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1.  Function of nonstructural 5A protein of genotype 2a in replication and infection of HCV with gene substitution 
AIM: To explore the function of Nonstructural 5A (NS5A) protein of genotype 2a (JFH1) in the replication and infection of hepatitis C virus (HCV).
METHODS: Intergenotypic chimera FL-J6JFH/J4NS5A was constructed by inserting NS5A gene from 1b stain HC-J4 by the overlapping polymerase chain reaction (PCR) method and the restriction enzyme reaction. In vitro RNA transcripts of chimera, prototype J6JFH and negative control J6JFH1 (GND) were prepared and transfected into Huh-7.5 cells with liposomes. Immunofluorescence assay (IFA), fluorescence quantitative PCR and infection assay were performed to determine the protein expression and gene replication in Huh-7.5 cells.
RESULTS: The HCV RNA levels in FL-J6JFH/J4NS5A chimera RNA transfected cells were significantly lower than the wild type at any indicated time point (2.58 ± 5.97 × 106 vs 4.27 ± 1.72 × 104, P = 0.032). The maximal level of HCV RNA in chimera was 5.6 ± 1.8 × 104 GE/μg RNA at day 34 after transfection, while the wild type reached a peak level at day 13 which was 126 folds higher (70.65 ± 14.11 × 105 vs 0.56 ± 0.90 × 105, P = 0.028). HCV proteins could also be detected by IFA in chimera-transfected cells with an obviously low level. Infection assay showed that FL-J6JFH/J4NS5A chimera could produce infectious virus particles, ranging from 10 ± 5 ffu/mL to 78.3 ± 23.6 ffu/mL, while that of FL-J6JFH1 ranged from 5.8 ± 1.5 × 102 ffu/mL to 2.5 ± 1.4 × 104 ffu/mL.
CONCLUSION: JFH1 NS5A might play an important role in the robust replication of J6JFH1. The establishment of FL-J6JFH/J4NS5A provided a useful platform for studying the function of other proteins of HCV.
PMCID: PMC3160566  PMID: 21876632
Hepatitis C virus; Nonstructural 5A; Chimera; Cell culture-produced virus; Replication; Infection
2.  Knockdown of survivin gene expression by RNAi induces apoptosis in human hepatocellular carcinoma cell line SMMC-7721 
AIM: To investigate the survivin gene expression in human hepatocellular carcinoma cell line SMMC-7721 and the effects of survivin gene RNA interference (RNAi) on cell apoptosis and biological behaviors of SMMC-7721 cells.
METHODS: Eukaryotic expression vector of survivin gene RNAi and recombinant plasmid pSuppressorNeo-survivin(pSuNeo-SVV), were constructed by ligating into the vector, pSupperssorNeo (pSuNeo) digested with restriction enzymes Xba I and Sal I and the designed double-chain RNAi primers. A cell model of SMMC-7721 after treatment with RNAi was prepared by transfecting SMMC-7721 cells with the lipofectin transfection method. Strept-avidin- biotin-complex (SABC) immunohistochemical staining and RT-PCR were used to detect survivin gene expressions in SMMC-7721 cells. Flow cytometry was used for the cell cycle analysis. Transmission electron microscopy was performed to determine whether RNAi induced cell apoptosis, and the method of measuring the cell growth curve was utilized to study the growth of SMMC-7721 cells before and after treatment with RNAi.
RESULTS: The eukaryotic expression vector of survivin gene RNAi and pSuNeo-SVV, were constructed successfully. The expression level of survivin gene in SMMC-7721 cells was observed. After the treatment of RNAi, the expression of survivin gene in SMMC-7721 cells was almost absent, apoptosis index was increased by 15.6%, and the number of cells was decreased in G2/M phase and the cell growth was inhibited.
CONCLUSION: RNAi can exert a knockdown of survivin gene expression in SMMC-7721 cells, and induce apoptosis and inhibit the growth of carcinoma cells.
PMCID: PMC4250756  PMID: 15655839
Hepatocellular carcinoma; Survivin; RNA interference; Apoptosis; Gene expression
3.  Intrinsic dynamics induce global symmetry in network controllability 
Scientific Reports  2015;5:8422.
Controlling complex networked systems to desired states is a key research goal in contemporary science. Despite recent advances in studying the impact of network topology on controllability, a comprehensive understanding of the synergistic effect of network topology and individual dynamics on controllability is still lacking. Here we offer a theoretical study with particular interest in the diversity of dynamic units characterized by different types of individual dynamics. Interestingly, we find a global symmetry accounting for the invariance of controllability with respect to exchanging the densities of any two different types of dynamic units, irrespective of the network topology. The highest controllability arises at the global symmetry point, at which different types of dynamic units are of the same density. The lowest controllability occurs when all self-loops are either completely absent or present with identical weights. These findings further improve our understanding of network controllability and have implications for devising the optimal control of complex networked systems in a wide range of fields.
PMCID: PMC4325315  PMID: 25672476
4.  Effects of whole cigarette smoke on human beta defensins expression and secretion by oral mucosal epithelial cells 
Tobacco Induced Diseases  2015;13(1):3.
Cigarette smoke a recognized risk factor for many systemic diseases and also oral diseases. Human beta defensins (HBDs), a group of important antimicrobial peptides expressed by the epithelium, are crucial for local defense and tissue homeostasis of oral cavity. The aim of this study was to evaluate potential effects of whole cigarette smoke (WCS) exposure on the expression and secretion of HBDs by oral mucosal epithelial cells.
Immortalized human oral mucosal epithelial (Leuk-1) cells were exposed to WCS for various time periods. HBD-1, -2 and -3 expression and subcellular localization were detected by real time qPCR, immunofluorescence assay and confocal microscopy. According to the relative fluorescent intensity, the expression levels of HBD-1, -2 and -3 were evaluated by digital image analysis system. The alteration of HBD-1, -2 and -3 secretion levels was measured by the Enzyme-Linked Immunosorbent Assay.
WCS exposure remarkably attenuated HBD-1 expression and secretion while clearly enhanced HBD-2, -3 expression levels and HBD-2 secretion by Leuk-l cells. It appeared that there was no significant effect of WCS exposure on HBD-3 secretion.
WCS exposure could modulate expression and secretion of HBDs by oral mucosal epithelial cells, establishing a link between cigarette smoke and abnormal levels of antimicrobial peptides. The present results may give a new perspective to investigate smoking-related local defense suppression and oral disease occurrence.
PMCID: PMC4310021  PMID: 25635179
Whole cigarette smoke; Human β defensin; Oral mucosa
5.  Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle 
Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis.
Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis.
Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5–8 μg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21–35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%).
Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.
PMCID: PMC4310348  PMID: 25653501
ionophore; methicillin-resistant Staphylococcus aureus; solid dispersion; intramammary drug delivery system; superbugs
6.  Lower irritation microemulsion-based rotigotine gel: formulation optimization and in vitro and in vivo studies 
Rotigotine is a potent and selective D1, D2, and D3 dopaminergic receptor agonist. Due to an extensive first-pass effect, it has a very low oral bioavailability (approximately 0.5% in rats).
The present investigation aimed to develop a microemulsion-based hydrogel for transdermal rotigotine delivery with lower application site reactions.
Pseudoternary phase diagrams were constructed to determine the region of oil in water (o/w)-type microemulsion. Central composite design was used to support the pseudoternary phase diagrams and to select homogeneous and stable microemulsions with an optimal amount of rotigotine permeation within 24 hours. In vitro skin permeation experiments were performed, using Franz diffusion cells, to compare rotigotine-loaded microemulsions with rotigotine solutions in oil. The optimized formulation was used to prepare a microemulsion-based hydrogel, which was subjected to bioavailability and skin irritancy studies.
The selected formulations of rotigotine-loaded microemulsions had enhanced flux and permeation coefficients compared with rotigotine in oil. The optimum microemulsion contained 68% water, 6.8% Labrafil®, 13.44% Cremophor® RH40, 6.72% Labrasol®, and 5.04% Transcutol® HP; the drug-loading rate was 2%. To form a microemulsion gel, 1% Carbomer 1342 was added to the microemulsion. The bioavailability of the rotigotine-loaded microemulsion gel was 105.76%±20.52% with respect to the marketed rotigotine patch (Neupro®). The microemulsion gel irritated the skin less than Neupro.
A rotigotine microemulsion-based hydrogel was successfully developed, and an optimal formulation for drug delivery was identified. This product could improve patient compliance and have broad marketability.
PMCID: PMC4298334  PMID: 25609965
pseudoternary phase diagrams; central composite design; transdermal
7.  Impact of removable dentures on oral health-related quality of life among elderly adults in Taiwan 
BMC Oral Health  2015;15:1.
Although the use of removable dentures can improve oral function and esthetics for elderly people, compared to those who do not wear removable dentures, those wearing removable dentures could have worse oral health related-quality of life (OHRQoL). Additional information is required to assess which factors related to denture wearing influence the OHRQoL of elderly individuals. The purpose of this study is to evaluate the association between denture wearing and OHRQoL in a sample of elderly individuals in Taiwan.
The study population included 277 elderly people wearing removable dentures (mean age = 76.0 years). Using face-to-face interviews, we collected data on the participants’ socio-demographic characteristics, dental care service usage (regular dental checkups, treatment during toothache, dental visits in the last year), and factors related to denture wearing (perceived oral pain, perceived loose denture, perceived oral ulcer, perceived halitosis, perceived dry mouth, and perceived total denture satisfaction scores). OHRQoL was measured using the Taiwanese version of the Geriatric Oral Health Assessment Index (GOHAI-T). The location and number of remaining natural teeth and the type of denture were also recorded. Hierarchical multiple regression analysis was performed using GOHAI-T scores as the dependent variable.
All the predictors together accounted for 50% of the variance in GOHAI-T scores. Further, education level, number of natural teeth, denture status, perceived loose denture, perceived oral ulcer, and perceived total denture satisfaction scores had statistically significant influences on OHRQoL. When compared with other variables, factors related to denture wearing, especially perceived total denture satisfaction scores, had the greatest impact on GOHAI-T scores.
Of the factors analyzed in this study, denture satisfaction was the strongest predictor of OHRQoL. This suggests that denture satisfaction is useful for assessing the effect of denture treatment on the OHRQoL of elderly individuals wearing removable dentures.
PMCID: PMC4298049  PMID: 25559722
Elderly; Removable dental prostheses; Oral health-related quality of life; Geriatric oral health assessment index; Denture satisfaction; Dental care service
8.  The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells 
Cytotechnology  2013;66(1):119-131.
Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72 h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.
Electronic supplementary material
The online version of this article (doi:10.1007/s10616-013-9544-8) contains supplementary material, which is available to authorized users.
PMCID: PMC3886541  PMID: 23579245
Simulated microgravity; Bone marrow mesenchymal stem cells; Pluripotency; Differentiation; OCT4
9.  Combination of interventional adenovirus-p53 introduction and ultrasonic irradiation in the treatment of liver cancer 
Oncology Letters  2014;9(3):1297-1302.
The aim of the present study was to investigate the effect of the combination of interventional adenovirus-p53 (Ad-p53) introduction and ultrasonic irradiation (CIAIUI) treatment for liver cancer, including evaluating the Ad-p53 transfection efficiency and the impact of the p53 gene on vascular endothelial growth factor (VEGF) and matrix metalloprotein 2 (MMP2) protein expression levels. Ad-p53 was arterially infused into the hepatic carcinoma via the interventional introduction of the hepatic tumor-bearing artery (IIHTBA) or the CIAIUI. Serum VEGF levels were determined by performing an enzyme-linked immunosorbent assay; immunohistochemical analysis was used to identify the expression levels of intratumoral p53, MMP2 and VEGF; and western blot analysis was used to determine the impact of different Ad-p53 administration methods on the expression of wild-type p53. The wild-type p53 expression level was significantly higher in the p53-treated group compared with the control group, and the p53 expression level in the CIAIUI group was significantly higher compared with the non-irradiation group. The CIAIUI could significantly reduce the serum VEGF levels. The two delivery methods caused a reduction in the intratumoral VEGF and MMP2 expression levels, and the effects of CIAIUI were most obvious. Ad-p53 infusion via IIHTBA promoted the protein expression levels of p53, however, it inhibited the protein expression levels of MMP2 and VEGF, indirectly indicating that the gene may inhibit the growth of liver cancer. Therefore, CIAIUI therapy exhibited an overall improved therapeutic effect compared with the more simple IIHTBA therapy.
PMCID: PMC4315071  PMID: 25663901
adenovirus-p53; liver cancer; interventional introduction; ultrasonic irradiation
10.  Levodopa/benserazide microsphere (LBM) prevents L-dopa induced dyskinesia by inactivation of the DR1/PKA/P-tau pathway in 6-OHDA-lesioned Parkinson's rats 
Scientific Reports  2014;4:7506.
L-3, 4-dihydroxyphenylalanine (L-dopa) is the gold standard for symptomatic treatment of Parkinson's disease (PD), but long-term therapy is associated with the emergence of L-dopa-induced dyskinesia (LID). In the present study, L-dopa and benserazide were loaded by poly (lactic-co-glycolic acid) microspheres (LBM), which can release levodopa and benserazide in a sustained manner in order to continuous stimulate dopaminergic receptors. We investigated the role of striatal DR1/PKA/P-tau signal transduction in the molecular event underlying LID in the 6-OHDA-lesioned rat model of PD. We found that animals rendered dyskinetic by L-dopa treatment, administration of LBM prevented the severity of AIM score, as well as improvement in motor function. Moreover, we also showed L-dopa elicits profound alterations in the activity of three LID molecular markers, namely DR1/PKA/P-tau (ser396). These modifications are totally prevented by LBM treatment, a similar way to achieve continuous dopaminergic delivery (CDD). In conclusion, our experiments provided evidence that intermittent administration of L-dopa, but not continuous delivery, and DR1/PKA/p-tau (ser396) activation played a critical role in the molecular and behavioural induction of LID in 6-OHDA-lesioned rats. In addition, LBM treatment prevented the development of LID by inhibiting the expression of DR1/PKA/p-tau, as well as PPEB mRNA in dyskintic rats.
PMCID: PMC4267205  PMID: 25511986
11.  Approaches for the Analysis of Chlorinated Lipids 
Analytical biochemistry  2013;443(2):10.1016/j.ab.2013.09.016.
Leukocytes are key cellular mediators of human diseases through their role in inflammation. Identifying unique molecules produced by leukocytes may provide new biomarkers and mechanistic insights into the role of leukocytes in disease. Chlorinated lipids are generated as a result of myeloperoxidase-containing leukocyte-derived hypochlorous acid targeting the vinyl ether bond of plasmalogens. The initial product of this reaction is α-chlorofatty aldehyde. α -Chlorofatty aldehyde is both oxidized to α-chlorofatty acid and reduced to α-chlorofatty alcohol by cellular metabolism. This review focuses on the separation techniques and quantitative analysis for these chlorinated lipids. For α-chlorofatty acid the negative charge of carboxylic acids is exploited to detect the chlorinated lipid species of these acids by electrospray ionization mass spectrometry in the negative ion mode. In contrast, α-chlorofatty aldehyde and α-chlorofatty alcohol are converted to pentafluorobenzyl oxime and pentafluorobenzoyl ester derivatives, which are detected by negative ion-chemical ionization mass spectrometry. These two detection methods coupled with the use of stable isotope internal standards and either liquid chromatography or gas chromatography provide highly sensitive analytical approaches to measure these novel lipids.
PMCID: PMC3839041  PMID: 24056259
fatty aldehyde; fatty acid; mass spectrometry; monocytes; chlorinated lipids; chromatography
12.  Caspase-12 Silencing Attenuates Inhibitory Effects of Cigarette Smoke Extract on NOD1 Signaling and hBDs Expression in Human Oral Mucosal Epithelial Cells 
PLoS ONE  2014;9(12):e115053.
Cigarette smoke exposure is associated with increased risk of various diseases. Epithelial cells-mediated innate immune responses to infectious pathogens are compromised by cigarette smoke. Although many studies have established that cigarette smoke exposure affects the expression of Toll-liked receptor (TLR), it remains unknown whether the nucleotide-binding oligomerization domain-containing protein 1 (NOD1) expression is affected by cigarette smoke exposure. In the study, we investigated effects of cigarette smoke extract (CSE) on NOD1 signaling in an immortalized human oral mucosal epithelial (Leuk-1) cell line. We first found that CSE inhibited NOD1 expression in a dose-dependent manner. Moreover, CSE modulated the expression of other crucial molecules in NOD1 signaling and human β defensin (hBD) 1, 2 and 3. We found that RNA interference-induced Caspase-12 silencing increased NOD1 and phospho-NF-κB (p-NF-κB) expression and down-regulated RIP2 expression. The inhibitory effects of CSE on NOD1 signaling can be attenuated partially through Caspase-12 silencing. Intriguingly, Caspase-12 silencing abrogated inhibitory effects of CSE on hBD1, 3 expression and augmented induced effect of CSE on hBD2 expression. Caspase-12 could play a vital role in the inhibitory effects of cigarette smoke on NOD1 signaling and hBDs expression in oral mucosal epithelial cells.
PMCID: PMC4263745  PMID: 25503380
13.  Molecular Mechanism of Yisui Shengxue Granule, a Complex Chinese Medicine, on Thalassemia Patients Suffering from Hemolysis and Anemia of Erythrocytes 
The objective of this study was to investigate the therapeutic biological mechanism of Yisui Shengxue Granule (YSSXG), a complex Chinese medicine, on the hemolysis and anemia of erythrocytes from patient with thalassemia disease. Sixteen patients with thalassemia (8 cases of α-thalassemia and 8 cases of β-thalassemia) disease were collected and treated with YSSXG for 3 months. The improvements of blood parameter demonstrated that YSSXG had a positive clinical effect on patients with thalassemia disease. For patients with α-thalassemia disease, RT-PCR showed that YSSXG upregulated the relative mRNA expression level of α-globin to β-globin and downregulated DNMT1, DNMT3a, and DNMT3b mRNA compared with pretreatment. Western blotting showed that YSSXG downregulated the expression of DNMT1 and DNMT3a. For patients with β-thalassemia disease, the relative expression level of Aγ-globin to α-globin had an increasing trend and the level of BCL11A mRNA expression obviously increased. For all patients, RT-PCR showed that YSSXG upregulated mRNA expression of SPTA1 and SPTB. Activities of SOD and GSH-Px significantly increased and MDA obviously reduced on erythrocyte and blood serum after YSSXG treatment. TEM showed that YSSXG decreased the content of inclusion bodies. Activities of Na+K+-ATPtase and T-ATPtase of erythrocyte increased significantly after YSSXG treatment. This study provides the basis for mechanisms of YSSXG on thalassemia suffering with hemolysis and anemia of erythrocytes from patient.
PMCID: PMC4276282  PMID: 25574177
14.  Understanding CELSRs - Cadherin EGF LAG seven-pass G-type receptors 
Journal of neurochemistry  2014;131(6):699-711.
The cadherin EGF LAG seven-pass G-type receptors (CELSRs) are a special subgroup of adhesion G protein-coupled receptors (GPCRs), which are pivotal regulators of many biological processes such as neuronal/endocrine cell differentiation, vessel valve formation and the control of planar cell polarity during embryonic development. All three members of the CELSR family (CELSR1-3) have large ecto-domains that form homophilic interactions and encompass more than 2,000 amino acids. Mutations in the ecto-domain or other gene locations of CELSRs are associated with neural tube defects (NTDs) and other diseases in humans. Celsr knockout (KO) animals have many developmental defects. Therefore, specific agonists or antagonists of CELSR members may have therapeutic potential. Although significant progress has been made regarding the functions and biochemical properties of CELSRs, our knowledge of these receptors is still lacking, especially considering that they are broadly distributed but have few characterized functions in a limited number of tissues. The dynamic activation and inactivation of CELSRs and the presence of endogenous ligands beyond homophilic interactions remain elusive, as do the regulatory mechanisms and downstream signaling of these receptors. Given this motivation, future studies with more advanced cell biology or biochemical tools, such as conditional KO mice, may provide further insights into the mechanisms underlying CELSR function, laying the foundation for the design of new CELSR-targeted therapeutic reagents.
PMCID: PMC4261025  PMID: 25280249
G Protein-Coupled Receptor (GPCR); Adhesion; CELSR; Development; Planar Cell Polarity (PCP)
15.  The Sheep Genome Illuminates Biology of the Rumen and Lipid Metabolism 
Science (New York, N.Y.)  2014;344(6188):1168-1173.
Sheep (Ovis aries) are a major source of meat, milk and fiber in the form of wool, and represent a distinct class of animals that have a specialized digestive organ, the rumen, which carries out the initial digestion of plant material. We have developed and analyzed a high quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants, compared to non-ruminant animals.
PMCID: PMC4157056  PMID: 24904168
16.  Circulating miRNAs in cancer: from detection to therapy 
Since the discovery of circulating microRNAs (miRNAs) in body fluids, an increasing number of studies have focused on their potential as non-invasive biomarkers and as therapeutic targets or tools for many diseases, particularly for cancers. Because of their stability, miRNAs are easily detectable in body fluids. Extracellular miRNAs have potential as biomarkers for the prediction and prognosis of cancer. Moreover, they also enable communication between cells within the tumor microenvironment, thereby influencing tumorigenesis. In this review, we summarize the progresses made over the past decade regarding circulating miRNAs, from the development of detection methods to their clinical application as biomarkers and therapeutic tools for cancer. We also discuss the advantages and limitations of different detection methods and the pathways of circulating miRNAs in cell-cell communication, in addition to their clinical pharmacokinetics and toxicity in human organs. Finally, we highlight the potential of circulating miRNAs in clinical applications for cancer.
PMCID: PMC4269921  PMID: 25476853
17.  PET Attenuation Correction using Synthetic CT from Ultrashort Echo-time MRI 
Integrated PET (Positron Emission Tomography)/MR(magnetic resonance) systems are becoming increasingly popular in clinical and research applications. Quantitative PET reconstruction requires correction for γ photon attenuations using an attenuation coefficient map (μ-map) that is a measure of the electron density. One challenge of PET/MR, in contrast to PET/CT, lies in the accurate computation of μ-maps. Unlike CT, MRI measures physical properties not directly related to electron density. Previous approaches have computed the attenuation coefficients using a segmentation of MR images or using deformable registration of atlas CT images to the space of the subject MRI.
In this work, we propose a patch-based method to generate whole head μ-maps from Ultra-short Echo Time (UTE) MR imaging sequences. UTE images are preferred to other MR sequences, because of the increased signal from bone). To generate a synthetic CT image, we use patches from a reference dataset, which consists of dual echo UTE images and a co-registered CT from the same subject. By matching patches between the reference and target images, corresponding patches from the reference CT are combined via a Bayesian framework. No registration or segmentation is required.
For evaluation, UTE, CT and PET data, acquired from 5 patients under an IRB approved protocol, were used. Another patient (with UTE and CT only) was selected to be the reference to generate synthetic CT images for these five patients. PET reconstructions were attenuation corrected using (1) the original CT, (2) our synthetic CT, and Siemens (3) Dixon- and (4) UTE-based μ-maps, and (5) a deformable registration based CT. Our synthetic CT based PET reconstruction shows higher correlation (average ρ = 0.99, R2 = 0.99) to the original CT based PET, as compared to the segmentation and registration based methods. Synthetic CT based reconstruction had minimal bias (regression slope 0.99) as compared to the segmentation based methods (regression slope 0.97). A peak signal-to-noise ratio of 36.0 dB in the reconstructed PET activity is observed, compared with 29.7, 29.3, 27.4 dB for Siemens Dixon, UTE, and registration based μ-maps.
A patch-matching approach to synthesize CT images from dual echo UTE images leads to significantly more accurate PET reconstruction as compared to actual CT scans. The PET reconstruction is improved over segmentation (Dixon and Siemens UTE) and registration based methods, even in subjects with pathology.
PMCID: PMC4254365  PMID: 25413135
attenuation correction; PET/CT; PET/MRI; CT; UTE
18.  Pleiotropy of the de novo-originated gene MDF1 
Scientific Reports  2014;4:7280.
MDF1 is a young de novo-originated gene from a non-coding sequence in baker's yeast, S. cerevisiae, which can suppress mating and promote vegetative growth. Our previous experiments successfully demonstrated how Mdf1p binds to the key mating pathway determinant MATα2 to suppress mating. However, how Mdf1p promotes growth and fulfills the crosstalk between the yeast mating and growth pathways are still open questions. Thus, the adaptive significance of this new de novo gene remains speculative. Here, we show that Mdf1p shortens the lag phase of S. cerevisiae by physically interacting with SNF1, the governing factor for nonfermentable carbon source utilization, and thereby confers a selective advantage on yeasts through the rapid consumption of glucose in the early generational stage in rich medium. Therefore, MDF1 functions in two important molecular pathways, mating and fermentation, and mediates the crosstalk between reproduction and vegetative growth. Together, our results provide a comprehensive example of how a de novo-originated gene organizes new regulatory circuits and thereby confers a selective advantage on S. cerevisiae to allow exquisite adaptation to the changing environment.
PMCID: PMC4250933  PMID: 25452167
19.  Intravoxel incoherent motion diffusion-weighted MR imaging of gliomas: efficacy in preoperative grading 
Scientific Reports  2014;4:7208.
The preoperative grading of gliomas, which is critical for guiding therapeutic strategies, remains unsatisfactory. We aimed to retrospectively assess the efficacy of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) in the grading of gliomas. Forty-two newly diagnosed glioma patients underwent conventional MR imaging, DWI, and contrast-enhanced MR imaging. Parameters of apparent diffusion coefficient (ADC), slow diffusion coefficient (D), fast diffusion coefficient (D*), and fraction of fast ADC (f) were generated. They were tested for differences between low- and high-grade gliomas based on one-way ANOVA. Receiver-operating characteristic (ROC) analyses were conducted to determine the optimal thresholds as well as the sensitivity and specificity for grading. ADC, D, and f were higher in the low-grade gliomas, whereas D* tended to be lower (all P<0.05). The AUC, sensitivity, specificity and the cutoff value, respectively, for differentiating low- from high-grade gliomas for ADC, D and f, and differentiating high- from low-grade gliomas for D* were as follows: ADC, 0.926, 100%, 82.8%, and 0.7 × 10−3 mm2/sec; D, 0.942, 92.3%, 86.2%, and 0.623 × 10−3 mm2/sec; f, 0.902, 92.3%, 86.2%, and 35.3%; D*, 0.798, 79.3%, 84.6%, and 0.303 × 10−3 mm2/sec. The IVIM DWI demonstrates efficacy in differentiating the low- from high-grade gliomas.
PMCID: PMC4248278  PMID: 25434593
20.  FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis 
Cell Research  2014;24(12):1403-1419.
The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.
PMCID: PMC4260349  PMID: 25412662
N6-methyladenosine (m6A); METTL3; FTO; mRNA splicing; adipogenesis
21.  Evaluation of Intraspecies Interactions in Biofilm Formation by Methylobacterium Species Isolated from Pink-Pigmented Household Biofilms 
Microbes and Environments  2014;29(4):388-392.
Concern regarding household biofilms has grown due to their widespread existence and potential to threaten human health by serving as pathogen reservoirs. Previous studies identified Methylobacterium as one of the dominant genera found in household biofilms. In the present study, we examined the mechanisms underlying biofilm formation by using the bacterial consortium found in household pink slime. A clone library analysis revealed that Methylobacterium was the predominant genus in household pink slime. In addition, 16 out of 21 pink-pigmented bacterial isolates were assigned to the genus Methylobacterium. Although all of the Methylobacterium isolates formed low-level biofilms, the amount of the biofilms formed by Methylobacterium sp. P-1M and P-18S was significantly increased by co-culturing with other Methylobacterium strains that belonged to a specific phylogenetic group. The single-species biofilm was easily washed from the glass surface, whereas the dual-species biofilm strongly adhered after washing. A confocal laser scanning microscopy analysis showed that the dual-species biofilms were significantly thicker and tighter than the single-species biofilms.
PMCID: PMC4262362
Methylobacterium; household biofilm; pink biofilm; intraspecies interaction
22.  Correlations of Ezrin Expression with Pathological Characteristics and Prognosis of Osteosarcoma: A Meta-Analysis 
The Scientific World Journal  2014;2014:837543.
We conducted a meta-analysis to comprehensively evaluate the correlations of ezrin expression with pathological characteristics and the prognosis of osteosarcoma. The MEDLINE (1966–2013), the Cochrane Library Database, EMBASE, CINAHL, Web of Science (1945–2013), and the Chinese Biomedical Database were searched without language restrictions. Meta-analyses conducted using STATA software were calculated. Ten studies met the inclusion criteria, including 459 patients with osteosarcoma. Meta-analysis results illustrated that ezrin expression may be closely associated with the recurrence of osteosarcoma or metastasis in osteosarcoma. Our findings also demonstrated that patients with grade III-IV osteosarcoma showed a higher frequency of ezrin expression than those with histological grade I-II osteosarcoma. Furthermore, we found that patients with positive expression of ezrin exhibited a shorter overall survival than those with negative ezrin expression. The results also indicated that positive ezrin expression was strongly correlated with poorer metastasis-free survival. Nevertheless, no significant relationships were observed between ezrin expression and clinical variables (age and gender). In the current meta-analysis, our results illustrated significant relationships of ezrin expression with pathological characteristics and prognosis of osteosarcoma. Thus, ezrin expression could be a promising marker in predicting the clinical outcome of patients with osteosarcoma.
PMCID: PMC4270117  PMID: 25544963
23.  Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen 
PLoS ONE  2014;9(11):e112602.
The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.
PMCID: PMC4236105  PMID: 25405618
24.  Usefulness of DWI in preoperative assessment of deep myometrial invasion in patients with endometrial carcinoma: a systematic review and meta-analysis 
Cancer Imaging  2014;14(1):32.
The objective of this study was to perform a systematic review and a meta-analysis in order to estimate the diagnostic accuracy of diffusion weighted imaging (DWI) in the preoperative assessment of deep myometrial invasion in patients with endometrial carcinoma.
Studies evaluating DWI for the detection of deep myometrial invasion in patients with endometrial carcinoma were systematically searched for in the MEDLINE, EMBASE, and Cochrane Library from January 1995 to January 2014. Methodologic quality was assessed by using the Quality Assessment of Diagnostic Accuracy Studies tool. Bivariate random-effects meta-analytic methods were used to obtain pooled estimates of sensitivity, specificity, diagnostic odds ratio (DOR) and receiver operating characteristic (ROC) curves. The study also evaluated the clinical utility of DWI in preoperative assessment of deep myometrial invasion.
Seven studies enrolling a total of 320 individuals met the study inclusion criteria. The summary area under the ROC curve was 0.91. There was no evidence of publication bias (P = 0.90, bias coefficient analysis). Sensitivity and specificity of DWI for detection of deep myometrial invasion across all studies were 0.90 and 0.89, respectively. Positive and negative likelihood ratios with DWI were 8 and 0.11 respectively. In patients with high pre-test probabilities, DWI enabled confirmation of deep myometrial invasion; in patients with low pre-test probabilities, DWI enabled exclusion of deep myometrial invasion. The worst case scenario (pre-test probability, 50%) post-test probabilities were 89% and 10% for positive and negative DWI results, respectively.
DWI has high sensitivity and specificity for detecting deep myometrial invasion and more importantly can reliably rule out deep myometrial invasion. Therefore, it would be worthwhile to add a DWI sequence to the standard MRI protocols in preoperative evaluation of endometrial cancer in order to detect deep myometrial invasion, which along with other poor prognostic factors like age, tumor grade, and LVSI would be useful in stratifying high risk groups thereby helping in the tailoring of surgical approach in patient with low risk of endometrial carcinoma.
PMCID: PMC4331837  PMID: 25608571
Diffusion-weighted imaging; Magnetic resonance imaging; Endometrial carcinoma; Myometrial invasion
25.  Predicting Subtypes of Thymic Epithelial Tumors Using CT: New Perspective based on a Comprehensive Analysis of 216 Patients 
Scientific Reports  2014;4:6984.
It is highly necessary to identify low versus high risk thymic epithelial tumors (TETs) before operation to guide optimal treatment strategies. Current CT diagnostic parameters could not effectively achieve this goal. We evaluated three parameters of CT scan in a cohort of 216 TETs patients. Parameters of contrast enhancement, risk of aggressiveness, and nodule with fibrous septum were evaluated in low (A, AB) versus high risk (B1, B2, B3 and thymic carcinoma) TETs. Grade of contrast enhancement showed predictive value in classifying low and high risk TETs well. A maximal contrast-enhanced range of 25.5 HU could produce 78.8% sensitivity and 68.5% specificity in determining low risk subtypes. Additionally, risk of aggressiveness parameter was demonstrated to be associated with TETs subtype (r = 0.801, P < 0.001) and may add confidence in determining low versus high risk subtypes. Furthermore, multiple nodule with fibrous septum could suggest subtype AB. Findings from this study support role of studied parameters of CT manifestations in predicting the low and high risk stages of TETs. These findings provide empirical evidence for incorporating these parameters in clinical practice for identifying TETs stage before operation, if validated in additional studies.
PMCID: PMC4225535  PMID: 25382196

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