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1.  Prediction of outcome following paraquat poisoning by arterial lactate concentration-time data 
The present study retrospectively analyzed 170 patients diagnosed with paraquat (PQ) poisoning with the aim of clarifying whether the arterial lactate-time (arterial lactate concentration × time between ingestion and arterial lactate measurement) was a good predictor of mortality in patients with acute PQ poisoning. The results indicated that there was a positive correlation between the arterial lactate-time and PQ concentration-time (ρ=0.485). In addition, the arterial lactate-time data exhibited a similar discriminative power to the plasma PQ concentration-time data (z=0.712; P=0.864). For the receiver operating characteristic curve analysis, the lactate-time data had an area of 0.782 with a cut-off value of 11.95 mmol/l.h (sensitivity, 64.52%; specificity, 84.42%). To calculate the predicted probability of survival for any specified time and initial arterial lactate concentration, the following formula was derived based on the logistic regression coefficients: Logit(p) = 3.066 − 0.139 × (time lag following PQ ingestion) − 0.177 × (initial arterial lactate concentration); where the probability of survivors = 1/1 + e−logit(p). Therefore, the arterial lactate-time data exhibited a good predictive power for evaluating the prognosis of patients with acute PQ poisoning.
PMCID: PMC4079403
arterial lactate; paraquat; poisoning; prognosis
2.  An on-chip, multichannel droplet sorter using standing surface acoustic waves (SSAW) 
Analytical chemistry  2013;85(11):5468-5474.
The emerging field of droplet microfluidics requires effective on-chip handling and sorting of droplets. In this work, we demonstrate a microfluidic device that is capable of sorting picoliter water-in-oil droplets into multiple outputs using standing surface acoustic waves (SSAW). This device integrates a single-layer microfluidic channel with interdigital transducers (IDTs) to achieve on-chip droplet generation and sorting. Within the SSAW field, water-in-oil droplets experience an acoustic radiation force and are pushed towards the acoustic pressure node. As a result, by tuning the frequency of the SSAW excitation, the position of the pressure nodes can be changed and droplets can be sorted to different outlets at rates up to 222 droplets s−1. With its advantages in simplicity, controllability, versatility, non-invasiveness, and capability to be integrated with other on-chip components such as droplet manipulation and optical detection units, the technique presented here could be valuable for the development of droplet-based micro total analysis systems (μTAS).
PMCID: PMC3988909  PMID: 23647057
Microfluidics; standing surface acoustic waves (SSAW); droplet sorting
3.  The effect of prone positioning on mortality in patients with acute respiratory distress syndrome: a meta-analysis of randomized controlled trials 
Critical Care  2014;18(3):R109.
Prone positioning (PP) has been reported to improve the survival of patients with severe acute respiratory distress syndrome (ARDS). However, it is uncertain whether the beneficial effects of PP are associated with positive end-expiratory pressure (PEEP) levels and long durations of PP. In this meta-analysis, we aimed to evaluate whether the effects of PP on mortality could be affected by PEEP level and PP duration and to identify which patients might benefit the most from PP.
Publications describing randomized controlled trials (RCTs) in which investigators have compared prone and supine ventilation were retrieved by searching the following electronic databases: PubMed/MEDLINE, the Cochrane Library, the Web of Science and Elsevier Science (inception to May 2013). Two investigators independently selected RCTs and assessed their quality. The data extracted from the RCTs were combined in a cumulative meta-analysis and analyzed using methods recommended by the Cochrane Collaboration.
A total of nine RCTs with an aggregate of 2,242 patients were included. All of the studies received scores of up to three points using the methods recommended by Jadad et al. One trial did not conceal allocation. This meta-analysis revealed that, compared with supine positioning, PP decreased the 28- to 30-day mortality of ARDS patients with a ratio of partial pressure of arterial oxygen/fraction of inspired oxygen ≤100 mmHg (n = 508, risk ratio (RR) = 0.71, 95 confidence interval (CI) = 0.57 to 0.89; P = 0.003). PP was shown to reduce both 60-day mortality (n = 518, RR = 0.82, 95% CI = 0.68 to 0.99; P = 0.04) and 90-day mortality (n = 516, RR = 0.57, 95% CI = 0.43 to 0.75; P < 0.0001) in ARDS patients ventilated with PEEP ≥10 cmH2O. Moreover, PP reduced 28- to 30-day mortality when the PP duration was >12 h/day (n = 1,067, RR = 0.73, 95% CI = 0.54 to 0.99; P = 0.04).
PP reduced mortality among patients with severe ARDS and patients receiving relatively high PEEP levels. Moreover, long-term PP improved the survival of ARDS patients.
PMCID: PMC4075407  PMID: 24887034
4.  Construction and verification of the targeted uPA-shRNA lentiviral vector and evaluation of the transfection and silencing rate 
Urokinase-type plasminogen activator (uPA) receptors, which are released by the synovial tissue, are responsible for the activation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). RNA interference (RNAi) technology has emerged as a potent tool to generate cellular knockdown phenotypes of a desired gene. The aims of the present study were to investigate the effect of siRNA specific to the uPA gene on chondrocytes and to investigate the possible mechanisms of OA. Firstly, four types of small hairpin RNA (shRNA) sequence (P1, P2, P3 and P4) were obtained from the targeted uPA gene of the New Zealand rabbit, based on siRNA theory. The sequences were designed, constructed and subjected to restriction enzyme digestion, transformation, polymerase chain reaction (PCR) identification, positive clone sequencing and lentivirus packaging. Secondly, primary culturing cartilage cells from the New Zealand rabbit were transfected with P1, P2, P3 or P4 to observe the transfection rate under a fluorescence microscope. The mRNA expression levels of uPA were analyzed in cartilage cells using quantitative PCR, while protein expression levels were analyzed in the cartilage cells using western blot technology. Four types of uPA-shRNA lentiviral vectors were constructed successfully, which were all able to be transfected into the primary culturing cartilage cells. The transfection rate was as high as 85% when the multiplicity of infection was 100, which demonstrated that P1, P2, P3 and P4 were all capable of inhibiting the mRNA and protein expression of uPA in cartilage cells. In addition, among the four sequences, the P2 sequence exhibited the highest silencing rate of 70%. Statistical significance (P<0.05) was observed when analyzing the silencing rate of P2 compared to the other three groups. The most efficient targeted uPA-shRNA sequence was identified following screening. The results strongly verified that siRNA lentiviral vectors can be transfected into cartilage cells to further inhibit the expression of the uPA gene efficiently and steadily. Thus, the results provide the foundation for further research on the role of uPA in the pathogenesis of OA.
PMCID: PMC4079424
urokinase-type plasminogen activator; RNA interference; lentiviral vector; osteoarthritis; chondrocytes
5.  Enhancement of radiotherapy efficacy by miR-200c-loaded gelatinase-stimuli PEG-Pep-PCL nanoparticles in gastric cancer cells 
Radiotherapy is the main locoregional control modality for many types of unresectable tumors, including gastric cancer. However, many patients fail radiotherapy due to intrinsic radioresistance of cancer cells, which has been found to be strongly associated with cancer stem cell (CSC)-like properties. In this study, we developed a nanoparticle formulation to deliver miR-200c, which is reported to inhibit CSC-like properties, and then evaluated its potential activity as a radiosensitizer. miR-200c nanoparticles significantly augmented radiosensitivity in three gastric cancer cell lines (sensitization enhancement ratio 1.13–1.25), but only slightly in GES-1 cells (1.06). In addition to radioenhancement, miR-200c nanoparticles reduced the expression of CD44, a putative CSC marker, and the percentage of CD44+ BGC823 cells. Meanwhile, other CSC-like properties, including invasiveness and resistance to apoptosis, could be suppressed by miR-200c nanoparticles. CSC-associated radioresistance mechanisms, involving reactive oxygen species levels and DNA repair capacity, were also attenuated. We have demonstrated that miR-200c nanoparticles are an effective radiosensitizer in gastric cancer cells and induce little radiosensitization in normal cells, which suggests that they are as a promising candidate for further preclinical and clinical evaluation.
PMCID: PMC4026568  PMID: 24872697
radiosensitizer; miR-200c; gelatinase-stimuli nanoparticles; cancer stem cell-like properties; gastric cancer
6.  A droplet-based, optofluidic device for high-throughput, quantitative bioanalysis 
Analytical chemistry  2012;84(24):10745-10749.
Analysis of chemical or biomolecular contents in a tiny amount of specimen presents a significant challenge in many biochemical studies and diagnostic applications. In this work, we present a single-layer, optofluidic device for real-time, high-throughput, quantitative analysis of droplet contents. Our device integrates an optical fiber-based, on-chip detection unit with a droplet-based microfluidic unit. It can quantitatively analyze the contents of individual droplets in real-time. It also achieves a detection throughput of 2000 droplets per second, a detection limit of 20 nM, and an excellent reproducibility in its detection results. In a proof-of-concept study, we demonstrate that our device can be used to perform detection of DNA and its mutations by monitoring the fluorescent signal changes of the target DNA/molecular beacon complex in single droplets. Our approach can be immediately extended to a real-time, high-throughput detection of other biomolecules (such as proteins and viruses) in droplets. With its advantages in throughput, functionality, cost, size, and reliability, the droplet-based optofluidic device presented here can be a valuable tool for many medical diagnostic applications.
PMCID: PMC4017855  PMID: 23140515
Microfluidics; droplet; optofluidics; DNA analysis; high-throughput detection
7.  Optoacoustic tweezers: a programmable, localized cell concentrator based on opto-thermally generated, acoustically activated, surface bubbles 
Lab on a chip  2013;13(9):1772-1779.
We present a programmable, biocompatible technique for dynamically concentrating and patterning particles and cells in a microfluidic device. Since our technique utilizes opto-thermally generated, acoustically activated, surface bubbles, we name it “optoacoustic tweezers.” The optoacoustic tweezers are capable of concentrating particles/cells at any prescribed locations in a microfluidic chamber without the use of permanent structures, rendering it particularly useful for the formation of flexible, complex cell patterns. Additionally, this technique has demonstrated excellent biocompatibility and can be conveniently integrated with other microfluidic units. In our experiments, micro-bubbles were generated by focusing a 405 nm diode laser onto a gold-coated glass chamber. By properly tuning the laser, we demonstrate precise control over the position and size of the generated bubbles. Acoustic waves were then applied to activate the surface bubbles, causing them to oscillate at an optimized frequency. The resulting acoustic radiation force allowed us to locally trap particles/cells, including 15 μm polystyrene beads and HeLa cells, around each bubble. Cell-adhesion tests were also conducted after cell concentrating to confirm the biocompatibility of this technique.
PMCID: PMC3988908  PMID: 23511348
8.  Ezetimibe Inhibits Hepatic Niemann-Pick C1-Like 1 to Facilitate Macrophage Reverse Cholesterol Transport in Mice 
Controversies have arisen from recent mouse studies regarding the essential role of biliary sterol secretion in reverse cholesterol transport (RCT). The objective of this study was to examine the role of biliary cholesterol secretion in modulating macrophage RCT in Niemann-Pick C1-Like 1 (NPC1L1) liver only (L1LivOnly) mice, an animal model that is defective in both biliary sterol secretion and intestinal sterol absorption, and to determine if NPC1L1 inhibitor ezetimibe facilitates macrophage RCT by inhibiting hepatic NPC1L1.
Approach and Results
L1LivOnly mice were generated by crossing NPC1L1 knockout (L1-KO) mice with transgenic mice overexpressing human NPC1L1 specifically in liver. Macrophage-to-feces RCT was assayed in L1-KO and L1LivOnly mice injected intraperitoneally with [3H]-cholesterol-labeled peritoneal macrophages isolated from C57BL/6 mice. Inhibition of biliary sterol secretion by hepatic overexpression of NPC1L1 substantially reduced transport of [3H]-cholesterol from primary peritoneal macrophages to the neutral sterol fraction in bile and feces in L1LivOnly mice without affecting tracer excretion in the bile acid fraction. Ezetimibe treatment for 2 weeks completely restored both biliary and fecal excretion of [3H]-tracer in the neutral sterol fraction in L1LivOnly mice. HDL kinetic studies showed that L1LivOnly relative L1-KO mice had a significantly reduced fractional catabolic rate without altered hepatic and intestinal uptake of HDL-cholesterol ether.
In mice lacking intestinal cholesterol absorption, macrophage-to-feces RCT depends on efficient biliary sterol secretion and ezetimibe promotes macrophage RCT by inhibiting hepatic NPC1L1 function.
PMCID: PMC3965670  PMID: 23471229
Biliary cholesterol secretion; NPC1L1; ezetimibe; reverse cholesterol transport; Fecal neutral sterol excretion; Transgenic
9.  Tunable Nanowire Patterning Using Standing Surface Acoustic Waves 
ACS nano  2013;7(4):3306-3314.
Patterning of nanowires in a controllable, tunable manner is important for the fabrication of functional nanodevices. Here we present a simple approach for tunable nanowire patterning using standing surface acoustic waves (SSAW). This technique allows for the construction of large-scale nanowire arrays with well-controlled patterning geometry and spacing within 5 seconds. In this approach, SSAWs were generated by interdigital transducers (IDTs), which induced a periodic alternating current (AC) electric field on the piezoelectric substrate and consequently patterned metallic nanowires in suspension. The patterns could be deposited onto the substrate after the liquid evaporated. By controlling the distribution of the SSAW field, metallic nanowires were assembled into different patterns including parallel and perpendicular arrays. The spacing of the nanowire arrays could be tuned by controlling the frequency of the surface acoustic waves. Additionally, we observed 3D spark-shape nanowire patterns in the SSAW field. The SSAW-based nanowire-patterning technique presented here possesses several advantages over alternative patterning approaches, including high versatility, tunability, and efficiency, making it promising for device applications.
PMCID: PMC3989880  PMID: 23540330
Surface Patterning; Nanowire; Standing Surface Acoustic Waves (SSAW); Virtual Electrodes
10.  Optofluidic imaging: now and beyond 
Lab on a chip  2012;13(1):17-24.
More than a decade of research work in optofluidics has yielded a large catalogue of optofluidic elements that can manipulate light at the micro-scale (e.g., lenses, prisms). Although these elements have proven useful for many on-chip processes (e.g., miniaturized flow cytometry, interferometry, and sample spectroscopy), certain deficiencies have precluded their use in micro-scale imaging. However, recent work in optofluidic imaging has avoided optofluidic elements entirely and focused instead on image capture and composition techniques, demonstrating impressive resolution in both 2D imagery and 3D tomography. In this Focus article, we will discuss some of the recent successes in optofluidic imaging, and will expound our expectations for the near future of the optofluidic imaging discipline.
PMCID: PMC3994168  PMID: 23138193
11.  Epidermal growth factor receptor inhibitor ameliorates excessive astrogliosis and improves the regeneration microenvironment and functional recovery in adult rats following spinal cord injury 
Astrogliosis is a common phenomenon after spinal cord injury (SCI). Although this process exerts positive effects on axonal regeneration, excessive astrogliosis imparts negative effects on neuronal repair and recovery. Epidermal growth factor receptor (EGFR) pathway is critical to the regulation of reactive astrogliosis, and therefore is a potential target of therapeutics to better control the response. In this report, we aim to investigate whether blocking EGFR signaling using an EGFR tyrosine kinase specific inhibitor can attenuate reactive astrogliosis and promote functional recovery after a traumatic SCI.
The astrocyte scratch injury model in vitro and the weight-drop SCI model in vivo were used as model systems. PD168393 was used to inhibit EGFR signaling activation. Astrocytic activation and phosphorylated EGFR (pEGFR) were observed after immunofluorescence staining and Western blot analysis. The rate of proliferation was determined by immunofluorescence detection of BrdU-incorporating cells located next to the wound. The levels of TNF-α, iNOS, COX-2 and IL-1β in the culture medium under different conditions were assayed by ELISA. Western blot was performed to semi-quantify the expression of EGFR/pEGFR, glial fibrillary acid protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs). Myelin was stained by Luxol Fast Blue Staining. Cresyl violet eosin staining was performed to analyze the lesion cavity volume and neuronal survival following injury. Finally, functional scoring and residual urine recording were performed to show the rats’ recovery.
EGFR phosphorylation was found to parallel astrocyte activation, and EGFR inhibitor PD168393 potently inhibited scratch-induced reactive astrogliosis and proinflammatory cytokine/mediator secretion of reactive astrocytes in vitro. Moreover, local administration of PD168393 in the injured area suppressed CSPGs production and glial scar formation, and resulted in reduced demyelination and neuronal loss, which correlated with remarkable hindlimb motor function and bladder improvement in SCI rats.
The specific EGFR inhibitor PD168393 can ameliorate excessive reactive astrogliosis and facilitate a more favorable environment for axonal regeneration after SCI. As such, EGFR inhibitor may be a promising therapeutic intervention in CNS injury.
PMCID: PMC4030311  PMID: 24708754
Epidermal growth factor receptor; Astrogliosis; Spinal cord injury; Regeneration microenvironment
12.  MicroRNA-424 Is Down-Regulated in Hepatocellular Carcinoma and Suppresses Cell Migration and Invasion through c-Myb 
PLoS ONE  2014;9(3):e91661.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs) are important regulators of multiple cellular processes, and the aberrant miRNAs expressions have been observed in different types of cancer including HCC. Their pathysiologic role and their relevance to tumorigenesis are still largely unknown. In this study, we demonstrated the down-regulation of miR-424 in HCC cell lines and tissues by quantitative RT-PCR analyses. Overexpression of miR-424 reduced the HCC cell prolifetation, migration, and invasion. Conversely, inhibiton of miR-424 expression significantly accelerated the cell proliferation, migration, and invasion. In addition, we further identified c-Myb as a functional downstream target of miR-424 by directly targeting the 3′UTR of c-Myb. Furthermore, overexpression of c-Myb impaired miR-424-induced inhibition of proliferation and invasion in HCC cells. Our results demonstrated that miR-424 was involved in tumorigenesis of HCC at least in part by suppression of c-Myb.
PMCID: PMC3968007  PMID: 24675898
13.  Chemical Constituents from the Fruits of Forsythia suspensa and Their Antimicrobial Activity 
BioMed Research International  2014;2014:304830.
Lignans and phenylethanoid glycosides purified from Forsythia suspensa were reported to display various bioactivities in the previous literature, including the antimicrobial activity. Therefore, the present research is aimed to purify and identify the chemical constituents of the methanol extracts of fruits of F. suspensa. The methanol extracts of fruits of F. suspensa were fractionated and further purified with the assistance of column chromatography to afford totally thirty-four compounds. Among these isolates, 3β-acetoxy-20α-hydroxyursan-28-oic acid (1) was reported from the natural sources for the first time. Some of the purified principles were subjected to the antimicrobial activity examinations against Escherichia coli to explore new natural lead compounds.
PMCID: PMC3972829  PMID: 24745011
14.  Intestinal Cgi-58 Deficiency Reduces Postprandial Lipid Absorption 
PLoS ONE  2014;9(3):e91652.
Comparative Gene Identification-58 (CGI-58), a lipid droplet (LD)-associated protein, promotes intracellular triglyceride (TG) hydrolysis in vitro. Mutations in human CGI-58 cause TG accumulation in numerous tissues including intestine. Enterocytes are thought not to store TG-rich LDs, but a fatty meal does induce temporary cytosolic accumulation of LDs. Accumulated LDs are eventually cleared out, implying existence of TG hydrolytic machinery in enterocytes. However, identities of proteins responsible for LD-TG hydrolysis remain unknown. Here we report that intestine-specific inactivation of CGI-58 in mice significantly reduces postprandial plasma TG concentrations and intestinal TG hydrolase activity, which is associated with a 4-fold increase in intestinal TG content and large cytosolic LD accumulation in absorptive enterocytes during the fasting state. Intestine-specific CGI-58 knockout mice also display mild yet significant decreases in intestinal fatty acid absorption and oxidation. Surprisingly, inactivation of CGI-58 in intestine significantly raises plasma and intestinal cholesterol, and reduces hepatic cholesterol, without altering intestinal cholesterol absorption and fecal neutral sterol excretion. In conclusion, intestinal CGI-58 is required for efficient postprandial lipoprotein-TG secretion and for maintaining hepatic and plasma lipid homeostasis. Our animal model will serve as a valuable tool to further define how intestinal fat metabolism influences the pathogenesis of metabolic disorders, such as obesity and type 2 diabetes.
PMCID: PMC3950255  PMID: 24618586
15.  Identification of candidate miRNA biomarkers from miRNA regulatory network with application to prostate cancer 
MicroRNAs (miRNAs) are a class of non-coding regulatory RNAs approximately 22 nucleotides in length that play a role in a wide range of biological processes. Abnormal miRNA function has been implicated in various human cancers including prostate cancer (PCa). Altered miRNA expression may serve as a biomarker for cancer diagnosis and treatment. However, limited data are available on the role of cancer-specific miRNAs. Integrative computational bioinformatics approaches are effective for the detection of potential outlier miRNAs in cancer.
The human miRNA-mRNA target network was reconstructed by integrating multiple miRNA-mRNA interaction datasets. Paired miRNA and mRNA expression profiling data in PCa versus benign prostate tissue samples were used as another source of information. These datasets were analyzed with an integrated bioinformatics framework to identify potential PCa miRNA signatures. In vitro q-PCR experiments and further systematic analysis were used to validate these prediction results.
Using this bioinformatics framework, we identified 39 miRNAs as potential PCa miRNA signatures. Among these miRNAs, 20 had previously been identified as PCa aberrant miRNAs by low-throughput methods, and 16 were shown to be deregulated in other cancers. In vitro q-PCR experiments verified the accuracy of these predictions. miR-648 was identified as a novel candidate PCa miRNA biomarker. Further functional and pathway enrichment analysis confirmed the association of the identified miRNAs with PCa progression.
Our analysis revealed the scale-free features of the human miRNA-mRNA interaction network and showed the distinctive topological features of existing cancer miRNA biomarkers from previously published studies. A novel cancer miRNA biomarker prediction framework was designed based on these observations and applied to prostate cancer study. This method could be applied for miRNA biomarker prediction in other cancers.
PMCID: PMC4007708  PMID: 24618011
miRNA biomarker; Gene expression; miRNA regulatory network; Prostate cancer
16.  Trajectories of Kinematic Risky Driving Among Novice Teenagers 
Elevated gravitational force event rates are associated with the likelihood of a crash or near crash and provide an objective measure of risky driving. The purpose of this research is to examine the patterns over time of kinematic measures of risky driving among novice teenage drivers.
Driving data were collected from 42 newly-licensed teenage drivers during the first 18 months of licensure. Data recording systems installed in participants’ vehicles provided information on driving performance and driver characteristics. Latent class and logistic regression models were used to analyze trajectories of elevated gravitational-force (g-force) event rates, called kinematic risky driving, with respect to risk groups and associated factors.
Kinematic risky driving over the 18-month study period was best characterized as two classes, a higher-risk and a lower-risk class. The rate of kinematic risky driving during the first 6 months generally maintained over 18 months. Indeed, of those classified by latent class analysis as higher risk, 88.9%, 94.4% and 94.4% had average event rates above the median in the 1st, 2nd, and 3rd 6-month periods, respectively, indicating substantial tracking over time. Friends’ risky driving, friends’ risky behavior, self-reported risky driving, and perceptions about risky driving and driving privileges were associated with trip-level rates of kinematic risky driving. However, none of these factors was associated with trip-level rates after stratifying by overall risk in a latent class model, although friend’s risky driving was marginally significant.
Kinematic risky driving tended to track over time within the lower and higher risky driving groups. Self-reported risky driving and having risky friends were predictors of kinematic risky driving rates, but these variables did not explain the heterogeneity within higher and lower classes of risky drivers.
PMCID: PMC3556237  PMID: 23182780
adolescence; risk taking; motor vehicle crashes; naturalistic driving
17.  Establishment of an inducing medium for type III effector secretion in Xanthomonas campestris pv. campestris 
Brazilian Journal of Microbiology  2014;44(3):945-952.
It is well known that the type III secretion system (T3SS) and type III (T3) effectors are essential for the pathogenicity of most bacterial phytopathogens and that the expression of T3SS and T3 effectors is suppressed in rich media but induced in minimal media and plants. To facilitate in-depth studies on T3SS and T3 effectors, it is crucial to establish a medium for T3 effector expression and secretion. Xanthomonas campestris pv. campestris (Xcc) is a model bacterium for studying plant-pathogen interactions. To date no medium for Xcc T3 effector secretion has been defined. Here, we compared four minimal media (MME, MMX, XVM2, and XOM2) which are reported for T3 expression induction in Xanthomonas spp. and found that MME is most efficient for expression and secretion of Xcc T3 effectors. By optimization of carbon and nitrogen sources and pH value based on MME, we established XCM1 medium, which is about 3 times stronger than MME for Xcc T3 effectors secretion. We further optimized the concentration of phosphate, calcium, and magnesium in XCM1 and found that XCM1 with a lower concentration of magnesium (renamed as XCM2) is about 10 times as efficient as XCM1 (meanwhile, about 30 times stronger than MME). Thus, we established an inducing medium XCM2 which is preferred for T3 effector secretion in Xcc.
PMCID: PMC3910216  PMID: 24516463
inducing medium; type III effector secretion; Xanthomonas
18.  Structural basis of PP2A activation by PTPA, an ATP-dependent activation chaperone 
Cell Research  2013;24(2):190-203.
Proper activation of protein phosphatase 2A (PP2A) catalytic subunit is central for the complex PP2A regulation and is crucial for broad aspects of cellular function. The crystal structure of PP2A bound to PP2A phosphatase activator (PTPA) and ATPγS reveals that PTPA makes broad contacts with the structural elements surrounding the PP2A active site and the adenine moiety of ATP. PTPA-binding stabilizes the protein fold of apo-PP2A required for activation, and orients ATP phosphoryl groups to bind directly to the PP2A active site. This allows ATP to modulate the metal-binding preferences of the PP2A active site and utilize the PP2A active site for ATP hydrolysis. In vitro, ATP selectively and drastically enhances binding of endogenous catalytic metal ions, which requires ATP hydrolysis and is crucial for acquisition of pSer/Thr-specific phosphatase activity. Furthermore, both PP2A- and ATP-binding are required for PTPA function in cell proliferation and survival. Our results suggest novel mechanisms of PTPA in PP2A activation with structural economy and a unique ATP-binding pocket that could potentially serve as a specific therapeutic target.
PMCID: PMC3915903  PMID: 24100351
phosphatase; chaperone; activation; PP2A; cell proliferation and survival; catalytic metal ions
19.  Mechanisms of the Scaffold Subunit in Facilitating Protein Phosphatase 2A Methylation 
PLoS ONE  2014;9(1):e86955.
The function of the biologically essential protein phosphatase 2A (PP2A) relies on formation of diverse heterotrimeric holoenzymes, which involves stable association between PP2A scaffold (A) and catalytic (C or PP2Ac) subunits and binding of variable regulatory subunits. Holoenzyme assembly is highly regulated by carboxyl methylation of PP2Ac-tail; methylation of PP2Ac and association of the A and C subunits are coupled to activation of PP2Ac. Here we showed that PP2A-specific methyltransferase, LCMT-1, exhibits a higher activity toward the core enzyme (A–C heterodimer) than free PP2Ac, and the A-subunit facilitates PP2A methylation via three distinct mechanisms: 1) stabilization of a proper protein fold and an active conformation of PP2Ac; 2) limiting the space of PP2Ac-tail movement for enhanced entry into the LCMT-1 active site; and 3) weak electrostatic interactions between LCMT-1 and the N-terminal HEAT repeats of the A-subunit. Our results revealed a new function and novel mechanisms of the A-subunit in PP2A methylation, and coherent control of PP2A activity, methylation, and holoenzyme assembly.
PMCID: PMC3900686  PMID: 24466300
20.  Small-cell extraskeletal osteosarcoma: case report and literature review 
Small-cell extraskeletal osteosarcoma is extremely rare and consists of sheets of small round cells with variable amounts of osteoid. This tumor is often difficult to diagnose when tissue samples do not include recognizable osteoid. Only four cases have been reported in English and none in Chinese. We report a typical case of small-cell extraskeletal osteosarcoma occurring in the left leg of a 40-year-old female. Laboratory results were within normal limits. Magnetic resonance imaging demonstrated a soft tissue mass measuring 36 mm × 18 mm in the medial lateral aspect of left limb. The initial histological findings led to a misdiagnosis because the first fine-needle biopsy was randomized and incomplete. However, an open surgical specimen showed recognizable osteoid, which enabled us to make a definitive diagnosis. We also present clinical, radiologic and pathologic features of this case.
PMCID: PMC3925930  PMID: 24551306
Small-cell extraskeletal osteosarcoma; osteosarcoma; soft tissue
21.  Gated F-18 FDG PET for Assessment of Left Ventricular Volumes and Ejection Fraction Using QGS and 4D-MSPECT in Patients with Heart Failure: A Comparison with Cardiac MRI 
PLoS ONE  2014;9(1):e80227.
Ventricular function is a powerful predictor of survival in patients with heart failure (HF). However, studies characterizing gated F-18 FDG PET for the assessment of the cardiac function are rare. The aim of this study was to prospectively compare gated F-18 FDG PET and cardiac MRI for the assessment of ventricular volume and ejection fraction (EF) in patients with HF.
Eighty-nine patients with diagnosed HF who underwent both gated F-18 FDG PET/CT and cardiac MRI within 3 days were included in the analysis. Left ventricular (LV) end-diastolic volume (EDV), end-systolic volume (ESV), and EF were obtained from gated F-18 FDG PET/CT using the Quantitative Gated SPECT (QGS) and 4D-MSPECT software.
LV EDV and LV ESV measured by QGS were significantly lower than those measured by cardiac MRI (both P<0.0001). In contrast, the corresponding values for LV EDV for 4D-MSPECT were comparable, and LV ESV was underestimated with borderline significance compared with cardiac MRI (P = 0.047). LV EF measured by QGS and cardiac MRI showed no significant differences, whereas the corresponding values for 4D-MSPECT were lower than for cardiac MRI (P<0.0001). The correlations of LV EDV, LV ESV, and LV EF between gated F-18 FDG PET/CT and cardiac MRI were excellent for both QGS (r = 0.92, 0.92, and 0.76, respectively) and 4D-MSPECT (r = 0.93, 0.94, and 0.75, respectively). However, Bland-Altman analysis revealed a significant systemic error, where LV EDV (−27.9±37.0 mL) and ESV (−18.6±33.8 mL) were underestimated by QGS.
Despite the observation that gated F-18 FDG PET/CT were well correlated with cardiac MRI for assessing LV function, variation was observed between the two imaging modalities, and so these imaging techniques should not be used interchangeably.
PMCID: PMC3880258  PMID: 24404123
22.  LPS-induced inflammation in the chicken is associated with CCAAT/enhancer binding protein beta-mediated fat mass and obesity associated gene down-regulation in the liver but not hypothalamus 
The fat mass and obesity associated gene (FTO) is widely investigated in humans regarding its important roles in obesity and type 2 diabetes. Studies in mammals demonstrate that FTO is also associated with inflammation markers. However, the association of FTO with inflammation in chickens remains unclear. In this study, male chickens on day 28 posthatching were injected intraperitoneally with lipopolysaccharide (LPS) or saline to investigate whether the FTO gene is involved in LPS-induced inflammation.
We detected significant down-regulation of FTO mRNA in the liver (P < 0.01), but not in the hypothalamus, 2 and 24 h after LPS challenge. Toll-like receptor (TLR) 2 (P < 0.01) and TLR4 (P < 0.01) followed the same pattern as FTO, being suppressed significantly in liver but not in hypothalamus. IL-1β was dramatically up-regulated (P < 0.01) in both liver and hypothalamus 2 h after LPS challenge, while activation of IL-6 was observed in the liver (P < 0.01), but not in hypothalamus. The 5′-flanking sequence of the chicken FTO gene contains nine predicted binding sites for CCAAT/enhancer binding protein beta (C/EBP beta) and one for signal transducer and activator of transcription 3 (STAT3). Significant elevation of C/EBP beta was detected in the liver (P < 0.01), but not in the hypothalamus, 2 h after LPS challenge. Lipopolysaccharide challenge increased the C/EBP beta binding to FTO promoter in the liver (P < 0.01 for fragment 1, P < 0.05 for fragment 2), although the protein content of C/EBP beta was not altered. Moreover, injection of LPS resulted in enhanced phosphorylation of liver STAT3, a downstream transcription factor in IL-6 signaling. Although phosphorylated STAT3 was not detected to directly bind to FTO promoter, it was found to interact with C/EBP beta.
Our results reveal that FTO expression in liver, but not in hypothalamus, is affected by the i.p. injection of LPS, which may be mediated through tissue-specific FTO transcriptional regulation by C/EBP beta and STAT3 interaction.
PMCID: PMC3892065  PMID: 24345215
FTO; LPS; Inflammation; Chicken
23.  Dual-Targeting Pro-apoptotic Peptide for Programmed Cancer Cell Death via Specific Mitochondria Damage 
Scientific Reports  2013;3:3468.
Mitochondria are vital organelles to eukaryotic cells. Damage to mitochondria will cause irreversible cell death or apoptosis. In this report, we aim at programmed cancer cell death via specific mitochondrial damage. Herein, a functionalized pro-apoptotic peptide demonstrates a dual-targeting capability using folic acid (FA) (targeting agent I) and triphenylphosphonium (TPP) cation (targeting agent II). FA is a cancer-targeting agent, which can increase the cellular uptake of the pro-apoptotic peptide via receptor-mediated endocytosis. And the TPP cation is the mitochondrial targeting agent, which specifically delivers the pro-apoptotic peptide to its particular subcellular mitochondria after internalized by cancer cells. Then the pro-apoptotic peptide accumulates in mitochondria and causes its serious damage. This dual-targeting strategy has the potential to effectively transport the pro-apoptotic peptide to targeted cancer cell mitochondria, inducing mitochondrial dysfunction and triggering the mitochondria-dependent apoptosis to efficiently eliminate cancer cells.
PMCID: PMC3863817  PMID: 24336626
24.  Domain of Dentine Sialoprotein Mediates Proliferation and Differentiation of Human Periodontal Ligament Stem Cells 
PLoS ONE  2013;8(12):e81655.
Classic embryological studies have documented the inductive role of root dentin on adjacent periodontal ligament differentiation.  The biochemical composition of root dentin includes collagens and cleavage products of dentin sialophosphoprotein (DSPP), such as dentin sialoprotein (DSP).  The high abundance of DSP in root dentin prompted us to ask the question whether DSP or peptides derived thereof would serve as potent biological matrix components to induce periodontal progenitors to further differentiate into periodontal ligament cells. Here, we test the hypothesis that domain of DSP influences cell fate. In situ hybridization and immunohistochemical analyses showed that the COOH-terminal DSP domain is expressed in mouse periodontium at various stages of root development. The recombinant COOH-terminal DSP fragment (rC-DSP) enhanced attachment and migration of human periodontal ligament stem cells (PDLSC), human primary PDL cells without cell toxicity. rC-DSP induced PDLSC cell proliferation as well as differentiation and mineralization of PDLSC and PDL cells by formation of mineralized tissue and ALPase activity. Effect of rC-DSP on cell proliferation and differentiation was to promote gene expression of tooth/bone-relate markers, transcription factors and growth factors. The results for the first time showed that rC-DSP may be one of the components of cell niche for stimulating stem/progenitor cell proliferation and differentiation and a natural scaffold for periodontal regeneration application.
PMCID: PMC3882282  PMID: 24400037
25.  Evaluation of Paeonol Skin-Target Delivery from Its Microsponge Formulation: In Vitro Skin Permeation and In Vivo Microdialysis 
PLoS ONE  2013;8(11):e79881.
The aim of the present study was to design a novel topical skin-target drug-delivery system, the paeonol microsponge, and to investigate its drug-release patterns in dosage form, both in vitro and in vivo. Paeonol microsponges were prepared using the quasi-emulsion solvent-diffusion method. In vitro release studies were carried out using Franz diffusion cells, while in vivo studies were investigated by microdialysis after the paeonol microsponges were incorporated into a cream base. In vitro release studies showed that the drug delivered via microsponges increased the paeonol permeation rate. Ex vivo drug-deposition studies showed that the microsponge formulation improved drug residence in skin. In addition, in vivo microdialysis showed that the values for the area under the concentration versus time curve (AUC) for the paeonol microsponge cream was much higher than that of paeonol cream without microsponges. Maximum time (Tmax) was 220 min for paeonol microsponge cream and 480 min for paeonol cream, while the half-life (t1/2) of paeonol microsponge cream (935.1 min) was almost twice that of paeonol cream (548.6 min) in the skin (n = 3). Meanwhile, in the plasma, the AUC value for paeonol microsponge cream was half that of the paeonol cream. Based on these results, paeonol-loaded microsponge formulations could be a better alternative for treating skin disease, as the formulation increases drug bioavailability by lengthening the time of drug residence in the skin and should reduce side-effects because of the lower levels of paeonol moving into the circulation.
PMCID: PMC3835837  PMID: 24278204

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