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2.  Preferential secretion of collagen type 3 versus type 1 from adventitial fibroblasts stimulated by TGF-β/Smad3-treated medial smooth muscle cells☆ 
Cellular signalling  2012;25(4):955-960.
Restenosis, or arterial lumen re-narrowing, occurs in 30–50% of the patients undergoing angioplasty. Adaptive remodeling is the compensatory enlargement of the vessel size, and has been reported to prevent the deleterious effects of restenosis. Our previous studies have shown that elevated transforming growth factor (TGF-β) and its signaling protein Smad3 in the media layer induce adaptive remodeling of angioplastied rat carotid artery accompanying an increase of total collagen in the adventitia. In order to gain insights into a possible role of collagen in Smad3-induced adaptive remodeling, here we have investigated a mechanism of cell–cell communication between medial smooth muscle cells (SMCs) and adventitial fibroblasts in regulating the secretion of two major collagen subtypes. We have identified a preferential collagen-3 versus collagen-1 secretion by adventitial fibroblasts following stimulation by the conditioned medium from the TGF-β1-treated/Smad3-expressing medial smooth muscle cells (SMCs), which contained higher levels of CTGF and IGF2 as compared to control medium. Treating the TGF-β/ Smad3-stimulated SMCs with an siRNA to either CTGF or IGF2 reversed the effect of conditioned media on preferential collagen-3 secretion from fibroblasts. Moreover, recombinant CTGF and IGF2 together stimulated adventitial fibroblasts to preferentially secrete collagen-3 versus collagen-1. This is the first study to identify a preferential secretion of collagen-3 versus collagen-1 from adventitial fibroblasts as a result of TGF-β/Smad3 stimulation of medial SMCs, and that CTGF and IGF2 function together to mediate this signaling communication between the two cell types.
PMCID: PMC3595331  PMID: 23280188
Collagen type I and III; Adventitial fibroblasts; Medial smooth muscle cells; CTGF; IGF2
3.  Survey of Culture, GoldenGate Assay, Universal Biosensor Assay, and 16S rRNA Gene Sequencing as Alternative Methods of Bacterial Pathogen Detection 
Journal of Clinical Microbiology  2013;51(10):3263-3269.
Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.
PMCID: PMC3811648  PMID: 23884998
4.  RNA-Seq-Based Analysis of Cold Shock Response in Thermoanaerobacter tengcongensis, a Bacterium Harboring a Single Cold Shock Protein Encoding Gene 
PLoS ONE  2014;9(3):e93289.
Although cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC), can survive from 50° to 80°C, but has poor natural competence under cold shock at 50°C. We therefore examined cold shock responses and their effect on natural competence in this bacterium.
The transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature.
Our study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic and molecular mechanisms associated with cold shock and acclimation at low temperature.
PMCID: PMC3965559  PMID: 24667527
5.  DBatVir: the database of bat-associated viruses 
Emerging infectious diseases remain a significant threat to public health. Most emerging infectious disease agents in humans are of zoonotic origin. Bats are important reservoir hosts of many highly lethal zoonotic viruses and have been implicated in numerous emerging infectious disease events in recent years. It is essential to enhance our knowledge and understanding of the genetic diversity of the bat-associated viruses to prevent future outbreaks. To facilitate further research, we constructed the database of bat-associated viruses (DBatVir). Known viral sequences detected in bat samples were manually collected and curated, along with the related metadata, such as the sampling time, location, bat species and specimen type. Additional information concerning the bats, including common names, diet type, geographic distribution and phylogeny were integrated into the database to bridge the gap between virologists and zoologists. The database currently covers >4100 bat-associated animal viruses of 23 viral families detected from 196 bat species in 69 countries worldwide. It provides an overview and snapshot of the current research regarding bat-associated viruses, which is essential now that the field is rapidly expanding. With a user-friendly interface and integrated online bioinformatics tools, DBatVir provides a convenient and powerful platform for virologists and zoologists to analyze the virome diversity of bats, as well as for epidemiologists and public health researchers to monitor and track current and future bat-related infectious diseases.
Database URL:
PMCID: PMC3958617  PMID: 24647629
6.  Monocyte Chemoattractant Protein-1 (MCP-1) Regulates Macrophage Cytotoxicity in Abdominal Aortic Aneurysm 
PLoS ONE  2014;9(3):e92053.
In abdominal aortic aneurysm (AAA), macrophages are detected in the proximity of aortic smooth muscle cells (SMCs). We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. Here we tested whether infiltrating macrophages also directly contribute to SMC apoptosis.
Methods and Results
Using a SMC/RAW264.7 macrophage co-culture system, we demonstrated that MCP-1-primed RAWs caused a significantly higher level of apoptosis in SMCs as compared to control macrophages. Next, we detected an enhanced Fas ligand (FasL) mRNA level and membrane FasL protein expression in MCP-1-primed RAWs. Neutralizing FasL blocked SMC apoptosis in the co-culture. In situ proximity ligation assay showed that SMCs exposed to primed macrophages contained higher levels of receptor interacting protein-1 (RIP1)/Caspase 8 containing cell death complexes. Silencing RIP1 conferred apoptosis resistance to SMCs. In the mouse elastase injury model of aneurysm, aneurysm induction increased the level of RIP1/Caspase 8 containing complexes in medial SMCs. Moreover, TUNEL-positive SMCs in aneurysmal tissues were frequently surrounded by CD68+/FasL+ macrophages. Conversely, elastase-treated arteries from MCP-1 knockout mice display a reduction of both macrophage infiltration and FasL expression, which was accompanied by diminished apoptosis of SMCs.
Our data suggest that MCP-1-primed macrophages are more cytotoxic. MCP-1 appears to modulate macrophage cytotoxicity by increasing the level of membrane bound FasL. Thus, we showed that MCP-1-primed macrophages kill SMCs through a FasL/Fas-Caspase8-RIP1 mediated mechanism.
PMCID: PMC3954911  PMID: 24632850
7.  Periadventitial Application of Rapamycin-Loaded Nanoparticles Produces Sustained Inhibition of Vascular Restenosis 
PLoS ONE  2014;9(2):e89227.
Open vascular reconstructions frequently fail due to the development of recurrent disease or intimal hyperplasia (IH). This paper reports a novel drug delivery method using a rapamycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs)/pluronic gel system that can be applied periadventitially around the carotid artery immediately following the open surgery. In vitro studies revealed that rapamycin dispersed in pluronic gel was rapidly released over 3 days whereas release of rapamycin from rapamycin-loaded PLGA NPs embedded in pluronic gel was more gradual over 4 weeks. In cultured rat vascular smooth muscle cells (SMCs), rapamycin-loaded NPs produced durable (14 days versus 3 days for free rapamycin) inhibition of phosphorylation of S6 kinase (S6K1), a downstream target in the mTOR pathway. In a rat balloon injury model, periadventitial delivery of rapamycin-loaded NPs produced inhibition of phospho-S6K1 14 days after balloon injury. Immunostaining revealed that rapamycin-loaded NPs reduced SMC proliferation at both 14 and 28 days whereas rapamycin alone suppressed proliferation at day 14 only. Moreover, rapamycin-loaded NPs sustainably suppressed IH for at least 28 days following treatment, whereas rapamycin alone produced suppression on day 14 with rebound of IH by day 28. Since rapamycin, PLGA, and pluronic gel have all been approved by the FDA for other human therapies, this drug delivery method could potentially be translated into human use quickly to prevent failure of open vascular reconstructions.
PMCID: PMC3931710  PMID: 24586612
8.  Twinning-like lattice reorientation without a crystallographic twinning plane 
Nature Communications  2014;5:3297.
Twinning on the plane is a common mode of plastic deformation for hexagonal-close-packed metals. Here we report, by monitoring the deformation of submicron-sized single-crystal magnesium compressed normal to its prismatic plane with transmission electron microscopy, the reorientation of the parent lattice to a ‘twin’ lattice, producing an orientational relationship akin to that of the conventional twinning, but without a crystallographic mirror plane, and giving plastic strain that is not simple shear. Aberration-corrected transmission electron microscopy observations reveal that the boundary between the parent lattice and the ‘twin’ lattice is composed predominantly of semicoherent basal/prismatic interfaces instead of the twinning plane. The migration of this boundary is dominated by the movement of these interfaces undergoing basal/prismatic transformation via local rearrangements of atoms. This newly discovered deformation mode by boundary motion mimics conventional deformation twinning but is distinct from the latter and, as such, broadens the known mechanisms of plasticity.
Deformation twinning and dislocations are known to govern the plastic behaviour of metals at room temperature. Here the authors demonstrate a new deformation mechanism in single-crystal magnesium characterized by twin-like crystal reorientation and special interfaces.
PMCID: PMC3929781  PMID: 24522756
9.  Regulation of DNA methyltransferase 1 transcription in BRCA1-mutated breast cancer: a novel crosstalk between E2F1 motif hypermethylation and loss of histone H3 lysine 9 acetylation 
Molecular Cancer  2014;13:26.
DNA methyltransferase 1 (DNMT1) plays a critical role in breast cancer progression. However, the epigenetic mechanism regulating DNMT1 expression remains largely unknown.
Epigenetic regulation of DNMT1 was assessed in 85 invasive ductal carcinomas from BRCA1 mutation carriers. Association between clinicopathological features and DNMT1 promoter methylation was determined using Fisher’s exact test. Univariate analysis of survival was performed using the Kaplan-Meier method. Multivariate Cox regression analysis was performed to identify the independent prognostic factors for overall survival.
Hypermethylated E2F transcription factor 1 (E2F1) motif is a key regulatory element for the DNMT1 gene in BRCA1-mutated breast cancer. Mechanistically, the abnormal E2F1 motif methylation-mediated loss of active histone H3 lysine 9 acetylation (H3K9ac) and transcription factor E2F1 enrichment synergistically inhibited the transcription of DNMT1. Clinicopathological data indicated that the hypermethylated E2F1 motif was associated with histological grade, lymph node, Ki67 and E-cadherin status; univariate survival and multivariate analyses demonstrated that lymph node metastasis was an independent and reliable prognostic factor for BRCA1-mutated breast cancer patients.
Our findings imply that genetic (such as BRCA1 mutation) and epigenetic mechanisms (such as DNA methylation, histone modification, transcription factor binding) are jointly involved in the malignant progression of DNMT1-related breast cancer.
PMCID: PMC3936805  PMID: 24502362
DNMT1; Histone modifications; E2F1; BRCA1; Breast cancer
10.  Three-Dimensional Magnetic Resonance Cholangiopancreatography for the Diagnosis of Biliary Atresia in Infants and Neonates 
PLoS ONE  2014;9(2):e88268.
Background and Objective
Magnetic resonance cholangiopancreatography (MRCP) is widely accepted for visualization of the biliary system. However, the sensitivity and specificity of MRCP for the diagnosis of biliary atresia (BA) are still not fully elucidated. This study aimed to investigate the diagnostic value of three-dimensional MRCP (3D-MRCP) for BA in a large cohort of cholestatic infants and neonates.
One hundred ninety patients with infant jaundice underwent 3D-MRCP and one or more of the following: (1) intraoperative cholangiography, (2) laparoscopic exploration and pathological examination, or/and (3) clinical therapy. Statistical analyses were performed to determine the diagnostic accuracy of 3D-MRCP for BA.
Our study demonstrated that 158 of 190 patients were interpreted as having BA by 3D-MRCP; of those, 103 patients were confirmed as having BA, whereas 55 patients did not have BA. Of the 32 patients interpreted as non-BA cases by 3D-MRCP, one patient was misdiagnosed. The diagnostic accuracy for 3D-MRCP was 70.53% (134 of 190), the sensitivity was 99.04% (103 of 104), the specificity was 36.05% (31 of 86), the negative predictive value was 96.88% (31 of 32), the positive predictive value was 65.19% (103 of 158), the positive likelihood ratio was 2.7473, the negative likelihood ratio was 0.0267, and the Youden index was 0.3509.
The sensitivity of 3D-MRCP in diagnosing BA was excellent, but the specificity was not as high as described in previous reports. 3D-MRCP can be an effective screening method but should be combined with other modalities to identify BA and distinguish it from other causes of infant jaundice.
PMCID: PMC3914942  PMID: 24505457
11.  Abdominal Aortic Intimal Flap Motion Characterization in Acute Aortic Dissection: Assessed with Retrospective ECG-Gated Thoracoabdominal Aorta Dual-Source CT Angiography 
PLoS ONE  2014;9(2):e87664.
To evaluate the feasibility of dose-modulated retrospective ECG-gated thoracoabdominal aorta CT angiography (CTA) assessing abdominal aortic intimal flap motion and investigate the motion characteristics of intimal flap in acute aortic dissection (AAD).
Materials and Methods
49 patients who had thoracoabdominal aorta retrospective ECG-gated CTA scan were enrolled. 20 datasets were reconstructed in 5% steps between 0 and 95% of the R-R interval in each case. The aortic intimal flap motion was assessed by measuring the short axis diameters of the true lumen and false lumen 2 cm above of celiac trunk ostium in different R-R intervals. Intimal flap motion and configuration was assessed by two independent observers.
In these 49 patients, 37 had AAD, 7 had intramural hematoma, and 5 had negative result for acute aortic disorder. 620 datasets of 31 patients who showed double lumens in abdominal aorta were enrolled in evaluating intimal flap motion. The maximum and minimum true lumen diameter were 12.2±4.1 mm (range 2.6∼17.4) and 6.7±4.1 mm (range 0∼15.3) respectively. The range of intimal flap motion in all patients was 5.5±2.6 mm (range 1.8∼10.2). The extent of maximum true lumen diameter decreased during a cardiac cycle was 49.5%±23.5% (range 12%∼100%). The maximum motion phase of true lumen diameter was in systolic phase (5%∼40% of R-R interval). Maximum and minimum intimal flap motion was at 15% and 75% of the R-R interval respectively. Intimal flap configuration had correlation with the phase of cardiac cycle.
Abdominal intimal flap position and configuration varied greatly during a cardiac cycle. Retrospective ECG-gated thoracoabdominal aorta CTA can reflect the actual status of the true lumen and provide more information about true lumen collapse. This information may be helpful to diagnosis and differential diagnosis of dynamic abstraction.
PMCID: PMC3913653  PMID: 24503676
12.  The principal fucosylated oligosaccharides of human milk exhibit prebiotic properties on cultured infant microbiota 
Glycobiology  2012;23(2):169-177.
Breast-fed infant microbiota is typically rich in bifidobacteria. Herein, major human milk oligosaccharides (HMOS) are assessed for their ability to promote the growth of bifidobacteria and to acidify their environment, key features of prebiotics. During in vitro anaerobic fermentation of infant microbiota, supplementation by HMOS significantly decreased the pH even greater than supplementation by fructooligosaccharide (FOS), a prebiotic positive control. HMOS elevated lactate concentrations, increased the proportion of Bifidobacterium spp. in culture, and through their fermentation into organic acids, decreased the proportion of Escherichia and Clostridium perfringens. Three principal components of HMOS, 2′-fucosyllactose, lactodifucotetraose and 3-fucosyllactose, were consumed in these cultures. These three principal oligosaccharides of human milk were then individually tested as supplements for in vitro growth of four individual representative strains of infant gut microbes. Bifidobacterium longum JCM7007 and B. longum ATCC15697 efficiently consumed oligosaccharides and produced abundant lactate and short-chain fatty acids, resulting in significant pH reduction. The specificity of fermentation differed by microbe species and strain and by oligosaccharide structure. Escherichia coli K12 and C. perfringens did not utilize appreciable fucosylated oligosaccharides, and a typical mixture of organic acid fermentation products inhibited their growth. In summary, 2′-fucosyllactose, lactodifucotetraose, and 3-fucosyllactose, when cultured with B. longum JCM7007 and B. longum ATCC15697, exhibit key characteristics of a prebiotic in vitro. If these bifidobacteria are representative of pioneering or keystone species for human microbiota, fucosylated HMOS could strongly promote colonization and maintenance of a mutualist symbiotic microbiome. Thus, these simple glycans could mediate beneficial effects of human milk on infant health.
PMCID: PMC3531294  PMID: 23028202
bifidobacteria; fucosylated oligosaccharides;  human milk; prebiotic
13.  Comparison of Patients Hospitalized With Influenza A Subtypes H7N9, H5N1, and 2009 Pandemic H1N1 
Hospitalization with H7N9 virus infection is associated with older age and chronic heart disease, and patients have a longer duration of hospitalization than patients with H5N1 or pH1N1. This suggests that host factors are an important contributor to H7N9 severity.
Background. Influenza A(H7N9) viruses isolated from humans show features suggesting partial adaptation to mammals. To provide insights into the pathogenesis of H7N9 virus infection, we compared risk factors, clinical presentation, and progression of patients hospitalized with H7N9, H5N1, and 2009 pandemic H1N1 (pH1N1) virus infections.
Methods. We compared individual-level data from patients hospitalized with infection by H7N9 (n = 123), H5N1 (n = 119; 43 China, 76 Vietnam), and pH1N1 (n = 3486) viruses. We assessed risk factors for hospitalization after adjustment for age- and sex-specific prevalence of risk factors in the general Chinese population.
Results. The median age of patients with H7N9 virus infection was older than other patient groups (63 years; P < .001) and a higher proportion was male (71%; P < .02). After adjustment for age and sex, chronic heart disease was associated with an increased risk of hospitalization with H7N9 (relative risk, 9.68; 95% confidence interval, 5.24–17.9). H7N9 patients had similar patterns of leukopenia, thrombocytopenia, and elevated alanine aminotransferase, creatinine kinase, C-reactive protein, and lactate dehydrogenase to those seen in H5N1 patients, which were all significantly different from pH1N1 patients (P < .005). H7N9 patients had a longer duration of hospitalization than either H5N1 or pH1N1 patients (P < .001), and the median time from onset to death was 18 days for H7N9 (P = .002) vs 11 days for H5N1 and 15 days for pH1N1 (P = .154).
Conclusions. The identification of known risk factors for severe seasonal influenza and the more protracted clinical course compared with that of H5N1 suggests that host factors are an important contributor to H7N9 severity.
PMCID: PMC3967826  PMID: 24488975
influenza A(H7N9); influenza A(H5N1); clinical epidemiology
14.  Seeds used for Bodhi beads in China 
Bodhi beads are a Buddhist prayer item made from seeds. Bodhi beads have a large and emerging market in China, and demand for the beads has particularly increased in Buddhism regions, especially Tibet. Many people have started to focus on and collect Bodhi beads and to develop a Bodhi bead culture. But no research has examined the source plants of Bodhi beads. Therefore, ethnobotanical surveys were conducted in six provinces of China to investigate and document Bodhi bead plants. Reasons for the development of Bodhi bead culture were also discussed.
Six provinces of China were selected for market surveys. Information was collected using semi-structured interviews, key informant interviews, and participatory observation with traders, tourists, and local residents. Barkhor Street in Lhasa was focused on during market surveys because it is one of the most popular streets in China.
Forty-seven species (including 2 varieties) in 19 families and 39 genera represented 52 types of Bodhi beads that were collected. The most popular Bodhi bead plants have a long history and religious significance. Most Bodhi bead plants can be used as medicine or food, and their seeds or fruits are the main elements in these uses. ‘Bodhi seeds’ have been historically used in other countries for making ornaments, especially seeds of the legume family. Many factors helped form Bodhi bead culture in China, but its foundation was in Indian Buddhist culture.
As one of the earliest adornment materials, seeds played an important role for human production and life. Complex sources of Bodhi beads have different cultural and historical significance. People bought and collected Bodhi beads to reflect their love and admiration for the plants. Thus, the documentation of Bodhi bead plants can serve as a basis for future investigation of Bodhi bead culture and modern Buddhist culture.
PMCID: PMC3914368  PMID: 24479788
Bodhi beads; Seeds; Buddhist culture
15.  Ionic Size Effects: Generalized Boltzmann Distributions, Counterion Stratification, and Modified Debye Length 
Nonlinearity  2013;26(10):2899-2922.
Near a charged surface, counterions of different valences and sizes cluster; and their concentration profiles stratify. At a distance from such a surface larger than the Debye length, the electric field is screened by counterions. Recent studies by a variational mean-field approach that includes ionic size effects and by Monte Carlo simulations both suggest that the counterion stratification is determined by the ionic valence-to-volume ratios. Central in the mean-field approach is a free-energy functional of ionic concentrations in which the ionic size effects are included through the entropic effect of solvent molecules. The corresponding equilibrium conditions define the generalized Boltzmann distributions relating the ionic concentrations to the electrostatic potential. This paper presents a detailed analysis and numerical calculations of such a free-energy functional to understand the dependence of the ionic charge density on the electrostatic potential through the generalized Boltzmann distributions, the role of ionic valence-to-volume ratios in the counterion stratification, and the modification of Debye length due to the effect of ionic sizes.
PMCID: PMC3899944  PMID: 24465094
16.  Genome Sequence of Bacillus sp. Strain FJAT-14515 
Genome Announcements  2014;2(1):e01123-13.
We report the draft genome sequence of Bacillus sp. strain FJAT-14515. The genome is 5.44 Mb in length. It covers 5,263 genes with an average length of 791 bp, has a G+C value of 37.06%, and contains 67 tRNAs, 31 small RNAs, and 5 rRNA loci.
PMCID: PMC3900888  PMID: 24459256
17.  SiO2 nanoparticles change colour preference and cause Parkinson's-like behaviour in zebrafish 
Scientific Reports  2014;4:3810.
With advances in the development of various disciplines, there is a need to decipher bio-behavioural mechanisms via interdisciplinary means. Here, we present an interdisciplinary study of the role of silica nanoparticles (SiO2-NPs) in disturbing the neural behaviours of zebrafish and a possible physiological mechanism for this phenomenon. We used adult zebrafish as an animal model to evaluate the roles of size (15-nm and 50-nm) and concentration (300 μg/mL and 1000 μg/mL) in SiO2-NP neurotoxicity via behavioural and physiological analyses. With the aid of video tracking and data mining, we detected changes in behavioural phenotypes. We found that compared with 50-nm nanosilica, 15-nm SiO2-NPs produced greater significant changes in advanced cognitive neurobehavioural patterns (colour preference) and caused potentially Parkinson's disease-like behaviour. Analyses at the tissue, cell and molecular levels corroborated the behavioural results, demonstrating that nanosilica acted on the retina and dopaminergic (DA) neurons to change colour preference and to cause potentially Parkinson's disease-like behaviour.
PMCID: PMC3898208  PMID: 24448416
18.  A Robot-Assisted Surgical System Using a Force-Image Control Method for Pedicle Screw Insertion 
PLoS ONE  2014;9(1):e86346.
To introduce a robot-assisted surgical system for spinal posterior fixation that can automatically recognize the drilling state and stop potential cortical penetration with force and image information and to further evaluate the accuracy and safety of the robot for sheep vertebra pedicle screw placement.
The Robotic Spinal Surgery System (RSSS) was composed of an optical tracking system, a navigation and planning system, and a surgical robot equipped with a 6-DOF force/torque sensor. The robot used the image message and force signals to sense the different operation states and to prevent potential cortical penetration in the pedicle screw insertion operation. To evaluate the accuracy and safety of the RSSS, 32 screw insertions were conducted. Furthermore, six trajectories were deliberately planned incorrectly to explore whether the robot could recognize the different drilling states and immediately prevent cortical penetration.
All 32 pedicle screws were placed in the pedicle without any broken pedicle walls. Compared with the preoperative planning, the average deviations of the entry points in the axial and sagittal views were 0.50±0.33 and 0.65±0.40 mm, and the average deviations of the angles in the axial and sagittal views were 1.9±0.82° and 1.48±1.2°. The robot successfully recognized the different drilling states and prevented potential cortical penetration. In the deliberately incorrectly planned trajectory experiments, the robot successfully prevented the cortical penetration.
These results verified the RSSS’s accuracy and safety, which supported its potential use for the spinal surgery.
PMCID: PMC3899254  PMID: 24466043
19.  Eight New Monoterpene Acylglucosides from Sibiraea angustata 
Phytochemistry letters  2013;6(3):461-466.
Chemical investigation of the aqueous extract from the aerial part of Sibiraea angustata, has led to the isolation of eight new monoterpene acylglucosides named sibiraglycoside A-H(1-8), together with two known monoterpenes, Sibiraic acid (9) and Sibiskoside (10). Their structures were elucidated by means of extensive spectroscopic data analysis (including 1D and 2D NMR, HRESIMS experiments), as well as compared with the literature data. The relative configuration was established by NOE studies. In the in vitro bioassay, all the compounds showed moderate hypolipemic effects, among them, compounds 7 and 9, showed distinctive activity on lowering lipid.
PMCID: PMC3896567  PMID: 24454588
Sibiraea angustata; sibiraglycoside A-H; monoterpene acylglucoside; hypolipemic effect
20.  Kernel Smoothed Profile Likelihood Estimation in the Accelerated Failure Time Frailty Model for Clustered Survival Data 
Biometrika  2013;100(3):741-755.
Clustered survival data frequently arise in biomedical applications, where event times of interest are clustered into groups such as families. In this article we consider an accelerated failure time frailty model for clustered survival data and develop nonparametric maximum likelihood estimation for it via a kernel smoother aided EM algorithm. We show that the proposed estimator for the regression coefficients is consistent, asymptotically normal and semiparametric efficient when the kernel bandwidth is properly chosen. An EM-aided numerical differentiation method is derived for estimating its variance. Simulation studies evaluate the finite sample performance of the estimator, and it is applied to the Diabetic Retinopathy data set.
PMCID: PMC3893096  PMID: 24443587
Accelerated failure time model; Clustered survival data; EM algorithm; Kernel smoothing; Profile likelihood estimation
21.  Pioglitazone Upregulates Angiotensin Converting Enzyme 2 Expression in Insulin-Sensitive Tissues in Rats with High-Fat Diet-Induced Nonalcoholic Steatohepatitis 
The Scientific World Journal  2014;2014:603409.
Background and Aim. Thiazolidinediones (TZDs) can improve hepatic steatosis in nonalcoholic steatohepatitis (NASH). Angiotensin (Ang) II, the primary effector of renin-angiotensin system (RAS), plays vital roles in the development and progression of NASH. And some AngII-mediated effects can be regulated by TZDs. Angiotensin-converting enzyme (ACE) 2, a new component of RAS, can degrade Ang II to attenuate its subsequent physiological actions. We aimed to evaluate the effects of TZDs on ACE2 expression in insulin-sensitive tissues in NASH rats. Methods. Forty rats were divided into the normal control, high-fat diet (HFD), pioglitazone control, and HFD plus pioglitazone groups. After 24 weeks of treatment, we evaluated changes in liver histology and tissue-specific ACE2 expression. Results. ACE2 gene and protein expression was significantly greater in liver and adipose tissue in the HFD group compared with normal control group, while was significantly reduced in skeletal muscle. Pioglitazone significantly reduced the degree of hepatic steatosis compared with the HFD group. Pioglitazone significantly increased ACE2 protein expression in liver, adipose tissue, and skeletal muscle compared with the HFD group. Conclusions. Pioglitazone improves hepatic steatosis in the rats with HFD-induced NASH and upregulates ACE2 expression in insulin-sensitive tissues.
PMCID: PMC3914411  PMID: 24558317
22.  The Predictive but Not Prognostic Value of MGMT Promoter Methylation Status in Elderly Glioblastoma Patients: A Meta-Analysis 
PLoS ONE  2014;9(1):e85102.
The clinical implication of O6-methylguanine-DNA methyltransferase (MGMT) promoter status is ill-defined in elderly glioblastoma patients. Here we report a meta-analysis to seek valid evidence for its clinical relevance in this subpopulation.
Literature were searched and reviewed in a systematic manner using the PubMed, EMBASE and Cochrane databases. Studies investigating the association between MGMT promoter status and survival data of elderly patients (≥65 years) were eligible for inclusion.
Totally 16 studies were identified, with 13 studies included in the final analyses. The aggregate proportion of MGMT promoter methylation in elderly patients was 47% (95% confidence interval [CI]: 42–52%), which was similar to the value for younger patients. The analyses showed differential effects of MGMT status on overall survival (OS) of elderly patients according to assigned treatments: methylated vs. unmethylated: (1) temozolomide (TMZ)-containing therapies: hazard ratio [HR] 0.49, 95% CI 0.41–0.58; (2) TMZ-free therapies: HR 0.97, 95% CI 0.77–1.21. More importantly, a useful predictive value was observed by an interaction analysis: TMZ-containing therapies vs. RT alone: (1) methylated tumors: HR 0.48, 95% CI 0.36–0.65; (2) unmethylated tumors: HR 1.14; 95% CI 0.90–1.44.
The meta-analysis reports an age-independent presence of MGMT promoter methylation. More importantly, the study encouraged routine testing of MGMT promoter status especially in elderly glioblastoma patients by indicating a direct linkage between biomarker test and individual treatment decision. Future studies are needed to justify the mandatory testing in younger patients.
PMCID: PMC3890309  PMID: 24454798
23.  Drugging a Stem Cell Compartment Using Wnt3a Protein as a Therapeutic 
PLoS ONE  2014;9(1):e83650.
The therapeutic potential of Wnt proteins has long been recognized but challenges associated with in vivo stability and delivery have hindered their development as drug candidates. By exploiting the hydrophobic nature of the protein we provide evidence that exogenous Wnt3a can be delivered in vivo if it is associated with a lipid vesicle. Recombinant Wnt3a associates with the external surface of the lipid membrane; this association stabilizes the protein and leads to prolonged activation of the Wnt pathway in primary cells. We demonstrate the consequences of Wnt pathway activation in vivo using a bone marrow engraftment assay. These data provide validation for the development of WNT3A as a therapeutic protein.
PMCID: PMC3882211  PMID: 24400074
24.  Reduction of Intimal Hyperplasia in Injured Rat Arteries Promoted by Catheter Balloons Coated with Polyelectrolyte Multilayers that Contain Plasmid DNA Encoding PKCδ 
Biomaterials  2012;34(1):226-236.
New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films (‘polyelectrolyte multilayers’, PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue 3 days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.
PMCID: PMC3483441  PMID: 23069712
Layer-by-Layer; Polyelectrolyte Multilayers; Thin Films; Gene Delivery; Intimal Hyperplasia
25.  PI3Kγ signaling promotes Campylobacter jejuni induced colitis through neutrophil recruitment in mice 
Crypt abscesses caused by excessive neutrophil accumulation are prominent features of human campylobacteriosis and its associated pathology. The molecular and cellular events responsible for this pathological situation are currently unknown. We investigated the contribution of PI3Kγ signaling in Campylobacter jejuni-induced neutrophil accumulation and intestinal inflammation. Germ-free and specific pathogen free Il10−/−and germ-free Il10−/−; Rag2−/− mice were infected with C. jejuni (109 CFU/mouse). PI3Kγ signaling was manipulated using either the pharmacological PI3Kγ inhibitor AS252424 (i.p. 10 mg/kg daily) or genetically using Pi3γ−/− mice. After up to 14 days, inflammation was assessed histologically and by measuring levels of colonic Il1β, Cxcl2 and Il17a mRNA. Neutrophils were depleted using anti-Gr1 antibody (i.p. 0.5 mg/mouse/every 3 days). Using germ-free Il10−/−; Rag2−/− mice, we observed that innate immune cells are the main cellular compartment responsible for campylobacteriosis. Pharmacological blockade of PI3Kγ signaling diminished C. jejuni-induced intestinal inflammation, neutrophil accumulation and NF-κB activity, which correlated with reduced Il1β (77%), Cxcl2 (73%) and Il17a (72%) mRNA accumulation. Moreover, Pi3kγ−/− mice pretreated with anti-IL-10R were resistant to C. jejuni-induced intestinal inflammation compared to Wt mice. This improvement was accompanied by a reduction of C. jejuni translocation into the colon and extra-intestinal tissues and by attenuation of neutrophil migratory capacity. Furthermore, neutrophil depletion attenuated C. jejuni-induced crypt abscesses and intestinal inflammation. Our findings indicate that C. jejuni-induced PI3Kγ signaling mediates neutrophil recruitment and intestinal inflammation in Il10−/− mice. Selective pharmacological inhibition of PI3Kγ may represent a novel means to alleviate severe cases of campylobacteriosis, especially in antibiotic-resistant strains.
PMCID: PMC3529774  PMID: 23180818
Innate immunity; NF-κB; inflammation; enteric infection; crypt abscesses

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