupling. [123I/125I]-labeled pyridyl benzofuran derivatives were readily prepared by an iododestannylation reaction. In vitro Aβ binding assays were carried out using Aβ(1–42) aggregates and postmortem human brain sections. Biodistribution experiments were conducted in normal mice at 2, 10, 30, and 60 min postinjection. Aβ labeling in vivo was evaluated by small-animal SPECT/CT in Tg2576 transgenic mice injected with [123I]8. Ex vivo autoradiography of the brain sections was performed after SPECT/CT. Iodinated pyridyl benzofuran derivatives showed excellent affinity for Aβ(1–42) aggregates (2.4 to 10.3 nM) and intensely labeled Aβ plaques in autoradiographs of postmortem AD brain sections. In biodistribution experiments using normal mice, all these derivatives displayed high initial uptake (4.03–5.49% ID/g at 10 min). [125I]8 displayed the quickest clearance from the brain (1.30% ID/g at 60 min). SPECT/CT with [123I]8 revealed higher uptake of radioactivity in the Tg2576 mouse brain than the wild-type mouse brain. Ex vivo autoradiography showed in vivo binding of [123I]8 to Aβ plaques in the Tg2576 mouse brain. These combined results warrant further investigation of [123I]8 as a SPECT imaging agent for visualizing Aβ plaques in the AD brain.
PMCID: PMC3772825  PMID: 24058519
10.  BODIPY-Based Molecular Probe for Imaging of Cerebral β-Amyloid Plaques 
ACS Chemical Neuroscience  2012;3(4):319-324.
We designed and synthesized a BODIPY-based probe (BAP-1) for the imaging of β-amyloid plaques in the brain. In binding experiments in vitro, BAP-1 showed excellent affinity for synthetic Aβ aggregates. β-Amyloid plaques in Tg2576 mouse brain were clearly visualized with BAP-1. In addition, the labeling of β-amyloid plaques was demonstrated in vivo in Tg2576 mice. These results suggest BAP-1 to be a useful fluorescent probe for the optical imaging of cerebral β-amyloid plaques in patients with Alzheimer’s disease.
PMCID: PMC3369805  PMID: 22860198
Alzheimer’s disease; β-amyloid plaque; BODIPY; optical imaging
11.  18F-Labeled Phenyldiazenyl Benzothiazole for in Vivo Imaging of Neurofibrillary Tangles in Alzheimer's Disease Brains 
We synthesized and evaluated (E)-4-((6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzo[d]thiazol-2-yl)diazenyl)-N,N-dimethylaniline (FPPDB) as a probe for the imaging of neurofibrillary tangles (NFTs) in patients with Alzheimer's disease (AD). In assays using thioflavin S (ThS) as a competitive ligand, FPPDB competed with ThS well and showed high affinity for both tau and Aβ1–42 aggregates (Ki = 13.0 and 20.0 nM, respectively). The results of saturation binding assays also verified that FPPDB bound to both tau and Aβ1–42 aggregates with high affinity (Kd = 44.8 nM and Bmax = 45.8 pmol/nmol protein for tau aggregates and Kd = 45.4 nM and Bmax = 38.9 pmol/nmol protein for Aβ1–42 aggregates). Furthermore, [18F]FPPDB substantially labeled NFTs and senile plaques in AD brain sections but not control brain sections. In biodistribution experiments using normal mice, [18F]FPPDB displayed higher uptake (4.28% ID/g at 2 min postinjection) into and washout (2.53% ID/g at 60 min postinjection) from the brain with time. On the basis of the chemical structure of FPPDB, further increases in selective binding to tau aggregates may lead to the development of more useful probes for the imaging of NFTs in AD brains.
PMCID: PMC4025873  PMID: 24900371
Alzheimer's disease (AD); neurofibrillary tangles (NFTs); imaging; benzothiazole; PET
12.  Radiolabelled probes for imaging of atherosclerotic plaques 
Cardiovascular disease is the leading cause of death worldwide. Unstable atherosclerotic plaques are prone to rupture followed by thrombus formation, vessel stenosis, and occlusion and frequently lead to acute myocardial infarction and brain infarction. As such, unstable plaques represent an important diagnostic target in clinical settings and the specific diagnosis of unstable plaques would enable preventive treatments for cardiovascular disease. To date, various imaging methods such as computed tomography (CT), magnetic resonance imaging (MRI), ultrasound (US), and intravascular ultrasound (IVUS) have been widely used clinically. Although these methods have advantages in terms of spatial resolution and the ability to make detailed identification of morphological alterations such as calcifications and vessel stenosis, these techniques require skill or expertise to discriminate plaque instability, which is essential for early diagnosis and treatment and can present difficulties for quantitative estimation. On the other hand, nuclear imaging techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) can noninvasively collect quantitative information on the expression levels of functional molecules and metabolic activities in vivo and thus provide functional diagnoses of unstable plaques with high sensitivity. Specifically, unstable plaques are characterized by an abundance of invasive inflammatory cells (macrophages), increased oxidative stress that increases oxidized LDL and its receptor expressed on cells in the lesions, increased occurrence of apoptosis of macrophages and other cells involved in disease progression, increased protease expression and activity, and finally thrombus formation triggered by plaque rupture, which is the most important mechanism leading to the onset of infarctions and ischemic sudden death. Therefore, these characteristics can all be targets for molecular imaging by PET and SPECT. In this paper, we review the present state and future of radiolabelled probes that have been developed for detecting atherosclerotic unstable plaques with nuclear imaging techniques.
PMCID: PMC3484420  PMID: 23145360
Molecular imaging; atherosclerosis; plaque; positron emission tomography; single photon emission computed tomography; 2-[18F]Fluoro-2-deoxy-D-glucose; lectin-like oxidized low density lipoprotein receptor-1; apoptosis; matrix metalloproteinase; thrombus
13.  In Vivo Visualization of Heterogeneous Intratumoral Distribution of Hypoxia-Inducible Factor-1α Activity by the Fusion of High-Resolution SPECT and Morphological Imaging Tests 
Purpose. We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α (HIF) activity in tumor tissues in vivo. Methods. We synthesized of 125I-IPOS, a 125I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of 125I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of 125I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtained in vivo SPECT-CT fusion images were compared with ex vivo images of excised tumors. Fusion imaging with MRI was also examined. Results. 125I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of 125I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of 125I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of 125I-IPOS. Conclusion. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of 125I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activity in vivo.
PMCID: PMC3385015  PMID: 22778544
14.  Rhodanine and Thiohydantoin Derivatives for Detecting Tau Pathology in Alzheimer’s Brains 
ACS Chemical Neuroscience  2011;2(5):269-275.
A novel series of rhodanin (RH) and thiohydantoin (TH) derivatives were designed and synthesized for detecting tau pathology in the brains of patients with Alzheimer’s disease (AD). In experiments in vitro using tau and β-amyloid (Aβ) aggregates, the TH derivative, TH2, showed high specific binding to tau aggregates. In hippocampal sections obtained from AD patients, TH2 intensely stained neurofibrillary tangles. In experiments using normal mice, [125I]TH2 showed good uptake (1.54%ID/g, 2 min postinjection) into and a rapid washout (0.25%ID/g, 60 min postinjection) from the brain. [123I]TH2 should be further investigated as a potential imaging agent for detecting tau pathology.
PMCID: PMC3369744  PMID: 22778869
Alzheimer’s disease; tau; imaging; rhodanine; thiohydantoin
15.  Advances in Drug Design of Radiometal-Based Imaging Agents for Bone Disorders 
Nuclear medicine bone imaging has been the optimum diagnosis for the detection of bone disorders because the lesion could be detectable before the appearance of symptomatic and radiographic changes. Over the past three decades, 99mTc-MDP and 99mTc-HMDP have been used as bone scintigraphic agents because of their superior biodistribution characteristics, although they are far from optimal from a chemical and pharmaceutical point of view. Recently, a more logical drug design has been proposed as a concept of bifunctional radiopharmaceuticals in which the carrier molecules (bisphosphonates) and radiometal chelating groups are separated within a molecule, specifically, 99mTc-mononuclear complex-conjugated bisphosphonate. Some of the 99mTc-mononuclear complex-conjugated bisphosphonate compounds showed superior biodistribution in preclinical studies. Moreover, the drug design concept could be applied to 68Ga PET bone imaging agents. These studies would provide useful information for the development of radiometal-based imaging and therapeutic agents for bone disorders such as bone metastases.
PMCID: PMC3246737  PMID: 22220275
16.  Novel Benzofurans with 99mTc Complexes as Probes for Imaging Cerebral β-Amyloid Plaques 
ACS Medicinal Chemistry Letters  2010;1(8):443-447.
Two novel benzofuran derivatives coupled with 99mTc complexes were tested as probes for imaging cerebral β-amyloid plaques using single photon emission tomography. Although both derivatives bound to Aβ(1−42) aggregates, 99mTc-BAT-BF showed higher affinity than 99mTc-MAMA-BF. In sections of brain tissue from an animal model of AD, 99mTc-BAT-BF clearly labeled β-amyloid plaques. In biodistribution experiments using normal mice, 99mTc-BAT-BF displayed high uptake soon after its injection and washed out from the brain rapidly, a highly desirable feature for an imaging agent. 99mTc-BAT-BF may be a potential probe for imaging β-amyloid plaques in Alzheimer's brains.
PMCID: PMC4007700  PMID: 24900230
Alzheimer's disease; β-amyloid plaque; Tc-99m; single photon emission computed tomography (SPECT); imaging
17.  Fluorinated Benzofuran Derivatives for PET Imaging of β-Amyloid Plaques in Alzheimer's Disease Brains 
ACS Medicinal Chemistry Letters  2010;1(7):321-325.
A series of fluorinated benzofuran derivatives as potential tracers for positron emission tomography (PET) targeting β-amyloid plaques in the brains of patients with Alzheimer's disease (AD) were synthesized and evaluated. The derivatives were produced using an intramolecular Wittig reaction. In experiments in vitro, all displayed high affinity for Aβ(1−42) aggregates with Ki values in the nanomolar range. Radiofluorinated 17, [18F]17, in particular labeled β-amyloid plaques in sections of Tg2576 mouse brain and displayed high uptake (5.66% ID/g) at 10 min postinjection, sufficient for PET imaging. In addition, in vivo β-amyloid plaque labeling can be clearly demonstrated with [18F]17 in Tg2576 mice. In conclusion, [18F]17 may be useful for detecting β-amyloid plaques in patients with AD.
PMCID: PMC4007906  PMID: 24900214
Alzheimer's disease; fluorine-18; benzofuran; positron emission tomography (PET)
18.  Synthesis and Evaluation of Novel Chalcone Derivatives with 99mTc/Re Complexes as Potential Probes for Detection of β-Amyloid Plaques 
ACS Chemical Neuroscience  2010;1(9):598-607.
Four 99mTc-labeled chalcone derivatives and their corresponding rhenium analogues were tested as potential probes for imaging β-amyloid plaques. The chalcones showed higher affinity for Aβ(1−42) aggregates than did 99mTc complexes. In sections of brain tissue from an animal model of AD, the four Re chalcones intensely stained β-amyloid plaques. In biodistribution experiments using normal mice, 99mTc-BAT-chalcone ([99mTc]17) displayed high uptake in the brain (1.48% ID/g) at 2 min postinjection. The radioactivity washed out from the brain rapidly (0.17% ID/g at 60 min), a highly desirable feature for an imaging agent. [99mTc]17 may be a potential probe for imaging β-amyloid plaques in Alzheimer’s brains.
PMCID: PMC3368688  PMID: 22778849
Alzheimer’s disease; β-amyloid plaque; 99mTc; single photon emission computed tomography (SPECT) imaging
19.  SPECT Imaging Agents for Detecting Cerebral β-Amyloid Plaques 
The development of radiotracers for use in vivo to image β-amyloid (Aβ) plaques in cases of Alzheimer's disease (AD) is an important, active area of research. The presence of Aβ aggregates in the brain is generally accepted as a hallmark of AD. Since the only definitive diagnosis of AD is by postmortem staining of affected brain tissue, the development of techniques which enable one to image Aβ plaques in vivo has been strongly desired. Furthermore, the quantitative evaluation of Aβ plaques in the brain could facilitate evaluation of the efficacy of antiamyloid therapies currently under development. This paper reviews the current situation in the development of agents for SPECT-based imaging of Aβ plaques in Alzheimer's brains.
PMCID: PMC3094870  PMID: 21603239

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