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1.  Functional Study of the Hap4-Like Genes Suggests That the Key Regulators of Carbon Metabolism HAP4 and Oxidative Stress Response YAP1 in Yeast Diverged from a Common Ancestor 
PLoS ONE  2014;9(12):e112263.
The transcriptional regulator HAP4, induced by respiratory substrates, is involved in the balance between fermentation and respiration in S. cerevisiae. We identified putative orthologues of the Hap4 protein in all ascomycetes, based only on a conserved sixteen amino acid-long motif. In addition to this motif, some of these proteins contain a DNA-binding motif of the bZIP type, while being nonetheless globally highly divergent. The genome of the yeast Hansenula polymorpha contains two HAP4-like genes encoding the protein HpHap4-A which, like ScHap4, is devoid of a bZIP motif, and HpHap4-B which contains it. This species has been chosen for a detailed examination of their respective properties. Based mostly on global gene expression studies performed in the S. cerevisiae HAP4 disruption mutant (ScΔhap4), we show here that HpHap4-A is functionally equivalent to ScHap4, whereas HpHap4-B is not. Moreover HpHAP4-B is able to complement the H2O2 hypersensitivity of the ScYap1 deletant, YAP1 being, in S. cerevisiae, the main regulator of oxidative stress. Finally, a transcriptomic analysis performed in the ScΔyap1 strain overexpressing HpHAP4-B shows that HpHap4-B acts both on oxidative stress response and carbohydrate metabolism in a manner different from both ScYap1 and ScHap4. Deletion of these two genes in their natural host, H. polymorpha, confirms that HpHAP4-A participates in the control of the fermentation/respiration balance, while HpHAP4-B is involved in oxidative stress since its deletion leads to hypersensitivity to H2O2. These data, placed in an evolutionary context, raise new questions concerning the evolution of the HAP4 transcriptional regulation function and suggest that Yap1 and Hap4 have diverged from a unique regulatory protein in the fungal ancestor.
PMCID: PMC4257542  PMID: 25479159
2.  Alternative splicing at GYNNGY 5′ splice sites: more noise, less regulation 
Nucleic Acids Research  2014;42(22):13969-13980.
Numerous eukaryotic genes are alternatively spliced. Recently, deep transcriptome sequencing has skyrocketed proportion of alternatively spliced genes; over 95% human multi-exon genes are alternatively spliced. One fundamental question is: are all these alternative splicing (AS) events functional? To look into this issue, we studied the most common form of alternative 5′ splice sites—GYNNGYs (Y = C/T), where both GYs can function as splice sites. Global analyses suggest that splicing noise (due to stochasticity of splicing process) can cause AS at GYNNGYs, evidenced by higher AS frequency in non-coding than in coding regions, in non-conserved than in conserved genes and in lowly expressed than in highly expressed genes. However, ∼20% AS GYNNGYs in humans and ∼3% in mice exhibit tissue-dependent regulation. Consistent with being functional, regulated GYNNGYs are more conserved than unregulated ones. And regulated GYNNGYs have distinctive sequence features which may confer regulation. Particularly, each regulated GYNNGY comprises two splice sites more resembling each other than unregulated GYNNGYs, and has more conserved downstream flanking intron. Intriguingly, most regulated GYNNGYs may tune gene expression through coupling with nonsense-mediated mRNA decay, rather than encode different proteins. In summary, AS at GYNNGY 5′ splice sites is primarily splicing noise, and secondarily a way of regulation.
PMCID: PMC4267661  PMID: 25428370
3.  Impairment of Atg5-Dependent Autophagic Flux Promotes Paraquat- and MPP+-Induced Apoptosis But Not Rotenone or 6-Hydroxydopamine Toxicity 
Toxicological Sciences  2013;136(1):166-182.
Controversial reports on the role of autophagy as a survival or cell death mechanism in dopaminergic cell death induced by parkinsonian toxins exist. We investigated the alterations in autophagic flux and the role of autophagy protein 5 (Atg5)-dependent autophagy in dopaminergic cell death induced by parkinsonian toxins. Dopaminergic cell death induced by the mitochondrial complex I inhibitors 1-methyl-4-phenylpyridinium (MPP+) and rotenone, the pesticide paraquat, and the dopamine analog 6-hydroxydopamine (6-OHDA) was paralleled by increased autophagosome accumulation. However, when compared with basal autophagy levels using chloroquine, autophagosome accumulation was a result of impaired autophagic flux. Only 6-OHDA induced an increase in autophagosome formation. Overexpression of a dominant negative form of Atg5 increased paraquat- and MPP+-induced cell death. Stimulation of mammalian target of rapamycin (mTOR)-dependent signaling protected against cell death induced by paraquat, whereas MPP+-induced toxicity was enhanced by wortmannin, a phosphoinositide 3-kinase class III inhibitor, rapamycin, and trehalose, an mTOR-independent autophagy activator. Modulation of autophagy by either pharmacological or genetic approaches had no effect on rotenone or 6-OHDA toxicity. Cell death induced by parkinsonian neurotoxins was inhibited by the pan caspase inhibitor (Z-VAD), but only caspase-3 inhibition was able to decrease MPP+-induced cell death. Finally, inhibition of the lysosomal hydrolases, cathepsins, increased the toxicity by paraquat and MPP+, supporting a protective role of Atg5-dependent autophagy and lysosomes degradation pathways on dopaminegic cell death. These results demonstrate that in dopaminergic cells, Atg5-dependent autophagy acts as a protective mechanism during apoptotic cell death induced by paraquat and MPP+ but not during rotenone or 6-OHDA toxicity.
PMCID: PMC3829573  PMID: 23997112
autophagy; apoptosis; Atg5; cathepsins; paraquat; rotenone; MPP+; 6-hydroxydopamine; neurodegeneration; Parkinson’s disease.
4.  Compartmentalized oxidative stress in dopaminergic cell death induced by pesticides and complex I inhibitors: Distinct roles of superoxide anion and superoxide dismutases 
The loss of dopaminergic neurons induced by the parkinsonian toxins paraquat, rotenone and 1-methyl-4-phenylpyridinium (MPP+) is associated with oxidative stress. However, controversial reports exist regarding the source/compartmentalization of reactive oxygen species (ROS) generation and its exact role in cell death. We aimed to determine in detail the role of superoxide anion (O2•−), oxidative stress and their subcellular compartmentalization in dopaminergic cell death induced by parkinsonian toxins. Oxidative stress and ROS formation was determined in the cytosol, intermembrane (IMS) and mitochondrial matrix compartments, using dihydroethidine derivatives, the redox sensor roGFP, as well as electron paramagnetic resonance spectroscopy. Paraquat induced an increase in ROS and oxidative stress in both the cytosol and mitochondrial matrix prior to cell death. MPP+ and rotenone primarily induced an increase in ROS and oxidative stress in the mitochondrial matrix. No oxidative stress was detected at the level of the IMS. In contrast to previous studies, overexpression of manganese superoxide dismutase (MnSOD) or copper/zinc SOD (CuZnSOD) had no effect on ROS steady state levels, lipid peroxidation, loss of mitochondrial membrane potential (ΔΨm) and dopaminergic cell death induced by MPP+ or rotenone. In contrast, paraquat-induced oxidative stress and cell death were selectively reduced by MnSOD overexpression, but not by CuZnSOD or manganese-porphyrins. However, MnSOD also failed to prevent ΔΨm loss. Finally, paraquat, but not MPP+ or rotenone, induced the transcriptional activation the redox-sensitive antioxidant response elements (ARE) and nuclear factor kappa-B (NF-κB). These results demonstrate a selective role of mitochondrial O2•− in dopaminergic cell death induced by paraquat, and show that toxicity induced by the complex I inhibitors rotenone and MPP+ does not depend directly on mitochondrial O2•− formation.
PMCID: PMC3883883  PMID: 23602909
Paraquat; MPP+; rotenone; roGFP; SOD; MnSOD; CuZnSOD; porphyrins; pesticides; environmental; Parkinson’s disease
5.  Diagnostic Efficacy of Cell Block Immunohistochemistry, Smear Cytology, and Liquid-Based Cytology in Endoscopic Ultrasound-Guided Fine-Needle Aspiration of Pancreatic Lesions: A Single-Institution Experience 
PLoS ONE  2014;9(9):e108762.
The diagnostic efficiency of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) cytology varies widely depending on the treatment method of the specimens. The present study aimed to evaluate the diagnostic efficacy of cell block (CB) immunohistochemistry, smear cytology (SC), and liquid-based cytology (LBC) in patients with pancreatic lesions without consulting an on-site cytopathologist.
This study prospectively enrolled 72 patients with pancreatic lesions. The EUS-FNA specimens were examined by SC, LBC, and CB immunohistochemistry. The diagnostic efficacy of the 3 methods was then compared. Patients’ final diagnosis was confirmed by surgical resection specimens, diagnostic imaging, and clinical follow-up.
Our results included 60 malignant and 12 benign pancreatic lesions. The diagnostic sensitivity (90%), negative predictive value (66.7%), and accuracy (91.7%) of CB immunohistochemistry were significantly higher than those of SC (70.0%, 30.0%, and 75.0%, respectively) and LBC (73.3%, 31.6%, and 77.8%, respectively) (all P<0.05). The combination of CB and SC, or CB and LBC, did not significantly increase the efficacy compared to CB immunohistochemistry alone.
Our findings suggest that in the absence of an on-site cytopathologist, CB immunohistochemistry on EUS-FNA specimens offers a higher diagnostic efficacy in patients with pancreatic lesions than does SC and LBC.
PMCID: PMC4178202  PMID: 25259861
6.  Discriminating between Lysine Sumoylation and Lysine Acetylation Using mRMR Feature Selection and Analysis 
PLoS ONE  2014;9(9):e107464.
Post-translational modifications (PTMs) are crucial steps in protein synthesis and are important factors contributing to protein diversity. PTMs play important roles in the regulation of gene expression, protein stability and metabolism. Lysine residues in protein sequences have been found to be targeted for both types of PTMs: sumoylations and acetylations; however, each PTM has a different cellular role. As experimental approaches are often laborious and time consuming, it is challenging to distinguish the two types of PTMs on lysine residues using computational methods. In this study, we developed a method to discriminate between sumoylated lysine residues and acetylated residues. The method incorporated several features: PSSM conservation scores, amino acid factors, secondary structures, solvent accessibilities and disorder scores. By using the mRMR (Maximum Relevance Minimum Redundancy) method and the IFS (Incremental Feature Selection) method, an optimal feature set was selected from all of the incorporated features, with which the classifier achieved 92.14% accuracy with an MCC value of 0.7322. Analysis of the optimal feature set revealed some differences between acetylation and sumoylation. The results from our study also supported the previous finding that there exist different consensus motifs for the two types of PTMs. The results could suggest possible dominant factors governing the acetylation and sumoylation of lysine residues, shedding some light on the modification dynamics and molecular mechanisms of the two types of PTMs, and provide guidelines for experimental validations.
PMCID: PMC4164654  PMID: 25222670
7.  KIF5B-RET fusion kinase promotes cell growth by multilevel activation of STAT3 in lung cancer 
Molecular Cancer  2014;13:176.
Lung cancer in nonsmokers tends to be driven by a single somatic mutation or a gene fusion. KIF5B-RET fusion is an oncogene identified in non-small cell lung cancers. In this study, we verified the oncogenic activity of KIF5B-RET fusion and investigated how KIF5B-RET activates the specific signaling pathways for cellular transformation. We aimed to provide a basis for the further development of the therapy for KIF5B-RET positive lung cancer patients.
RT-PCR was used to screen for KIF5B-RET fusions in Chinese lung cancer patients. To verify the oncogenic activity of KIF5B-RET kinase in lung cancer cells, we manipulated its expression genetically followed by colony formation and tumor formation assays. The mechanism by which KIF5B-RET kinase induces proliferation was investigated by western blot, coimmunoprecipitation, and administration of RET, MAPK and STAT3 inhibitors.
Our study identified a KIF5B-RET fusion in Chinese NSCLC patients and demonstrated that KIF5B-RET transfected cells showed a significantly increased proliferation rate and colony-forming ability. Furthermore, we found that KIF5B-RET fusion kinase induced multilevel activation of STAT3 at both Tyr705 and Ser727, and KIF5B-RET-STAT3 signaling related inhibitors repressed the proliferation and tumorigenicity of lung cancer cells significantly.
Our data suggest that KIF5B-RET promotes the cell growth and tumorigenicity of non-small cell lung cancers through multilevel activation of STAT3 signaling, providing possible strategies for the treatment of KIF5B-RET positive lung cancers.
PMCID: PMC4114102  PMID: 25047660
KIF5B-RET; Lung cancer; Cell growth; STAT3 pathway
8.  Combined Analysis with Copy Number Variation Identifies Risk Loci in Lung Cancer 
BioMed Research International  2014;2014:469103.
Background. Lung cancer is the most important cause of cancer mortality worldwide, but the underlying mechanisms of this disease are not fully understood. Copy number variations (CNVs) are promising genetic variations to study because of their potential effects on cancer. Methodology/Principal Findings. Here we conducted a pilot study in which we systematically analyzed the association of CNVs in two lung cancer datasets: the Environment And Genetics in Lung cancer Etiology (EAGLE) and the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial datasets. We used a preestablished association method to test the datasets separately and conducted a combined analysis to test the association accordance between the two datasets. Finally, we identified 167 risk SNP loci and 22 CNVs associated with lung cancer and linked them with recombination hotspots. Functional annotation and biological relevance analyses implied that some of our predicted risk loci were supported by other studies and might be potential candidate loci for lung cancer studies. Conclusions/Significance. Our results further emphasized the importance of copy number variations in cancer and might be a valuable complement to current genome-wide association studies on cancer.
PMCID: PMC4100386  PMID: 25093167
9.  A prospective study of midfoot osteotomy combined with adjacent joint sparing internal fixation in treatment of rigid pes cavus deformity 
Midfoot osteotomy has been previously confirmed to be a good method to correct pes cavus. How to fix the osteotomy and which point to choose for the procedure has been a focus for most surgeons. The aim of this study was to analyse the outcomes of a series of patients who had been treated for pes cavus deformity using midfoot osteotomy combined with adjacent joint sparing internal fixation.
Materials and methods
Between 2008 and 2012, 17 patients with a mean age of 16.8 years (12–36 years) were tracked after treatment by midfoot osteotomy combined with adjacent joint sparing internal fixation with three cannulated screws between the Lisfranc line and Cyma line. Clinical outcomes were assessed by measuring improvements of appearance and function, American Orthopedic Foot and Ankle Society (AOFAS) scores, and radiographic changes.
The mean follow-up time was 25.3 months (range, 10–50). The mean healing time from the osteotomy was 7.8 weeks (range, 6–12). The appearance and weight-bearing function were significantly improved in all patients. At a final follow-up, the mean AOFAS score was 75.8/100 points (range, 63–90). The mean Meary's angle, calcaneal pitch angle, tibiotalar angle, and Hibb's angle values improved from 26.3 to 5.5, 44.5 to 28.3, 133.1 to 100.8 and 66.9 to 41.1, respectively. Adjacent joints presented no obviously arthritic degeneration at the follow-up. Subjectively, 94.1% of patients were very satisfied or satisfied with minor reservations. Objective outcomes were excellent or good in 88.2% of feet.
For the treatment of rigid pes cavus deformity, extra-articular midfoot osteotomy combined with adjacent joint sparing internal fixation is effective and safe. This surgical technique is especially effective with low rates of arthritic degeneration and joint stiffness in the adjacent joints and little reduction of ankle and foot flexibility.
PMCID: PMC4067371  PMID: 24898481
Pes cavus; Midfoot osteotomy; Treatment; Adjacent joint sparing internal fixation
10.  IL-1β and TNF-α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase 
Journal of neurochemistry  2013;125(6):897-908.
Glutaminase 1 is the main enzyme responsible for glutamate production in mammalian cells. The roles of macrophage and microglia glutaminases in brain injury, infection, and inflammation are well documented. However, little is known about the regulation of neuronal glutaminase, despite neurons being a predominant cell type of glutaminase expression. Using primary rat and human neuronal cultures, we confirmed that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), two proinflammatory cytokines that are typically elevated in neurodegenerative disease states, induced neuronal death and apoptosis in vitro. Furthermore, both intracellular and extracellular glutamate levels were significantly elevated following IL-1β and/or TNF-α treatment. Pretreatment with N-Methyl-D-aspartate (NMDA) receptor antagonist MK-801 blocked cytokine-induced glutamate production and alleviated the neurotoxicity, indicating that IL-1β and/or TNF-α induce neurotoxicity through glutamate. To determine the potential source of excess glutamate production in the culture during inflammation, we investigated the neuronal glutaminase and found that treatment with IL-1β or TNF-α significantly upregulated the kidney type glutaminase (KGA), a glutaminase 1 isoform, in primary human neurons. The upregulation of neuronal glutaminase was also demonstrated in situ in a murine model of HIV-1 encephalitis. In addition, IL-1β or TNF-α treatment increased the levels of KGA in cytosol and TNF-α specifically increased KGA levels in the extracellular fluid, away from its main residence in mitochondria. Together, these findings support neuronal glutaminase as a potential component of neurotoxicity during inflammation and that modulation of glutaminase may provide therapeutic avenues for neurodegenerative diseases.
PMCID: PMC3747774  PMID: 23578284
Inflammation; glutaminase; glutamate; neurotoxicity
11.  Polymeric Nanoparticles Containing Combination Antiretroviral Drugs for HIV Type 1 Treatment 
The use of combination antiretroviral nanoparticles (cART NPs) was investigated as a novel treatment approach for the inhibition of HIV-1 replication. We developed nanoparticles of biodegradable polymer, poly-(dl-lactide-co-glycolic acid; PLGA) containing efavirenz (EFV) and boosted lopinavir (lopinavir/ritonavir; LPV/r) by a high-pressure homogenization method. The method resulted in >79% drug entrapment efficiency for each of the three drugs. The average size of cART NPs was 138.3±55.4 nm as measured by dynamic light scanning, confirmed by scanning electron microscopy (SEM) with an average surface charge of −13.7±4.5. Lissamine-rhodamine-labeled fluorescent PLGA NPs exhibited efficient uptake in nonimmune (HeLa cells) and immune (H9 T cells) cells as measured by confocal microscopy. Cells treated with cART NPs resulted in minimal loss of cell viability over 28 days. Subcellular fractionation studies demonstrated that HIV-1-infected H9 monocytic cells treated with cART NPs contained significantly (p<0.05) higher nuclear, cytoskeleton, and membrane antiretroviral drug levels compared to cells treated with drug solutions alone. Finally, cART NPs efficiently inhibited HIV-1 infection and transduction. The IC50 for each of the three drugs in the cART NPs was <31 nM. These experiments demonstrate the efficacy of a novel PLGA NPs formulation for the delivery of cART to inhibit HIV-1 replication.
PMCID: PMC3636590  PMID: 23289671
12.  Histone preconditioning protects against obstructive jaundice-induced liver injury in rats 
A major consequence of obstructive jaundice (OJ) in clinical practice is the development of severe liver injury, and at present, no effective treatments have been developed to protect against it. Preconditioning with damage-associated molecular pattern (DAMP) molecules has been demonstrated to protect multiple organs from injury, and histones have been recently identified as DAMP molecules. The aim of the present study was to investigate the protective effect of histone preconditioning against OJ-induced liver injury in rats and the involvement of Toll-like receptors. Rats were administered histone proteins (200 μg/kg; 1 ml) or physiological saline (1 ml) intraperitoneally 24 h prior to being subjected to bile duct ligation (BDL). The serum levels of liver enzymes and bilirubin, as well as the histopathology were analyzed. The mRNA expression of interleukin-6 (IL-6) in the liver tissue was analyzed using quantitative polymerase chain reaction. BDL in the control group caused severe OJ-induced liver injury, as indicated by the significantly elevated levels of liver enzymes and mRNA levels of IL-6, and confirmed by histopathological alterations. However, histone preconditioning significantly ameliorated the OJ-induced liver injury caused by BDL, as shown by an improvement in the levels of liver enzymes, a suppression of IL-6 production, as well as histopathological alterations. Therefore, these results suggested that histone preconditioning is able to protect against OJ-induced liver injury in rats.
PMCID: PMC4061184  PMID: 24944590
histone; precondition; obstructive jaundice; liver injury
13.  Th22 cells are associated with hepatocellular carcinoma development and progression 
IL-22-producing CD4+ T helper cells (Th22 cells) have been identified as major inducers of tissue inflammation and immune responses. Currently, no previous study explored the role of Th22 cells in the pathogenesis of hepatocellular carcinoma (HCC). The study aimed to determine the biological function of Th22 cells and its effector IL-22 in HCC patients.
Forty-five HCC patients and 19 healthy controls were recruited and their peripheral blood was collected. The fresh HCC tissues, adjacent HCC tissues and ten normal liver tissues were also collected. Flow cytometry analysis was used to determine the frequencies of circulating Th22 cells and Th17 cells. Serum IL-22 levels were tested by enzyme-linked immunosorbent assay (ELISA). Immunohistochemical staining and real-time polymerase chain reaction (PCR) were used to detect IL-22 protein and mRNA in tissues specimens, respectively.
Circulating Th22 cells, Th17 cells and serum IL-22 levels were significantly elevated in HCC patients compared with those of healthy controls (P<0.001). Th22 cells were showed to be positively correlated with IL-22 in HCC patients (P<0.05), but not in healthy controls. No significant differences were found in HCC patients with HBeAg positivity or negativity in term of Th22 cells and serum IL-22 levels. The expression of IL-22 protein and mRNA was highest in HCC tissues, followed by adjacent HCC tissues and normal liver tissues. Furthermore, Th22 cells, serum IL-22 levels and IL-22 mRNA were elevated at stage III-IV compared with stage I-II of HCC (P<0.05).
Elevation of circulating Th22 cells and IL-22 may be implicated in the pathogenesis of HCC, and potentially be cellular targets for therapeutic intervention.
PMCID: PMC4000908  PMID: 24826053
Th22; IL-22; hepatocellular carcinoma (HCC)
14.  Direct Staining with Major Histocompatibility Complex Class II Dextramers Permits Detection of Antigen-Specific, Autoreactive CD4 T Cells In Situ 
PLoS ONE  2014;9(1):e87519.
We report here the utility of major histocompatibility complex (MHC) class II dextramers for in situ detection of self-reactive CD4 T cells in two target organs, the brain and heart. We optimized the conditions for in situ detection of antigen-specific CD4 T cells using brain sections obtained from SJL mice immunized with myelin proteolipid protein (PLP) 139–151; the sections were costained with IAs/PLP 139–151 (specific) or Theiler's murine encephalomyelitis virus (TMEV) 70–86 (control) dextramers and anti-CD4. Analysis of sections by laser scanning confocal microscope revealed detection of cells positive for PLP 139–151 but not for TMEV 70–86 dextramers to be colocalized with CD4-expressing T cells, indicating that the staining was specific to PLP 139–151 dextramers. Further, we devised a method to reliably enumerate the frequencies of antigen-specific T cells by counting the number of dextramer+ CD4+ T cells in the ‘Z’ serial images acquired sequentially. We next extended these observations to detect cardiac myosin-specific T cells in autoimmune myocarditis induced in A/J mice by immunizing with cardiac myosin heavy chain-α (Myhc) 334–352. Heart sections prepared from immunized mice were costained with Myhc 334–352 (specific) or bovine ribonuclease 43–56 (control) dextramers together with anti-CD4; the sections showed the infiltrations of Myhc-specific CD4 T cells. The data suggest that MHC class II dextramers are useful tools for enumerating the frequencies of antigen-specific CD4 T cells in situ by direct staining without having to amplify the fluorescent signals, an approach commonly employed with conventional MHC tetramers.
PMCID: PMC3903673  PMID: 24475302
15.  Insulin-Like Growth Factor-1 and Bone Morphogenetic Protein-2 Jointly Mediate Prostaglandin E2-Induced Adipogenic Differentiation of Rat Tendon Stem Cells 
PLoS ONE  2014;9(1):e85469.
Tendinopathy is characterized histopathologically by lipid accumulation and tissue calcification. Adipogenic and osteogenic differentiation of tendon stem cells (TSCs) are believed to play key roles in these processes. The major inflammatory mediator prostaglandin E2 (PGE2) has been shown to induce osteogenic differentiation of TSCs via bone morphogenetic protein-2 (BMP-2), and BMP-2 has also been implicated in adipogenic differentiation of stem cells. We therefore examined the mechanisms responsible for PGE2-induced adipogenesis in rat TSCs in vitro. Insulin-like growth factor-1 (IGF-1) mRNA and protein were significantly up-regulated in PGE2-stimulated TSCs, measured by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Incubation with specific inhibitors of cAMP, cAMP-dependent protein kinase A (PKA), and CCAAT/enhancer binding protein-δ (CEBPδ) demonstrated that IGF-1 up-regulation occurred via a cAMP/PKA/CEBPδ pathway. Furthermore, neither IGF-1 nor BMP-2 alone was able to mediate adipogenic differentiation of TSCs, but IGF-1 together with BMP-2 significantly increased adipogenesis, indicated by Oil Red O staining. Moreover, knock-down of endogenous IGF-1 and BMP2 abolished PGE2-induced adipogenic differentiation. Phosphorylation of CREB and Smad by IGF-1 and BMP-2, respectively, were required for induction of the adipogenesis-related peroxisome proliferator-activated receptor γ2 (PPARγ2) gene and for adipogenic differentiation. In conclusion, IGF-1 and BMP-2 together mediate PGE2-induced adipogenic differentiation of TSCs in vitro via a CREB- and Smad-dependent mechanism. This improved understanding of the mechanisms responsible for tendinopathies may help the development of more effective therapies.
PMCID: PMC3887066  PMID: 24416413
16.  Association of interleukin-10 polymorphisms with risk of irritable bowel syndrome: A meta-analysis 
AIM: To clarify the current understanding of the association between interleukin-10 (IL-10) polymorphisms and the risk of irritable bowel syndrome (IBS).
METHODS: We searched for studies in any language recorded in PubMed, Embase and Cochrane library before August 2013. The associations under allele contrast model, codominant model, dominant model, and recessive model were analyzed. The strengths of the association between IL-10 polymorphisms and IBS risk were estimated using odds ratios (OR) with 95% confidence interval (CI). Fixed effects model was used to pool the result if the test of heterogeneity was not significant, otherwise the random-effect model was selected.
RESULTS: Eight case-control studies analyzing three single-nucleotide polymorphisms rs1800870 (-1082 A/G), rs1800871 (-819C/T), and rs1800872 (-592A/C) of the IL-10 gene, which involved 928 cases and 1363 controls, were eligible for our analysis. The results showed that rs1800870 polymorphisms were associated with a decreased risk of IBS (GG+GA vs AA: OR = 0.80, 95%CI: 0.66-0.96), (AA+GA vs GG: OR = 0.68, 95%CI: 0.52-0.90). Subgroup analysis revealed such association only existed in Caucasian ethnicity (AA+GA vs GG, OR = 0.70, 95%CI: 0.55-0.89). The rs1800872 polymorphisms were associated with an increased risk of IBS in Asian ethnicity (CC vs GG: OR = 1.29, 95%CI: 1.01-1.16). There were no associations between rs1800871 polymorphisms and the IBS risk.
CONCLUSION: The results suggest that IL-10 rs1800870 confers susceptibility to the risk of IBS in Caucasian ethnicity, and the rs1800872 may associate with IBS risk in Asians. However, no significant associations are found between rs1800871 and IBS risk.
PMCID: PMC3882424  PMID: 24409078
Interleukin-10; Irritable bowel syndrome; Gene polymorphism; Case-control; Meta-analysis
17.  Reproductive and Exogenous Hormone Factors in Relation to Risk of Meningioma in Women: A Meta-Analysis 
PLoS ONE  2013;8(12):e83261.
Background and Objective
A number of studies have focused on the association between oral contraceptive (OC), hormonal replacement therapy (HRT) and reproductive factors and meningioma risk, but the results were inconsistent. Thus, a meta-analysis was performed to obtain more precise estimates of risk.
We conducted a literature search using PubMed and EMBASE databases to July2013, without any limitations. Random effects models were used to summarize results.
Twelve case-control and six cohort studies were included in this meta-analysis. We found that an increased risk of meningioma was associated with HRT use(RR = 1.19, 95% CI = 1.01–1.40), postmenopausal women(RR = 1.32, 95% CI = 1.07–1.64) and parity(RR = 1.18, 95% CI = 1.00–1.40).No significant associations were observed for OC use (RR = 0.93, 95% CI = 0.83–1.03), age at menarche(RR = 1.06, 95% CI = 0.92–1.21), age at menopause(RR = 1.03, 95% CI = 0.81–1.30), or age at first birth(RR = 0.94, 95% CI = 0.80–1.10).
In conclusion, the results of our study support the hypothesis that longer exposure to effect of female sex hormones may increase the risk of meningioma in women, yet additional studies are warranted to confirm our findings and identify the underlying biological mechanisms.
PMCID: PMC3873952  PMID: 24386167
18.  Molecular Characterization of Host-Specific Biofilm Formation in a Vertebrate Gut Symbiont 
PLoS Genetics  2013;9(12):e1004057.
Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process.
Author Summary
The bacterial communities found in the vertebrate gastrointestinal tract are remarkably stable and host-specific. However, the ecological and molecular processes that facilitate the selection of microbial symbionts and the exclusion of detrimental bacteria are not well understood. Here, we explore the mechanisms that underlie colonization and biofilm formation in specific strains of the gut symbiont Lactobacillus reuteri. When previously germ-free mice are colonized by individual strains of L. reuteri, only strains originating from rodents formed biofilms on the forestomach epithelium. Genomic, proteomic, and molecular analysis provide a detailed look into the host-specific molecular processes, such as adhesion, that contribute to colonization and biofilm formation. Our findings demonstrate high fidelity in the epithelial selection of a bacterial gut inhabitant, which can differentiate even between strains of the same species, strengthening the notion that some relationships between vertebrates and their microbial symbionts are highly coevolved and exclusive.
PMCID: PMC3873254  PMID: 24385934
19.  SMPDB 2.0: Big Improvements to the Small Molecule Pathway Database 
Nucleic Acids Research  2013;42(Database issue):D478-D484.
The Small Molecule Pathway Database (SMPDB, is a comprehensive, colorful, fully searchable and highly interactive database for visualizing human metabolic, drug action, drug metabolism, physiological activity and metabolic disease pathways. SMPDB contains >600 pathways with nearly 75% of its pathways not found in any other database. All SMPDB pathway diagrams are extensively hyperlinked and include detailed information on the relevant tissues, organs, organelles, subcellular compartments, protein cofactors, protein locations, metabolite locations, chemical structures and protein quaternary structures. Since its last release in 2010, SMPDB has undergone substantial upgrades and significant expansion. In particular, the total number of pathways in SMPDB has grown by >70%. Additionally, every previously entered pathway has been completely redrawn, standardized, corrected, updated and enhanced with additional molecular or cellular information. Many SMPDB pathways now include transporter proteins as well as much more physiological, tissue, target organ and reaction compartment data. Thanks to the development of a standardized pathway drawing tool (called PathWhiz) all SMPDB pathways are now much more easily drawn and far more rapidly updated. PathWhiz has also allowed all SMPDB pathways to be saved in a BioPAX format. Significant improvements to SMPDB’s visualization interface now make the browsing, selection, recoloring and zooming of pathways far easier and far more intuitive. Because of its utility and breadth of coverage, SMPDB is now integrated into several other databases including HMDB and DrugBank.
PMCID: PMC3965088  PMID: 24203708
20.  DrugBank 4.0: shedding new light on drug metabolism 
Nucleic Acids Research  2013;42(Database issue):D1091-D1097.
DrugBank ( is a comprehensive online database containing extensive biochemical and pharmacological information about drugs, their mechanisms and their targets. Since it was first described in 2006, DrugBank has rapidly evolved, both in response to user requests and in response to changing trends in drug research and development. Previous versions of DrugBank have been widely used to facilitate drug and in silico drug target discovery. The latest update, DrugBank 4.0, has been further expanded to contain data on drug metabolism, absorption, distribution, metabolism, excretion and toxicity (ADMET) and other kinds of quantitative structure activity relationships (QSAR) information. These enhancements are intended to facilitate research in xenobiotic metabolism (both prediction and characterization), pharmacokinetics, pharmacodynamics and drug design/discovery. For this release, >1200 drug metabolites (including their structures, names, activity, abundance and other detailed data) have been added along with >1300 drug metabolism reactions (including metabolizing enzymes and reaction types) and dozens of drug metabolism pathways. Another 30 predicted or measured ADMET parameters have been added to each DrugCard, bringing the average number of quantitative ADMET values for Food and Drug Administration-approved drugs close to 40. Referential nuclear magnetic resonance and MS spectra have been added for almost 400 drugs as well as spectral and mass matching tools to facilitate compound identification. This expanded collection of drug information is complemented by a number of new or improved search tools, including one that provides a simple analyses of drug–target, –enzyme and –transporter associations to provide insight on drug–drug interactions.
PMCID: PMC3965102  PMID: 24203711
21.  The Association of Diabetes Mellitus with Clinical Outcomes after Coronary Stenting: A Meta-Analysis 
PLoS ONE  2013;8(9):e72710.
Previous studies have shown inconsistent results on the association between diabetes mellitus (DM) and some clinical outcomes. We conducted a meta-analysis of observational studies to assess effect of DM on clinical outcomes after coronary stenting.
We searched for studies without language restriction in PubMed, Embase and Cochrane library prior to 2012. The clinical outcomes including in-stent restenosis (ISR), major adverse cardiac events (MACE), stent thrombosis (ST), target lesion revascularization (TLR) and target vessel revascularization (TVR). Adjusted odds ratio (OR), and the corresponding 95% confidence interval (95% CI) was summarized.
55 studies involving 128,084 total patients (38,416 DM patients and 89,668 controls) were eligible for our analysis. Overall, there were significant associations between DM and ISR (OR = 1.70, 95% CI: 1.53–1.89, I2 = 0.0%), MACE (OR = 1.54, 95% CI: 1.36–1.73, I2 = 29.0%), ST (OR = 2.01, 95% CI: 1.36–2.97, I2 = 47.7%), TLR (OR = 1.46, 95% CI: 1.26–1.68, I2 = 43.3%) as well as TVR (OR = 1.33, 95% CI: 1.17–1.51, I2 = 48.3). Subgroup analysis showed that the associations were similar between BMS and DES implantation. Moreover, there was no significant association in the ST subgroup after 1–3 years follow-up.
Our meta-analysis suggests that after coronary stent implantation, DM is associated with ISR, MACE, ST, TLR and TVR. DM appears to be a vital risk factor of these clinical outcomes.
PMCID: PMC3774683  PMID: 24066025
22.  Fish intake and risk of heart failure: A meta-analysis of five prospective cohort studies 
The findings on the association between fish intake and the risk of heart failure (HF) have been inconsistent. The purpose of this study was to clarify this potential association. We searched for relevant studies in the PubMed database through January 2012 and manually reviewed references. Five independent prospective cohort studies involving 5,273 cases and 144,917 participants were included. The summary relative risk estimates (SRRE) based on the highest compared with the lowest category of fish consumption were estimated by variance-based meta-analysis. In addition, we performed sensitivity and dose-response analyses to examine the association. Overall, an absence of an association between fish intake and HF was observed (SRRE=1.00; 95% CI, 0.81–1.24). However, fried fish intake positively associated with HF (SRRE=1.40; 95% CI, 1.22–1.61). In addition, dose-response analysis of fried fish suggested that each increment of six fried fish per month corresponded to a 37% increase of HF rate (RR=1.37; 95% CI, 1.20–1.56). In conclusion, our findings suggest that there is no significant association between fish intake and risk of HF, with the exception of a possible positive correlation with individuals comsuming fried fish, based on a limited number of studies. Future studies are required to confirm these findings.
PMCID: PMC3503629  PMID: 23181122
fish; nutrition; heart failure; meta-analysis
23.  GSTP1 Ile105Val Polymorphism and Prostate Cancer Risk: Evidence from a Meta-Analysis 
PLoS ONE  2013;8(8):e71640.
Glutathione S-transferase P1 (GSTP1) is thought to be involved in the detoxification of reactive carcinogen metabolites. Numerous epidemiological studies have evaluated the association of GSTP1 Ile105Val polymorphism with the risk of prostate cancer. However, the results remain inconclusive. To derive a more precise estimation, a meta-analysis was performed.
Methodology/Principal Findings
A comprehensive search was conducted to identify the eligible studies. We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the relationship. The overall association was not significant (Val/Val vs. Ile/Ile OR = 1.06, 95% CI = 0.90–1.25, P = 0.50; Val/Val vs. Val/Ile+Ile/Ile: OR = 1.07, 95% CI = 0.91–1.25, P = 0.44). In subgroup analyses by ethnicity and prostate cancer grade, the similar results were observed. However, in stratified analysis by clinical stage, we found a significant association with low-stage prostate cancer (Val/Val vs. Ile/Ile: OR = 2.70, 95% CI = 1.73–4.22, P<0.001; Val/Val vs. Val/Ile+Ile/Ile: OR = 2.14, 95% CI = 1.38–3.33, P = 0.001). Moreover, there was no statistically significant evidence of multiplicative interactions neither between the GSTP1 Ile105Val polymorphism and GSTM1, nor between smoking status and GSTP1 on prostate cancer risk.
This meta-analysis showed that GSTP1 Ile105Val polymorphism might not be significantly associated with overall prostate cancer risk. Further stratified analyses showed a significant association with low-stage prostate cancer.
PMCID: PMC3747220  PMID: 23977100
24.  Low Molecular Weight Heparin Relieves Experimental Colitis in Mice by Downregulating IL-1β and Inhibiting Syndecan-1 Shedding in the Intestinal Mucosa 
PLoS ONE  2013;8(7):e66397.
Low molecular weight heparin (LMWH) exhibits anti-inflammatory properties, but its effect on inflammation in colitis remains unclear. This study aimed to evaluate the therapeutic effects of LMWH on dextran sulfate sodium (DSS)-induced colitis in mice, in which acute colitis progresses to chronic colitis, and to explore the potential mechanism involved in this process. C57BL/6 mice were randomly divided into control, DSS, and DSS plus LMWH groups (n = 18). Disease activity was scored by a disease activity index (DAI). Histological changes were evaluated by hematoxylin and eosin (HE) staining. The mRNA levels of syndecan-1, interleukin (IL)-1β, and IL-10 were determined by quantitative reverse transcription polymerase chain reaction. Protein expression of syndecan-1 was detected by immunohistochemistry. The serum syndecan-1 level was examined by a dot immunobinding assay. LMWH ameliorated the disease activity of colitis induced by DSS administration in mice. Colon destruction with the appearance of crypt damage, goblet cell loss, and a larger ulcer was found on day 12 after DSS administration, which was greatly relieved by the treatment of LMWH. LMWH upregulated syndecan-1 expression in the intestinal mucosa and reduced the serum syndecan-1 level on days 12 and 20 after DSS administration (P<0.05 vs. DSS group). In addition, LMWH significantly decreased the expression of both IL-1β and IL-10 mRNA on days 12 and 20 (P<0.05 vs. DSS group). LMWH has therapeutic effects on colitis by downregulating inflammatory cytokines and inhibiting syndecan-1 shedding in the intestinal mucosa.
PMCID: PMC3715511  PMID: 23874391
25.  Capturing Amplitude Changes of Low-Frequency Fluctuations in Functional Magnetic Resonance Imaging Signal: A Pilot Acupuncture Study on NeiGuan (PC6) 
This study aims to examine amplitude changes of low-frequency oscillations (fALFF) in the blood-oxygen level-dependent (BOLD) signal associated with acupuncture on NeiGuan (PC6).
Experimental design
Ten (10) healthy adults participated in a functional magnetic resonance imaging (i.e., nuclear medicine; fMRI) study. During the brain-imaging procedure, the participants were instructed to lie quietly; they did not perform any cognitive task.
Main outcome measures
Three (3) fMRI scans were conducted for each participant: a first resting-state scan (R1), a stimulating-acupoint scan (AP), and a second resting-state scan (R2) after AP. Individual fALFF maps were calculated for each scan.
During R1, consistent with previous studies, the default network regions showed significantly detectable fALFF amplitudes. Acupuncture on PC6 increased fALFF amplitudes within the anterior cingulate cortex (ACC), occipital fusiform gyrus, posterior cingulate cortex, and precuneus (PCC/PCU). In contrast, during R2, fALFF within PCC is still significantly higher than R1 while ACC and cerebellum showed decreased fALFF.
These findings imply that stimulating PC6 can change the amplitude of the intrinsic cortical activity of the brain. In particular, a continuous and temporally consistent effect of acupuncture within PCC not the common brain circuit of pain including ACC and cerebellum was observed. Considering the cognitive functions and deficits of the relevant areas in mild cognitive impairment and Alzheimer disease, acupuncture on PC6 could potentially affect both psychiatric and neurological disorders. Thus, stimulating PC6 may be a candidate method for improving cognitive impairment.
PMCID: PMC3326268  PMID: 22515798

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