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1.  Over-expression of BCAT1, a c-Myc target gene, induces cell proliferation, migration and invasion in nasopharyngeal carcinoma 
Molecular Cancer  2013;12:53.
Background
Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but its molecular mechanisms of pathogenesis are poorly understood. Our previous work has demonstrated that BCAT1 mRNA is over expressed in NPC and knocking down its expression in 5-8F NPC cell line can potently inhibit cell cycle progression and cell proliferation. However, the mechanism of BCAT1 up-regulation and its functional role in NPC development remain to be elucidated yet.
Methods
Immunohistochemistry (IHC) method was utilized to detect the expression of BCAT1 protein in NPC at different pathological stages. The roles of gene mutation, DNA amplification and transcription factor c-Myc in regulating BCAT1 expression were analyzed using PCR-sequencing, quantitative polymerase chain reaction (qPCR), IHC, ChIP and luciferase reporter system, respectively. The functions of BCAT1 in colony formation, cell migration and invasion properties were evaluated by RNA interference (RNAi).
Results
The positive rates of BCAT1 protein expression in normal epithelia, low-to-moderate grade atypical hyperplasia tissues, high-grade atypical hyperplasia tissues and NPC tissues were 23.6% (17/72), 75% (18/24 ), 88.9% (8/9) and 88.8% (71/80), respectively. Only one SNP site in exon1 was detected, and 42.4% (12/28) of the NPC tissues displayed the amplification of microsatellite loci in BCAT1. C-Myc could directly bind to the c-Myc binding site in promoter region of BCAT1 and up-regulate its expression. The mRNA and protein of c-Myc and BCAT1 were co-expressed in 53.6% (15/28) and 59.1% (13/22) of NPC tissues, respectively, and BCAT1 mRNA expression was also down-regulated in c-Myc knockdown cell lines. In addition, BCAT1 knockdown cells demonstrated reduced proliferation and decreased cell migration and invasion abilities.
Conclusions
Our study indicates that gene amplification and c-Myc up-regulation are responsible for BCAT1 overexpression in primary NPC, and overexpression of BCAT1 induces cell proliferation, migration and invasion. The results suggest that BCAT1 may be a novel molecular target for the diagnosis and treatment of NPC.
doi:10.1186/1476-4598-12-53
PMCID: PMC3698204  PMID: 23758864
Nasopharyngeal carcinoma; BCAT1; c-Myc; Proliferation; Migration; Invasion; Gene amplification; Gene regulation
2.  Genome sequence of the date palm Phoenix dactylifera L 
Nature Communications  2013;4:2274.
Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4 Mb in size and covers >90% of the genome (~671 Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palm’s unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.
The date palm is one of the most economically important plants of the palm family. Here, the authors present a high-quality genome assembly of the date palm Phoenix dactylifera, and reveal insights into the unique sugar metabolism underlying fruit ripening.
doi:10.1038/ncomms3274
PMCID: PMC3741641  PMID: 23917264
3.  The Limbic-Prefrontal Network Modulated by Electroacupuncture at CV4 and CV12 
fMRI studies showed that acupuncture could induce hemodynamic changes in brain networks. Many of these studies focused on whether specific acupoints could activate specific brain regions and were often limited to manual acupuncture at acupoints on the limbs. In this fMRI study, we investigated acupuncture's modulation effects on brain functional networks by electroacupuncture (EA) at acupoints on the midline of abdomen. Acupoints Guanyuan (CV4) and Zhongwan (CV12) were stimulated in 21 healthy volunteers. The needling sensations, brain activation, and functional connectivity were studied. We found that the limbic-prefrontal functional network was deactivated by EA at CV4 and CV12. More importantly, the local functional connectivity was significantly changed during EA stimulation, and the change persisted during the period after the stimulation. Although minor differences existed, both acupoints similarly modulated the limbic-prefrontal functional network, which is overlapped with the functional circuits associated with emotional and cognitive regulation.
doi:10.1155/2012/515893
PMCID: PMC3265182  PMID: 22291848
4.  Genomic and Proteomic Analyses of the Fungus Arthrobotrys oligospora Provide Insights into Nematode-Trap Formation 
PLoS Pathogens  2011;7(9):e1002179.
Nematode-trapping fungi are “carnivorous” and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.
Author Summary
The fungus Arthrobotrys oligospora has multiple lifestyles. It's not only a nematode pathogen, but also a saprophyte, a pathogen of other fungi, and a colonizer of plant roots. As a nematode pathogen, A. oligospora forms adhesive networks to capture nematodes and is a model organism for understanding the interaction between these fungi and their host nematodes. In this study, the whole genome sequence of A. oligospora was reported. Our analyses of the proteome profiles of intracellular proteins from cells treated with nematode extracts for 10 h and 48 h revealed a key set of genes involved in trap formation. The changes in protein levels for some trap formation related genes were further confirmed by qPCR. The combined genome and proteome analysis identified the major genetic and metabolic pathways involved in trap formation in A. oligospora. Our results provide the first glimpse into the genome and proteome of this fascinating group of carnivorous fungi. The data should serve as a roadmap for further investigations into the interaction between nematode-trapping fungi and their host nematodes, providing broad foundations for research on the biocontrol of pathogenic nematodes.
doi:10.1371/journal.ppat.1002179
PMCID: PMC3164635  PMID: 21909256
5.  Fresh raspberry phytochemical extract inhibits hepatic lesion in a Wistar rat model 
Background
Red raspberry possesses potent antioxidant capacity and antiproliferative activity against cancer in vitro.
Methods
The objective of this study was to determine the protective effects of raspberry 80% acetone extract in a rat hepatic lesions model induced by diethylnitrosamine (DEN). Rats were treated with the red raspberry extract (0.75, 1.5 or 3.0 g/kg of body weight) by gavage starting 2 h after DEN administration and continuing for 20 weeks.
Results
A dose-dependent inhibition by red raspberry extract of DEN-induced hepatic nodule formation which stands for hepatic lesions was observed. Corresponding hepatic nodule incidence rates were 45.0, 40.0, 25.0 and 5.0% in positive control, low, middle and high groups, respectively (P < 0.01 or 0.05). Gross findings, histopathological and ultrastructural evaluations of hepatic lesion were performed on 9, 8, 5 and 1 hepatic nodule in positive control, low, middle and high doses of groups, respectively, identified in rats from the respective groups of 20. A decreasing trend of proportions of hepatocellular carcinoma masses accompanied the increasing doses of red raspberry extract.
Conclusions
These findings demonstrate that the potent capacity of red raspberry diet could not only suppress DEN-induced hepatic lesions in rats, but also reduce the definite diagnostic features of neoplasm.
doi:10.1186/1743-7075-7-84
PMCID: PMC2999596  PMID: 21108811
6.  Differential expression of proteomics models of colorectal cancer, colorectal benign disease and healthy controls 
Proteome Science  2010;8:16.
Background
Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. The hypersensitive analytical technique of proteomics can detect molecular changes before the tumor is palpable. The surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS) is a newly-developed technique of evaluating protein separation in recent years. The protein chips have established the expression of tumor protein in the serum specimens and become the newly discovered markers for tumor diagnosis. The objective of this study was to find new markers of the diagnosis among groups of CRC, colorectal benign diseases (CBD) and healthy controls. The assay of SELDI-TOF-MS with analytical technique of protein-chip bioinformatics was used to detect the expression of protein mass peaks in the sera of patients or controls. One hundred serum samples, including 52 cases of colorectal cancer, 27 cases of colorectal benign disease, and 21 cases of healthy controls, were examined by SELDI-TOF-MS with WCX2 protein-chips.
Results
The diagnostic models (I, II and III) were setup by analyzed the data and sieved markers using Ciphergen - Protein-Chip-Software 5.1. These models were combined with 3 protein mass peaks to discriminate CRC, CBD, and healthy controls. The accuracy, the sensitivity and the particularity of cross verification of these models are all highly over 80%.
Conclusions
The SELDI-TOF-MS is a useful tool to help diagnose colorectal cancer, especially during the early stage. However, identification of the significantly differentiated proteins needs further study.
doi:10.1186/1477-5956-8-16
PMCID: PMC2862023  PMID: 20334691
7.  Effects of Yinchenhao Tang and related decoctions on DMN-induced cirrhosis/fibrosis in rats 
Chinese Medicine  2008;3:1.
Background
Chinese medicine decoctions such as Yinchenhao Tang (YCHT), Xiayuxue Tang (XYXT), Huangqi Tang (HQT), Yiguan Jian (YGJ) and Xiaochaihu Tang (XCHT)) were used to treat liver cirrhosis. The present study evaluates the effects of these decoctions on fibrosis in rats induced by dimethylnitrosamine (DMN).
Methods
DMN solution (0.5%) was injected to rats for three consecutive days per week for four weeks. At the beginning of week 3, rats were randomly divided into 4-week DMN control group, YCHT, XYXT, HQT, YGJ, XCHT and vehicle groups. Each group was orally administered with specific decoctions daily for two weeks. Rats in the vehicle group were orally administered with only water.
Results
Liver fibrosis and cirrhosis were observed in weeks 2 and 4 in DMN-intoxicated rats. Compared with normal rats, alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) activities and level of total bilirubin acid (TBA) in serum and content of Hydroxyproline (Hyp) in liver tissue of model group rats rose significantly. However, the albumin (Alb) level in serum decreased significantly. Compared with the 4-week DMN group, the pathological conditions and functions of the liver in the YCHT group improved significantly, and the content of Hyp decreased remarkably: only one rat in this group developed liver cirrhosis and the ratio of cirrhosis was only 8.3%. On the other hand, the other decoctions did not show remarkable effects. YCHT inhibited α-SMA activation, including its gene expression into mRNA and protein.
Conclusion
Among the five Chinese medicine decoctions, YCHT exerted the most significant therapeutic effects on DMN-induced cirrhosis/fibrosis in rats.
doi:10.1186/1749-8546-3-1
PMCID: PMC2267793  PMID: 18237412
8.  DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease▿ †  
Applied and Environmental Microbiology  2007;73(12):4082-4088.
Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 103 CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.
doi:10.1128/AEM.01820-06
PMCID: PMC1932722  PMID: 17449692
9.  Development of a Serotype-Specific DNA Microarray for Identification of Some Shigella and Pathogenic Escherichia coli Strains▿  
Journal of Clinical Microbiology  2006;44(12):4376-4383.
Shigella and pathogenic Escherichia coli are major causes of human infectious diseases and are responsible for millions of cases of diarrhea worldwide every year. A convenient and rapid method to identify highly pathogenic serotypes of Shigella and E. coli is needed for large-scale epidemiologic study, timely clinical diagnosis, and reliable quarantine of the pathogens. In this study, a DNA microarray targeting O-serotype-specific genes was developed to detect 15 serotypes of Shigella and E. coli, including Shigella sonnei; Shigella flexneri type 2a; Shigella boydii types 7, 9, 13, 16, and 18; Shigella dysenteriae types 4, 8, and 10; and E. coli O55, O111, O114, O128, and O157. The microarray was tested against 186 representative strains of all Shigella and E. coli O serotypes, 38 clinical isolates, and 9 strains of other bacterial species that are commonly present in stool samples and was shown to be specific and reproducible. The detection sensitivity was 50 ng genomic DNA or 104 CFU per ml in mock stool specimens. This is the first report of a microarray for serotyping Shigella and pathogenic E. coli. The method has a number of advantages over traditional bacterial culture and antiserum agglutination methods and is promising for applications in basic microbiological research, clinical diagnosis, food safety, and epidemiological surveillance.
doi:10.1128/JCM.01389-06
PMCID: PMC1698391  PMID: 17021058
10.  Increased ARPP-19 Expression Is Associated with Hepatocellular Carcinoma 
The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.
doi:10.3390/ijms16010178
PMCID: PMC4307242  PMID: 25547487
hepatocellular carcinoma; ARPP-19; cell proliferation; cell cycle
11.  Sympathetic Denervation-Induced MSC Mobilization in Distraction Osteogenesis Associates with Inhibition of MSC Migration and Osteogenesis by Norepinephrine/adrb3 
PLoS ONE  2014;9(8):e105976.
The sympathetic nervous system regulates bone formation and resorption under physiological conditions. However, it is still unclear how the sympathetic nerves affect stem cell migration and differentiation in bone regeneration. Distraction osteogenesis is an ideal model of bone regeneration due to its special nature as a self-engineering tissue. In this study, a rat model of mandibular distraction osteogenesis with transection of cervical sympathetic trunk was used to demonstrate that sympathetic denervation can deplete norepinephrine (NE) in distraction-induced bone callus, down-regulate β3-adrenergic receptor (adrb3) in bone marrow mesenchymal stem cells (MSCs), and promote MSC migration from perivascular regions to bone-forming units. An in vitro Transwell assay was here used to demonstrate that NE can inhibit stroma-derived factor-1 (SDF-1)-induced MSC migration and expression of the migration-related gene matrix metalloproteinase-2 (MMP-2) and downregulate that of the anti-migration gene tissue inhibitor of metalloproteinase-3 (TIMP-3). Knockdown of adrb3 using siRNA abolishes inhibition of MSC migration. An in vitro osteogenic assay was used to show that NE can inhibit the formation of MSC bone nodules and expression of the osteogenic marker genes alkaline phosphatase (ALP), osteocalcin (OCN), and runt-related transcription factor-2 (RUNX2), but knockdown of adrb3 by siRNA can abolish such inhibition of the osteogenic differentiation of MSCs. It is here concluded that sympathetic denervation-induced MSC mobilization in rat mandibular distraction osteogenesis is associated with inhibition of MSC migration and osteogenic differentiation by NE/adrb3 in vitro. These findings may facilitate understanding of the relationship of MSC mobilization and sympathetic nervous system across a wide spectrum of tissue regeneration processes.
doi:10.1371/journal.pone.0105976
PMCID: PMC4140837  PMID: 25144690
12.  Analysis of the Transcriptional Regulator GlpR, Promoter Elements, and Posttranscriptional Processing Involved in Fructose-Induced Activation of the Phosphoenolpyruvate-Dependent Sugar Phosphotransferase System in Haloferax mediterranei 
Among all known archaeal strains, the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for fructose utilization is used primarily by haloarchaea, which thrive in hypersaline environments, whereas the molecular details of the regulation of the archaeal PTS under fructose induction remain unclear. In this study, we present a comprehensive examination of the regulatory mechanism of the fructose PTS in the haloarchaeon Haloferax mediterranei. With gene knockout and complementation, microarray analysis, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), we revealed that GlpR is the indispensable activator, which specifically binds to the PTS promoter (PPTS) during fructose induction. Further promoter-scanning mutation indicated that three sites located upstream of the H. mediterranei PPTS, which are conserved in most haloarchaeal PPTSs, are involved in this induction. Interestingly, two PTS transcripts (named T8 and T17) with different lengths of 5′ untranslated region (UTR) were observed, and promoter or 5′ UTR swap experiments indicated that the shorter 5′ UTR was most likely generated from the longer one. Notably, the translation efficiency of the transcript with this shorter 5′ UTR was significantly higher and the ratio of T8 (with the shorter 5′ UTR) to T17 increased during fructose induction, implying that a posttranscriptional mechanism is also involved in PTS activation. With these insights into the molecular regulation of the haloarchaeal PTS, we have proposed a working model for haloarchaea in response to environmental fructose.
doi:10.1128/AEM.03372-13
PMCID: PMC3911062  PMID: 24334671
13.  Differentially Expressed Plasma MicroRNAs and the Potential Regulatory Function of Let-7b in Chronic Thromboembolic Pulmonary Hypertension 
PLoS ONE  2014;9(6):e101055.
Chronic thromboembolic pulmonary hypertension (CTEPH) is a progressive disease characterized by misguided thrombolysis and remodeling of pulmonary arteries. MicroRNAs are small non-coding RNAs involved in multiple cell processes and functions. During CTEPH, circulating microRNA profile endued with characteristics of diseased cells could be identified as a biomarker, and might help in recognition of pathogenesis. Thus, in this study, we compared the differentially expressed microRNAs in plasma of CTEPH patients and healthy controls and investigated their potential functions. Microarray was used to identify microRNA expression profile and qRT-PCR for validation. The targets of differentially expressed microRNAs were identified in silico, and the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway database were used for functional investigation of target gene profile. Targets of let-7b were validated by fluorescence reporter assay. Protein expression of target genes was determined by ELISA or western blotting. Cell migration was evaluated by wound healing assay. The results showed that 1) thirty five microRNAs were differentially expressed in CTEPH patients, among which, a signature of 17 microRNAs, which was shown to be related to the disease pathogenesis by in silico analysis, gave diagnostic efficacy of both sensitivity and specificity >0.9. 2) Let-7b, one of the down-regulated anti-oncogenic microRNAs in the signature, was validated to decrease to about 0.25 fold in CTEPH patients. 3) ET-1 and TGFBR1 were direct targets of let-7b. Altering let-7b level influenced ET-1 and TGFBR1 expression in pulmonary arterial endothelial cells (PAECs) as well as the migration of PAECs and pulmonary arterial smooth muscle cells (PASMCs). These results suggested that CTEPH patients had aberrant microRNA signature which might provide some clue for pathogenesis study and biomarker screening. Reduced let-7b might be involved in the pathogenesis of CTEPH by affecting ET-1 expression and the function of PAECs and PASMCs.
doi:10.1371/journal.pone.0101055
PMCID: PMC4076206  PMID: 24978044
14.  Preoperative Aspiration Culture for Preoperative Diagnosis of Infection in Total Hip or Knee Arthroplasty 
Journal of Clinical Microbiology  2013;51(11):3830-3834.
This meta-analysis evaluated preoperative aspiration culture for diagnosing prosthetic joint infection (PJI) in total hip arthroplasty (THA) and total knee arthroplasty (TKA). The pooled sensitivity and specificity were 0.72 (95% confidence interval, 0.65 to 0.78) and 0.95 (0.93 to 0.97), respectively. Subgroup analyses revealed nonsignificant worse diagnostic performance for THA than for TKA (sensitivity, 0.70 versus 0.78; specificity, 0.94 versus 0.96). Preoperative aspiration culture has moderate to high sensitivity and very high specificity for diagnosing PJI.
doi:10.1128/JCM.01467-13
PMCID: PMC3889774  PMID: 23946521
15.  Suxiao Jiuxin Pill Induces Potent Relaxation and Inhibition on Contraction in Human Artery and the Mechanism 
Suxiao Jiuxin Pill, a compound Chinese traditional medicine with main components of tetramethylpyrazine and borneol, is widely used for antiangina treatment in China but its pharmacological effect on human blood vessels is unknown. We investigated the effect and possible mechanism of SJP in the human internal mammary artery (IMA, n = 78) taken from patients undergoing coronary surgery. SJP caused full relaxation in KCl- (99.4 ± 10.5%, n = 6) and U46619- (99.9 ± 5.6%, n = 6) contracted IMA. Pretreatment of IMA with plasma concentrations of SJP (1 mg/mL), calculated from the plasma concentration of its major component borneol, significantly depressed the maximal contraction to KCl (from 35.8 ± 6.0 mN to 12.6 ± 5.6 mN, P = 0.03) and U46619 (from 19.4 ± 2.9 mN to 5.7 ± 2.4 mN, P = 0.007) while SJP at 10 mg/mL abolished the subsequent contraction. Endothelium denudation and inhibition of eNOS significantly altered the SJP-induced relaxation without changes of eNOS expression. We conclude that SJP has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors in human arteries. The vasorelaxation involves both endothelium-dependent and -independent mechanisms. Thus, the effect of SJP on human arteries demonstrated in this study may prove to be particularly important in vasorelaxing therapy in cardiovascular disease.
doi:10.1155/2014/956924
PMCID: PMC3997901  PMID: 24808920
16.  Electrically facilitated translocation of protein through solid nanopore 
Nanoscale Research Letters  2014;9(1):140.
Nanopores have been proven as versatile single-molecule sensors for individual unlabeled biopolymer detection and characterization. In the present work, a relative large nanopore with a diameter of about 60 nm has been used to detect protein translocation driven by a series of applied voltages. Compared with previous studied small nanopores, a distinct profile of protein translocation through a larger nanopore has been characterized. First, a higher threshold voltage is required to drive proteins into the large nanopore. With the increase of voltages, the capture frequency of protein into the nanopore has been markedly enhanced. And the distribution of current blockage events is characterized as a function of biased voltages. Due to the large dimension of the nanopore, the adsorption and desorption phenomenon of proteins observed with a prolonged dwell time has been weakened in our work. Nevertheless, the protein can still be stretched into an unfolded state by increased electric forces at high voltages. In consideration of the high throughput of the large nanopore, a couple of proteins passing through the nanopore simultaneously occur at high voltage. As a new feature, the feasibility and specificity of a nanopore with distinct geometry have been demonstrated for sensing protein translocation, which broadly expand the application of nanopore devices.
doi:10.1186/1556-276X-9-140
PMCID: PMC3976542  PMID: 24661490
Protein translocation; Solid state nanopore; Current blockage; Translocation time
17.  Complete Genome Sequence of Magnetospirillum gryphiswaldense MSR-1 
Genome Announcements  2014;2(2):e00171-14.
We report the complete genomic sequence of Magnetospirillum gryphiswaldense MSR-1 (DSM 6361), a type strain of the genus Magnetospirillum belonging to the Alphaproteobacteria. Compared to the reported draft sequence, extensive rearrangements and differences were found, indicating high genomic flexibility and “domestication” by accelerated evolution of the strain upon repeated passaging.
doi:10.1128/genomeA.00171-14
PMCID: PMC3953193  PMID: 24625872
18.  GSK3β Overexpression Indicates Poor Prognosis and Its Inhibition Reduces Cell Proliferation and Survival of Non-Small Cell Lung Cancer Cells 
PLoS ONE  2014;9(3):e91231.
Background
Glycogen synthase kinase 3 beta (GSK3β) is centrally involved in diverse cellular processes, including proliferation and apoptosis. This study aimed to investigate the influence of GSK3β expression on the prognosis of human non-small cell lung cancer (NSCLC) and the effects of GSK3β inhibition in NSCLC cell lines.
Methods
Immunohistochemical and western blot assays were used to evaluate the GSK3β expression level in human NSCLC tissues. Lentiviral RNA interference was performed to inhibit the expression of GSK3β in the A549, H292, H1299 and SK-MES-1 cell lines. Cell survival, apoptosis and motility were evaluated in vivo and in vitro.
Results
The levels of GSK3β were greater in NSCLC tissues (n = 211) than in control tissues (n = 194) (P<0.001). The 5-year follow-up analysis showed that positive GSK3β expression was indicative of poor prognosis (P = 0.006). Furthermore, knockdown of GSK3β in NSCLC cell lines suppressed cell proliferation, arrested tumor cells in G0/G1 phase, induced apoptosis and reduced cell motility. A xenograft model showed that the deregulation of GSK3β attenuated tumorigenesis, as confirmed by reduced cell proliferation based on Ki-67 and significantly increased apoptotic cell death. The inhibition of GSK3β had inconsistent effects on the expression of β-catenin, depending on the cell type examined.
Conclusion
Aberrant expression of GSK3β serves as an independent marker of poor prognosis for NSCLC. The inhibition of GSK3β suppressed tumorigenesis by attenuating cell proliferation, increasing apoptosis and restraining cell motility. These results identify GSK3β as a tumor promoter and a potential therapeutic target in NSCLC.
doi:10.1371/journal.pone.0091231
PMCID: PMC3949982  PMID: 24618715
19.  Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups 
Applied and Environmental Microbiology  2013;79(11):3392-3399.
Salmonella is a major cause of food-borne disease in many countries. Serotype determination of Salmonella is important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46 Salmonella O serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293 Salmonella strains, 186 Shigella strains, representative Escherichia coli strains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of the Salmonella strains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46 Salmonella O serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multiple Salmonella O serogroups simultaneously.
doi:10.1128/AEM.00225-13
PMCID: PMC3648052  PMID: 23524674
20.  Adsorption of Cu(II) on Oxidized Multi-Walled Carbon Nanotubes in the Presence of Hydroxylated and Carboxylated Fullerenes 
PLoS ONE  2013;8(8):e72475.
The adsorption of Cu(II) on oxidized multi-walled carbon nanotubes (oMWCNTs) as a function of contact time, pH, ionic strength, temperature, and hydroxylated fullerene (C60(OH)n) and carboxylated fullerene (C60(C(COOH)2)n) were studied under ambient conditions using batch techniques. The results showed that the adsorption of Cu(II) had rapidly reached equilibrium and the kinetic process was well described by a pseudo-second-order rate model. Cu(II) adsorption on oMWCNTs was dependent on pH but independent of ionic strength. Compared with the Freundlich model, the Langmuir model was more suitable for analyzing the adsorption isotherms. The thermodynamic parameters calculated from temperature-dependent adsorption isotherms suggested that Cu(II) adsorption on oMWCNTs was spontaneous and endothermic. The effect of C60(OH)n on Cu(II) adsorption of oMWCNTs was not significant at low C60(OH)n concentration, whereas a negative effect was observed at higher concentration. The adsorption of Cu(II) on oMWCNTs was enhanced with increasing pH values at pH < 5, but decreased at pH ≥ 5. The presence of C60(C(COOH)2)n inhibited the adsorption of Cu(II) onto oMWCNTs at pH 4–6. The double sorption site model was applied to simulate the adsorption isotherms of Cu(II) in the presence of C60(OH)n and fitted the experimental data well.
doi:10.1371/journal.pone.0072475
PMCID: PMC3756995  PMID: 24009683
21.  The application of hyaluronic acid-derivatized carbon nanotubes in hematoporphyrin monomethyl ether-based photodynamic therapy for in vivo and in vitro cancer treatment 
Carbon nanotubes (CNTs) have shown great potential in both photothermal therapy and drug delivery. In this study, a CNT derivative, hyaluronic acid-derivatized CNTs (HA-CNTs) with high aqueous solubility, neutral pH, and tumor-targeting activity, were synthesized and characterized, and then a new photodynamic therapy agent, hematoporphyrin monomethyl ether (HMME), was adsorbed onto the functionalized CNTs to develop HMME-HA-CNTs. Tumor growth inhibition was investigated both in vivo and in vitro by a combination of photothermal therapy and photodynamic therapy using HMME-HA-CNTs. The ability of HMME-HA-CNT nanoparticles to combine local specific photodynamic therapy with external near-infrared photothermal therapy significantly improved the therapeutic efficacy of cancer treatment. Compared with photodynamic therapy or photothermal therapy alone, the combined treatment demonstrated a synergistic effect, resulting in higher therapeutic efficacy without obvious toxic effects to normal organs. Overall, it was demonstrated that HMME-HA-CNTs could be successfully applied to photodynamic therapy and photothermal therapy simultaneously in future tumor therapy.
doi:10.2147/IJN.S45407
PMCID: PMC3702246  PMID: 23843694
photodynamic therapy; photothermal therapy; HA-derivatized carbon nanotubes; tumor targeting; synergistic effect; hematoporphyrin monomethyl ether
22.  PTEN Loss Increases PD-L1 Protein Expression and Affects the Correlation between PD-L1 Expression and Clinical Parameters in Colorectal Cancer 
PLoS ONE  2013;8(6):e65821.
Background
Programmed death ligand-1 (PD-L1) has been identified as a factor associated with poor prognosis in a range of cancers, and was reported to be mainly induced by PTEN loss in gliomas. However, the clinical effect of PD-L1 and its regulation by PTEN has not yet been determined in colorectal cancer (CRC). In the present study, we verified the regulation of PTEN on PD-L1 and further determined the effect of PTEN on the correlation between PD-L1 expression and clinical parameters in CRC.
Methods/Results
RNA interference approach was used to down-regulate PTEN expression in SW480, SW620 and HCT116 cells. It was showed that PD-L1 protein, but not mRNA, was significantly increased in cells transfected with siRNA PTEN compared with the negative control. Moreover, the capacity of PTEN to regulate PD-L1 expression was not obviously affected by IFN-γ, the main inducer of PD-L1. Tissue microarray immunohistochemistry was used to detect PD-L1 and PTEN in 404 CRC patient samples. Overexpression of PD-L1 was significantly correlated with distant metastasis (P<0.001), TNM stage (P<0.01), metastatic progression (P<0.01) and PTEN expression (P<0.001). Univariate analysis revealed that patients with high PD-L1 expression had a poor overall survival (P<0.001). However, multivariate analysis did not support PD-L1 as an independent prognostic factor (P = 0.548). Univariate (P<0.001) and multivariate survival (P<0.001) analysis of 310 located CRC patients revealed that high level of PD-L1 expression was associated with increased risks of metastatic progression. Furthermore, the clinical effect of PD-L1 on CRC was not statistically significant in a subset of 39 patients with no PTEN expression (distant metastasis: P = 0.102; TNM stage: P = 0.634, overall survival: P = 0.482).
Conclusions
PD-L1 can be used to identify CRC patients with high risk of metastasis and poor prognosis. This clinical manifestation may be partly associated with PTEN expression.
doi:10.1371/journal.pone.0065821
PMCID: PMC3681867  PMID: 23785454
23.  Activated N-Ras signaling regulates arterial-venous specification in zebrafish 
Background
The aberrant activation of Ras signaling is associated with human diseases including hematological malignancies and vascular disorders. So far the pathological roles of activated Ras signaling in hematopoiesis and vasculogenesis are largely unknown.
Methods
A conditional Cre/loxP transgenic strategy was used to mediate the specific expression of a constitutively active form of human N-Ras in zebrafish endothelial and hematopoietic cells driven by the zebrafish lmo2 promoter. The expression of hematopoietic and endothelial marker genes was analyzed both via whole mount in situ hybridization (WISH) assay and real-time quantitative PCR (qPCR). The embryonic vascular morphogenesis was characterized both by living imaging and immunofluorescence on the sections with a confocal microscopy, and the number of endothelial cells in the embryos was quantified by flow cytometry. The functional analyses of the blood circulation were carried out by fluorescence microangiography assay and morpholino injection.
Results
In the activated N-Ras transgenic embryos, the primitive hematopoiesis appeared normal, however, the definitive hematopoiesis of these embryos was completely absent. Further analysis of endothelial cell markers confirmed that transcription of arterial marker ephrinB2 was significantly decreased and expression of venous marker flt4 excessively increased, indicating the activated N-Ras signaling promotes the venous development at the expense of arteriogenesis during zebrafish embryogenesis. The activated N-Ras-expressing embryos showed atrophic axial arteries and expansive axial veins, leading to no definitive hematopoietic stem cell formation, the blood circulation failure and subsequently embryonic lethality.
Conclusions
Our studies revealed for the first time that activated N-Ras signaling during the endothelial differentiation in vertebrates can disrupt the balance of arterial-venous specification, thus providing new insights into the pathogenesis of the congenital human vascular disease and tumorigenic angiogenesis.
doi:10.1186/1756-8722-6-34
PMCID: PMC3658992  PMID: 23663822
Vasculogenesis; Arteriogenesis; N-Ras
24.  Characterization of the Interaction between Cadmium and Chlorpyrifos with Integrative Techniques in Incurring Synergistic Hepatoxicity 
PLoS ONE  2013;8(3):e59553.
Mixture toxicity is an important issue for the risk assessment of environmental pollutants, for which an extensive amount of data are necessary in evaluating their potential adverse health effects. However, it is very hard to decipher the interaction between compounds due to limited techniques. Contamination of heavy metals and organophosphoric insecticides under the environmental and biological settings poses substantial health risk to humans. Although previous studies demonstrated the co-occurrence of cadmium (Cd) and chlorpyrifos (CPF) in environmental medium and food chains, their interaction and potentially synergistic toxicity remain elusive thus far. Here we integrated the approaches of thin-layer chromatography and 1H NMR to study the interaction between Cd2+ and CPF in inducing hepatoxicity. A novel interaction was identified between Cd2+ and CPF, which might be the bonding between Cd2+ and nitrogen atom in the pyridine ring of CPF, or the chelation formation between one Cd2+ and two CPF molecules. The Cd-CPF complex was conferred with distinct biological fate and toxicological performances from its parental components. We further demonstrated that the joint hepatoxicity of Cd ion and CPF was chiefly due to the Cd-CPF complex-facilitated intracellular transport associated with oxidative stress.
doi:10.1371/journal.pone.0059553
PMCID: PMC3597607  PMID: 23516638
25.  Characterization of De Qi with Electroacupuncture at Acupoints with Different Properties 
Abstract
Objectives
This study sought to explore the characteristics of de qi using electroacupuncture at acupoints with different properties in the meridian category, histological type, and nerve innervations.
Methods
Electroacupuncture was performed on 21 healthy volunteers at paired acupoints of ST36-GB34, CV4-CV12, ST36-ST28, PC6-PC7, and ST36-CV4. Upon acupuncture de qi, the intensities and the prevalence of individual sensations, sensation transmission, and the amplitude of electrical current were recorded. Chi-square, Fischer's exact test, Wilcoxon test, and two-sample paired t test were used to compare the components of de qi within each paired group.
Results
Overall intensities and prevalence of individual sensations are fullness, numbness, soreness, tingling, heaviness, pressure, dull pain, warmness, and coolness in decreasing order. No significant difference was found in the prevalence of needling sensations between the two paired points (p>0.05). However, significant intensity differences (p<0.05) were showed in soreness, fullness, and heaviness between ST36-ST28, in fullness and numbness between ST36-CV4, and in fullness between CV4-CV12. Tingling sensation was stronger than heaviness and pressure at acupoints PC6-PC7 and ST36 (as paired with CV4). Sharp pain occurred in 10/216 tests (4.63%). Sensation transmission occurred highly (77.78%) on participants, and sensations mostly propagate over one joint but no further than two joints. Overall electrical current was 2.35±0.07 mA (mean±standard error). No significant difference was found between paired acupoints for the distance of sensation transmission and the amplitude of electrical current.
Conclusions
Fullness, numbness, and soreness were the most common and obvious sensations associated with electroacupuncture. The difference of sensation intensities may be associated with different nerve innervations.
doi:10.1089/acm.2010.0652
PMCID: PMC3221509  PMID: 21999154

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