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1.  Evaluation of 99mTc-peptide-ZHER2:342 Affibody® molecule for in vivo molecular imaging 
The British Journal of Radiology  2013;87(1033):20130484.
Objective:
The aim of this study was to develop an improved method for labelling ZHER2:342 with Technetium-99m (99mTc) using Gly-(d) Ala-Gly-Gly as a chelator and to evaluate the feasibility of its use for visualization of HER2 expression in vivo.
Methods:
The Affibody® molecule ZHER2:342 was synthesized by Fmoc/tBu solid phase synthesis. The chelator, Gly-(d) Ala-Gly-Gly, was introduced by manual synthesis as the N-terminal extensions of ZHER2:342. ZHER2:342 was labelled with 99mTc. The labelling efficiency, radiochemical purity and in vitro stability of the labelled molecular probe were analysed by reversed-phase high performance liquid chromatography. Biodistribution and molecular imaging using 99mTc-peptide-ZHER2:342 were performed.
Results:
The molecular probe was successfully synthesized and labelled with 99mTc with the labelling efficiency of 98.10 ± 1.73% (n = 5). The radiolabelled molecular probe remained highly stable in vitro. The molecular imaging showed high uptake in HER2-expressing SKOV-3 xenografts, whereas the MDA-MB-231 xenografts with low HER2 expression were not clearly imaged at any time after the injection of 99mTc-peptide-ZHER2:342. The predominant clearance pathway for 99mTc-peptide-ZHER2:342 was through the kidneys.
Conculsion:
99mTc-peptide-ZHER2:342 using Gly-(d) Ala-Gly-Gly as a chelator is a promising tracer agent with favourable biodistribution and imaging properties that may be developed as a radiopharmaceutical for the detection of HER2-positive malignant tumours.
Advances in knowledge:
The 99mTc-peptide-ZHER2:342 molecular probe is a promising tracer agent, and the results in this study provide a foundation for future development of protocols for earlier visual detection of cancer in the clinical setting.
doi:10.1259/bjr.20130484
PMCID: PMC3898972  PMID: 24273251
2.  Liraglutide inhibits autophagy and apoptosis induced by high glucose through GLP-1R in renal tubular epithelial cells 
ZHAO, X | LIU, G | SHEN, H | GAO, B | LI, X | FU, J | ZHOU, J | JI, Q
Tubular atrophy and dysfunction is a critical process underlying diabetic nephropathy (DN). Understanding the mechanisms underlying renal tubular epithelial cell survival is important for the prevention of kidney failure associated with glucotoxicity. Autophagy is a cellular pathway involved in protein and organelle degradation. It is associated with many types of cellular homeostasis and human diseases. To date, little is known of the association between high concentrations of glucose and autophagy in renal tubular cells. In the present study, we investigated high glucose-induced toxicity in renal tubular epithelial cells by means of several complementary assays, including cell viability, cell death assays and changes in ultrastructure in an immortalized human kidney cell line, HK-2 cells. The extent of apoptosis was significantly increased in the HK-2 cells following treatment with high levels of glucose. In addition, in in vivo experiments using diabetic rats, high glucose exerted harmful effects on the tissue structure of the kidneys in the diabetic rats. Chronic exposure of the HK-2 cells and tubular epithelial cells of nephritic rats to high levels of glucose induced autophagy. Liraglutide inhibited these effects; however, treatment witht a glucagon-like peptide-1 receptor (GLP-1R) antagonist enhanced these effects. Our results also indicated that the exposure of the renal tubular epithelial cells to high glucose concentrations in vitro led to the downregulation of GLP-1R expression. Liraglutide reversed this effect, while the GLP-1R antagonist promoted it, promoting autophagy, suggesting that liraglutide exerts a renoprotective effect in the presence of high glucose, at least in part, by inhibiting autophagy and increasing GLP-1R expression in the HK-2 cells and kidneys of diabetic rats.
doi:10.3892/ijmm.2014.2052
PMCID: PMC4314412  PMID: 25573030
high glucose; autophagy; autophagy-related gene; glucagon-like peptide-1 receptor; liraglutide
3.  SRF expedites metastasis and modulates the epithelial to mesenchymal transition by regulating miR-199a-5p expression in human gastric cancer 
Zhao, X | He, L | Li, T | Lu, Y | Miao, Y | Liang, S | Guo, H | Bai, M | Xie, H | Luo, G | Zhou, L | Shen, G | Guo, C | Bai, F | Sun, S | Wu, K | Nie, Y | Fan, D
Cell Death and Differentiation  2014;21(12):1900-1913.
Dysregulation of transcription factors (TFs) is associated with tumor progression, but little is known about TF expression patterns in the context of gastric cancer (GC) metastasis. Using array-based profile analysis, we found that 22 TFs showed differential activities between GC cell lines with low- and high-metastatic potential. Of this group of TFs, serum response factor (SRF) was significantly upregulated in metastatic GC cells. SRF expression was frequently elevated in a panel of metastatic GC cells and tissues, and high-level expression of SRF was significantly associated with a more aggressive phenotype and poor prognosis in patients with GC. In GC cell lines, overexpression of SRF potently promoted cell migration and invasion in vitro as well as the formation of intrahepatic and pulmonary metastases in vivo, whereas loss of SRF inhibited GC cell invasion and metastasis. We also performed a microRNA microarray to screen for transcriptional targets of SRF and found that SRF transactivates miR-199a-5p and miR-199a-3p by directly binding to their promoters. We further determined that overexpression of miR-199a-5p, but not miR-199a-3p, increased GC cell invasion and metastasis. In contrast, inhibition of miR-199a-5p impaired the metastatic potential of GC cells in vitro and in vivo, and E-cadherin was identified as a direct and functional target of miR-199a-5p in GC cells. Specifically, our results showed that SRF promotes GC metastasis and the epithelial to mesenchymal transition (EMT) though miR-199a-5p-mediated downregulation of E-cadherin. The present study thus provides insight into the specific biological behavior of SRF in GC metastasis. As increased activity of the SRF/miR-199a-5p/E-cadherin pathway appears to promote GC cell EMT and metastasis, these regulators may therefore be developed as therapeutic targets or biomarkers for GC progression.
doi:10.1038/cdd.2014.109
PMCID: PMC4227147  PMID: 25080937
4.  MiR-125b acts as an oncogene in glioblastoma cells and inhibits cell apoptosis through p53 and p38MAPK-independent pathways 
Wu, N | Lin, X | Zhao, X | Zheng, L | Xiao, L | Liu, J | Ge, L | Cao, S
British Journal of Cancer  2013;109(11):2853-2863.
Background:
We have recently identified miR-125b upregulation in glioblastoma (GMB). The aim of this study is to determine the correlation between miR-125b expression and malignant grades of glioma and the genes targeted by miR-125b.
Methods:
Real-time PCR was employed to measure the expression level of miR-125b. Cell viability was evaluated by cell growth and colony formation in soft-agar assays. Cell apoptosis was determined by Hoechst 33342 staining and AnnexinV-FITC assay. The Luciferase assay was used to confirm the actual binding sites of p38MAPK mRNA. Western blot was used to detect the gene expression level.
Results:
The expression level of miR-125b is positively correlated with the malignant grade of glioma. Ectopic expression of miR-125b promotes the proliferation of GMB cells. Knockdown of endogenous miR-125b inhibits cell proliferation and promotes cell apoptosis. Further studies reveal that p53 is regulated by miR-125b. However, downregulation of the endogenous miR-125b also results in p53-independent apoptotic pathway leading to apoptosis in p53 mutated U251 cells and p53 knockdown U87 cells. Moreover, p38MAPK is also regulated by miR-125b and downregulation of miR-125b activates the p38MAPK-induced mitochondria apoptotic pathway.
Conclusion:
High-level expression of miR-125b is associated with poor outcomes of GMB. MiR-125b may have an oncogenic role in GMB cells by promoting cell proliferation and inhibiting apoptosis.
doi:10.1038/bjc.2013.672
PMCID: PMC3844918  PMID: 24169356
microRNA; miR-125b; glioblastoma; cell apoptosis; p53; p38MAPK
5.  Localized micro- and nano-scale remodelling in the diabetic aorta 
Acta Biomaterialia  2014;10(11):4843-4851.
Graphical abstract
Diabetes is strongly associated with cardiovascular disease, but the mechanisms, structural and biomechanical consequences of aberrant blood vessel remodelling remain poorly defined. Using an experimental (streptozotocin, STZ) rat model of diabetes, we hypothesized that diabetes enhances extracellular protease activity in the aorta and induces morphological, compositional and localized micromechanical tissue remodelling. We found that the medial aortic layer underwent significant thickening in diabetic animals but without significant changes in collagen or elastin (abundance). Scanning acoustic microscopy demonstrated that such tissue remodelling was associated with a significant decrease in acoustic wave speed (an indicator of reduced material stiffness) in the inter-lamellar spaces of the vessel wall. This index of decreased stiffness was also linked to increased extracellular protease activity (assessed by semi-quantitative in situ gelatin zymography). Such a proteolytically active environment may affect the macromolecular structure of long-lived extracellular matrix molecules. To test this hypothesis, we also characterized the effects of diabetes on the ultrastructure of an important elastic fibre component: the fibrillin microfibril. Using size exclusion chromatography and atomic force microscopy, we isolated and imaged microfibrils from both healthy and diabetic aortas. Microfibrils derived from diabetic tissues were fragmented, morphologically disrupted and weakened (as assessed following molecular combing). These structural and functional abnormalities were not replicated by in vitro glycation. Our data suggest that proteolysis may be a key driver of localized mechanical change in the inter-lamellar space of diabetic rat aortas and that structural proteins (such as fibrillin microfbrils) may be biomarkers of diabetes induced damage.
doi:10.1016/j.actbio.2014.07.001
PMCID: PMC4199142  PMID: 25014552
Arterial stiffening; Fibrillin microfibrils; Type 1 diabetes; Extracellular matrix; Mechanical properties
6.  Effects of the Cowpea chlorotic mottle bromovirus β-hexamer structure on virion assembly 
Virology  2003;306(2):280-288.
The X-ray crystal structure of Cowpea chlorotic mottle bromovirus (CCMV) revealed a unique tubular structure formed by the interaction of the N-termini from six coat protein subunits at each three-fold axis of the assembled virion. This structure, termed the β-hexamer, consists of six short β-strands. The β-hexamer was postulated to play a critical role in the assembly and stability of the virion by stabilizing hexameric capsomers (Speir et al., 1995). Mutational analyses of the β-hexamer structure, utilizing both in vitro and in vivo assembly assays, demonstrate that this structure is not required for virion formation devoid of nucleic acids in vitro or for RNA-containing virions in vivo. However, the β-hexamer structure does contribute to virion stability in vitro and modulates disease expression in vivo. These results support a model for CCMV assembly through pentamer intermediates.
PMCID: PMC4191912  PMID: 12642101
Virus structure; Virus assembly; Virus stability; Symptom expression
7.  A new method for determining gastric acid output using a wireless ph sensing capsule 
Alimentary pharmacology & therapeutics  2013;37(12):1198-1209.
BACKGROUND
Gastroesophageal reflux disease (GERD) and gastric acid hypersecretion respond well to suppression of gastric acid secretion. However, clinical management and research in diseases of acid secretion have been hindered by the lack of a non-invasive, accurate and reproducible tool to measure gastric acid output (GAO). Thus, symptoms or, in refractory cases, invasive testing may guide acid suppression therapy.
AIM
To present and validate a novel, non-invasive method of GAO analysis in healthy subjects using a wireless pH sensor, SmartPill® (SP) (SmartPill® Corporation, Buffalo, NY).
METHODS
Twenty healthy subjects underwent conventional GAO studies with a nasogastric tube. Variables impacting liquid meal-stimulated GAO analysis were assessed by modeling and in vitro verification. Buffering capacity of Ensure Plus® was empirically determined. SP GAO was calculated using the rate of acidification of the Ensure Plus® meal. Gastric emptying scintigraphy and GAO studies with radiolabeled Ensure Plus® and SP assessed emptying time, acidification rate and mixing. Twelve subjects had a second SP GAO study to assess reproducibility.
RESULTS
Meal stimulated SP GAO analysis was dependent on acid secretion rate and meal buffering capacity but not on gastric emptying time. On repeated studies, SP GAO strongly correlated with conventional BAO (r=0.51, P=0.02), MAO (r=0.72, P=0.0004) and PAO; (r=0.60, P=0.006). The SP sampled the stomach well during meal acidification.
CONCLUSIONS
SP GAO analysis is a non-invasive, accurate and reproducible method for the quantitative measurement of GAO in healthy subjects. SP GAO analysis could facilitate research and clinical management of GERD and other disorders of gastric acid secretion.
doi:10.1111/apt.12325
PMCID: PMC3703786  PMID: 23639004
acid output; wireless capsule; SmartPill®; gastroesophageal reflux disease
8.  Extranodal Marginal Zone Lymphoma Presenting within the Meckel Diverticulum as Diverticulitis: A Case Report 
Case Reports in Pathology  2014;2014:374814.
Meckel diverticulum is the most common congenital defect of the gastrointestinal tract. It can be asymptomatic or mimic appendicitis and may be complicated by bleeding, diverticulitis, obstruction, and, rarely, neoplasia. We report the first case of extranodal marginal zone lymphoma occupying a Meckel diverticulum. A 44-year-old man with history of colonic diverticulitis presented to the emergency department for evaluation of acute abdominal pain. Radiography showed enteric obstruction, prompting diagnostic laparoscopy. Above the level of mid-ileum an intact Meckel diverticulum was identified. Microscopy showed extensive infiltration of sheets of small lymphocytes with abundant cytoplasm (monocytoid B-cells) prominently in submucosa and focally transmural involving serosal adipose tissue with multiple reactive germinal centers. The immunostains showed positivity for CD20, BCL-2, and CD43 (weak) and negativity for CD3, CD5, BCL-1, CD10, and BCL-6 in monocytoid B-cells. Fluorescence in situ hybridization studies revealed API2-MALT1 fusion signals consistent with t(11;18)(q21;q21), which confirmed the diagnosis of extranodal marginal zone lymphoma, also known as mucosa associated lymphoid tissue lymphoma.
doi:10.1155/2014/374814
PMCID: PMC4020529  PMID: 24868477
9.  Effects of single-dose atorvastatin on interleukin-6, interferon gamma, and myocardial no-reflow in a rabbit model of acute myocardial infarction and reperfusion 
The mechanisms of statins relieving the no-reflow phenomenon and the effects of single-dose statins on it are not well known. This study sought to investigate the effects of inflammation on the no-reflow phenomenon in a rabbit model of acute myocardial infarction and reperfusion (AMI/R) and to evaluate the effects of single-dose atorvastatin on inflammation and myocardial no-reflow. Twenty-four New Zealand white male rabbits (5-6 months old) were randomized to three groups of eight: a sham-operated group, an AMI/R group, and an atorvastatin-treated group (10 mg/kg). Animals in the latter two groups were subjected to 4 h of coronary occlusion followed by 2 h of reperfusion. Serum levels of interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay. The expression of interferon gamma (IFN-γ) in normal and infarcted (reflow and no-reflow) myocardial tissue was determined by immunohistochemical methods. The area of no-reflow and necrosis was evaluated pathologically. Levels of serum IL-6 were significantly lower in the atorvastatin group than in the AMI/R group (P<0.01). Expression of IFN-γ in infarcted reflow and no-reflow myocardial tissue was also significantly lower in the atorvastatin group than in the AMI/R group. The mean area of no-reflow [47.01% of ligation area (LA)] was significantly smaller in the atorvastatin group than in the AMI/R group (85.67% of LA; P<0.01). The necrosis area was also significantly smaller in the atorvastatin group (85.94% of LA) than in the AMI/R group (96.56% of LA; P<0.01). In a secondary analysis, rabbits in the atorvastatin and AMI/R groups were divided into two groups based on necrosis area (90% of LA): a small group (<90% of LA) and a large group (>90% of LA). There was no significant difference in the area of no-reflow between the small (61.40% of LA) and large groups (69.87% of LA; P>0.05). Single-dose atorvastatin protected against inflammation and myocardial no-reflow and reduced infarct size during AMI/R in rabbits. No-reflow was not dependent on the reduction of infarct size.
doi:10.1590/1414-431X20132999
PMCID: PMC3982946  PMID: 24554037
Myocardial infarction; No-reflow phenomenon; Inflammation; Atorvastatin
10.  Expression Profiles of Cellular Retinol-binding Protein, Type II (CRBP II) in Erlang Mountainous Chickens 
Cellular retinol-binding protein II (CRBP II) belongs to the family of cellular retinol-binding proteins and plays a major role in absorption, transport, and metabolism of vitamin A. In addition, because vitamin A is correlated with reproductive performance, we measured CRBP II mRNA abundance in erlang mountainous chickens by real-time PCR using the relative quantification method. The expression of CRBP II showed a tissue-specific pattern and egg production rate-dependent changes. The expression was very high (p<0.05) in jejunum and liver, intermediate in kidney, ovary, and oviduct, and lowest (p<0.05) in heart, hypothalamus, and pituitary. In the hypothalamus, oviduct, ovary, and pituitary, CRBP II mRNA abundance were correlated to egg production rate, which increased from 12 wk to 32 wk, peaked at 32 wk relative to the other time points, and then decreased from 32 wk to 45 wk. In contrast, the expression of CRBP II mRNA in heart, jejunum, kidney, and liver was not different at any of the ages evaluated in this study. These data may help to understand the genetic basis of vitamin A metabolism, and suggest that CRBP II may be a candidate gene to affect egg production traits in chickens.
doi:10.5713/ajas.2013.13469
PMCID: PMC4093264  PMID: 25049956
CRBP II; mRNA Expression; Chicken; Egg Laying Rate
11.  Enhanced tumor suppression in vitro and in vivo by co-expression of survivin-specific siRNA and wild-type p53 protein 
Shao, Y | Liu, Y | Shao, C | Hu, J | Li, X | Li, F | Zhang, L | Zhao, D | Sun, L | Zhao, X | Kopecko, DJ | Kalvakolanu, DV | Li, Y | Xu, DQ
Cancer gene therapy  2010;17(12):844-854.
The development of malignant prostate cancer involves multiple genetic alterations. For example, alterations in both survivin and p53 are reported to have crucial roles in prostate cancer progression. However, little is known regarding the interrelationships between p53 and survivin in prostate cancer. Our data demonstrate that the expression of survivin is inversely correlated with that of wtp53 protein (rs=0.548) in prostate cancer and in normal prostate tissues. We have developed a therapeutic strategy, in which two antitumor factors, small interfering RNA-survivin and p53 protein, are co-expressed from the same plasmid, and have examined their effects on the growth of PC3, an androgen-independent prostate cancer cell line. When p53 was expressed along with a survivin-specific short hairpin RNA (shRNA), tumor cell proliferation was significantly suppressed and apoptosis occurred. In addition, this combination also abrogated the expression of downstream target molecules such as cyclin-dependent kinase 4 and c-Myc, while enhancing the expression of GRIM19. These changes in gene expression occurred distinctly in the presence of survivin-shRNA/wtp53 compared with control or single treatment groups. Intratumoral injection of the co-expressed construct inhibited the growth and survival of tumor xenografts in a nude mouse model. These studies revealed evidence of an interaction between p53 and survivin proteins plus a complex signaling network operating downstream of the wtp53-survivin pathway that actively controls tumor cell proliferation, survival and apoptosis.
doi:10.1038/cgt.2010.41
PMCID: PMC3915357  PMID: 20706288
prostate cancer; p53; survivin; siRNA
12.  Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development 
PLoS Genetics  2014;10(1):e1004105.
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.
Author Summary
Control of gene expression on the translational level is critical for proper function of major cellular processes and deregulation of translation can promote cellular transformation. Emerging actors in this post-transcriptional gene regulation are small non-coding RNAs referred to as microRNAs (miRNAs). We established that miR-520c-3p represses tumor growth through the repression of eIF4GII, a major structural component of the translation initiation complex. Since translation of most cellular mRNAs is primarily regulated at the level of initiation, this node is becoming a potential target for therapeutic intervention. Identified in this study, tumor suppressor function of miR-520c-3p is mediated through the inhibition of translational factor eIF4GII, resulting in the repression of global translational machinery and induction of senescence in tumor cells. While aging and senescence has been shown to be associated with reduced translation the linkage between translational deregulation and senescence in malignant cells has not been previously described. Lending further clinical significance to our findings, we were able to demonstrate that primary DLBCL samples had elevated levels of eIF4GII while having reciprocally low miR-520c-3p expression.
doi:10.1371/journal.pgen.1004105
PMCID: PMC3907297  PMID: 24497838
13.  Targeted therapy via oral administration of attenuated Salmonella expression plasmid-vectored Stat3-shRNA cures orthotopically transplanted mouse HCC 
Cancer gene therapy  2012;19(6):393-401.
The development of RNA interference-based cancer gene therapies has been delayed due to the lack of effective tumor-targeting delivery systems. Attenuated Salmonella enterica serovar Typhimurium (S. Typhimurium) has a natural tropism for solid tumors. We report here the use of attenuated S. Typhimurium as a vector to deliver shRNA directly into tumor cells. Constitutively activated signal transducer and activator of transcription 3 (Stat3) is a key transcription factor involved in both hepatocellular carcinoma (HCC) growth and metastasis. In this study, attenuated S. Typhimurium was capable of delivering shRNA-expressing vectors to the targeted cancer cells and inducing RNA interference in vivo. More importantly, a single oral dose of attenuated S. Typhimurium carrying shRNA-expressing vectors targeting Stat3 induced remarkably delayed and reduced HCC (in 70% of mice). Cancer in these cured mice did not recur over 2 years following treatment. These data demonstrated that RNA interference combined with Salmonella as a delivery system may offer a novel clinical approach for cancer gene therapy.
doi:10.1038/cgt.2012.12
PMCID: PMC3891655  PMID: 22555509
HCC; immune response; RNA interference; Stat3
14.  Cardiopulmonary Complications Leading to Premature Deaths in Adult Patients with Sickle Cell Disease 
American journal of hematology  2010;85(1):10.1002/ajh.21569.
Sickle cell disease (SCD) is associated with early mortality. We sought to determine the incidence, cause, and risk factors for death in an adult population of patients with SCD. All patients aged ≥18 years seen at the Adult Sickle Cell Center at Duke University Medical Center between January 2000 and April 2005 were enrolled. Forty-three patients (21 males and 22 females) died during the study period. Median age of survival was 39 years for females (95% CI 34–56), 40 years for males (95% CI 34–48), and 40 years overall (95% CI 35–48). Cardiac causes of death accounted for 25.6% (11/43 patients); pulmonary, 14.0% (6 patients); other SCD related, 32.6% (14 patients); unknown, 14.0% (6 patients); and others, 14.0% (6 patients). Pulseless electrical activity arrest, pulmonary emboli, multi-organ failure, and stroke were the most frequent causes of death. Among the deceased patients, the most common pre-morbid conditions were cardiopulmonary: ACS/pneumonia (58.1%), pHTN (41.9%), systemic hypertension (HTN) (25.6%), congestive heart failure (CHF) (25.6%), myocardial infarction (20.9%), and arrhythmias (14.0%). Tricuspid regurgitant jet velocity (TRv) was significantly higher (3.1 m/s vs. 2.6 m/s, p<0.001) and hemoglobin significantly lower (8.3 g/dL vs. 9.2 g/dL, p<0.05) in deceased patients as compared to patients who lived, respectively. With improved preventive and therapeutic advances, including hydroxyurea therapy, acute complications such as infection are no longer the leading cause of death; instead causes of death and pre-morbid conditions are shifting to chronic cardiopulmonary complications. Further, arrhythmia leading to premature death is under-recognized in SCD and warrants further investigation.
doi:10.1002/ajh.21569
PMCID: PMC3865703  PMID: 20029950
sickle cell disease; adult; mortality; risk factors; cardiopulmonary complications
15.  SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies 
Bioorganic & medicinal chemistry  2012;20(24):6929-6939.
A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [131I]SIB-DOTA in 27.1 ± 6.2% (n = 2) conjugation yields and its targeting properties to the same mAb labeled with [125I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [131I]SIB-DOTA-L8A4 was 21.4 ± 0.5% and 26.2 ± 1.1% of initially bound radioactivity at 16 and 24 h, respectively. In comparison, these values for [125I]SGMIB-L8A4 were 16.7 ± 0.5% and 14.9 ± 1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.
doi:10.1016/j.bmc.2012.10.025
PMCID: PMC3508367  PMID: 23159039
Monoclonal antibody; Internalization; Residualizing Labels; Radioiodine
16.  Dichloroacetate induces protective autophagy in LoVo cells: involvement of cathepsin D/thioredoxin-like protein 1 and Akt-mTOR-mediated signaling 
Gong, F | Peng, X | Sang, Y | Qiu, M | Luo, C | He, Z | Zhao, X | Tong, A
Cell Death & Disease  2013;4(11):e913-.
Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), and recently it has been shown as a promising nontoxic antineoplastic agent. In this study, we demonstrated that DCA could induce autophagy in LoVo cells, which were confirmed by the formation of autophagosomes, appearance of punctate patterns of LC3 immunoreactivity and activation of autophagy associated proteins. Moreover, autophagy inhibition by 3-methyladenine (3-MA) or Atg7 siRNA treatment can significantly enhance DCA-induced apoptosis. To determine the underlying mechanism of DCA-induced autophagy, target identification using drug affinity responsive target stability (DARTS) coupled with ESI-Q-TOF MS/MS analysis were utilized to profile differentially expressed proteins between control and DCA-treated LoVo cells. As a result, Cathepsin D (CTSD) and thioredoxin-like protein 1 (TXNL1) were identified with significant alterations compared with control. Further study indicated that DCA treatment significantly promoted abnormal reactive oxygen species (ROS) production. On the other hand, DCA-triggered autophagy could be attenuated by N-acetyl cysteine (NAC), a ROS inhibitor. Finally, we demonstrated that the Akt-mTOR signaling pathway, a major negative regulator of autophagy, was suppressed by DCA treatment. To our knowledge, it was the first study to show that DCA induced protective autophagy in LoVo cells, and the potential mechanisms were involved in ROS imbalance and Akt-mTOR signaling pathway suppression.
doi:10.1038/cddis.2013.438
PMCID: PMC3847316  PMID: 24201812
dichloroacetate; autophagy; DARTS; ROS; LoVo cells
17.  MiRNA-296-3p-ICAM-1 axis promotes metastasis of prostate cancer by possible enhancing survival of natural killer cell-resistant circulating tumour cells 
Liu, X | Chen, Q | Yan, J | Wang, Y | Zhu, C | Chen, C | Zhao, X | Xu, M | Sun, Q | Deng, R | Zhang, H | Qu, Y | Huang, J | Jiang, B | Yu, J
Cell Death & Disease  2013;4(11):e928-.
Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.
doi:10.1038/cddis.2013.458
PMCID: PMC3847334  PMID: 24263102
NK cells; ICAM-1; prostate cancer; miR-296-3p; metastasis
18.  Implication of transcriptional repression in compound C-induced apoptosis in cancer cells 
Cell Death & Disease  2013;4(10):e883-.
Compound C, a well-known inhibitor of AMP-activated protein kinase (AMPK), has been reported to induce apoptosis in some types of cells. However, the underlying mechanisms remain largely unclear. Using a DNA microarray analysis, we found that the expression of many genes was downregulated upon treatment with compound C. Importantly, compound C caused transcriptional repression with the induction of p53, a well-known marker of transcriptional stress response, in several cancer cell lines. Compound C did not induce the phosphorylation of p53 but dramatically increased the protein level of p53 similar to some other transcriptional inhibitors, including 5,6-dichloro-1-β-D-ribobenzimidazole (DRB). Consistent with previous reports, we found that compound C initiated apoptotic death of cancer cells in an AMPK-independent manner. Similar to DRB and actinomycin D (ActD), two classic transcription inhibitors, compound C not only resulted in the loss of Bcl-2 and Bcl-xl protein but also induced the phosphorylation of eukaryotic initiation factor-alpha (eIF2α) on Ser51. Hence, the phosphorylation of eIF2α might be a novel marker of transcriptional inhibition. It is noteworthy that compound C-mediated apoptosis of cancer cells is correlated with decreased expression of Bcl-2 and Bcl-xl and the phosphorylation of eIF2α on Ser51. Remarkably, compound C exhibits potent anticancer activities in vivo. Taken together, our data suggest that compound C may be an attractive candidate for anticancer drug development.
doi:10.1038/cddis.2013.419
PMCID: PMC3920957  PMID: 24157877
compound C; transcriptional inhibition; apoptosis; cancer
19.  Effect of Hybridization on Carcass Traits and Meat Quality of Erlang Mountainous Chickens 
Native chickens hold a significant share of the market in China. In response to the huge demand from the market, the productivity of Chinese native chickens needs to be improved. Cross breeding is an effective method to increase productivity, although it might affect meat quality. In this study, two pure lines (SD02 and SD03) of Erlang mountainous chickens were hybridized with a yellow feather and faster growing line (SD01). The effect of hybridization on carcass and meat quality (physiochemical and textural traits) was measured in the F1 population at d 91 of age. The hybrids exhibited higher body weight and dressed weight, and amount of semi-eviscerated, eviscerated, breast muscle and abdominal fat (p<0.05). Abdominal fat yield also increased (p<0.05) compared to the offspring of the two pure-lines. Meanwhile, there was no significant difference in meat quality traits except for the myofiber diameter and density and the shear force of the breast muscle. Overall, the offspring of cross-lines were similar to pure lines in meat color, pH value, inosinic acid, crude protein, crude fat, dry matter, moisture content and amino acid composition in the breast muscle. These results suggest that productivity can be improved via cross-breeding while maintaining meat quality of the Erlang mountainous chicken.
doi:10.5713/ajas.2013.13097
PMCID: PMC4093072  PMID: 25049734
Hybridization; Carcass Traits; Meat Quality; Erlang Mountainous Chicken
20.  Antimetastatic activity isolated from Colocasia esculenta (Taro) 
Anti-cancer drugs  2012;23(2):200-211.
Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E2 (PGE2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.
doi:10.1097/CAD.0b013e32834b85e8
PMCID: PMC3769987  PMID: 21934603
Taro; Breast cancer; Antimetastatic activity; Tumor; Cancer therapy
21.  A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease 
Heredity  2012;109(3):156-162.
A type of lower motor neuron (LMN) disease inherited as autosomal recessive in Romney sheep was characterized with normal appearance at birth, but with progressive weakness and tetraparesis after the first week of life. Here, we carried out genome-wide homozygosity mapping using Illumina Ovine SNP50 BeadChips on lambs descended from one carrier ram, including 19 sheep diagnosed as affected and 11 of their parents that were therefore known carriers. A homozygous region of 136 consecutive single-nucleotide polymorphism (SNP) loci on chromosome 2 was common to all affected sheep and it was the basis for searching for the positional candidate genes. Other homozygous regions shared by all affected sheep spanned eight or fewer SNP loci. The 136-SNP region contained the sheep ATP/GTP-binding protein 1 (AGTPBP1) gene. Mutations in this gene have been shown to be related to Purkinje cell degeneration (pcd) phenotypes including ataxia in mice. One missense mutation c.2909G>C on exon 21 of AGTPBP1 was discovered, which induces an Arg to Pro substitution (p.Arg970Pro) at amino-acid 970, a conserved residue for the catalytic activity of AGTPBP1. Genotyping of this mutation showed 100% concordant rate with the recessive pattern of inheritance in affected, carrier, phenotypically normal and unrelated normal individuals. This is the first report showing a mutant AGTPBP1 is associated with a LMN disease in a large mammal animal model. Our finding raises the possibility of human patients with the same etiology caused by this gene or other genes in the same pathway of neuronal development.
doi:10.1038/hdy.2012.23
PMCID: PMC3752038  PMID: 22588130
AGTPBP1; lower motor neuron disease; missense mutation; Purkinje cell degeneration; sheep
22.  Renal tubular dysfunction during long-term adefovir or tenofovir therapy in chronic hepatitis B 
Alimentary pharmacology & therapeutics  2012;35(11):1317-1325.
SUMMARY
Background
Adefovir and tenofovir are nucleotide analogues used as long-term therapy of chronic hepatitis B. Side effects are few, but prolonged and high-dose therapy has been associated with proximal renal tubular dysfunction (RTD).
Aim
To assess the incidence of RTD during long-term nucleotide therapy of chronic hepatitis B.
Methods
A total of 51 patients being treated at the Clinical Center, National Institutes of Health were studied. Diagnosis of RTD required de novo appearance of at least three of five features: hypophosphataemia, hypouricaemia, serum creatinine elevation, proteinuria or glucosuria.
Results
Among 51 patients treated for 1–10 (mean 7.4) years with adefovir (n = 42), tenofovir (n = 4) or adefovir followed by tenofovir (n = 5), 7 (14%) developed RTD. Time to onset ranged from 22 to 94 (mean 49) months with an estimated 10-year cumulative rate of 15%. All seven had low urinary percent maximal tubular reabsorption of phosphate (<82%). Patients with RTD were older (58 vs. 44 years; P = 0.01) and had lower baseline glomerular filtration rates (82 vs. 97 cc/min; P = 0.08) compared to those without; but did not differ in other features. Six patients with RTD were switched to entecavir, all subsequently had improvements in serum phosphate (2.0–3.0 mg/dL), creatinine (1.6–1.1 mg/dL), uric acid (2.7 –3.8 mg/dL) and proteinuria.
Conclusions
Renal tubular dysfunction develops in 15% of patients treated with adefovir or tenofovir for 2–9 years and is partially reversible with change to other antivirals. Monitoring for serum phosphate, creatinine and urinalysis is prudent during long-term adefovir and tenofovir therapy.
doi:10.1111/j.1365-2036.2012.05093.x
PMCID: PMC3443969  PMID: 22506503
23.  Paradoxical role of autophagy in the dysplastic and tumor-forming stages of hepatocarcinoma development in rats 
Cell Death & Disease  2013;4(2):e501-.
Many reports have shown that autophagy has a role as both a promoter and inhibitor in tumor development. However, the mechanism of this paradox is unknown. Tumor development is a multistep process. Therefore, we investigated whether the role of autophagy in hepatocarcinoma formation depended on the stage of tumor development. Based on our results, autophagy inhibition by chloroquine had a tumor-promotive effect in the rat model with N-diethylnitrosamine-induced hepatocarcinogenesis in its dysplastic stage (Ds) and a tumor-suppressive effect in its tumor-forming stage (Ts). In the Ds, autophagy inhibition enhanced cell proliferation, DNA damage and inflammatory cytokines expression in liver. These changes were dependent on the upregulation of reactive oxygen species (ROS) that was resulted from autophagy inhibition, and ultimately accelerated the process of hepatocarcinogenesis. However, in the Ts, autophagy inhibition restrained tumor formation by decreasing tumor cell survival and proliferation. In this stage, autophagy inhibition led to excessive ROS accumulation in the tumor, which promoted cell apoptosis, and prominently suppressed tumor cell metabolism. Taken together, our data suggested that autophagy suppressed hepatocarcinogenesis in the Ds by protecting normal cell stability and promoted hepatocarcinogenesis in the Ts by supporting tumor cells growth. Autophagy always had a role as a protector throughout the process of hepatocarcinoma development.
doi:10.1038/cddis.2013.35
PMCID: PMC3734842  PMID: 23429287
autophagy; hepatocarcinoma development; different stages; paradoxical role; ROS
24.  Plasma membrane-associated sialidase (NEU3) regulates progression of prostate cancer to androgen-independent growth through modulation of androgen receptor signaling 
Cell Death and Differentiation  2011;19(1):170-179.
Prostate cancers generally become androgen-independent and resistant to hormone therapy with progression. To understand the underlying mechanisms and facilitate the development of novel treatments for androgen-independent prostate cancer, we have investigated plasma membrane-associated sialidase (NEU3), the key enzyme for ganglioside hydrolysis participating in transmembrane signaling. We have discovered NEU3 to be upregulated in human prostate cancer compared with non-cancerous tissue, correlating with the Gleason score. NEU3 silencing with siRNA in prostate cancer PC-3 and LNCaP cells resulted in increased expression of differentiation markers and in cell apoptosis, but decrease in Bcl-2 as well as a progression-related transcription factor, early growth response gene (EGR-1). In androgen-sensitive LNCaP cells, forced overexpression of NEU3 significantly induced expression of EGR-1, androgen receptor (AR) and PSA both with and without androgen, the cells becoming sensitive to androgen. The NEU3-mediated induction was abrogated by inhibitors for PI-3 kinase and MAP kinase and more specifically by their silencing in the absence of androgen, being confirmed by increased phosphorylation of AKT and ERK1/2 in NEU3 overexpressing cells. NEU3 siRNA introduction caused reduction of cell growth of an androgen-independent PC-3 cells in culture and of transplanted tumors in nude mice. These data suggest that NEU3 regulates tumor progression through AR signaling, and thus be a potential tool for diagnosis and therapy of androgen-independent prostate cancer.
doi:10.1038/cdd.2011.83
PMCID: PMC3252839  PMID: 21681193
sialidase; prostate cancer; androgen receptor; EGR-1; PSA
25.  AUC-based biomarker ensemble with an application on gene scores predicting low bone mineral density 
Bioinformatics  2011;27(21):3050-3055.
Motivation: The area under the receiver operating characteristic (ROC) curve (AUC), long regarded as a ‘golden’ measure for the predictiveness of a continuous score, has propelled the need to develop AUC-based predictors. However, the AUC-based ensemble methods are rather scant, largely due to the fact that the associated objective function is neither continuous nor concave. Indeed, there is no reliable numerical algorithm identifying optimal combination of a set of biomarkers to maximize the AUC, especially when the number of biomarkers is large.
Results: We have proposed a novel AUC-based statistical ensemble methods for combining multiple biomarkers to differentiate a binary response of interest. Specifically, we propose to replace the non-continuous and non-convex AUC objective function by a convex surrogate loss function, whose minimizer can be efficiently identified. With the established framework, the lasso and other regularization techniques enable feature selections. Extensive simulations have demonstrated the superiority of the new methods to the existing methods. The proposal has been applied to a gene expression dataset to construct gene expression scores to differentiate elderly women with low bone mineral density (BMD) and those with normal BMD. The AUCs of the resulting scores in the independent test dataset has been satisfactory.
Conclusion: Aiming for directly maximizing AUC, the proposed AUC-based ensemble method provides an efficient means of generating a stable combination of multiple biomarkers, which is especially useful under the high-dimensional settings.
Contact: lutian@stanford.edu
Supplementary Information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btr516
PMCID: PMC3198577  PMID: 21908541

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