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1.  Early serodiagnosis of trichinellosis by ELISA using excretory–secretory antigens of Trichinella spiralis adult worms 
Parasites & Vectors  2015;8:484.
The excretory–secretory (ES) antigens of Trichinella spiralis muscle larvae (ML) are the most commonly used diagnostic antigens for trichinellosis. Their main disadvantage for the detection of anti-Trichinella IgG is false-negative results during the early stage of infection. Additionally, there is an obvious window between clinical symptoms and positive serology.
ELISA with adult worm (AW) ES antigens was used to detect anti-Trichinella IgG in the sera of experimentally infected mice and patients with trichinellosis. The sensitivity and specificity were compared with ELISAs with AW crude antigens and ML ES antigens.
In mice infected with 100 ML, anti-Trichinella IgG were first detected by ELISA with the AW ES antigens, crude antigens and ML ES antigens 8, 12 and 12 days post-infection (dpi), respectively. In mice infected with 500 ML, specific antibodies were first detected by ELISA with the three antigen preparations at 10, 8 and 10 dpi, respectively. The sensitivity of the ELISA with the three antigen preparations for the detection of sera from patients with trichinellosis at 35 dpi was 100 %. However, when the patients’ sera were collected at 19 dpi, the sensitivities of the ELISAs with the three antigen preparations were 100 % (20/20), 100 % (20/20) and 75 % (15/20), respectively (P < 0.05). The specificities of the ELISAs with the three antigen preparations were 98.11, 95.60 and 89.31 %, respectively (P < 0.05).
The sensitivity and specificity of the T. spiralis AW ES antigens were superior to those of the AW crude antigens and ML ES antigens. Thus, the AW ES antigens might serve as potential antigens for the early and specific serodiagnosis of trichinellosis.
PMCID: PMC4579640  PMID: 26394626
Trichinella spiralis; Trichinellosis; Adult worm; Excretory–secretory (ES) antigens; Serodiagnosis
2.  Sparse Regression by Projection and Sparse Discriminant Analysis 
Recent years have seen active developments of various penalized regression methods, such as LASSO and elastic net, to analyze high dimensional data. In these approaches, the direction and length of the regression coefficients are determined simultaneously. Due to the introduction of penalties, the length of the estimates can be far from being optimal for accurate predictions. We introduce a new framework, regression by projection, and its sparse version to analyze high dimensional data. The unique nature of this framework is that the directions of the regression coefficients are inferred first, and the lengths and the tuning parameters are determined by a cross validation procedure to achieve the largest prediction accuracy. We provide a theoretical result for simultaneous model selection consistency and parameter estimation consistency of our method in high dimension. This new framework is then generalized such that it can be applied to principal components analysis, partial least squares and canonical correlation analysis. We also adapt this framework for discriminant analysis. Compared to the existing methods, where there is relatively little control of the dependency among the sparse components, our method can control the relationships among the components. We present efficient algorithms and related theory for solving the sparse regression by projection problem. Based on extensive simulations and real data analysis, we demonstrate that our method achieves good predictive performance and variable selection in the regression setting, and the ability to control relationships between the sparse components leads to more accurate classification. In supplemental materials available online, the details of the algorithms and theoretical proofs, and R codes for all simulation studies are provided.
PMCID: PMC4560121  PMID: 26345204
Discriminant analysis; Sparse discriminant analysis; Sparse regression by projection; Zero within-class and between-class correlations
3.  Gene expression profiling leads to discovery of correlation of matrix metalloproteinase 11 and heparanase 2 in breast cancer progression 
BMC Cancer  2015;15:473.
In order to identify biomarkers involved in breast cancer, gene expression profiling was conducted using human breast cancer tissues.
Total RNAs were extracted from 150 clinical patient tissues covering three breast cancer subtypes (Luminal A, Luminal B, and Triple negative) as well as normal tissues. The expression profiles of a total of 50,739 genes were established from a training set of 32 samples using the Agilent Sure Print G3 Human Gene Expression Microarray technology. Data were analyzed using Agilent Gene Spring GX 12.6 software. The expression of several genes was validated using real-time RT-qPCR.
Data analysis with Agilent GeneSpring GX 12.6 software showed distinct expression patterns between cancer and normal tissue samples. A group of 28 promising genes were identified with ≥ 10-fold changes of expression level and p-values < 0.05. In particular, MMP11 and HPSE2 were closely examined due to the important roles they play in cancer cell growth and migration. Real-time RT-qPCR analyses of both training and testing sets validated the gene expression profiles of MMP11 and HPSE2.
Our findings identified these 2 genes as a novel breast cancer biomarker gene set, which may facilitate the diagnosis and treatment in breast cancer clinical therapies.
PMCID: PMC4477316  PMID: 26084486
Breast cancer; Gene expression profiling; Biomarker; MMP11; HPSE2
4.  Characterization of Spirometra erinaceieuropaei Plerocercoid Cysteine Protease and Potential Application for Serodiagnosis of Sparganosis 
PLoS Neglected Tropical Diseases  2015;9(6):e0003807.
Sparganosis is a neglected but important food-borne parasitic zoonosis. Clinical diagnosis of sparganosis is difficult because there are no specific manifestations. ELISA using plerocercoid crude or excretory–secretory (ES) antigens has high sensitivity but has cross-reactions with other helminthiases. The aim of this study was to characterize Spirometra erinaceieuropaei cysteine protease (SeCP) and to evaluate its potential application for serodiagnosis of sparganosis.
Methodology/Principal Findings
The full length SeCP gene was cloned, and recombinant SeCP (rSeCP) was expressed and purified. Western blotting showed that rSeCP was recognized by the serum of sparganum-infected mice, and anti-rSeCP serum recognized the native SeCP protein of plerocercoid crude or ES antigens. Expression of SeCP was observed at plerocercoid stages but not at the adult and egg stages. Immunolocalization identified SeCP in plerocercoid tegument and parenchymal tissue. The rSeCP had CP activity, and the optimum pH and temperature were 5.5 and 37°C, respectively. Enzymatic activity was significantly inhibited by E-64. rSeCP functions to degrade different proteins and the function was inhibited by anti-rSeCP serum and E-64. Immunization of mice with rSeCP induced Th2-predominant immune responses and anti-rSeCP antibodies had the potential capabilities to kill plerocercoids in an ADCC assay. The sensitivity of rSeCP-ELISA and ES antigen ELISA was 100% when performed on sera of patients with sparganosis. The specificity of rSeCP-ELISA and ES antigen ELISA was 98.22% (166/169) and 87.57% (148/169), respectively (P<0.05).
The rSeCP had the CP enzymatic activity and SeCP seems to be important for the survival of plerocercoids in host. The rSeCP is a potential diagnostic antigen for sparganosis.
Author Summary
Sparganosis is a neglected tropical disease; its diagnosis is difficult and it is often misdiagnosed. ELISA using the crude or ES antigens of plerocercoids cross reacts with other helminthiases. Cysteine protease is a type of hydrolase and plays important roles in the development and survival of parasites; it has been used for diagnostic markers and vaccine targets for some parasitic diseases. In this study, a 36 kDa Spirometra erinaceieuropaei cysteine protease (SeCP) was expressed and purified. The results showed that SeCP was a plerocercoid stage-specific protein located in the teguments and parenchymal tissue. The rSeCP had cysteine protease activity and functioned to degrade host proteins. Vaccination of mice with rSeCP induced high levels of IgG1 and anti-rSeCP antibodies with the ability to kill plerocercoids in an ADCC assay. The rSeCP had a high sensitivity and specificity for detecting anti-plerocercoid antibodies, and could be used as a potential antigen for serodiagnosis of sparganosis.
PMCID: PMC4457932  PMID: 26046773
5.  OSM Enhances Angiogenesis and Improves Cardiac Function after Myocardial Infarction 
BioMed Research International  2015;2015:317905.
Oncostatin M (OSM) has been reported to stimulate angiogenesis by upregulating VEGF and bFGF, implying that it could be a therapeutic strategy in treating ischemic diseases. The present study was aimed at investigating whether OSM could improve cardiac function via prompting angiogenesis following myocardial infarction (MI). Wild type (WT) and Oβ knock-out (Oβ−/−) mice were, respectively, randomized into sham group, MI + vehicle group, and MI + OSM group. WT mice displayed significantly impaired cardiac function after MI. OSM treatment attenuated cardiac dysfunction in WT MI mice, while Oβ deletion abrogated the protective effects. Besides, OSM attenuated heart hypertrophy and pulmonary congestion evidenced by decreased heart weight/body weight and lung weight/body weight ratio. Further, reduction of apoptosis and fibrosis in infarct border zone was observed in OSM treated WT MI mice compared with vehicle. Moreover, in WT mice subjected to MI, OSM treatment significantly increased capillary density along with upregulation of p-Akt and angiogenic factors VEGF and bFGF in comparison with vehicle, and this phenomenon was not found in Oβ−/− mice. In conclusion, OSM treatment preserved cardiac function, inhibited apoptosis and fibrosis, and stimulated angiogenesis via upregulating VEGF and bFGF in infarct border zone of ischemic myocardium, indicating that OSM could be a novel therapeutic target for MI.
PMCID: PMC4471304  PMID: 26146616
6.  Extracellular Matrix can Recover the Downregulation of Adhesion Molecules after Cell Detachment and Enhance Endothelial Cell Engraftment 
Scientific Reports  2015;5:10902.
The low cell engraftment after transplantation limits the successful application of stem cell therapy and the exact pathway leading to acute donor cell death following transplantation is still unknown. Here we investigated if processes involved in cell preparation could initiate downregulation of adhesion-related survival signals, and further affect cell engraftment after transplantation. Human embryonic stem cell-derived endothelial cells (hESC-ECs) were suspended in PBS or Matrigel and kept at 4 °C. Quantitative RT-PCR analysis was used to test the adhesion and apoptosis genes’ expression of hESC-ECs. We demonstrated that cell detachment can cause downregulation of cell adhesion and extracellular matrix (ECM) molecules, but no obvious cell anoikis, a form of apoptosis after cell detachment, was observed. The downregulation of adhesion and ECM molecules could be regained in the presence of Matrigel. Finally, we transplanted hESC-ECs into a mouse myocardial ischemia model. When transplanted with Matrigel, the long-term engraftment of hESC-ECs was increased through promoting angiogenesis and inhibiting apoptosis, and this was confirmed by bioluminescence imaging. In conclusion, ECM could rescue the functional genes expression after cell detached from culture dish, and this finding highlights the importance of increasing stem cell engraftment by mimicking stem cell niches through ECM application.
PMCID: PMC4454140  PMID: 26039874
7.  O-GlcNAc Modification of Transcription Factor Sp1 Mediates Hyperglycemia-Induced VEGF-A Upregulation in Retinal Cells 
Proangiogenic protein VEGF-A contributes significantly to retinal lesions and neovascularization in diabetic retinopathy (DR). In preclinical DR, hyperglycemia can upregulate VEGF-A in retinal cells. The VEGF-A promoter is responsive to the transcription factor specificity protein 1 (Sp1). The O-GlcNAc modification is driven by glucose concentration and has a profound effect on Sp1 activity. This study investigated the effects of hyperglycemia on Sp1-mediated expression of VEGF-A in the retinal endothelium and pigment epithelium.
Hyperglycemia-exposed ARPE-19 (human retinal pigment epithelial cells) and TR-iBRB (rat retinal microendothelial cells) were assayed for levels of VEGF-A by qRT-PCR, Western blot, and ELISA. Small molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) were used to manipulate O-GlcNAc levels. Vascular endothelial growth factor–A protein and transcript were measured in cells depleted of OGT or Sp1 by shRNA. The proximal VEGF-A promoter was analyzed for glucose sensitivity by luciferase assay. Chromatin immunoprecipitation (ChIP) was used to assess Sp1 occupancy on the VEGF-A promoter.
Hyperglycemia increased VEGF-A promoter activity and upregulated VEGF-A transcript and protein. Elevation of O-GlcNAc by OGA inhibitors was sufficient to increase VEGF-A. O-GlcNAc transferase inhibition abrogated glucose-driven VEGF-A. Cellular depletion of OGT or Sp1 by shRNA significantly abrogated glucose-induced changes in VEGF-A. ChIP analysis showed that hyperglycemia significantly increased binding of Sp1 to the VEGF-A promoter.
Hyperglycemia-driven VEGF-A production is mediated by elevated O-GlcNAc modification of the Sp1 transcription factor. This mechanism may be significant in the pathogenesis of preclinical DR through VEGF-A upregulation.
We show that hyperglycemia-driven O-GlcNAc modification of transcription factor Sp1 upregulates VEGF-A in retinal endothelial and pigment epithelial cells. We propose that O-GlcNAc transferase (OGT) and Sp1 mediate VEGF-A production in preclinical diabetic retinopathy.
PMCID: PMC4541483  PMID: 25352121
VEGF-A; Sp1; O-GlcNAc; diabetic retinopathy; OGT
8.  Identification of jasmonic acid-associated microRNAs and characterization of the regulatory roles of the miR319/TCP4 module under root-knot nematode stress in tomato 
Journal of Experimental Botany  2015;66(15):4653-4667.
Screening revealed that the action of miR319/TCP4 in serving as a systemic defensive responder and regulator that modulated the RKN systemic defensive response was mediated via JA.
MicroRNAs (miRNAs) are important transcriptional and post-transcriptional modulators of gene expression that play crucial roles in the responses to diverse stresses. To explore jasmonic acid (JA)-dependent miRNA-mediated regulatory networks that are responsive to root-knot nematode (RKN), two small RNA libraries were constructed from wild-type (WT) and JA mutant (spr2) plants. A total of 263 known miRNAs and 441 novel miRNAs were significantly regulated under RKN stress in the two libraries. The spatio-temporal expression of candidate miRNAs and their corresponding targets were analysed by qRT-PCR under RKN stress. A clear negative correlation was observed between miR319 and its target TEOSINTE BRANCHED1/CYCLOIDEA/PRO-LIFERATING CELL FACTOR 4 (TCP4) in leaf, stem, and root under RKN stress, implying that the miR319/TCP4 module is involved in the systemic defensive response. Reverse genetics demonstrated that the miR319/TCP4 module affected JA synthetic genes and the endogenous JA level in leaves, thereby mediating RKN resistance. These results suggested that the action of miR319 in serving as a systemic signal responder and regulator that modulated the RKN systemic defensive response was mediated via JA. The potential cross-talk between miR319/TCP4 and miR396/GRF (GROWTH RESPONDING FACTOR) in roots under RKN invasion is discussed, and a predictive model regarding miR319/TCP4-mediated RKN resistance is proposed.
PMCID: PMC4507771  PMID: 26002970
Deep sequencing; jasmonic acid; miRNAs; miR319/TCP4; root-knot nematode; tomato.
9.  Associations between climate variability, unemployment and suicide in Australia: a multicity study 
BMC Psychiatry  2015;15:114.
A number of studies have examined the associations of suicide with meteorological variables (MVs) and socioeconomic status but the results are inconsistent. This study assessed whether MVs and unemployment were associated with suicide in eight Australian capital cities.
Data on suicide, population and unemployment rate (UER) between 1985 and 2005 were from the Australian Bureau of Statistics. MVs was provided by Australian Bureau of Meteorology. A generalized linear regression model with Poisson link was applied to explore the association of suicide with MVs and UER.
Temperature difference (ΔT, the difference in mean temperature between current month and previous one month) was positively associated with suicide in Sydney, Melbourne, Brisbane and Hobart. There was also a significant and positive association between UER and suicide in Sydney, Melbourne, Brisbane and Perth. MVs had more significant associations with violent suicide than that of non-violent suicide. There were no consistent associations between other MVs and suicide. A significant interaction between ΔT and UER on suicide was found in Sydney, Melbourne and Brisbane, such that increased temperature amplified the magnitude of the association between UER and suicide.
ΔT and UER appeared to jointly influence the occurrence of suicide in Australian capital cities. This finding may have implications for developing effective suicide prevention strategies.
PMCID: PMC4488118  PMID: 25964132
Suicide; Australia; Cities; Climate; Unemployment
10.  Osteoinduction and proliferation of bone-marrow stromal cells in three-dimensional poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds 
Osteoinduction and proliferation of bone-marrow stromal cells (BMSCs) in three-dimensional (3D) poly(ε-caprolactone) (PCL) scaffolds have not been studied throughly and are technically challenging. This study aimed to optimize nanocomposites of 3D PCL scaffolds to provide superior adhesion, proliferation and differentiation environment for BMSCs in this scenario.
BMSCs were isolated and cultured in a novel 3D tissue culture poly(ε-caprolactone) (PCL) scaffold coated with poly-lysine, hydroxyapatite (HAp), collagen and HAp/collagen. Cell morphology was observed and BMSC biomarkers for osteogenesis, osteoblast differentiation and activation were analyzed.
Scanning Electron Microscope (SEM) micrographs showed that coating materials were uniformly deposited on the surface of PCL scaffolds and BMSCs grew and aggregated to form clusters during 3D culture. Both mRNA and protein levels of the key players of osteogenesis and osteoblast differentiation and activation, including runt-related transcription factor 2 (Runx2), alkaline phosphates (ALP), osterix, osteocalcin, and RANKL, were significantly higher in BMSCs seeded in PCL scaffolds coated with HAp or HAp/collagen than those seeded in uncoated PCL scaffolds, whereas the expression levels were not significantly different in collagen or poly-lysine coated PCL scaffolds. In addition, poly-lysine, collagen, HAp/collagen, and HAp coated PCL scaffolds had significantly more viable cells than uncoated PCL scaffolds, especially scaffolds with HAp/collagen and collagen-alone coatings. That BMSCs in HAp or HAp/collagen PCL scaffolds had remarkably higher ALP activities than those in collagen-coated alone or uncoated PCL scaffolds indicating higher osteogenic differentiation levels of BMSCs in HAp or HAp/collagen PCL scaffolds. Moreover, morphological changes of BMSCs after four-week of 3D culture confirmed that BMSCs successfully differentiated into osteoblast with spread-out phenotype in HAp/collagen coated PCL scaffolds.
This study showed a proof of concept for preparing biomimetic 3D poly (ε-caprolactone)/ hydroxyapatite/collagen scaffolds with excellent osteoinduction and proliferation capacity for bone regeneration.
PMCID: PMC4429830  PMID: 25952675
Poly(ε-caprolactone); Hydroxyapatite and collagen coating; 3D culture
11.  Incremental value of resting three-dimensional speckle-tracking echocardiography in detecting coronary artery disease 
The aim of the present study was to investigate the incremental value of resting three-dimensional speckle-tracking echocardiography (3D-STE) in the detection of early-stage left ventricular dysfunction in patients with coronary artery disease (CAD). A total of 110 patients suspected of having CAD were recruited. All patients underwent 3D-STE and coronary artery angiography (CAG). They were divided to a CAD group and a normal group according to the results of CAG. Using 3D-STE software, the peak values of longitudinal strain (LS), circumferential strain (CS), radial strain (RS) and area strain (AS) and the time to peak value of these strains (T-LS, T-CS, T-RS and T-AS) were measured. A receiver operator characteristic curve (ROC) was used to analyze the sensitivity of these strains for the diagnosis of CAD. ROC analysis indicated that T-LS and composite indices combining the peak strain value and time to peak of LS, CS and AS have diagnostic value for the early detection of CAD; the area under the curve (AUC) values were 0.667, 0.692, 0.621 and 0.672 respectively (P<0.005). The composite index of longitudinal strain demonstrated the highest diagnostic value for CAD with 62% sensitivity and 76% specificity. These results indicate that 3D-STE has incremental value for the diagnosis of CAD in patients at rest.
PMCID: PMC4473507  PMID: 26136933
three-dimensional speckle-tracking echocardiography; coronary artery disease; strain; left ventricular dysfunction
12.  Parallel content-based sub-image retrieval using hierarchical searching 
Bioinformatics  2013;30(7):996-1002.
Motivation: The capacity to systematically search through large image collections and ensembles and detect regions exhibiting similar morphological characteristics is central to pathology diagnosis. Unfortunately, the primary methods used to search digitized, whole-slide histopathology specimens are slow and prone to inter- and intra-observer variability. The central objective of this research was to design, develop, and evaluate a content-based image retrieval system to assist doctors for quick and reliable content-based comparative search of similar prostate image patches.
Method: Given a representative image patch (sub-image), the algorithm will return a ranked ensemble of image patches throughout the entire whole-slide histology section which exhibits the most similar morphologic characteristics. This is accomplished by first performing hierarchical searching based on a newly developed hierarchical annular histogram (HAH). The set of candidates is then further refined in the second stage of processing by computing a color histogram from eight equally divided segments within each square annular bin defined in the original HAH. A demand-driven master-worker parallelization approach is employed to speed up the searching procedure. Using this strategy, the query patch is broadcasted to all worker processes. Each worker process is dynamically assigned an image by the master process to search for and return a ranked list of similar patches in the image.
Results: The algorithm was tested using digitized hematoxylin and eosin (H&E) stained prostate cancer specimens. We have achieved an excellent image retrieval performance. The recall rate within the first 40 rank retrieved image patches is ∼90%.
Availability and implementation: Both the testing data and source code can be downloaded from
PMCID: PMC3967099  PMID: 24215030
13.  Drp1 stabilizes p53 on the mitochondria to trigger necrosis under oxidative stress conditions in vitro and in vivo 
The Biochemical journal  2014;461(1):137-146.
Oxidative-stress-induced necrosis is considered to be one of the main pathological mediators in various neurological disorders, such as brain ischaemia. However, little is known about the mechanism by which cells modulate necrosis in response to oxidative stress. In the present study, we showed that Drp1 (dynamin-related protein 1), a primary mitochondrial fission protein, stabilizes the well-known stress gene p53 and is required for p53 translocation to the mitochondria under conditions of oxidative stress. We found that Drp1 binding to p53 induced mitochondria-related necrosis. In contrast, inhibition of Drp1 hyperactivation by Drp1 siRNA reduced necrotic cell death in cell cultures exposed to oxidative stress. Most significantly, we demonstrated that inhibition of Drp1 by the Drp1 peptide inhibitor P110, which was developed recently by our group, abolished p53 association with the mitochondria and reduced brain infarction in rats subjected to brain ischaemia/reperfusion injury. Taken together, these findings reveal a novel mechanism of Drp1 hyperactivation in the induction of mitochondrial damage and subsequent cell death. We propose that a Drp1 inhibitor such as P110 is a possible therapeutic agent for diseases in which hyperactivated Drp1 contributes to the pathology.
PMCID: PMC4381936  PMID: 24758576
dynamin-related protein 1 (Drp1); mitochondrion; necrosis; oxidative stress; p53; ubiquitination
14.  Genetic Structure Analysis of Spirometra erinaceieuropaei Isolates from Central and Southern China 
PLoS ONE  2015;10(3):e0119295.
Sparganosis caused by invasion of the plerocercoid larvae (spargana) of Spirometra erinaceieuropaei have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) to analyze genetic variation and phylogeographic structure of the S. erinaceieuropaei populations.
Methodology/Principal Findings
A total of 88 S. erinaceieuropaei isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete cytb and cox1 genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise FST values supported great genetic differentiation between S. erinaceieuropaei populations. Both negative Fu’s FS value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago.
S. erinaceieuropaei from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.
PMCID: PMC4368571  PMID: 25793277
15.  Identification of epipolythiodioxopiperazines HDN-1 and chaetocin as novel inhibitor of heat shock protein 90 
Oncotarget  2015;6(7):5263-5274.
The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an important target for cancer treatment. HDN-1, an epipolythiopiperazine-2, 5-diones (ETPs) compound, was here identified as a new Hsp90 inhibitor. HDN-1 bound directly to C-terminus of Hsp90α, resulting in a potential conformational change that interfered with the binding of 17-AAG and novobiocin to Hsp90α. In contrast, association of 17-AAG, novobiocin or ATP with Hsp90α did not prevent the binding HDN-1 to Hsp90α. HDN-1 in combination with 17-AAG exhibited an enhanced inhibitory effect on non-small lung cancer cell proliferation. Molecular docking analyses revealed that HDN-1 bound to Hsp90α at C-terminal 526–570 region. In addition, HDN-1 degraded multiple oncoproteins and promoted EGF-induced wild type and mutated EGFR downregulation. Notably, chaetocin, used as a SUV39H1 inhibitor with similar structure to HDN-1, bound to Hsp90 and degraded Hsp90 client proteins and SUV39H1 as did HDN-1. These results indicate that HDN-1 and chaetocin are inhibitors of Hsp90 and that SUV39H1 is a novel client protein of Hsp90.
PMCID: PMC4467147  PMID: 25742791
pipolythiodioxopiperazines; HDN-1; chaetocin; inhibitor; Hsp90
16.  Development of Monoclonal Antibodies against Human CYP11B1 and CYP11B2 
The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate.Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme.The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.
PMCID: PMC3939805  PMID: 24325867
CYP11B1; CYP11B2; Adrenal cortex; Monoclonal antibodies; 17α-Hydroxylase; Immunofluorescence
17.  High-Density Genetic Linkage Map Construction and QTL Mapping of Grain Shape and Size in the Wheat Population Yanda1817 × Beinong6 
PLoS ONE  2015;10(2):e0118144.
High-density genetic linkage maps are necessary for precisely mapping quantitative trait loci (QTLs) controlling grain shape and size in wheat. By applying the Infinium iSelect 9K SNP assay, we have constructed a high-density genetic linkage map with 269 F 8 recombinant inbred lines (RILs) developed between a Chinese cornerstone wheat breeding parental line Yanda1817 and a high-yielding line Beinong6. The map contains 2431 SNPs and 128 SSR & EST-SSR markers in a total coverage of 3213.2 cM with an average interval of 1.26 cM per marker. Eighty-eight QTLs for thousand-grain weight (TGW), grain length (GL), grain width (GW) and grain thickness (GT) were detected in nine ecological environments (Beijing, Shijiazhuang and Kaifeng) during five years between 2010–2014 by inclusive composite interval mapping (ICIM) (LOD≥2.5). Among which, 17 QTLs for TGW were mapped on chromosomes 1A, 1B, 2A, 2B, 3A, 3B, 3D, 4A, 4D, 5A, 5B and 6B with phenotypic variations ranging from 2.62% to 12.08%. Four stable QTLs for TGW could be detected in five and seven environments, respectively. Thirty-two QTLs for GL were mapped on chromosomes 1B, 1D, 2A, 2B, 2D, 3B, 3D, 4A, 4B, 4D, 5A, 5B, 6B, 7A and 7B, with phenotypic variations ranging from 2.62% to 44.39%. QGl.cau-2A.2 can be detected in all the environments with the largest phenotypic variations, indicating that it is a major and stable QTL. For GW, 12 QTLs were identified with phenotypic variations range from 3.69% to 12.30%. We found 27 QTLs for GT with phenotypic variations ranged from 2.55% to 36.42%. In particular, QTL QGt.cau-5A.1 with phenotypic variations of 6.82–23.59% was detected in all the nine environments. Moreover, pleiotropic effects were detected for several QTL loci responsible for grain shape and size that could serve as target regions for fine mapping and marker assisted selection in wheat breeding programs.
PMCID: PMC4326355  PMID: 25675376
18.  Two new species of Stenochironomus Kieffer (Diptera, Chironomidae) from Zhejiang, China 
ZooKeys  2015;109-119.
Two new species of Stenochironomus Kieffer (Diptera: Chironomidae: Chironominae), Stenochironomus brevissimus sp. n. and Stenochironomus linanensis sp. n., are described from China and the male imagines are illustrated. Stenochironomus brevissimus sp. n. can be separated from the so far known species by having very short and small, spatulate superior volsella with two long setae, whereas Stenochironomus linanensis sp. n. is easily separated from the other species of Stenochironomus by the following characters: wings transparent, body yellow, superior volsella finger-like, with nine long setae, elongated inferior volsella with four long setae and one well developed terminal spine; tergite IX with 10−15 long setae medially. A key to the males of Stenochironomus occurring in China is given.
PMCID: PMC4319065  PMID: 25685018
Stenochironomus; new species; key; China
19.  Distinct Conformational Transition Patterns of Noncoding 7SK snRNA and HIV TAR RNAs upon Tat Binding 
Biochemistry  2014;53(4):675-681.
Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information.
PMCID: PMC3985858  PMID: 24422492
20.  Optimal contouring of seminal vesicle for definitive radiotherapy of localized prostate cancer: comparison between EORTC prostate cancer radiotherapy guideline, RTOG0815 protocol and actual anatomy 
Intermediate- to-high-risk prostate cancer can locally invade seminal vesicle (SV). It is recommended that anatomic proximal 1-cm to 2-cm SV be included in the clinical target volume (CTV) for definitive radiotherapy based on pathology studies. However, it remains unclear whether the pathology indicated SV extent is included into the CTV defined by current guidelines. The purpose of this study is to compare the volume of proximal SV included in CTV defined by EORTC prostate cancer radiotherapy guideline and RTOG0815 protocol with the actual anatomic volume.
Radiotherapy planning CT images from 114 patients with intermediate- (36.8%) or high-risk (63.2%) prostate cancer were reconstructed with 1-mm-thick sections. The starting and ending points of SV and the cross sections of SV at 1-cm and 2-cm from the starting point were determined using 3D-view. Maximum (D1H, D2H) and minimum (D1L, D2L) vertical distance from these cross sections to the starting point were measured. Then, CTV of proximal SV defined by actual anatomy, EORTC guideline and RTOG0815 protocol were contoured and compared (paired t test).
Median length of D1H, D1L, D2H and D2L was 10.8 mm, 2.1 mm, 17.6 mm and 8.8 mm (95th percentile: 13.5mm, 5.0mm, 21.5mm and 13.5mm, respectively). For intermediate-risk patients, the proximal 1-cm SV CTV defined by EORTC guideline and RTOG0815 protocol inadequately included the anatomic proximal 1-cm SV in 62.3% (71/114) and 71.0% (81/114) cases, respectively. While for high-risk patients, the proximal 2-cm SV CTV defined by EORTC guideline inadequately included the anatomic proximal 2-cm SV in 17.5% (20/114) cases.
SV involvement indicated by pathology studies was not completely included in the CTV defined by current guidelines. Delineation of proximal 1.4 cm and 2.2 cm SV in axial plane may be adequate to include the anatomic proximal 1-cm and 2-cm SV. However, part of SV may be over-contoured.
PMCID: PMC4299806  PMID: 25526901
Prostate cancer; Radiotherapy; Seminal vesicle; Target delineation; CT reconstruction
21.  Anticancer efficacy and absorption, distribution, metabolism, and toxicity studies of Aspergiolide A in early drug development 
Since the first anthracycline was discovered, many other related compounds have been studied in order to overcome its defects and improve efficacy. In the present paper, we investigated the anticancer effects of a new anthracycline, aspergiolide A (ASP-A), from a marine-derived fungus in vitro and in vivo, and we evaluated the absorption, distribution, metabolism, and toxicity drug properties in early drug development. We found that ASP-A had activity against topoisomerase II that was comparable to adriamycin. ASP-A decreased the growth of various human cancer cells in vitro and induced apoptosis in BEL-7402 cells via a caspase-dependent pathway. The anticancer efficacy of ASP-A on the growth of hepatocellular carcinoma xenografts was further assessed in vivo. Results showed that, compared with the vehicle group, ASP-A exhibited significant anticancer activity with less loss of body weight. A pharmacokinetics and tissue distribution study revealed that ASP-A was rapidly cleared in a first order reaction kinetics manner, and was enriched in cancer tissue. The maximal tolerable dose (MTD) of ASP-A was more than 400 mg/kg, and ASP-A was not considered to be potentially genotoxic or cardiotoxic, as no significant increase of micronucleus rates or inhibition of the hERG channel was seen. Finally, an uptake and transport assay of ASP-A was performed in monolayers of Caco-2 cells, and ASP-A was shown to be absorbed through the active transport pathway. Altogether, these results indicate that ASP-A has anticancer activity targeting topoisomerase II, with a similar structure and mechanism to adriamycin, but with much lower toxicity. Nonetheless, further molecular structure optimization is necessary.
PMCID: PMC4207554  PMID: 25378909
aspergiolide A; anticancer; anthracyclines; ADMT; drug activity
22.  The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening 
Future medicinal chemistry  2014;6(7):809-823.
G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity.
PMCID: PMC4172322  PMID: 24941874
23.  Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters 
Acta Pharmacologica Sinica  2014;35(9):1188-1198.
To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.
Male SD rats were administered TA3 (100 mg·kg−1·d−1, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.
TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.
TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.
PMCID: PMC4155534  PMID: 25087997
timosaponin A3; hepatotoxicity; cholestasis; bile acid; transporter; Cyp7a1; ROS; N-acetyl-L- cysteine; mangiferin
24.  A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission 
Molecular Biology of the Cell  2014;25(12):1905-1915.
Drp1 catalyzes mitochondrial division, but the mechanisms remain elusive. The mitochondrial lipid cardiolipin stimulates Drp1 activity and supports membrane constriction. In addition, Drp1 populates two polymeric states that equilibrate via a dimeric intermediate. Dimers nucleate Drp1 reassembly on mitochondria for fission.
The GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial division, but the mechanisms remain poorly understood. Much of what is attributed to Drp1’s mechanism of action in mitochondrial membrane fission parallels that of prototypical dynamin in endocytic vesicle scission. Unlike the case for dynamin, however, no lipid target for Drp1 activation at the mitochondria has been identified. In addition, the oligomerization properties of Drp1 have not been well established. We show that the mitochondria-specific lipid cardiolipin is a potent stimulator of Drp1 GTPase activity, as well as of membrane tubulation. We establish further that under physiological conditions, Drp1 coexists as two morphologically distinct polymeric species, one nucleotide bound in solution and the other membrane associated, which equilibrate via a dimeric assembly intermediate. With two mutations, C300A and C505A, that shift Drp1 polymerization equilibria in opposite directions, we demonstrate that dimers, and not multimers, potentiate the reassembly and reorganization of Drp1 for mitochondrial membrane remodeling both in vitro and in vivo.
PMCID: PMC4055269  PMID: 24790094
25.  Content-based histopathology image retrieval using CometCloud 
BMC Bioinformatics  2014;15(1):287.
The development of digital imaging technology is creating extraordinary levels of accuracy that provide support for improved reliability in different aspects of the image analysis, such as content-based image retrieval, image segmentation, and classification. This has dramatically increased the volume and rate at which data are generated. Together these facts make querying and sharing non-trivial and render centralized solutions unfeasible. Moreover, in many cases this data is often distributed and must be shared across multiple institutions requiring decentralized solutions. In this context, a new generation of data/information driven applications must be developed to take advantage of the national advanced cyber-infrastructure (ACI) which enable investigators to seamlessly and securely interact with information/data which is distributed across geographically disparate resources. This paper presents the development and evaluation of a novel content-based image retrieval (CBIR) framework. The methods were tested extensively using both peripheral blood smears and renal glomeruli specimens. The datasets and performance were evaluated by two pathologists to determine the concordance.
The CBIR algorithms that were developed can reliably retrieve the candidate image patches exhibiting intensity and morphological characteristics that are most similar to a given query image. The methods described in this paper are able to reliably discriminate among subtle staining differences and spatial pattern distributions. By integrating a newly developed dual-similarity relevance feedback module into the CBIR framework, the CBIR results were improved substantially. By aggregating the computational power of high performance computing (HPC) and cloud resources, we demonstrated that the method can be successfully executed in minutes on the Cloud compared to weeks using standard computers.
In this paper, we present a set of newly developed CBIR algorithms and validate them using two different pathology applications, which are regularly evaluated in the practice of pathology. Comparative experimental results demonstrate excellent performance throughout the course of a set of systematic studies. Additionally, we present and evaluate a framework to enable the execution of these algorithms across distributed resources. We show how parallel searching of content-wise similar images in the dataset significantly reduces the overall computational time to ensure the practical utility of the proposed CBIR algorithms.
PMCID: PMC4161917  PMID: 25155691
Histopathology; Digital pathology; Content-based image retrieval; High performance computing

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