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1.  Pre-treatment with IL-1β enhances the efficacy of MSC transplantation in DSS-induced colitis 
Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly due to their immunosuppressive properties. Although interleukin-1β (IL-1β) is one of the most important inflammatory mediators, growing evidence indicates that IL-1β signaling elicits the immunosuppressive properties of MSCs. However, it remains unclear how IL-1β signaling accomplishes this activity. Here, we focus on the therapeutic efficacy of IL-1β-primed MSCs in the dextran sulfate sodium (DSS)-induced colitis model, in addition to the underlining mechanisms. We first found that IL-1β-primed MSCs, without any observable phenotype change in vitro, significantly attenuated the development of DSS-induced murine colitis. Moreover, IL-1β-primed MSCs modulated the balance of immune cells in the spleen and the mesenteric lymph nodes (MLNs) through elevating cyclooxygenase-2 (COX-2), IL-6 and IL-8 expression and influencing the polarization of peritoneal macrophages. Importantly, IL-1β-primed MSCs possessed an enhanced ability to migrate to the inflammatory site of the gut via upregulation of chemokine receptor type 4 (CXCR4) expression. In summary, IL-1β-primed MSCs have improved efficacy in treating DSS-induced colitis, which at least partly depends on their increased immunosuppressive capacities and enhanced migration ability.
doi:10.1038/cmi.2012.40
PMCID: PMC4002219  PMID: 23085948
IL-1β; mesenchymal stem cells; ulcerative colitis
2.  Evaluation of several adjuvants in avian influenza vaccine to chickens and ducks 
Virology Journal  2011;8:321.
The effects of three different adjuvants, mineral oil, Montanide™ ISA 70M VG, and Montanide™ ISA 206 VG, were evaluated on reverse genetics H5N3 avian influenza virus cell cultured vaccine. The immune results of SPF chickens after challenging with highly pathogenic avian influenza (HPAI) virus demonstrated that mineral oil adjuvant group and 70M adjuvant group provided 100% protection efficiency, but 206 adjuvant group provided only 40%. Statistical analysis indicated that the protection effects of mineral oil adjuvant group and the 70M adjuvant showed no significant difference to each other, but with significant difference to 206 adjuvant group. All three groups could induce high titres of antibody after immunizing SPF ducks, but there was no significant difference among them. The immunization effect of 70M adjuvant group on SPF chickens were the best and showed significant difference compared with optimized 70Mi Montanide™ eight series adjuvants groups. These results suggest that 70M adjuvant could be a novel adjuvant for preparing avian influenza vaccine.
doi:10.1186/1743-422X-8-321
PMCID: PMC3141683  PMID: 21703008
3.  Genomic and transcriptomic analysis of NDM-1 Klebsiella pneumoniae in spaceflight reveal mechanisms underlying environmental adaptability 
Scientific Reports  2014;4:6216.
The emergence and rapid spread of New Delhi Metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae strains has caused a great concern worldwide. To better understand the mechanisms underlying environmental adaptation of those highly drug-resistant K. pneumoniae strains, we took advantage of the China's Shenzhou 10 spacecraft mission to conduct comparative genomic and transcriptomic analysis of a NDM-1 K. pneumoniae strain (ATCC BAA-2146) being cultivated under different conditions. The samples were recovered from semisolid medium placed on the ground (D strain), in simulated space condition (M strain), or in Shenzhou 10 spacecraft (T strain) for analysis. Our data revealed multiple variations underlying pathogen adaptation into different environments in terms of changes in morphology, H2O2 tolerance and biofilm formation ability, genomic stability and regulation of metabolic pathways. Additionally, we found a few non-coding RNAs to be differentially regulated. The results are helpful for better understanding the adaptive mechanisms of drug-resistant bacterial pathogens.
doi:10.1038/srep06216
PMCID: PMC4147364  PMID: 25163721
4.  Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains 
Protein & Cell  2014;5(8):603-615.
ABSTRACT
The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0060-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0060-1
PMCID: PMC4130922  PMID: 24866699
TCR repertoire; next-generation sequencing; V/J usage; V-J pairing; CDR3; D-D fusion
5.  Characterization of human αβTCR repertoire and discovery of D-D fusion in TCRβ chains 
Protein & Cell  2014;5(8):603-615.
ABSTRACT
The characterization of the human T-cell receptor (TCR) repertoire has made remarkable progress, with most of the work focusing on the TCRβ chains. Here, we analyzed the diversity and complexity of both the TCRα and TCRβ repertoires of three healthy donors. We found that the diversity of the TCRα repertoire is higher than that of the TCRβ repertoire, whereas the usages of the V and J genes tended to be preferential with similar TRAV and TRAJ patterns in all three donors. The V-J pairings, like the V and J gene usages, were slightly preferential. We also found that the TRDV1 gene rearranges with the majority of TRAJ genes, suggesting that TRDV1 is a shared TRAV/DV gene (TRAV42/DV1). Moreover, we uncovered the presence of tandem TRBD (TRB D gene) usage in ~2% of the productive human TCRβ CDR3 sequences.
Electronic supplementary material
The online version of this article (doi:10.1007/s13238-014-0060-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s13238-014-0060-1
PMCID: PMC4130922  PMID: 24866699
TCR repertoire; next-generation sequencing; V/J usage; V-J pairing; CDR3; D-D fusion
6.  Evolutionary dynamics of ecological niche in three Rhinogobio fishes from the upper Yangtze River inferred from morphological traits 
Ecology and Evolution  2015;5(3):567-577.
In the past decades, it has been debated whether ecological niche should be conserved among closely related species (phylogenetic niche conservatism, PNC) or largely divergent (traditional ecological niche theory and ecological speciation) and whether niche specialist and generalist might remain in equilibrium or niche generalist could not appear. In this study, we employed morphological traits to describe ecological niche and test whether different niche dimensions exhibit disparate evolutionary patterns. We conducted our analysis on three Rhinogobio fish species (R. typus,R. cylindricus, and R. ventralis) from the upper Yangtze River, China. Among the 32 measured morphological traits except body length, PCA extracted the first four principal components with their loading scores >1.000. To find the PNC among species, Mantel tests were conducted with the Euclidean distances calculated from the four principal components (representing different niche dimensions) against the pairwise distances calculated from mitochondrial cytochrome b sequence variations. The results showed that the second and the third niche dimension, both related to swimming ability and behavior, exhibited phylogenetic conservatism. Further comparison on niche breadth among these three species revealed that the fourth dimension of R. typus showed the greatest width, indicating that this dimension exhibited niche generalism. In conclusion, our results suggested that different niche dimensions could show different evolutionary dynamic patterns: they may exhibit PNC or not, and some dimensions may evolve generalism.
doi:10.1002/ece3.1386
PMCID: PMC4328762
Ecological niche; generalist; morphological trait; niche difference; phylogenetic niche conservatism; Rhinogobio; specialist; the Yangtze River
7.  Molecular Epidemiology and Clinical Characteristics of Drug-Resistant Mycobacterium tuberculosis in a Tuberculosis Referral Hospital in China 
PLoS ONE  2014;9(10):e110209.
Background
Despite the large number of drug-resistant tuberculosis (TB) cases in China, few studies have comprehensively analyzed the drug resistance-associated gene mutations and genotypes in relation to the clinical characteristics of M. tuberculosis (Mtb) isolates.
Methodology/Principal Findings
We thus analyzed the phenotypic and genotypic drug resistance profiles of 115 Mtb clinical isolates recovered from a tuberculosis referral hospital in Beijing, China. We also performed genotyping by 28 loci MIRU-VNTR analysis. Socio-demographic and clinical data were retrieved from medical records and analyzed. In total, 78 types of mutations (including 42 previously reported and 36 newly identified ones) were identified in 115 Mtb clinical isolates. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P<0.001). Genotyping revealed 101 MIRU-VNTR types, with 20 isolates (17.4%) being clustered and 95 isolates (82.6%) having unique genotypes. Higher proportion of re-treatment cases was observed among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). Moreover, clinical epidemiological links were identified among patients infected by Mtb strains belonging to the same clusters, suggesting a potential of transmission among patients.
Conclusions/Significance
Our study provided information on novel potential drug resistance-associated mutations in Mtb. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.
doi:10.1371/journal.pone.0110209
PMCID: PMC4193878  PMID: 25302501
8.  Carinatines A and B, Lycopodium Alkaloids from Phlegmariurus carinatus 
Carinatine A (1), a C16N2-type Lycopodium alkaloid possessing a 5/6/6/6 ring system formed by a new C-4/C-12 bond, and carinatine B (2), the first derivative of lycojaponicumin C, along 16 known compounds, were isolated from the whole plant of Phlegmariurus carinatus. Their structures were elucidated based on the spectroscopic data. The two new isolates were no inhibitory activity for the acetylcholinesterase (AChE).
doi:10.1007/s13659-014-0030-6
PMCID: PMC4111872  PMID: 25089240
Lycopodium alkaloid; Phlegmariurus carinatus; Carinatines A and B; Acetylcholinesterase (AChE) inhibitory activity
9.  Lycopodine-Type Alkaloids from Lycopodium japonicum 
Three new lycopodine-type alkaloids, 4α-hydroxyanhydrolycodoline (1), 4α,6α-dihydroxyanhydrolycodoline (2), and 6-epi-8β-acetoxylycoclavine (3), and an artifact, lycoposerramine G nitrate (4), along with seventeen related known compounds, were isolated from the club moss Lycopodiumjaponicum Thunb. ex Murray (Lycopodiaceae). Their structures were elucidated by extensive spectroscopic methods as well as X-ray analysis. Compounds 1–4 were evaluated for their acetylcholine esterase inhibitory activity.
Electronic supplementary material
The online version of this article (doi:10.1007/s13659-014-0027-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s13659-014-0027-1
PMCID: PMC4111880  PMID: 25089239
Lycopodium japonicum; Lycopodine-type alkaloids; 4α-Hydroxyanhydrolycodoline; 4α,6α-Dihydroxyanhydrolycodoline; 6-epi-8β-Acetoxylycoclavine; Lycoposerramine G nitrate
10.  Carinatines A and B, Lycopodium Alkaloids from Phlegmariurus carinatus 
Carinatine A (1), a C16N2-type Lycopodium alkaloid possessing a 5/6/6/6 ring system formed by a new C-4/C-12 bond, and carinatine B (2), the first derivative of lycojaponicumin C, along 16 known compounds, were isolated from the whole plant of Phlegmariurus carinatus. Their structures were elucidated based on the spectroscopic data. The two new isolates were no inhibitory activity for the acetylcholinesterase (AChE).
doi:10.1007/s13659-014-0030-6
PMCID: PMC4111872  PMID: 25089240
Lycopodium alkaloid; Phlegmariurus carinatus; Carinatines A and B; Acetylcholinesterase (AChE) inhibitory activity
11.  Lycopodine-Type Alkaloids from Lycopodium japonicum 
Three new lycopodine-type alkaloids, 4α-hydroxyanhydrolycodoline (1), 4α,6α-dihydroxyanhydrolycodoline (2), and 6-epi-8β-acetoxylycoclavine (3), and an artifact, lycoposerramine G nitrate (4), along with seventeen related known compounds, were isolated from the club moss Lycopodiumjaponicum Thunb. ex Murray (Lycopodiaceae). Their structures were elucidated by extensive spectroscopic methods as well as X-ray analysis. Compounds 1–4 were evaluated for their acetylcholine esterase inhibitory activity.
Electronic supplementary material
The online version of this article (doi:10.1007/s13659-014-0027-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s13659-014-0027-1
PMCID: PMC4111880  PMID: 25089239
Lycopodium japonicum; Lycopodine-type alkaloids; 4α-Hydroxyanhydrolycodoline; 4α,6α-Dihydroxyanhydrolycodoline; 6-epi-8β-Acetoxylycoclavine; Lycoposerramine G nitrate
12.  Comparative genomic analysis of Mycobacterium tuberculosis clinical isolates 
BMC Genomics  2014;15(1):469.
Background
Due to excessive antibiotic use, drug-resistant Mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. To better understand the evolution of drug-resistant M. tuberculosis strains, we performed whole genome sequencing for 7 M. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.
Results
We observed that all 7 M. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of SNPs, ranging from 1409–1464. The numbers of insertion/deletions (Indels) identified in the 7 isolates were also similar, ranging from 56 to 101. A total of 39 types of mutations were identified in drug resistance-associated loci, including 14 previously reported ones and 25 newly identified ones. Sixteen of the identified large Indels spanned PE-PPE-PGRS genes, which represents a major source of antigenic variability. Aside from SNPs and Indels, a CRISPR locus with varied spacers was observed in all 7 clinical isolates, suggesting that they might play an important role in plasticity of the M. tuberculosis genome. The nucleotide diversity (Л value) and selection intensity (dN/dS value) of the whole genome sequences of the 7 isolates were similar. The dN/dS values were less than 1 for all 7 isolates (range from 0.608885 to 0.637365), supporting the notion that M. tuberculosis genomes undergo purifying selection. The Л values and dN/dS values were comparable between drug-susceptible and drug-resistant strains.
Conclusions
In this study, we show that clinical M. tuberculosis isolates exhibit distinct variations in terms of the distribution of SNP, Indels, CRISPR-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. Our study provides evidence strengthening the notion that the evolution of drug resistance among clinical M. tuberculosis isolates is clearly a complex and diversified process.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-469) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-469
PMCID: PMC4070564  PMID: 24923884
Mycobacterium tuberculosis; Drug resistance; Single nucleotide polymorphisms; Whole genome sequencing; Evolution
13.  Luteolin Inhibits Behavioral Sensitization by Blocking Methamphetamine-Induced MAPK Pathway Activation in the Caudate Putamen in Mice 
PLoS ONE  2014;9(6):e98981.
Goal
To investigate the effect of luteolin on methamphetamine (MA)-induced behavioral sensitization and mitogen-activated protein kinase (MAPK) signal transduction pathway activation in mice.
Methods
Mice received a single dose of MA to induce hyperactivity or repeated intermittent intraperitoneal injections of MA to establish an MA-induced behavioral sensitization mouse model. The effect of luteolin on the development and expression of MA-induced hyperactivity and behavioral sensitization was examined. The expression and activity of ΔFosB and the levels of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), phosphorylated c-Jun N-terminal kinase (pJNK), and phosphorylated p38 mitogen-activated protein kinase (pp38) in the caudate putamen (CPu) were measured by western blot.
Results
Luteolin significantly decreased hyperactivity as well as the development and expression of MA-induced behavioral sensitization in mice. ΔFosB, pERK1/2, and pJNK levels in the CPu were higher in MA-treated mice than in control mice, whereas the pp38 level did not change. Injection of luteolin inhibited the MA-induced increase in ΔFosB, pERK1/2, and pJNK levels, but did not affect the pp38 level.
Conclusions
Luteolin inhibits MA-induced hyperactivity and behavioral sensitization in mice through the ERK1/2/ΔFosB pathway. Furthermore, the JNK signaling pathway might be involved in MA-induced neurodegeneration in the CPu, and luteolin inhibits this process.
doi:10.1371/journal.pone.0098981
PMCID: PMC4047057  PMID: 24901319
14.  Cessation of Epithelial Bmp Signaling Switches the Differentiation of Crown Epithelia to the Root Lineage in a β-Catenin-Dependent Manner 
Molecular and Cellular Biology  2013;33(23):4732-4744.
The differentiation of dental epithelia into enamel-producing ameloblasts or the root epithelial lineage compartmentalizes teeth into crowns and roots. Bmp signaling has been linked to enamel formation, but its role in root epithelial lineage differentiation is unclear. Here we show that cessation of epithelial Bmp signaling by Bmpr1a depletion during the differentiation stage switched differentiation of crown epithelia into the root lineage and led to formation of ectopic cementum-like structures. This phenotype is related to the upregulation of Wnt/β-catenin signaling and epithelial-mesenchymal transition (EMT). Although epithelial β-catenin depletion during the differentiation stage also led to variable enamel defect and precocious/ectopic formation of fragmented root epithelia in some teeth, it did not cause ectopic cementogenesis and inhibited EMT in cultured dental epithelia. Concomitant epithelial β-catenin depletion rescued EMT and ectopic cementogenesis caused by Bmpr1a depletion. These data suggested that Bmp and Wnt/β-catenin pathways interact antagonistically in dental epithelia to regulate the root lineage differentiation and EMT. These findings will aid in the design of new strategies to promote functional differentiation in the regeneration and tissue engineering of teeth and will provide new insights into the dynamic interactions between the Bmp and Wnt/β-catenin pathways during cell fate decisions.
doi:10.1128/MCB.00456-13
PMCID: PMC3838012  PMID: 24081330
15.  Association of ERCC1 and ERCC2 polymorphisms with colorectal cancer risk in a Chinese population 
Scientific Reports  2014;4:4112.
The ERCC1 and ERCC2 genes are important in repairing DNA damage and genomic instability, and are involved in the nucleotide excision repair pathway. We hypothesized that single nucleotide polymorphisms (SNPs) in ERCC1 and ERCC2 are associated with the risk of colorectal cancer in a Chinese population. To test this hypothesis, we genotyped four functional SNPs (ERCC1 Asn118Asn, C8092A, ERCC2 Asp312Asn, and Lys751Gln) in a case-control study with 213 colorectal cancer cases and 240 cancer-free controls. We found that the ERCC1 C8092A polymorphism AA and CA/AA variant genotypes were associated with a significantly increased risk of colorectal cancer, compared with the CC genotype (OR = 2.50, 95% CI = 1.10–5.70 for AA versus CC, and OR = 1.58, 95% CI = 1.08–2.30 for CA/AA versus CC). Furthermore, the effect appeared to be more prominent among men, smokers, drinkers, and patients with rectal cancer. However, no other SNPs were observed for any significant association with colorectal cancer risk. These results suggest that the ERCC1 C8092A polymorphism may contribute to colorectal cancer susceptibility in the Chinese population. Further large and functional studies are needed to confirm our findings.
doi:10.1038/srep04112
PMCID: PMC3925949  PMID: 24531312
16.  Identification of latent tuberculosis infection-related microRNAs in human U937 macrophages expressing Mycobacterium tuberculosis Hsp16.3 
BMC Microbiology  2014;14:37.
Background
Latent tuberculosis infection (LTBI) relies on a homeostasis of macrophages and Mycobacterium tuberculosis (Mtb). The small heat shock protein, Mtb Hsp16.3 (also known as latency-associated antigen), plays an important role in Mtb persistence within macrophages. However, the mechanism of LTBI remains elusive. The aim of this study was to delineate LTBI-related miRNA expression in U937 macrophages expressing Mtb Hsp16.3 protein. U937 macrophages were infected with an integrase-deficient Lentivirus vector to transiently express Mtb Hsp16.3, and green fluorescent protein (GFP) as a control. We used a microRNA (miRNA) microarray chip containing more than 1000 probes to identify the significant differentially expressed miRNAs in the infected U937 cells, and employed real-time quantitative polymerase chain reaction (qRT-PCR) for validation. Furthermore, we confirmed these candidate LTBI-related miRNAs in peripheral blood mononuclear cells from subjects with LTBI and in healthy control individuals. Functional annotation prediction of miRNA target genes and pathway enrichment analyses were used to explore the putative links between these miRNAs and LTBI.
Results
Analysis of the miRNA expression profile identified 149 miRNAs that were differentially expressed in U937 macrophages expressing Mtb Hsp16.3 compared with the control expressing GFP. The expression level of seven miRNAs (miR-424-5p, miR-493-5p, miR-296-5p, miR-27b-3p, miR-377-5p, miR-3680-5p, miR-191-5p) were validated by qRT-PCR. The expression level of four miRNAs (miR-424-5p, miR-27b-3p, miR-377-5p, miR-3680-5p) in the peripheral blood mononuclear cells samples from LTBI and healthy participants reflected the altered patterns observed in the microarray profile. The bioinformatic analyses suggest that the miRNAs may regulate Mtb latent infection by affecting the development of macrophage cells.
Conclusions
The results suggest that miRNA expression may play a considerable role in the pathogenesis of LTBI, and this would increase our understanding of the molecular basis of Hsp16.3-facilitated Mtb survival in macrophages.
doi:10.1186/1471-2180-14-37
PMCID: PMC3925440  PMID: 24521422
microRNAs; Macrophages; Mycobacterium; Tuberculosis; Small heat shock protein; Latent tuberculosis infection
17.  Chronic cerebral hypoperfusion causes decrease of O-GlcNAcylation, hyperphosphorylation of tau and behavioral deficits in mice 
Chronic cerebral hypoperfusion (CCH) is one of the causes of vascular dementia (VaD) and is also an etiological factor for Alzheimer’s disease (AD). However, how CCH causes cognitive impairment and contributes to Alzheimer’s pathology is poorly understood. Here we produced a mouse model of CCH by unilateral common carotid artery occlusion (UCCAO) and studied the behavioral changes and brain abnormalities in mice 2.5 months after UCCAO. We found that CCH caused significant short-term memory deficits and mild long-term spatial memory impairment, as well as decreased level of protein O-GlcNAcylation, increased level of tau phosphorylation, dysregulated synaptic proteins and insulin signaling, and selective neurodegeneration in the brain. These findings provide mechanistic insight into the effects of CCH on memory and cognition and the likely link between AD and VaD.
doi:10.3389/fnagi.2014.00010
PMCID: PMC3918671  PMID: 24575038
chronic cerebral hypoperfusion; Alzheimer’s disease; cognitive impairment; O-GlcNAcylation; tau phosphorylation; synaptic plasticity markers; brain insulin signaling; neurodegeneration
18.  8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin 
AIM: To evaluate whether 8-bromo-7-methoxychrysin (BrMC), a synthetic analogue of chrysin, inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.
METHODS: CD133+ cells were sorted from the MHCC97 cell line by magnetic activated cell sorting, and amplified in stem cell-conditioned medium to obtain the enriched CD133+ sphere forming cells (SFCs). The stem cell properties of CD133+ SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo, and termed liver cancer stem cells (LCSCs). The effects of BrMC on LCSCs in vitro were evaluated by MTT assay, tumorsphere formation assay and transwell chamber assay. The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice. Expressions of the stem cell markers, epithelial-mesenchymal transition (EMT) markers and β-catenin protein were analyzed by western blotting or immunohistochemical analysis.
RESULTS: CD133+ SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells. We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs (P < 0.05). Furthermore, BrMC significantly suppressed EMT and invasion of LCSCs. Moreover, BrMC could efficaciously eliminate LCSCs in vivo. Interestingly, we showed that BrMC decreased the expression of β-catenin in LCSCs. Silencing of β-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC, while Wnt3a treatment antagonized the inhibitory effects of BrMC.
CONCLUSION: BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation of β-catenin expression.
doi:10.3748/wjg.v19.i43.7680
PMCID: PMC3837267  PMID: 24431896
Liver cancer; Cancer stem cell; 8-bromo-7-methoxychrysin; Self-renewal; β-catenin
19.  Hypoxia Promotes Epithelial - Mesenchymal Transition of Hepatocellular Carcinoma Cells via Inducing GLIPR-2 Expression 
PLoS ONE  2013;8(10):e77497.
Glioma pathogenesis related-2 (GLIPR-2) belongs to pathogenesis related-1 (PR-1) family whose function remains unknown. In our previous studies, GLIPR-2 was found to be a novel potent stimulator of epithelial-to-mesenchymal transition (EMT) in renal fibrosis which has been classified as type 2 EMT. However, whether GLIPR-2 could induce type 3 EMT in carcinogenesis needs further investigation. In this study, we showed that GLIPR-2 was expressed in hepatocellular carcinoma (HCC) tissues, hypoxia could upregulate the expression of GLIPR-2 in HepG2 and PLC/PRF/5 cells in vitro, overexpression of this protein promoted migration and invasion via EMT, knockdown of GLIPR-2 attenuated migration and invasion of HepG2 and PLC/PRF/5 cells in hypoxia. Moreover, extracellular signal-regulated kinases 1 and 2 (ERK1/2) are positively regulated by GLIPR-2. Taken together, we provide evidence for a hypoxia/GLIPR-2/EMT/migration and invasion axis in HCC cells and it provides novel insights into the mechanism of migration and invasion of hepatocellular carcinoma cells in hypoxia condition.
doi:10.1371/journal.pone.0077497
PMCID: PMC3812270  PMID: 24204846
20.  Gadoxetic Acid Disodium-Enhanced Magnetic Resonance Imaging for the Detection of Hepatocellular Carcinoma: A Meta-Analysis 
PLoS ONE  2013;8(8):e70896.
Objective
To determine the accuracy of MR imaging with gadoxetic acid disodium (Gd-EOB-DTPA) for the detection of hepatocelluar carcinoma (HCC).
Materials and Methods
A systematic search was performed in PUBMED, EMBASE, Web of Science, Cochrane Library and the Chinese Biomedical Literature Database up to March 2013 to identify studies about evaluation of Gd-EOB-DTPA enhanced MR imaging in patients suspected of having HCC. The data were extracted to perform heterogeneity test and threshold effect test and to calculate sensitivity, specificity, diagnostic odds ratio, predictive value, and areas under summary receiver operating characteristic curve (AUC).
Results
From 601 citations, 10 were included in the meta-analysis. The methodological quality of the 10 studies was good. Overall HCC: There was significant heterogeneity in the pooled analysis (I2 = 69.4%, P = 0.0005), and the pooled weighted values were determined to be sensitivity: 0.91 (95% confidence interval (CI): 0.89, 0. 93); specificity: 0.95 (95% CI: 0.94, 0.96); diagnostic odds ratio: 169.94 (95% CI: 108.84, 265.36); positive likelihood ratio: 15.75 (95% CI: 7.45, 33.31); negative likelihood ratio: 0.10 (95% CI: 0.06, 0.15). The AUC was 0.9778. HCC in cirrhosis: The estimates were to be sensitivity: 0.91 (95% CI: 0.88, 0.93); specificity: 0.93 (95% CI: 0.89, 0.95); diagnostic odds ratio: 234.24 (95% CI: 33.47, 1639.25); positive likelihood ratio: 15.08 (95% CI: 2.20, 103.40); negative likelihood ratio: 0.08 (95% CI: 0.03, 0.21). The AUC was 0.9814. ≤20 mm HCC: The AUC was 0.9936. There was no notable publication bias.
Conclusions
This meta-analysis suggests that MR imaging with Gd-EOB-DTPA has high diagnostic accuracy for the detection of HCC, especially for ≤20 mm HCC. This technique shows good prospect in diagnosis of HCC.
doi:10.1371/journal.pone.0070896
PMCID: PMC3744536  PMID: 23967130
21.  GLIPR-2 Overexpression in HK-2 Cells Promotes Cell EMT and Migration through ERK1/2 Activation 
PLoS ONE  2013;8(3):e58574.
The epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in the adult kidney is one of the key events in renal interstitial fibrosis. Glioma pathogenesis related-2 (GLIPR-2) has been shown to be up-regulated in proximal tubular cells (PTCs) in the fibrotic kidney. However, the biological function of GLIPR-2 remains unknown. In this study, we found that GLIPR-2 expression is elevated in the kidney tissue samples of patients with diabetic nephropathy (DN). Human proximal renal tubular epithelial cells (HK-2 cells) were transfected with pcDNA3.0-GLIPR-2 and selected with G418. To identify the biological function of GLIPR-2, an epithelial-to-mesenchymal transition (EMT) PCR array analysis was performed, and genes that had statistically significantly altered expression levels with more than a two-fold difference compared with the pcDNA3.0-transfected HK-2 cells were considered. Key elements of the EMT process, such as E-cadherin and vimentin, were transcriptionally activated in the pcDNA3.0-GLIPR-2-transfected sublines. In addition, α-SMA gene expression, which is a marker of myofibroblasts, increased in the pcDNA3.0-GLIPR-2-transfected HK-2 cells. The cell migration assay demonstrated that the transfection of HK-2 with GLIPR-2 promoted cell migration following an EMT. Additionally, consistent with the effects of increased EGFR expression levels, we found that the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was highly elevated in the pcDNA3.0-GLIPR-2-transfected group. Our study demonstrates that GLIPR-2 overexpression in HK-2 cells can potentiate EMT-like processes in this cell type through the ERK1/2 signaling pathway. GLIPR-2 may be responsible for the development of renal fibrosis by increasing the accumulation of interstitial fibroblasts.
doi:10.1371/journal.pone.0058574
PMCID: PMC3596275  PMID: 23516513
22.  HTQC: a fast quality control toolkit for Illumina sequencing data 
BMC Bioinformatics  2013;14:33.
Background
Illumina sequencing platform is widely used in genome research. Sequence reads quality assessment and control are needed for downstream analysis. However, software that provides efficient quality assessment and versatile filtration methods is still lacking.
Results
We have developed a toolkit named HTQC – abbreviation of High-Throughput Quality Control – for sequence reads quality control, which consists of six programs for reads quality assessment, reads filtration and generation of graphic reports.
Conclusions
The HTQC toolkit can generate reads quality assessment faster than existing tools, providing guidance for reads filtration utilities that allow users to choose different strategies to remove low quality reads.
doi:10.1186/1471-2105-14-33
PMCID: PMC3571943  PMID: 23363224
23.  Amelioration of Albuminuria in ROCK1 Knockout Mice with Streptozotocin-Induced Diabetic Kidney Disease 
American Journal of Nephrology  2011;34(5):468-475.
Background
Although blockade of Rho kinase with pharmacologic inhibitors ameliorates renal fibrosis and diabetic kidney disease (DKD), the underlined mechanisms remain largely unclear. The present study tested the hypothesis that ROCK1 may regulate the early development of albuminuria via the megalin/cubilin-dependent mechanism.
Methods
A DKD model was induced in ROCK1 knockout and wild-type mice by streptozotocin (STZ). The effect of deleted ROCK1 on urinary albumin excretion and the expression of megalin/cubilin were examined. In addition, the effect of blocking ROCK activities with an inhibitor (Y-27632) on tubular albumin reabsorption was tested in a normal rat tubular epithelial cell line (NRK52E) under high-glucose conditions. Expression of transforming growth factor (TGF)-β1, interleukin-1β and collagen-1 was also been examined.
Results
Urinary albumin excretion was significantly increased in ROCK1 WT mice at 8 weeks after STZ injection. In contrast, mice lacking ROCK1 gene were protected against the development of albuminuria. This was associated with the protection against the loss of megalin/cubilin and an increase in TGF-β1, IL-1β, and fibrosis in the kidney. In vitro, we also found that blockade of Rho kinase with inhibitor Y-27632 prevented high-glucose-induced loss of megalin expression and an increase of TGF-β1, thereby increasing the absorption rate of FITC-labeled albumin by tubular epithelial cells.
Conclusion
ROCK1 may play a role in the development of albuminuria in DKD by downregulating the endocytosis receptors complex – megalin/cubilin.
Copyright © 2011 S. Karger AG, Basel
doi:10.1159/000332040
PMCID: PMC3691875  PMID: 21986457
Diabetic kidney disease; Tubular cells, albuminuria; ROCK; Megalin; Cubilin
24.  Developmental Regulation of Protein O-GlcNAcylation, O-GlcNAc Transferase, and O-GlcNAcase in Mammalian Brain 
PLoS ONE  2012;7(8):e43724.
O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins by β-N-acetylglucosamine (GlcNAc). The dynamic addition and removal of O-GlcNAc groups to and from proteins are catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA), respectively. O-GlcNAcylation often modulates protein phosphorylation and regulates several cellular signaling and functions, especially in the brain. However, its developmental regulation is not well known. Here, we studied protein O-GlcNAcylation, OGT, and OGA in the rat brain at various ages from embryonic day 15 to the age of 2 years. We found a gradual decline of global protein O-GlcNAcylation during developmental stages and adulthood. This decline correlated positively to the total protein phosphorylation at serine residues, but not at threonine residues. The expression of OGT and OGA isoforms was regulated differently at various ages. Immunohistochemical studies revealed ubiquitous distribution of O-GlcNAcylation at all ages. Strong immunostaining of O-GlcNAc, OGT, and OGA was observed mostly in neuronal cell bodies and processes, further suggesting the role of O-GlcNAc modification of neuronal proteins in the brain. These studies provide fundamental knowledge of age-dependent protein modification by O-GlcNAc and will help guide future studies on the role of O-GlcNAcylation in the mammalian brain.
doi:10.1371/journal.pone.0043724
PMCID: PMC3425547  PMID: 22928023
25.  The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation 
PLoS ONE  2012;7(8):e43204.
Objective
Hepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.
Methods
We established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.
Results
HBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.
Conclusion
miR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.
doi:10.1371/journal.pone.0043204
PMCID: PMC3422285  PMID: 22912826

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