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1.  Bicyclic 1-Hydroxy-2-oxo-1,2-dihydropyridine-3-carboxamide-Containing HIV-1 Integrase Inhibitors Having High Antiviral Potency against Cells Harboring Raltegravir-Resistant Integrase Mutants 
Journal of Medicinal Chemistry  2014;57(4):1573-1582.
Integrase (IN) inhibitors are the newest class of antiretroviral agents developed for the treatment of HIV-1 infections. Merck’s Raltegravir (RAL) (October 2007) and Gilead’s Elvitegravir (EVG) (August 2012), which act as IN strand transfer inhibitors (INSTIs), were the first anti-IN drugs to be approved by the FDA. However, the virus develops resistance to both RAL and EVG, and there is extensive cross-resistance to these two drugs. New “2nd-generation” INSTIs are needed that will have greater efficacy against RAL- and EVG-resistant strains of IN. The FDA has recently approved the first second generation INSTI, GSK’s Dolutegravir (DTG) (August 2013). Our current article describes the design, synthesis, and evaluation of a series of 1,8-dihydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamides, 1,4-dihydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides, and 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides. This resulted in the identification of noncytotoxic inhibitors that exhibited single digit nanomolar EC50 values against HIV-1 vectors harboring wild-type IN in cell-based assays. Importantly, some of these new inhibitors retain greater antiviral efficacy compared to that of RAL when tested against a panel of IN mutants that included Y143R, N155H, G140S/Q148H, G118R, and E138K/Q148K.
doi:10.1021/jm401902n
PMCID: PMC3983366  PMID: 24471816
2.  Carotid magnetic resonance imaging for monitoring atherosclerotic plaque progression: a multicenter reproducibility study 
This study sought to determine the multicenter reproducibility of magnetic resonance imaging (MRI) and the compatibility of different scanner platforms in assessing carotid plaque morphology and composition. A standardized multi-contrast MRI protocol was implemented at 16 imaging sites (GE: 8; Philips: 8). Sixty-eight subjects (61 ± 8 years; 52 males) were dispersedly recruited and scanned twice within 2 weeks on the same magnet. Images were reviewed centrally using a streamlined semiautomatic approach. Quantitative volumetric measurements on plaque morphology (lumen, wall, and outer wall) and plaque tissue composition [lipid-rich necrotic core (LRNC), calcification, and fibrous tissue] were obtained. Inter-scan reproducibility was summarized using the within-subject standard deviation, coefficient of variation (CV) and intraclass correlation coefficient (ICC). Good to excellent reproducibility was observed for both morphological (ICC range 0.98–0.99) and compositional (ICC range 0.88–0.96) measurements. Measurement precision was related to the size of structures (CV range 2.5–4.9 % for morphology, 36–44 % for LRNC and calcification). Comparable measurement variability was found between the two platforms on both plaque morphology and tissue composition. In conclusion, good to excellent inter-scan reproducibility of carotid MRI can be achieved in multicenter settings with comparable measurement precision between platforms, which may facilitate future multicenter endeavors that use serial MRI to monitor atherosclerotic plaque progression.
doi:10.1007/s10554-014-0532-7
PMCID: PMC4297722  PMID: 25216871
Magnetic resonance imaging; Carotid artery; Atherosclerosis; Reproducibility; Multicenter study
3.  High levels of SIRT1 expression enhance tumorigenesis and associate with a poor prognosis of colorectal carcinoma patients 
Scientific Reports  2014;4:7481.
SIRT1, a NAD+ dependent class III deacetylase, takes part in many important biological processes. Previous studies show that SIRT1 is overexpressed in some cancers and plays an essential role in tumorigenesis. However, the association between SIRT1 and colorectal cancer (CRC) is still unclear. We found that many CRC specimens had strong SIRT1 expression, which had an obvious correlation with poor prognosis of CRC patients. Meanwhile, SIRT1 expression had a co-localization with CD133, a current universal marker to characterize colorectal cancer stem cells (CSCs). In vitro studies also revealed that SIRT1 was overexpressed in colorectal CSC-like cells. Moreover, SIRT1 deficiency decreased percentage of CD133+ cells, attenuated the abilities of colony and sphere formation, and inhibited tumorigenicity in vivo in CRC cells. Further study demonstrated that the expressions of several stemness-associated genes, including Oct4, Nanog, Cripto, Tert and Lin28, were reduced by SIRT1 knockdown in CRC cells. Taken together, our findings suggest that SIRT1 plays a crucial role in keeping the characteristics of CSCs cells. SIRT1 is a potential independent prognostic factor of CRC patients after tumor resection with curative intent, and will contribute to providing a promising new approach to target at CSCs in CRC treatment.
doi:10.1038/srep07481
PMCID: PMC4265776  PMID: 25500546
4.  Biological mechanism analysis of acute renal allograft rejection: integrated of mRNA and microRNA expression profiles 
Objectives: Renal transplantation is the preferred method for most patients with end-stage renal disease, however, acute renal allograft rejection is still a major risk factor for recipients leading to renal injury. To improve the early diagnosis and treatment of acute rejection, study on the molecular mechanism of it is urgent. Methods: MicroRNA (miRNA) expression profile and mRNA expression profile of acute renal allograft rejection and well-functioning allograft downloaded from ArrayExpress database were applied to identify differentially expressed (DE) miRNAs and DE mRNAs. DE miRNAs targets were predicted by combining five algorithm. By overlapping the DE mRNAs and DE miRNAs targets, common genes were obtained. Differentially co-expressed genes (DCGs) were identified by differential co-expression profile (DCp) and differential co-expression enrichment (DCe) methods in Differentially Co-expressed Genes and Links (DCGL) package. Then, co-expression network of DCGs and the cluster analysis were performed. Functional enrichment analysis for DCGs was undergone. Results: A total of 1270 miRNA targets were predicted and 698 DE mRNAs were obtained. While overlapping miRNA targets and DE mRNAs, 59 common genes were gained. We obtained 103 DCGs and 5 transcription factors (TFs) based on regulatory impact factors (RIF), then built the regulation network of miRNA targets and DE mRNAs. By clustering the co-expression network, 5 modules were obtained. Thereinto, module 1 had the highest degree and module 2 showed the most number of DCGs and common genes. TF CEBPB and several common genes, such as RXRA, BASP1 and AKAP10, were mapped on the co-expression network. C1R showed the highest degree in the network. These genes might be associated with human acute renal allograft rejection. Conclusions: We conducted biological analysis on integration of DE mRNA and DE miRNA in acute renal allograft rejection, displayed gene expression patterns and screened out genes and TFs that may be related to acute renal allograft rejection.
PMCID: PMC4307466  PMID: 25664019
Renal transplantation; acute rejection; microRNA; mRNA; transcription factor
5.  An Efficient Diagnosis System for Parkinson's Disease Using Kernel-Based Extreme Learning Machine with Subtractive Clustering Features Weighting Approach 
A novel hybrid method named SCFW-KELM, which integrates effective subtractive clustering features weighting and a fast classifier kernel-based extreme learning machine (KELM), has been introduced for the diagnosis of PD. In the proposed method, SCFW is used as a data preprocessing tool, which aims at decreasing the variance in features of the PD dataset, in order to further improve the diagnostic accuracy of the KELM classifier. The impact of the type of kernel functions on the performance of KELM has been investigated in detail. The efficiency and effectiveness of the proposed method have been rigorously evaluated against the PD dataset in terms of classification accuracy, sensitivity, specificity, area under the receiver operating characteristic (ROC) curve (AUC), f-measure, and kappa statistics value. Experimental results have demonstrated that the proposed SCFW-KELM significantly outperforms SVM-based, KNN-based, and ELM-based approaches and other methods in the literature and achieved highest classification results reported so far via 10-fold cross validation scheme, with the classification accuracy of 99.49%, the sensitivity of 100%, the specificity of 99.39%, AUC of 99.69%, the f-measure value of 0.9964, and kappa value of 0.9867. Promisingly, the proposed method might serve as a new candidate of powerful methods for the diagnosis of PD with excellent performance.
doi:10.1155/2014/985789
PMCID: PMC4251425  PMID: 25484912
6.  Effect of Danshao Huaxian capsule on Gremlin and bone morphogenetic protein-7 expression in hepatic fibrosis in rats 
World Journal of Gastroenterology : WJG  2014;20(40):14875-14883.
AIM: To observe the effect of Danshao Huaxian capsule (DHC) on the expression of Gremlin and bone morphogenetic protein-7 (BMP-7) in the liver of hepatic fibrosis rats.
METHODS: A total of 75 male Wistar rats were randomly divided into a normal control group (A), a CCl4-induced hepatic fibrosis model group (B), a natural recovery group (C), a low-dose DHC-treated group (D), and a high-dose DHC-treated group (E), with 15 rats in each group. Liver fibrosis was induced by subcutaneous injections of carbon tetrachloride (CCl4) and a high-lipid/low-protein diet for 8 wk, except for the rats in group A. Then, the rats in the two DHC-treated groups were administered 0.5 and 1.0 g/kg DHC by gastrogavage once per day for 8 successive weeks, respectively. By the end of the experiment, the level of transforming growth factor β1 (TGF-β1) in the liver homogenate was determined by an enzyme-linked immunosorbent assay. The mRNA and protein expression of Gremlin and BMP-7 in the liver tissue was determined by reverse-transcription polymerase chain reaction, an immunohistochemical assay, and Western blot analysis.
RESULTS: Compared with group A, the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly higher in group B (TGF-β1: 736.30 ± 24.40 μg/g vs 284.20 ± 18.32 μg/g, P < 0.01; mRNA of Gremlin: 80.40 ± 5.46 vs 49.83 ± 4.20, P < 0.01; positive protein expression rate of Gremlin: 38.46% ± 1.70% vs 3.83% ± 0.88%, P < 0.01; relative protein expression of Gremlin: 2.81 ± 0.24 vs 0.24 ± 0.06, P < 0.01), and the mRNA and protein expression of BMP-7 was significantly lower in group B (mRNA: 54.00 ± 4.34 vs 93.99 ± 7.03, P < 0.01; positive protein expression rate: 28.97% ± 3.14% vs 58.29% ± 6.02, P < 0.01; relative protein expression: 0.48 ± 0.31 vs 1.05 ± 0.12, P < 0.01). Compared with groups B and C, the degree of hepatic fibrosis was significantly improved, and the level of TGF-β1 and the mRNA and protein expression of Gremlin were significantly lowered in the two DHC-treated groups (TGF-β1: 523.14 ± 21.29 μg/g, 441.86 ± 23.18 μg/g vs 736.30 ± 24.40 μg/g, 651.13 ± 15.75 μg/g, P < 0.01; mRNA of Gremlin: 64.86 ± 2.83, 55.82 ± 5.39 vs 80.40 ± 5.46, 70.37 ± 4.01, P < 0.01; positive protein expression rate of Gremlin: 20.78% ± 1.60%, 17.43% ± 2.02% vs 38.46% ± 1.70%, 29.50% ± 2.64%, P < 0.01; relative protein expression of Gremlin: 1.95 ± 0.26, 1.65 ± 0.20 vs 2.81 ± 0.24, 2.22 ± 0.63, P < 0.01), and the mRNA and protein expression of BMP-7 was higher in the two DHC-treated groups (mRNA: 73.52 ± 4.56, 81.78 ± 5.38 vs 54.00 ± 4.34, 62.28 ± 4.51, P < 0.01; positive protein expression rate: 41.44% ± 4.77%, 47.49% ± 4.59% vs 28.97% ± 3.14%, 35.85% ± 3.50%, P < 0.01; relative protein expression: 0.71 ± 0.06, 0.81 ± 0.07 vs 0.48 ± 0.31, 0.60 ± 0.37, P < 0.01).
CONCLUSION: The therapeutic mechanism of DHC for hepatic fibrosis in rats may be associated with inhibition of the expression of Gremlin and up-regulation of the expression of BMP-7.
doi:10.3748/wjg.v20.i40.14875
PMCID: PMC4209550  PMID: 25356047
Hepatic fibrosis; Bone morphogenetic protein; Gremlin; Transforming growth factor; Traditional Chinese herbs
7.  Relationship of Lipoproteins to Cardiovascular Events in the Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides and Impact on Global Health Outcomes (AIM-HIGH) Trial 
Journal of the American College of Cardiology  2013;62(17):10.1016/j.jacc.2013.07.023.
Objectives
In this secondary analysis of the AIM-HIGH trial, the objectives were to examine the relationship between niacin treatment, lipoproteins, and cardiovascular (CV) outcomes.
Background
During 3-year follow-up in 3,414 patients with established CV disease and low HDL-C, combined niacin + LDL-lowering therapy did not reduce CV events versus LDL-lowering therapy alone.
Methods
Subjects taking simvastatin + ezetimibe were randomized to extended-release (ER) niacin 1500–2000 mg or minimal immediate-release niacin (<150 mg) as placebo at bedtime. LDL-C in both groups was maintained from 40 to 80 mg/dL. Hazard ratios (HR) were estimated by Cox proportional hazards for relationships between lipoproteins and the composite endpoint of CV death, myocardial infarction, acute coronary syndrome, ischemic stroke, or symptom-driven revascularization.
Results
CV outcomes were not associated with ER niacin in any baseline lipoprotein tertile. In a subset of patients in both the highest triglyceride (>198 mg/dl) and lowest HDL-C (<33 mg/dl) tertiles, ER niacin showed a trend toward benefit (HR=0.74, p=0.073). In-trial LDL-C, nonHDL-C, and TC/HDL-C ratio were positively associated with CV events in the control group, but these relationships were absent in the ER niacin group.
Conclusions
Baseline lipoprotein tertiles did not predict differential benefit or harm with ER niacin added to LDL-lowering therapy, but a small dyslipidemic subgroup may benefit. ER niacin attenuated expected relationships of lipoprotein risk factors with CV events, raising the possibility that nonlipoprotein actions of niacin could impact risk.
Clinical trial info
AIM-HIGH; NCT00120289
doi:10.1016/j.jacc.2013.07.023
PMCID: PMC3862446  PMID: 23916935
niacin; cardiovascular events; clinical trial; lipoproteins; GPR109A
8.  Clinical experiences of NBI laryngoscope in diagnosis of laryngeal lesions 
Endoscopy is essential for the diagnosis and treatment of cancers derived from the larynx. However, a laryngoscope with conventional white light (CWL) has technical limitations in detecting small or superficial lesions on the mucosa. Narrow band imaging especially combined with magnifying endoscopy (ME) is useful for the detection of superficial squamous cell carcinoma (SCC) within the oropharynx, hypopharynx, and oral cavity. A total of 3675 patients who have come to the outpatient clinic and complained of inspiratory stridor, dyspnea, phonation problems or foreign body sensation, were enrolled in this study. We describe the glottic conditions of the patients. All 3675 patients underwent laryngoscopy equipped with conventional white light (CWL) and NBI system. 1149 patients received a biopsy process. And 1153 lesions were classified into different groups according to their histopathological results. Among all the 1149 patients, 346 patients (312 males, 34 females; mean age 62.2±10.5 years) were suspected of having a total of 347 precancerous or cancerous (T1 or T2 without lymphnode involvement) lesions of the larynx under the CWL. Thus, we expected to attain a complete vision of what laryngeal lesions look like under the NBI view of a laryngoscope. The aim was to develop a complete description list of each laryngeal conditions (e.g. polyps, papilloma, leukoplakia, etc.), which can serve as a criteria for further laryngoscopic examinations and diagnosis.
PMCID: PMC4238487  PMID: 25419362
Narrow band imaging; diagnosis; endoscopy; laryngeal lesion
9.  Mechanisms for Cellular NO Oxidation and Nitrite Formation in Lung Epithelial Cells 
Airway lining fluid contains relatively high concentrations of nitrite and arterial blood levels of nitrite are higher than venous levels, suggesting the lung epithelium may represent an important source of nitrite in vivo. To investigate whether lung epithelial cells possess the ability to convert NO to nitrite by oxidation, and the effect of oxygen reactions on nitrite formation, the NO donor DETA NONOate was incubated with or without A549 cells or primary human bronchial epithelial (HBE) cells for 24 hrs under normoxic (21% O2) and hypoxic (1% O2) conditions. Nitrite production was significantly increased under all conditions in the presence of A549 or HBE cells, suggesting that both A549 and HBE cells have the capacity to oxidize NO to nitrite even under low oxygen conditions. The addition of oxy-hemoglobin (oxy-Hb) to the A549 cell media decreased the production of nitrite, consistent with NO scavenging limiting nitrite formation. Heat-denatured A549 cells produced much lower nitrite and bitrate, suggesting an enzymatic activity is required. This NO oxidation activity was found to be highest in membrane bound proteins with molecular sizes < 100 kDa. In addition, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one] (ODQ) and cyanide inhibited formation of nitrite in A549 cells. It has been shown that ceruloplasmin (Cp) possesses an NO oxidase and nitrite synthase activity in plasma based on NO oxidation to nitrosonium cation (NO+). We observed that Cp is expressed intracellularly in lung epithelial A549 cells and secreted into medium under basal conditions and during cytokine stimulation. However, an analysis of Cp expression level and activity measured via ρ-phenylenediamine oxidase activity assay revealed very low activity compared with plasma, suggesting that there is insufficient Cp to contribute to detectable NO oxidation to nitrite in A549 cells. Additionally, Cp levels were knocked down using siRNA by more than 75% in A549 cells, with no significant change in either nitrite or cellular S-nitrosothiol (SNO) formation compared to scrambled siRNA control under basal conditions or cytokine stimulation. These data suggest that lung epithelial cells possess NO oxidase activity, which is enhanced in cell membrane associated proteins and not regulated by intracellular or secreted Cp, indicating that alternative NO oxidases determine hypoxic and normoxic nitrite formation from NO in human lung epithelial cells.
doi:10.1016/j.freeradbiomed.2013.04.031
PMCID: PMC3883890  PMID: 23639566
ceruloplasmin; NO oxidase; nitrite; nitric oxide; human lung epithelial cells; hypoxia
10.  Cell-based therapy for acute and chronic liver failures: Distinct diseases, different choices 
Scientific Reports  2014;4:6494.
Cell-based therapies (CBTs) are considered the effective approaches to treat liver failure. However, which cell type is the most suitable source of CBTs for acute liver failure (ALF) or chronic liver failure (CLF) remains unclear. To investigate this, mature hepatocytes in adult liver (adult HCs), fetal liver cells (FLCs), induced hepatic stem cells (iHepSCs) and bone marrow derived mesenchymal stromal cells (BMSCs) were used to CBTs for ConA-induced ALF and Fah-deficient induced CLF in mice. The results showed that only BMSCs remitted liver damage and rescued ALF in ConA-treated mice. In this process, BMSCs inhibited ConA-induced inflammatory response by decreasing the mRNA expressions of TNF-α, IFN-γ and FasL and increasing IL-10 mRNA expression. However, in the CLF model, not BMSCs but adult HCs transplantation lessened liver injury, recovered liver function and rescued the life of Fah-/- mice after NTBC withdrawal. Further study showed that adult HCs offered more effective liver regeneration compared to other cells in Fah-/- mice without NTBC. These results demonstrated that BMSCs and adult HCs are the optimal sources of CBTs for ConA-induced ALF and Fah-deficient induced CLF in mice, respectively. This finding deepens our understanding about how to select a proper CBT for different liver failure.
doi:10.1038/srep06494
PMCID: PMC4178291  PMID: 25263068
11.  Econazole Nitrate Induces Apoptosis in MCF-7 Cells via Mitochondrial and Caspase Pathways 
Econazole nitrate (EN), a synthetic compound, is now in use as a routine antifungal drug. EN was shown to have antitumor effect, the tumor cell killing mechanisms, however, remain unclear. In this research, the apoptosis-inducing effect of EN on MCF-7 cells was investigated. The results showed that EN inhibited the proliferation of MCF-7 cells in a time- and dose-dependent manner by MTT method and colony forming assay. MCF-7 cells treated with EN showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Meanwhile, the loss of mitochondrial membrane potential was showed by flow cytometry. In addition, western blot analysis showed that EN resulted in the decrease expression of procaspase-3, procaspase-9 and bcl-2. In conclusion, these findings suggest that EN may be an effective way for treating human breast cancer. The anti-tumor mechanisms of EN might involve mitochondrial and caspase pathways.
PMCID: PMC4232799  PMID: 25587322
Econazole nitrate; Apoptosis; MCF-7 cell; Caspase
12.  Expression quantitative trait loci infer the regulation of isoflavone accumulation in soybean (Glycine max L. Merr.) seed 
BMC Genomics  2014;15(1):680.
Background
Mapping expression quantitative trait loci (eQTL) of targeted genes represents a powerful and widely adopted approach to identify putative regulatory variants. Linking regulation differences to specific genes might assist in the identification of networks and interactions. The objective of this study is to identify eQTL underlying expression of four gene families encoding isoflavone synthetic enzymes involved in the phenylpropanoid pathway, which are phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), 2-hydroxyisoflavanone synthase (IFS; EC1.14.13.136) and flavanone 3-hydroxylase (F3H; EC 1.14.11.9). A population of 130 recombinant inbred lines (F5:11), derived from a cross between soybean cultivar ‘Zhongdou 27’ (high isoflavone) and ‘Jiunong 20’ (low isoflavone), and a total of 194 simple sequence repeat (SSR) markers were used in this study. Overlapped loci of eQTLs and phenotypic QTLs (pQTLs) were analyzed to identify the potential candidate genes underlying the accumulation of isoflavone in soybean seed.
Results
Thirty three eQTLs (thirteen cis-eQTLs and twenty trans-eQTLs) underlying the transcript abundance of the four gene families were identified on fifteen chromosomes. The eQTLs between Satt278-Sat_134, Sat_134-Sct_010 and Satt149-Sat_234 underlie the expression of both IFS and CHS genes. Five eQTL intervals were overlapped with pQTLs. A total of eleven candidate genes within the overlapped eQTL and pQTL were identified.
Conclusions
These results will be useful for the development of marker-assisted selection to breed soybean cultivars with high or low isoflavone contents and for map-based cloning of new isoflavone related genes.
doi:10.1186/1471-2164-15-680
PMCID: PMC4138391  PMID: 25124843
Soybean; eQTL; Isoflavone; pQTL; Candidate genes
13.  Monoclonal Antibodies Directed Against the Outer Membrane Protein of Bordetella avium 
Bordetella avium is the etiologic agent of coryza and rhinotracheitis in poultry. This respiratory disease is responsible for substantial economic losses in the poultry industry. Monoclonal antibodies (MAbs) were produced against the outer membrane proteins (OMPs) of B. avium isolated from diseased chickens. BALB/c mice were immunized with the extracted B. avium OMPs. Then the splenocytes from immunized mice and SP2/0 myeloma cells were fused using PEG 4000. Three stable hybridoma clones (designated as 3G10, 4A3, and 4E8) were produced via indirect ELISA and three rounds of subcloning. The MAbs were classified as IgG1, and can recognize the 58 kDa OMP band by Western blot assays. No MAb cross-reactivity with chicken Proteus mirabilis, Escherichia coli, and Salmonella was observed. A double antibody sandwich ELISA (DAS-ELISA) was developed using the rabbit polyclonal antibodies as the capture antibody and MAb 4A3 as the detection antibody. Under the DAS-ELISA, the minimum detectable concentration of B. avium was 1×104 CFU/mL, and no cross-reactivity occurred with chicken Proteus mirabilis, Escherichia coli, and Salmonella. Results showed that the DAS-ELISA has good sensitivity and specificity. Clinical application showed the DAS-ELISA was more sensitive than the plate agglutination test. This study may be used to develop a quick and specific diagnostic kit, analyze epitopes, and establish systems for typing B. avium.
doi:10.1089/mab.2012.0124
PMCID: PMC3733320  PMID: 23909425
14.  Scan-rescan reproducibility of quantitative assessment of inflammatory carotid atherosclerotic plaque using dynamic contrast-enhanced 3T CMR in a multi-center study 
Background
The aim of this study is to investigate the inter-scan reproducibility of kinetic parameters in atherosclerotic plaque using dynamic contrast-enhanced (DCE) cardiovascular magnetic resonance (CMR) in a multi-center setting at 3T.
Methods
Carotid arteries of 51 subjects from 15 sites were scanned twice within two weeks on 3T scanners using a previously described DCE-CMR protocol. Imaging data with protocol compliance and sufficient image quality were analyzed to generate kinetic parameters of vessel wall, expressed as transfer constant (Ktrans) and plasma volume (vp). The inter-scan reproducibility was evaluated using intra-class correlation coefficient (ICC) and coefficient of variation (CV). Power analysis was carried out to provide sample size estimations for future prospective study.
Results
Ten (19.6%) subjects were found to suffer from protocol violation, and another 6 (11.8%) had poor image quality (n = 6) in at least one scan. In the 35 (68.6%) subjects with complete data, the ICCs of Ktrans and vp were 0.65 and 0.28, respectively. The CVs were 25% and 62%, respectively. The ICC and CV for vp improved to 0.73 and 28% in larger lesions with analyzed area larger than 25 mm2. Power analysis based on the measured CV showed that 50 subjects per arm are sufficient to detect a 20% difference in change of Ktrans over time between treatment arms with 80% power without consideration of the dropout rate.
Conclusion
The result of this study indicates that quantitative measurement from DCE-CMR is feasible to detect changes with a relatively modest sample size in a prospective multi-center study despite the limitations. The relative high dropout rate suggested the critical needs for intensive operator training, optimized imaging protocol, and strict quality control in future studies.
doi:10.1186/s12968-014-0051-7
PMCID: PMC4237824  PMID: 25084698
Carotid artery; Atherosclerosis; Reproducibility; Dynamic contrast-enhanced cardiovascular magnetic resonance
15.  On-line separation and characterization of hyaluronan oligosaccharides derived from radical depolymerization 
Carbohydrate polymers  2013;96(2):503-509.
Hydroxyl radicals are widely implicated in the oxidation of carbohydrates in biological and industrial processes and are often responsible for their structural modification resulting in functional damage. In this study, the radical depolymerization of the polysaccharide hyaluronan was studied in a reaction with hydroxyl radicals generated by Fenton Chemistry. A simple method for isolation and identification of the resulting non-sulfated oligosaccharide products of oxidative depolymerization was established. Hyaluronan oligosaccharides were analyzed using ion-pairing reversed phase high performance liquid chromotography coupled with tandem electrospray mass spectrometry. The sequence of saturated hyaluronan oligosaccharides having even- and odd-numbers of saccharide units, afforded through oxidative depolymerization, were identified. This study represents a simple, effective ‘fingerprinting’ protocol for detecting the damage done to hyaluronan by oxidative radicals. This study should help reveal the potential biological outcome of reactive-oxygen radical-mediated depolymerization of hyaluronan.
doi:10.1016/j.carbpol.2013.04.009
PMCID: PMC3711257  PMID: 23768593
Hyaluronan; oligosaccharides; free radical depolymerization; mass spectrometry
16.  Lycopene ameliorates renal function in rats with streptozotocin-induced diabetes 
Aim: To study the effect of lycopene on ameliorating renal function of diabetic nephropathy. Methods: Sixty male SD rats were divided into four groups: normal untreated (NC-U), normal treatment (NC-L), diabetes untreated (DM-U) and diabetes treatment (DM-L). DM was prepared by a single injection of STZ (70 mg/kg, intraperitoneally) dissolved in 0.1 M citrate buffer (pH 4.5). DM-U and NC-U rats received control diet; DM-L and NC-L rats received lycopene. After treated with lycopene for 8 weeks, blood was obtained for analyzing plasma lipid profiles, glucose and renal function. The kidneys were used to determine SOD activity, malondialdehyde (MDA) level, processed for histological examination and western blot. Results: Treatment of diabetic rats with lycopene decreased the values of blood urea nitrogen, 24 h urea protein and creatinine. The serum lipids like TC, TG, and LDL were decreased and HDL was increased in DM-L rats when compared with those of diabetic rats. Administration of lycopene decreased the levels of MDA content and expression of CTGF, increased Akt/PKB phosphorylation and SOD activity in diabetic renal tissues. Conclusions: Lycopene protects against development of diabetic nephropathy and ameliorates renal function via improving oxidative status and regulating p-Akt and CTGF.
PMCID: PMC4152062  PMID: 25197372
Diabetic nephropathy; lycopene; oxidative stress
17.  Early decrease in carotid plaque lipid content as assessed by magnetic resonance imaging during treatment of rosuvastatin 
Background
Statin therapy has shown to deplete atherosclerotic plaque lipid content and induce plaque regression. However, how early the plaque lipid depletion can occur with low-density lipoprotein cholesterol (LDL-C) lowering in humans in vivo has not been fully described.
Methods
We enrolled 43 lipid treatment naïve subjects with asymptomatic carotid atherosclerosis and LDL-C ≥ 100 and ≤ 250 mg/dl. Rosuvastatin 5–20 mg/day was used to lower LDL-C levels to < 80 mg/dl. Lipid profile and carotid MRI scans were obtained at baseline, 3, 12, and 24 months. Carotid plaque lipid-rich necrotic core (LRNC) and plaque burden were measured and compared between baseline and during treatment.
Results
Among the 32 subjects who completed the study, at 3 months, an average dose of rosuvastatin of 11 mg/day lowered LDL-C levels by 47% (125.2 ± 24.4 mg/dl vs. 66.7 ± 17.3 mg/dl, p < 0.001). There were no statistically significant changes in total wall volume, percent wall volume or lumen volume. However, LRNC volume was significantly decreased by 7.9 mm3, a reduction of 7.3% (111.5 ± 104.2 mm3 vs. 103.6 ± 95.8 mm3, p = 0.044). Similarly, % LRNC was also significantly decreased from 18.9 ± 11.9% to 17.9 ± 11.5% (p = 0.02) at 3 months. Both LRNC volume and % LRNC continued to decrease moderately at 12 and 24 months, although this trend was not significant.
Conclusions
Among a small number of lipid treatment naïve subjects, rosuvastatin therapy may induce a rapid and lasting decrease in carotid plaque lipid content as assessed by MRI.
Trial registration
ClinicalTrials.Gov numbers NCT00885872
doi:10.1186/1471-2261-14-83
PMCID: PMC4107586  PMID: 25022285
Atherosclerosis; Plaque lipid content; Statin; Magnetic resonance imaging
18.  HIV Integrase Inhibitors Block Replication of Alpha-, Beta-, and Gammaherpesviruses 
mBio  2014;5(4):e01318-14.
ABSTRACT
The catalytic site of the HIV integrase is contained within an RNase H-like fold, and numerous drugs have been developed that bind to this site and inhibit its activity. Herpes simplex virus (HSV) encodes two proteins with potential RNase H-like folds, the infected cell protein 8 (ICP8) DNA-binding protein, which is necessary for viral DNA replication and exhibits recombinase activity in vitro, and the viral terminase, which is essential for viral DNA cleavage and packaging. Therefore, we hypothesized that HIV integrase inhibitors might also inhibit HSV replication by targeting ICP8 and/or the terminase. To test this, we evaluated the effect of 118-D-24, a potent HIV integrase inhibitor, on HSV replication. We found that 118-D-24 inhibited HSV-1 replication in cell culture at submillimolar concentrations. To identify more potent inhibitors of HSV replication, we screened a panel of integrase inhibitors, and one compound with greater anti-HSV-1 activity, XZ45, was chosen for further analysis. XZ45 significantly inhibited HSV-1 and HSV-2 replication in different cell types, with 50% inhibitory concentrations that were approximately 1 µM, but exhibited low cytotoxicity, with a 50% cytotoxic concentration greater than 500 µM. XZ45 blocked HSV viral DNA replication and late gene expression. XZ45 also inhibited viral recombination in infected cells and ICP8 recombinase activity in vitro. Furthermore, XZ45 inhibited human cytomegalovirus replication and induction of Kaposi’s sarcoma herpesvirus from latent infection. Our results argue that inhibitors of enzymes with RNase H-like folds may represent a general antiviral strategy, which is useful not only against HIV but also against herpesviruses.
IMPORTANCE
The herpesviruses cause considerable morbidity and mortality. Nucleoside analogs have served as effective antiviral agents against the herpesviruses, but resistance can arise through viral mutation. Second-line anti-herpes drugs have limitations in terms of pharmacokinetic properties and/or toxicity, so there is a great need for additional drugs for treatment of herpesviral infections. This study showed that the HIV integrase inhibitors also block herpesviral infection, raising the important potential of a new class of anti-herpes drugs and the prospect of drugs that combat both HIV and the herpesviruses.
doi:10.1128/mBio.01318-14
PMCID: PMC4161245  PMID: 24987091
19.  Modified posterior soft tissue repair for the prevention of early postoperative dislocation in total hip arthroplasty 
International Orthopaedics  2013;37(6):1039-1044.
Purpose
Dislocation following total hip arthroplasty (THA) with the posterior approach has been quite a common and bothering complication. Previous researches suggest that careful repair of the posterior structures significantly reduces this risk. The purposes of the present study were to describe a modified posterior soft tissue repair procedure in THA using a suture anchor (TwinFix Ti 5.0, Smith & Nephew, Andover, MA) and evaluate the early postoperative dislocation rate.
Methods
From July 2004 to June 2008, 220 consecutive primary total hip arthroplasties were performed using the modified surgical approach. The average age in the group was 46.4 years (range from 21 to 90) at the time of the procedure. The rate of postoperative hip dislocation, as well as any signs of complications related to the technique, has been observed and analyzed in this study.
Results
There was no postoperative dislocation following primary THA in 220 cases, and no signs of complications related to the technique, such as greater trochanteric fractures and sciatic nerve palsy, have been noted in any of the cases at their most recent follow-up.
Conclusions
These initial results demonstrate that the modified repair in THA using the suture anchor can serve as an effective and reliable mean for prevention of early postoperative dislocation
doi:10.1007/s00264-013-1874-9
PMCID: PMC3664159  PMID: 23549842
20.  Autophagy protects against palmitate-induced apoptosis in hepatocytes 
Cell & Bioscience  2014;4:28.
Background
Non-alcoholic fatty liver disease, one of the most common liver diseases, has obtained increasing attention. Palmitate (PA)-induced liver injury is considered a risk factor for the development of non-alcoholic fatty liver disease. Autophagy, a cellular degradative pathway, is an important self-defense mechanism in response to various stresses. In this study, we investigated whether autophagy plays a protective role in the progression of PA-induced hepatocytes injury.
Results
Annexin V-FITC/PI staining by FCM analysis, TUNEL assay and the detection of PARP and cleaved caspase3 expression levels demonstrated that PA treatment prominently induced the apoptosis of hepatocytes. Meanwhile, treatment of PA strongly induced the formation of GFP-LC3 dots, the conversion from LC3I to LC3II, the decrease of p62 protein levels and the increase of autophagosomes. These results indicated that PA also induced autophagy activation. Autophagy inhibition through chloroquine pretreatment or Atg5shRNA infection led to the increase of cell apoptosis after PA treatment. Moreover, induction of autophagy by pretreatment with rapamycin resulted in distinct decrease of PA-induced apoptosis. Therefore, autophagy can prevent hepatocytes from PA-induced apoptosis. In the further study, we explored pathway of autophagy activation in PA-treated hepatocytes. We found that PA activated PKCα in hepatocytes, and had no influence on mammalian target of rapamycin and endoplasmic reticulum stress pathways.
Conclusions
These results demonstrated that autophagy plays a protective role in PA-induced hepatocytes apoptosis. And PA might induce autophagy through activating PKCα pathway in hepatocytes.
doi:10.1186/2045-3701-4-28
PMCID: PMC4045884  PMID: 24904743
Autophagy; Palmitate; Hepatocytes; Apoptosis; Protector
21.  Reduced growth and proliferation dynamics of nasal epithelial stem/progenitor cells in nasal polyps in vitro 
Scientific Reports  2014;4:4619.
Basal cells in nasal epithelium have stemness/progenitor characters and play essential roles in the epithelial remodeling in nasal polyps (NP). We investigate whether the human nasal epithelial stem/progenitor cells (hNESPCs) from patients with NP are inherently distinct from those obtained from healthy controls. Epithelial basal cells were isolated and cultured for four passages from NP tissues and control nasal mucosa. hNESPCs from controls were stained positively with stem cell marker p63 and KRT5 and presented a consistent high Ki67 expression level over four passages. In contrast, hNESPCs from NP patients showed: i). a reduced growth and proliferation rate at each passage by evaluating colony-forming efficiency and doubling time; ii). a lower percentage of Ki67+ cells among p63+ cells in the colonies in late passages, which was also confirmed by immunostaining in the NP tissues. Thus reduced growth/proliferation dynamics in hNESPCs from NP could be an important pathological phenomenon in NP development.
doi:10.1038/srep04619
PMCID: PMC3980221  PMID: 24714674
22.  Circulating miR-208b and miR-34a Are Associated with Left Ventricular Remodeling after Acute Myocardial Infarction 
Left ventricular remodeling after acute myocardial infarction (AMI) is associated with adverse prognosis. It is becoming increasingly clear that circulating miRNAs could be promising biomarkers for various pathological processes in the heart, including myocardial infarction, myocardial remodeling and progression to heart failure. In the present study, a total of 359 consecutive patients were recruited. Plasma samples were collected on admission. Echocardiographic studies were performed during the admission and at six months follow-up after AMI. Remodeling was defined as an at least 10% increase from baseline in the left ventricular end-diastolic volume. Plasma miRNA levels were assessed for association with six months mortality or development of heart failure. Results showed that levels of plasma miR-208b and miR-34a were significantly higher in patients with remodeling than those without. Increased miRNA levels were strongly associated with increased risk of mortality or heart failure within six months for miR-208b (OR 17.91, 95% confidence interval = 2.07–98.81, p = 0.003), miR-34a (OR 4.18, 95% confidence interval = 1.36–12.83, p = 0.012) and combination of the two miRNAs (OR 18.73, 95% confidence interval = 1.96–101.23, p = 0.000). The two miRNA panels reclassified a significant proportion of patients with a net reclassification improvement of 11.7% (p = 0.025) and an integrated discrimination improvement of 7.7% (p = 0.002). These results demonstrated that circulating miR-208b and miR-34a could be useful biomarkers for predicting left ventricular remodeling after AMI, and the miRNA levels are associated with increased risk of mortality or heart failure.
doi:10.3390/ijms15045774
PMCID: PMC4013595  PMID: 24714087
microRNAs; myocardial infarction; left ventricular remodeling; prognosis; circulating biomarkers
23.  Microscale separation of heparosan, heparan sulfate and heparin 
Analytical biochemistry  2012;434(2):215-217.
The separation and quantification of glycosaminoglycan (GAG) chains, with different levels of sulfation, from cells, media and prepared through chemoenzymatic synthesis or metabolic engineering, poses a major challenge in glycomics analysis. A method for microscale separation and quantification of heparin, heparan sulfate and heparosan from cells is reported. This separation relies on a mini-strong anion exchange spin column eluted stepwise with different concentrations of sodium chloride. Disaccharide analysis by LC-MS was used to monitor the chemical structure of the different GAG chains that were recovered.
doi:10.1016/j.ab.2012.12.009
PMCID: PMC3572195  PMID: 23262074
heparosan; heparan sulfate; heparin; glycosaminoglycan; anion exchange chromatography; disaccharide analysis
24.  Variations in the MHC Region Confer Risk to Esophageal Squamous Cell Carcinoma on the Subjects from High-Incidence Area in Northern China 
PLoS ONE  2014;9(3):e90438.
Background
The human major histocompatibility complex (MHC) is the most important region in vertebrate genome, and is crucial in innate immunity. Recent studies have demonstrated the possible role of polymorphisms in the MHC region to high risk for esophageal squamous cell carcinoma (ESCC). Our previous genome-wide association study (GWAS) has indicated that the MHC region may confer important risk loci for ESCC, but without further fine mapping. The aim of this study is to further identify the risk loci in the MHC region for ESCC in Chinese population.
Methods
Conditional logistic regression analysis (CLRA) was performed on 24 single nucleotide polymorphisms (SNPs) within the MHC region, which were obtained from the genetically matched 937 cases and 692 controls of Chinese Han population. The identified promising SNPs were further correlated with clinical and clinicopathology characteristics. Immunohistochemistry was performed to explore the protein expression pattern of the related genes in ESCC and neighboring normal tissues.
Results
Of the 24 promising SNPs analyzed, we identified three independent SNPs in the MHC region associated with ESCC: rs35399661 (P = 6.07E-06, OR = 1.71, 95%CI = 1.36–2.17), rs3763338 (P = 1.62E-05, OR = 0.63, 95%CI = 0.50–0.78) and rs2844695 (P = 7.60E-05, OR = 0.74, 95%CI = 0.64–0.86). These three SNPs were located at the genes of HLA-DQA1, TRIM27, and DPCR1, respectively. Further analyses showed that rs2844695 was preferentially associated with younger ESCC cases (P = 0.009). The positive immunostaining rates both for HLA-DQA1 and TRIM27 were much higher in ESCC tissues than in neighboring normal tissues (69.4% vs. 26.8% for HLA-DQA1 and 77.6% vs. 47.8% for TRIM27, P<0.001). Furthermore, the overexpression of HLA-DQA1 is correlated significantly with age (P = 0.001) and family history (P<0.001).
Conclusion
This study for the first time provides evidence that multiple genetic factors within the MHC region confer risk to ESCC on the subjects from high-risk area in northern China.
doi:10.1371/journal.pone.0090438
PMCID: PMC3942432  PMID: 24595008
25.  Co-Circulation of Multiple Hemorrhagic Fever Diseases with Distinct Clinical Characteristics in Dandong, China 
PLoS ONE  2014;9(2):e89896.
Hemorrhagic fevers (HF) caused by viruses and bacteria are a major public health problem in China and characterized by variable clinical manifestations, such that it is often difficult to achieve accurate diagnosis and treatment. The causes of HF in 85 patients admitted to Dandong hospital, China, between 2011–2012 were determined by serological and PCR tests. Of these, 34 patients were diagnosed with Huaiyangshan hemorrhagic fever (HYSHF), 34 with Hemorrhagic Fever with Renal Syndrome (HFRS), one with murine typhus, and one with scrub typhus. Etiologic agents could not be determined in the 15 remaining patients. Phylogenetic analyses of recovered bacterial and viral sequences revealed that the causative infectious agents were closely related to those described in other geographical regions. As these diseases have no distinctive clinical features in their early stage, only 13 patients were initially accurately diagnosed. The distinctive clinical features of HFRS and HYSHF developed during disease progression. Enlarged lymph nodes, cough, sputum, and diarrhea were more common in HYSHF patients, while more HFRS cases presented with headache, sore throat, oliguria, percussion pain kidney area, and petechiae. Additionally, HYSHF patients displayed significantly lower levels of white blood cells (WBC), higher levels of creations kinase (CK) and alanine aminotransferase (ALT), while HFRS patients presented with an elevation of blood urea nitrogen (BUN) and creatinine (CREA). These clinical features will assist in the accurate diagnosis of both HYSHF and HFRS. Overall, our data reveal the complexity of pathogens causing HFs in a single Chinese hospital, and highlight the need for accurate early diagnosis and a better understanding of their distinctive clinical features.
doi:10.1371/journal.pone.0089896
PMCID: PMC3937409  PMID: 24587107

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