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1.  Characterization of Constitutive Promoters for piggyBac Transposon-Mediated Stable Transgene Expression in Mesenchymal Stem Cells (MSCs) 
PLoS ONE  2014;9(4):e94397.
Multipotent mesenchymal stem cells (MSCs) can undergo self-renewal and give rise to multi-lineages under given differentiation cues. It is frequently desirable to achieve a stable and high level of transgene expression in MSCs in order to elucidate possible molecular mechanisms through which MSC self-renewal and lineage commitment are regulated. Retroviral or lentiviral vector-mediated gene expression in MSCs usually decreases over time. Here, we choose to use the piggyBac transposon system and conduct a systematic comparison of six commonly-used constitutive promoters for their abilities to drive RFP or firefly luciferase expression in somatic HEK-293 cells and MSC iMEF cells. The analyzed promoters include three viral promoters (CMV, CMV-IVS, and SV40), one housekeeping gene promoter (UbC), and two composite promoters of viral and housekeeping gene promoters (hEFH and CAG-hEFH). CMV-derived promoters are shown to drive the highest transgene expression in HEK-293 cells, which is however significantly reduced in MSCs. Conversely, the composite promoter hEFH exhibits the highest transgene expression in MSCs whereas its promoter activity is modest in HEK-293 cells. The reduced transgene expression driven by CMV promoters in MSCs may be at least in part caused by DNA methylation, or to a lesser extent histone deacetlyation. However, the hEFH promoter is not significantly affected by these epigenetic modifications. Taken together, our results demonstrate that the hEFH composite promoter may be an ideal promoter to drive long-term and high level transgene expression using the piggyBac transposon vector in progenitor cells such as MSCs.
doi:10.1371/journal.pone.0094397
PMCID: PMC3979777  PMID: 24714676
2.  Transcriptome Sequencing and Gene Expression Analysis of Trichoderma brevicompactum under Different Culture Conditions 
PLoS ONE  2014;9(4):e94203.
Background
Trichoderma brevicompactum is the Trichoderma species producing simple trichothecenes-trichodermin, a potential antifungal antibiotic and a protein synthesis inhibitor. However, the biosynthetic pathway of trichodermin in Trichoderma is not completely clarified. Therefore, transcriptome and gene expression profiling data for this species are needed as an important resource to better understand the mechanism of the trichodermin biosynthesis and provide a blueprint for further study of T. brevicompactum.
Results
In this study, de novo assembly of the T. brevicompactum transcriptome using the short-read sequencing technology (Illumina) was performed. In addition, two digital gene expression (DGE) libraries of T. brevicompactum under the trichodermin-producing and trichodermin-nonproducing culture conditions, respectively, were constructed to identify the differences in gene expression. A total of 23,351 unique transcripts with a mean length of 856 bp were obtained by a new Trinity de novo assembler. The variations of the gene expression under different culture conditions were also identified. The expression profiling data revealed that 3,282 unique transcripts had a significantly differential expression under the trichodermin-producing condition, as compared to the trichodermin-nonproducing condition. This study provides a large amount of transcript sequence data that will contribute to the study of the trichodermin biosynthesis in T. brevicompactum. Furthermore, quantitative real-time PCR (qRT-PCR) was found to be useful to confirm the differential expression of the unique transcripts.
Conclusion
Our study provides considerable gene expression information of T. brevicompactum at the transcriptional level,which will help accelerate the research on the trichodermin biosynthesis. Additionally, we have demonstrated the feasibility of using the Illumina sequencing based DGE system for gene expression profiling, and have shed new light on functional studies of the genes involved in T. brevicompactum biosynthesis.
doi:10.1371/journal.pone.0094203
PMCID: PMC3978026  PMID: 24710600
3.  Adenovirus-Mediated Efficient Gene Transfer into Cultured Three-Dimensional Organoids 
PLoS ONE  2014;9(4):e93608.
Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell–based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured “mini-gut” organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D “mini-gut” organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.
doi:10.1371/journal.pone.0093608
PMCID: PMC3973564  PMID: 24695466
4.  Unprotected sex with their clients among low-paying female sex workers in southwest China 
AIDS care  2012;25(4):503-506.
Background
China has been experiencing a rapid increase in the HIV epidemic for decades. Commercial sex plays a critical role in heterosexual transmission of HIV. Limited studies suggested that low-paying female sex workers (FSWs) faced a higher risk of HIV infection. Low-paying FSWs are women who usually encounter their clients on the street or small establishments in rural or less-developed areas, or who charge low fees for each sexual service.
Methods
A total of 720 low-paying FSWs from 130 commercial sex venues/locations in southwest China were included in the data analysis. Multivariate regression models were employed to examine the associations of unprotected sex with a number of exploratory variables among the study sample.
Results
About 33.9% and 61.5% of low-paying FSWs reported unprotected sex with clients in the last sex act and in the last month, respectively. After controlling for confounders, women’s HIV knowledge, risk perception, experience of police arrest, and venue types were significantly associated with unprotected sex among low-paying FSWs.
Discussion
Low-paying FSWs are at an alarmingly high risk of HIV infection. HIV prevention programs are urgently needed to address risk factors posit in both individual and contextual levels among this most-at-risk population in order to curb the HIV epidemic in China.
doi:10.1080/09540121.2012.726345
PMCID: PMC3594428  PMID: 23062062
Low-paying FSWs; HIV risks; Unprotected sex; China
5.  The enforced expression of c-Myc in pig fibroblasts triggers mesenchymal-epithelial transition (MET) via F-actin reorganization and RhoA/Rock pathway inactivation 
Cell Cycle  2013;12(7):1119-1127.
In previous studies from other labs it has been well demonstrated that the ectopic expression of c-Myc in mammary epithelial cells can induce epithelial-mesenchymal transition (EMT), whereas in our pilot experiment, epithelial-like morphological changes were unexpectedly observed in c-Myc-expressing pig fibroblasts [i.e., porcine embryonic fibroblasts (PEFs) and porcine dermal fibroblasts (PDFs)] and pig mesenchymal stem cells, suggesting that the same c-Myc gene is entitled to trigger EMT in epithelial cells and mesenchymal-epithelial transition (MET) in fibroblasts. This prompted us to characterize the existence of a MET in c-Myc-expressing PEFs and PDFs at the molecular level. qRT-PCR, immunofluorescence and western blot analysis illustrated that epithelial-like morphological changes were accompanied by the increased expression of epithelial markers [such as cell adhesion proteins (E-cadherin, α-catenin and Bves), tight junction protein occludin and cytokeratins (Krt8 and Krt18)], the reduced expression of mesenchymal markers [vimentin, fibronectin 1 (FN1), snail1, collagen family of proteins (COL1A1, COL5A2) and matrix metalloproteinase (MMP) family (MMP12 and MMP14)] and the decreased cell motility and increased cell adhesion in c-Myc-expressing PEFs and PDFs. Furthermore, the ectopic expression of c-Myc in pig fibroblasts disrupted the stress fiber network, suppressed the formation of filopodia and lamellipodia, and resulted in RhoA/Rock pathway inactivation, which finally participates in epithelial-like morphological conversion. Taken together, these findings demonstrate, for the first time, that the enforced expression of c-Myc in fibroblasts can trigger MET, to which cytoskeleton depolymerization and RhoA/Rock pathway inactivation contribute.
doi:10.4161/cc.24164
PMCID: PMC3646867  PMID: 23466707
RhoA/Rock pathway; Tibetan miniature pigs; c-Myc; cytoskeleton reorganization; dermal fibroblasts; embryonic fibroblasts; mesenchymal stem cells; mesenchymal-epithelial transition (MET)
6.  Broad Distribution of Diverse Anaerobic Ammonium-Oxidizing Bacteria in Chinese Agricultural Soils 
Applied and Environmental Microbiology  2013;79(19):6167-6172.
Anaerobic ammonium-oxidizing (anammox) bacteria have been detected in many marine and freshwater ecosystems. However, little is known about the distribution, diversity, and abundance of anammox bacteria in terrestrial ecosystems. In this study, anammox bacteria were found to be present in various agricultural soils collected from 32 different locations in China. Phylogenetic analysis of the 16S rRNA genes showed “Candidatus Brocadia,” “Candidatus Kuenenia,” “Candidatus Anammoxoglobus,” and “Candidatus Jettenia” in the collected soils, with “Candidatus Brocadia” being the dominant genus. Quantitative PCR showed that the abundance of anammox bacteria ranged from 6.38 × 104 ± 0.42 × 104 to 3.69 × 106 ± 0.25 × 106 copies per gram of dry weight. Different levels of diversity, composition, and abundance of the anammox bacterial communities were observed, and redundancy analysis indicated that the soil organic content and the distribution of anammox communities were correlated in the soils examined. Furthermore, Pearson correlation analysis showed that the diversity of the anammox bacteria was positively correlated with the soil ammonium content and the organic content, while the anammox bacterial abundance was positively correlated with the soil ammonium content. These results demonstrate the broad distribution of diverse anammox bacteria and its correlation with the soil environmental conditions within an extensive range of Chinese agricultural soils.
doi:10.1128/AEM.00884-13
PMCID: PMC3811364  PMID: 23747706
7.  Effect of Intracellular Expression of Antimicrobial Peptide LL-37 on Growth of Escherichia coli Strain TOP10 under Aerobic and Anaerobic Conditions 
Antimicrobial Agents and Chemotherapy  2013;57(10):4707-4716.
Antimicrobial peptides (AMPs) can cause lysis of target bacteria by directly inserting themselves into the lipid bilayer. This killing mechanism confounds the identification of the intracellular targets of AMPs. To circumvent this, we used a shuttle vector containing the inducible expression of a human cathelicidin-related AMP, LL-37, to examine its effect on Escherichia coli TOP10 under aerobic and anaerobic growth conditions. Induction of LL-37 caused growth inhibition and alteration in cell morphology to a filamentous phenotype. Further examination of the E. coli cell division protein FtsZ revealed that LL-37 did not interact with FtsZ. Moreover, intracellular expression of LL-37 results in the enhanced production of reactive oxygen species (ROS), causing lethal membrane depolarization under aerobic conditions. Additionally, the membrane permeability was increased after intracellular expression of LL37 under both aerobic and anaerobic conditions. Transcriptomic analysis revealed that intracellular LL-37 mainly affected the expression of genes related to energy production and carbohydrate metabolism. More specifically, genes related to oxidative phosphorylation under both aerobic and anaerobic growth conditions were affected. Collectively, our current study demonstrates that intracellular expression of LL-37 in E. coli can inhibit growth under aerobic and anaerobic conditions. While we confirmed that the generation of ROS is a bactericidal mechanism for LL-37 under aerobic growth conditions, we also found that the intracellular accumulation of cationic LL-37 influences the redox and ion status of the cells under both growth conditions. These data suggest that there is a new AMP-mediated bacterial killing mechanism that targets energy metabolism.
doi:10.1128/AAC.00825-13
PMCID: PMC3811468  PMID: 23856776
8.  Oxidative aliphatic C-H fluorination with manganese catalysts and fluoride ion 
Nature protocols  2013;8(12):2348-2354.
Fluorination is a reaction that is useful in improving the chemical stability and changing the binding affinity of biologically active compounds. The protocol described here can be used to replace aliphatic, C(sp3)-H hydrogen in small molecules with fluorine. Notably, isolated methylene groups and unactivated benzylic sites are accessible. The method uses readily available manganese porphyrin and manganese salen catalysts and various fluoride ion reagents, including silver fluoride (AgF), tetrabutylammonium fluoride and triethylamine trihydrofluoride (TREAT·HF), as the source of fluorine. Typically, the reactions afford 50–70% yield of mono-fluorinated products in one step. Two representative examples, the fragrance component celestolide and the nonsteroidal anti-inflammatory drug ibuprofen, are described; they produced useful isolated quantities (250–300 mg, ~50% yield) of fluorinated material over periods of 1–8 h. The procedures are performed in a typical fume hood using ordinary laboratory glassware. No special precautions to rigorously exclude water are required.
doi:10.1038/nprot.2013.144
PMCID: PMC3972251  PMID: 24177292
9.  Adenovirus-Mediated Gene Transfer in Mesenchymal Stem Cells Can Be Significantly Enhanced by the Cationic Polymer Polybrene 
PLoS ONE  2014;9(3):e92908.
Mesenchymal stem cells (MSCs) are multipotent progenitors, which can undergo self-renewal and give rise to multi-lineages. A great deal of attentions have been paid to their potential use in regenerative medicine as potential therapeutic genes can be introduced into MSCs. Genetic manipulations in MSCs requires effective gene deliveries. Recombinant adenoviruses are widely used gene transfer vectors. We have found that although MSCs can be infected in vitro by adenoviruses, high virus titers are needed to achieve high efficiency. Here, we investigate if the commonly-used cationic polymer Polybrene can potentiate adenovirus-mediated transgene delivery into MSCs, such as C2C12 cells and iMEFs. Using the AdRFP adenovirus, we find that AdRFP transduction efficiency is significantly increased by Polybrene in a dose-dependent fashion peaking at 8 μg/ml in C2C12 and iMEFs cells. Quantitative luciferase assay reveals that Polybrene significantly enhances AdFLuc-mediated luciferase activity in C2C12 and iMEFs at as low as 4 μg/ml and 2 μg/ml, respectively. FACS analysis indicates that Polybrene (at 4 μg/ml) increases the percentage of RFP-positive cells by approximately 430 folds in AdRFP-transduced iMEFs, suggesting Polybrene may increase adenovirus infection efficiency. Furthermore, Polybrene can enhance AdBMP9-induced osteogenic differentiation of MSCs as early osteogenic marker alkaline phosphatase activity can be increased more than 73 folds by Polybrene (4 μg/ml) in AdBMP9-transduced iMEFs. No cytotoxicity was observed in C2C12 and iMEFs at Polybrene up to 40 μg/ml, which is about 10-fold higher than the effective concentration required to enhance adenovirus transduction in MSCs. Taken together, our results demonstrate that Polybrene should be routinely used as a safe, effective and inexpensive augmenting agent for adenovirus-mediated gene transfer in MSCs, as well as other types of mammalian cells.
doi:10.1371/journal.pone.0092908
PMCID: PMC3962475  PMID: 24658746
10.  Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors 
Bioscience Reports  2014;34(2):e00097.
The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors.
We conducted kinetic analysis of the relatively unexplored PKN family and effects of lipids, and identified potent inhibitors with various isoform selectivity. The kinetic mechanism, lipid activators and inhibitors could be useful for understanding PKN biology and developing PKN-targeted therapies.
doi:10.1042/BSR20140010
PMCID: PMC3958129  PMID: 24476049
AGC kinase; cancer; kinase inhibitor; kinetic mechanism; lipid; protein kinase N (PKN); CaM, calmodulin; Cdk1/2, cyclin-dependent kinase 1/2; DAG, 1,2-dioctanoyl-sn-glycerol; DTT, dithiothreitol; HEK-293 cells, human embryonic kidney 293 cells; IP3, D-myo-inositol-1,3,5-triphosphate; MAPK, mitogen-activated protein kinase; MS/MS, tandem MS; PDK1, phosphoinositide-dependent kinase 1; PIF, PDK1-interacting fragment; PIP2, phosphatidylinositol-4,5-bisphosphate; PIP3, phosphatidylinositol-3,4,5-triphosphate; PKA, protein kinase A; PKC, protein kinase C; PKG, protein kinase G; PKN, protein kinase N; ROCK, Rho-associated kinase; S6K, S6 kinase
11.  An Efficient Strategy of Screening for Pathogens in Wild-Caught Ticks and Mosquitoes by Reusing Small RNA Deep Sequencing Data 
PLoS ONE  2014;9(3):e90831.
This paper explored our hypothesis that sRNA (18∼30 bp) deep sequencing technique can be used as an efficient strategy to identify microorganisms other than viruses, such as prokaryotic and eukaryotic pathogens. In the study, the clean reads derived from the sRNA deep sequencing data of wild-caught ticks and mosquitoes were compared against the NCBI nucleotide collection (non-redundant nt database) using Blastn. The blast results were then analyzed with in-house Python scripts. An empirical formula was proposed to identify the putative pathogens. Results showed that not only viruses but also prokaryotic and eukaryotic species of interest can be screened out and were subsequently confirmed with experiments. Specially, a novel Rickettsia spp. was indicated to exist in Haemaphysalis longicornis ticks collected in Beijing. Our study demonstrated the reuse of sRNA deep sequencing data would have the potential to trace the origin of pathogens or discover novel agents of emerging/re-emerging infectious diseases.
doi:10.1371/journal.pone.0090831
PMCID: PMC3949703  PMID: 24618575
12.  Carbon nanotubes as VEGF carriers to improve the early vascularization of porcine small intestinal submucosa in abdominal wall defect repair 
Insufficient early vascularization in biological meshes, resulting in limited host tissue incorporation, is thought to be the primary cause for the failure of abdominal wall defect repair after implantation. The sustained release of exogenous angiogenic factors from a biocompatible nanomaterial might be a way to overcome this limitation. In the study reported here, multiwalled carbon nanotubes (MWNT) were functionalized by plasma polymerization to deliver vascular endothelial growth factor165 (VEGF165). The novel VEGF165-controlled released system was incorporated into porcine small intestinal submucosa (PSIS) to construct a composite scaffold. Scaffolds incorporating varying amounts of VEGF165-loaded functionalized MWNT were characterized in vitro. At 5 weight percent MWNT, the scaffolds exhibited optimal properties and were implanted in rats to repair abdominal wall defects. PSIS scaffolds incorporating VEGF165-loaded MWNT (VEGF–MWNT–PSIS) contributed to early vascularization from 2–12 weeks postimplantation and obtained more effective collagen deposition and exhibited improved tensile strength at 24 weeks postimplantation compared to PSIS or PSIS scaffolds, incorporating MWNT without VEGF165 loading (MWNT–PSIS).
doi:10.2147/IJN.S58626
PMCID: PMC3956480  PMID: 24648727
vascular endothelial growth factor165; controlled release; multi-walled carbon nanotube; early vascularization
13.  Diode laser transscleral cyclophotocoagulation followed by phacotrabeculectomy on medically unresponsive acute primary angle closure eyes: the long-term result 
BMC Ophthalmology  2014;14:26.
Background
To explore the intraocular pressure-lowering effect and complications of diode laser transscleral cyclophotocoagulation (DLTSC) followed by phacotrabeculectomy on medically unresponsive acute primary angle closure eyes.
Methods
Nine eyes of nine medically unresponsive acute primary angle closure patients were enrolled. All the patients underwent cyclophotocoagulation followed by phacotrabeculectomy to control the prolonged acute attack. Data were recorded prospectively and then analyzed retrospectively. The reduction in intraocular pressure, improvement of vision and the complications were evaluated.
Results
After DLTSC, the IOP of all the patients were reduced, but all were above 21 mmHg under topical anti-glaucoma medications. After phacotrabeculectomy, the IOP of all the patients was decreased. At the final visit, the vision of all the patients was improved and the IOP of all the patients was below 21 mmHg without anti-glaucoma medications. There were no complications during the DLTSC and phacotrabeculectomy. Uveitis was the common complications after the both procedures, which were resolved by medication treatment.
Conclusion
Diode laser transscleral cyclophotocoagulation followed by phacotrabeculectomy is an alternative procedure to control the intraocular pressure of medically unresponsive acute primary angle closure eyes with few complications.
doi:10.1186/1471-2415-14-26
PMCID: PMC3975279  PMID: 24606842
Diode laser transscleral cyclophotocoagulation; Acute primary angle closure; Phacotrabeculectomy
14.  Comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens 
The ISME Journal  2012;7(3):615-621.
Segmented filamentous bacteria (SFB) are indigenous gut commensal bacteria. They are commonly detected in the gastrointestinal tracts of both vertebrates and invertebrates. Despite the significant role they have in the modulation of the development of host immune systems, little information exists regarding the presence of SFB in humans. The aim of this study was to investigate the distribution and diversity of SFB in humans and to determine their phylogenetic relationships with their hosts. Gut contents from 251 humans, 92 mice and 72 chickens were collected for bacterial genomic DNA extraction and subjected to SFB 16S rRNA-specific PCR detection. The results showed SFB colonization to be age-dependent in humans, with the majority of individuals colonized within the first 2 years of life, but this colonization disappeared by the age of 3 years. Results of 16S rRNA sequencing showed that multiple operational taxonomic units of SFB could exist in the same individuals. Cross-species comparison among human, mouse and chicken samples demonstrated that each host possessed an exclusive predominant SFB sequence. In summary, our results showed that SFB display host specificity, and SFB colonization, which occurs early in human life, declines in an age-dependent manner.
doi:10.1038/ismej.2012.128
PMCID: PMC3578561  PMID: 23151642
distribution; diversity; phylogenetic relationships; segmented filamentous bacteria
15.  Silencing of CXCR4 Inhibits Tumor Cell Proliferation and Neural Invasion in Human Hilar Cholangiocarcinoma 
Gut and Liver  2013;8(2):196-204.
Background/Aims
To evaluate the expression of CXC motif chemokine receptor 4 (CXCR4) in the tissues of patients with hilar cholangiocarcinoma (hilar-CCA) and to investigate the cell proliferation and frequency of neural invasion (NI) influenced by RNAi-mediated CXCR4 silencing.
Methods
An immunohistochemical technique was used to detect the expression of CXCR4 in 41 clinical tissues, including hilar-CCA, cholangitis, and normal bile duct tissues. The effects of small interference RNA (siRNA)-mediated CXCR4 silencing were detected in the hilar-CCA cell line QBC939. Cell proliferation was determined by MTT. Expression of CXCR4 was monitored by quantitative real time polymerase chain reaction and Western blot analysis. The NI ability of hilar-CCA cells was evaluated using a perineural cell and hilar-CCA cell coculture migration assay.
Results
The expression of CXCR4 was significantly induced in clinical hilar-CCA tissue. There was a positive correlation between the expression of CXCR4 and lymph node metastasis/NI in hilar-CCA patients (p<0.05). Silencing of CXCR4 in tumor cell lines by siRNA led to significantly decreased NI (p<0.05) and slightly decreased cell proliferation.
Conclusions
CXCR4 is likely correlated with clinical recurrence of hilar-CCA. CXCR4 is involved in the invasion and proliferation of human hilar-CCA cell line QBC939, indicating that CXCR4 could be a promising therapeutic target for hilar-CCA.
doi:10.5009/gnl.2014.8.2.196
PMCID: PMC3964271  PMID: 24672662
Hilar cholangiocarcinoma; Neural invasion; CXCR4; RNA interference
16.  Nanomedicine-Based Neuroprotective Strategies in Patient Specific-iPSC and Personalized Medicine 
In recent decades, nanotechnology has attracted major interests in view of drug delivery systems and therapies against diseases, such as cancer, neurodegenerative diseases, and many others. Nanotechnology provides the opportunity for nanoscale particles or molecules (so called “Nanomedicine”) to be delivered to the targeted sites, thereby, reducing toxicity (or side effects) and improving drug bioavailability. Nowadays, a great deal of nano-structured particles/vehicles has been discovered, including polymeric nanoparticles, lipid-based nanoparticles, and mesoporous silica nanoparticles. Nanomedical utilizations have already been well developed in many different aspects, including disease treatment, diagnostic, medical devices designing, and visualization (i.e., cell trafficking). However, while quite a few successful progressions on chemotherapy using nanotechnology have been developed, the implementations of nanoparticles on stem cell research are still sparsely populated. Stem cell applications and therapies are being considered to offer an outstanding potential in the treatment for numbers of maladies. Human induced pluripotent stem cells (iPSCs) are adult cells that have been genetically reprogrammed to an embryonic stem cell-like state. Although the exact mechanisms underlying are still unclear, iPSCs are already being considered as useful tools for drug development/screening and modeling of diseases. Recently, personalized medicines have drawn great attentions in biological and pharmaceutical studies. Generally speaking, personalized medicine is a therapeutic model that offers a customized healthcare/cure being tailored to a specific patient based on his own genetic information. Consequently, the combination of nanomedicine and iPSCs could actually be the potent arms for remedies in transplantation medicine and personalized medicine. This review will focus on current use of nanoparticles on therapeutical applications, nanomedicine-based neuroprotective manipulations in patient specific-iPSCs and personalized medicine.
doi:10.3390/ijms15033904
PMCID: PMC3975375  PMID: 24599081
induced pluripotent stem cells; personalized medicine; neurodegenerative; nanoparticles
17.  Nanostructured lipid carriers as a novel oral delivery system for triptolide: induced changes in pharmacokinetics profile associated with reduced toxicity in male rats 
After oral administration in rodents, triptolide (TP), a diterpenoid triepoxide compound, active as anti-inflammatory, immunosuppressive, anti-fertility, anti-cystogenesis, and anticancer agent, is rapidly absorbed into the blood circulation (from 5.0 to 19.5 minutes after dosing, depending on the rodent species) followed by a short elimination half-life (from about 20 minutes to less than 1 hour). Such significant and rapid fluctuations of TP in plasma likely contribute to its toxicity, which is characterized by injury to hepatic, renal, digestive, reproductive, and hematological systems. With the aim of prolonging drug release and improving its safety, TP-loaded nanostructured lipid carriers (TP-NLCs), composed of Compritol® 888 ATO (solid lipid) and Capryol™ 90 (liquid lipid), were developed using a microemulsion technique. The formulated TP-NLCs were also characterized and in vitro release was evaluated using the dialysis bag diffusion technique. In addition, the pharmacokinetics and toxicology profiles of TP-NLCs were compared to free TP and TP-loaded solid lipid nanoparticles (TP-SLNs; containing Compritol 888 ATO only). Results demonstrate that TP-NLCs had mean particle size of 231.8 nm, increased drug encapsulation with a 71.6% efficiency, and stable drug incorporation for over 1-month. TP-NLCs manifested a better in vitro sustained-release pattern compared to TP-SLNs. Furthermore, TP-NLCs prolonged mean residence time (MRT)0–t (P<0.001, P<0.001), delayed Tmax (P<0.01, P<0.05) and decreased Cmax (P<0.01, P<0.05) compared to free TP and TP-SLNs, respectively, which was associated with reduced subacute toxicity in male rats. In conclusion, our data suggest that TP-NLCs are superior to TP-SLNs and could be a promising oral delivery system for a safer use of TP.
doi:10.2147/IJN.S55144
PMCID: PMC3934590  PMID: 24591827
triptolide; microemulsion technique; in vivo pharmacokinetics; sustained-release; rat subacute toxicity
18.  Epigallocatechin-3-gallate Ameliorates Seawater Aspiration-Induced Acute Lung Injury via Regulating Inflammatory Cytokines and Inhibiting JAK/STAT1 Pathway in Rats 
Mediators of Inflammation  2014;2014:612593.
Signal transducers and activators of transcriptions 1 (STAT1) play an important role in the inflammation process of acute lung injury (ALI). Epigallocatechin-3-gallate (EGCG) exhibits a specific and strong anti-STAT1 activity. Therefore, our study is to explore whether EGCG pretreatment can ameliorate seawater aspiration-induced ALI and its possible mechanisms. We detected the arterial partial pressure of oxygen, lung wet/dry weight ratios, protein content in bronchoalveolar lavage fluid, and the histopathologic and ultrastructure staining of the lung. The levels of IL-1, TNF-α, and IL-10 and the total and the phosphorylated protein level of STAT1, JAK1, and JAK2 were assessed in vitro and in vivo. The results showed that EGCG pretreatment significantly improved hypoxemia and histopathologic changes, alleviated pulmonary edema and lung vascular leak, reduced the production of TNF-α and IL-1, and increased the production of IL-10 in seawater aspiration-induced ALI rats. EGCG also prevented the seawater aspiration-induced increase of TNF-α and IL-1 and decrease of IL-10 in NR8383 cell line. Moreover, EGCG pretreatment reduced the total and the phosphorylated protein level of STAT1 in vivo and in vitro and reduced the phosphorylated protein level of JAK1 and JAK2. The present study demonstrates that EGCG ameliorates seawater aspiration-induced ALI via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats.
doi:10.1155/2014/612593
PMCID: PMC3945896
19.  Prospective Screening of Family Members with Moyamoya Disease Patients 
PLoS ONE  2014;9(2):e88765.
Background
Genetic factors play an important role in the pathogenesis of moyamoya disease (MMD). Previous studies concentrated on familial MMD patients. In this study, we focused on family members of sporadic MMD patients, and aimed to gain a clearer understanding of the role that genetic factors play in MMD.
Methods
The immediate family members of MMD patients were initially screened by transcranial Doppler sonography (TCD) and positive cases were verified by magnetic resonance angiography (MRA).
Results
From July 2011 to March 2013, there were 527 MMD patients managed in our hospital, including 38 familial MMD cases. In this study, 285 immediate family members of 245 sporadic MMD patients were screened. Another 41 cases of familial MMD cases were identified, which included 21 family members and 20 corresponding sporadic MMD patients who had family members confirmed positive with MMD. As a result, the proportion of familial MMD patients increased from 7% (38/527) to 15% (79/527) in this period. For the main segments of the circle of Willis, Kappa values between TCD and MRA for the anterior cerebral arteries, middle cerebral arteries and posterior cerebral arteries were 0.91, 0.72, and 0.47, respectively. Familial cases confirmed by our screening showed a significantly higher percentage of asymptomatic patients (57%) compared with 9% from the control group who had a clear family history before.
Conclusions
Familial MMD patients may account for a higher percentage among all cases than previously thought. Some family members of MMD patients may also have MMD, but not have any obvious symptoms. Routine screening should be implemented for all family members of MMD patients to improve the detection rate for this part of the patient base. TCD has a high diagnostic agreement with MRA for MMD. TCD may be the preferred choice for screening because it is inexpensive and safe.
doi:10.1371/journal.pone.0088765
PMCID: PMC3929504  PMID: 24586386
20.  Measurement of retinal thickness in macular region of high myopic eyes using spectral domain OCT 
AIM
To investigate the changes of retinal thickness in macula of high myopic eyes using spectral domain optical coherence tomography (OCT).
METHODS
Middle-aged and young myopic patients were divided into three groups according to their refractive error/axial length: low and medium myopia group (LMMG), high myopia group (HMG) and super high myopia group (SHMG). Cirrus HD-OCT was used to evaluate total average macular thickness, central subfield thickness, inner/outer macular thickness and macular volume. The differences among experimental groups were analyzed by one-factor analysis of variance. Associations between macular thickness and refractive error/axial length were analyzed by Pearson correlation analysis.
RESULTS
There was no significant difference in age among the three groups (P=0.2789). The mean refraction error in the LMMG, HMG, and SHMG groups was -2.49±1.38D, -8.53±1.95D and -13.88±1.76D, respectively (P<0.001). The central subfield thickness of three groups was 244.56±12.19µm, 254.33±11.61µm and 261.75±11.83µm, respectively, and there were statistically significance between random two groups. The total average macular thickness, inner/outer macular thickness, and macular volume decreased with increased myopia/axial length. Average foveal thickness had negative correlations with refractive error (P<0.001), and positive correlations with axial length. The inferior and temporal inner macular thickness, all the quadrants of outer ring, total average macular thickness and macular volume featured positive correlations with refractive error, and negative correlations with axial length. Average foveal thickness, superior and temporal inner macular thicknesses, and temporal outer macular thickness was lower in females compared to males.
CONCLUSION
With an increase in myopia degree/axial length, the average foveal thickness increased and the inner/outer macular thickness decreased. Females featured thicker average foveal thickness, and thinner macular thickness compared to males.
doi:10.3980/j.issn.2222-3959.2014.01.23
PMCID: PMC3949472  PMID: 24634877
optical coherence tomography; retinal thickness; high myopic eyes
21.  Inactivation of miR-34a by aberrant CpG methylation in Kazakh patients with esophageal carcinoma 
Background
Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with dismal prognosis and high incidence and mortality in Kazakh population. MiR-34a, a direct p53 target gene, possesses tumor-suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. The reduced expression of miR-34a by methylation in various cancers has been reported.
Methods
To determine whether aberrant miR-34a methylation occurs in esophageal cancer, the DNA methylation of 23 CpGs sites in the miR-34a promoter was quantitatively analyzed in relation to the translation initiation site by MALDI -TOF mass spectrometry in 59 ESCC tissues and 34 normal tissues from the Kazakh population. Real-time PCR was used to detect the inhibition of miR-34a expression levels and to evaluate their association with methylation.
Results
We found that miR-34a is more frequently methylated in ESCC (0.133 ± 0.040) than in controls (0.066 ± 0.045, P < 0.01). A nearly two-fold increase in miR-34a expression for the hypomethylated promoter was found in normal esophageal tissues than ESCC with hypermethylation (P <0.0001), pointing to a negative relationship between miR-34a CpG sites methylation and expression(r = −0.594, P = 0.042). The hypermethylation of miR-34a CpG_8.9 was associated with the advanced UICC stage III/IV of the esophageal cancers, and the hypermethylation of CpG_8.9 and CpG_5 of miR-34a was significantly correlated with lymph node metastasis.
Conclusions
Our findings suggest that miR-34a is involved in the etiology of ESCC and that hypermethylated miR-34a is a potential biomarker for ESCC diagnosis and prognosis. Moreover, targeting miR-34a methylation by demethylating agents may offer a novel strategy for anticancer therapy of ESCC.
doi:10.1186/1756-9966-33-20
PMCID: PMC3931274  PMID: 24528540
MiR-34a; Esophageal squamous cell carcinoma; Kazakh; Methylation
22.  Chinese Patent Medicine Tongxinluo Capsule for Hypertension: A Systematic Review of Randomised Controlled Trials 
Background. This study was intended to evaluate the efficacy and safety of Tongxinluo capsule for hypertension. Search Strategy. We searched the Cochrane Central Register of Controlled Trials (CENTRAL) in the Cochrane Library, The PubMed, EMBASE, Chinese Bio-Medical Literature Database (CBM), Chinese National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database, and Wan-fang Data started from the first of database to October 28, 2013. No language restriction was applied. We included randomized clinical trials testing Tongxinluo capsule against western medicine, Tongxinluo capsule versus placebo, and Tongxinluo capsule combined with western medicine versus western medicine. Study selection, data extraction, quality assessment, and data analyses were conducted according to the Cochrane standards. Results. 25 trials with 1958 participants were included. The methodological quality of the included trials was evaluated as generally low. The blood pressure (BP) lowering effect of Tongxinluo capsule plus western medicine was significantly higher than that of western medicine (systolic blood pressure (SBP): −3.87, −5.32 to −2.41, P < 0.00001; and diastolic blood pressure (DBP): −2.72, −4.19 to −1.24, P = 0.0003). The BP also decreased significantly from baseline with Tongxinluo capsule than placebo (SBP: −9.40, −10.90 to −7.90, P < 0.00001; and DBP: −11.80, −12.40 to −11.20, P < 0.00001) or western medicine (SBP: −3.90, −4.93 to −2.87, P < 0.00001; and DBP: −3.70, −3.83 to −3.57, P < 0.00001). 12 trials reported adverse events without details. Conclusions. There is some but weak evidence about the effectiveness of TXL in treating patients with hypertension.
doi:10.1155/2014/187979
PMCID: PMC3947843
23.  Prenatal Exposure to Organophosphate Pesticides and Neurobehavioral Development of Neonates: A Birth Cohort Study in Shenyang, China 
PLoS ONE  2014;9(2):e88491.
Background
A large amount of organophosphate pesticides (OPs) are used in agriculture in China every year, contributing to exposure of OPs through dietary consumption among the general population. However, the level of exposure to OPs in China is still uncertain.
Objective
To investigate the effect of the exposure to OPs on the neonatal neurodevelopment during pregnancy in Shenyang, China.
Methods
249 pregnant women enrolled in the Central Hospital Affiliated to Shenyang Medical College from February 2011 to August 2012. A cohort of the mothers and their neonates participated in the study and information on each subject was obtained by questionnaire. Dialkyl phosphate (DAP) metabolites were detected in the urine of mothers during pregnancy to evaluate the exposure level to OPs. Neonate neurobehavioral developmental levels were assessed according to the standards of the Neonatal Behavioral Neurological Assessment (NBNA). Multiple linear regressions were utilized to analyze the association between pregnancy exposure to OPs and neonatal neurobehavioral development.
Results
The geometric means (GM) of urinary metabolites for dimethyl phosphate (DMP), dimethyl thiophosphate (DMTP), diethyl phosphate (DEP), and diethyl thiophosphate (DETP) in pregnant women were 18.03, 8.53, 7.14, and 5.64 µg/L, respectively. Results from multiple linear regressions showed that prenatal OP exposure was one of the most important factors affecting NBNA scores. Prenatal total DAP concentrations were inversely associated with scores on the NBNA scales.?Additionally, a 10-fold increase in DAP concentrations was associated with a decrease of 1.78 regarding the Summary NBNA (95% CI, −2.12 to −1.45). And there was an estimated 2.11-point difference in summary NBNA scores between neonates in the highest quintile of prenatal OP exposure and the lowest quintile group.
Conclusion
The high exposure of pregnant women to OPs in Shenyang, China was the predominant risk factor for neonatal neurobehavioral development.
doi:10.1371/journal.pone.0088491
PMCID: PMC3923780  PMID: 24551109
24.  Traditional Chinese Medicine Syndromes for Essential Hypertension: A Literature Analysis of 13,272 Patients 
Background. To simplify traditional Chinese medicine syndrome differentiation and allow researchers to master syndrome differentiation for hypertension, this paper retrospectively studied the literature and analyzed syndrome elements corresponding to hypertension syndromes. Methods. Six databases including PubMed, EMBASE, Chinese Bio-Medical Literature Database, Chinese National Knowledge Infrastructure, Chinese Scientific Journal Database, and Wan-fang Data were searched from 1/January/2003 to 30/October/2013. We included all clinical literature testing hypertension syndromes and retrospectively studied the hypertension literature published from 2003 to 2013. Descriptive statistics calculated frequencies and percentages. Results. 13,272 patients with essential hypertension were included. Clinical features of hypertension could be attributed to 11 kinds of syndrome factors. Among them, seven syndrome factors were excess, while four syndrome factors were deficient. Syndrome targets were mainly in the liver and related to the kidney and spleen. There were 33 combination syndromes. Frequency of single-factor syndromes was 31.77% and frequency of two-factor syndromes was 62.26%. Conclusions. Excess syndrome factors of hypertension patients include yang hyperactivity, blood stasis, phlegm turbidity, internal dampness, and internal fire. Deficient syndrome factors of hypertension patients are yin deficiency and yang deficiency. Yin deficiency with yang hyperactivity, phlegm-dampness retention, and deficiency of both yin and yang were the three most common syndromes in clinical combination.
doi:10.1155/2014/418206
PMCID: PMC3934631  PMID: 24660016
25.  Pseudomonas pyocyanin stimulates IL-8 expression through MAPK and NF-κB pathways in differentiated U937 cells 
BMC Microbiology  2014;14:26.
Background
Pyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined.
Results
It was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-κB inhibitors inhibited PCN-induced NF-κB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression.
Conclusions
It is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-κΒ signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.
doi:10.1186/1471-2180-14-26
PMCID: PMC3925954  PMID: 24499192
Pyocyanin; IL-8; U937 cell; p38; ERK; NF-κB

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