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1.  UBE3C Promotes Growth and Metastasis of Renal Cell Carcinoma via Activating Wnt/β-Catenin Pathway 
PLoS ONE  2015;10(2):e0115622.
Renal cell carcinoma (RCC) is the most common primary malignancy of the kidney and one of the most lethal genitourinary malignancies. Clear-cell renal cell carcinoma (ccRCC) has an extremely poor prognosis because of a high potential for tumor growth, vascular invasion, metastasis and recurrence. Unfortunately, the mechanism of RCC growth and metastasis is not well understood. In this report, we for the first time demonstrated ubiquitin protein ligase E3C (UBE3C) as a driving factor for RCC growth and metastasis. UBE3C expression was increased in ccRCC tissues compared with adjacent normal tissues. ccRCC patients with high UBE3C protein expression in tumors were associated with significantly worse postoperative survival. Knockdown of UBE3C expression in ACHN cells inhibited cell proliferation, migrations and invasiveness in vitro while overexpression of UBE3C in 786-O cells exerted the opposite effects. UBE3C up-regulated β-catenin protein levels and promoted β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signal pathway in RCC cells. Collectively, these observations suggest that UBE3C plays an important role in RCC development and progression, and UBE3C may be a novel target for prevention and treatment of ccRCC.
doi:10.1371/journal.pone.0115622
PMCID: PMC4319909  PMID: 25658088
2.  Rapid publication-ready MS-Word tables for two-way ANOVA 
SpringerPlus  2015;4:33.
Background
Statistical tables are an essential component of scientific papers and reports in biomedical and agricultural sciences. Measurements in these tables are summarized as mean ± SEM for each treatment group. Results from pairwise-comparison tests are often included using letter displays, in which treatment means that are not significantly different, are followed by a common letter. However, the traditional manual processes for computation and presentation of statistically significant outcomes in MS Word tables using a letter-based algorithm are tedious and prone to errors.
Results
Using the R package ‘Shiny’, we present a web-based program freely available online, at https://houssein-assaad.shinyapps.io/TwoWayANOVA/. No download is required. The program is capable of rapidly generating publication-ready tables containing two-way analysis of variance (ANOVA) results. Additionally, the software can perform multiple comparisons of means using the Duncan, Student-Newman-Keuls, Tukey Kramer, Westfall, and Fisher’s least significant difference (LSD) tests. If the LSD test is selected, multiple methods (e.g., Bonferroni and Holm) are available for adjusting p-values. Significance statements resulting from all pairwise comparisons are included in the table using the popular letter display algorithm. With the application of our software, the procedures of ANOVA can be completed within seconds using a web-browser, preferably Mozilla Firefox or Google Chrome, and a few mouse clicks. To our awareness, none of the currently available commercial (e.g., Stata, SPSS and SAS) or open-source software (e.g., R and Python) can perform such a rapid task without advanced knowledge of the corresponding programming language.
Conclusions
The new and user-friendly program described in this paper should help scientists perform statistical analysis and rapidly generate publication-ready MS-Word tables for two-way ANOVA. Our software is expected to facilitate research in agriculture, biomedicine, and other fields of life sciences.
doi:10.1186/s40064-015-0795-z
PMCID: PMC4305362  PMID: 25635246
Two-way ANOVA; Multiple comparisons; Online software; Statistical analysis; Biology; Agriculture; R; Shiny
3.  Gene Expression Profiling Analysis of Castration-Resistant Prostate Cancer 
Background
Prostate cancer is a global health issue. Usually, men with metastatic disease will progress to castration-resistant prostate cancer (CRPC). We aimed to identify the differentially expressed genes (DEGs) in tumor samples from non-castrated and castrated men from LNCaP Orthotopic xenograft models of prostate cancer and to study the mechanisms of CRPC.
Material/Methods
In this work, GSE46218 containing 4 samples from non-castrated men and 4 samples from castrated men was downloaded from Gene Expression Omnibus. We identified DEGs using limma Geoquery in R, the Robust Multi-array Average (RMA) method in Bioconductor, and Bias methods, followed by constructing an integrated regulatory network involving DEGs, miRNAs, and TFs using Cytoscape. Then, we analyzed network motifs of the integrated gene regulatory network using FANMOD. We selected regulatory modules corresponding to network motifs from the integrated regulatory network by Perl script. We preformed gene ontology (GO) and pathway enrichment analysis of DEGs in the regulatory modules using DAVID.
Results
We identified total 443 DEGs. We built an integrated regulatory network, found three motifs (motif 1, motif 2 and motif 3), and got two function modules (module 1 corresponded to motif 1, and module 2 corresponded to motif 2). Several GO terms (such as regulation of cell proliferation, positive regulation of macromolecule metabolic process, phosphorylation, and phosphorus metabolic process) and two pathways (pathway in cancer and Melanoma) were enriched. Furthermore, some significant DEGs (such as CAV1, LYN, FGFR3 and FGFR3) were related to CPRC development.
Conclusions
These genes might play important roles in the development and progression of CRPC.
doi:10.12659/MSM.891193
PMCID: PMC4306671  PMID: 25592164
Genes, abl; Neurology; Prostatic Neoplasms
4.  Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions 
Biological Chemistry  2004;385(5):373-379.
We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2–8, the carboxy-terminal residues 313–327, and the linker (amino acids 179–184) between the two functional domains – the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related ‘dimer’ with the largest dimer interface area for the group-β MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.
PMCID: PMC506909  PMID: 15195996
DNA methyltransferase; evolutionary link; protein-protein interactions; Type I and II restriction-modification systems
5.  Destabilization of Heterologous Proteins Mediated by the GSK3β Phosphorylation Domain of the β-Catenin Protein 
Background and Aims
Wnt/β-catenin signaling plays important roles in development and cellular processes. The hallmark of canonical Wnt signaling activation is the stabilization of β-catenin protein in cytoplasm and/or nucleus. The stability of β-catenin is the key to its biological functions and is controlled by the phosphorylation of its amino-terminal degradation domain. Aberrant activation of β-catenin signaling has been implicated in the development of human cancers. It has been recently suggested that GSK3β may play an essential role in regulating global protein turnover. Here, we investigate if the GSK3β phosphorylation site-containing degradation domain of β-catenin is sufficient to destabilize heterologous proteins.
Methods and Results
We engineer chimeric proteins by fusing β-catenin degradation domain at the N- and/or C-termini of the enhanced green fluorescent protein (eGFP). In both transient and stable expression experiments, the chimeric GFP proteins exhibit a significantly decreased stability, which can be effectively antagonized by lithium and Wnt1. An activating mutation in the destruction domain significantly stabilizes the fusion protein. Furthermore, GSK3 inhibitor SB-216763 effectively increases the GFP signal of the fusion protein. Conversely, the inhibition of Wnt signaling with tankyrase inhibitor XAV939 results in a decrease in GFP signal of the fusion proteins, while these small molecules have no significant effects on the mutant destruction domain-GFP fusion protein.
Conclusion
Our findings strongly suggest that the β-catenin degradation domain may be sufficient to destabilize heterologous proteins in Wnt signaling-dependent manner. It is conceivable that the chimeric GFP proteins may be used as a functional reporter to measure the dynamic status of β-catenin signaling, and to identify potential anticancer drugs that target β-catenin signaling.
doi:10.1159/000354518
PMCID: PMC4064945  PMID: 24335169
β-catenin; Wnt signal; Human cancer; Protein degradation; Green fluorescent protein; Destabilized GFP; Chimeric proteins
6.  An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules 
PLoS ONE  2014;9(9):e108535.
Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion.
doi:10.1371/journal.pone.0108535
PMCID: PMC4177923  PMID: 25255288
7.  Rapid publication-ready MS-Word tables for one-way ANOVA 
SpringerPlus  2014;3:474.
Background
Statistical tables are an important component of data analysis and reports in biological sciences. However, the traditional manual processes for computation and presentation of statistically significant results using a letter-based algorithm are tedious and prone to errors.
Results
Based on the R language, we present two web-based software for individual and summary data, freely available online, at http://shiny.stat.tamu.edu:3838/hassaad/Table_report1/ and http://shiny.stat.tamu.edu:3838/hassaad/SumAOV1/, respectively. The software are capable of rapidly generating publication-ready tables containing one-way analysis of variance (ANOVA) results. No download is required. Additionally, the software can perform multiple comparisons of means using the Duncan, Student-Newman-Keuls, Tukey Kramer, and Fisher’s least significant difference (LSD) tests. If the LSD test is selected, multiple methods (e.g., Bonferroni and Holm) are available for adjusting p-values. Using the software, the procedures of ANOVA can be completed within seconds using a web-browser, preferably Mozilla Firefox or Google Chrome, and a few mouse clicks. Furthermore, the software can handle one-way ANOVA for summary data (i.e. sample size, mean, and SD or SEM per treatment group) with post-hoc multiple comparisons among treatment means. To our awareness, none of the currently available commercial (e.g., SPSS and SAS) or open-source software (e.g., R and Python) can perform such a rapid task without advanced knowledge of the corresponding programming language.
Conclusions
Our new and user-friendly software to perform statistical analysis and generate publication-ready MS-Word tables for one-way ANOVA are expected to facilitate research in agriculture, biomedicine, and other fields of life sciences.
doi:10.1186/2193-1801-3-474
PMCID: PMC4153873  PMID: 25191639
Statistical analysis; Multiple comparisons; Online software; Computation; Biology; R; Shiny
8.  Potential Use of Sox9 Gene Therapy for Intervertebral Degenerative Disc Disease 
Spine  2003;28(8):755-763.
Study Design
A new recombinant adenoviral vector expressing Sox9, a chondrocyte-specific transcription factor, was tested in a chondroblastic cell line and primary human intervertebral disc cells in vitro. Direct infection of intervertebral disc cells then was assessed in a rabbit model.
Objectives
To deliver a potentially therapeutic viral vector expressing Sox9 to degenerative human and rabbit intervertebral discs cells, and to assess the effect of Sox9 expression on Type 2 collagen production.
Summary of the Background Data
The concentration of competent Type 2 collagen, an essential constituent of the healthy nucleus pulposus, declines with intervertebral disc degeneration. Recent studies suggest that Sox9 upregulates Type 2 collagen production. Interventions that augment Type 2 collagen production by intervertebral disc cells may represent a novel therapeutic method for patients with degenerative disc disease.
Methods
Adenoviral delivery vectors expressing Sox9 and green fluorescent protein were constructed using the AdEasy system. The chondroblastic cell line, HTB-94, and cultured human degenerated intervertebral disc cells were infected with the vectors. Reverse transcriptase-polymerase chain reaction and immunohistochemical analyses were performed to document increased Type 2 collagen expression. The AdSox9 virus then was injected directly into the intervertebral discs of three rabbits. After 5 weeks, the injected discs were evaluated histologically.
Results
The AdSox9 virus efficiently transduced HTB-94 cells and degenerated human disc cells. Western blot analysis confirmed increased Sox9 production. Increased Type 2 collagen production was demonstrated in infected HTB-94 and human disc cells using both reverse transcriptase-polymerase chain reaction and immunohistochemical staining. In the rabbit model, cells infected with AdSox9 maintained a chondrocytic phenotype, and the architecture of the nucleus pulposus was preserved over a 5-week study period compared to control discs.
Conclusions
A novel adenoviral vector efficiently increased Sox9 and Type 2 collagen synthesis in cultured chondroblastic cells and human degenerated disc cells. In a rabbit model, sustained Sox9 production preserved the histologic appearance of the nucleus pulposus cells in vivo. These findings suggest a potential role for Sox9 gene therapy in the treatment of human degenerative disc disease.
PMCID: PMC4123440  PMID: 12698117
degenerative disc disease; gene therapy; nucleus pulposus; Sox9; type 2 collagen
9.  CYTOPLASMIC AND/OR NUCLEAR ACCUMULATION OF THE β-CATENIN PROTEIN IS A FREQUENT EVENT IN HUMAN OSTEOSARCOMA 
The molecular events that precede the development of osteosarcoma, the most common primary malignancy of bone, are unclear, and concurrent molecular and genetic alterations associated with its pathogenesis have yet to be identified. Recent studies suggest that activation of β-catenin signaling may play an important role in human tumorigenesis. To investigate the potential role of β-catenin deregulation in human osteosarcoma, we analyzed a panel of 47 osteosarcoma samples for β-catenin accumulation using immunohistochemistry. Potential activating mutations were investigated by sequencing exon 3 of the β-catenin gene in genomic DNA isolated from tumor samples. Our findings revealed cytoplasmic and/or nuclear accumulation of β-catenin in 33 of 47 samples (70.2%); however, mutation analysis failed to detect any genetic alterations within exon 3, suggesting that other regulatory mechanisms may play an important role in activating β-catenin signaling in osteosarcoma. In our survival analysis, β-catenin deregulation conferred a hazard ratio of 1.05, indicating that β-catenin accumulation does not appear to be of prognostic value for osteosarcoma patients. When analyzed against other clinicopathologic parameters, β-catenin accumulation correlated only with younger age at presentation (26.4 vs. 39.8 years). Nevertheless, our results demonstrate that the deregulation of β-catenin signaling is a common occurrence in osteosarcoma that is implicated in the pathogenesis of osteosarcoma.
doi:10.1002/ijc.10719
PMCID: PMC4122310  PMID: 12402302
Wnt signal; β-catenin; osteosarcoma; tumorigenesis; bone tumor
10.  Bone morphogenetic protein 2 inhibits the proliferation and growth of human colorectal cancer cells 
Oncology Reports  2014;32(3):1013-1020.
Colorectal cancer (CRC) is one of the most deadly cancers worldwide. Significant progress has been made in understanding the molecular pathogenesis of CRC, which has led to successful early diagnosis, surgical intervention and combination chemotherapy. However, limited therapeutic options are available for metastatic and/or drug-resistant CRC. While the aberrantly activated Wnt/β-catenin pathway plays a critical initiating role in CRC development, disruption of the bone morphogenetic protein (BMP) pathway causes juvenile polyposis syndrome, suggesting that BMP signaling may play a role in CRC development. However, conflicting results have been reported concerning the possible roles of BMP signaling in sporadic colon cancer. Here, we investigated the effect of BMP2 on the proliferation, migration, invasiveness and tumor growth capability of human CRC cells. Using an adenovirus vector that overexpresses BMP2 and the piggyBac transposon-mediated stable BMP2 overexpression CRC line, we found that exogenous BMP2 effectively inhibited HCT116 cell proliferation and colony formation. BMP2 was shown to suppress colon cancer cell migration and invasiveness. Under a low serum culture condition, forced expression of BMP2 induced a significantly increased level of apoptosis in HCT116 cells. Using a xenograft tumor model, we found that forced expression of BMP2 in HCT116 cells suppressed tumor growth, accompanied by decreased cell proliferation activity. Taken together, our results strongly suggest that BMP2 plays an important inhibitory role in governing the proliferation and aggressive features of human CRC cells.
doi:10.3892/or.2014.3308
PMCID: PMC4121423  PMID: 24993644
colorectal cancer; BMP2; BMP signaling; tumorigenesis; proliferation; intestinal epithelial cells
11.  Analysis of energy expenditure in diet-induced obese rats 
Development of obesity in animals is affected by energy intake, dietary composition, and metabolism. Useful models for studying this metabolic problem are Sprague-Dawley rats fed low-fat (LF) or high-fat (HF) diets beginning at 28 days of age. Through experimental design, their dietary intakes of energy, protein, vitamins, and minerals per kg body weight (BW) do not differ in order to eliminate confounding factors in data interpretation. The 24-h energy expenditure of rats is measured using indirect calorimetry. A regression model is constructed to accurately predict BW gain based on diet, initial BW gain, and the principal component scores of respiratory quotient and heat production. Time-course data on metabolism (including energy expenditure) are analyzed using a mixed effect model that fits both fixed and random effects. Cluster analysis is employed to classify rats as normal-weight or obese. HF-fed rats are heavier than LF-fed rats, but rates of their heat production per kg non-fat mass do not differ. We conclude that metabolic conversion of dietary lipids into body fat primarily contributes to obesity in HF-fed rats.
PMCID: PMC4048864  PMID: 24896330
Obesity; Energy Expenditure; Indirect Calorimetry; Statistical Analysis; Review
12.  Structure of the bacteriophage T4 DNA adenine methyltransferase 
Nature structural biology  2003;10(10):849-855.
DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a β-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.
doi:10.1038/nsb973
PMCID: PMC4030375  PMID: 12937411
13.  BMP9 Inhibits Proliferation and Metastasis of HER2-Positive SK-BR-3 Breast Cancer Cells through ERK1/2 and PI3K/AKT Pathways 
PLoS ONE  2014;9(5):e96816.
Bone morphogenetic protein 9 (BMP9), a member of TGF-β superfamily, is reported to inhibit the growth and migration of prostate cancer, osteosarcoma and triple-negative MDA-MB-231 breast cancer cells. However, little is known about the effect of on the biological behaviors of HER2-positive SK-BR-3 breast cancer cells and the underlying mechanisms. This study aimed to investigate the effects of BMP9 on the proliferation and metastasis of SK-BR-3 cells with BMP9 over-expression or BMP9 down-regulated expression. Results indicated that exogenously expressed BMP9 inhibited the proliferation and metastasis of SK-BR-3 cells while decreased endogenous BMP9 expression in SK-BR-3 cells promoted the proliferation and migration of breast cancer cells in vitro and in vivo. In SK-BR-3 cells with BMP9 over-expression, the phosphorylation of HER2, ERK1/2 and AKT was markedly suppressed and the HER2 expression decreased at both mRNA and protein levels, while opposite results were observed in SK-BR-3 cells with BMP9 knock down. When the phosphorylation of ERK1/2 and PI3K/AKT was inhibited by PD98059 and LY294002, respectively, the decreased proliferation and invasion induced by BMP9 knock down were eliminated. These findings suggest that BMP9 can inhibit the proliferation and metastasis of SK-BR-3 cells via inactivating ERK1/2 and PI3K/AKT signaling pathways. Thus, BMP9 may serve as a useful agent in the treatment of HER-2 positive breast cancer.
doi:10.1371/journal.pone.0096816
PMCID: PMC4013047  PMID: 24805814
14.  Anatomical Evidence that the Superior Colliculus Controls Saccades through Central Mesencephalic Reticular Formation Gating of Omnipause Neuron Activity 
The Journal of Neuroscience  2013;33(41):16285-16296.
Omnipause neurons (OPNs) within the nucleus raphe interpositus (RIP) help gate the transition between fixation and saccadic eye movements by monosynaptically suppressing activity in premotor burst neurons during fixation, and releasing them during saccades. Premotor neuron activity is initiated by excitatory input from the superior colliculus (SC), but how the tectum's saccade-related activity turns off OPNs is not known. Since the central mesencephalic reticular formation (cMRF) is a major SC target, we explored whether this nucleus has the appropriate connections to support tectal gating of OPN activity. In dual-tracer experiments undertaken in macaque monkeys (Macaca fascicularis), cMRF neurons labeled retrogradely from injections into RIP had numerous anterogradely labeled terminals closely associated with them following SC injections. This suggested the presence of an SC–cMRF–RIP pathway. Furthermore, anterograde tracers injected into the cMRF of other macaques labeled axonal terminals in RIP, confirming this cMRF projection. To determine whether the cMRF projections gate OPN activity, postembedding electron microscopic immunochemistry was performed on anterogradely labeled cMRF terminals with antibody to GABA or glycine. Of the terminals analyzed, 51.4% were GABA positive, 35.5% were GABA negative, and most contacted glycinergic cells. In summary, a trans-cMRF pathway connecting the SC to the RIP is present. This pathway contains inhibitory elements that could help gate omnipause activity and allow other tectal drives to induce the bursts of firing in premotor neurons that are necessary for saccades. The non-GABAergic cMRF terminals may derive from fixation units in the cMRF.
doi:10.1523/JNEUROSCI.2726-11.2013
PMCID: PMC3792464  PMID: 24107960
15.  Utilization of CDX2 Expression in Diagnosing Pancreatic Ductal Adenocarcinoma and Predicting Prognosis 
PLoS ONE  2014;9(1):e86853.
CDX2, a master transcriptional regulator of intestinal cell differentiation and survival, has been used as a marker to indicate colorectal lineage in adenocarcinomas of unknown origin. Pancreatic ductal adenocarcinoma (PDAC) is one of the most common causes for adenocarcinomas of unknown origin, but CDX2 expression in pancreatic disease remains unclear. In this study, we systemically and extensively investigated the expression and role of CDX2 in PDAC. We reported that CDX2 expression is weak and heterogeneous is all normal pancreas and chronic pancreatitis. It is largely expressed in epithelial-lining cells of pancreatic ducts including main ducts, inter-lobular ducts, intra-lobular ducts, intercalated ducts and centroacinar cells, but not in acinar cells or islet cells. CDX2 expression is down regulated during the transformation process from PanIN to PDAC. Only one third of PDACs retain some degree of CDX2 expression, and this group of PDACs have reduced median survival time compared to that of CDX2 negative group (308 days vs. 586 days, p = 0.0065). Metastatic PDACs remain similar expression pattern to that of the primary sites. Our study clearly demonstrates CDX2 expression in pancreatic diseases including PDAC, which is practically important when CDX2 is used to establish the primary sites of adenocarcinomas of unknown origin. In addition, our study also provides CDX2 as a prognostic marker for PDAC and implicates an important role of CDX2 in the development of normal pancreas and PDAC.
doi:10.1371/journal.pone.0086853
PMCID: PMC3906088  PMID: 24489794
16.  Activation of the PI3K/Akt/mTOR/p70S6K Pathway is Involved in S100A4-induced Viability and Migration in Colorectal Cancer Cells 
The S100 protein family member S100A4 regulates various cellular functions. Previous studies have shown that elevated expression of S100A4 is associated with progression and metastasis of colorectal cancer (CRC). However, little is known about whether and how S100A4 contributes to CRC development. In our present study, the elevated expression of S100A4 in CRC tissues compared to matched adjacent normal tissues was confirmed by immunohistochemistry, semi-quantitative RT-PCR and Western blot. Adenovirus-mediated S100A4 overexpression obviously enhanced viability and migration of CRC cells, which was detected by MTT assay and transwell assay, respectively. Additionally, S100A4 overexpression increased the phosphorylation levels of Akt, mTOR and p70S6K. These effects of S100A4 were abolished by treatment with either the specific PI3K/Akt inhibitor LY294002, or the specific mTOR/p70S6K inhibitor rapamycin. Furthermore, overexpression of S100A4 resulted in upregulation of VEGF and downregulation of E-cadherin, which were strongly reversed by either LY294002 or rapamycin. Altogether, our results demonstrate that activation of the PI3K/Akt/mTOR/p70S6K signaling pathway is involved in S100A4-induced viability, migration, upregulation of VEGF and downregulation of E-cadherin in CRC cells.
doi:10.7150/ijms.8128
PMCID: PMC4057487  PMID: 24936148
colorectal cancer; S100A4; PI3K/Akt/mTOR/p70S6K; viability; migration.
17.  Modulation of β-Catenin Signaling by the Inhibitors of MAP Kinase, Tyrosine Kinase, and PI3-Kinase Pathways 
Aberrant activation of β-catenin signaling plays an important role in human tumorigenesis. However, molecular mechanisms behind the β-catenin signaling deregulation are mostly unknown because genetic alterations in this pathway only account for a small fraction of tumors. Here, we investigator if other major pathways can regulate β-catenin signaling activity. By employing a panel of chemical activators and/or inhibitors of several cellular signaling pathways, we assess these modulators' effects on luciferase reporter driven by β-catenin/TCF4-responsive elements. We find that lithium-stimulated β-catenin activity is synergistically enhanced by protein kinase C activator PMA. However, β-catenin-regulated transcriptional (CRT) activity is significantly inhibited by casein kinase II inhibitor DRB, MEK inhibitor PD98059, G-proteins and their receptor uncoupling agent suramin, protein tyrosine kinase inhibitor genistein, and PI-3 kinase inhibitor wortmannin, suggesting that these cellular pathways may participate in regulating β-catenin signaling. Interestingly, the Ca++/calmodulin kinase II inhibitor HDBA is shown to activate β-catenin activity at low doses. Furthermore, Wnt3A-stimulated and constitutively activated CRT activities, as well as the intracellular accumulation of β-catenin protein in human colon cancer cells, are effectively suppressed by PD98059, genistein, and wortmannin. We further demonstrate that EGF can activate TCF4/β-catenin activity and induce the tyrosine phosphorylation of β-catenin protein. Thus, our results should provide important insights into the molecular mechanisms underlying Wnt/β-catenin activation. This knowledge should facilitate our efforts to develop efficacious and novel therapeutics by targeting these pathways.
doi:10.7150/ijms.6019
PMCID: PMC3856380  PMID: 24324366
β-catenin; G protein; MEK kinase; PI3 kinase; protein kinase C; tumorigenesis; tyrosine phosphorylation; Wnt signaling.
18.  ERCC1 mRNA levels can predict the response to cisplatin-based concurrent chemoradiotherapy of locally advanced cervical squamous cell carcinoma 
Background
The purpose of this study was to investigate whether the excision repair cross-complementation group 1 (ERCC1) mRNA expression could predict treatment response of patients with locally advanced cervical squamous cell carcinoma (LACSCC) who underwent cisplatin-based concurrent chemoradiotherapy (CCCRT).
Methods
A total of sixty LACSCC patients, treated with radical CCCRT from a single institution were evaluated. ERCC1 mRNA expression was determined by quantitative real-time RT-PCR in pre-treatment tumor tissues. The association of ERCC1 status with clinicopathological characteristics (age, histological grade, tumor size, parametrial invasion, lymph node metastasis and FIGO stage) and treatment response were analyzed.
Results
No significant association between ERCC1 mRNA expression and clinicopathological characteristics were observed. Patients with low ERCC1 mRNA level had a significantly higher rate of complete response (86.21%) than patients with high level of ERCC1 expression (19.36%; p < 0.001). In the logistic regression analysis, low ERCC1 mRNA level retained an independent role in predicting complete response to CCCRT (P < 0.001). An ERCC1 expression level of 0.0901 was determined as an optimal cutoff value to identify complete response patients to CCCRT treatment. The sensitivity for detection of a complete response was 81.48% with a specificity of 96.97% (area under the curve, 0.893; 95% confidence interval, 0.804–0.983).
Conclusions
This is the first analysis of the association between ERCC1 mRNA levels and treatment response in patients with LACSCC. Low ERCC1 mRNA level appears to be a highly specific predictor of response to CCCRT in LACSCC.
doi:10.1186/1748-717X-7-221
PMCID: PMC3536645  PMID: 23259415
Excision repair cross-complementation group 1 (ERCC1); Cervical squamous cell carcinoma; Chemoradiotherapy; Response prediction
19.  RATE AND INFLUENCE OF RESPIRATORY VIRUS CO-INFECTION ON PANDEMIC (H1N1) INFLUENZA DISEASE 
The Journal of infection  2011;63(4):260-266.
Objectives
Many patients with influenza have more than one viral agent with co-infection frequencies reported as high as 20%. The impact of respiratory virus copathogens on influenza disease is unclear. We sought to determine if respiratory virus co-infection with pandemic H1N1 altered clinical disease.
Methods
Respiratory samples from 229 and 267 patients identified with and without H1N1 influenza respectively were screened for the presence of 13 seasonal respiratory viruses by multiplex RT-PCR. Disease severity between coinfected and monoinfected H1N1 patients were quantified using a standardized clinical severity scale. Influenza viral load was calculated by quantitative RT-PCR.
Results
Thirty (13.1%) influenza samples screened positive for the presence of 31 viral copathogens. The most prominent copathogens included rhinovirus (61.3%), and coronaviruses (16.1%). Median clinical severity of both monoinfected and co-infected groups were 1. Patients coinfected with rhinovirus tended to have lower clinical severity (median 0), whereas non rhinovirus co-infections had substantially higher clinical severity (median 2). No difference in H1N1 viral load was observed between co-infected and mono infected groups.
Conclusions
Respiratory viruses co-infect patients with influenza disease. Patients coinfected with rhinovirus had less severe disease while non-rhinovirus co-infections were associated with substantially higher severity without changes in influenza viral titer.
doi:10.1016/j.jinf.2011.04.004
PMCID: PMC3153592  PMID: 21546090
Influenza; Influenza Co-infection; Co-infection; Dual Infection; Respiratory Virus Co-infection; Viral Co-infection; Pneumonia; Respiratory Disease
20.  Clock-controlled mir-142-3p can target its activator, Bmal1 
BMC Molecular Biology  2012;13:27.
Background
microRNAs (miRNAs) are shown to be involved in the regulation of circadian clock. However, it remains largely unknown whether miRNAs can regulate the core clock genes (Clock and Bmal1).
Results
In this study, we found that mir-142-3p directly targeted the 3’UTR of human BMAL1 and mouse Bmal1. The over-expression (in 293ET and NIH3T3 cells) and knockdown (in U87MG cells) of mir-142-3p reduced and up-regulated the Bmal1/BMAL1 mRNA and protein levels, respectively. Moreover, the expression level of mir-142-3p oscillated in serum-shocked NIH3T3 cells and the results of ChIP and luciferase reporter assays suggested that the expression of mir-142-3p was directly controlled by CLOCK/BMAL1 heterodimers in NIH3T3 cells.
Conclusions
Our study demonstrates that mir-142-3p can directly target the 3’UTR of Bmal1. In addition, the expression of mir-142-3p is controlled by CLOCK/BMAL1 heterodimers, suggesting a potential negative feedback loop consisting of the miRNAs and the core clock genes. These findings open new perspective for studying the molecular mechanism of circadian clock.
doi:10.1186/1471-2199-13-27
PMCID: PMC3482555  PMID: 22958478
mir-142-3p; Bmal1; Circadian clock
21.  Sympathetic Blocks Provided Sustained Pain Relief in a Patient with Refractory Painful Diabetic Neuropathy 
Case Reports in Anesthesiology  2012;2012:285328.
The sympathetic nervous system has been implicated in pain associated with painful diabetic neuropathy. However, therapeutic intervention targeted at the sympathetic nervous system has not been established. We thus tested the hypothesis that sympathetic nerve blocks significantly reduce pain in a patient with painful diabetic neuropathy who has failed multiple pharmacological treatments. The diagnosis of small fiber sensory neuropathy was based on clinical presentations and confirmed by skin biopsies. A series of 9 lumbar sympathetic blocks over a 26-month period provided sustained pain relief in his legs. Additional thoracic paravertebral blocks further provided control of the pain in the trunk which can occasionally be seen in severe diabetic neuropathy cases, consequent to extensive involvement of the intercostal nerves. These blocks provided sustained and significant pain relief and improvement of quality of life over a period of more than two years. We thus provided the first clinical evidence supporting the notion that sympathetic nervous system plays a critical role in painful diabetic neuropathy and sympathetic blocks can be an effective management modality of painful diabetic neuropathy. We concluded that the sympathetic nervous system is a valuable therapeutic target of pharmacological and interventional modalities of treatments in painful diabetic neuropathy patients.
doi:10.1155/2012/285328
PMCID: PMC3350298  PMID: 22606406
22.  Bone Morphogenetic Protein-9 Induces Osteogenic Differentiation of Rat Dental Follicle Stem Cells in P38 and ERK1/2 MAPK Dependent Manner 
Dental follicle stem cells are a group of cells possessing osteogenic, adipogenetic and neurogenic differentiations, but the specific mechanism underlying the multilineage differentiation remains still unclear. Great attention has been paid to bone morphogenetic protein-9 (BMP-9) due to its potent osteogenic activity. In the present study, rat dental follicle stem cells were isolated and purified, and cells of passage 3 underwent adenovirus mediated BMP-9 gene transfection to prepare dental follicle stem cells with stable BMP-9 expression. Detection of alkaline phosphatase (ALP) and calcium deposition showed dental follicle stem cells transfected with BMP-9 gene could significantly promote the osteogenesis. In addition, SB203580 and PD98059 were employed to block the p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK1/2), respectively. Detection of ALP and calcium deposition revealed the BMP-9 induced osteogenic differentiation of dental follicle stem cells depended on MAPK signaling pathway.
doi:10.7150/ijms.5027
PMCID: PMC3498751  PMID: 23155360
Dental follicle stem cells; rat recombinant adenovirus transfection; bone morphogenetic protein-9; osteogenic differentiation; signaling pathway.
23.  Fucose-Deficient Hematopoietic Stem Cells Have Decreased Self-Renewal and Aberrant Marrow Niche Occupancy 
Transfusion  2010;50(12):2660-2669.
BACKGROUND
Modification of Notch receptors by O-linked fucose and its further elongation by the Fringe family of glycosyltransferase has been shown to be important for Notch signaling activation. Our recent studies disclose a myeloproliferative phenotype, hematopoietic stem cell (HSC) dysfunction, and abnormal Notch signaling in mice deficient in FX, which is required for fucosylation of a number of proteins including Notch. The purpose of this study is to assess the self-renewal and stem cell niche features of fucose-deficient HSCs.
STUDY DESIGN AND METHODS
Homeostasis and maintenance of HSCs derived from FX-/- mice were studied by serial bone marrow transplantation, homing assay, and cell cycle analysis. Two-photon intravital microscopy was performed to visualize and compare the in vivo marrow niche occupancy by fucose-deficient and wild type (WT) HSCs.
RESULTS
Marrow progenitors from FX-/- mice had mild homing defects that could be partially prevented by exogenous fucose supplementation. Fucose-deficient HSCs from FX-/- mice displayed decreased self-renewal capability compared with the WT controls. This is accompanied with their increased cell cycling activity and suppressed Notch ligand binding. When tracked in vivo by 2-photon intravital imaging, the fucose-deficient HSCs were found localized further from the endosteum of the calvarium marrow than the WT HSCs.
CONCLUSION
The current reported aberrant niche occupancy by HSCs from FX-/- mice, in the context of a faulty blood lineage homeostasis and HSC dysfunction in mice expressing Notch receptors deficient in O-fucosylation, suggests that fucosylation modified Notch receptor may represent a novel extrinsic regulator for HSC engraftment and HSC niche maintenance.
doi:10.1111/j.1537-2995.2010.02745.x
PMCID: PMC2946970  PMID: 20573072
24.  A new category of autoinflammatory disease associated with NOD2 gene mutations 
Arthritis Research & Therapy  2011;13(5):R148.
Introduction
Autoinflammatory diseases are characterized by seemingly unprovoked episodes of inflammation, without high titers of autoantibodies or antigen-specific T cells, and derive from genetic variants of the innate immune system. This study characterized a cohort of patients with similar phenotypes and nucleotide oligomerization domain 2 (NOD2) gene mutations.
Methods
Diagnostically challenging patients with the following clinical and genetic characteristics were prospectively studied between January 2009 and April 2011: periodic fever, dermatitis, polyarthritis, serositis, negative serum autoantibodies and additional positive NOD2 IVS8+158 gene mutation. Genetic testing for gene mutations of NOD2, tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS) and familial Mediterranean fever (FMF) was performed.
Results
All seven patients with the disease were Caucasians, with four being male. The mean age at disease onset was 40.7 years and disease duration was 3.2 years. These patients characteristically presented with periodic fever, dermatitis and inflammatory polyarthritis. There were gastrointestinal symptoms in three patients, granulomas of the skin and gut in two, and recurrent chest pain in two, with one having pleuritis and pericarditis. Three patients had sicca-like symptoms. Five patients had increased acute phase reactants. All seven patients had negative tests for autoantibodies but carried the NOD2 gene mutation IVS8+158 with four having concurrent R702W mutation.
Conclusions
Our cohort may represent a new disease category of autoinflammatory disease with characteristic clinical phenotypes and genotypes. It may somewhat resemble pediatric Blau's syndrome.
doi:10.1186/ar3462
PMCID: PMC3308076  PMID: 21914217
25.  Targeting Fibrosis in Duchenne Muscular Dystrophy 
Duchenne muscular dystrophy (DMD) is the most common genetic muscle disease affecting 1 in 3,500 live male births. It is an X-linked recessive disease caused by a defective dystrophin gene. The disease is characterized by progressive limb weakness, respiratory and cardiac failure and premature death. Fibrosis is a prominent pathological feature of muscle biopsies from patients with DMD. It directly causes muscle dysfunction and contributes to the lethal DMD phenotype. Although gene therapy and cell therapy may ultimately provide a cure for DMD, currently the disease is devastating, with no effective therapies. Recent studies have demonstrated that ameliorating muscle fibrosis may represent a viable therapeutic approach for DMD. By reducing scar formation, antifibrotic therapies may not only improve muscle function but also enhance muscle regeneration and promote gene and stem cell engraftment. Antifibrotic therapy may serve as a necessary addition to gene and cell therapies to treat DMD in the future. Therefore, understanding cellular and molecular mechanisms underlying muscle fibrogenesis associated with dystrophin deficiency is key to the development of effective antifibrotic therapies for DMD.
doi:10.1097/NEN.0b013e3181e9a34b
PMCID: PMC2916968  PMID: 20613637
Antifibrotic therapy; Duchenne muscular dystrophy; Muscle fibrosis

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