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1.  Associations of Genetic Variants in the PSCA, MUC1 and PLCE1 Genes with Stomach Cancer Susceptibility in a Chinese Population 
PLoS ONE  2015;10(2):e0117576.
Several genetic variants including PSCA rs2294008 C>T and rs2976392 G>A, MUC1 rs4072037 T>C, and PLCE1 rs2274223 A>G have shown significant association with stomach cancer risk in the previous genome-wide association studies (GWASs).
To evaluate associations of these SNPs in the Han Chinese, an independent hospital based case-control study was performed by genotyping these four polymorphisms in a total of 692 stomach cancer cases and 774 healthy controls acquired by using frequency matching for age and gender. False-positive report probability (FPRP) analysis was also performed to validate all statistically significant findings.
In the current study, significant association with stomach cancer susceptibility was observed for all the four polymorphisms of interest. Specifically, a significant increased stomach cancer risk was associated with PSCA rs2294008 (CT vs. CC: adjusted OR = 1.37, 95% CI = 1.07–1.74, and CT/TT vs.CC: adjusted OR = 1.30, 95% CI = 1.03–1.63), PSCA rs2976392 (AG vs. GG: adjusted OR = 1.30, 95% CI = 1.02–1.65, and AG/AA vs. GG: adjusted OR = 1.26, 95% CI = 1.00–1.59), or PLCE1 rs2274223 (AG vs. AA: adjusted OR = 1.48, 95% CI = 1.15–1.90, and AG/GG vs. AA: adjusted OR = 1.45, 95% CI = 1.14–1.84), respectively. In contrast, MUC1 rs4072037 was shown to decrease the cancer risk (CT vs. TT: adjusted OR = 0.77, 95% CI = 0.60–0.98). Patients with more than one risk genotypes had significant increased risk to develop stomach cancer (adjusted OR = 1.30, 95% CI = 1.03–1.64), when compared with those having 0–1 risk genotypes. Stratified analysis indicated that the increased risk was more pronounced in younger subjects, men, ever smokers, smokers with pack years ≤ 27, patients with high BMI, or non-cardia stomach cancer.
This study substantiated the associations between four previous reported genetic variants and stomach cancer susceptibility in an independent Han Chinese population. Further studies with larger sample size and different ethnicities are warranted to validate our findings.
PMCID: PMC4319726  PMID: 25658482
2.  NLRP1 inflammasome is activated in patients with medial temporal lobe epilepsy and contributes to neuronal pyroptosis in amygdala kindling-induced rat model 
Temporal lobe epilepsy (TLE) is often characterized pathologically by severe neuronal loss in the hippocampus. Understanding the mechanisms of neuron death is key to preventing the neurodegeneration associated with TLE. However, the involvement of neuronal loss to the epileptogenic process has yet to be fully determined. Recent studies have shown that the activation of NLRP1 can generate a functional caspase-1-containing inflammasome in vivo to drive the proinflammatory programmed cell death termed ‘pyroptosis’, which has a key role in the pathogenesis of neurological disorders. To the best of our knowledge, there are no reported studies that performed detailed identification and validation of NLRP1 inflammasome during the epileptogenic process.
We first compared expression of NLRP1 and caspase-1 in resected hippocampus from patients with intractable mesial temporal lobe epilepsy (mTLE) with that of matched control samples. To further examine whether the activation of NLRP1 inflammasome contributes to neuronal pyroptosis, we employed a nonviral strategy to knock down the expression of NLRP1 and caspase-1 in the amygdala kindling-induced rat model. Proinflammatory cytokines levels and hippocampal neuronal loss were evaluated after 6 weeks of treatment in these NLRP1 or caspase-1 deficiency TLE rats.
Western blotting detected upregulated NLRP1 levels and active caspase-1 in mTLE patients in comparison to those levels seen in the controls, suggesting a role for this inflammasome in mTLE. Moreover, we employed direct in vivo infusion of nonviral small interfering RNA to knockdown NLRP1 or caspase-1 in the amygdala kindling-induced rat model, and discovered that these NLRP1 or caspase-1 silencing rats resulted in significantly reduced neuronal pyroptosis.
Our data suggest that NLRP1/caspase-1 signaling participates in the seizure-induced degenerative process in humans and in the animal model of TLE and points to the silencing of NLRP1 inflammasome as a promising strategy for TLE therapy.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0233-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4314732  PMID: 25626361
NLRP1; pyroptosis; inflammasome; Caspase-1; temporal lobe epilepsy
3.  Distributional Variations in Trabecular Architecture of the Mandibular Bone: An In Vivo Micro-CT Analysis in Rats 
PLoS ONE  2015;10(1):e0116194.
To evaluate the effect of trabecular thickness and trabecular separation on modulating the trabecular architecture of the mandibular bone in ovariectomized rats.
Materials and Methods
Fourteen 12-week-old adult female Wistar rats were divided into an ovariectomy group (OVX) and a sham-ovariectomy group (sham). Five months after the surgery, the mandibles from 14 rats (seven OVX and seven sham) were analyzed by micro-CT. Images of inter-radicular alveolar bone of the mandibular first molars underwent three-dimensional reconstruction and were analyzed.
Compared to the sham group, trabecular thickness in OVX alveolar bone decreased by 27% (P = 0.012), but trabecular separation in OVX alveolar bone increased by 59% (P = 0.005). A thickness and separation map showed that trabeculae of less than 100μm increased by 46%, whereas trabeculae of more than 200μm decreased by more than 40% in the OVX group compared to those in the sham group. Furthermore, the OVX separation of those trabecular of more than 200μm was 65% higher compared to the sham group. Bone mineral density (P = 0.028) and bone volume fraction (p = 0.001) were also significantly decreased in the OVX group compared to the sham group.
Ovariectomy-induced bone loss in mandibular bone may be related to the distributional variations in trabecular thickness and separation which profoundly impact the modulation of the trabecular architecture.
PMCID: PMC4307973  PMID: 25625431
4.  Abnormal serine phosphorylation of insulin receptor substrate 1 is associated with tau pathology in Alzheimer's disease and tauopathies 
Acta neuropathologica  2014;128(5):679-689.
Neuronal insulin signaling abnormalities have been associated with Alzheimer's disease (AD). However, the specificity of this association and its underlying mechanisms have been unclear. This study investigated the expression of abnormal serine phosphorylation of insulin receptor substrate 1 (IRS1) in 157 human brain autopsy cases that included AD, tauopathies, α-synucleinopathies, TDP-43 proteinopathies, and normal aging. IRS1-pS616, IRS1-pS312 and downstream target Akt-pS473 measures were most elevated in AD but were also significantly increased in the tauopathies: Pick's disease, corticobasal degeneration and progressive supranuclear palsy. Double immunofluorescence labeling showed frequent co-expression of IRS1-pS616 with pathologic tau in neurons and dystrophic neurites. To further investigate an association between tau and abnormal serine phosphorylation of IRS1, we examined the presence of abnormal IRS1-pS616 expression in pathological tau-expressing transgenic mice and demonstrated that abnormal IRS1-pS616 frequently co-localizes in tangle-bearing neurons. Conversely, we observed increased levels of hyperphosphorylated tau in the high-fat diet-fed mouse, a model of insulin resistance. These results provide confirmation and specificity that abnormal phosphorylation of IRS1 is a pathological feature of AD and other tauopathies, and provide support for an association between insulin resistance and abnormal tau as well as amyloid-β.
PMCID: PMC4304658  PMID: 25107476
Alzheimer's disease; Tau; Synuclein; TDP-43; Insulin resistance; Insulin receptor substrate 1
5.  Association between epidermal growth factor receptor mutations and the expression of excision repair cross-complementing protein 1 and ribonucleotide reductase subunit M1 mRNA in patients with non-small cell lung cancer 
The present study aimed to investigate the association between epidermal growth factor receptor (EGFR) gene mutations and excision repair cross-complementing protein 1 (ERCC1) and ribonucleotide reductase subunit M1 (RRM1) mRNA expression in non-small cell lung cancer (NSCLC) tissue. The quantitative polymerase chain reaction was used to detect EGFR mutations, and ERCC1 and RRM1 mRNA expression in 257 cases of NSCLC. In the NSCLC samples the EGFR mutation rate was 49.03% (126/257). The rate was higher in females and non-smoking patients (P<0.05). High expression of ERCC1 mRNA was observed in 47.47% of the samples (122/257), while a high RRM1 mRNA expression was observed in 61.87% of the samples (159/257). In comparison with patients with NSCLC without EGFR mutations, patients with EGFR mutations had significantly lower levels of ERCC1 mRNA expression (P<0.05); however, EGFR mutations and expression levels of RRM1 mRNA were not correlated in NSCLC tissues (P>0.05). In addition, ERCC1 mRNA expression was not correlated with the expression levels of RRM1 mRNA (P>0.05). In conclusion, patients with NSCLC with EGFR mutations tend to have a low expression of ERCC1 mRNA and may potentially benefit from platinum-based chemotherapy.
PMCID: PMC4316980  PMID: 25667646
non-small cell lung cancer; epidermal growth factor receptor; excision repair cross-complementing protein 1; ribonucleotide reductase subunit M1; molecular detection; individualized treatment
6.  Association between Serum C-Peptide as a Risk Factor for Cardiovascular Disease and High-Density Lipoprotein Cholesterol Levels in Nondiabetic Individuals 
PLoS ONE  2015;10(1):e112281.
Objective: Although serum C-peptide has increasingly received attention as a new and important risk factor for cardiovascular disease (CVD), the potential mechanisms remain unclear. This study aimed to investigate the association between serum C-peptide as a risk factor for CVD and high-density lipoprotein cholesterol (HDL-C) levels.
The present study included 13,185 participants aged ≥20 years. Serum C-peptide and HDL-C levels were measured according to a standard protocol. Stratified analysis of covariance was used to compare serum HDL-C levels between different quartiles of serum C-peptide levels. Logistic regression analysis was used to determine the association between serum C-peptide and HDL-C levels. Cox proportional hazard regression analysis was conducted to determine the hazard ratio of serum HDL-C for CVD-related mortality.
The results of the ANCOVA analysis showed a significant linear trend between the mean serum HDL-C level and the different quartiles of serum C-peptide. Compared to the first quartile (25th percentile), the second, third, and fourth quartiles had gradual reduction in serum HDL-C levels. Logistic regression analyses showed a strong negative association between serum C-peptide levels and HDL-C levels; the p value for the linear trend was <0.001. In men, compared with the lowest quartile of the serum C-peptide level, the relative risk was 1.75, 2.79, and 3.07 for the upper three quartiles of the serum C-peptide level. The relative risk was 1.60, 2.61, and 3.67 for women. The results of the survival analysis showed that serum HDL-C levels were negatively associated with CVD-related death in both men and women.
Serum C-peptide as a risk factor for CVD was significantly and negatively associated with serum HDL-C levels in individuals without diabetes. These findings suggest that serum C-peptide levels association with CVD death can be caused, at least in part, by the low serum HDL-C level.
PMCID: PMC4283961  PMID: 25559358
7.  Chloroplast genome of Aconitum barbatum var. puberulum (Ranunculaceae) derived from CCS reads using the PacBio RS platform 
The chloroplast genome (cp genome) of Aconitum barbatum var. puberulum was sequenced using the third-generation sequencing platform based on the single-molecule real-time (SMRT) sequencing approach. To our knowledge, this is the first reported complete cp genome of Aconitum, and we anticipate that it will have great value for phylogenetic studies of the Ranunculaceae family. In total, 23,498 CCS reads and 20,685,462 base pairs were generated, the mean read length was 880 bp, and the longest read was 2,261 bp. Genome coverage of 100% was achieved with a mean coverage of 132× and no gaps. The accuracy of the assembled genome is 99.973%; the assembly was validated using Sanger sequencing of six selected genes from the cp genome. The complete cp genome of A. barbatum var. puberulum is 156,749 bp in length, including a large single-copy region of 87,630 bp and a small single-copy region of 16,941 bp separated by two inverted repeats of 26,089 bp. The cp genome contains 130 genes, including 84 protein-coding genes, 34 tRNA genes and eight rRNA genes. Four forward, five inverted and eight tandem repeats were identified. According to the SSR analysis, the longest poly structure is a 20-T repeat. Our results presented in this paper will facilitate the phylogenetic studies and molecular authentication on Aconitum.
PMCID: PMC4319492
chloroplast genome; Aconitum; circular consensus sequencing; PacBio RS; the third generation sequencing
8.  Patterns of Evolutionary Conservation of Ascorbic Acid-Related Genes Following Whole-Genome Triplication in Brassica rapa 
Genome Biology and Evolution  2014;7(1):299-313.
Ascorbic acid (AsA) is an important antioxidant in plants and an essential vitamin for humans. Extending the study of AsA-related genes from Arabidopsis thaliana to Brassica rapa could shed light on the evolution of AsA in plants and inform crop breeding. In this study, we conducted whole-genome annotation, molecular-evolution and gene-expression analyses of all known AsA-related genes in B. rapa. The nucleobase–ascorbate transporter (NAT) gene family and AsA l-galactose pathway genes were also compared among plant species. Four important insights gained are that: 1) 102 AsA-related gene were identified in B. rapa and they mainly diverged 12–18 Ma accompanied by the Brassica-specific genome triplication event; 2) during their evolution, these AsA-related genes were preferentially retained, consistent with the gene dosage hypothesis; 3) the putative proteins were highly conserved, but their expression patterns varied; and 4) although the number of AsA-related genes is higher in B. rapa than in A. thaliana, the AsA contents and the numbers of expressed genes in leaves of both species are similar, the genes that are not generally expressed may serve as substitutes during emergencies. In summary, this study provides genome-wide insights into evolutionary history and mechanisms of AsA-related genes following whole-genome triplication in B. rapa.
PMCID: PMC4316640  PMID: 25552535
AsA-related genes; Brassica rapa; evolutionary conservation; synteny analysis; gene dosage hypothesis; expression pattern
9.  Controlled Release of Dutasteride from Biodegradable Microspheres: In Vitro and In Vivo Studies 
PLoS ONE  2014;9(12):e114835.
The aim of the present work was to study the in vitro/in vivo characteristics of dutasteride loaded biodegradable microspheres designed for sustained release of dutasteride over four weeks. An O/W emulsion-solvent evaporation method was used to incorporate dutasteride, which is of interest in the treatment of benign prostatic hyperplasia (BPH), into poly(lactide-co-glycolide) (PLGA). A response surface method (RSM) with central composite design (CCD) was employed to optimize the formulation variables. A prolonged in vitro drug release profile was observed, with a complete release of the entrapped drug within 28 days. The pharmacokinetics study showed sustained plasma drug concentration-time profile of dutasteride loaded microspheres after subcutaneous injection into rats. The in vitro drug release in rats correlated well with the in vivo pharmacokinetics profile. The pharmacodynamics evaluated by determination of the BPH inhibition in the rat models also showed a prolonged pharmacological response. These results suggest the potential use of dutasteride loaded biodegradable microspheres for the management of BPH over long periods.
PMCID: PMC4277280  PMID: 25541985
10.  MicroRNA-302a Functions as a Putative Tumor Suppressor in Colon Cancer by Targeting Akt 
PLoS ONE  2014;9(12):e115980.
Micro RNAs (miRNAs) are important regulators involved in various physical and pathological processes, including cancer. The miRNA-302 family has been documented as playing a critical role in carcinogenesis. In this study, we investigated the role of miRNA-302a in colon cancer. MiRNA-302a expression was detected in 44 colon cancer tissues and 10 normal colon tissues, and their clinicopathological significance was analyzed. Cell proliferation and cell cycle analysis were performed on colon cancer cells that stably expressed miRNA-302a. The target gene of miRNA-302a and the downstream pathway were further investigated. Compared with normal colon tissues, miRNA-302a expression was downregulated in colon cancer tissues. Overexpression of miRNA-302a induced G1/S cell cycle arrest in colon cancer cells, and suppressed colon cancer cell proliferation both in vitro and in vivo. Furthermore, miRNA-302a inhibited AKT expression by directly binding to its 3′ untranslated region, resulting in subsequent alterations of the AKT-GSK3β-cyclin D1 pathway. These results reveal miRNA-302a as a tumor suppressor in colon cancer, suggesting that miRNA-302a may be used as a potential target for therapeutic intervention in colon cancer.
PMCID: PMC4277419  PMID: 25542007
11.  Biodegradable Nasal Packings for Endoscopic Sinonasal Surgery: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(12):e115458.
To assess biodegradable nasal packing effectiveness for improving postoperative symptoms and mucosal healing after endoscopic sinonasal surgery as compared with conventional/non-packing groups.
Relevant articles were searched on PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. We included randomized controlled trials (RCTs) that compared biodegradable packings with conventional packings or no packing, reporting postoperative symptoms and/or mucosal healing outcomes.
This review included 19 studies, of which 11 compared biodegradable packings with conventional packings. Meta-analysis found that biodegradable packings significantly improved postoperative symptoms: bleeding at removal, pain at removal, pain in situ, and nasal blockage. Mucosal healing outcomes were inconsistent within studies, with no data could be pooled. Eight studies compared biodegradable packings with non-packing group. Postoperative symptom data in this comparison could not be pooled: A protective or equal effect on postoperative bleeding was reported in different studies; no difference was reported in pain status and nasal blockage. As for mucosal healing, meta-analysis showed that two arms of comparison had similar effect on synechiae, edema, infection and granulation at each time point.
The limiting evidence suggests that biodegradable nasal packings are statistically better than conventional packings in postoperative symptoms, and probably comparable to non-packing group, as in this comparison we could not carry out meta-analysis. No beneficial or detrimental effect on postoperative mucosal healing could be determined based on existing evidence.
PMCID: PMC4272281  PMID: 25526585
12.  Altered expression of T cell Immunoglobulin-Mucin (Tim) molecules in peripheral blood mononuclear cells in aplastic anemia 
Cancer Cell International  2014;14(1):144.
To evaluate the balance between T-cell immunoglobulin and mucin domain (Tim) molecules(Tim)-1 and Tim-3 in patients with aplastic anemia (AA), plasma IFN-γ and IL-4 levels were measured in patients with active AA (n = 41), AA in remission (n = 29) and in healthy subjects (n = 40) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression of IFN-γ, IL-4, Tim-1 and Tim-3 were studied in all subjects. The results showed that the expression of Tim-3 in newly diagnosed patients was significantly deceased, compared with the controls. Meanwhile, Tim-1 mRNA expression in the active AA group was not significantly reduced, which resulted in a declined ratio of Tim-3/Tim-1 in patients with active disease. During the remission stages, the levels of these transcription factors were comparable with those observed in the healthy controls. These findings are the first data on the expression of the Tim-1 and Tim-3 molecules in AA. The reduced levels of Tim-3/Tim-1 in PBMCs during the active stages of disease suggest that they may play a possible role in the pathogenesis and course of AA.
PMCID: PMC4278261  PMID: 25548541
Aplastic anemia; T-cell immunoglobulin and mucin domain; IFN-γ; Interleukin 4; Cytokines; RT-PCR
13.  APOBEC3B upregulation and genomic mutation patterns in serous ovarian carcinoma 
Cancer research  2013;73(24):10.1158/0008-5472.CAN-13-1753.
Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here we report wide variation in expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in a majority of ovarian cancer cell lines (3 standard deviations above the mean of normal ovarian surface epithelial cells) and high grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5′TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5′TC dinucleotide motifs in early-stage high grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA ‘repair’ enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability.
PMCID: PMC3867573  PMID: 24154874
APOBEC3B; DNA cytosine deamination; genomic uracil; ovarian cancer; transversion mutations
14.  Association of Upregulated HMGB1 and c-IAP2 Proteins With Hepatocellular Carcinoma Development and Progression 
Hepatitis Monthly  2014;14(12):e23552.
Hepatocellular carcinoma (HCC) is one of the most important health problems in China.
This study analyzed expression of high-mobility group protein B1 (HMGB1) and inhibitor of apoptosis protein-2 (c-IAP2) proteins in HCC compared to paired para-tumor tissue samples to assess the association with HCC pathogenesis and progression.
Materials and Methods:
Sixty-eight HCC and para-tumor tissue samples were collected for Western blot, qRT-PCR and immunohistochemical analyses of HMGB1 and c-IAP2.
HMGB1 and c-IAP2 proteins were highly expressed in HCC tissue samples [85.3% (58/68) and 82.4% (56/68), respectively] compared to para-tumor tissue samples [32.3% and 27.9%, respectively]. Furthermore, expression of HMGB1 was significantly associated with enhanced c-IAP2 expression in HCC tissue samples (r = 0.878, P < 0.01). Expression of HMGB1 was associated with tumor multiplicity and size, alpha-fetoprotein (AFP) level and advanced TNM stage, while expression of c-IAP2 was associated with tumor size, AFP level and advanced TNM stage.
Expression of HMGB1 and c-IAP2 proteins was associated with HCC development and progression, and the expression of HMGB1 and c-IAP2 proteins in HCC were significantly associated with each other. Additionally, these proteins may show promise as biomarkers to predict HCC progression.
PMCID: PMC4310020
Hepatocellular Carcinoma; HMGB1; c-IAP2 Protein; Biomarkers
15.  Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma 
Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL) conjugated with anti-CD40 antibody (anti-CD40-BFL) showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm3 versus 270 mm3, respectively); the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic immunity while blocking systemic toxicity.
PMCID: PMC4260685  PMID: 25506218
liposomes; bufalin; anti-CD40; chemoimmunotherapy
16.  Indole-3-Acetic Acid-Producing Yeasts in the Phyllosphere of the Carnivorous Plant Drosera indica L 
PLoS ONE  2014;9(12):e114196.
Yeasts are widely distributed in nature and exist in association with other microorganisms as normal inhabitants of soil, vegetation, and aqueous environments. In this study, 12 yeast strains were enriched and isolated from leaf samples of the carnivorous plant Drosera indica L., which is currently threatened because of restricted habitats and use in herbal industries. According to similarities in large subunit and small subunit ribosomal RNA gene sequences, we identified 2 yeast species in 2 genera of the phylum Ascomycota, and 5 yeast species in 5 genera of the phylum Basidiomycota. All of the isolated yeasts produced indole-3-acetic acid (IAA) when cultivated in YPD broth supplemented with 0.1% L-tryptophan. Growth conditions, such as the pH and temperature of the medium, influenced yeast IAA production. Our results also suggested the existence of a tryptophan-independent IAA biosynthetic pathway. We evaluated the effects of various concentrations of exogenous IAA on yeast growth and observed that IAA produced by wild yeasts modifies auxin-inducible gene expression in Arabidopsis. Our data suggest that yeasts can promote plant growth and support ongoing prospecting of yeast strains for inclusion into biofertilizer for sustainable agriculture.
PMCID: PMC4252105  PMID: 25464336
17.  Gene Arrangements in Expression Vector Affect 3-Hydroxypropionic Acid Production in Klebsiella pneumoniae 
Indian Journal of Microbiology  2013;53(4):418-424.
Biosynthesis of 3-hydroxypropionic acid (3-HP) typically involves two sequential reactions catalyzed by glycerol dehydratase (DhaB) and aldehyde dehydrogenase (AldH). Although plasmid-dependent over-expression of the two enzymes is common, systematic investigation of gene arrangement in vector has not been reported. Here we show that gene arrangements have a noticeable influence on 3-HP production. Using Klebsiella pneumoniae as a host, three AldH-coding genes: ald4 from Saccharomyces cerevisiae, aldh from Escherichia coli, and puuC from host K. pneumoniae, were respectively ligated to dhaB. The recombinant Kp/pET-pk-ald4-dhaB (Kp refers to as K. pneumoniae, pk is a native promoter) produced the highest yield of 3-HP in comparison to both Kp/pET-pk-dhaB-ald4 and Kp/pET-pk-dhaB-pk-ald4, suggesting that the preferential expression of AldH can increase 3-HP production. Additionally, when different AldH-coding genes were respectively ligated downstream of dhaB, the recombinant Kp/pET-pk-dhaB-puuC produced more 3-HP than that by Kp/pET-pk-dhaB-aldh or Kp/pET-pk-dhaB-ald4, implying the intrinsic compatibility of native gene puuC with its host. These findings indicate the applicability of native AldH-coding gene and provide insights into strategies for metabolic engineering of multiple genes.
Electronic supplementary material
The online version of this article (doi:10.1007/s12088-013-0390-3) contains supplementary material, which is available to authorized users.
PMCID: PMC3779304  PMID: 24426145
Klebsiella pneumoniae; 3-Hydroxypropionic acid; Gene arrangement; Glycerol dehydratase; Aldehyde dehydrogenase
18.  Molecular profiling and computational network analysis of TAZ-mediated mammary tumorigenesis identifies actionable therapeutic targets 
Oncotarget  2014;5(23):12166-12176.
Triple-negative breast cancer (TNBC) accounts for approximately 15–20% of all breast cancer (BC) cases and contributes disproportionately to BC mortality. TAZ, a key transducer of the Hippo pathway, has recently been demonstrated to confer breast cancer stem cell (CSC) traits. However, TAZ target genes and the underlying transcriptional regulatory pathways responsible for the CSC phenomenon remain unknown. Here, we demonstrate that the oncogenic activity of TAZ is essential for propagation of the malignant phenotype. We further show that constitutively active TAZ tumor-derived cells exhibit unique tumor-initiating properties, including increased self-renewal and metastatic seeding potential, acquired chemotherapy resistance and the ability to efficiently regenerate tumor formation in vivo. Combined digital RNA expression analysis and computational network approaches identify several signaling pathways that distinguish breast cancer tumor-initiating cells (T-ICs) from bulk tumor cells. We demonstrate the utility of this approach by repositioning the small molecule tyrosine kinase inhibitor, Dasatinib, which selectively targets T-ICs and inhibits TNBC growth in vivo.
PMCID: PMC4323005  PMID: 25361000
breast cancer; TAZ; tumor-initiating cells; RNA sequencing; Dasatinib
19.  Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target 
Scientific Reports  2014;4:7245.
N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.
PMCID: PMC4246208  PMID: 25430794
20.  Laparoscopic vs open D2 gastrectomy for locally advanced gastric cancer: A meta-analysis 
World Journal of Gastroenterology : WJG  2014;20(44):16750-16764.
AIM: To conduct a meta-analysis comparing laparoscopic (LGD2) and open D2 gastrectomies (OGD2) for the treatment of advanced gastric cancer (AGC).
METHODS: Randomized controlled trials (RCTs) and non-RCTs comparing LGD2 with OGD2 for AGC treatment, published between 1 January 2000 and 12 January 2013, were identified in the PubMed, Embase, and Cochrane Library databases. Primary endpoints included operative outcomes (operative time, intraoperative blood loss, and conversion rate), postoperative outcomes (postoperative analgesic consumption, time to first ambulation, time to first flatus, time to first oral intake, postoperative hospital stay length, postoperative morbidity, incidence of reoperation, and postoperative mortality), and oncologic outcomes (the number of lymph nodes harvested, tumor recurrence and metastasis, disease-free rates, and overall survival rates). The Cochrane Collaboration tools and the modified Newcastle-Ottawa scale were used to assess the quality and risk of bias of RCTs and non-RCTs in the study. Subgroup analyses were conducted to explore the incidence rate of various postoperative morbidities as well as recurrence and metastasis patterns. A Begg’s test was used to evaluate the publication bias.
RESULTS: One RCT and 13 non-RCTs totaling 2596 patients were included in the meta-analysis. LGD2 in comparison to OGD2 showed lower intraoperative blood loss [weighted mean difference (WMD) = -137.87 mL, 95%CI: -164.41--111.33; P < 0.01], lower analgesic consumption (WMD = -1.94, 95%CI: -2.50--1.38; P < 0.01), shorter times to first ambulation (WMD = -1.03 d, 95%CI: -1.90--0.16; P < 0.05), flatus (WMD = -0.98 d, 95%CI: -1.30--0.66; P < 0.01), and oral intake (WMD = -0.85 d, 95%CI: -1.67--0.03; P < 0.05), shorter hospitalization (WMD = -3.08 d, 95%CI: -4.38--1.78; P < 0.01), and lower postoperative morbidity (odds ratio = 0.78, 95%CI: 0.61-0.99; P < 0.05). No significant differences were observed between LGD2 and OGD2 for the following criteria: reoperation incidence, postoperative mortality, number of harvested lymph nodes, tumor recurrence/metastasis, or three- or five-year disease-free and overall survival rates. However, LGD2 had longer operative times (WMD = 57.06 min, 95%CI: 41.87-72.25; P < 0.01).
CONCLUSION: Although a technically demanding and time-consuming procedure, LGD2 may be safe and effective, and offer some advantages over OGD2 for treatment of locally AGC.
PMCID: PMC4248223  PMID: 25469048
D2 lymph node dissection; Gastrectomy; Gastric cancer; Laparoscopy; Meta-analysis
21.  Subcellular location and photodynamic therapeutic effect of chlorin e6 in the human tongue squamous cell cancer Tca8113 cell line 
Oncology Letters  2014;9(2):551-556.
The present study aimed to investigate the distribution and photodynamic therapeutic effect of chlorin e6 (Ce6) in the human tongue squamous cell carcinoma Tca8113 cell line in vitro. The distribution of Ce6 in the Tca8113 cells was observed in situ combined with mitochondrial and lysosomal fluorescent probes. Next, 630-nm semiconductor laser irradiation was performed. The MTS colorimetric method was used to determine cell survival. Annexin V fluorescein isothiocyanate/propidium iodide (PI) double staining was used to detect early apoptosis following photodynamic therapy (PDT). The flow cytometer was used to analyze the DNA content subsequent to PI-staining. It was observed that Ce6 could combine with the cellular membrane following 30 min of incubation with the Tca8113 cells. As the length of incubation increased, Ce6 gradually entered the cells in a particular distribution and reached saturation by 3 h. Co-localization analysis demonstrated that Ce6 was more likely to be present in the mitochondria than in the lysosomes. The cells incubated with 5 μg/ml Ce6 for 24 h exhibited a low toxicity of 5%, however, following light irradiation, Ce6-PDT was able to kill the Tca8113 cells in vitro. The cell toxicity was positively correlated with Ce6 concentration and light dose, therefore, the effect of Ce6 was concentration/dose-dependent (P<0.01). The lower Ce6 concentrations and light doses could significantly induce apoptosis in the Tca8113 cells, while higher doses increased necrosis/percentage of dead cells. In summary, Ce6 saturated the Tca8113 cells following 3 h of incubation. Furthermore, Ce6-PDT effectively killed the cultured Tca8113 cells in vitro at a safe concentration. At a low concentration and light dose, Ce6 is more likely to induce cell apoptosis via the mitochondria than the lysosomes.
PMCID: PMC4301477  PMID: 25621023
photodynamic therapy; chlorin; mitochondria; human tongue squamous cell carcinoma; cell death
22.  Hepatoprotective and antioxidant activities of extracts from Salvia–Nelumbinis naturalis against nonalcoholic steatohepatitis induced by methionine- and choline-deficient diet in mice 
Nonalcoholic steatohepatitis (NASH), the advanced stage of nonalcoholic fatty liver disease that is characterized by both steatosis and severe injury in liver, still lacks efficient treatment. The traditional Chinese formula Salvia–Nelumbinis naturalis (SNN) is effectively applied to improve the symptoms of nonalcoholic simple fatty liver (NAFL) patients. Previous studies have confirmed that SNN could reduce the liver lipid deposition and serum transaminases of NAFL experimental models. This study aims to determine whether SNN is effective for murine NASH model and investigate the underlying pharmacological mechanisms.
C57BL/6 J mice were fed with methionine- and choline-deficient (MCD) diet for six weeks to induce NASH. Simultaneously, SNN or saline was intragastrically administered daily to the mice in the SNN or model group, respectively. A standard diet was given to the control mice. Serum biochemical indices and tumor necrosis factor-α were measured. Liver histopathology was observed, and the contents of triglycerides and lipid peroxide malondialdehyde (MDA) in liver homogenates were evaluated. The hepatic expression and/or activation of genes associated with inflammation, apoptosis, and oxidative stress were determined by quantitative RT-PCR or Western blot analysis.
The prominent liver steatosis displayed in the NASH model was prevented by SNN. The liver injury of NASH mice was obviously manifested by the increased levels of serum transaminases and bilirubin, as well as the lobular inflammation, elevated pro-inflammatory cytokines, and upregulated apoptosis in liver tissues. SNN administration improved the aforementioned pathological changes. The increased hepatic levels of MDA and cytochrome P450 2E1 of the model confirmed the unregulated balance of oxidative stress. The hepatic expression of nuclear factor erythroid 2-related factor 2 and its target genes decreased, whereas c-Jun N-terminal kinase activation in the model mice increased. Treating the mice with SNN significantly improved oxidative stress-related harmful factors.
This study shows that SNN can protect the liver from severe steatosis and damage induced by MCD diet, which suggests the potential use of SNN on the treatment of NASH patient. The results also indicate that improving the hepatic antioxidant capability of the liver may contribute to the underlying hepatoprotective mechanism.
PMCID: PMC4239328  PMID: 25406833
Salvia–Nelumbinis naturalis; NASH; Liver injury; Oxidative stress
23.  Reciprocal Changes in Vanilloid (TRPV1) and Endocannabinoid (CB1) Receptors Contribute to Visceral Hyperalgesia in the Water Avoidance Stressed Rat 
Gut  2008;58(2):202-210.
Increasing evidence suggests that chronic stress plays an important role in the pathophysiology of several functional gastrointestinal disorders. We investigated whether CB1 and TRPV1 receptors are involved in stress induced visceral hyperalgesia.
Male rats were exposed to 1-hour water avoidance (WA) stress daily for 10 consecutive days. The visceromotor response (VMR) to colorectal distension (CRD) was measured. Immunofluorescence and Western blot analysis were used to assess the expression of CB1 and TRPV1 receptors in DRG neurons.
WA stressed rats demonstrated a significant increase in the serum corticosterone levels and fecal pellet output compared to controls supporting stimulation of the HPA axis. The VMR increased significantly at pressures of 40 and 60 mmHg in WA stress rats compared with controls, respectively, and was associated with hyperalgesia. The endogenous CB1 agonist anandamide was increased significantly in DRGs from stressed rats. Immunofluorescence and Western blot analysis showed a significant decrease in CB1 and reciprocal increase in TRPV1 expression and phosphorylation in DRG neurons from stressed rats. These reciprocal changes in CB1 and TRPV1 were reproduced by treatment of control DRGs with anandamide in vitro. In contrast, treatment of control DRGs in vitro with the CB1 receptor agonist WIN 55,212-2 decreased the levels of TRPV1 and TRPV1 phosphorylation. Treatment of WA stress rats in situ with WIN or the TRPV1 antagonist capsazepine prevented the development of visceral hyperalgesia and blocked the up-regulation of TRPV1.
These results suggest that the endocannabinoid (CB1) and TRP (TRPV1) pathways may play a potentially important role in stress-induced visceral hyperalgesia.
PMCID: PMC4236191  PMID: 18936104
TRPV1; CB1; visceral hyperalgesia; stress; dorsal root ganglion
24.  Computed tomography-guided cutting needle pleural biopsy: Accuracy and complications 
In cases of pleural lesion, tissue samples can be obtained through thoracoscopy or closed needle biopsy for histopathological analysis. Cutting needle biopsy is a relatively recent addition to these techniques. The aim of this study was to evaluate the diagnostic accuracy and safety of computed tomography-guided cutting needle pleural biopsy (CT-CNPB), as well as the associated complications, in patients with pleural lesion. This study was a retrospective analysis of 92 percutaneous CT-CNPBs on 90 patients between March 2008 and May 2013. For group comparisons, χ2 tests were used to detect the risk factors for diagnostic accuracy (false-negative rate). Of the 92 CT-CNPBs, malignant lesions were diagnosed in 55 cases (mesothelioma in 12, metastatic pleural disease in 36, synoviosarcoma in one, indeterminate-origin disease in one and false-negative lesion in five) and benign pleural disease was diagnosed in 37 cases (inflammation in 15, tuberculosis in 10, granuloma in three, solitary fibrous tumor in two, hematoma in one, fungus in one and indeterminate-origin disease in five). The sensitivity of diagnostic malignant lesion was 90.9%, and the specificity and positive and negative predictive values were 100, 100 and 88.1%, respectively. The overall diagnostic accuracy was 94.6%. A specific diagnosis was achieved in 89.1% of malignant lesions and 86.4% of benign lesions. Univariate analysis of the risk factors affecting accuracy (false-negative rate) did not reveal any significant differences (all P>0.05). The complication rates were 6.5% for pneumothorax, 8.7% for hemorrhage and 1.1% for hemothorax. In conclusion, CT-CNPB is a safe and accurate diagnostic technique that can be recommended as the primary method of diagnosis in patients with pleural thickening or lesions observed by CT scan.
PMCID: PMC4247294  PMID: 25452813
pleural biopsy; pleural disease; cutting needle; computed tomography-guided
25.  Multifunctional Core/Shell Nanoparticles Cross-linked Polyetherimide-folic Acid as Efficient Notch-1 siRNA Carrier for Targeted Killing of Breast Cancer 
Scientific Reports  2014;4:7072.
In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis.
PMCID: PMC4233336  PMID: 25400232

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