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1.  Human Ghrelin Mitigates Intestinal Injury and Mortality after Whole Body Irradiation in Rats 
PLoS ONE  2015;10(2):e0118213.
Widespread use of ionizing radiation has led to the realization of the danger associated with radiation exposure. Although studies in radiation countermeasures were initiated a half century ago, an effective therapy for a radiomitigator has not been identified. Ghrelin is a gastrointestinal hormone, and administration of ghrelin is protective in animal models of injuries including radiation combined injury. To test whether ghrelin can be protective in whole body irradiaton (WBI) alone, male Sprague Dawley (SD) rats were treated with human ghrelin (20 nmol/rat) daily for 6 days starting at either 24 h or 48 h after 10 Gray (Gy) WBI and survival outcome was examined. The 10 Gy WBI produced a LD70/30 model in SD rats (30% survival in 30 days). The survival rate in rats treated with ghrelin starting at 24 h was significantly improved to 63% and when treatment was initiated at 48 h, the survival remained at 61%. At 7 days post WBI, plasma ghrelin was significantly reduced from the control value. Ghrelin treatment starting at 24 h after WBI daily for 6 days improved histological appearance of the intestine, reduced gut permeability, serum endotoxin levels and bacterial translocation to the liver by 38%, 42% and 61%, respectively at day 7 post WBI. Serum glucose and albumin were restored to near control levels with treatment. Ghrelin treatment also attenuated WBI-induced intestinal apoptosis by 62% as evidenced by TUNEL staining. The expression of anti-apoptotic cell regulator Bcl-xl was decreased by 38% in the vehicle and restored to 75% of the control with ghrelin treatment. Increased expression of intestinal CD73 and pAkt were observed with ghrelin treatment, indicating protection of the intestinal epithelium after WBI. These results indicate that human ghrelin attenuates intestinal injury and mortality after WBI. Thus, human ghrelin can be developed as a novel mitigator for radiation injury.
PMCID: PMC4325005  PMID: 25671547
2.  Reduced expression levels of let-7c in human breast cancer patients 
Oncology Letters  2015;9(3):1207-1212.
Circulating microRNAs (miRNAs) are important in the diagnosis of a number of diseases, since serum or plasma miRNAs are more stable compared with miRNA isolated from blood samples. The aim of the present study was to investigate the association between the expression levels of serum let-7c miRNA and the clinical diagnosis of breast cancer (BC). The circulating let-7c levels of 90 BC patients and 64 healthy controls were determined by performing a reverse transcription-quantitative polymerase chain reaction assay. The results demonstrated that let-7c expression was downregulated in the BC tissues compared with the paracarcinoma control tissues. In addition, the let-7c expression in the serum of BC patients was significantly lower compared with the healthy controls (P<0.01). Using a cutoff value of 0.374×103 copies/ml, the serum expression levels of let-7c exhibited 87.5% sensitivity and 78.9% specificity for distinguishing BC patients from healthy controls (area under the receiver operating characteristic curve, 0.848; 95% confidence interval, 0.785–0.911). Furthermore, the results demonstrated that the serum expression levels of let-7c were significantly higher in premenopausal compared with postmenopausal patients (P<0.05), supporting the hypothesis that postmenopausal status may affect the serum expression levels of let-7c. However, no statistically significant differences were detected in the serum levels of let-7c between ER (or PR)-positive and -negative patients. Therefore, the current study hypothesized that serum let-7c may be used as a novel and valuable biomarker for the diagnosis of BC.
PMCID: PMC4315068  PMID: 25663883
circulating miRNA; breast cancer; let-7c; predictive factor; receiver operating characteristic analysis
3.  Nitric Oxide and Cellular Maturity Are Key Components of Pro-Inflammatory Cytokine-Induced Apoptosis of Human Fetal Lung Epithelial Cells 
Inflammation is a major contributor to the pathogenesis of bronchopulmonary dysplasia (BPD). BPD is associated with prematurity of birth, sepsis, with increased production of both cytokines and nitric oxide, and with the shedding of bronchial epithelial cells. The pathological mechanisms involved in this disease remain unclear, in particular the role that epithelial maturity plays. The effects of pro-inflammatory cytokines upon immature and mature cells are examined within this study, using primary culture of human lung epithelial cells. Pro-inflammatory cytokines increase inducible nitric oxide synthase (iNOS) expression and raise NO production, irrespective of cellular maturity. Pre-incubation with 1400W, a specific iNOS inhibitor, abrogated pro-inflammatory cytokine-induced NO generation and apoptosis. However, immature fetal lung epithelial cells were uniquely sensitive to cellular injury in response to cytokine exposure. These observations suggest that pro-inflammatory cytokines, which are present within BPD, may cause apoptosis of lung epithelial cells via de novo generation of NO. Furthermore, the prematurity of lung epithelial cells may be a factor in free radical mediated pulmonary damage.
PMCID: PMC4288020  PMID: 25580166
iNOS; apoptosis; caspase-3; maturity; nitric oxide; type II cell
4.  Sensitivity and Source of Amine Proton EXchange (APEX) and Amide Proton Transfer (APT) MRI in Cerebral Ischemia 
Amide proton transfer (APT) and amine-water proton exchange (APEX) can be viable to map pH-decreasing ischemic regions. However, their exact contributions are unclear.
We measured APEX- and APT-weighted magnetization transfer ratio asymmetry (denoted as APEXw and APTw), ADC, T2 and T1 images, and localized proton spectra in rats with permanent middle cerebral artery occlusion at 9.4 T. Phantoms and theoretical studies were also performed.
Within one hour post-occlusion, APEXw and APTw maps showed hyperintensity (3.1% of M0) and hypointensity (−1.8%), respectively, in regions with decreased ADC. Ischemia increased lactate and gamma aminobutyric acid (GABA) concentrations, but decreased glutamate and taurine concentrations. Over time, the APEXw contrast decreased with glutamate, taurine and creatine, while the APTw contrast and lactate level were similar. Phantom and theoretical studies suggest that the source of APEXw signal is mainly from proteins at normal pH, while at decreased pH, GABA and glutamate contributions increase, inducing the positive APEXw contrast in ischemic regions. The APTw contrast is sensitive to lactate concentration and pH, but contaminated from contributions of the faster amine-water proton exchange processes.
Positive APEXw contrast is more sensitive to ischemia than negative APTw contrast. They may provide complementary tissue metabolic information.
PMCID: PMC3655131  PMID: 23401310
Cerebral Ischemia; ADC; Amide; Amine; Chemical Exchange Saturation Transfer; Spin Locking; Stroke; MR Spectroscopy
5.  Pharmacological Postconditioning Treatment of Myocardial Infarction with Netrin-1 
The present study investigated whether pharmacological postconditoning with netrin-1 is cardioprotective against ischemia reperfusion (I/R) injury, and the underlying signaling mechanisms. Langendorff perfused hearts isolated from wild-type (WT) C57BL/6 or DCC+/− mice underwent a 20 min of ischemia, followed by a 60 min of reperfusion, in the presence or absence of netrin-1, or netrin-1 in combination with U0126 (MEK1/2 inhibitor), or PTIO (nitric oxide/NO scavenger). In WT mice, netrin-1 postconditioning dramatically reduced infarct size to 17.0±2.5%, from 40.5±4.2% in the untreated I/R group. U0126 or PTIO alone had no effect on infarct size but abolished the effects of netrin-1. The protective effect of netrin-1 was markedly diminished in DCC+/− mice (44.5±2% vs. 15±2.6 % for infract size in DCC+/− vs. DCC+/+ group). Our results indicate that netrin-1, given as a pharmacological postconditioning agent, induces cardioprotection via a DCC-dependent mechanism that involves ERK1/2 activation and NO production. Combined with our previous findings, netrin-1 treatment proves to be extremely and consistently beneficial whenever delivered to the heart, establishing its substantial promises for being developed into a robust therapeutic strategy for acute myocardial infarction.
PMCID: PMC4053198  PMID: 24389204
Netrin-1; Pharmacological postconditioning; Cardioprotection; Ischemia reperfusion (I/R) injury; deleted in colorectal cancer (DCC); ERK1/2; nitric oxide (NO)
6.  A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices 
PLoS ONE  2014;9(12):e114622.
Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.
PMCID: PMC4275185  PMID: 25536105
7.  Changes in Birth Weight between 2002 and 2012 in Guangzhou, China 
PLoS ONE  2014;9(12):e115703.
Recent surveillance data suggest that mean birth weight has begun to decline in several developed countries. The aim of this study is to examine the changes in birth weight among singleton live births from 2002 to 2012 in Guangzhou, one of the most rapidly developed cities in China.
We used data from the Guangzhou Perinatal Health Care and Delivery Surveillance System for 34108 and 54575 singleton live births with 28–41 weeks of gestation, who were born to local mothers, in 2002 and 2012, respectively. The trends in birth weight, small (SGA) and large (LGA) for gestational age and gestational length were explored in the overall population and gestational age subgroups.
The mean birth weight decreased from 3162 g in 2002 to 3137 g in 2012 (crude mean difference, −25 g; 95% CI, −30 to −19). The adjusted change in mean birth weight appeared to be slight (−6 g from 2002 to 2012) after controlling for maternal age, gestational age, educational level, parity, newborn's gender and delivery mode. The percentages of SGA and LGA in 2012 were 0.6% and 1.5% lower than those in 2002, respectively. The mean gestational age dropped from 39.2 weeks in 2002 to 38.9 weeks in 2012. In the stratified analysis, we observed the changes in birth weight differed among gestational age groups. The mean birth weight decreased among very preterm births (28–31 weeks), while remained relatively stable among other gestational age subcategories.
Among local population in Guangzhou from 2002 to 2012, birth weight appeared to slightly decrease. The percentage of SGA and LGA also simultaneously dropped, indicating that newborns might gain a healthier weight for gestational age.
PMCID: PMC4274089  PMID: 25531295
8.  Glucose transporter 3 is a rab11-dependent trafficking cargo and its transport to the cell surface is reduced in neurons of CAG140 Huntington’s disease mice 
Huntington’s disease (HD) disturbs glucose metabolism in the brain by poorly understood mechanisms. HD neurons have defective glucose uptake, which is attenuated upon enhancing rab11 activity. Rab11 regulates numerous receptors and transporters trafficking onto cell surfaces; its diminished activity in HD cells affects the recycling of transferrin receptor and neuronal glutamate/cysteine transporter EAAC1. Glucose transporter 3 (Glut3) handles most glucose uptake in neurons. Here we investigated rab11 involvement in Glut3 trafficking. Glut3 was localized to rab11 positive puncta in primary neurons and immortalized striatal cells by immunofluorescence labeling and detected in rab11-enriched endosomes immuno-isolated from mouse brain by Western blot. Expression of dominant active and negative rab11 mutants in clonal striatal cells altered the levels of cell surface Glut3 suggesting a regulation by rab11. About 4% of total Glut3 occurred at the cell surface of primary WT neurons. HD140Q/140Q neurons had significantly less cell surface Glut3 than did WT neurons. Western blot analysis revealed comparable levels of Glut3 in the striatum and cortex of WT and HD140Q/140Q mice. However, brain slices immunolabeled with an antibody recognizing an extracellular epitope to Glut3 showed reduced surface expression of Glut3 in the striatum and cortex of HD140Q/140Q mice compared to that of WT mice. Surface labeling of GABAα1 receptor, which is not dependent on rab11, was not different between WT and HD140Q/140Q mouse brain slices. These data define Glut3 to be a rab11-dependent trafficking cargo and suggest that impaired Glut3 trafficking arising from rab11 dysfunction underlies the glucose hypometabolism observed in HD.
PMCID: PMC4297405  PMID: 25526803
Huntington’s disease; Glucose transporter 3; Rab11; Recycling endosomes
9.  Comparison of long-term mortality of acute ST-segment elevation myocardial infarction and non-ST-segment elevation acute coronary syndrome patients after percutaneous coronary intervention 
Background and aims: This study is to compare the short-term and long-term mortality in patients with ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation acute coronary syndrome (NSTE-ACS) after percutaneous coronary intervention (PCI). Methods and results: A total of 266 STEMI patients and 140 NSTE-ACS patients received PCI. Patients were followed up by telephone or at medical record or case statistics center and were followed up for 4 years. Descriptive statistics and multivariate survival analyses were employed to compare the mortality in STEMI and NSTE-ACS. All statistical analyses were performed by SPSS19.0 software package. NSTE-ACS patients had significantly higher clinical and angiographic risk profiles at baseline. During the 4-year follow-up, all-cause mortality in STEMI was significantly higher than that in NSTE-ACS after coronary stent placement (HR 1.496, 95% CI 1.019-2.197). In a landmark analysis no difference was seen in all-cause mortality for both STEMI and NSTE-ACS between 6 month and 4 years of follow-up (HR 1.173, 95% CI 0.758-1.813). Conclusions: Patients with STEMI have a worse long-term prognosis compared to patients with NSTE-ACS after PCI, due to higher short-term mortality. However, NSTE-ACS patients have a worse long-term survival after 6 months.
PMCID: PMC4307524  PMID: 25664077
Coronary stent placement; long-term prognosis; long-term survival; mortality
10.  Identification of a Novel Homozygous Mutation, TMPRSS3: c.535G>A, in a Tibetan Family with Autosomal Recessive Non-Syndromic Hearing Loss 
PLoS ONE  2014;9(12):e114136.
Different ethnic groups have distinct mutation spectrums associated with inheritable deafness. In order to identify the mutations responsible for congenital hearing loss in the Tibetan population, mutation screening for 98 deafness-related genes by microarray and massively parallel sequencing of captured target exons was conducted in one Tibetan family with familiar hearing loss. A homozygous mutation, TMPRSS3: c.535G>A, was identified in two affected brothers. Both parents are heterozygotes and an unaffected sister carries wild type alleles. The same mutation was not detected in 101 control Tibetan individuals. This missense mutation results in an amino acid change (p.Ala179Thr) at a highly conserved site in the scavenger receptor cysteine rich (SRCR) domain of the TMPRSS3 protein, which is essential for protein-protein interactions. Thus, this mutation likely affects the interactions of this transmembrane protein with extracellular molecules. According to our bioinformatic analyses, the TMPRSS3: c.535G>A mutation might damage protein function and lead to hearing loss. These data suggest that the homozygous mutation TMPRSS3: c.535G>A causes prelingual hearing loss in this Tibetan family. This is the first TMPRSS3 mutation found in the Chinese Tibetan population.
PMCID: PMC4256404  PMID: 25474651
11.  Label-free Quantitative imaging of Cholesterol in Intact Tissues by Hyperspectral Stimulated Raman Scattering Microscopy** 
Current molecular analysis of cells and tissues routinely relies on separation, enrichment, and subsequent measurements by various assays. Here we demonstrate a platform of hyperspectral stimulated Raman scattering microscopy for fast, quantitative and label-free imaging of biomolecules in intact tissues using spectroscopic fingerprints as the contrast mechanism.
PMCID: PMC3932421  PMID: 24127161
Vibrational spectroscopy; Stimulated Raman scattering imaging; Lipids
12.  The Role of Placental Protein 14 in the Pathogenesis of Endometriosis 
Reproductive Sciences  2013;20(12):1465-1470.
Placental protein 14 (PP-14) is the principal secretory phase product of endometrium and has been shown to inhibit cell immune function. But its role in the pathogenesis of endometriosis is controversy. The objective of this study is to determine the concentrations of PP-14 in peritoneal fluid (PF) and serum and PP-14 protein expression in endometriotic lesions in women with ovarian endometriosis (n = 75) when compared to women without endometriosis (n = 49) between day 7 and day 20 of their menstrual cycle. Concentrations of PP-14 in PF and serum as well as PP-14 protein expression in endometriotic lesions in women with and without endometriosis were evaluated by using enzyme-linked immunosorbent assay and immunohistochemical staining, respectively. Serum PP-14 concentrations were significantly increased in women with endometriosis (7.5 ± 1.4 ng/mL) compared to those in women without endometriosis (5.8 ± 0.9 ng/mL; P < .05) and statistically decreased after surgery and further reduced by using gonadotropin-releasing hormone agonist therapy (P < .05). However, the concentrations of PP-14 in PF did not reach a significant difference between women with and without endometriosis (P > .05). In women with endometriosis, scores of PP-14 protein expression in the lesions (n = 50, 2.2 [0∼5.8]) were significantly correlated with serum PP-14 concentrations (n = 50, 7.6 ± 1.3 ng/mL; P < .01). Our results suggest that PP-14 may play an important role in the pathogenesis of endometriosis.
PMCID: PMC3817670  PMID: 23670949
endometriosis; placental protein 14; serum; peritoneal fluid; endometrium
13.  Growth Arrest-Specific Protein 6 (Gas6) Attenuates Neutrophil Migration and Acute Lung Injury in Sepsis 
Shock (Augusta, Ga.)  2013;40(6):10.1097/SHK.0b013e3182a588c1.
Sepsis is an acute inflammatory condition that can result in multiple organ failure and acute lung injury (ALI). Growth arrest-specific protein 6 (Gas6) is a broad regulator of the innate immune response involved with the NF-κB signaling pathway. We hypothesized that Gas6 could have a protective role in attenuating the severity of ALI and sepsis. Male mice were subjected to sepsis by cecal ligation and puncture (CLP) after which recombinant murine Gas6 (rmGas6; 5 μg/mouse) or normal saline (vehicle) was administered intravenously. Blood and lung tissues were collected at 20 h after CLP for various measurements. Treatment with rmGas6 significantly reduced serum levels of the injury markers AST, ALT and LDH as well as proinflammatory cytokines IL-6 and IL-17, compared to the vehicle group (P<0.05). The parenchyma of the lungs damaged by CLP was attenuated by rmGas6 treatment. Lung mRNA levels of TNF-α, IL-1β, IL-6, IL-17 and MIP-2 were decreased by 60%, 86%, 82%, 93% and 82%, respectively, with rmGas6 treatment as determined by real time RT-PCR (P<0.05). The degradation of IκB-α induced by CLP in the lungs was inhibited by rmGas6 treatment. The number of neutrophils and myeloperoxidase activity in the lungs were significantly reduced in the rmGas6 group. Moreover, rmGas6 reduced the in-vitro migration of differentiated human promyelocytic HL60 cells by 64%. Finally, the 10-day survival rate of mice subjected to CLP was increased from 31% in the vehicle group to 67% in the rmGas6 group (P<0.05). Thus, Gas6 has potential to be developed as a novel therapeutic agent to treat patients with sepsis and acute lung injury.
PMCID: PMC3834042  PMID: 23881260
Gas6; acute lung injury; sepsis; inflammation; neutrophil
14.  Enhanced Cellular Responses and Distinct Gene Profiles in Human Fetoplacental Artery Endothelial Cells under Chronic Low Oxygen1  
Biology of Reproduction  2013;89(6):133.
Fetoplacental endothelial cells are exposed to oxygen levels ranging from 2% to 8% in vivo. However, little is known regarding endothelial function within this range of oxygen because most laboratories use ambient air (21% O2) as a standard culture condition (SCN). We asked whether human umbilical artery endothelial cells (HUAECs) that were steadily exposed to the physiological chronic normoxia (PCN, 3% O2) for ∼20–25 days differed in their proliferative and migratory responses to FGF2 and VEGFA as well as in their global gene expression compared with those in the SCN. We observed that PCN enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. In oxygen reversal experiments (i.e., when PCN cells were exposed to SCN for 24 h and vice versa), we found that preexposure to 21% O2 decreased the migratory ability, but not the proliferative ability, of the PCN-HUAECs in response to FGF2 and VEGFA. These PCN-enhanced cellular responses were associated with increased protein levels of HIF1A and NOS3, but not FGFR1, VEGFR1, and VEGFR2. Microarray analysis demonstrated that PCN up-regulated 74 genes and down-regulated 86, 14 of which were directly regulated by hypoxia-inducible factors as evaluated using in silico analysis. Gene function analysis further indicated that the PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from our functional assays. Given that PCN significantly alters cellular responses to FGF2 and VEGFA as well as transcription in HUAECs, it is likely that we may need to reexamine the current cellular and molecular mechanisms controlling fetoplacental endothelial functions, which were largely derived from endothelial models established under ambient O2.
Chronic low oxygen enhances human endothelial cell proliferation and migration and alters gene expression.
PMCID: PMC4076354  PMID: 24152727
angiogenesis; artery endothelial cells; growth factors; physiological chronic low oxygen; transcriptome
15.  When is antidepressant polypharmacy appropriate in the treatment of depression? 
Shanghai Archives of Psychiatry  2014;26(6):357-359.
Depression is a serious medical condition that is often only partially improved or completely unchanged after standard treatment with antidepressant medications. Various approaches have been developed to treat this subgroup of individuals with ‘treatment-resistant’ depression; but many individuals continue to live with chronic depressive symptoms that seriously affect their quality of life and overall functioning. One relatively new strategy is ‘antidepressant polypharmacy’ – simultaneously administering two or more antidepressant medications. Given the heterogeneity of the etiology of depression, this approach could improve therapeutic outcomes by concurrently activating multiple neurological pathways with different mechanisms of action, but there is also the risk that using multiple antidepressants would increase the prevalence and severity of side effects. Further work is needed to assess the potential benefits and risks of this strategy to managing treatmentresistant depression.
PMCID: PMC4311109  PMID: 25642110
depression; treatment-resistant depression; antidepressants; polypharmacy; adjunctive treatment; multimodal treatment
16.  TGF-β-induced CD4+Foxp3+ T cells attenuate acute graft-versus-host disease via suppressing expansion and killing of effector CD8+ cells 
TGF-β-induced CD4+Foxp3+ T cells (iTregs) have been identified as important prevention and treatment strategies for cell therapy in autoimmune diseases and other disorders. However, the potential use of iTregs as a treatment modality for acute graft-verse-host disease (GVHD) has not been realized because iTregs may be unstable and less suppressive in this disease. Here we restudied the ability of iTregs to prevent and treat acute GVHD in two different mouse models. Our results showed that so long as an appropriate iTreg-generation protocol is used, these iTregs consistently displayed a potent ability to control acute GVHD development and reduce mortality in the acute GVHD animal models. iTreg infusion markedly suppressed the engraftment of donor CD8+ cells and CD4+ cells, the expression of Granzyme A and B, the cytotoxic effect of donor CD8+ cells and the production of T cell cytokines in acute GVHD. We therefore conclude that so long as the right methods for generating iTreg cells have been employed, iTregs can indeed prevent and even treat acute GVHD.
PMCID: PMC4247987  PMID: 25156367
17.  Experimental study on the regulation of erlotinib-induced radiosensitization with an anti-c-MET monoclonal antibody 
Cancer Cell International  2014;14(1):109.
Erlotinib is a novel therapeutic agent for cancer treatment. This study was performed to investigate the role of c-MET-PI3K-AKT pathway in the regulation of erlotinib-induced radiosensitization.
A973 lung adenocarcinoma cells treated with 6 Gy of radiation were incubated in the presence of erlotinib. The apoptotic rate after 24 hours, the colony-formating rate after 14 days, and changes in the c-MET expression levels after 14 days of irradiation were examined. Surviving fractions in different treatment groups (blank control, radiation alone, erlotinib alone, anti-c-MET monoclonal antibody alone, combined erlotinib and radiation, and combined erlotinib and radiation with anti-c-MET monoclonal antibody groups) were determined, the survival curves were plotted, and the sensitizer enhancement ratio was calculated using colony formation assays. Expressions of c-MET, p-c-MET, PI3K, AKT, and p-AKT in cells in different treatment groups were examined by Western blot analysis.
The apoptotic rate in the combined erlotinib and radiation group was higher than those in single treatment groups; however, the colony-forming rate remained approximately 2.04 ± 1.02%. The expression of c-MET in colony-forming cells in the combined group significantly increased, and the blockade of c-MET activity significantly enhanced the radiosensitizing effect of erlotinib. The expression of c-Met, p-c-MET, PI3K, AKT, and p-AKT among colony-forming cells significantly decreased upon the inhibition of c-MET.
Upregulated activity of the c-MET-PI3K-AKT pathway was found to be important for cell survival under combined the treatment with erlotinib and radiation. The blockade of the c-MET-PI3K-AKT signaling pathway enhanced the radiosensitizing effect of erlotinib.
PMCID: PMC4263203  PMID: 25505849
Tyrosine kinase inhibitor; Radiosensitization; Radiation resistance; Acquired drug resistance; PI3K pathway
18.  Ras-related C3 botulinum toxin substrate 1 activation is involved in the pathogenesis of diabetic retinopathy 
This study used a streptozotocin (STZ)-induced rat model of diabetes to investigate whether Ras-related C3 botulinum toxin substrate 1 (Rac1) was involved in the pathogenesis of diabetic retinopathy. The effects of Rac1 inhibition on vascular endothelial (VE)-cadherin and β-catenin expression in high glucose-induced rat retinal endothelial cells (RRECs) were additionally examined. Rac1 activation in the retinas from STZ-induced diabetic rats and in high glucose-induced RRECs was measured by reverse transcription-quantitative polymerase chain reaction analysis, immunohistochemistry and western blot analysis. The expression levels of VE-cadherin and β-catenin were also examined with or without Rac1 inhibition through small interfering (si)RNA transfection. STZ-induced diabetes was associated with an increase in the vascular permeability of the retina. Furthermore, Rac1 activation was increased in the retina of STZ-induced diabetic rats and in high glucose-induced RRECs compared with that in the controls. Immunohistochemistry showed that immunostaining of Rac1 was localized in the outer plexiform, inner nuclear, inner plexiform and ganglion cell layers and in the retinal microvasculature of rats. The expression of β-catenin was increased in the retinas of the diabetic rats at four, eight and 12 weeks after the induction of diabetes compared with that in the controls. Additionally, Rac1 activation was required for the high glucose-induced VE-cadherin expression decrease and for β-catenin expression in high glucose-induced RRECs. Rac1 inhibition by Rac1-siRNA transfection effectively prevented hyperpermeability, β-catenin expression and the VE-cadherin expression decrease in high glucose-induced RRECs. In conclusion, diabetes affects the expression of Rac1 in the retina. Rac1 may be involved in the diabetes-induced damage and/or alterations to the blood-retinal barrier through changes in VE-cadherin and β-catenin expression.
PMCID: PMC4247314  PMID: 25452781
Rac1; β-catenin; VE-cadherin; diabetes; retinopathy; rat; rat retinal endothelial cell
19.  Comprehensive analysis of the association of EGFR, CALM3 and SMARCD1 gene polymorphisms with BMD in Caucasian women 
PLoS ONE  2014;9(11):e112358.
Three genes, including EGFR (epidermal growth factor receptor), CALM3 (calmodulin 3, calcium-modulated protein 3) and SMARCD1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily d member 1), play different roles in bone and/or fat metabolism in Caucasian women. In this population-based investigation of 870 unrelated postmenopausal Caucasian women, CALM3 polymorphisms were significantly associated with femoral neck bone mineral density (FNK BMD), hip BMD and spine BMD. Age and tobacco status also affected BMD levels and were therefore corrected for in our statistical analysis.
EGFR, CALM3 and SMARCD1 play roles in bone and/or fat metabolism. However, the correlations between the polymorphisms of these three genes and body composition levels, including BMD, remain to be determined.
Materials and Methods
A population-based investigation of 870 white women was conducted. Forty-four SNPs (single nucleotide polymorphisms) in EGFR, CALM3 and SMARCD1 were chosen by the software, including those of potential functional importance. The candidate SNPs were genotyped by the KASPar assay for an association analysis with body composition levels. The correlation analysis was assessed by the Pearson's product-moment correlation coefficient and Spearman rank-order correlation tests, and the family-wise error was corrected using the Wald test implemented in PLINK.
The SNP rs12461917 in the 3′-flanking region of the CALM3 gene was significantly associated with FNK BMD (P = 0.001), hip BMD (P<0.001) and spine BMD (P = 0.001); rs11083838 in the 5′-flanking region of CALM3 gene was associated with spine BMD (P = 0.009). After adjusting for multiple comparisons, rs12461917 remained significant (P-adjusted  = 0.033 for FNK BMD, P-adjusted  = 0.006 for hip BMD and P-adjusted  = 0.018 for spine BMD).
Our data show that polymorphisms of the CALM3 gene in Caucasian women may contribute to variations in the BMD of the hip, spine and femoral neck.
PMCID: PMC4232396  PMID: 25396734
20.  Transplantation of ATP7B–Transduced Bone Marrow Mesenchymal Stem Cells Decreases Copper Overload in Rats 
PLoS ONE  2014;9(11):e111425.
Recent studies have demonstrated that transplantation of ATP7B-transduced hepatocytes ameliorates disease progression in LEC (Long-Evans Cinnamon) rats, a model of Wilson's disease (WD). However, the inability of transplanted cells to proliferate in a normal liver hampers long-term treatment. In the current study, we investigated whether transplantation of ATP7B-transduced bone marrow mesenchymal stem cells (BM-MSCs) could decrease copper overload in LEC rats.
Materials and Methods
The livers of LEC rats were preconditioned with radiation (RT) and/or ischemia-reperfusion (IRP) before portal vein infusion of ATP7B-transduced MSCs (MSCsATP7B). The volumes of MSCsATP7B or saline injected as controls were identical. The expression of ATP7B was analyzed by real-time quantitative polymerase chain reaction (RT-PCR) at 4, 12 and 24 weeks post-transplantation. MSCATP7B repopulation, liver copper concentrations, serum ceruloplasmin levels, and alanine transaminase (ALT) and aspartate transaminase (AST) levels were also analyzed at each time-point post-transplantation.
IRP-plus-RT preconditioning was the most effective strategy for enhancing the engraftment and repopulation of transplanted MSCsATP7B. This strategy resulted in higher ATP7B expression and serum ceruloplasmin, and lower copper concentration in this doubly preconditioned group compared with the saline control group, the IRP group, and the RT group at all three time-points post-transplantation (p<0.05 for all). Moreover, 24 weeks post-transplantation, the levels of ALT and AST in the IRP group, the RT group, and the IRP-plus-RT group were all significantly decreased compared to those of the saline group (p<0.05 compared with the IRP group and RT group, p<0.01 compared with IRP-plus-RT group); ALT and AST levels were significantly lower in the IRP-plus-RT group compared to either the IRP group or the RT group (p<0.01 and p<0.05. respectively).
These results demonstrate that transplantation of MSCsATP7B into IRP-plus-RT preconditioned LEC rats decreased copper overload and was associated with an increase in MSC engraftment and repopulation.
PMCID: PMC4222898  PMID: 25375371
21.  Risk factor analysis for central nodal metastasis in papillary thyroid carcinoma 
Oncology Letters  2014;9(1):103-107.
Lymph node involvement is associated with recurrence in papillary thyroid carcinoma (PTC). The central neck compartment (level VI) lymph nodes are at the greatest risk of metastases from PTC, but the role of central neck dissection (CND) remains controversial, particularly in PTC without clinical cervical lymph node metastasis (cN0). The present study aimed to identify risk factors of central cervical nodal metastasis and the safety of CND in patients with cN0 PTC. The current study retrospectively investigated 389 patients who had been followed up for 12.0–25.5 months after surgery, and were divided into positive or negative lymph node involvement groups according to the pathological results subsequent to this surgery. Univariate and multivariate analyses were used to study the risk factor of central node involvement. The mean tumor size was 0.71±0.35 cm (range, 0.1–2.0 cm). There was no significant difference in the rate of central lymph node involvement based on age (<45 or ≥45 years) or tumor focality (unifocal or multifocal). However, there were significant differences based on gender, extra-thyroid invasion and tumor size (P<0.05). The incidence of transient hypoparathyroidism and transient vocal cord paralysis following CND was 12.34 and 4.11%, respectively. No patient experienced permanent hypoparathyroidism or vocal cord paralysis. One patient (1/389; 0.23%) experienced disease recurrence during the follow-up. A larger tumor size and the male gender were significantly associated with the central nodal metastasis rate for cN0 PTC with a tumor size of <2.0 cm. CND for cN0 PTC patients was safe and the tumor-associated recurrence rate following CND plus total thyroidectomy was low. The present study suggests that CND should be conducted for male cN0 PTC patients with a larger tumor size (≥0.5 cm).
PMCID: PMC4246692  PMID: 25435941
papillary thyroid carcinoma; central neck dissection; total thyroidectomy; hypoparathyroidism; vocal cord paralysis
22.  What Is Normal Femoral Head/Neck Anatomy? An Analysis of Radial CT Reconstructions in Adolescents 
Cam morphology in femoroacetabular impingement has been implicated in the development of osteoarthritis. The alpha angle and femoral head/neck offset are widely used to determine femoral head asphericity. To our knowledge, no study has evaluated the alpha angle circumferentially using three-dimensional imaging in a population of healthy individuals of adolescent age.
We sought to (1) determine normal values for the alpha angle in adolescents, (2) define the location along the neck with the highest alpha angle, and (3) determine normal femoral head and neck radii and femoral head/neck offset.
Fifty CT scans from a database of scans obtained for reasons not related to hip pain were studied. The average age of the subjects was 15 years (range, 14–16 years). Alpha angle and femoral head/neck offset were measured circumferentially.
The alpha angle averaged 40.66 ± 4.46 mm for males and 37.77 ± 5.65 mm for females. The alpha angle generally was highest between the 11:40 and 12:40 o’clock and between the 6:00 and 7:40 o’clock positions. The femoral head radius was 24.53 ± 1.74 mm for males and 21.94 ± 1.13 mm for females, and the femoral neck radius was 16.14 ± 2.32 mm for males and 13.82 ± 2.38 mm for females. The mean femoral head/neck offset was 8.39 ± 1.97 mm for males and 8.13 ± 2.27 mm for females.
In this healthy population of 14- to 16-year-old subjects, the highest alpha angle was at the superior and inferior aspects of the heads rather than at the anterosuperior aspect. This information will provide benchmark values for distinction between normal and abnormal morphologic features of the femoral head.
Level of Evidence
Level III, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.
PMCID: PMC3792287  PMID: 23922189
24.  Gender-specific genetic associations of polymorphisms in ACE, AKR1C2, FTO and MMP2 with weight gain over a 10-year period 
Genes & Nutrition  2014;9(6):434.
Weight gain, when it leads to overweight or obesity, is nowadays one of the major health problems. ACE, FTO, AKR1C2, TIMP4 and MMP2 genes have been implicated in previous studies on weight regulation. This study investigated the contribution of polymorphisms in these five candidate genes to the risk of weight gain over a 10-year time period. Two groups were selected from participants of the Doetinchem cohort study who were followed over a 10-year period: A stable weight group (±2 kg/10 year; n = 259) and a weight gainers group who increased their body weight by roughly 10 % (>8 kg/10 year; n = 237). Starting BMI was between 20 and 35 kg/m2 and baseline age between 20 and 45 years. Selected SNPs and insert/deletion in candidate genes were measured in each group. In men, the allelic distribution of FTO rs9939609 (χ2p = 0.005), ACE rs4340 (χ2p = 0.006) and AKR1C2 rs12249281 (χ2p = 0.019) differed between the weight stable and weight gainers group. Interaction between FTO rs9939609 and ACE rs4340 was observed. In women, the allelic distribution of MMP2 rs1132896 differed between the weight stable and weight gainers group (χ2p = 0.00001). The A-allele of FTO was associated with a 1.99× higher risk of gaining weight in men (OR 1.99, p = 0.020), while in women, the C-allele of MMP2 was associated with a 2.50× higher risk of weight gain (OR 2.50, p = 0.001) over the 10-year period. We found that FTO in men and MMP2 in women are associated with weight gain over a 10-year follow-up period.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0434-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4235831  PMID: 25322899
Weight gain; Overweight; SNP; Genotyping
25.  Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba 
BMB Reports  2014;47(10):546-551.
The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]
PMCID: PMC4261511  PMID: 24286331
Aedes aegypti; Bacillus thuringiensis; Cry4Ba toxin; Larvicidal activity; Peritrophic membrane

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