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1.  Peri-operative use of sorafenib in liver transplantation: A time-to-event meta-analysis 
AIM: To evaluate whether the application of sorafenib during the peri-operative period of liver transplantation improves prognosis in liver cancer patients.
METHODS: We searched PubMed, EMBASE and MEDLINE for eligible articles. A total of 4 studies were found that fulfilled the previously agreed-upon standards. We then performed a systematic review and meta-analysis on the enrolled trials that met the inclusion criteria.
RESULTS: Out of the 104 studies identified in the database, 82 were not clinical experiments, and 18 did not fit the inclusion standards. Among the remaining 4 articles, only 1 was related to the preoperative use of sorafenib, whereas the other 3 were related to its postoperative use. As the heterogeneity among the 4 studies was high, with an I2 of 86%, a randomized effect model was applied to pool the data. The application of sorafenib before liver transplantation had a hazard ratio (HR) of 3.29 with a 95% confidence interval (CI) of 0.33-32.56. The use of sorafenib after liver transplantation had an HR of 1.44 (95%CI: 0.27-7.71). The overall pooled HR was 1.68 (95%CI: 0.41-6.91).
CONCLUSION: The results showed that the use of sorafenib during the peri-operative period of liver transplantation did not improve patient survival significantly. In fact, sorafenib could even lead to a worse prognosis, as its use may increase the hazard of poor survival.
doi:10.3748/wjg.v21.i5.1636
PMCID: PMC4316107  PMID: 25663784
Liver transplantation; Sorafenib; Peri-operative period; Kaplan-Meier curve; Hazard ratio
2.  Retrospective review using targeted deep sequencing reveals mutational differences between gastroesophageal junction and gastric carcinomas 
BMC Cancer  2015;15:32.
Background
Adenocarcinomas of both the gastroesophageal junction and stomach are molecularly complex, but differ with respect to epidemiology, etiology and survival. There are few data directly comparing the frequencies of single nucleotide mutations in cancer-related genes between the two sites. Sequencing of targeted gene panels may be useful in uncovering multiple genomic aberrations using a single test.
Methods
DNA from 92 gastroesophageal junction and 75 gastric adenocarcinoma resection specimens was extracted from formalin-fixed paraffin-embedded tissue. Targeted deep sequencing of 46 cancer-related genes was performed through emulsion PCR followed by semiconductor-based sequencing. Gastroesophageal junction and gastric carcinomas were contrasted with respect to mutational profiles, immunohistochemistry and in situ hybridization, as well as corresponding clinicopathologic data.
Results
Gastroesophageal junction carcinomas were associated with younger age, more frequent intestinal-type histology, more frequent p53 overexpression, and worse disease-free survival on multivariable analysis. Among all cases, 145 mutations were detected in 31 genes. TP53 mutations were the most common abnormality detected, and were more common in gastroesophageal junction carcinomas (42% vs. 27%, p = 0.036). Mutations in the Wnt pathway components APC and CTNNB1 were more common among gastric carcinomas (16% vs. 3%, p = 0.006), and gastric carcinomas were more likely to have ≥3 driver mutations detected (11% vs. 2%, p = 0.044). Twenty percent of cases had potentially actionable mutations identified. R132H and R132C missense mutations in the IDH1 gene were observed, and are the first reported mutations of their kind in gastric carcinoma.
Conclusions
Panel sequencing of routine pathology material can yield mutational information on several driver genes, including some for which targeted therapies are available. Differing rates of mutations and clinicopathologic differences support a distinction between adenocarcinomas that arise in the gastroesophageal junction and those that arise in the stomach proper.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1021-7) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1021-7
PMCID: PMC4322811  PMID: 25656989
Gastric cancer; Gastroesophageal junction cancer; Gastric cancer genomics; Gastric cancer sequencing
3.  Gadd45b is a Novel Mediator of Neuronal Apoptosis in Ischemic Stroke 
Apoptosis plays an essential role in ischemic stroke pathogenesis. Research on the process of neuronal apoptosis in models of ischemic brain injury seems promising. The role of growth arrest and DNA-damage-inducible protein 45 beta (Gadd45b) in brain ischemia has not been fully examined to date. This study aims to investigate the function of Gadd45b in ischemia-induced apoptosis. Adult male Sprague-Dawley rats were subjected to brain ischemia by middle cerebral artery occlusion (MCAO). RNA interference (RNAi) system, which is mediated by a lentiviral vector (LV), was stereotaxically injected into the ipsilateral lateral ventricle to knockdown Gadd45b expression. Neurologic scores and infarct volumes were assessed 24 h after reperfusion. Apoptosis-related molecules were studied using immunohistochemistry and Western blot analysis. We found that Gadd45b-RNAi significantly increased infarct volumes and worsened the outcome of transient focal cerebral ischemia. Gadd45b-RNAi also significantly increased neuronal apoptosis as indicated by increased levels of Bax and active caspase-3, and decreased levels of Bcl-2. These results indicate that Gadd45b is a beneficial mediator of neuronal apoptosis.
doi:10.7150/ijbs.9813
PMCID: PMC4323375
MCAO; Gadd45b; BDNF; Apoptosis
4.  Clinical trial with traditional Chinese medicine intervention ''tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment'' for chronic hepatitis B-associated liver failure 
World Journal of Gastroenterology : WJG  2014;20(48):18458-18465.
AIM: To study the clinical efficacy of traditional Chinese medicine (TCM) intervention “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) for treating liver failure due to chronic hepatitis B.
METHODS: We designed the study as a randomized controlled clinical trial. Registration number of Chinese Clinical Trial Registry is ChiCTR-TRC-12002961. A total of 144 patients with liver failure due to infection with chronic hepatitis B virus were enrolled in this randomized controlled clinical study. Participants were randomly assigned to the following three groups: (1) a modern medicine control group (MMC group, 36 patients); (2) a “tonifying qi and detoxification” (“TQD”) group (72 patients); and (3) a “tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”) group (36 patients). Patients in the MMC group received general internal medicine treatment; patients in the “TQD” group were given a TCM formula “tonifying qi and detoxification” and general internal medicine treatment; patients in the “TTK” group were given a TCM formula of “TTK” and general internal medicine treatment. All participants were treated for 8 wk and then followed at 48 wk following their final treatment. The primary efficacy end point was the patient fatality rate in each group. Measurements of various virological and biochemical indicators served as secondary endpoints. The one-way analysis of variance and the t-test were used to compare patient outcomes in the different treatment groups.
RESULTS: At the 48-wk post-treatment time point, the patient fatality rates in the MMC, “TQD”, and “TTK” groups were 51.61%, 35.38%, and 16.67%, respectively, and the differences between groups were statistically significant (P < 0.05). However, there were no significant differences in the levels of hepatitis B virus DNA or prothrombin activity among the three groups (P > 0.05). Patients in the “TTK” group had significantly higher levels of serum total bilirubin compared to MMC subjects (339.40 μmol/L ± 270.09 μmol/L vs 176.13 μmol/L ± 185.70 μmol/L, P = 0.014). Serum albumin levels were significantly increased in both the “TQD” group and “TTK” group as compared with the MMC group (31.30 g/L ± 4.77 g/L, 30.72 g/L ± 2.89 g/L vs 28.57 g/L ± 4.56 g/L, P < 0.05). There were no significant differences in levels of alanine transaminase among the three groups (P > 0.05). Safety data showed that there was one case of stomachache in the “TQD” group and one case of gastrointestinal side effect in the “TTK” group.
CONCLUSION: Treatment with “TTK” improved the survival rates of patients with liver failure due to chronic hepatitis B. Additionally, liver tissue was regenerated and liver function was restored.
doi:10.3748/wjg.v20.i48.18458
PMCID: PMC4277987  PMID: 25561817
Clinical study; “Tonifying the kidney to promote liver regeneration and repair by affecting stem cells and their microenvironment” (“TTK”); Liver regeneration; Treatment with integrated traditional and Western medicine; Chronic hepatitis B-associated liver failure
5.  Phosphorylation of Ribosomal Protein S6 Kinase 1 at Thr421/Ser424 and Dephosphorylation at Thr389 Regulates SP600125-Induced Polyploidization of Megakaryocytic Cell Lines 
PLoS ONE  2014;9(12):e114389.
Megakaryocytes (MKs) are one of the few cell types that become polyploid; however, the mechanisms by which these cells are designated to become polyploid are not fully understood. In this investigation, we successfully established two relatively synchronous polyploid cell models by inducing Dami and CMK cells with SP600125. We found that SP600125 induced the polyploidization of Dami and CMK cells, concomitant with the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) at Thr421/Ser424 and dephosphorylation at Thr389. The polyploidization was partially blocked by H-89, a cAMP-dependent protein kinase (PKA) inhibitor, through direct binding to S6K1, leading to dephosphorylation at Thr421/Ser424 and phosphorylation at Thr389, independent of PKA. Overexpression of a rapamycin-resistant mutant of S6K1 further enhanced the inhibitory effect of LY294002 on the SP600125-induced polyploidization of Dami and CMK cells. SP600125 also induced the polyploidization of Meg-01 cells, which are derived from a patient with chronic myelogenous leukemia, without causing a significant change in S6K1 phosphorylation. Additionally, SP600125 induced the polyploidization of HEL cells, which are derived from a patient with erythroleukemia, and phosphorylation at Thr389 of S6K1 was detected. However, the polyploidization of both Meg-01 cells and HEL cells as a result of SP600125 treatment was lower than that of SP600125-induced Dami and CMK cells, and it was not blocked by H-89 despite the increased phosphorylation of S6K1 at Thr389 in both cell lines in response to H-89. Given that the Dami and CMK cell lines were derived from patients with acute megakaryocytic leukemia (AMKL) and expressed high levels of platelet-specific antigens, our data suggested that SP600125-induced polyploidization is cell-type specific, that these cell lines were more differentiated, and that phosphorylation at Thr421/Ser424 and dephosphorylation at Thr389 of S6K1 may play an important role in the SP600125-induced polyploidization of these cell lines synergistically with other signaling pathways.
doi:10.1371/journal.pone.0114389
PMCID: PMC4259319  PMID: 25486532
6.  Pre-treatment with a Xingnaojing preparation ameliorates sevoflurane-induced neuroapoptosis in the infant rat striatum 
Molecular Medicine Reports  2014;11(3):1615-1622.
Xingnaojing (XNJ), is a standardized Chinese herbal medicine product derived from An Gong Niu Huang Pill. It may be involved in neuroprotection in a number of neurological disorders. Exposure to anesthetic agents during the brain growth spurt may trigger widespread neuroapoptosis in the developing brain. Thus the present study aimed to identify whether there was a neuroprotective effect of XNJ on anesthesia-induced neuroapoptosis. Seven-day-old rats received treatment with 2.1% sevoflurane for 6 h. Rat pups were injected intraperitoneally with 1 or 10 ml/kg XNJ at 0.2, 24 and 48 h prior to sevoflurane exposure. The striata of neonatal rats were collected following administration of anesthesia. Western blotting and immunohistochemistry were used to analyze the expression of activated caspase 3, Bax and phosphorylated protein kinase B (p-AKT) in the striatum. It was found that activated caspase 3 and Bax expression were upregulated in the striatum following sevoflurane treatment. Preconditioning with XNJ attenuated the neuronal apoptosis induced by sevoflurane in a dose-dependent manner. Anesthesia reduced the expression of p-AKT (phosphorylated at sites Thr308 and Ser473) and phosphorylated extracellular-regulated protein kinase (p-ERK) in the striatum. Pre-treatment with XNJ reversed the reduction in p-AKT, but not p-ERK expression. These data suggest that XNJ has an antiapoptotic effect against sevoflurane-induced cell loss in the striatum. It thus holds promise as a safe and effective neuroprotective agent. The action of XNJ on p-AKT may make a significant contribution to its neuroprotective effect.
doi:10.3892/mmr.2014.2934
PMCID: PMC4270333  PMID: 25395182
chinese medicine; sevoflurane; apoptosis; signal transduction; protein kinase B signaling; striatum
7.  Association between Hemorrhagic Fever with Renal Syndrome Epidemic and Climate Factors in Heilongjiang Province, China 
The purpose of this study was to quantify the relationship between climate variation and transmission of hemorrhagic fever with renal syndrome (HFRS) in Heilongjiang Province, a highly endemic area for HFRS in China. Monthly notified HFRS cases and climatic data for 2001–2009 in Heilongjiang Province were collected. Using a seasonal autoregressive integrated moving average model, we found that relative humidity with a one-month lag (β = –0.010, P = 0.003) and a three-month lag (β = 0.008, P = 0.003), maximum temperature with a two-month lag (β = 0.082, P = 0.028), and southern oscillation index with a two-month lag (β = –0.048, P = 0.019) were significantly associated with HFRS transmission. Our study also showed that predicted values expected under the seasonal autoregressive integrated moving average model were highly consistent with observed values (Adjusted R2 = 83%, root mean squared error = 108). Thus, findings may help add to the knowledge gap of the role of climate factors in HFRS transmission in China and also assist national local health authorities in the development/refinement of a better strategy to prevent HFRS transmission.
doi:10.4269/ajtmh.12-0473
PMCID: PMC3820312  PMID: 24019443
8.  Can Serum Glypican-3 Be a Biomarker for Effective Diagnosis of Hepatocellular Carcinoma? A Meta-Analysis of the Literature 
Disease Markers  2014;2014:127831.
Objective. This review is to evaluate the diagnostic value of serum GPC3 for hepatocellular carcinoma (HCC) due to conflicting results reported. Methods. NCBI PubMed and Embase were comprehensively searched for studies that have used serum GPC3 level as a diagnostic index for HCC. The quality of the included studies was assessed. Subgroup analyses were conducted to evaluate the sensitivity and specificity of GPC3 as a HCC marker. Statistical analysis was performed with the software STATA version 12.0. Results. A total of 22 studies were included. The qualities of included studies were relatively poor. Among them, 18 studies have shown that serum GPC3 is a specific biomarker for HCC, and the pooled sensitivity and specificity of these studies were 69 and 93%, respectively. The other 4 studies have reported conflicting results, which were not caused by races, infection status of HBV and HCV, or assay reagents but due to one common experimental design of enrolling liver cirrhosis patients as control subjects. Conclusions. This meta-analysis indicates that serum GPC3 is elevated in HCC patients compared with healthy individuals, but more studies are needed to evaluate its effectiveness to differentially diagnose HCC and liver cirrhosis.
doi:10.1155/2014/127831
PMCID: PMC4214040  PMID: 25378766
9.  Hepatoma upregulated protein expression is involved in the pathogenesis of human breast carcinogenesis 
Oncology Letters  2014;8(6):2543-2548.
In the last decade, the overexpression of hepatoma upregulated protein (HURP) has been reported in hepatocellular carcinoma, adrenocortical tumors and urogenital carcinoma. However, the role of HURP in breast cancer remains unknown. In the present study, a comprehensive analysis was performed to examine the HURP expression level in 43 breast cancer tumor samples and paired adjacent normal tissues. The correlation between the HURP expression level and the clinicopathological characteristics was evaluated. The role of HURP in breast cancer was investigated by quantitative polymerase chain reaction, western blot analysis and cell proliferation assays. HURP expression was found to be significantly increased in the breast cancer samples. The HURP expression level was higher in the tumors with advanced-grade metastasis and was strongly associated with tumor-node-metastasis staging (P=0.003). Transfection and cell proliferation assays suggested that the suppression of HURP expression or the interference in HURP activity in the breast cancer cells inhibited cell proliferation significantly. These data suggest that HURP is associated with the degree of malignancy and the proliferation of breast cancer. HURP could be a tumor biomarker for prognosis and a potential therapeutic drug target for human breast cancer.
doi:10.3892/ol.2014.2614
PMCID: PMC4214440  PMID: 25364424
hepatoma upregulated protein expression; breast carcinogenesis; cell proliferation
10.  Reversible Hydrophobic to Hydrophilic Transition in Graphene via Water Splitting Induced by UV Irradiation 
Scientific Reports  2014;4:6450.
Although the reversible wettability transition between hydrophobic and hydrophilic graphene under ultraviolet (UV) irradiation has been observed, the mechanism for this phenomenon remains unclear. In this work, experimental and theoretical investigations demonstrate that the H2O molecules are split into hydrogen and hydroxyl radicals, which are then captured by the graphene surface through chemical binding in an ambient environment under UV irradiation. The dissociative adsorption of H2O molecules induces the wettability transition in graphene from hydrophobic to hydrophilic. Our discovery may hold promise for the potential application of graphene in water splitting.
doi:10.1038/srep06450
PMCID: PMC4171696  PMID: 25245110
11.  Investigation of the mechanism of dural arteriovenous fistula formation induced by high intracranial venous pressure in a rabbit model 
BMC Neuroscience  2014;15(1):101.
Background
The causes of dural arteriovenous fistula have not been clearly defined. The aim of this study was to investigate the mechanism of dural arteriovenous fistula formation induced by high intracranial venous pressure using a rabbit model.
Results
By using rabbit model, dural arteriovenous fistula formation induced by high intracranial venous pressure could be produced by end-to-end and end-to-side anastomosis of the right side common carotid artery with the posterior facial vein plus ligation of the contralateral external jugular vein. As compared the post arteriovenous fistula formation among 1 week, 2 weeks, 3 weeks, and 90 days, the expression level of vascular endothelial growth factor in the 1- and 2-weeks groups was significantly higher compared with the control group, 3 weeks and 90 days groups (p ≤0.002). There was significantly higher hypoxia inducible factor-1α expression in the one week group compared with the control, 2 weeks, 3 weeks, and 90 days groups (p ≤0.002). The results of Western blotting showed that vascular endothelial growth factor expression level was highest in the 1 week group. The expression level of vascular endothelial growth factor was significantly different between all groups.
Conclusions
The results of the experiments in our rabbit model indicate that high intracranial venous pressure is a key for dural arteriovenous fistula formation. Cerebral ischemia caused by lack of cerebral perfusion pressure plays a key role in the process that leads from high intracranial venous pressure to increased hypoxia inducible factor-1α expression and then increased vascular endothelial growth factor expression.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2202-15-101) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2202-15-101
PMCID: PMC4152575  PMID: 25160131
Dural arteriovenous fistula; Rabbit model; High intracranial venous pressure; Hypoxia inducible factor-1α; Vascular endothelial growth factor
12.  Plant-Produced Human Recombinant Erythropoietic Growth Factors Support Erythroid Differentiation In Vitro 
Stem Cells and Development  2013;22(16):2326-2340.
Clinically available red blood cells (RBCs) for transfusions are at high demand, but in vitro generation of RBCs from hematopoietic stem cells requires significant quantities of growth factors. Here, we describe the production of four human growth factors: erythropoietin (EPO), stem cell factor (SCF), interleukin 3 (IL-3), and insulin-like growth factor-1 (IGF-1), either as non-fused proteins or as fusions with a carrier molecule (lichenase), in plants, using a Tobacco mosaic virus vector-based transient expression system. All growth factors were purified and their identity was confirmed by western blotting and peptide mapping. The potency of these plant-produced cytokines was assessed using TF1 cell (responsive to EPO, IL-3 and SCF) or MCF-7 cell (responsive to IGF-1) proliferation assays. The biological activity estimated here for the cytokines produced in plants was slightly lower or within the range cited in commercial sources and published literature. By comparing EC50 values of plant-produced cytokines with standards, we have demonstrated that all four plant-produced growth factors stimulated the expansion of umbilical cord blood-derived CD34+ cells and their differentiation toward erythropoietic precursors with the same potency as commercially available growth factors. To the best of our knowledge, this is the first report on the generation of all key bioactive cytokines required for the erythroid development in a cost-effective manner using a plant-based expression system.
doi:10.1089/scd.2012.0489
PMCID: PMC3730378  PMID: 23517237
13.  Inhibitory and Acceleratory Effects of Inonotus obliquus on Tyrosinase Activity and Melanin Formation in B16 Melanoma Cells 
The aim of the present study is to preliminarily investigate the antimelanogenesis effect of Inonotus obliquus extracts by cell-free mushroom tyrosinase assay. It was found that petroleum ether and n-butanol extracts might contain unknown potential tyrosinase inhibitors, while its ethyl acetate extract might contain some unknown accelerators. Six compounds were isolated and their structures were identified by interpretation of NMR data and nicotinic acid was first discovered in Inonotus obliquus. In cells testing, betulin and trametenolic acid decreased tyrosinase activity and melanin content, while inotodiol and lanosterol significantly increased tyrosinase activity and melanin content, showing an AC⁡50 of 9.74 and 8.43 μM, respectively. Nicotinie acid, 3β,22,25-trihydroxy-lanosta-8-ene, had a little or no effect on tyrosinase. Betulin exhibited a mode of noncompetitive inhibition with a KI = KIS of 0.4 μM on tyrosinase activity showing an IC50 of 5.13 μM and being more effective than kojic acid (6.43 μM), and trametenolic acid exhibited a mode of mixed inhibition with a KI of 0.9 μM, KIS of 0.5 μM, and an IC50 of 7.25 μM. We proposed betulin and trametenolic acid as a new candidate of potent tyrosinase inhibitors and inotodiol and lanosterol as accelerators that could be used as therapeutic agent.
doi:10.1155/2014/259836
PMCID: PMC4145793  PMID: 25197307
14.  Elevated levels of plasma D-dimer predict a worse outcome in patients with nasopharyngeal carcinoma 
BMC Cancer  2014;14(1):583.
Background
Hemostatic alterations occur during the development of cancer. Plasma D-dimer is a hypercoagulability and fibrinolytic system marker that is increased in patients with various solid tumours. The aim of this study was to evaluate the hemostatic status of nasopharyngeal carcinoma (NPC) patients by assessing plasma D-dimer levels to investigate its value as a prognostic marker.
Methods
We retrospectively analysed 717 patients with nasopharyngeal carcinoma, and we applied Cox regression and log-rank tests to assess the association of D-dimer levels with disease-free survival (DFS), distant metastasis-free survival (DMFS), and overall survival (OS). D-dimer levels were measured using a quantitative D-dimer latex agglutination assay.
Results
Using the 3rd quartile values (0.8 μg/L) as the optimal cut-offs, we found that patients with high D-dimer levels have a shorter 3-year DFS, (79%, 95%CI (73.1–84.9)) vs. (69%, 95%CI (59.2–78.8)), DMFS (87%, 95%CI (83.1–90.9)) vs. (77%, 95%CI (69.2–84.8)), and overall survival (82%, 95%CI (76.1–87.9)) vs. (76%, 95%CI (66.2–85.8)). Multivariate analysis revealed that pre-treatment D-dimer levels and EBV DNA were significant independent factors for DFS, DMFS, and OS in NPC patients. Subgroup analyses indicated that the plasma D-dimer levels could effectively stratify patient prognosis for early cancer, advanced stage cancer, and patients with EBV DNA ≥4000 copies/ml.
Conclusions
High D-dimer levels were associated with poor disease-free survival, distant metastasis-free survival, overall survival, and increased risk of mortality in NPC patients. Prospective trials are required to assess the prognostic value of D-dimer levels.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-583) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-583
PMCID: PMC4242497  PMID: 25109220
Nasopharyngeal carcinoma; D-dimer; Survival
15.  Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus, Aspergillus versicolor ZBY-3 
Marine Drugs  2014;12(8):4326-4352.
A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.
doi:10.3390/md12084326
PMCID: PMC4145319  PMID: 25076061
Aspergillus versicolor ZBY-3; deep-sea fungus; neomycin resistance; ultrasound; antitumor activity; secondary metabolite production
16.  Ablation of EIF5A2 induces tumor vasculature remodeling and improves tumor response to chemotherapy via regulation of matrix metalloproteinase 2 expression 
Oncotarget  2014;5(16):6716-6733.
Hepatocellular carcinoma (HCC) is a highly vascularized tumor with poor clinical outcome. Our previous work has shown that eukaryotic initiation factor 5A2 (EIF5A2) over-expression enhances HCC cell metastasis. In this study, EIF5A2 was identified to be an independent risk factor for poor disease-specific survival among HCC patients. Both in vitro and in vivo assays indicated that ablation of endogenous EIF5A2 inhibited tumor angiogenesis by reducing matrix metalloproteinase 2 (MMP-2) expression. Given that MMP-2 degrades collagen IV, a main component of the vascular basement membrane (BM), we subsequently investigated the effect of EIF5A2 on tumor vasculature remodeling using complementary approaches, including fluorescent immunostaining, transmission electron microscopy, tumor perfusion assays and tumor hypoxia assays. Taken together, our results indicate that EIF5A2 silencing increases tumor vessel wall continuity, increases blood perfusion and improves tumor oxygenation. Additionally, we found that ablation of EIF5A2 enhanced the chemosensitivity of HCC cells to 5-Fluorouracil (5-FU). Finally, we demonstrated that EIF5A2 might exert these functions by enhancing MMP-2 activity via activation of p38 MAPK and JNK/c-Jun pathways. Conclusion: This study highlights an important role of EIF5A2 in HCC tumor vessel remodeling and indicates that EIF5A2 represents a potential therapeutic target in the treatment of HCC.
PMCID: PMC4196158  PMID: 25071013
Hepatocellular carcinoma; Eukaryotic translation initiation factor 5A2; Matrix Metalloproteinase 2; Vasculature remodeling; Chemotherapy
17.  Imine Resveratrol Analogues: Molecular Design, Nrf2 Activation and SAR Analysis 
PLoS ONE  2014;9(7):e101455.
Resveratrol is a natural phenol with protective effects against cancer and inflammation-related diseases. Its mechanism of action involves the activation of nuclear factor E2 p45-related factor 2 (Nrf2), which plays a key role in regulation of genes driven by antioxidant response element (ARE). Inspired by the effect of resveratrol, here we synthesized a series of imine resveratrol analogs (IRAs), evaluated their abilities to activate Nrf2 by using cell based ARE-reporter assay. After the first-round screening, preliminary and quantitative structure-activity relationship (SAR) was analyzed, and the structural features determining Nrf2 activation ability were proposed. Two novel IRAs were designed and subsequently synthesized, namely 2-methoxyl-3,6-dihydroxyl-IRA and 2,3,6-trihydroxyl-IRA. They were proved to be the most potent Nrf2 activators among the IRAs.
doi:10.1371/journal.pone.0101455
PMCID: PMC4100753  PMID: 25028928
18.  Melatonin facilitates extinction, but not acquisition or expression, of conditional cued fear in rats 
BMC Neuroscience  2014;15(1):86.
Background
Previous studies have shown that melatonin is involved in the processes that contribute to learning and memory. At present study, we tested the effects of exogenous melatonin (2.5 mg/kg) on the acquisition, expression and extinction of cued fear in rats.
Results
Results showed that a single afternoon administration 30 min before conditioning has no effect on the acquisition of cued fear. Compared to rats injected with vehicle, rats injected with melatonin 30 min before extinction training presented a significant lower freezing during both extinction training and extinction test phases, however, freezing response did not differ for the initial four trials during extinction training. Melatonin injected immediately after extinction training was ineffective on extinction learning.
Conclusions
These results suggest that melatonin, at the dose applied in this study, facilitates the extinction of conditional cued fear without affecting its acquisition or expression, and melatonin facilitates cued fear extinction only when it is present during extinction training. These findings extend previous research on the melatonin effects on learning and memory and suggest that melatonin may serve as an agent for the treatment of anxiety disorders such as posttraumatic stress disorder (PTSD).
doi:10.1186/1471-2202-15-86
PMCID: PMC4111846  PMID: 25026909
Melatonin; Cued fear; Fear conditioning; Fear extinction; PTSD; Rats
19.  Activation Effects of Polysaccharides of Flammulina velutipes Mycorrhizae on the T Lymphocyte Immune Function 
Journal of Immunology Research  2014;2014:285421.
Flammulina velutipes mycorrhizae have increasingly been produced with increasing of F. velutipes production. A mouse model was thus used to examine potential effect of F. velutipes mycorrhizae on the immune function. Fifty female Wistar mice (5-weeks-old) weighed 15–20 g were randomly allocated into five groups. Polysaccharide of F. velutipes mycorrhizae were treated with mice and mice spleen lymphocytes. The levels of CD3+, CD4+, and CD8+ T lymphocyte, interleukin-2 (IL-2), and tumor necrosis factor-a (TNF-α) were determined. The results showed that the proportions of CD3+, and CD4+ T lymphocyte, the ratio of CD4+/CD8+, and the levels of IL-2 and TNF-a were significantly increased in polysaccharide of F. velutipes mycorrhizae, while the proportion of CD8+ T lymphocyte was decreased in polysaccharide of F. velutipes mycorrhizae-dose dependent manner. Our findings indicated that a long term exposure of polysaccharide of F. velutipes mycorrhizae could activate the T lymphocyte immune function. Polysaccharide of F. velutipes mycorrhizae was expected to develop into the immune health products.
doi:10.1155/2014/285421
PMCID: PMC4122143  PMID: 25133194
20.  ER stress and ER stress-induced apoptosis are activated in gastric SMCs in diabetic rats 
AIM: To investigate the gastric muscle injury caused by endoplasmic reticulum (ER) stress in rats with diabetic gastroparesis.
METHODS: Forty rats were randomly divided into two groups: a control group and a diabetic group. Diabetes was induced by intraperitoneal injection of 60 mg/kg of streptozotocin. Gastric emptying was determined at the 4th and 12th week. The ultrastructural changes in gastric smooth muscle cells (SMCs) were investigated by transmission electron microscopy. TdT-mediated dUTP nick end labeling (TUNEL) assay was performed to assess apoptosis of SMCs. Expression of the ER stress marker, glucose-regulated protein 78 (GRP78), and the ER-specific apoptosis mediator, caspase-12 protein, was determined by immunohistochemistry.
RESULTS: Gastric emptying was significantly lower in the diabetic rats than in the control rats at the 12th wk (40.71% ± 2.50%, control rats vs 54.65% ± 5.22%, diabetic rats; P < 0.05). Swollen and distended ER with an irregular shape was observed in gastric SMCs in diabetic rats. Apoptosis of gastric SMCs increased in the diabetic rats in addition to increased expression of GRP78 and caspase-12 proteins.
CONCLUSION: ER stress and ER stress-mediated apoptosis are activated in gastric SMCs in diabetic rats with gastroparesis.
doi:10.3748/wjg.v20.i25.8260
PMCID: PMC4081701  PMID: 25009401
Diabetic gastroparesis; Apoptosis; Endoplasmic reticulum stress; glucose-regulated protein 78 kD; Caspase-12
21.  Learning Curve and Interobserver Agreement of Confocal Laser Endomicroscopy for Detecting Precancerous or Early-Stage Esophageal Squamous Cancer 
PLoS ONE  2014;9(6):e99089.
Background
Confocal laser endomicroscopy (CLE) can provide in vivo subcellular resolution images of esophageal lesions. However, the learning curve in interpreting CLE images of precancerous or early-stage esophageal squamous cancer is unknown. The goal of this study is to evaluate the diagnostic accuracy and inter-observer agreement for differentiating esophageal lesions in CLE images among experienced and inexperienced observers and to assess the learning curve.
Method
After a short training, 8 experienced and 14 inexperienced endoscopists evaluated in sequence 4 sets of high-quality CLE images. Their diagnoses were corrected and discussed after each set. For each image, the diagnostic results, confidence in diagnosis, quality and time to evaluate were recorded.
Results
Overall, diagnostic accuracy was greater for the second, third, fourth set of images as compared with the initial set (odds ratio [OR] 2.01, 95% CI 1.22–3.31; 7.95, 3.74–16.87; and 6.45, 3.14–13.27), respectively, with no difference between the third and fourth sets in accuracy (p = 0.67). Previous experience affected the diagnostic accuracy only in the first set of images (OR 3.70, 1.87–7.29, p<0.001). Inter-observer agreement was higher for experienced than inexperienced endoscopists (0.732 vs. 0.666, p<0.01)
Conclusion
CLE is a promising technology that can be quickly learned after a short training period; previous experience is associated with diagnostic accuracy only at the initial stage of learning.
doi:10.1371/journal.pone.0099089
PMCID: PMC4045985  PMID: 24897112
22.  Nine New and Five Known Polyketides Derived from a Deep Sea-Sourced Aspergillus sp. 16-02-1 
Marine Drugs  2014;12(6):3116-3137.
Nine new C9 polyketides, named aspiketolactonol (1), aspilactonols A–F (2–7), aspyronol (9) and epiaspinonediol (11), were isolated together with five known polyketides, (S)-2-(2′-hydroxyethyl)-4-methyl-γ-butyrolactone (8), dihydroaspyrone (10), aspinotriol A (12), aspinotriol B (13) and chaetoquadrin F (14), from the secondary metabolites of an Aspergillus sp. 16-02-1 that was isolated from a deep-sea sediment sample. Structures of the new compounds, including their absolute configurations, were determined by spectroscopic methods, especially the 2D NMR, circular dichroism (CD), Mo2-induced CD and Mosher’s 1H NMR analyses. Compound 8 was isolated from natural sources for the first time, and the possible biosynthetic pathways for 1–14 were also proposed and discussed. Compounds 1–14 inhibited human cancer cell lines, K562, HL-60, HeLa and BGC-823, to varying extents.
doi:10.3390/md12063116
PMCID: PMC4071568  PMID: 24871461
Aspergillus sp. 16-02-1; fungal strain from deep sea sediment; aspiketolactonol; aspilactonol; aspyronol; lactone; epiaspinonediol; polyketide; structure; cytotoxicity
23.  Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate 
Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.
doi:10.3892/etm.2014.1741
PMCID: PMC4079424  PMID: 25009597
urokinase-type plasminogen activator; RNA interference; lentiviral vector; osteoarthritis; chondrocytes
24.  Megakaryocytic Differentiation of K562 Cells Induced by PMA Reduced the Activity of Respiratory Chain Complex IV 
PLoS ONE  2014;9(5):e96246.
Mitochondria are involved in the regulation of cell differentiation processes, but its function changes and molecular mechanisms are not yet clear. In this study, we found that mitochondrial functions changed obviously when K562 cells were induced to megakaryocytic differentiation by phorbol 12-myristate 13-acetate (PMA). During the cell differentiation, the reactive oxygen species (ROS) level was increased, mitochondrial membrane potential declined and respiratory chain complex IV activity was decreased. Treatment with specific inhibitor of mitochondrial respiratory chain complex IV led to a significant inhibition in mitochondrial membrane potential and reduction of PMA-induced cell differentiation. However, treatment with cyclosporine A, a stabilization reagent of mitochondrial membrane potential, did not improve the down-regulation of mitochondrial respiratory chain complex IV induced by PMA. Furthermore, we found that the level of the complex IV core subunit COX3 and mitochondrial transport-related proteins Tim9 and Tim10 were decreased during the differentiation of K562 cells induced by PMA, suggesting an important role of these factors in mitochondrial functional changes. Our results suggest that changes in mitochondrial functions are involved in the PMA-induced K562 cell differentiation process, and the maintenance of the steady-state of mitochondrial functions plays a critical role in the regulation of cell differentiation.
doi:10.1371/journal.pone.0096246
PMCID: PMC4015910  PMID: 24817082
25.  Novel designed polyoxyethylene nonionic surfactant with improved safety and efficiency for anticancer drug delivery 
In order to limit the adverse reactions caused by polysorbate 80 in Taxotere®, a widely used formulation of docetaxel, a safe and effective nanocarrier for this drug has been developed based on micelles formed by a new class of well-defined polyoxyethylene sorbitol oleate (PSO) with sorbitol as the matrix in aqueous solution. The physicochemical properties of the amphiphilic surfactant and the resulting micelles can be easily fine-tuned by the homogeneous sorbitol matrix and pure oleic acid. Composition, critical micelle concentration, and entrapment efficiency were investigated by ultraviolet visible spectroscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, fluorospectrophotometry, and high-performance liquid chromatography. In vitro and in vivo evaluation revealed that PSO had exceptionally low hemolysis and histamine release rates compared with commercial polysorbate 80. Moreover, the tumor targeting delivery of PSO was investigated by in vivo imaging in S180 tumor-bearing mice. The results suggest that this novel delivery system, PSO, provides an acceptable alternative to polysorbate 80 for delivery of docetaxel. Further, due to the hypoallergenic nature of PSO, the mechanism of pseudoallergy caused by the polyoxyethylene nonionic surfactant was investigated. Based on in vitro cell analysis, it was assumed that the initial contact of polyoxyethylene nonionic surfactant with mast cells provoked pseudoallergy via polyamine receptor-mediated endocytosis.
doi:10.2147/IJN.S60667
PMCID: PMC4010632  PMID: 24812509
polyoxyethylene nonionic surfactant; sorbitol; isosorbide; pseudoallergy; docetaxel

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