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author:("Wang, lima")
1.  Active Targeting Using HER-2-affibody-conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER-2 Positive Ovarian Tumors 
Despite advances in cancer diagnosis and treatment, ovarian cancer remains one of the most fatal cancer types. The development of targeted nanoparticle imaging probes and therapeutics offers promising approaches for early detection and effective treatment of ovarian cancer. In this study, we have developed HER-2 targeted magnetic iron oxide nanoparticles (IONPs) by conjugating a high affinity and small size HER-2 affibody that is labeled with a unique near infrared dye (NIR-830) to the nanoparticles. Using a clinically relevant orthotopic human ovarian tumor xenograft model, we have shown that HER-2 targeted IONPs are selectively delivered into both primary and disseminated ovarian tumors, enabling non-invasive optical and MR imaging of the tumors as small as 1 mm in the peritoneal cavity. We have determined that HER-2 targeted delivery of the IONPs is essential for specific and sensitive imaging of the HER-2 positive tumor since we are unable to detect the imaging signal in the tumors following systemic delivery of non-targeted IONPs into the mice bearing HER-2 positive SKOV3 tumors. Furthermore, imaging signals and the IONPs are not detected in HER-2 low expressing OVCAR3 tumors after systemic delivery of HER-2 targeted-IONPs. Since HER-2 is expressed in a high percentage of ovarian cancers, the HER-2 targeted dual imaging modality IONPs have potential for the development of novel targeted imaging and therapeutic nanoparticles for ovarian cancer detection, targeted drug delivery, and image-guided therapy and surgery.
PMCID: PMC3946402  PMID: 24038985
HER-2 targeted nanoparticles; HER-2 affibody; NIR-830 dye; orthotopic human ovarian tumor xenograft model
2.  Facile non-hydrothermal synthesis of oligosaccharides coated sub-5 nm magnetic iron oxide nanoparticles with dual MRI contrast enhancement effect 
Ultrafine sub-5 nm magnetic iron oxide nanoparticles coated with oligosaccharides (SIO) with dual T1-T2 weighted contrast enhancing effect and fast clearance has been developed as magnetic resonance imaging (MRI) contrast agent. Excellent water solubility, biocompatibility and high stability of such sub-5 nm SIO nanoparticles were achieved by using the “in-situ polymerization” coating method, which enables glucose forming oligosaccharides directly on the surface of hydrophobic iron oxide nanocrystals. Reported ultrafine SIO nanoparticles exhibit a longitudinal relaxivity (r1) of 4.1 mM−1s−1 and a r1/r2 ratio of 0.25 at 3 T (clinical field strength), rendering improved T1 or “brighter” contrast enhancement in T1-weighted MRI in addition to typical T2 or “darkening” contrast of conventional iron oxide nanoparticles. Such dual contrast effect can be demonstrated in liver imaging with T2 “darkening” contrast in the liver parenchyma but T1 “bright” contrast in the hepatic vasculature. More importantly, this new class of ultrafine sub-5 nm iron oxide nanoparticles showed much faster body clearance than those with larger sizes, promising better safety for clinical applications.
PMCID: PMC4147377  PMID: 25181490
3.  Theranostic Nanoparticles Carrying Doxorubicin Attenuate Targeting Ligand Specific Antibody Responses Following Systemic Delivery 
Theranostics  2015;5(1):43-61.
Understanding the effects of immune responses on targeted delivery of nanoparticles is important for clinical translations of new cancer imaging and therapeutic nanoparticles. In this study, we found that repeated administrations of magnetic iron oxide nanoparticles (IONPs) conjugated with mouse or human derived targeting ligands induced high levels of ligand specific antibody responses in normal and tumor bearing mice while injections of unconjugated mouse ligands were weakly immunogenic and induced a very low level of antibody response in mice. Mice that received intravenous injections of targeted and polyethylene glycol (PEG)-coated IONPs further increased the ligand specific antibody production due to differential uptake of PEG-coated nanoparticles by macrophages and dendritic cells. However, the production of ligand specific antibodies was markedly inhibited following systemic delivery of theranostic nanoparticles carrying a chemotherapy drug, doxorubicin. Targeted imaging and histological analysis revealed that lack of the ligand specific antibodies led to an increase in intratumoral delivery of targeted nanoparticles. Results of this study support the potential of further development of targeted theranostic nanoparticles for the treatment of human cancers.
PMCID: PMC4265747  PMID: 25553097
Targeting ligands; nanoparticles; antibody; immune response; tumor imaging; nanoparticle delivery.
4.  Age Distribution of Various Corneal Diseases in China by Histopathological Examination of 3112 Surgical Specimens 
To analyze the age distribution of corneal disease in China by histopathological examination.
A retrospective histopathological analysis of the prevalence and age distribution of corneal disease was performed on corneal specimens from 3112 patients who underwent corneal surgery between 2001 and 2012 in the Henan Eye Institute.
Infectious keratitis (2212 specimens, 71.08%) was the most common corneal disease. The distribution of age groups was significantly higher after age 30 (16.29%), reaching a peak by age 40 (22.04%) and remaining high until age 59 (21.05%, P < 0.01). The highest age-specific percentage of corneal infection was between the ages of 50 and 59 (83.21%, P < 0.01). Noninfectious corneal diseases occurred mainly in the age range of 10 to 19 years (42.47%) and 20 to 29 years (40.78%). Between the ages of 10 and 19, the leading pathologic diagnosis was keratoconus, accounting for 21.29% of cases. In patients aged 80 and older, the most common noninfectious corneal diseases were Terrien-marginal degeneration (8.33%) and Mooren's ulcer (4.17%). The highest incidence of corneal tumor was seen between the ages of 0 and 9 years (65.83%) and age 80 and older (45.86%; P < 0.01).
The age distribution of corneal diseases in the Chinese population is distinct from other countries. The corneal diseases tend to increase in prevalence in middle age, especially for infectious corneal disease. The prevalence of corneal tumor is high in early and late life. Corneal degenerative disease occurs predominately in seniors.
We found significant differences in the age distributions of common corneal diseases, such as corneal infection, corneal tumor, and degeneration.
PMCID: PMC4015267  PMID: 24722694
age; corneal disease; histopathology; infection; tumor
5.  Detection of “oncometabolite” 2-hydroxyglutarate by magnetic resonance analysis as a biomarker of IDH1/2 mutations in glioma 
Somatic mutations in isocitrate dehydrogenase (IDH)1 and 2 have been identified in a subset of gliomas, rendering these tumors with elevated levels of “oncometabolite,” D-2-hydroxyglutarate (2HG). Herein, we report that 2HG can be precisely detected by magnetic resonance (MR) in human glioma specimens and used as a reliable biomarker to identify this subset of tumors. Specifically, we developed a two-dimensional correlation spectroscopy resonance method to reveal the distinctive cross-peak pattern of 2HG in the complex metabolite nuclear MR spectra of brain tumor tissues. This study demonstrates the feasibility, specificity, and selectivity of using MR detection and quantification of 2HG for the diagnosis and classification of IDH1/2 mutation-positive brain tumors. It further opens up the possibility of developing analogous non-invasive MR-based imaging and spectroscopy studies directly in humans in the neuro-oncology clinic.
PMCID: PMC4147374  PMID: 22426639
Cancer; 2-Hydroxyglutarate; Isocitrate dehydrogenase; Nuclear magnetic resonance; Biomarker
6.  Development of Receptor Targeted Magnetic Iron Oxide Nanoparticles for Efficient Drug Delivery and Tumor Imaging 
The development of multifunctional nanoparticles that have dual capabilities of tumor imaging and delivering therapeutic agents into tumor cells holds great promises for novel approaches for tumor imaging and therapy. We have engineered urokinase plasminogen activator receptor (uPAR) targeted biodegradable nanoparticles using a size uniform and amphiphilic polymer-coated magnetic iron oxide (IO) nanoparticle conjugated with the amino-terminal fragment (ATF) of urokinase plasminogen activator (uPA), which is a high affinity natural ligand for uPAR. We further developed methods to encapsulate hydrophobic chemotherapeutic drugs into the polymer layer on the IO nanoparticles, making these targeted magnetic resonance imaging (MRI) sensitive nanoparticles drug delivery vehicles. Using a fluorescent drug doxorubicin (Dox) as a model system, we showed that this hydrophobic drug can be efficiently encapsulated into the uPAR-targeted IO nanoparticles. This class of Dox-loaded nanoparticles has a compact size and is stable in pH 7.4 buffer. However, encapsulated Doxcan be released from the nanoparticles at pH 4.0 to 5.0 within 2 hrs. In comparison with the effect of equivalent dosage of free drug or non-targeted IO-Dox nanoparticles, uPAR-targeted IO-Dox nanoparticles deliver higher levels of Dox into breast cancer cells and produce a stronger inhibitory effect on tumor cell growth. Importantly, Dox-loaded IO nanoparticles maintain their T2 MRI contrast effect after being internalized into the tumor cells due to their significant susceptibility effect in the cells, indicating that this drug delivery nanoparticle has the potential to be used as targeted therapeutic imaging probes for monitoring the drug delivery using MRI.
PMCID: PMC4139059  PMID: 25152701
Magnetic Iron Oxide Nanoparticles; uPAR; Targeted Nanoparticle; Breast Cancer; Drug Delivery Nanoparticle; Doxorubicin
7.  Casein-coated Iron Oxide Nanoparticles for High MRI Contrast Enhancement and Efficient Cell Targeting 
ACS applied materials & interfaces  2013;5(11):4632-4639.
Surface properties, as well as inherent physicochemical properties, of the engineered nanomaterials play important roles in their interactions with the biological systems, which eventually affect their efficiency in diagnostic and therapeutic applications. Here we report a new class MRI contrast agent based on milk casein protein coated iron oxide nanoparticles (CNIOs) with a core size of 15 nm and hydrodynamic diameter ~30 nm. These CNIOs exhibited excellent water-solubility, colloidal stability, and biocompatibility. Importantly, CNIOs exhibited prominent T2 enhancing capability with a transverse relaxivity r2 of 273 mM−1s−1 at 3 Tesla. The transverse relaxivity is ~2.5-fold higher than that of iron oxide nanoparticles with the same core but an amphiphilic polymer coating. CNIOs showed pH-responsive properties, formed loose and soluble aggregates near the pI (pH~4.0). The aggregates could be dissociated reversibly when the solution pH was adjusted away from the pI. The transverse relaxation property and MRI contrast enhancing effect of CNIOs remained unchanged in the pH range of 2.0 to 8.0. Further functionalization of CNIOs can be achieved via surface modification of the protein coating. Bio-affinitive ligands, such as a single chain fragment from the antibody of epidermal growth factor receptor (ScFvEGFR), could be readily conjugated onto the protein coating, enabling specific targeting to MDA-MB-231 breast cancer cells over-expressing EGFR. T2-weighted MRI of mice intravenously administered with CNIOs demonstrated strong contrast enhancement in the liver and spleen. These favorable properties suggest CNIOs as a class of biomarker targeted magnetic nanoparticles for MRI contrast enhancement and related biomedical applications.
PMCID: PMC3699787  PMID: 23633522
iron; oxide; nanoparticles; magnetic resonance; imaging; casein; contrast agent; targeting
8.  Theranostic Nanoparticles with Controlled Release of Gemcitabine for Targeted Therapy and MRI of Pancreatic Cancer 
ACS nano  2013;7(3):2078-2089.
The tumor stroma in human cancers significantly limits the delivery of therapeutic agents into cancer cells. To develop an effective therapeutic approach overcoming the physical barrier of the stroma, we engineered urokinase plasminogen activator receptor (uPAR)-targeted magnetic iron oxide nanoparticles (IONPs) carrying gemcitabine (Gem) as a chemotherapy drug for targeted delivery into uPAR-expressing tumor and stromal cells. The uPAR-targeted nanoparticle construct, ATF-IONP-Gem, was prepared by conjugating IONPs with the amino-terminal fragment (ATF) peptide of the receptor-binding domain of uPA, a natural ligand of uPAR, and Gem via a lysosomally cleavable tetrapeptide linker. These theranostic nanoparticles enable intracellular release of Gem following receptor-mediated endocytosis of ATF-IONP-Gem into tumor cells, and also allow in vivo magnetic resonance imaging (MRI) of tumors. Our results demonstrated the pH- and lysosomal enzyme-dependent release of gemcitabine, preventing the drug from enzymatic degradation. Systemic administrations of ATF-IONP-Gem significantly inhibited the growth of orthotopic human pancreatic cancer xenografts in nude mice. With MRI contrast enhancement by IONPs, we detected the presence of IONPs in the residual tumor lesions following the treatment, suggesting the possibility of monitoring drug delivery and assessing drug resistant tumors by MRI. The theranostic ATF-IONP-Gem nanoparticle has great potential for the development of targeted therapeutic and imaging approaches that are capable of overcoming the tumor stromal barrier, thus enhancing the therapeutic effect of nanoparticle drugs on pancreatic cancers.
PMCID: PMC3609912  PMID: 23402593
targeted cancer therapy; theranostic nanoparticle; uPAR; pancreatic cancer; gemcitabine; controlled drug release; magnetic resonance imaging; drug delivery
9.  Exome Sequencing Analysis Identifies Compound Heterozygous Mutation in ABCA4 in a Chinese Family with Stargardt Disease 
PLoS ONE  2014;9(3):e91962.
Stargardt disease is the most common cause of juvenile macular dystrophy. Five subjects from a two-generation Chinese family with Stargardt disease are reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family initiated the disease during childhood, developing progressively impaired central vision and bilateral atrophic macular lesions in the retinal pigmental epithelium (RPE) that resembled a “beaten-bronze” appearance. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. Exome sequencing was used to analyze the exome of two patients II1, II2. A total of 50709 variations shared by the two patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in all family members by PCR and Sanger sequencing. Compound heterozygous variants p.Y808X and p.G607R of the ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) gene, which encodes the ABCA4 protein, a member of the ATP-binding cassette (ABC) transport superfamily, were identified as causative mutations for Stargardt disease of this family. Our findings provide one novel ABCA4 mutation in Chinese patients with Stargardt disease.
PMCID: PMC3954841  PMID: 24632595
10.  The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress 
Nucleic Acids Research  2014;42(8):4972-4984.
In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.
PMCID: PMC4005647  PMID: 24561807
11.  Geographic Co-distribution of Influenza Virus Subtypes H7N9 and H5N1 in Humans, China 
Emerging Infectious Diseases  2013;19(11):1898-1900.
PMCID: PMC3837643  PMID: 24206620
geographic co-distribution; influenza; subtypes; H7N9; H5N1; humans; China; avian influenza; influenza A; viruses; low pathogenicity; highly pathogenic
12.  Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer 
Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs.
PMCID: PMC3794963  PMID: 24124366
magnetic nanoparticles; active targeting; antifouling; breast cancer; magnetic resonance imaging
13.  T1-Weighted Ultrashort Echo Time Method for Positive Contrast Imaging of Magnetic Nanoparticles and Cancer Cells Bound With the Targeted Nanoparticles 
To obtain positive contrast based on T1 weighting from magnetic iron oxide nanoparticle (IONP) using ultrashort echo time (UTE) imaging and investigate quantitative relationship between positive contrast and the core size and concentration of IONPs.
Materials and Methods
Solutions of IONPs with different core sizes and concentrations were prepared. T1 and T2 relaxation times of IONPs were measured using the inversion recovery turbo spin echo (TSE) and multi-echo spin echo sequences at 3 Tesla. T1-weighted UTE gradient echo and T2-weighted TSE sequences were used to image IONP samples. U87MG glioblastoma cells bound with arginine-glycine-aspartic acid (RGD) peptide and IONP conjugates were scanned using UTE, T1 and T2-weighted sequences.
Positive contrast was obtained by UTE imaging from IONPs with different core sizes and concentrations. The relative-contrast-to-water ratio of UTE images was three to four times higher than those of T2-weighted TSE images. The signal intensity increases as the function of the core size and concentration. Positive contrast was also evident in cell samples bound with RGD-IONPs.
UTE imaging allows for imaging of IONPs and IONP bound tumor cells with positive contrast and provides contrast enhancement and potential quantification of IONPs in molecular imaging applications.
PMCID: PMC3785614  PMID: 21182139
magnetic nanoparticle; magnetic resonance imaging; iron oxide; ultrashort TE; molecular Imaging
14.  Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs 
BMC Microbiology  2013;13:184.
Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates.
Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources.
We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent inhibitors of Mpn growth and that the mechanism of inhibition are most likely due to inhibition of enzymes in the nucleotide biosynthesis pathway and nucleoside transporter. Our results suggest that enzymes in Mycoplasma nucleotide biosynthesis are potential targets for future design of antibiotics against Mycoplasma infection.
PMCID: PMC3750255  PMID: 23919755
Mycoplasma pneumoniae; Growth inhibition; 6-thioguanine; Trifluorothymidine; Hypoxanthine guanine phosphoribosyl transferase; Thymidine kinase
15.  Molecular Imaging of Pancreatic Cancer in an Animal Model Using Targeted Multifunctional Nanoparticles 
Gastroenterology  2009;136(5):1514-25.e2.
Background & Aims
Identification of a ligand/receptor system that enables functionalized nanoparticles to efficiently target pancreatic cancer holds great promise for the development of novel approaches for the detection and treatment of pancreatic cancer. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging of pancreatic cancer using multifunctional nanoparticles.
The uPAR-targeted dual-modality molecular imaging nanoparticle probe is designed and prepared by conjugating a near-infrared dye-labeled amino-terminal fragment of the receptor binding domain of urokinase plasminogen activator to the surface of functionalized magnetic iron oxide nanoparticles.
We have shown that the systemic delivery of uPAR-targeted nanoparticles leads to their selective accumulation within tumors of orthotopically xenografted human pancreatic cancer in nude mice. The uPAR-targeted nanoparticle probe binds to and is subsequently internalized by uPAR-expressing tumor cells and tumor-associated stromal cells, which facilitates the intratumoral distribution of the nanoparticles and increases the amount and retention of the nanoparticles in a tumor mass. Imaging properties of the nanoparticles enable in vivo optical and magnetic resonance imaging of uPAR-elevated pancreatic cancer lesions.
Targeting uPAR using biodegradable multifunctional nanoparticles allows for the selective delivery of the nanoparticles into primary and metastatic pancreatic cancer lesions. This novel receptor-targeted nanoparticle is a potential molecular imaging agent for the detection of pancreatic cancer.
PMCID: PMC3651919  PMID: 19208341
16.  Group Independent Component Analysis and Functional MRI Examination of Changes in Language Areas Associated with Brain Tumors at Different Locations 
PLoS ONE  2013;8(3):e59657.
This study investigates the effect of tumor location on alterations of language network by brain tumors at different locations using blood oxygenation level dependent (BOLD) fMRI and group independent component analysis (ICA).
Subjects and Methods
BOLD fMRI data were obtained from 43 right handed brain tumor patients. Presurgical mapping of language areas was performed on all 43 patients with a picture naming task. All data were retrospectively analyzed using group ICA. Patents were divided into three groups based on tumor locations, i.e., left frontal region, left temporal region or right hemisphere. Laterality index (LI) was used to assess language lateralization in each group.
The results from BOLD fMRI and ICA revealed the different language activation patterns in patients with brain tumors located in different brain regions. Language areas, such as Broca’s and Wernicke’s areas, were intact in patients with tumors in the right hemisphere. Significant functional changes were observed in patients with tumor in the left frontal and temporal areas. More specifically, the tumors in the left frontal region affect both Broca’s and Wernicke’s areas, while tumors in the left temporal lobe affect mainly Wernicke’s area. The compensated activation increase was observed in the right frontal areas in patients with left hemisphere tumors.
Group ICA provides a model free alternative approach for mapping functional networks in brain tumor patients. Altered language activation by different tumor locations suggested reorganization of language functions in brain tumor patients and may help better understanding of the language plasticity.
PMCID: PMC3608667  PMID: 23555736
17.  Modeling the evolution dynamics of exon-intron structure with a general random fragmentation process 
Most eukaryotic genes are interrupted by spliceosomal introns. The evolution of exon-intron structure remains mysterious despite rapid advance in genome sequencing technique. In this work, a novel approach is taken based on the assumptions that the evolution of exon-intron structure is a stochastic process, and that the characteristics of this process can be understood by examining its historical outcome, the present-day size distribution of internal translated exons (exon). Through the combination of simulation and modeling the size distribution of exons in different species, we propose a general random fragmentation process (GRFP) to characterize the evolution dynamics of exon-intron structure. This model accurately predicts the probability that an exon will be split by a new intron and the distribution of novel insertions along the length of the exon.
As the first observation from this model, we show that the chance for an exon to obtain an intron is proportional to its size to the 3rd power. We also show that such size dependence is nearly constant across gene, with the exception of the exons adjacent to the 5′ UTR. As the second conclusion from the model, we show that intron insertion loci follow a normal distribution with a mean of 0.5 (center of the exon) and a standard deviation of 0.11. Finally, we show that intron insertions within a gene are independent of each other for vertebrates, but are more negatively correlated for non-vertebrate. We use simulation to demonstrate that the negative correlation might result from significant intron loss during evolution, which could be explained by selection against multi-intron genes in these organisms.
The GRFP model suggests that intron gain is dynamic with a higher chance for longer exons; introns are inserted into exons randomly with the highest probability at the center of the exon. GRFP estimates that there are 78 introns in every 10 kb coding sequences for vertebrate genomes, agreeing with empirical observations. GRFP also estimates that there are significant intron losses in the evolution of non-vertebrate genomes, with extreme cases of around 57% intron loss in Drosophila melanogaster, 28% in Caenorhabditis elegans, and 24% in Oryza sativa.
PMCID: PMC3732091  PMID: 23448166
Evolution of exon-intron structure; General random fragmentation process; Simulation
18.  PEGylation of Protein-based MRI Contrast Agents Improves Relaxivities and Biocompatibilities 
Journal of Inorganic Biochemistry  2011;107(1):111-118.
Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd3+ binding sites into a stable protein resulting in significantly increased longitudinal (r1) and transverse (r2) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r1 and r2 relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.
PMCID: PMC3273044  PMID: 22178673
Contrast agent; Magnet Resonance Imaging; PEGylation; Relaxivity
19.  Canine Model of Convection-Enhanced Delivery of Cetuximab Conjugated Iron-Oxide Nanoparticles Monitored with Magnetic Resonance Imaging 
Clinical neurosurgery  2012;59:107-113.
Visualizing distribution of infused therapeutic agents into the brain by convection-enhanced delivery (CED) is necessary to ensure accurate delivery into target sites. Recently, bioconjugated magnetic iron-oxide nanoparticles (IONPs) have been shown to produce a magnetic resonance imaging (MRI) contrast in the rodent brain after CED permitting direct visualization of nanoparticle distribution and dispersion over time. We have now studied the CED of IONPs in the larger, more clinically relevant, canine brain for assessment of distribution, dispersion, toxicity, and clearance.
Eight healthy laboratory dogs were infused with either free IONPs (n=4) or cetuximab-conjugated IONPs (cetuximab-IONPs; n=4) at different infusion rates (0.5, 1.0, 3.0, and 5.0 microliters/min) and volumes (180, 300, 360, and 720 microliters). IONP CED was monitored by sequential MRIs (pre-operative, within 12 h, 5 d, 7 d, and 30 d post-operative) and volumes of distribution and dispersion were calculated from the MR images. Toxicity assessment was based on MRI, clinical examination, hematologic/cerebrospinal fluid (CSF) analysis, and brain histopathological evaluation.
Robust delivery and monitoring of IONP distribution in the grey and white matter of the canine brain was achieved by CED and MRI. Quantitative measurements of IONP distribution volumes was achieved by MRI. Distribution volumes were linearly proportional to infusion volumes and dispersion of IONPs occurred 5 d after CED. Use of the slower infusion rates allowed for more uniform initial distribution of IONPs and low infusate leakback of IONPs along the catheter track. No signs of toxicity were found in any animals that underwent IONP or cetuximab-conjugated IONP CED based on physical examination and hematologic/CSF analysis. MRI and histopathologic analysis of brains 30 d after CED revealed near complete clearance of IONPs. Uptake of IONPs by astrocytes and microglia was found adjacent to the catheter sites.
CED of either free or cetuximab-conjugated IONPs in the canine brain is safe and represents an effective delivery method in a larger animal model. MRI monitoring of distribution and dispersion of IONPs is possible and quantitative after CED. Future studies involving CED of bioconjugated IONPs in canines with spontaneous gliomas may provide a unique and more clinical relevant animal model for targeting infiltrative cancer cells responsible for tumor recurrence.
PMCID: PMC3473085  PMID: 22960522
Glioblastoma; Magnetic Nanoparticles; Convection-Enhanced Delivery; MRI; EGFR; Cetuximab; Canine
20.  Protein-Based MRI Contrast Agents for Molecular Imaging of Prostate Cancer 
The purpose of this study was to demonstrate a novel protein-based magnetic resonance imaging (MRI) contrast agent that has the capability of targeting prostate cancer and which provides high-sensitivity MR imaging in tumor cells and mouse models.
A fragment of gastrin-releasing peptide (GRP) was fused into a protein-based MRI contrast agent (ProCA1) at different regions. MR imaging was obtained in both tumor cells (PC3 and H441) and a tumor mouse model administrated with ProCA1.GRP.
PC3 and DU145 cells treated with ProCA1.GRPs exhibited enhanced signal in MRI. Intratumoral injection of ProCA1.GRP in a PC3 tumor model displayed enhanced MRI signal. The contrast agent was retained in the PC3 tumor up to 48 h post-injection.
Protein-based MRI contrast agent with tumor targeting modality can specifically target GRPR-positive prostate cancer. Intratumoral injection of the ProCA1 agent in the prostate cancer mouse model verified the targeting capability of ProCA1.GRP and showed a prolonged retention time in tumors.
PMCID: PMC3463956  PMID: 20574851
MRI; Contrast agents; Prostate cancer; Molecular imaging; Relaxivity
21.  Biomechanical evaluation of three surgical scenarios of posterior lumbar interbody fusion by finite element analysis 
For the treatment of low back pain, the following three scenarios of posterior lumbar interbody fusion (PLIF) were usually used, i.e., PLIF procedure with autogenous iliac bone (PAIB model), PLIF with cages made of PEEK (PCP model) or titanium (Ti) (PCT model) materiel. But the benefits or adverse effects among the three surgical scenarios were still not fully understood.
Finite element analysis (FEA), as an efficient tool for the analysis of lumbar diseases, was used to establish a three-dimensional nonlinear L1-S1 FE model (intact model) with the ligaments of solid elements. Then it was modified to simulate the three scenarios of PLIF. 10 Nm moments with 400 N preload were applied to the upper L1 vertebral body under the loading conditions of extension, flexion, lateral bending and torsion, respectively.
Different mechanical parameters were calculated to evaluate the differences among the three surgical models. The lowest stresses on the bone grafts and the greatest stresses on endplate were found in the PCT model. The PCP model obtained considerable stresses on the bone grafts and less stresses on ligaments. But the changes of stresses on the adjacent discs and endplate were minimal in the PAIB model.
The PCT model was inferior to the other two models. Both the PCP and PAIB models had their own relative merits. The findings provide theoretical basis for the choice of a suitable surgical scenario for different patients.
PMCID: PMC3430554  PMID: 22709659
Spine; Cage; PEEK; Autogenous iliac bone; Ligaments
22.  Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism 
PLoS ONE  2012;7(5):e37724.
To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design.
PMCID: PMC3360617  PMID: 22662200
23.  White Matter Hyperintensities and Changes in White Matter Integrity in Patients with Alzheimer’s Disease 
Neuroradiology  2010;53(5):373-381.
White matter hyperintensities (WMHs) are a risk factor for Alzheimer’s disease (AD). This study investigated the relationship between WMHs and white matter changes in AD using diffusion tensor imaging (DTI) and the sensitivity of each DTI index in distinguishing AD with WMHs.
Subjects and Methods
Forty-four subjects with WMHs were included. Subjects were classified into three groups based on the Scheltens rating scale: 15 AD patients with mild WMHs, 12 AD patients with severe WMHs, and 17 controls with mild WMHs. Fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (DR) and axial diffusivity (DA) were analyzed using the region of interest and Tract-Based Spatial Statistics methods. Sensitivity and specificity of DTI indices in distinguishing AD groups from the controls were evaluated.
AD patients with mild WMHs exhibited differences from control subjects in most DTI indices in the medial temporal and frontal areas; however, differences in DTI indices from AD patients with mild WMHs and AD patients with severe WMHs were found in the parietal and occipital areas. FA and DR were more sensitive measurements than MD and DA in differentiating AD patients from controls, while MD was a more sensitive measurement in distinguishing AD patients with severe WMHs from those with mild WMHs.
WMHs may contribute to the white matter changes in AD brains, specifically in temporal and frontal areas. Changes in parietal and occipital lobes may be related to the severity of WMHs. DR may serve as an imaging marker of myelin deficits associated with AD.
PMCID: PMC3332065  PMID: 21152911
Magnetic Resonance Imaging; White Matter Hyperintensity; Diffusion Tensor; Alzheimer’s Disease; Diffusivity; White Matter
24.  Improving the Magnetic Resonance Imaging Contrast and Detection Methods with Engineered Magnetic Nanoparticles 
Theranostics  2012;2(1):86-102.
Engineering and functionalizing magnetic nanoparticles have been an area of the extensive research and development in the biomedical and nanomedicine fields. Because their biocompatibility and toxicity are well investigated and better understood, magnetic nanoparticles, especially iron oxide nanoparticles, are better suited materials as contrast agents for magnetic resonance imaging (MRI) and for image-directed delivery of therapeutics. Given tunable magnetic properties and various surface chemistries from the coating materials, most applications of engineered magnetic nanoparticles take advantages of their superb MRI contrast enhancing capability as well as surface functionalities. It has been found that MRI contrast enhancement by magnetic nanoparticles is highly dependent on the composition, size and surface properties as well as the degree of aggregation of the nanoparticles. Therefore, understanding the relationships between these intrinsic parameters and the relaxivities that contribute to MRI contrast can lead to establishing essential guidance that may direct the design of engineered magnetic nanoparticles for theranostics applications. On the other hand, new contrast mechanism and imaging strategy can be developed based on the novel properties of engineered magnetic nanoparticles. This review will focus on discussing the recent findings on some chemical and physical properties of engineered magnetic nanoparticles affecting the relaxivities as well as the impact on MRI contrast. Furthermore, MRI methods for imaging magnetic nanoparticles including several newly developed MRI approaches aiming at improving the detection and quantification of the engineered magnetic nanoparticles are described.
PMCID: PMC3263519  PMID: 22272222
magnetic nanoparticles; engineering; functionalizing; magnetic resonance imaging
25.  The Kinetic Effects on Thymidine Kinase 2 by Enzyme-Bound dTTP May Explain the Mitochondrial Side Effects of Antiviral Thymidine Analogs▿† 
Mitochondrial thymidine kinase 2 (TK2) is a key enzyme in the salvage of pyrimidine deoxynucleosides needed for mitochondrial DNA synthesis. TK2 phosphorylates thymidine (dThd), deoxycytidine (dCyd), and many other antiviral pyrimidine nucleoside analogs. Zidovudine (AZT) is the first nucleoside analog approved for anti-HIV therapy, and it is still used in combination with other drugs. One of the side effects of long-term treatment with nucleoside analogs is mitochondrial DNA depletion, which has been ascribed to competition by AZT for the endogenous dThd phosphorylation carried out by TK2. Here we studied the kinetics of AZT and 3′-fluorothymidine phosphorylation by recombinant human TK2 and the effects of these and other pyrimidine nucleoside analogs on the phosphorylation of dThd and dCyd. Thymidine analogs strongly inhibited dThd phosphorylation but not dCyd phosphorylation, which instead was stimulated ∼30%. We found that recombinant human TK2 contained the feedback inhibitor dTTP in a 1:1 molar ratio and that incubation with dThd and AZT could completely remove the enzyme-bound dTTP, but dCyd was less efficient in this regard. The release of feedback inhibitor by dThd and dThd analogs most likely accounts for the observed kinetics. Similar effects were also observed with native rat liver mitochondrial TK2, strongly indicating a physiologic role for this process, which most likely is an important factor in the mitochondrial toxicity observed with antiviral nucleoside analogs.
PMCID: PMC3101437  PMID: 21444706

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