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1.  Peginterferon plus weight-based ribavirin for treatment-naïve hepatitis C virus genotype 2 patients not achieving rapid virologic response: a randomized trial 
Scientific Reports  2015;5:11710.
Hepatitis C virus genotype 2 (HCV-2) slow responders poorly respond to 24 weeks of peginterferon (Peg-IFN) plus ribavirin (RBV). We evaluated the efficacy of extended 48-week regimen and the role of interleukin-28B (IL-28B) genotype in this clinical setting. Treatment-naïve HCV-2 patients not achieving rapid virologic response (RVR) by Peg-IFN alfa-2a 180 μg/week plus weight-based RBV (1,000–1,200 mg/day, cutoff body weight of 75 kg) were randomly assigned to receive a total duration of 48 (n = 94) or 24 (n = 93) weeks of therapy. The primary endpoint was sustained virologic response (SVR). Baseline patient characteristics to predict SVR were analyzed. Patients receiving 48 weeks of treatment had a greater SVR rate than those receiving 24 weeks of treatment (70.2% versus 46.2%, P = 0.001). Compared to patients treated for 24 weeks, the SVR rate in those treated for 48 weeks increased by 10.9% [95% CI: −5.9% to 27.7%] and 65.6% [95% CI: 44.5% to 86.7%] if they had IL-28B rs8099917 TT genotype, and GT/GG genotype, respectively (interaction P = 0.002). In conclusion, 48-week treatment with Peg-IFN plus weight-based RBV provides a greater SVR rate than 24-week treatment in treatment-naïve HCV-2 patients with unfavorable IL-28B genotypes who fail to achieve RVR.
doi:10.1038/srep11710
PMCID: PMC4486927  PMID: 26130141
2.  The vascularized periosteum flap as novel tissue engineering model for repair of cartilage defects 
Periosteum is a promising tissue engineering scaffold in research of cartilage repair; so far however, periosteum transfers have not been realized successfully because of insufficient nourishment of the graft. In a translational approach we, for the first time, designed a vascularized periosteum flap as ‘independent’ biomaterial with its own blood supply to address this problem and to reconstruct circumscript cartilage defects. In six 3-month-old New Zealand rabbits, a critical size cartilage defect of the medial femur condyle was created and covered by a vascularized periosteum flap pedicled on the saphenous vessels. After 28 days, formation of newly built cartilage was assessed macroscopically, histologically and qualitatively via biomechanical compression testing, as well as on molecular biological level via immunohistochemistry. All wounds healed completely, all joints were stable and had full range of motion. All flaps survived and were perfused through their pulsating pedicles. They showed a stable attachment to the bone, although partially incomplete adherence. Hyaline cartilage with typical columnar cell distribution and positive Collagen II staining was formed in the transferred flaps. Biomechanical testing revealed a significantly higher maximum load than the positive control, but a low elasticity. This study proved that vascularization of the periosteum flap is the essential step for flap survival and enables the flap to transform into cartilage. Reconstruction of circumscript cartilage defects seems to be possible. Although these are the first results out of a pilot project, this technique, we believe, can have a wide range of potential applications and high relevance in the clinical field.
doi:10.1111/jcmm.12485
PMCID: PMC4459843  PMID: 25754287
cartilage repair; tissue engineering; vascularized periosteum flap; translational research; osteoarthritis; cartilage defects
3.  Nonautophagic cytoplasmic vacuolation death induction in human PC-3M prostate cancer by curcumin through reactive oxygen species -mediated endoplasmic reticulum stress 
Scientific Reports  2015;5:10420.
The antiapoptotic and antiautophagic abilities of cancer cells constitute a major challenge for anticancer drug treatment. Strategies for triggering nonapoptotic or nonautophagic cell death may improve therapeutic efficacy against cancer. Curcumin has been reported to exhibit cancer chemopreventive properties. Herein, we report that curcumin induced apoptosis in LNCaP, DU145, and PC-3 cells but triggered extensive cytoplasmic vacuolation in PC-3M cells. Electron microscopic images showed that the vacuoles lacked intracellular organelles and were derived from the endoplasmic reticulum (ER). Moreover, curcumin-induced vacuolation was not reversed by an apoptosis- or autophagy-related inhibitor, suggesting that vacuolation-mediated cell death differs from classical apoptotic and autophagic cell death. Mechanistic investigations revealed that curcumin treatment upregulated the ER stress markers CHOP and Bip/GRP78 and the autophagic marker LC3-II. In addition, curcumin induced ER stress by triggering ROS generation, which was supported by the finding that treating cells with the antioxidant NAC alleviated curcumin-mediated ER stress and vacuolation-mediated death. An in vivo PC-3M orthotopic prostate cancer model revealed that curcumin reduced tumor growth by inducing ROS production followed by vacuolation-mediated cell death. Overall, our results indicated that curcumin acts as an inducer of ROS production, which leads to nonapoptotic and nonautophagic cell death via increased ER stress.
doi:10.1038/srep10420
PMCID: PMC4445067  PMID: 26013662
4.  Regulators Associated with Clinical Outcomes Revealed by DNA Methylation Data in Breast Cancer 
PLoS Computational Biology  2015;11(5):e1004269.
The regulatory architecture of breast cancer is extraordinarily complex and gene misregulation can occur at many levels, with transcriptional malfunction being a major cause. This dysfunctional process typically involves additional regulatory modulators including DNA methylation. Thus, the interplay between transcription factor (TF) binding and DNA methylation are two components of a cancer regulatory interactome presumed to display correlated signals. As proof of concept, we performed a systematic motif-based in silico analysis to infer all potential TFs that are involved in breast cancer prognosis through an association with DNA methylation changes. Using breast cancer DNA methylation and clinical data derived from The Cancer Genome Atlas (TCGA), we carried out a systematic inference of TFs whose misregulation underlie different clinical subtypes of breast cancer. Our analysis identified TFs known to be associated with clinical outcomes of p53 and ER (estrogen receptor) subtypes of breast cancer, while also predicting new TFs that may also be involved. Furthermore, our results suggest that misregulation in breast cancer can be caused by the binding of alternative factors to the binding sites of TFs whose activity has been ablated. Overall, this study provides a comprehensive analysis that links DNA methylation to TF binding to patient prognosis.
Author Summary
DNA methylation is a ubiquitous and simple covalent modification that occurs directly on genetic material whereby a simple methyl group (CH3) is attached to Cytosine nucleotides in the context of CpG sites. Modifications of these sites have been postulated to function in gene regulation, potentially via interactions with transcription factors. In this study, we hypothesized that DNA methylation signals contain valuable information that can help infer transcription factors that may be associated with a given disease. Here, we utilize the vast repository of breast cancer data that is available in the public domain, and which contains a rich resource for DNA methylation and clinical data on breast cancer patients. In this guilt-by-association analysis, we postulated that conserved transcription factor binding motifs that are statistically enriched in regions near methylated CpG sites that are correlated with breast cancer patient survival would suggest that their cognate transcription factors would play a role in the initiation, growth, metastasis, or even suppression of the tumor. This integrative approach supports the claim that DNA methylation profiling of patient tumors in the clinic may contain valuable information that can guide the development of treatment regimens for individual patients; thus contributing to the progression of precision medicine.
doi:10.1371/journal.pcbi.1004269
PMCID: PMC4440643  PMID: 25996148
5.  Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection 
PLoS ONE  2015;10(5):e0126121.
Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.
doi:10.1371/journal.pone.0126121
PMCID: PMC4418827  PMID: 25938761
6.  Osteopontin inhibits HIF-2α mRNA expression in osteoarthritic chondrocytes 
The aim of the present study was to investigate the in vitro effect of osteopontin (OPN) on the expression of hypoxia-inducible factor-2α (HIF-2α) in chondrocytes and the role of OPN in osteoarthritis (OA). Cartilage was purified from the tibial surfaces of patients with OA of the knee and cultured in vitro to obtain chondrocytes. Recombinant human OPN (rhOPN) and OPN small interfering RNA (siRNA) were used to treat the chondrocytes, and the changes in the expression levels of the HIF-2α gene were measured. An anti-CD44 blocking monoclonal antibody (mAb) was used to determine the probable ligand-receptor interactions. Reverse transcription-quantitative polymerase chain reaction assays were designed and validated with SYBR® Green dyes for the simultaneous quantification of the mRNA expression levels of OPN and HIF-2α. The mRNA expression level of HIF-2α was markedly decreased in the rhOPN-treated group compared with that in the control group; by contrast, OPN siRNA increased HIF-2α gene expression. CD44 blocking mAb suppressed the inhibitory effect of OPN on HIF-2α mRNA expression. The results of the present study suggest that OPN may play a protective role in OA by inhibiting HIF-2α gene expression in osteoarthritic chondrocytes through CD44 interaction.
doi:10.3892/etm.2015.2434
PMCID: PMC4473661  PMID: 26136997
chondrocyte; CD44; HIF-2α; osteopontin; osteoarthritis
7.  Loregic: A Method to Characterize the Cooperative Logic of Regulatory Factors 
PLoS Computational Biology  2015;11(4):e1004132.
The topology of the gene-regulatory network has been extensively analyzed. Now, given the large amount of available functional genomic data, it is possible to go beyond this and systematically study regulatory circuits in terms of logic elements. To this end, we present Loregic, a computational method integrating gene expression and regulatory network data, to characterize the cooperativity of regulatory factors. Loregic uses all 16 possible two-input-one-output logic gates (e.g. AND or XOR) to describe triplets of two factors regulating a common target. We attempt to find the gate that best matches each triplet’s observed gene expression pattern across many conditions. We make Loregic available as a general-purpose tool (github.com/gersteinlab/loregic). We validate it with known yeast transcription-factor knockout experiments. Next, using human ENCODE ChIP-Seq and TCGA RNA-Seq data, we are able to demonstrate how Loregic characterizes complex circuits involving both proximally and distally regulating transcription factors (TFs) and also miRNAs. Furthermore, we show that MYC, a well-known oncogenic driving TF, can be modeled as acting independently from other TFs (e.g., using OR gates) but antagonistically with repressing miRNAs. Finally, we inter-relate Loregic’s gate logic with other aspects of regulation, such as indirect binding via protein-protein interactions, feed-forward loop motifs and global regulatory hierarchy.
Author Summary
Gene expression is controlled by various gene regulatory factors. Those factors work cooperatively forming a complex regulatory circuit genome wide. Corruptions of regulatory cooperativity may lead to abnormal gene expression activity such as cancer. Traditional experimental methods, however, can only identify small-scale regulatory activity. Thus, to systematically understand the cooperativity between and among different types of regulatory factors, we need the efficient and systematic computational methods. Regulatory circuits have been suggested to behave analogously to the electronic circuits in which a wide variety of electronic elements work coordinately to function correctly. Recently, an increasing amount of next generation sequencing data provides a great resource to study regulatory activity. Thus, we developed a general-purpose computational method using logic-circuit models from electronics and applied it to a human leukemia dataset, identifying the genome-wide cooperativity of transcription factors and microRNAs.
doi:10.1371/journal.pcbi.1004132
PMCID: PMC4401777  PMID: 25884877
8.  Effect of estrogen receptor α binding on functional DNA methylation in breast cancer 
Epigenetics  2014;9(4):523-532.
Epigenetic modifications introduce an additional layer of regulation that drastically expands the instructional capability of the human genome. The regulatory consequences of DNA methylation is context dependent; it can induce, enhance, and suppress gene expression, or have no effect on gene regulation. Therefore, it is essential to account for the genomic location of its occurrence and the protein factors it associates with to improve our understanding of its function and effects. Here, we use ENCODE ChIP-seq and DNase I hypersensitivity data, along with large-scale breast cancer genomic data from The Cancer Genome Atlas (TCGA) to computationally dissect the intricacies of DNA methylation in regulation of cancer transcriptomes. In particular, we identified a relationship between estrogen receptor α (ERα) activity and DNA methylation patterning in breast cancer. We found compelling evidence that methylation status of DNA sequences at ERα binding sites is tightly coupled with ERα activity. Furthermore, we predicted several transcription factors including FOXA1, GATA1, and SUZ12 to be associated with breast cancer by examining the methylation status of their binding sites in breast cancer. Lastly, we determine that methylated CpGs highly correlated with gene expression are enriched in regions 1kb or more downstream of TSSs, suggesting more significant regulatory roles for CpGs distal to gene TSSs. Our study provides novel insights into the role of ERα in breast cancers.
doi:10.4161/epi.27688
PMCID: PMC4121363  PMID: 24434785
ChIP-seq; DNase I hypersensitivity; breast cancer; differential gene expression; differential methylation; estrogen receptor α; transcription factor
9.  An approach for determining and measuring network hierarchy applied to comparing the phosphorylome and the regulome 
Genome Biology  2015;16(1):63.
Many biological networks naturally form a hierarchy with a preponderance of downward information flow. In this study, we define a score to quantify the degree of hierarchy in a network and develop a simulated-annealing algorithm to maximize the hierarchical score globally over a network. We apply our algorithm to determine the hierarchical structure of the phosphorylome in detail and investigate the correlation between its hierarchy and kinase properties. We also compare it to the regulatory network, finding that the phosphorylome is more hierarchical than the regulome.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0624-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s13059-015-0624-2
PMCID: PMC4404648  PMID: 25880651
10.  Integrative analysis of survival-associated gene sets in breast cancer 
BMC Medical Genomics  2015;8:11.
Background
Patient gene expression information has recently become a clinical feature used to evaluate breast cancer prognosis. The emergence of prognostic gene sets that take advantage of these data has led to a rich library of information that can be used to characterize the molecular nature of a patient’s cancer. Identifying robust gene sets that are consistently predictive of a patient’s clinical outcome has become one of the main challenges in the field.
Methods
We inputted our previously established BASE algorithm with patient gene expression data and gene sets from MSigDB to develop the gene set activity score (GSAS), a metric that quantitatively assesses a gene set’s activity level in a given patient. We utilized this metric, along with patient time-to-event data, to perform survival analyses to identify the gene sets that were significantly correlated with patient survival. We then performed cross-dataset analyses to identify robust prognostic gene sets and to classify patients by metastasis status. Additionally, we created a gene set network based on component gene overlap to explore the relationship between gene sets derived from MSigDB. We developed a novel gene set based on this network’s topology and applied the GSAS metric to characterize its role in patient survival.
Results
Using the GSAS metric, we identified 120 gene sets that were significantly associated with patient survival in all datasets tested. The gene overlap network analysis yielded a novel gene set enriched in genes shared by the robustly predictive gene sets. This gene set was highly correlated to patient survival when used alone. Most interestingly, removal of the genes in this gene set from the gene pool on MSigDB resulted in a large reduction in the number of predictive gene sets, suggesting a prominent role for these genes in breast cancer progression.
Conclusions
The GSAS metric provided a useful medium by which we systematically investigated how gene sets from MSigDB relate to breast cancer patient survival. We used this metric to identify predictive gene sets and to construct a novel gene set containing genes heavily involved in cancer progression.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-015-0086-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12920-015-0086-0
PMCID: PMC4359519  PMID: 25881247
Breast cancer; Gene sets; Prognosis; Survival prediction
11.  Danggui Buxue Tang Inhibits 2,4-Dinitrochlorobenzene: Induced Atopic Dermatitis in Mice 
Danggui Buxue Tang (DBT) is a herbal decoction that has been used in Chinese medicine to enhance qi and blood circulation. Previously, we found that DBT can suppress allergy-related asthma in mice, leading us to hypothesize that DBT might ameliorate allergy disease. In this study, we evaluated whether DBT can attenuate atopic dermatitis (AD) symptoms and have an anti-inflammatory effect on AD-like mice. The dorsal skin of female mice was shaved and sensitized cutaneously (skin smear) with 1-chloro-2,4-dinitrobenzene. Mice were then given various doses of DBT from days 14 to 29 cutaneously. DBT treatment suppressed ear swelling and skin inflammation and decreased mast cell and eosinophil infiltration into skin and ear tissue. DBT also inhibited levels of IgE and Th2-associated cytokine levels in serum. These results demonstrate that cutaneous administration of DBT reduced the development of AD-like skin lesions in mice.
doi:10.1155/2015/672891
PMCID: PMC4377538  PMID: 25861366
12.  Association of Gamma-Aminobutyric Acid A Receptor α2 Gene (GABRA2) with Alcohol Use Disorder 
Neuropsychopharmacology  2013;39(4):907-918.
Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in mammalian brain. GABA receptor are involved in a number of complex disorders, including substance abuse. No variants of the commonly studied GABA receptor genes that have been associated with substance dependence have been determined to be functional or pathogenic. To reconcile the conflicting associations with substance dependence traits, we performed a meta-analysis of variants in the GABAA receptor genes (GABRB2, GABRA6, GABRA1, and GABRG2 on chromosome 5q and GABRA2 on chromosome 4p12) using genotype data from 4739 cases of alcohol, opioid, or methamphetamine dependence and 4924 controls. Then, we combined the data from candidate gene association studies in the literature with two alcohol dependence (AD) samples, including 1691 cases and 1712 controls from the Study of Addiction: Genetics and Environment (SAGE), and 2644 cases and 494 controls from our own study. Using a Bonferroni-corrected threshold of 0.007, we found strong associations between GABRA2 and AD (P=9 × 10−6 and odds ratio (OR) 95% confidence interval (CI)=1.27 (1.15, 1.4) for rs567926, P=4 × 10−5 and OR=1.21 (1.1, 1.32) for rs279858), and between GABRG2 and both dependence on alcohol and dependence on heroin (P=0.0005 and OR=1.22 (1.09, 1.37) for rs211014). Significant association was also observed between GABRA6 rs3219151 and AD. The GABRA2 rs279858 association was observed in the SAGE data sets with a combined P of 9 × 10−6 (OR=1.17 (1.09, 1.26)). When all of these data sets, including our samples, were meta-analyzed, associations of both GABRA2 single-nucleotide polymorphisms remained (for rs567926, P=7 × 10−5 (OR=1.18 (1.09, 1.29)) in all the studies, and P=8 × 10−6 (OR=1.25 (1.13, 1.38)) in subjects of European ancestry and for rs279858, P=5 × 10−6 (OR=1.18 (1.1, 1.26)) in subjects of European ancestry. Findings from this extensive meta-analysis of five GABAA receptor genes and substance abuse support their involvement (with the best evidence for GABRA2) in the pathogenesis of AD. Further replications with larger samples are warranted.
doi:10.1038/npp.2013.291
PMCID: PMC3924525  PMID: 24136292
addiction and substance abuse; alcohol and alcoholism; association; biological psychiatry; GABA; gamma-aminobutyric acid receptor; meta-analysis; addiction; gamma-aminobutyric acid receptor; association; meta-analysis; susceptibility gene
13.  Comparative Analysis of the Transcriptome across Distant Species 
Gerstein, Mark B. | Rozowsky, Joel | Yan, Koon-Kiu | Wang, Daifeng | Cheng, Chao | Brown, James B. | Davis, Carrie A | Hillier, LaDeana | Sisu, Cristina | Li, Jingyi Jessica | Pei, Baikang | Harmanci, Arif O. | Duff, Michael O. | Djebali, Sarah | Alexander, Roger P. | Alver, Burak H. | Auerbach, Raymond | Bell, Kimberly | Bickel, Peter J. | Boeck, Max E. | Boley, Nathan P. | Booth, Benjamin W. | Cherbas, Lucy | Cherbas, Peter | Di, Chao | Dobin, Alex | Drenkow, Jorg | Ewing, Brent | Fang, Gang | Fastuca, Megan | Feingold, Elise A. | Frankish, Adam | Gao, Guanjun | Good, Peter J. | Guigó, Roderic | Hammonds, Ann | Harrow, Jen | Hoskins, Roger A. | Howald, Cédric | Hu, Long | Huang, Haiyan | Hubbard, Tim J. P. | Huynh, Chau | Jha, Sonali | Kasper, Dionna | Kato, Masaomi | Kaufman, Thomas C. | Kitchen, Robert R. | Ladewig, Erik | Lagarde, Julien | Lai, Eric | Leng, Jing | Lu, Zhi | MacCoss, Michael | May, Gemma | McWhirter, Rebecca | Merrihew, Gennifer | Miller, David M. | Mortazavi, Ali | Murad, Rabi | Oliver, Brian | Olson, Sara | Park, Peter J. | Pazin, Michael J. | Perrimon, Norbert | Pervouchine, Dmitri | Reinke, Valerie | Reymond, Alexandre | Robinson, Garrett | Samsonova, Anastasia | Saunders, Gary I. | Schlesinger, Felix | Sethi, Anurag | Slack, Frank J. | Spencer, William C. | Stoiber, Marcus H. | Strasbourger, Pnina | Tanzer, Andrea | Thompson, Owen A. | Wan, Kenneth H. | Wang, Guilin | Wang, Huaien | Watkins, Kathie L. | Wen, Jiayu | Wen, Kejia | Xue, Chenghai | Yang, Li | Yip, Kevin | Zaleski, Chris | Zhang, Yan | Zheng, Henry | Brenner, Steven E. | Graveley, Brenton R. | Celniker, Susan E. | Gingeras, Thomas R | Waterston, Robert
Nature  2014;512(7515):445-448.
doi:10.1038/nature13424
PMCID: PMC4155737  PMID: 25164755
14.  Comparative analysis of regulatory information and circuits across distant species 
Nature  2014;512(7515):453-456.
Summary
Despite the large evolutionary distances, metazoan species show remarkable commonalities, which has helped establish fly and worm as model organisms for human biology1,2. Although studies of individual elements and factors have explored similarities in gene regulation, a large-scale comparative analysis of basic principles of transcriptional regulatory features is lacking. We mapped the genome-wide binding locations of 165 human, 93 worm, and 52 fly transcription-regulatory factors (RFs) generating a total of 1,019 data sets from diverse cell-types, developmental stages, or conditions in the three species, of which 498 (48.9%) are presented here for the first time. We find that structural properties of regulatory networks are remarkably conserved and that orthologous RF families recognize similar binding motifs in vivo and show some similar co-associations. Our results suggest that gene-regulatory properties previously observed for individual factors are general principles of metazoan regulation that are remarkably well-preserved despite extensive functional divergence of individual network connections. The comparative maps of regulatory circuitry provided here will drive an improved understanding in the regulatory underpinnings of model organism biology and how these relate to human biology, development, and disease.
doi:10.1038/nature13668
PMCID: PMC4336544  PMID: 25164757
Transcription Factor; Regulatory Information; Gene Regulation; Single Nucleotide Polymorphisms; ChIP-seq
15.  Effect of osteopontin on the mRNA expression of ADAMTS4 and ADAMTS5 in chondrocytes from patients with knee osteoarthritis 
Previous studies have demonstrated that osteopontin (OPN) levels are elevated in the synovial fluid and articular cartilage, and are associated with the severity of knee osteoarthritis (OA). However, the role of OPN in the pathogenesis of OA has yet to be elucidated. The present study aimed to investigate the effects of OPN on the expression of the aggrecanases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5, in human OA chondrocytes, as they serve a key function in aggrecan degradation. Human OA chondrocytes were obtained from the knees of 16 patients with OA, and subsequently cultured in a monolayer. The chondrocytes were divided into three groups, which included the control (no treatment), N-OPN (treated with 100 ng/ml OPN, the normal circulating OPN concentration) and the H-OPN groups (treated with 1 µg/ml OPN, a high OPN concentration). Reverse transcription-quantitative polymerase chain reaction was performed to quantify the relative mRNA expression levels of ADAMTS4, ADAMTS5 and aggrecan in the chondrocytes. The mRNA expression levels of ADAMTS4 were significantly reduced in the N-OPN and H-OPN groups when compared with the control group (P<0.0001). In addition, the mRNA expression levels of ADAMTS4 were lower in the H-OPN group when compared with the N-OPN group (P<0.001). However, no statistically significant difference was observed in the relative mRNA expression levels of ADAMTS5 among the three groups (P>0.05). Furthermore, the mRNA expression levels of aggrecan were higher in the N-OPN and H-OPN groups when compared with the control group (P<0.0001), and a statistically significant difference was observed between the N-OPN and H-OPN groups with regard to the mRNA expression of aggrecan (P<0.0001). These results demonstrated that OPN may exert a protective effect in human OA chondrocytes against aggrecan degradation by suppressing the expression of ADAMTS4.
doi:10.3892/etm.2015.2310
PMCID: PMC4471803  PMID: 26136925
osteopontin; osteoarthritis; a disintegrin and metalloproteinase with thrombospondin motifs 4; a disintegrin and metalloproteinase with thrombospondin motifs 5; aggrecan
16.  SATB1 Overexpression Regulates the Development and Progression in Bladder Cancer through EMT 
PLoS ONE  2015;10(2):e0117518.
The global gene regulator Special AT-rich sequence-binding protein-1 (SATB1) has been reported to induce EMT-like changes and be associated with poor clinical outcome in several cancers. This study aims to evaluate whether SATB1 affects the biological behaviors of bladder transitional cell carcinoma (BTCC) and further elucidate if this effect works through an epithelial-mesenchymal transition (EMT) pathway. The expression of SATB1, E-cadherin (epithelial markers), vimentin (mesenchymal markers) in BTCC tissues and adjacent noncancerous tissues, as well as in two cell lines of bladder cancer were investigated. Whether the SATB1 expression is associated with clinicopathological factors or not was statistically analyzed. Cell invasion and migration, cell cycle, cell proliferation and apoptosis were evaluated in SATB1 knockdown and overexpressed cell lines. Our results showed that the expression of SATB1 was remarkably up-regulated both in BTCC tissues and in bladder cancer cell lines with high potential of metastasis. The results were also associated with EMT markers and poor prognosis of BTCC patients. Moreover, SATB1 induced EMT processes through downregulation of E-cadherin, upregulation of E-cadherin repressors (Snail, Slug and vimentin). SATB1 also promoted cell cycle progression, cell proliferation, cell invasion and cell migration, but did not alter cell survival. In conclusion, our results suggest that SATB1 plays a crucial role in the progression of bladder cancer by regulating genes controlling EMT processes. Further, it may be a novel therapeutic target for aggressive bladder cancers.
doi:10.1371/journal.pone.0117518
PMCID: PMC4338074  PMID: 25706386
17.  Huntingtin-Associated Protein 1 Interacts with Breakpoint Cluster Region Protein to Regulate Neuronal Differentiation 
PLoS ONE  2015;10(2):e0116372.
Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.
doi:10.1371/journal.pone.0116372
PMCID: PMC4324908  PMID: 25671650
18.  Protein mistranslation protects bacteria against oxidative stress 
Nucleic Acids Research  2015;43(3):1740-1748.
Accurate flow of genetic information from DNA to protein requires faithful translation. An increased level of translational errors (mistranslation) has therefore been widely considered harmful to cells. Here we demonstrate that surprisingly, moderate levels of mistranslation indeed increase tolerance to oxidative stress in Escherichia coli. Our RNA sequencing analyses revealed that two antioxidant genes katE and osmC, both controlled by the general stress response activator RpoS, were upregulated by a ribosomal error-prone mutation. Mistranslation-induced tolerance to hydrogen peroxide required rpoS, katE and osmC. We further show that both translational and post-translational regulation of RpoS contribute to peroxide tolerance in the error-prone strain, and a small RNA DsrA, which controls translation of RpoS, is critical for the improved tolerance to oxidative stress through mistranslation. Our work thus challenges the prevailing view that mistranslation is always detrimental, and provides a mechanism by which mistranslation benefits bacteria under stress conditions.
doi:10.1093/nar/gku1404
PMCID: PMC4330365  PMID: 25578967
19.  APPLICATIONS OF BIOINFORMATICS TO NON-CODING RNAS IN THE ERA OF NEXT-GENERATION SEQUENCING 
The human genome encodes a large number of non-coding RNAs, which employ a new and crucial layer of biological regulation in addition to proteins. Technical advancement in recent years, particularly, the wide application of next generation sequencing analysis, provide an unprecedented opportunity to identify new non-coding RNAs and investigate their functions and regulatory mechanisms. The aim of this workshop is to bring together experimental and computational biologist to exchange ideas on non-coding RNA studies.
PMCID: PMC3900277  PMID: 24297566
20.  Dendritic cells respond to nasopharygeal carcinoma cells through annexin A2-recognizing DC-SIGN 
Oncotarget  2014;6(1):159-170.
Dendritic cells (DCs) play an essential role in immunity and are used in cancer immunotherapy. However, these cells can be tuned by tumors with immunosuppressive responses. DC-specific intercellular adhesion molecule 3-Grabbing Nonintegrin (DC-SIGN), a C-type lectin expressed on DCs, recognizes certain carbohydrate structures which can be found on cancer cells. Nasopharyngeal carcinoma (NPC) is an epithelial cell-derived malignant tumor, in which immune response remains unclear. This research is to reveal the molecular link on NPC cells that induces the immunosuppressive responses in DCs. In this article, we report identification of annexin A2 (ANXA2) on NPC cells as a ligand for DC-SIGN on DCs. N-linked mannose-rich glycan on ANXA2 may mediate the interaction. ANXA2 was abundantly expressed in NPC, and knockdown of ANXA2 suppressed NPC xenograft in mice, suggesting a crucial role of ANXA2 in NPC growth. Interaction with NPC cells caused DC-SIGN activation in DCs. Consequently DC maturation and the proinflammatory interleukin (IL)-12 production were inhibited, and the immunosuppressive IL-10 production was promoted. Blockage of either DC-SIGN or ANXA2 eliminated the production of IL-10 from DCs. This report suggests that suppression of ANXA2 at its expression or glycosylation on NPC may improve DC-mediated immunotherapy for the tumor.
PMCID: PMC4381585  PMID: 25402728
dendritic cell; DC-SIGN; NPC; annexin A2; IL-10; immunosuppression
21.  E2F4 regulatory program predicts patient survival prognosis in breast cancer 
Introduction
Genetic and molecular signatures have been incorporated into cancer prognosis prediction and treatment decisions with good success over the past decade. Clinically, these signatures are usually used in early-stage cancers to evaluate whether they require adjuvant therapy following surgical resection. A molecular signature that is prognostic across more clinical contexts would be a useful addition to current signatures.
Methods
We defined a signature for the ubiquitous tissue factor, E2F4, based on its shared target genes in multiple tissues. These target genes were identified by chromatin immunoprecipitation sequencing (ChIP-seq) experiments using a probabilistic method. We then computationally calculated the regulatory activity score (RAS) of E2F4 in cancer tissues, and examined how E2F4 RAS correlates with patient survival.
Results
Genes in our E2F4 signature were 21-fold more likely to be correlated with breast cancer patient survival time compared to randomly selected genes. Using eight independent breast cancer datasets containing over 1,900 unique samples, we stratified patients into low and high E2F4 RAS groups. E2F4 activity stratification was highly predictive of patient outcome, and our results remained robust even when controlling for many factors including patient age, tumor size, grade, estrogen receptor (ER) status, lymph node (LN) status, whether the patient received adjuvant therapy, and the patient’s other prognostic indices such as Adjuvant! and the Nottingham Prognostic Index scores. Furthermore, the fractions of samples with positive E2F4 RAS vary in different intrinsic breast cancer subtypes, consistent with the different survival profiles of these subtypes.
Conclusions
We defined a prognostic signature, the E2F4 regulatory activity score, and showed it to be significantly predictive of patient outcome in breast cancer regardless of treatment status and the states of many other clinicopathological variables. It can be used in conjunction with other breast cancer classification methods such as Oncotype DX to improve clinical outcome prediction.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0486-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0486-7
PMCID: PMC4303196  PMID: 25440089
22.  Mechanotransduction in intervertebral discs 
Mechanotransduction plays a critical role in intracellular functioning—it allows cells to translate external physical forces into internal biochemical activities, thereby affecting processes ranging from proliferation and apoptosis to gene expression and protein synthesis in a complex web of interactions and reactions. Accordingly, aberrant mechanotransduction can either lead to, or be a result of, a variety of diseases or degenerative states. In this review, we provide an overview of mechanotransduction in the context of intervertebral discs, with a focus on the latest methods of investigating mechanotransduction and the most recent findings regarding the means and effects of mechanotransduction in healthy and degenerative discs. We also provide some discussion of potential directions for future research and treatments.
doi:10.1111/jcmm.12377
PMCID: PMC4302640  PMID: 25267492
mechanotransduction; intervertebral discs; physical forces; gene expression; biochemical activities; protein synthesis
23.  Rho-kinase inhibition is involved in the activation of PI3-kinase/Akt during ischemic-preconditioning-induced cardiomyocyte apoptosis 
We and others have reported that Rho-kinase plays an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. Studies also have demonstrated that the activation of Rho-kinase was reversed in ischemic preconditioning (IPC). This study aimed to explain the mechanism of Rho-kinase-mediated cardiomyocyte apoptosis increased in I/R and reversed in IPC. Materials and methods: Studies were performed with female Wistar rats. The I/R rats were created by ligating the left anterior descending branch (LAD) for 30 min and releasing the ligature for 180 min. The IPC rats underwent IPC (two cycles of 5 min ligation of the LAD and 5 min reflow) before I/R. Results: Ischemia followed by reperfusion caused a significant increase in Rho-kinase and a decrease in Akt phosphorylation. Administration of fasudil, an inhibitor of Rho-kinase, decreased myocardial infarction size and cardiomyocyte apoptosis and increased Akt activation. IPC also caused the reduced Rho-kinase activity and cardiomyocyte apoptosis and a significant increase in Akt activity (P<0.05 vs I/R). Conclusion: Rho-kinase inhibition by IPC leads to reduced cardiomyocyte apoptosis may be mediated by activation of PI3-kinase/Akt.
PMCID: PMC4276178  PMID: 25550920
Rho-kinase; heart ischemia/reperfusion; ischemic preconditioning; apoptosis; PI3-kinase/Akt
24.  Peroxisome Proliferator-Activated Receptor γ Deficiency in T Cells Accelerates Chronic Rejection by Influencing the Differentiation of CD4+ T Cells and Alternatively Activated Macrophages 
PLoS ONE  2014;9(11):e112953.
Background
In a previous study, activation of the peroxisome proliferator–activated receptor γ (PPARγ) inhibited chronic cardiac rejection. However, because of the complexity of chronic rejection and the fact that PPARγ is widely expressed in immune cells, the mechanism of the PPARγ - induced protective effect was unclear.
Materials and Methods
A chronic rejection model was established using B6.C-H-2bm12KhEg (H-2bm12) mice as donors, and MHC II-mismatched T-cell-specific PPARγ knockout mice or wild type (WT) littermates as recipients. The allograft lesion was assessed by histology and immunohistochemistry. T cells infiltrates in the allograft were isolated, and cytokines and subpopulations were detected using cytokine arrays and flow cytometry. Transcription levels in the allograft were measured by RT-PCR. In vitro, the T cell subset differentiation was investigated after culture in various polarizing conditions. PPARγ-deficient regularory T cells (Treg) were cocultured with monocytes to test their ability to induce alternatively activated macrophages (AAM).
Results
T cell-specific PPARγ knockout recipients displayed reduced cardiac allograft survival and an increased degree of pathology compared with WT littermates. T cell-specific PPARγ knockout resulted in more CD4+ T cells infiltrating into the allograft and altered the Th1/Th2 and Th17/Treg ratios. The polarization of AAM was also reduced by PPARγ deficiency in T cells through the action of Th2 and Treg. PPARγ-deficient T cells eliminated the pioglitazone-induced polarization of AAM and reduced allograft survival.
Conclusions
PPARγ-deficient T cells influenced the T cell subset and AAM polarization in chronic allograft rejection. The mechanism of PPARγ activation in transplantation tolerance could yield a novel treatment without side effects.
doi:10.1371/journal.pone.0112953
PMCID: PMC4226585  PMID: 25383620
25.  ChIP-seq and In Vivo Transcriptome Analyses of the Aspergillus fumigatus SREBP SrbA Reveals a New Regulator of the Fungal Hypoxia Response and Virulence 
PLoS Pathogens  2014;10(11):e1004487.
The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence.
Author Summary
Despite improvements in diagnostics and antifungal drug treatments, mortality rates from invasive mold infections remain high. Defining the fungal adaptation and growth mechanisms at the infection site microenvironment is one research focus that is expected to improve treatment of established invasive fungal infections. The Aspergillus fumigatus transcription factor SrbA is a major regulator of the fungal response to hypoxia found at sites of invasive fungal growth in vivo. In this study, new insights into how SrbA mediates hypoxia adaptation and virulence were revealed through identification of direct transcriptional targets of SrbA under hypoxic conditions. A major novel finding from these studies is the identification of a critical role in fungal hypoxia adaptation and virulence of an SrbA target gene, srbB, which is also in the SREBP family. SrbB plays a major role in regulation of heme biosynthesis and carbohydrate metabolism early in the response to hypoxia. The discovery of SrbA-dependent regulation of srbB gene expression, and the target genes they regulate opens new avenues to understand how SREBPs and their target genes mediate adaptation to the in vivo infection site microenvironment and responses to current antifungal therapies.
doi:10.1371/journal.ppat.1004487
PMCID: PMC4223079  PMID: 25375670

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