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1.  Sperm, but Not Oocyte, DNA Methylome Is Inherited by Zebrafish Early Embryos 
Cell  2013;153(4):773-784.
5-methylcytosine is a major epigenetic modification that is sometimes called “the fifth nucleotide.” However, our knowledge of how offspring inherit the DNA methylome from parents is limited. We generated nine single-base resolution DNA methylomes, including zebrafish gametes and early embryos. The oocyte methylome is significantly hypomethylated compared to sperm. Strikingly, the paternal DNA methylation pattern is maintained throughout early embryogenesis. The maternal DNA methylation pattern is maintained until the 16-cell stage. Then, the oocyte methylome is gradually discarded through cell division and is progressively reprogrammed to a pattern similar to that of the sperm methylome. The passive demethylation rate and the de novo methylation rate are similar in the maternal DNA. By the midblastula stage, the embryo’s methylome is virtually identical to the sperm methylome. Moreover, inheritance of the sperm methylome facilitates the epigenetic regulation of embryogenesis. Therefore, besides DNA sequences, sperm DNA methylome is also inherited in zebrafish early embryos.
PMCID: PMC4081501  PMID: 23663777
2.  Investigating Migration Inhibition and Apoptotic Effects of Fomitopsis pinicola Chloroform Extract on Human Colorectal Cancer SW-480 Cells 
PLoS ONE  2014;9(7):e101303.
Fomitopsis pinicola (Sw. Ex Fr.m) Karst (FPK) which belongs to the Basidiomycota fungal class is one of the most popular medical fungi in China. It has been used for many diseases: cancer, heart diseases, diabetes and so on. However, little study on the pro-apoptotic effect and migration inhibition of FPK chloroform extract (FPKc) has been reported and the possible involved mechanism has not been illuminated.
Methodology/Principal Findings
Chemical analysis was performed by HPLC which showed ergosterol (ES) concentration was 105 µg/mg. MTT assay revealed that FPKc could selectively inhibit SW-480 cells viability with the IC50 of 190.28 µg/ml. Wound healing and transwell assay indicated that FPKc could inhibit the migration of SW-480 cells obviously, FPKc could also dramatically decreased the matrix metalloproteinases-2, 9 (MMP-2 and MMP-9) expression. Annexin V–FITC/PI staining, nuclear Hoechst 33342 staining and DNA fragmentation analysis revealed that FPKc and ES could induce SW-480 cells apoptosis. The apoptosis process closely involved in ROS accumulation and depletion of GSH, activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation. FPKc could also up-regulate P53 expression and thus lead to G1 phase arrest. When SW-480 cells were pretreated with N-acetylcysteine (NAC), the ROS generation, cell viability and apoptotic ratio were partially declined, which indicated that ROS was vertical in the pro-apoptosis process induced by FPKc. Moreover, in the whole process, ES which has been previously found in FPKc had the similar effect to FPKc. Thus we could conclude that ES, as one of the highest abundant components in FPKc, might also be one of the active constituents.
FPKc could inhibit the migration of SW-480 cells, induce SW-480 cells G1 phase arrest and cause ROS-mediated apoptosis effect. And ES might be one of the effective constituents in the whole process.
PMCID: PMC4081521  PMID: 24992193
3.  Pharmacological Preconditioning with Vitamin C Attenuates Intestinal Injury via the Induction of Heme Oxygenase-1 after Hemorrhagic Shock in Rats 
PLoS ONE  2014;9(6):e99134.
Pre-induction of heme oxygenase (HO)-1, which is regarded as an effective method of “organ preconditioning”, exerts beneficial effects during hemorrhagic shock (HS). However, the available HO-1 inducers exhibit disadvantages such as toxicity or complex technical requirements. Therefore, a safe and convenient HO-1 inducer would be promising and could be exploited in the treatment of foreseeable hemorrhaging, such as prior to major surgery. Here we investigated the effect of vitamin C (VitC), a common antioxidant, on intestinal HO-1 expression and examined whether VitC pretreatment prevented HS related intestinal tissue injuries after HO-1 induction. First, we conducted an in vitro study and found that HO-1 expression in rat intestinal epithelial cells (IEC-6) was induced by non-toxic VitC in a time and concentration dependent manner, and the mechanism was related to the activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Next, we conducted an in vivo study and found that VitC induced intestinal HO-1 protein expression (mainly observed in the intestinal epithelial cells) and HO-1 activity in normal SD rats, and that these HO-1 levels were further enhanced by VitC in a rat model of HS. The HS related intestinal injuries, including histological damage, pro-inflammatory cytokine levels (tumor necrosis factor and interleukin-6), neutrophil infiltration and apoptosis decreased after VitC pretreatment, and this alleviating of organ injuries was abrogated after the inhibition of HO-1 activity by zinc protoporphyrin-IX. It was of note that VitC did little histological damage to the intestine of the sham rats. These data suggested that VitC might be applied as a safe inducer of intestinal HO-1 and that VitC pretreatment attenuated HS related intestinal injuries via the induction of HO-1.
PMCID: PMC4057195  PMID: 24927128
4.  Screening for Acute HIV Infections and Estimating HIV Incidence among Female Sex Workers from Low-Grade Venues in Guangxi, China 
PLoS ONE  2014;9(6):e99522.
Guangxi has become one of the provinces with the most severe HIV-1 epidemic in China, where heterosexual contact is the dominant transmission route. However, data of acute HIV infections and HIV incidence among female sex workers (FSWs) from low-grade venues are scant.
A cross-sectional survey was performed among FSWs from low-grade venues in Guangxi. HIV antibody screening was performed by rapid testing (RT). HIV antibody–negative specimens were screened by pooled nucleic acid amplification testing (NAAT) for acute HIV infections. HIV antibody-positive specimens were further analyzed by Western blot (WB), followed by an HIV-1 BED capture enzyme immunoassay (BED-CEIA) to identify the recent infections. HIV-1 incidence was estimated by the data of pooled NAAT and BED-CEIA, respectively.
A total of 7936 FSWs were recruited and answered the questionnaires. We successfully collected the blood samples from 6469 (81.5%) participants, of which 139 (2.1%) were HIV antibody–positive and 6330 (97.9%) were HIV antibody-negative by RT. With pooled NAAT, 7 cases were found to be HIV RNA positive, representing an additional 5.0% of HIV-infected persons and an estimated HIV incidence of 1.45 (95% CI: 1.17–1.76) per 100 person years. There were 137 positive and 2 indeterminate by WB, of which 124 (90.5%) positive specimens were subjected to BED-CEIA testing identifying 28 recent infections. The HIV incidence determined by BED-CEIA testing was 1.04 (95% CI: 0.65–1.43) per 100 person years. The overall prevalence of HIV among FSWs from low-grade venues in Guangxi was 2.2% (95% CI: 1.9–2.6).
We found that the addition of HIV RNA screening to routine HIV antibody testing significantly improved the detection of HIV infection among FSWs from low-grade venues in Guangxi. Our findings also provided the useful baseline data of HIV incidence among this population for targeting local HIV prevention, intervention, monitoring and treatment.
PMCID: PMC4053442  PMID: 24918933
5.  PIK3CA and TP53 Gene Mutations in Human Breast Cancer Tumors Frequently Detected by Ion Torrent DNA Sequencing 
PLoS ONE  2014;9(6):e99306.
Breast cancer is the most common malignancy and the leading cause of cancer deaths in women worldwide. While specific genetic mutations have been linked to 5–10% of breast cancer cases, other environmental and epigenetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive breast cancer molecular profile is needed to develop more effective target therapies. Until recently, identifying genetic cancer mutations via personalized DNA sequencing was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 105 human breast cancer samples. The sequencing analysis revealed missense mutations in PIK3CA, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.
PMCID: PMC4053449  PMID: 24918944
6.  miR-145 downregulates the expression of cyclin-dependent kinase 6 in human cervical carcinoma cells 
The present study aimed to investigate the effect of the inhibition of miR-145 on cyclin-dependent protein kinase 6 (CDK6) and the proliferation of human cervical carcinoma cells. The miR-145 sequence was synthesized and cloned into pcDNA™6.2-GW to construct the recombinant plasmid pcDNA6.2-GW-miR-145. HeLa cells were divided into the micro (mi)R-145, normal control and blank groups. The transcription levels of miR-145 and CDK6 were detected using quantitative polymerase chin reaction and western blot analysis was used to examine the CDK6 protein expression. In addition, the inhibitory effect of miR-145 on the proliferation of HeLa cells was measured by an MTT assay. The recombinant plasmid pcDNA6.2-GW-miR-145 was successfully constructed and used to transfect the HeLa cells in the MiR-145 group. The miR-145 expression level in the miR-145 group was significantly higher than that in the blank group. The CDK6 expression level in miR-145 group was significantly lower than that in the blank group. Furthermore, miR-145 inhibited the proliferation of HeLa cells. In conclusion, miR-145 overexpression suppresses the expression of CDK6 and inhibits the proliferative ability of HeLa cells.
PMCID: PMC4079406
cervical carcinoma; miRNA-145; eukaryotic expression vector; cyclin-dependent kinase 6
7.  MiR-106a is an independent prognostic marker in patients with glioblastoma 
Neuro-Oncology  2013;15(6):707-717.
Very little is known regarding correlation of micro RNA (miR)–106a with clinical outcomes of patients with glioblastoma multiforme (GBM). This study determined whether miR-106a could be used as an independent prognostic biomarker in those patients.
A total of 156 GBM patients were divided into 2 cohorts. In the first cohort, matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples were collected from 24 GBM patients, while in the second cohort, only FFPE samples were collected from 132 GBM patients. MiR-106a expression levels were examined by quantitative real-time PCR in the 2 cohorts and further validated by in situ hybridization assay in the second cohort. The correlation between miR-106a expression levels and overall survival was evaluated in the second cohort of 114 GBM patients available for follow-up by a log-rank test and a multivariate Cox proportional hazards model.
Our data showed a very good correlation of miR-106a or U6 expression between fresh frozen and FFPE GBM specimens, with Pearson's correlation coefficients of 0.849 and 0.823, respectively (P < .001). Their expression levels in archival FFPE samples were quite stable for at least 7 years when stored at room temperature. Multivariate analysis revealed that the expression level of miR-106a was an independent and significant predictor of overall survival in GBM patients (P = .011).
MiR-106a expression was relatively abundant and stable in a large cohort of archival FFPE GBM specimens and could be used as an independent prognostic biomarker in those patients. Thus, miR-106a can be used to predict prognosis and treatment response in individual GBM patients.
PMCID: PMC3661090  PMID: 23416698
FFPE; glioblastoma; miR-106a; prognosis; prognostic marker
8.  Hemodynamic, Autonomic, and Vascular Effects of Exposure to Coarse Particulate Matter Air Pollution from a Rural Location 
Environmental Health Perspectives  2014;122(6):624-630.
Background: Fine particulate matter (PM) air pollution is associated with numerous adverse health effects, including increased blood pressure (BP) and vascular dysfunction. Coarse PM substantially contributes to global air pollution, yet differs in characteristics from fine particles and is currently not regulated. However, the cardiovascular (CV) impacts of coarse PM exposure remain largely unknown.
Objectives: Our goal was to elucidate whether coarse PM, like fine PM, is itself capable of eliciting adverse CV responses.
Methods: We performed a randomized double-blind crossover study in which 32 healthy adults (25.9 ± 6.6 years of age) were exposed to concentrated ambient coarse particles (CAP; 76.2 ± 51.5 μg/m3) in a rural location and filtered air (FA) for 2 hr. We measured CV outcomes during, immediately after, and 2 hr postexposures.
Results: Both systolic (mean difference = 0.32 mmHg; 95% CI: 0.05, 0.58; p = 0.021) and diastolic BP (0.27 mmHg; 95% CI: 0.003, 0.53; p = 0.05) linearly increased per 10 min of exposure during the inhalation of coarse CAP when compared with changes during FA exposure. Heart rate was on average higher (4.1 bpm; 95% CI: 3.06, 5.12; p < 0.0001) and the ratio of low-to-high frequency heart rate variability increased (0.24; 95% CI: 0.07, 0.41; p = 0.007) during coarse particle versus FA exposure. Other outcomes (brachial flow-mediated dilatation, microvascular reactive hyperemia index, aortic hemodynamics, pulse wave velocity) were not differentially altered by the exposures.
Conclusions: Inhalation of coarse PM from a rural location is associated with a rapid elevation in BP and heart rate during exposure, likely due to the triggering of autonomic imbalance. These findings add mechanistic evidence supporting the biological plausibility that coarse particles could contribute to the triggering of acute CV events.
Citation: Brook RD, Bard RL, Morishita M, Dvonch JT, Wang L, Yang HY, Spino C, Mukherjee B, Kaplan MJ, Yalavarthi S, Oral EA, Ajluni N, Sun Q, Brook JR, Harkema J, Rajagopalan S. 2014. Hemodynamic, autonomic, and vascular effects of exposure to coarse particulate matter air pollution from a rural location. Environ Health Perspect 122:624–630;
PMCID: PMC4050508  PMID: 24618231
9.  Remodeling of rat pulmonary artery induced by chronic smoking exposure 
Journal of Thoracic Disease  2014;6(6):818-828.
To evaluate the dominant role in rat pulmonary artery (PA) remodeling induced by chronic smoking exposure (CSE).
Thirty-five male Sprague-Dawley (SD) rats were exposed to 36 cigarettes per day, 6 days per week, for 1, 3, or 5 months. Another 35 SD rats were sham-exposed during the same period. Hemodynamic measurement, evaluation of the right ventricular hypertrophy index (RVHI) plus right ventricle-to-weight ratio, and hematoxylin eosin staining was performed. Wall thickness, artery radius, luminal area, and total area were measured morphometrically. Western blotting assessed expression of PPAR-γ BMP4, BMPR2, and TRPC1/4/6 in the artery and lung. Store-operated calcium entry (SOCE) and [Ca2+]i were measured using Fura-2 as dye.
Mean right ventricular pressure increased after 3 months of smoking exposure and continued to increase through 5 months. Right ventricular systolic pressure (RVSP) increased after 3 months of exposure and then stabilized. RVHI increased after 5 months; right ventricle-to-weight ratio was elevated after 3 months and further increased after 5 months. Wall thickness-to-radius ratio does-dependently increased after 3 months through 5 months, in parallel with the decreased luminal area/total area ratio after 5 months. Other changes included the development of inflammatory responses, enlargement of the alveolar spaces, and reductions in the endothelial lining of PAs, proliferative smooth muscle cells, fibroblasts, and adventitia. Moreover, BMP4 and TRPC1/4/6 expression increased to varying degrees in the arteries and lungs of smoking-exposed animals, whereas BMPR expression and SOCE increased only in the arteries, and PPAR-γ was downregulated in both the arteries and lungs.
In SD rats, smoking exposure induces pulmonary vascular remodeling. The consequences of increased SOCE include increase in TRPC1/4/6, probably via augmented BMP4 expression, which also contribute to inflammatory responses in the lung. Moreover, interactions between BMP4 and PPAR-γ may play a role in preventing inflammation under normal physiological conditions.
PMCID: PMC4073362  PMID: 24977008
Pulmonary artery hypertension (PAH); smoking; remodeling
10.  Evodiamine Synergizes with Doxorubicin in the Treatment of Chemoresistant Human Breast Cancer without Inhibiting P-Glycoprotein 
PLoS ONE  2014;9(5):e97512.
Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO), a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX)-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose) polymerase (PARP), caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs). Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp). Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.
PMCID: PMC4022620  PMID: 24830744
11.  Emotional Body-Word Conflict Evokes Enhanced N450 and Slow Potential 
PLoS ONE  2014;9(5):e95198.
Emotional conflict refers to the influence of task irrelevant affective stimuli on current task set. Previously used emotional face-word tasks have produced certain electrophysiological phenomena, such as an enhanced N450 and slow potential; however, it remains unknown whether these effects emerge in other tasks. The present study used an emotional body-word conflict task to investigate the neural dynamics of emotional conflict as reflected by response time, accuracy, and event-related potentials, which were recorded with the aim of replicating the previously observed N450 and slow potential effect. Results indicated increased response time and decreased accuracy in the incongruent condition relative to the congruent condition, indicating a robust interference effect. Furthermore, the incongruent condition evoked pronounced N450 amplitudes and a more positive slow potential, which might be associated with conflict-monitoring and conflict resolution. The present findings extend our understanding of emotional conflict to the body-word domain.
PMCID: PMC4018289  PMID: 24819150
12.  MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma 
PLoS ONE  2014;9(5):e96765.
Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in vivo animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.
PMCID: PMC4016005  PMID: 24816813
13.  Lipid modulatory activities of Cichorium glandulosum Boiss et Huet are mediated by multiple components within hepatocytes 
Scientific Reports  2014;4:4715.
To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of non-alcoholic fatty liver disease (NAFLD), we have made use of Cichorium glandulosum Boiss et Huet (CG), a traditional Chinese herbal medicine that has been proven to be effective in treating hepatic diseases. Here, we report that the extract of CG effectively reduced lipid accumulation under conditions of lipid overloading in vivo and in vitro (in a rat high-fat diet model and a hepG2 cell model of free fatty acid treatment). CG extract also protected hepatocytes from injury and inflammation to aid its lipid-lowering properties (in a rat high-fat diet model and a L02 cell model of acetaminophen treatment). Serum chemistry analysis accompanied by in vitro drug screening confirmed that CG-4, CG-10 and CG-14 are the lipo-effective components of CG. Western blotting analysis revealed that these components can regulate key lipid targets at the molecular level, including CD36, FATP5 and PPAR-α, thus the lipid oxidation and lipid absorption pathways. Finally, we adopted the experimental design and statistical method to calculate the best combination proportion (CG-4: CG-10: CG-14 = 2.065: 1.782: 2.153) to optimize its therapeutic effect.
PMCID: PMC4010933  PMID: 24797163
14.  Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice 
Rice  2014;7(1):5.
The type II clustered, regularly interspaced, short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) system is a novel molecular tool for site-specific genome modification. The CRISPR-Cas9 system was recently introduced into plants by transient or stable transformation.
Here, we report gene targeting in rice via the Agrobacterium tumefaciens-mediated CRISPR-Cas9 system. Three 20-nt CRISPR RNAs were designed to pair with diverse sites followed by the protospacer adjacent motif (PAM) of the rice herbicide resistance gene BEL. After integrating the single-guide RNA (sgRNA) and Cas9 cassette in a single binary vector, transgenic rice plants harboring sgRNA:Cas9 were generated by A. tumefaciens-mediated stable transformation. By analyzing the targeting site on the genome of corresponding transgenic plants, the mutations were determined. The mutagenesis efficiency was varied from ~2% to ~16%. Furthermore, phenotypic analysis revealed that the biallelic mutated transgenic plant was sensitive to bentazon.
Our results indicate that the agricultural trait could be purposely modified by sgRNA:Cas9-induced gene targeting. CRISPR-Cas9 system could be exploited as a powerful tool for trait improvements in crop breeding.
PMCID: PMC4052633  PMID: 24920971
Gene targeting; CRISPR/Cas9; Rice; Agrobacterium; Stable transformation; Genome editing
15.  Blood 25-Hydroxy Vitamin D Levels and Incident Type 2 Diabetes 
Diabetes Care  2013;36(5):1422-1428.
To quantitatively assess the strength and shape of the association between blood 25-hydroxy vitamin D [25(OH)D] levels and incident risk of type 2 diabetes.
A systematic search of the MEDLINE and Embase databases and a hand search of references from original reports were conducted up to 31 October 2012. Prospective observational studies that assessed the association between blood levels of 25(OH)D and risk of incident type 2 diabetes were included for meta-analysis. DerSimonian and Laird’s random-effects model was used. A quadratic spline regression analysis was used to examine the shape of the association with a generalized least-squares trend test performed for the dose-response relation.
A total of 21 prospective studies involving 76,220 participants and 4,996 incident type 2 diabetes cases were included for meta-analysis. Comparing the highest to the lowest category of 25(OH)D levels, the summary relative risk for type 2 diabetes was 0.62 (95% CI 0.54–0.70). A spline regression model showed that higher 25(OH)D levels were monotonically associated with a lower diabetes risk. This inverse association did not differ by sex, duration of follow-up, study sample size, diabetes diagnostic criteria, or 25(OH)D assay method. A linear trend analysis showed that each 10 nmol/L increment in 25(OH)D levels was associated with a 4% lower risk of type 2 diabetes (95% CI 3–6; P for linear trend < 0.0001).
Our meta-analysis showed an inverse and significant association between circulating 25(OH)D levels and risk of type 2 diabetes across a broad range of blood 25(OH)D levels in diverse populations.
PMCID: PMC3631862  PMID: 23613602
16.  Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway 
H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.
Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.
We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.
PMCID: PMC4012743  PMID: 24884338
H-rev107; HRASLS3; PLA2G16; H-RAS; Phospholipase A/acyltransferase; Acyl-biotin exchange assay
17.  MADS-Box Transcription Factor SsMADS Is Involved in Regulating Growth and Virulence in Sclerotinia sclerotiorum 
MADS-box proteins, a well-conserved family of transcription factors in eukaryotic organisms, specifically regulate a wide range of cellular functions, including primary metabolism, cell cycle, and cell identity. However, little is known about roles of the MADS-box protein family in the fungal pathogen Sclerotinia sclerotiorum. In this research, the S. sclerotiorum MADS-box gene SsMADS was cloned; it encodes a protein that is highly similar to Mcm1 orthologs from Saccharomyces cerevisiae and other fungi, and includes a highly conserved DNA-binding domain. MADS is a member of the MADS box protein SRF (serum response factor) lineage. SsMADS function was investigated using RNA interference. Silenced strains were obtained using genetic transformation of the RNA interference vectors pS1-SsMADS and pSD-SsMADS. SsMADS expression levels in silenced strains were analyzed using RT-PCR. The results showed that SsMADS mRNA expression in these silenced strains was reduced to different degrees, and growth rate in these silenced strains was significantly decreased. Infecting tomato leaflets with silenced strains indicated that SsMADS was required for leaf pathogenesis in a susceptible host. Our results suggest that the MADS-box transcription factor SsMADS is involved in S. sclerotiorum growth and virulence.
PMCID: PMC4057718  PMID: 24815067
Sclerotinia sclerotiorum; RNA interference; transcription factor; MADS-box
18.  Helicobacter pylori isolates from ethnic minority patients in Guangxi: Resistance rates, mechanisms, and genotype 
AIM: To investigate the rate of Helicobacter pylori (H. pylori) resistance to clarithromycin among ethnic minority patients in Guangxi, explore the underlying mechanisms, and analyze factors influencing genotype distribution of H. pylori isolates.
METHODS: H. pylori strains were isolated, cultured and subjected to drug sensitivity testing. The 23S rRNA gene of H. pylori isolates was amplified by PCR and analyzed by PCR-RFLP and direct sequencing to detect point mutations. REP-PCR was used for genotyping of H. pylori isolates, and NTsys_2 software was used for clustering analysis based on REP-PCR DNA fingerprints. Factors potentially influencing genotype distribution of H. pylori isolates were analyzed.
RESULTS: The rate of clarithromycin resistance was 31.3%. A2143G and A2144G mutations were detected in the 23S rRNA gene of all clarithromycin-resistant H. pylori isolates. At a genetic distance of 78%, clarithromycin-resistant H. pylori isolates could be divided into six groups. Significant clustering was noted among H. pylori isolates from patients with peptic ulcer or gastritis.
CONCLUSION: The rate of clarithromycin resistance is relatively high in ethnic minority patients in Guangxi. Main mechanisms of clarithromycin resistance are A2143G and A2144G mutations in the 23S rRNA gene. Clarithromycin-resistant H. pylori isolates can be divided into six groups based on REP-PCR DNA fingerprints. Several factors such as disease type may influence the genotype distribution of H. pylori isolates.
PMCID: PMC4000514  PMID: 24782630
Helicobacter pylori; Antibiotic resistance; Mechanism; Clarithromycin; Genotype
Phthalates and bisphenol A (BPA) are ubiquitous environmental toxicants, present in high concentrations in numerous consumer products. We hypothesized that maternal exposure to phthalates and BPA in pregnancy is associated with shortened gestation.
Urinary phthalate and BPA metabolites from 72 pregnant women were measured at the last obstetric clinic visit prior to delivery. Using linear regression models, we estimated the change in gestational age associated with each interquartile range (IQR) increase in phthalate and BPA metabolite concentration.
IQR increases in urinary mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and BPA concentrations were associated with 4.2 and 1.1 day decreases in gestation, respectively. When stratified by gender, these alterations were found only in male infants.
We conclude that MEHHP and BPA (free + glucuronide) are associated with reductions in gestation, with effects observed only in males. Our findings are consistent with the idea that these agents induce gender-specific alterations in signaling via PPAR-γ transcription factor, androgen precursors, and/or inflammatory mediators during the initiation of labor.
PMCID: PMC3996679  PMID: 23795657
phthalates; bisphenol A; labor; prematurity; genital; maternal; neonatal
20.  Frequent Mutations in EGFR, KRAS and TP53 Genes in Human Lung Cancer Tumors Detected by Ion Torrent DNA Sequencing 
PLoS ONE  2014;9(4):e95228.
Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.
PMCID: PMC3997391  PMID: 24760004
21.  MicroRNA-338 Inhibits Growth, Invasion and Metastasis of Gastric Cancer by Targeting NRP1 Expression 
PLoS ONE  2014;9(4):e94422.
NRP1 as multifunctional non-tyrosine-kinase receptors play critical roles in tumor progression. MicroRNAs (miRNAs) are an important class of pervasive genes that are involved in a variety of biological functions, particularly cancer. It remains unclear whether miRNAs can regulate the expression of NRP1. The goal of this study was to identify miRNAs that could inhibit the growth, invasion and metastasis of gastric cancer by targeting NRP1 expression. We found that miR-338 expression was reduced in gastric cancer cell lines and in gastric cancer tissues. Moreover, we found that miR-338 inhibited gastric cancer cell migration, invasion, proliferation and promoted apoptosis by targeting NRP1 expression. As an upstream regulator of NRP1, miR-338 directly targets NRP1. The forced expression of miR-338 inhibited the phosphorylation of Erk1/2, P38 MAPK and Akt; however, the expression of phosphorylated Erk1/2, P38 MAPK and Akt was restored by the overexpression of NRP1. In AGS cells infected with miR-338 or transfected with SiNRP1, the protein levels of fibronectin, vimentin, N-cadherin and SNAIL were decreased, but the expression of E-cadherin was increased. The expression of mesenchymal markers in miR-338-expressing cells was restored to normal levels by the restoration of NRP1 expression. In vivo, miR-338 also decreased tumor growth and suppressed D-MVA by targeting NRP1. Therefore, we conclude that miR-338 acts as a novel tumor suppressor gene in gastric cancer. miR-338 can decrease migratory, invasive, proliferative and apoptotic behaviors, as well as gastric cancer EMT, by attenuating the expression of NRP1.
PMCID: PMC3988047  PMID: 24736504
22.  Parent-adolescent interaction and risk of adolescent internet addiction: a population-based study in Shanghai 
BMC Psychiatry  2014;14:112.
Family-based intervention is essential for adolescents with behavioral problems. However, limited data are available on the relationship between family-based factors and adolescent internet addiction (AIA). We aimed to examine this relationship using a representative sample of Shanghai adolescents.
In October 2007, a total of 5122 adolescents were investigated from 16 high schools via stratified-random sampling in Shanghai. Self-reported and anonymous questionnaires were used to assess parent-adolescent interaction and family environments. AIA was assessed by DRM-52 Scale, developed from Young’s Internet-addiction Scale, using seven subscales to evaluate psychological symptoms of AIA.
Adjusting for adolescents’ ages, genders, socio-economic status, school performances and levels of the consumption expenditure, strong parental disapproval of internet-use was associated with AIA (vs. parental approval, OR = 2.20, 95% CI: 1.24-3.91). Worse mother-adolescent relationships were more significantly associated with AIA (OR = 3.79, 95% CI: 2.22-6.48) than worse father-adolescent relationships (OR = 1.76, 95% CI: 1.10-2.80). Marital status of “married-but-separated” and family structure of “left-behind adolescents” were associated with symptoms of some subscales. When having high monthly allowance, resident students tended to develop AIA but commuter students did not. Family social-economic status was not associated with the development of AIA.
The quality of parent-adolescent relationship/communication was closely associated with the development of AIA, and maternal factors were more significantly associated with development of AIA than paternal factors. Family social-economic status moderated adolescent internet-use levels but not the development of AIA.
PMCID: PMC3999889  PMID: 24731648
Adolescents; Internet addiction; Mother-child relations; Father-child relations; China; Marital status; Family structure
23.  Hepatoid adenocarcinoma of the lung: Presenting mediastinal metastasis without transfer to the liver 
Oncology Letters  2014;8(1):105-110.
Hepatoid adenocarcinoma of the lung (HAL) is a rare type of lung cancer. Its diagnosis and treatment may be difficult due to the varied presentation; however, immunohistochemical analysis facilitates the diagnosis. The present study presents a case of HAL. The patient was a 48-year-old male who presented with a primary complaint of back pain. A chest computed-tomography scan revealed a lobulated soft-tissue mass that extended from the left lung apex to the middle and posterior mediastinum. The area of the largest cross-section was 7.9×10.0 cm and the lymph nodes did not demonstrate metastasis. Immunohistochemical staining of a transbronchial lung biopsy revealed that the tumor cells were α-fetoprotein (AFP)(positive) and hepatocytes(positive) and a diagnosis of hepatoid carcinoma of the left lung was established. The level of serum AFP, a tumor marker, was elevated (6,283 ng/ml). The patient presented with mediastinal metastases and was classified as stage IIIA (N2); following diagnosis, the patient received concurrent chemoradiation. Subsequent to chemoradiation, the left lung lump with the largest cross-section was 3.3×4.2 cm and the serum AFP had fallen to its lowest level (23.11 ng/ml). However, when the patient relapsed, the serum AFP level elevated markedly (57,800 ng/ml). Furthermore, the nodules of metastasis increased in number and enlarged, with the largest measuring 2.1 cm. The patient succumbed as a result of a lung infection.
PMCID: PMC4063569  PMID: 24959228
hepatoid adenocarcinoma; lung; α-fetoprotein; immunohistochemistry; metastasis; chemoradiation
24.  Disaster resilience in tertiary hospitals: a cross-sectional survey in Shandong Province, China 
Hospital disaster resilience can be defined as a hospital’s ability to resist, absorb, and respond to the shock of disasters while maintaining critical functions, and then to recover to its original state or adapt to a new one. This study aims to explore the status of resilience among tertiary hospitals in Shandong Province, China.
A stratified random sample (n = 50) was derived from tertiary A, tertiary B, and tertiary C hospitals in Shandong Province, and was surveyed by questionnaire. Data on hospital characteristics and 8 key domains of hospital resilience were collected and analysed. Variables were binary, and analysed using descriptive statistics such as frequencies.
A response rate of 82% (n = 41) was attained. Factor analysis identified four key factors from eight domains which appear to reflect the overall level of disaster resilience. These were hospital safety, disaster management mechanisms, disaster resources and disaster medical care capability. The survey demonstrated that in regard to hospital safety, 93% had syndromic surveillance systems for infectious diseases and 68% had evaluated their safety standards. In regard to disaster management mechanisms, all had general plans, while only 20% had specific plans for individual hazards. 49% had a public communication protocol and 43.9% attended the local coordination meetings. In regard to disaster resources, 75.6% and 87.5% stockpiled emergency drugs and materials respectively, while less than a third (30%) had a signed Memorandum of Understanding with other hospitals to share these resources. Finally in regard to medical care, 66% could dispatch an on-site medical rescue team, but only 5% had a ‘portable hospital’ function and 36.6% and 12% of the hospitals could surge their beds and staff capacity respectively. The average beds surge capacity within 1 day was 13%.
This study validated the broad utility of a framework for understanding and measuring the level of hospital resilience. The survey demonstrated considerable variability in disaster resilience arrangements of tertiary hospitals in Shandong province, and the difference between tertiary A hospitals and tertiary B hospitals was also identified in essential areas.
PMCID: PMC3987831  PMID: 24661641
China; Current status; Disaster management; Emergency; Evaluation framework; Hospital resilience
25.  Can Tongue Acupuncture Enhance Body Acupuncture? First Results from Heart Rate Variability and Clinical Scores in Patients with Depression 
Tongue acupuncture (TA) is a method which is not used in western medicine and even in China it is applied very rarely in clinical practice. This study aimed at investigating whether additional TA can improve the efficacy of body acupuncture (BA) in patients with depression. Twenty patients with a mean age of ± SD of 42.9 ± 11.2 years were randomly divided into two groups (n = 10 patients each), one group receiving BA (Zusanli, Sanyinjiao, Neiguan, Shenting, Yintang, and Baihui) and the other receiving BA and TA (Liver, Heart, and Brain). The quantitative and qualitative outcome measures were heart rate (HR), heart rate variability (HRV), and different clinical scores. We found that in both groups all scores and HR improved significantly, whereas HRV increased partly significantly. It seems that TA can enhance acute and treatment effects of BA in patients with depression. The investigation of de qi sensation in TA needs further attention.
PMCID: PMC3980924  PMID: 24782907

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