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author:("Sun, nankai")
1.  The Role of Cdk5 in Neuroendocrine Thyroid Cancer 
Cancer cell  2013;24(4):10.1016/j.ccr.2013.08.027.
SUMMARY
Medullary thyroid carcinoma (MTC) is a neuroendocrine cancer that originates from calcitonin-secreting parafollicular cells, or C cells. We found that Cdk5 and its cofactors, p35 and p25, are highly expressed in human MTC and that Cdk5 activity promotes MTC proliferation. A conditional MTC mouse model was generated and corroborated the role of aberrant Cdk5 activation in MTC. C cell-specific overexpression of p25 caused rapid C cell hyperplasia leading to lethal MTC, which was arrested by repressing p25 overexpression. A comparative phosphoproteomic screen between proliferating and arrested MTC identified the retinoblastoma protein (Rb) as a crucial Cdk5 downstream target. Prevention of Rb phosphorylation at Ser807/811 attenuated MTC proliferation. These findings implicate Cdk5 signaling via Rb as critical to MTC tumorigenesis and progression.
doi:10.1016/j.ccr.2013.08.027
PMCID: PMC3849320  PMID: 24135281
p25/Cdk5; medullary thyroid carcinoma; tumorigenesis; MEN2A
2.  The Effect of Erythropoietin on Autologous Stem Cell-Mediated Bone Regeneration 
Biomaterials  2013;34(30):7364-7371.
Mesenchymal stem cells (MSCs) although used for bone tissue engineering are limited by the requirement of isolation and culture prior to transplantation. Our recent studies have shown that biomaterial implants can be engineered to facilitate the recruitment of MSCs. In this study, we explore the ability of these implants to direct the recruitment and the differentiation of MSCs in the setting of a bone defect. We initially determined that both stromal derived factor-1alpha (SDF-1α) and erythropoietin (Epo) prompted different degrees of MSC recruitment. Additionally, we found that Epo and bone morphogenetic protein-2 (BMP-2), but not SDF-1α, triggered the osteogenic differentiation of MSCs in vitro. We then investigated the possibility of directing autologous MSC-mediated bone regeneration using a murine calvaria model. Consistent with our in vitro observations, Epo-releasing scaffolds were found to be more potent in bridging the defect than BMP-2 loaded scaffolds, as determined by Computed Tomography (CT) scanning, fluorescent imaging and histological analyses. These results demonstrate the tremendous potential, directing the recruitment and differentiation of autologous MSCs has in the field of tissue regeneration.
doi:10.1016/j.biomaterials.2013.06.031
PMCID: PMC3753364  PMID: 23831188
Autologous stem cells; Scaffold; Erythropoietin; Stromal derived factor-1α; Bone morphogenetic protein-2; Bone; Calvaria; Bone Tissue Engineering
3.  A Multivalent Approach of Imaging Probe Design to Overcome an Endogenous Anion Binding Competition for Noninvasive Assessment of Prostate Specific Membrane Antigen 
Molecular pharmaceutics  2013;10(8):2975-2985.
2[(3-amino-3-carboxypropyl)(hydroxy)(phosphinyl)-methyl]pentane-1,5-dioic acid) (GPI) is a highly potent inhibitor of prostate specific membrane antigen (PSMA) with a rapid in vivo clearance profile from non-target organs including kidneys, but its use for imaging of PSMA is blocked by an endogenous anion (serum phosphate) competition, which compromises its specific binding to the antigen. Multi-presentation of a targeting molecule on a single entity has been recognized as a practical way for imaging sensitivity enhancement. Herein, we demonstrate a multivalent approach based on a 64Cu-specific bifunctional chelator scaffold to overcome the endogenous phosphate competition thus enabling the utility of GPI conjugates for in vivo detection of PSMA and imaging quantification. Both monomeric (H2CBT1G) and dimeric (H2CBT2G) conjugates were synthesized and labeled with 64Cu for in vitro and in vivo evaluations. A 4-fold enhancement of PSMA binding affinity was observed for H2CBT2G as compared to H2CBT1G from the PSMA competitive binding assays performed on LNCaP cells. In vivo PET imaging studies were conducted on mouse xenograft models established with a PSMA+ cell line, LNCaP, and PSMA− PC3 and H2009 cell lines. 64Cu-CBT2G showed significantly higher LNCaP tumor uptake than 64Cu-CBT1G at 1, 4, and 24 h post-injection (p.i.) (p < 0.05). In addition, tumor uptake of 64Cu-CBT2G remained steady out to 24 h p.i. (1.46 ± 0.54, 1.12 ± 0.56, and 1.00 ± 0.50 %ID/g at 1, 4 and 24 h p.i., respectively), while 64Cu-CBT1G showed a great decrease from 1 h to 4 h p.i. The PSMA imaging specificity of both H2CBT1G and H2CBT2G was demonstrated by their low uptake in PSMA− tumors (PC3 and H2009) and further confirmed by a significant signal reduction in PSMA+ LNCaP tumors in the blockade study. In addition, the LNCaP tumor uptake (%ID/g) of 64Cu-CBT2G was found to be in a positive linear correlation with the tumor size (R2 = 0.92, 0.94, and 0.93 for 1 h, 4 h, and 24 h p.i.). This may render the probe with potential application in the management of patients with prostate cancer.
doi:10.1021/mp4000844
PMCID: PMC3757929  PMID: 23768233
PSMA; prostate cancer; PET; 64Cu; multivalency
4.  Dendrimers Terminated with Dichlorotriazine Groups Provide a Route to Compositional Diversity 
Organic letters  2013;15(15):3808-3811.
Triazine dendrimers terminated with either four or eight dichlorotriazines can be prepared in high yields by reacting an amine-terminated dendrimer with cyanuric chloride. These materials exist as white powders and are stable to storage at room temperature. Sequential nucleophilic aromatic substitution with two different amine nucleophiles yields compounds that display the desired compositional diversity. Reactions conditions for the substitution were developed using a model dichlorotriazine with amine nucleophiles at -20 °C, 0 °C and 25 °C. Selective substitution is favored at lower temperatures and with more nucleophilic amine groups.
doi:10.1021/ol400811h
PMCID: PMC4091816  PMID: 23869522
5.  Synthesis and characterization of a high-affinity αvβ6-specific ligand for in vitro and in vivo applications 
Molecular cancer therapeutics  2009;8(5):1239-1249.
The αvβ6 integrin is an attractive therapeutic target for several cancers due to its role in metastasis and its negligible expression in normal tissues. We previously identified a peptide from a phage-displayed peptide library that binds specifically to αvβ6. The tetrameric version of the peptide has higher affinity for its cellular targets than the corresponding monomers. However, the inefficient synthesis limits its clinical potential. We report here a convergent synthesis producing the tetrameric peptide in high yield and purity. The ease of the synthesis allows for rapid optimization of the peptide. We have optimized this αvβ6 integrin–binding peptide, determining the minimal binding domain and valency. Importantly, the half-maximal binding affinity of the optimal peptide for its target cell is in the 40 to 60 pmol/L range, rivaling the affinity of commonly used antibody-targeting reagents. This peptide mediates cell-specific uptake, is functional in diagnostic formats, is stable in sera, and can home to a tumor in an animal. We anticipate that this high-affinity ligand for αvβ6 will find clinical use as a diagnostic and therapeutic reagent.
doi:10.1158/1535-7163.MCT-08-1098
PMCID: PMC4053473  PMID: 19435868
6.  In vivo evaluation of medical device-associated inflammation using a macrophage-specific Positron Emission Tomography (PET) imaging probe 
To image implant-surrounding activated macrophages, a macrophage-specific PET probe was prepared by conjugating folic acid (FA) and 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl) tetracetic acid (DOTA) to polyethylene glycol (PEG) and then labeling the conjugate with Ga-68. In vivo PET imaging evaluations demonstrate that the probe is able to detect foreign body reactions, and more importantly, quantify the degree of inflammatory responses to an implanted medical device. These results were further validated by histological analysis.
doi:10.1016/j.bmcl.2013.02.004
PMCID: PMC3602365  PMID: 23481649
Folic acid; Biomaterial implant; Inflammation; Biocompatibility; PET imaging
7.  Dendrimer Nanoscaffolds for Potential Theranostics of Prostate Cancer with a Focus on Radiochemistry 
Molecular pharmaceutics  2013;10(3):793-812.
Dendrimers are a class of structurally defined macromolecules featured with a central core, a low-density interior formed by repetitive branching units, and a high-density exterior terminated with surface functional groups. In contrast to their polymeric counterparts, dendrimers are nano-sized and symmetrically shaped, which can be reproducibly synthesized in a large scale with monodispersity. These unique features have made dendrimers of increasing interest for drug delivery and other biomedical applications as a nanoscaffold system. Intended to address the potential use of dendrimers for the development of theranostic agents, which combines therapeutics and diagnostics in a single entity for personalized medicine, this review focuses on the reported methodologies of using dendrimer nanoscaffolds for targeted imaging and therapy of prostate cancer. Of particular interest, relevant chemistry strategies are discussed due to their important roles in the design and synthesis of diagnostic and therapeutic dendrimer-based nanoconjugates and potential theranostic agents, targeted or non-targeted. Given the developing status of nanoscaffolded theranostics, major challenges and potential hurdles are discussed along with the examples representing current advances.
doi:10.1021/mp3005325
PMCID: PMC3599782  PMID: 23294202
Dendrimer; Theranostics; Prostate Cancer; Molecular Imaging; Drug Delivery
8.  Dimerization of a Phage-Display Selected Peptide for Imaging of αvβ6- Integrin: Two Approaches to the Multivalent Effect 
Theranostics  2014;4(7):745-760.
The integrin αvβ6 is an emerging biomarker for non-small cell lung cancer (NSCLC). An αvβ6-binding peptide was previously selected from a phage-displayed peptide library. Here, we utilize a multivalent design to develop a peptidic probe for positron emission tomography (PET) imaging of αvβ6+ NSCLC tumors. Multimeric presentation of this peptide, RGDLATLRQL, on a bifunctional copper chelator was achieved using two approaches: dimerization of the peptide followed by conjugation to the chelator (H2-D10) and direct presentation of two copies of the peptide on the chelator scaffold (H2-(M10)2). Binding affinities of the divalent peptide conjugates are four-fold higher than their monovalent counterpart (H2-M10), suggestive of multivalent binding. PET imaging using the bivalent 64Cu-labeled conjugates showed rapid and persistent accumulation in αvβ6+ tumors. By contrast, no significant accumulation was observed in αvβ6- tumors. Irrespective of the dimerization approach, all divalent probes showed three-fold higher tumor uptake than the monovalent probe, indicating the role of valency in signal enhancement. However, the divalent probes have elevated uptake in non-target organs, especially the kidneys. To abrogate nonspecific uptake, the peptide's N-terminus was acetylated. The resultant bivalent probe, 64Cu- AcD10, showed drastic decrease of kidney accumulation while maintaining tumor uptake. In conclusion, we developed an αvβ6-integrin specific probe with optimized biodistribution for noninvasive PET imaging of NSCLC. Further, we have demonstrated that use of multivalent scaffolds is a plausible method to improve library selected peptides, which would be suboptimal or useless otherwise, for imaging probe development.
doi:10.7150/thno.7811
PMCID: PMC4038756  PMID: 24883124
Lung Cancer; Molecular Imaging; Positron Emission Tomography; Integrin; Peptide.
9.  Highly Specific PET Imaging of Prostate Tumors in Mice with an Iodine-124-Labeled Antibody Fragment That Targets Phosphatidylserine 
PLoS ONE  2013;8(12):e84864.
Phosphatidylserine (PS) is an attractive target for imaging agents that identify tumors and assess their response to therapy. PS is absent from the surface of most cell types, but becomes exposed on tumor cells and tumor vasculature in response to oxidative stresses in the tumor microenvironment and increases in response to therapy. To image exposed PS, we used a fully human PS-targeting antibody fragment, PGN635 F(ab’)2, that binds to complexes of PS and β2-glycoprotein I. PGN635 F(ab’)2 was labeled with the positron-emitting isotope iodine-124 (124I) and the resulting probe was injected into nude mice bearing subcutaneous or orthotopic human PC3 prostate tumors. Biodistribution studies showed that 124I-PGN635 F(ab’)2 localized with remarkable specificity to the tumors with little uptake in other organs, including the liver and kidneys. Clear delineation of the tumors was achieved by PET 48 hours after injection. Radiation of the tumors with 15 Gy or systemic treatment of the mice with 10 mg/kg docetaxel increased localization in the tumors. Tumor-to-normal (T/N) ratios were inversely correlated with tumor growth measured over 28 days. These data indicate that 124I-PGN635 F(ab’)2 is a promising new imaging agent for predicting tumor response to therapy.
doi:10.1371/journal.pone.0084864
PMCID: PMC3868598  PMID: 24367699
10.  Glucose Metabolism via the Pentose Phosphate Pathway, Glycolysis and Krebs Cycle in an Orthotopic Mouse Model of Human Brain Tumors 
NMR in biomedicine  2012;25(10):1177-1186.
It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using an orthotopic mouse model of primary human glioblastoma (GBM) and a brain metastatic renal tumor of clear cell renal cell carcinoma (CCRCC) histology, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-13C2]glucose. The [3-13C]lactate/[2,3-13C2]lactate ratio was similar for both the GBM and renal tumor and their respective surrounding brains (GBM: 0.197 ± 0.011 and 0.195 ± 0.033 (p=1); CCRCC: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than PPP flux in these tumors, and that PPP flux into the lactate pool was similar in both tissues. Remarkably, 13C-13C coupling was observed in molecules derived from Krebs cycle intermediates in both tumors, denoting glucose oxidation. In the renal tumor, in contrast to GBM and surrounding brain, 13C multiplets of GABA differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. Additionally, the orthotopic renal tumor, the patient’s primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a CCRCC tissue microarray suggesting that GABA synthesis is cell-autonomous in at least a subset of renal tumors. Taken together, these data demonstrate that 13C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.
doi:10.1002/nbm.2787
PMCID: PMC3670098  PMID: 22383401
Intermediary metabolism; Mouse model; 13C NMR; brain; glioblastoma; renal cell carcinoma
11.  Nonlinear optical effects of ultrahigh-Q silicon photonic nanocavities immersed in superfluid helium 
Scientific Reports  2013;3:1436.
Photonic nanocavities are a key component in many applications because of their capability of trapping and storing photons and enhancing interactions of light with various functional materials and structures. The maximal number of photons that can be stored in silicon photonic cavities is limited by the free-carrier and thermo-optic effects at room temperature. To reduce such effects, we performed the first experimental study of optical nonlinearities in ultrahigh-Q silicon disk nanocavities at cryogenic temperatures in a superfluid helium environment. At elevated input power, the cavity transmission spectra exhibit distinct blue-shifted bistability behavior when temperature crosses the liquid helium lambda point. At even lower temperatures, the spectra restore to symmetric Lorentzian shapes. Under this condition, we obtain a large intracavity photon number of about 40,000, which is limited ultimately by the local helium phase transition. These new discoveries are explained by theoretical calculations and numerical simulations.
doi:10.1038/srep01436
PMCID: PMC3595694  PMID: 23486445
12.  Serial Non-Invasive Assessment of Antibody Induced Nephritis in Mice Using Positron Emission Tomography 
PLoS ONE  2013;8(2):e57418.
Mouse models of experimental anti-glomerular basement membrane (anti-GBM) nephritis provide an analytical tool for studying spontaneous lupus nephritis. The potential of Positron Emission Tomography (PET) was evaluated using 2-deoxy-2-[18F]fluoro-d-glucose (FDG) as a probe to monitor the progression of anti-GBM induced nephritis in a mouse model. The imaging results were compared to conventional measures of renal function and pathological changes. Serum and urinary vascular cell adhesion molecule-1 (VCAM-1) levels were used as measures of endothelial cell activation and inflammation. Following a challenge with anti-glomerular antibodies, mice exhibited peak changes in serum creatinine, proteinuria, and glomerulonephritis score at 14 days post-challenge (p.c.). In contrast, VCAM levels peaked at day 7 p.c. On dynamic PET images (0–60 min) of day 7, kidneys of the anti-GBM nephritis mice demonstrated a unique pattern of FDG uptake. Compared to the time activity curve (TAC) prior to challenge, a rightward shift was observed after the challenge. By day 10 p.c., kidney FDG uptake was lower than baseline and remained so until the study ended at 21 days p.c. During this time frame measures of renal dysfunction remained high but VCAM-1 levels declined. These changes were accompanied by an increase in kidney volume as measured by Computed Tomography (CT) and intra-abdominal fluid collection. Our results suggest that FDG-PET-CT can be used as a non-invasive imaging tool to longitudinally monitor the progression of renal disease activity in antibody mediated nephritis and the magnitude of renal FDG retention correlates better with early markers of renal inflammation than renal dysfunction.
doi:10.1371/journal.pone.0057418
PMCID: PMC3584026  PMID: 23460853
14.  Positron Emission Tomography of Copper Metabolism in the Atp7b−/− Knock-out Mouse Model of Wilson’s Disease 
Molecular Imaging and Biology  2012;14(1):70-78.
Purpose
This study aims to determine feasibility and utility of copper-64(II) chloride (64CuCl2) as a tracer for positron emission tomography (PET) of copper metabolism imbalance in human Wilson’s disease (WD).
Procedures
Atp7b−/− mice, a mouse model of human WD, were injected with 64CuCl2 intravenously and subjected to PET scanning using a hybrid PET-CT (computerized tomography) scanner, with the wild-type C57BL mice as a normal control. Quantitative PET analysis was performed to determine biodistribution of 64Cu radioactivity and radiation dosimetry estimates of 64Cu were calculated for PET of copper metabolism in humans.
Results
Dynamic PET analysis revealed increased accumulation and markedly reduced clearance of 64Cu from the liver of the Atp7b−/− mice, compared to hepatic uptake and clearance of 64Cu in the wild-type C57BL mice. Kinetics of copper clearance and retention was also altered for kidneys, heart, and lungs in the Atp7b−/− mice. Based on biodistribution of 64Cu in wild-type C57BL mice, radiation dosimetry estimates of 64Cu in normal human subjects were obtained, showing an effective dose (ED) of 32.2 μ (micro)Sv/MBq (weighted dose over 22 organs) and the small intestine as the critical organ for radiation dose (61 μGy/MBq for males and 69 μGy/MBq for females). Radiation dosimetry estimates for the patients with WD, based on biodistribution of 64Cu in the Atp7b−/− mice, showed a similar ED of 32.8 μ (micro)Sv/MBq (p= 0.53), with the liver as the critical organ for radiation dose (120 μSv/MBq for male and 161 μSv/MBq for female).
Conclusions
Quantitative PET analysis demonstrates abnormal copper metabolism in the mouse model of WD with improved time–resolution. Human radiation dosimetry estimates obtained in this preclinical study encourage direct radiation dosimetry of 64CuCl2 in human subjects. The results suggest feasibility of utilizing 64CuCl2 as a tracer for noninvasive assessment of copper metabolism in WD with PET.
doi:10.1007/s11307-011-0476-4
PMCID: PMC3157550  PMID: 21327972
Copper metabolism; Wilson’s disease; ATP7B copper transporter; Positron emission tomography; Copper-64 (II) chloride; Radiation dosimetry
15.  Multivalent Bifunctional Chelator Scaffolds for Gallium-68 Based Positron Emission Tomography Imaging Probe Design: Signal Amplification via Multivalency 
Bioconjugate chemistry  2011;22(8):1650-1662.
The role of the multivalent effect has been well recognized in the design of molecular imaging probes towards the desired imaging signal amplification. Recently we reported a bifunctional chelator (BFC) scaffold design, which provides a simple and versatile approach to impart multivalency to radiometal based nuclear imaging probes. In this work, we report a series of BFC scaffolds (tBu3-1-COOH, tBu3-2-(COOH)2 and tBu3-3-(COOH)3) constructed on the framework of 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) for 68Ga-based PET probe design and signal amplification via multivalent effect. For proof of principle, a known integrin αvβ3 specific ligand (c(RGDyK)) was used to build the corresponding NOTA conjugates (H31, H32, and H33), which present 1 – 3 copies of c(RGDyK) peptide, respectively, in a systematic manner. Using the integrin αvβ3 binding affinities (IC50 values), the enhanced specific binding was observed for multivalent conjugates (H32: 43.9 ± 16.1 nM; H33: 14.7 ± 5.0 nM) as compared to their monovalent counterpart (H31: 171 ± 60 nM) and the intact c(RGDyK) peptide (204 ± 76 nM). The obtained conjugates were efficiently labeled with 68Ga3+ within 30 min at room temperature in high radiochemical yields (> 95%). The in vivo evaluation of the labeled conjugates, 68Ga-1, 68Ga-2 and 68Ga-3, was performed using male severe combined immunodeficiency (SCID) mice bearing integrin αvβ3 positive PC-3 tumor xenografts (n = 3). All 68Ga -labeled conjugates showed high in vivo stability with no detectable metabolites found by radio-HPLC within 2 h post-injection (p.i.). The PET signal amplification in PC-3 tumor by multivalent effect was clearly displayed by the tumor uptake of the 68Ga-labeled conjugates (68Ga-3: 2.55 ± 0.50%ID/g; 68Ga-2: 1.90 ± 0.10 %ID/g; 68Ga-1: 1.66 ± 0.15 %ID/g) at 2 h p.i. In summary, we have designed and synthesized a series of NOTA-based BFC scaffolds with signal amplification properties, which may find potential applications in diagnostic gallium radiopharmaceuticals.
doi:10.1021/bc200227d
PMCID: PMC3170926  PMID: 21740059
16.  Upregulation of TRAG3 gene in urothelial carcinoma of the bladder 
Conventional chemotherapy is commonly used for advanced stages of bladder cancer with modest success and high morbidity. Identifying markers of resistance will allow clinicians to tailor treatment to a specific patient population. T24-tumorigenic cell line was grown orthotopically in nude mice and monitored using bioluminescence imaging and microcomputed tomography until they developed metastases. Stable sublines were then developed from primary bladder (T24-P), lung (T24-L) and bone (T24-B) tissues. Chromosomal analysis and DNA microarray were used to characterize these sublines. qRT-PCR and immunohistochemistry (IHC) were used for validation. Epigenetic modifiers were used to study gene regulation. The cell viability was quantified with MTT assay. Chromosomal analysis revealed multiple alterations in metastatic cell lines compared to T24-P. DNA microarray analysis showed that Taxol-Resistance-Associated-Gene-3 (TRAG3) gene was the most upregulated gene. From qRT-PCR and IHC, TRAG3 was significantly higher in T24-L and T24-B than T24-P. TRAG3 gene expression is likely controlled by DNA methylation, but not histone acetylation. Interestingly, T24-B and T24-L cells were more resistant than T24-P to treatment with anti-microtubule agents such as docetaxel, paclitaxel and vinblastine. TRAG3 mRNA expression was higher in 20% of patients with ≤pT2 (n=10) and 60% of patients with ≥pT3 (n=20) compared to normal adjacent tissue (p=0.05). In addition, the median TRAG3 expression was 6.7-fold higher in ≥pT3 tumors compared to ≤pT2 tumors. Knowing the status of TRAG3 expression could help clinicians tailor treatment to a particular patient population that could benefit from treatment, while allocating patients with resistant tumors to new experimental therapies.
doi:10.1002/ijc.25631
PMCID: PMC3082622  PMID: 20734393
urothelial carcinoma; bladder; TRAG3; resistance
18.  Attention-Deficit/Hyperactivity Phenotype in Mice Lacking the Cyclin-dependent Kinase 5 Cofactor p35 
Biological psychiatry  2010;68(12):1163-1171.
Background
Attention-deficit/hyperactivity disorder (ADHD) may result from delayed establishment of corticolimbic circuitry or perturbed dopamine (DA) neurotransmission. Despite the widespread use of stimulants to treat ADHD, little is known regarding their long-term effects on neurotransmitter levels and metabolism. Cyclin-dependent kinase 5 (Cdk5) regulates DA signaling through control of synthesis, postsynaptic responses, and vesicle release. Mice lacking the Cdk5-activating cofactor p35 are deficient in cortical lamination, suggesting altered motor/reward circuitry.
Methods
We employed mice lacking p35 to study the effect of altered circuitry in vivo. Positron emission tomography measured glucose metabolism in the cerebral cortex using 2-deoxy-2-[18F]fluoro-D-glucose as the radiotracer. Retrograde dye tracing and tyrosine hydroxylase immunostains assessed the effect of p35 knockout on the medial prefrontal cortex (PFC), especially in relation to mesolimbic circuit formation. We defined the influence of Cdk5/p35 activity on catecholaminergic neurotransmission and motor activity via examination of locomotor responses to psychostimulants, monoamine neurotransmitter levels, and DA signal transduction.
Results
Here, we report that mice deficient in p35 display increased glucose uptake in the cerebral cortex, basal hyperactivity, and paradoxical decreased locomotion in response to chronic injection of cocaine or methylphenidate. Knockout mice also exhibited an increased susceptibility to changes in PFC neurotransmitter content after chronic methylphenidate exposure, and altered basal DAergic activity in acute striatal and PFC slices.
Conclusions
Our findings suggest that dysregulation of Cdk5/p35 activity during development may contribute to ADHD pathology, as indicated by the behavioral phenotype, improperly established mesolimbic circuitry, and aberrations in striatal and PFC catecholaminergic signaling in p35 knockout mice.
doi:10.1016/j.biopsych.2010.07.016
PMCID: PMC2997929  PMID: 20832057
Cdk5; p35; dopamine; prefrontal cortex; methylphenidate; ADHD
19.  MRI detection of VEGFR2 in vivo using a low molecular weight peptoid-(Gd)8-dendron for targeting 
Journal of the American Chemical Society  2010;132(37):12829-12831.
The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r1 relaxivity of ~48 mM−1s−1 is shown to enhance MR images of tumors grown in mice in vivo.
doi:10.1021/ja105563a
PMCID: PMC2967214  PMID: 20795620
20.  Transplantation of mesenchymal stem cells from young donors delays aging in mice 
Scientific Reports  2011;1:67.
Increasing evidence suggests that the loss of functional stem cells may be important in the aging process. Our experiments were originally aimed at testing the idea that, in the specific case of age-related osteoporosis, declining function of osteogenic precursor cells might be at least partially responsible. To test this, aging female mice were transplanted with mesenchymal stem cells from aged or young male donors. We find that transplantation of young mesenchymal stem cells significantly slows the loss of bone density and, surprisingly, prolongs the life span of old mice. These observations lend further support to the idea that age-related diminution of stem cell number or function may play a critical role in age-related loss of bone density in aging animals and may be one determinant of overall longevity.
doi:10.1038/srep00067
PMCID: PMC3216554  PMID: 22355586
21.  Biological Assessment of Triazine Dendrimers as Candidate Platforms for Nanomedicine: Toxicological Profiles, Solution Behavior, Biodistribution, and Drug Release and Efficacy in a PEGylated, Paclitaxel Construct 
Molecular pharmaceutics  2010;7(4):993-1006.
The physicochemical characteristics, in vitro properties, and in vivo toxicity and efficacy of a third generation triazine dendrimer bearing approximately nine 2 kDa polyethylene glycol chains and twelve ester linked paclitaxel groups are reported. The hydrodynamic diameter of the neutral construct varies slightly with aqueous solvent ranging from 15.6–19.4 nm. Mass spectrometry and light scattering suggest radically different molecular weights with the former ~40 kDa mass consistent with expectation, and the latter 400 kDa mass consistent with a decameric structure and the observed hydrodynamic radii. HPLC can be used to assess purity as well as paclitaxel release, which is insignificant in organic solvents or aqueous solutions at neutral and low pH. Paclitaxel release occurs in vitro in human, rat, and mouse plasma and is non-linear, ranging from 7–20% cumulative release over a 48 hour incubation period. The construct is 2–3 orders of magnitude less toxic than Taxol® by weight in human hepatocarcinoma (Hep G2), porcine renal proximal tubule (LLC-PK1), and human colon carcinoma (LS174T) cells, but shows similar cytotoxicity to Abraxane® in LS174T cells. Both Taxol® and the construct appear to induce caspase 3-dependent apoptosis. The construct shows a low level of endotoxin, is not hemolytic and does not induce platelet aggregation in vitro, but does appear to reduce collagen-induced platelet aggregation in vitro. Furthermore, the dendrimer formulation slightly activates the complement system in vitro due most likely to the presence of trace amounts (<1%) of free paclitaxel. An animal study provided insight into the maximum tolerated dose (MTD) wherein 10, 25, 50 and 100 mg paclitaxel/kg of construct or Abraxane were administered once per week for three consecutive weeks to non-tumor bearing athymic nude mice. The construct showed in vivo toxicity comparable to Abraxane. Both formulations were found to be non-toxic at the administered doses, and the dendrimer had an acute MTD greater than the highest dose administered. In a prostate tumor model (PC-3-luc), efficacy was observed over 70 days with an arrest of tumor growth and lack of luciferase activity observed in the twice treated cohort.
doi:10.1021/mp100104x
PMCID: PMC2914493  PMID: 20481608
drug delivery; paclitaxel; dendrimer; triazine; melamine; prostate cancer; colon cancer; in vivo; therapy
22.  Peptoid-based PET imaging of vascular endothelial growth factor receptor (VEGFR) expression 
Non-invasive detection of vascular endothelial growth factor receptor 2 (VEGFR2) by positron emission tomography (PET) would allow the evaluation of tumor vascular activity in vivo. Recently, a dimeric peptoid, GU40C4, was reported as a highly potent antagonist of VEGFR2 activation inhibiting angiogenesis and tumor growth in vivo. The purpose of this work was to evaluate the potential of this peptoid for PET imaging of VEGFR2 expression. To label GU40C4 and a control peptoid with a positron emitter, 64Cu (t1/2 = 12.7 h; β+: 0.653 MeV, 17.4%), a cysteine was introduced to the C-terminus of the peptoids and then conjugated to a bifunctional chelator (DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) through the maleimide-thiol coupling chemistry. The in vitro binding assay showed a negligible effect of the DOTA conjugation on the VEGFR2 binding affinity of GU40C4. Both peptoid conjugates were efficiently labeled with 64Cu in high radiochemical yields (> 90%); the specific activity was in the range of 10 – 80 GBq/μmol. PET imaging evaluation using a prostate cancer xenograft (PC3) mouse model showed that 64Cu-DOTA-GU40C4 had a prominent and steady accumulation in the VEGFR2 positive PC3 tumors (2.25 ± 0.24, 2.15 ± 0.34, and 1.90 ± 0.18 %ID/g at 1, 4, and 20 h p.i., respectively; n = 3), which is significantly higher than the control peptoid conjugate (0.3 – 0.5 %ID/g; p < 0.001 at 1, 4, and 20 h p.i.). Interestingly, the mouse salivary glands were also clearly visualized by 64Cu-DOTA-GU40C4 (3.17 ± 0.25, 3.00 ± 0.36, and 1.83 ± 0.21 %ID/g at 1, 4, and 20 h p.i., respectively; n = 3) rather than its control peptoid conjugate. VEGFR2 expression in the salivary glands was shown by polymerase chain reaction (PCR) assay. Our results demonstrate that 64Cu-DOTA-GU40C4 can be used to image the expression of VEGFR2 in vivo.
PMCID: PMC3477717  PMID: 23133797
VEGFR2; peptoid; PET; 64Cu; prostate cancer; tumor angiogenesis
23.  Trapping Iron Oxide into Hollow Gold Nanoparticles 
Synthesis of the core/shell-structured Fe3O4/Au nanoparticles by trapping Fe3O4 inside hollow Au nanoparticles is described. The produced composite nanoparticles are strongly magnetic with their surface plasmon resonance peaks in the near infrared region (wavelength from 700 to 800 nm), combining desirable magnetic and plasmonic properties into one nanoparticle. They are particularly suitable for in vivo diagnostic and therapeutic applications. The intact Au surface provides convenient anchorage sites for attachment of targeting molecules, and the particles can be activated by both near infrared lights and magnetic fields. As more and more hollow nanoparticles become available, this synthetic method would find general applications in the fabrication of core–shell multifunctional nanostructures.
doi:10.1007/s11671-010-9792-x
PMCID: PMC3211847
Gold nanoparticles; Iron oxide nanoparticles; Core/shell nanoparticles; Hollow nanoparticles; Porous nanoparticles; Plasmonics
24.  Synthesis and evaluation of lanthanide ion DOTA-tetraamide complexes bearing peripheral hydroxyl groups 
The use of lanthanide-based contrast agents for magnetic resonance imaging (MRI) has become an integral component of this important diagnostic modality. These inert chelates typically possess high thermodynamic stability constants that serve as a predictor for in vivo stability and low toxicity. Recently a new class of contrast agents was reported having a significantly lower degree thermodynamic stability while exhibiting biodistribution profiles indicative of high stability under biological conditions. These observations are suggestive that the nature of contrast agent stability is also dependent upon the kinetics of complex dissociation; a feature of potential importance when contemplating the design of new chelates for in vivo use. In this paper we present a study of the kinetics of acid catalyzed dissociation, thermodynamic stability, serum stability and biodistribution of a series of DOTA-tetraamide complexes that have been substituted with peripheral hydroxyl groups. The data indicate that these non-traditional contrast agents exhibit in vivo stability comparable to agents with much higher log KML values demonstrating the important contribution of kinetic inertness.
doi:10.1007/s00775-008-0459-3
PMCID: PMC2796837  PMID: 19083028
Biodistribution; Toxicity; Lanthanides complexes; DOTA-tetraamide derivatives; MRI contrast agents; Lutetium-177
25.  ADVANCES IN MOLECULAR IMAGING OF PANCREATIC BETA CELLS 
The development of non-invasive imaging methods for early diagnosis of the beta cell associated metabolic diseases, including type 1 and type 2 diabetes (T1D and T2D), has recently drawn considerable interest from the molecular imaging community as well as clinical investigators. Due to the challenges imposed by the location of the pancreas, the sparsely dispersed beta cell population within the pancreas, and the poor understanding of the pathogenesis of the diseases, clinical diagnosis of beta cell abnormalities is still limited. Current diagnostic methods are invasive, often inaccurate, and usually performed post-onset of the disease. Advances in imaging techniques for probing beta cell mass and function are needed to address this critical health care problem. A variety of currently available imaging techniques have been tested for the assessment of the pancreatic beta cell islets. Here we discuss the current advances in magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging for the study of beta cell diseases. Spurred by early successes in nuclear imaging techniques for beta cells, especially positron emission tomography (PET), the need for beta cell specific ligands has expanded. Progress in the field for obtaining such ligands is presented. Additionally, we report our preliminary efforts of developing such a peptidic ligand for PET imaging of the pancreatic beta cells.
PMCID: PMC2790725  PMID: 18508529

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