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1.  Cytosolic carboxypeptidase CCP6 is required for megakaryopoiesis by modulating Mad2 polyglutamylation 
The Journal of Experimental Medicine  2014;211(12):2439-2454.
Ye et al. identify cytosolic carboxypeptidase CCP6 as a protein required for the regulation of bone marrow megakaryopoiesis in mice. The authors find that Mad2 (a core component of spindle checkpoint in mitosis) is a substrate of CCP6 in megakaryocytes and is polyglutamylated by proteins TTLL6 and TTLL4, subsequently affecting the activity of Aurora B kinase. Mad2 is thus additionally implicated in megakaryopoiesis regulation.
Bone marrow progenitor cells develop into mature megakaryocytes (MKs) to produce platelets for hemostasis and other physiological functions. However, the molecular mechanisms underlying megakaryopoiesis are not completely defined. We show that cytosolic carboxypeptidase (CCP) 6 deficiency in mice causes enlarged spleens and increased platelet counts with underdeveloped MKs and dysfunctional platelets. The prominent phenotypes of CCP6 deficiency are different from those of CCP1-deficient mice. We found that CCP6 and tubulin tyrosine ligase-like family (TTLL) members TTLL4 and TTLL6 are highly expressed in MKs. We identify Mad2 (mitotic arrest deficient 2) as a novel substrate for CCP6 and not CCP1. Mad2 can be polyglutamylated by TTLL4 and TTLL6 to modulate the maturation of MKs. CCP6 deficiency causes hyperglutamylation of Mad2 to promote activation of Aurora B, leading to suppression of MK maturation. We reveal that Mad2 polyglutamylation plays a critical role in the regulation of megakaryopoiesis.
PMCID: PMC4235637  PMID: 25332286
2.  Expression of folate receptors alpha and beta in normal and cancerous gynecologic tissues: correlation of expression of the beta isoform with macrophage markers 
Folate receptor alpha (FOLR1/FRA) is expressed in a number of epithelial cancers and in particular epithelial ovarian cancer (EOC), especially of the serous histotype. Recent studies have shown that EOC originates from the fallopian tube fimbriae rather than from epithelial cells lining the ovary. We have previously shown by immunohistochemistry a strong correlation between FRA expression in EOC and normal and fallopian adenocarcinoma. Folate receptor beta (FOLR2/FRB) has been described to be expressed by macrophages both in inflammatory disorders and certain epithelial cancers. Given the high sequence identity of these two folate receptor family members we sought to investigate the architectural and cell-specific expression of these two receptors in gynecologic tissues.
RNA scope, a novel chromogenic in situ hybridization assay tool, was used to examine expression of the alpha (FOLR1) and beta (FOLR2) isoforms of folate receptor relative to each other as well as to the macrophage markers CD11b and CD68, in samples of normal fallopian tube and fallopian adenocarcinoma as well as normal ovary and EOC.
We demonstrated expression of both FOLR1 and FOLR2 in EOC, normal fallopian tube and fallopian adenocarcinoma tissue while very little expression of either marker was observed in normal ovary. Furthermore, FOLR2 was shown to be expressed almost exclusively in macrophages, of both the M1 and M2 lineages, as determined by co-expression of CD11b and/or CD68, with little or no expression in epithelial cells.
These findings further substantiate the hypothesis that the cell of origin of EOC is tubal epithelium and that the beta isoform of folate receptor is primarily restricted to macrophages. Further, macrophages expressing FOLR2 may represent tumor associated or infiltrating macrophages (TAMs) in epithelial cancers.
PMCID: PMC4464638  PMID: 25971554
FOLR1; FOLR2; CD68; CD11b; Epithelial ovarian cancer; Fallopian adenocarcinoma; Tumor microenvironment; Stromal cells
3.  Association between MUC5B and TERT Polymorphisms and Different Interstitial Lung Disease Phenotypes 
TERT and MUC5B polymorphisms have been consistently associated with idiopathic pulmonary fibrosis (IPF) in recent genome-wide genetic studies. However, it remains unclear how both loci contribute to the susceptibility to different entities of sporadic interstitial lung disease (ILD). We sought to test the associations of the two polymorphisms with IPF and non-IPF ILD entities in a Caucasian population. Associations between two polymorphisms in TERT (rs2736100) and MUC5B (rs35705950) and IPF or non-IPF sporadic ILD were tested using 227 ILD patients and 689 controls. Genotypic data were also correlated with pulmonary functions measured in ILD patients. As a result, rs2736100 and rs35705950 were significantly and independently associated with ILD as a single phenotype [Odds ratio (OR)=1.29, 95% Confidence Interval (CI): 1.04–1.60, p= 2×10−2 and OR=2.22, 95%CI: 1.69–2.92, p=7×10−9, respectively). When considering IPF and “other ILD” (non-IPF) separately, rs35705950 had a stronger association with IPF (OR=3.2, 95%CI: 2.21–4.63, p=1.2×10−10) than other ILD (OR=1.72, 95%CI: 1.22–2.42, p=1.2×10−3). In contrast, rs2736100 was associated with other ILD (OR=1.43, 95%CI: 1.11–1.85, p=6.2×10−3) but not IPF (OR=1.08, 95%CI: 0.78–1.49, p>0.05). Rs35705950 was significantly correlated with increased pulmonary function (p<0.05). It was also associated with ILD without airflow obstruction in both IPF and other ILD groups (p<0.01 for both), and conferred the highest risk for IPF without airflow obstruction (OR=4.46, 95%CI: 2.60–7.66, p=4.5×10−9). Our study suggests that while both loci confer independent risks for ILD, rs35705950 may particularly contribute differentially to IPF and other ILD entities. Our study further highlighted the genetic and phenotypic heterogeneity of ILD.
PMCID: PMC4074379  PMID: 24434656
interstitial lung disease; idiopathic pulmonary fibrosis; TERT; MUC5B; polymorphism
4.  Polymorphisms of the Ovine BMPR-IB, BMP-15 and FSHR and Their Associations with Litter Size in Two Chinese Indigenous Sheep Breeds 
The Small Tailed Han sheep and Hu sheep are two prolific local sheep in China. In this study, the polymorphisms of BMPR-IB (Bone morphogenetic protein receptor IB), BMP-15 (Bone morphogenetic protein 15) and FSHR (follicle stimulating hormone receptor) were investigated to check whether they are associated with litter size in Small Tailed Han sheep and Hu sheep. Consequently, three polymorphisms, FecB mutation in BMPR-IB (c.746A>G), FecG mutation in BMP-15 (c.718C>T) and the mutation (g. 47C>T) in FSHR were found in the above two sheep breeds with a total number of 1630 individuals. The single marker association analysis showed that the three mutations were significantly associated with litter size. The ewes with genotype FecBB/FecBB and FecBB/FecB+ had 0.78 and 0.58 more lambs (p < 0.01) than those with genotype FecB+/FecB+, respectively. The heterozygous Han and Hu ewes with FecXG/FecX+ genotype showed 0.30 (p = 0.05) more lambs than those with the FecX+/FecX+ genotype. For FSHR gene, the ewes with genotype CC had 0.52 (p < 0.01) and 0.75 (p < 0.01) more lambs than those with genotypes TC and TT, respectively. Combined effect analyses indicated an extremely significant interaction (p < 0.01) between the random combinations of BMPR-IB, BMP-15 and FSHR genes on litter size. In addition, the Han and Hu ewes with BB/G+/CC genotype harbor the highest litter size among ewes analyzed in current study. In conclusion, BMPR-IB, BMP-15 and FSHR polymorphisms could be used as genetic markers in multi-gene pyramiding for improving litter size in sheep husbandry.
PMCID: PMC4463706  PMID: 25993301
BMPR-IB gene; BMP-15 gene; FSHR gene; litter size; Small Tailed Han sheep; Hu sheep
5.  SIRT1 expression is associated with poor prognosis of lung adenocarcinoma 
OncoTargets and therapy  2015;8:977-984.
Several studies have reported that the overexpression of Sirtuin 1 (SIRT1) was associated with poor prognosis in various human cancers. However, little is known regarding the prognostic value of SIRT1 in lung adenocarcinoma. Therefore, the aim of this study is to evaluate the role of SIRT1 in the prognosis of lung adenocarcinoma patients. Using a tissue microarray, we detected SIRT1 expression by immunohistochemistry in lung adenocarcinoma tissue, as well as in corresponding noncancerous tissues (NCTs). A high expression level of SIRT1 was observed in 74.7% (56/75) of patients with lung adenocarcinoma and 6.7% (5/75) of NCTs (P<0.001). SIRT1 expression was significantly associated with high pathological stage. Importantly, we found that SIRT1 expression was associated with worse overall survival in these lung adenocarcinoma patients (67.0 months vs 104.5 months; P=0.005). In addition, anaplastic lymphoma kinase, epidermal growth factor receptor, vascular endothelial growth factor (VEGF), and Survivin expression were evaluated by fluorescent in situ hybridization or immunohistochemistry, respectively. We found that VEGF and Survivin were both highly expressed in the lung adenocarcinoma tissues, as compared to NCTs. Moreover, the SIRT1 and VEGF expression statuses were significantly positively correlated (r=0.238, P=0.039), while SIRT1 and Survivin expression status were not significantly correlated (r=0.220, P=0.058). Correlation analysis showed a positive correlation between VEGF and Survivin expression (r=0.436, P<0.001). However, we found that VEGF and Survivin expression were not associated with the prognosis of lung adenocarcinoma patients (P=0.334; P=0.433, respectively). Taken together, our findings suggest that SIRT1 plays a role in the progression of lung adenocarcinoma and may be a significant prognostic indicator for lung adenocarcinoma patients.
PMCID: PMC4425340  PMID: 25995644
SIRT1; VEGF; Survivin; prognosis; lung adenocarcinoma
6.  Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress 
PLoS ONE  2015;10(3):e0121341.
The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5.
PMCID: PMC4373758  PMID: 25806814
7.  Catalytic activities of noble metal atoms on WO3 (001): nitric oxide adsorption 
Using first-principles density functional theory calculations within the generalized gradient approximation, we investigate the adsorption of NO molecule on a clean WO3(001) surface as well as on the noble metal atom (Cu, Ag, and Au)-deposited WO3(001) surfaces. We find that on a clean WO3 (001) surface, the NO molecule binds to the W atom with an adsorption energy (Eads) of −0.48 eV. On the Cu- and Ag-deposited WO3(001) surface where such noble metal atoms prefer to adsorb on the hollow site, the NO molecule also binds to the W atom with Eads = −1.69 and −1.41 eV, respectively. This relatively stronger bonding of NO to the W atom is found to be associated with the larger charge transfer of 0.43 e (Cu) and 0.33 e (Ag) from the surface to adsorbed NO. However, unlike the cases of Cu-WO3(001) and Ag-WO3(001), Au atoms prefer to adsorb on the top of W atom. On such an Au-WO3(001) complex, the NO molecule is found to form a bond to the Au atom with Eads = −1.32 eV. Because of a large electronegativity of Au atom, the adsorbed NO molecule captures the less electrons (0.04 e) from the surface compared to the Cu and Ag catalysts. Our findings not only provide useful information about the NO adsorption on a clean WO3(001) surface as well as on the noble metal atoms deposited WO3(001) surfaces but also shed light on a higher sensitive WO3 sensor for NO detection employing noble metal catalysts.
PMCID: PMC4385050  PMID: 25852357
Surface; Catalytic; Charge transfer; Bond length
8.  Minimally invasive image-guided keyhole aspiration of cerebral abscesses 
Despite the low incidence of brain abscesses in Western nations (1-2%), the incidence in developing countries is as high as 8%. We evaluate a minimally invasive image-guided keyhole aspiration of cerebral abscesses and compare it with a series of cases treated with surgical excision. 23 patients (20 male and 3 female, aged 7-67 years) underwent image-guided burr hole aspiration of single or multiple cerebral abscesses. Patient characteristics, perioperative, and postoperative data were analyzed and compared with a second group of 22 patients (14 male and 8 female, aged 12-72) treated for cerebral abscesses with open surgical excision. In all cases, the surgical procedure was performed successfully without complication. 8 of the 23 aspiration cases were performed with the aid of iMRI. A comparison of patient demographics, duration of hospital stay, duration of antibiotic therapy, postoperative neurological recovery time, intraoperative blood loss, operative duration, length of incision, postoperative fever, repeat surgery, and mortality was performed between the aspiration and excision groups. Intraoperative blood loss, operative duration, length of incision, and postoperative fever were all significantly reduced in the aspiration group. Though, duration of hospital stay and antibiotic therapy and postoperative neurological recovery time were all increased in the aspiration group, and statistical significance was observed in all except the duration of hospital stay. This technique is a feasible and comparable minimally invasive alternative to open surgical excision and may provide reduced intraoperative blood loss, shortened operative duration, improved cosmetic outcomes, and a lessened incidence of postoperative fever.
PMCID: PMC4358439  PMID: 25784984
Brain abscess; burr hole; neuronavigation; drainage; aspiration; minimally invasive
9.  Serum lemur tyrosine kinase-3: a novel biomarker for screening primary non-small cell lung cancer and predicting cancer progression 
Purpose: We aimed to determine the expression level of serum soluble lemur tyrosine kinase-3 (sLMTK3) in human non-small cell lung cancer (NSCLC), and to examine whether the s sLMTK3 level could be used as a biomarker to screen primary NSCLC and to predict lung cancer progression. Methods: Serum levels of sLMTK3 in 67 patients with primary NSCLC, 28 patients with lung benign lesion, and 53 healthy volunteers were measured by sandwich ELISA. LMTK3 protein expression in NSCLC tissues and normal lung tissues was also detected by using immunohistochemical staining. Receiver operating characteristic (ROC) curve was selected to evaluate the sensitivity and the specificity of serum sLMTK3 level. Results: The mean concentration of sLMTK3 in NSCLC group was significantly higher than in the lung benign lesion group (P < 0.001) and the healthy control group (P < 0.001). Higher sLMTK3 level was correlated with age (P = 0.013), tumor-node-metastasis (TNM) stage (P < 0.001), and lymph node metastasis (P < 0.001) of NSCLC. In contrast to the normal lung tissues, increased LMTK3 expression was found in the NSCLC tissues, and was mainly located on the cytoplasm and the nuclei of cancer cells. For separating NSCLC from control group, the corresponding areas under the ROC curve (AUC) were 0.947 for sLMTK3 and 0.804 for CEA. With cutoffs of 10.05 ng/ml for sLMTK3 and 5.0 ng/ml for CEA respectively, the sensitivity and the specificity of sLMTK3 and CEA were, 80.60% and 97.53%, 35.82% and 96.30%, respectively, indicating better diagnostic value of sLMTK3. Conclusions: The sLMTK3 level was significantly increased in human NSCLC, and could be used as a potential and valuable biomarker for screening primary NSCLC and for predicting the progression of patients with this malignancy.
PMCID: PMC4348877  PMID: 25755755
Lemur tyrosine kinase-3; non-small cell lung cancer; estrogen receptor; tumor biomarker
10.  AlGaInP LED with low-speed spin-coating silver nanowires as transparent conductive layer 
The low-speed spin-coating method was developed to prepare uniform and interconnected silver nanowires (AgNWs) film with the transmittance of 95% and sheet resistance of 20Ω/sq on glass, which was comparable to ITO. The fitting value of σdc/σop of 299.3 was attributed to the spin-coating process. Advantages of this solution-processed AgNW film on AlGaInP light-emitting diodes (LEDs) as transparent conductive layer were explored. The optical output power enhanced 100%, and the wavelength redshift decreased from 12 to 3 nm, which indicated the AgNW films prepared by low-speed spin-coating possessed attractive features for large-scale TCL applications in optoelectronic devices.
PMCID: PMC4493991  PMID: 26089002
AlGaInP; Light-emitting diodes (LEDs); Low-speed spin-coating; Silver nanowires; Transparent conductive layer
11.  Effects of Combined Anisodamine and Neostigmine Treatment on the Inflammatory Response and Liver Regeneration of Obstructive Jaundice Rats after Hepatectomy 
BioMed Research International  2014;2014:362024.
Background. Cholestasis is associated with high rates of morbidity and mortality in patients undergoing major liver resection. This study aimed to evaluate the effects of a combined anisodamine and neostigmine (Ani+Neo) treatment on the inflammatory response and liver regeneration in rats with obstructive jaundice (OJ) after partial hepatectomy. Materials and Methods. OJ was induced in the rats by bile duct ligation. After 7 days biliary drainage and partial hepatectomy were performed. These rats were assigned to a saline group or an Ani+Neo treatment group. The expressions of inflammatory mediators, liver regeneration, and liver damage were assessed at 48 h after hepatectomy. Results. The mRNA levels of TNF-α, IL-1β, IL-6, MCP-1, and MIP-1α, in the remnant livers, and the serum levels of TNF-α and IL-1β were substantially reduced in the Ani+Neo group compared with saline group (P < 0.05). The Ani+Neo treatment obviously promoted liver regeneration as indicated by the liver weights and Ki-67 labeling index (P < 0.05). The serum albumin and γ-GT levels and liver neutrophil infiltration also significantly improved in the Ani+Neo group (P < 0.05) compared with the saline group. Conclusions. These results demonstrate that the combined anisodamine and neostigmine treatment is able to improve the liver regeneration in rats with OJ by substantially alleviating the inflammatory response.
PMCID: PMC4244971  PMID: 25478569
12.  Interferon-stimulated Gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma 
Oncotarget  2014;5(18):8429-8441.
Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with poor prognosis. IFN-stimulated genes 15 (ISG15) is an ubiquitin-like molecule that is strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. However, the role of ISG15 in HCC remains unclear. In this study, we investigated the function of ISG15 during HCC progression and related mechanism using clinicopathological data, cell line and xenograft model. Our results indicated that ISG15 is highly expressed in HCC tissues and multiple HCC cell lines. ISG15 expression is significantly associated with the differentiation grade, metastatic of tumor and survival of HCC patients. However, the expression of ISG15 is not affected by HBV infection. ISG15 promotes the proliferation and migration of hepatocarcinoma cells through maintaining Survivin protein stabilization via sequestering XIAP from interacting with Survivin. Knowing down ISG15 with SiRNA inhibited the xenografted tumor growth and prolonged the lifespan of tumor-bearing mice. All these results support that ISG15 high expression is an intrinsic feature for HCC and a trigger for tumorigenesis and metastasis. ISG15 may be a prognostic biomarker and the inhibition of ISG15 could provide a therapeutic advantage for HCC patients over-expressing ISG15.
PMCID: PMC4226694  PMID: 25238261
ISG15; HCC; Metastasis; Tumorigenesis
Blood cells, molecules & diseases  2008;41(3):255-258.
Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of β-thalassemia major and sickle cell anemia. Recent genome wide association studies reported that single nucleotide polymorphisms (SNPs) in BCL11A gene on chromosome 2p16.1 were correlated with F-cells among healthy northern Europeans, and HbF among Sardinians with β-thalassemias. In this study, we showed that SNPs in BCL11A were associated with F-cell numbers in Chinese with β-thalassemia trait, and with HbF levels in Thais with either β-thalassemia or HbE trait and in African Americans with sickle cell anemia. Taken together, the data suggest that the functional motifs responsible for modulating F-cells and HbF levels reside within a 3 kb region in the second intron of BCL11A.
PMCID: PMC4100606  PMID: 18691915
14.  Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53-MDM2 interaction1 
Bioorganic & medicinal chemistry  2013;21(14):4045-4050.
Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable D-isomers to recapitulate biological activities of their parent L-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent L-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical L-peptide on its target protein binding has been estimated to be 3.0-3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX – the two negative regulators of p53. We also prepared the retro isomer and D-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of L-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of L-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an L-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural levela.
PMCID: PMC3726206  PMID: 23660015
Retro-inverso peptide; p53; MDM2; MDMX; D-peptide; Apamin; Stingin
15.  Decreased Expression of C10orf10 and Its Prognostic Significance in Human Breast Cancer 
PLoS ONE  2014;9(6):e99730.
Breast cancer is a common malignant tumor, which severely threatens the health of women with an increasing incidence in many countries. Here, we identified C10orf10 as a novel differentially expression gene using expression microarray screening. The expression analysis indicated that C10orf10 was frequently decreased in human breast cancers compared to noncancerous breast tissues (81/95, P = 0.0063). Kaplan-Meier analysis indicated that patients with low C10orf10 expression showed a poorer prognosis both in mRNA (n = 1115, P = 0.0013) and protein (n = 100, P = 0.003) levels. Univariate and multivariate analysis showed that the C10orf10 expression was an independent prognostic factor for overall survival of breast cancer patients. Further analysis revealed that low expression of C10orf10 was an unfavorable factor for the prognosis of the patients who were luminal A, luminal B, Her2+ subtypes, at histological grade 2, lymph node negative and ER positive. Our data provided the first evidence that C10orf10 expression was frequently decreased in breast cancer tissues, and low expression of C10orf10 may be an important prognostic factor for poorer survival time of breast cancer patients.
PMCID: PMC4061027  PMID: 24936657
16.  Imaging RAGE expression in atherosclerotic plaques in hyperlipidemic pigs 
EJNMMI Research  2014;4:26.
Receptor for advanced glycated end product (RAGE) expression is a prominent feature of atherosclerosis. We have previously shown in apoE null mice uptake of a radiolabeled anti-RAGE antibody in atherosclerotic plaque and now evaluate RAGE-directed imaging to identify advanced plaques in a large animal model.
Nine hyperlipidemic (HL) pigs were injected with 603.1 ± 129.5 MBq of 99mTc-anti-RAGE F(ab′)2, and after 6 h (blood pool clearance), they underwent single-photon emission computed tomography/computed tomography (SPECT/CT) imaging of the neck, thorax, and hind limbs. Two HL pigs received 99mTc non-immune IgG F(ab′)2, and three farm pigs were injected with 99mTc-anti-RAGE F(ab′)2. After imaging, the pigs were euthanized. The aorta from the root to bifurcation was dissected, and the innominates, proximal carotids, and coronaries were dissected and counted, stained for H&E and RAGE, and AHA-classified.
On pathology, 24% of the arterial segments showed AHA class III or IV lesions, and these lesions were confined almost exclusively to coronaries and carotids with % stenosis from 15% to 65%. Scatter plots of %ID/g for class III/IV vs. I/II lesions showed almost complete separation. Focal vascular uptake of tracer visualized on SPECT scans corresponded to class III/IV lesions in the coronary and carotid vessels. In addition, uptake in the hind limbs was noted in the HL pigs and corresponded to RAGE staining of small arteries in the muscle sections. Correlations for the vascular lesions were r = 0.747, P = 0.001 for %ID vs. %ID/g and r = 0.83, P = 0.002 for %ID/g vs. % RAGE staining.
Uptake of radiolabeled anti-RAGE antibody in coronary and carotid fibroatheroma and in the small arteries of the hind limbs in a relevant large animal model of atherosclerosis supports the important role of RAGE in atherosclerosis and peripheral artery disease as a target for imaging and treatment.
PMCID: PMC4078320  PMID: 25006545
RAGE; Atherosclerosis; Hyperlipidemic pigs; Imaging
17.  The Microglial Sensome Revealed by Direct RNA Sequencing 
Nature neuroscience  2013;16(12):10.1038/nn.3554.
Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings by fluorescent dual in-situ hybridization, unbiased proteomic analysis and quantitative PCR. We report here that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we term the “sensome”. With aging, sensome transcripts for endogenous ligand recognition are downregulated, whereas those involved in microbe recognition and host defense are upregulated. In addition, aging is associated with an overall increase in expression of microglial genes involved in neuroprotection.
PMCID: PMC3840123  PMID: 24162652
Microglia; Sensome; Direct RNA Sequencing; Aging; Macrophages; classical activation; Microbes; Alternative activation; quantitative transcriptome
18.  Epidermal Growth Factor Receptor (EGFR) Pathway Genes and Interstitial Lung Disease: An Association Study 
Scientific Reports  2014;4:4893.
The etiology and pathogenesis of idiopathic interstitial lung disease (ILD) remain incompletely understood. Genetic susceptibility to ILD has been demonstrated in previous studies. It is well known that EGFR inhibitors can induce ILD in human lung cancer patient with ethnic differences, which prompted us to hypothesize that genetic variation in EGFR pathway genes confer susceptibility to ILD. We aimed in this study to investigate whether functional polymorphisms of EGFR and its ligands genes (EGF and TGFA) were associated with ILD. Three EGFR [−216G/T (rs712830), −191A/C (rs712829), 497R > K(A/G) (rs2227983)], one EGF [61A/G, (rs4444903)] and one TGFA (rs3821262C/T) polymorphisms previously demonstrated to alter gene functions were genotyped in 229 sporadic idiopathic ILD patients and 693 normal healthy individuals. Allelic and genotypic association tests between these polymorphisms and ILD were performed. The EGF 61A/G polymorphism was significantly associated with elevated risk of ILD, with the frequency of G allele significantly increased in the ILD patient population (OR = 1.33, 95%CI = 1.07–1.66, P = 0.0099). None of the other polymorphisms were associated with risk of ILD. Our study suggested that the EGF 61A/G polymorphism may be associated with sporadic ILD. While a false positive finding cannot be excluded, independent studies are warranted to further validate this result.
PMCID: PMC4018612  PMID: 24819665
19.  Insulin Regulates Glucose Consumption and Lactate Production through Reactive Oxygen Species and Pyruvate Kinase M2 
Although insulin is known to regulate glucose metabolism and closely associate with liver cancer, the molecular mechanisms still remain to be elucidated. In this study, we attempt to understand the mechanism of insulin in promotion of liver cancer metabolism. We found that insulin increased pyruvate kinase M2 (PKM2) expression through reactive oxygen species (ROS) for regulating glucose consumption and lactate production, key process of glycolysis in hepatocellular carcinoma HepG2 and Bel7402 cells. Interestingly, insulin-induced ROS was found responsible for the suppression of miR-145 and miR-128, and forced expression of either miR-145 or miR-128 was sufficient to abolish insulin-induced PKM2 expression. Furthermore, the knockdown of PKM2 expression also inhibited cancer cell growth and insulin-induced glucose consumption and lactate production, suggesting that PKM2 is a functional downstream effecter of insulin. Taken together, this study would provide a new insight into the mechanism of insulin-induced glycolysis.
PMCID: PMC4034658  PMID: 24895527
20.  MicroRNA-143 inhibits tumor growth and angiogenesis and sensitizes chemosensitivity to oxaliplatin in colorectal cancers 
Cell Cycle  2013;12(9):1385-1394.
Colorectal cancer (CRC) is one of the leading cancer-related causes of death in the world. Recently, downregulation of microRNA-143 (miR-143) has been observed in CRC tissues. Here in this study, we found that miR-143 expression was downregulated both in CRC patients’ blood samples and tumor specimens. MiR-143 expression levels were strongly correlated with clinical stages and lymph node metastasis. Furthermore, insulin-like growth factor-I receptor (IGF-IR), a known oncogene, was a novel direct target of miR-143, whose expression levels were inversely correlated with miR-143 expression in human CRC specimens. Overexpression of miR-143 inhibited cell proliferation, migration, tumor growth and angiogenesis and increased chemosensitivity to oxaliplatin treatment in an IGF-IR-dependent manner. Taken together, these results revealed that miR-143 levels in human blood and tumor tissues are associated with CRC cancer occurrence, metastasis and drug resistance, and miR-143 levels may be used as a new diagnostic marker and therapeutic target for CRC in the future.
PMCID: PMC3674066  PMID: 23574723
microRNA-143; tumorigenesis; angiogenesis; IGF-IR; chemotherapy
21.  Hyperoxia arrests pulmonary development in newborn rats via disruption of endothelial tight junctions and downregulation of Cx40 
Molecular Medicine Reports  2014;10(1):61-67.
This study investigated changes in vascular endothelial cell tight junction structure and the expression of the gene encoding connexin 40 (Cx40) at the early pneumonedema stage of hyperoxia-induced bronchopulmonary dysplasia (BPD) in a newborn rat model. A total of 96 newborn rats were randomly assigned to one of the following two groups, the hyperoxia group (n=48) and the control group (n=48). A hyperoxia-induced BPD model was established for the first group, while rats in the control group were maintained under normoxic conditions. Extravasation of Evans Blue (EB) was measured; the severity of lung injury was assessed; a transmission electron microscope (TEM) was used to examine the vascular endothelial cell tight junction structures, and immunohistochemical assay, western blotting and reverse transcription-polymerase chain reaction (RT-PCR) were used to evaluate the expression of Cx40 at the mRNA and protein level. Our findings showed that injuries due to BPD are progressively intensified during the time-course of exposure to hyperoxic conditions. Pulmonary vascular permeability in the hyperoxia group reached the highest level at day 5, and was significantly higher compared to the control group. TEM observations demonstrated tight junctions between endothelial cells were extremely tight. In the hyperoxia group, no marked changes in the tight junction structure were found at days 1 and 3; paracellular gaps were visible between endothelial cells at days 5 and 7. Immunohistochemical staining revealed that the Cx40 protein is mainly expressed in the vascular endothelial cells of lung tissue. Western blotting and RT-PCR assays showed a gradual decrease in Cx40 expression, depending on the exposure time to hyperoxic conditions. However, the Cx40 mRNA level reached a trough at 5 days. Overall, our study demonstrated that exposure to hyperoxia damages the tight junction structures between vascular endothelial cells and downregulates Cx40. We therefore conclude that hyperoxia may participate in the regulation of pulmonary vascular endothelial permeability.
PMCID: PMC4068730  PMID: 24789212
hyperoxia; tight junction; Cx40; permeability; newborn; bronchopulmonary dysplasia
22.  Identification of Pummelo Cultivars by Using a Panel of 25 Selected SNPs and 12 DNA Segments 
PLoS ONE  2014;9(4):e94506.
Pummelo cultivars are usually difficult to identify morphologically, especially when fruits are unavailable. The problem was addressed in this study with the use of two methods: high resolution melting analysis of SNPs and sequencing of DNA segments. In the first method, a set of 25 SNPs with high polymorphic information content were selected from SNPs predicted by analyzing ESTs and sequenced DNA segments. High resolution melting analysis was then used to genotype 260 accessions including 55 from Myanmar, and 178 different genotypes were thus identified. A total of 99 cultivars were assigned to 86 different genotypes since the known somatic mutants were identical to their original genotypes at the analyzed SNP loci. The Myanmar samples were genotypically different from each other and from all other samples, indicating they were derived from sexual propagation. Statistical analysis showed that the set of SNPs was powerful enough for identifying at least 1000 pummelo genotypes, though the discrimination power varied in different pummelo groups and populations. In the second method, 12 genomic DNA segments of 24 representative pummelo accessions were sequenced. Analysis of the sequences revealed the existence of a high haplotype polymorphism in pummelo, and statistical analysis showed that the segments could be used as genetic barcodes that should be informative enough to allow reliable identification of 1200 pummelo cultivars. The high level of haplotype diversity and an apparent population structure shown by DNA segments and by SNP genotypes, respectively, were discussed in relation to the origin and domestication of the pummelo species.
PMCID: PMC3986212  PMID: 24732455
23.  Beneficial Effect of Glucose Control on Atherosclerosis Progression in Diabetic ApoE−/− Mice: Shown by Rage Directed Imaging 
Objective. Receptor for advanced glycated endproducts (RAGE) plays an important role in atherogenesis in diabetes. We imaged RAGE to investigate the effect of glucose control to suppress RAGE and reduce atherosclerosis in apolipoprotein E null (apoE−/−) diabetic mice. Methods and Results. Thirty-three apoE−/− mice received streptozotocin and 6 weeks later 15 began treatment with insulin implants. Blood glucose measurements during study averaged: 140 ± 23 mg/dL (treated) and 354 ± 14 mg/dL (untreated). After 15 wk 30 mice were injected with 99mTc-anti-RAGE F(ab′)2, 3 with 99mTc-nonimmune IgG F(ab′)2, and all with CT contrast agent and underwent SPECT/CT imaging. At necropsy, the proximal aorta was weighed, counted, and sectioned and the % injected dose per gram (%ID/g) was calculated. From the merged SPECT/CT scans, tracer uptake localized to arteries was lower in the treated mice: 3.15 ± 1.82 × 10−3 versus 8.69 ± 4.58 × 10−3%ID (P = 0.001). Percent cross-sectional lesion area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Lesion size as percent cross-sectional area was smaller in the treated (14.3 ± 7.8% versus 29.5 ± 10.9%) (P = 0.03). RAGE uptake on scans (%ID) correlated with quantitative RAGE staining in the atheroma and with %ID/g (R = 0.6887; P = 0.01). Conclusions. These results support the importance of suppressing RAGE to reduce atherosclerotic complications of diabetes and value of molecular imaging to assess treatment effect.
PMCID: PMC4009322  PMID: 24829796
24.  RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma 
The 'gold standard' for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current 'gold standard' method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.
PMCID: PMC4145807  PMID: 24637627
Medicine; Issue 85; RNAscope; Head and Neck Squamous Cell Carcinoma (HNSCC); Oropharyngeal Squamous Cell Carcinoma (OPSCC); Human Papillomavirus (HPV); E6/ E7 mRNA; in situ hybridization; tumor
25.  Turning a Scorpion Toxin into an Antitumor Miniprotein 
Journal of the American Chemical Society  2008;130(41):10.1021/ja8042036.
PMCID: PMC3810402  PMID: 18798622

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