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1.  Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release 
PLoS ONE  2014;9(4):e94574.
Protein tyrosine kinases, which are highly expressed in the central nervous system, are implicated in many neural processes. However, the relationship between protein tyrosine kinases and neurotransmitter release remains unknown. In this study, we found that ionomycin, a Ca2+ ionophore, concurrently induced asynchronous neurotransmitter release and phosphorylation of a non-receptor protein tyrosine kinase, proline-rich tyrosine kinase 2 (Pyk2), in clonal rat pheochromocytoma PC12 cells and cerebellar granule cells, whereas introduction of Pyk2 siRNA dramatically suppressed ionomycin-induced neurotransmitter release. Further study indicated that Tyr-402 (Y402) in Pyk2, instead of other tyrosine sites, underwent rapid phosphorylation after ionomycin induction in 1 min to 2 min. We demonstrated that the mutant of Pyk2 Y402 could abolish ionomycin-induced dopamine (DA) release by transfecting cells with recombinant Pyk2 and its mutants (Y402F, Y579F, Y580F, and Y881F). In addition, Src inhibition could prolong phosphorylation of Pyk2 Y402 and increase DA release. These findings suggested that Pyk2 was involved in ionomycin-induced neurotransmitter release through phosphorylation of Y402.
doi:10.1371/journal.pone.0094574
PMCID: PMC3981813  PMID: 24718602
2.  An Insulin-to-Insulin Regulatory Network Orchestrates Phenotypic Specificity in Development and Physiology 
PLoS Genetics  2014;10(3):e1004225.
Insulin-like peptides (ILPs) play highly conserved roles in development and physiology. Most animal genomes encode multiple ILPs. Here we identify mechanisms for how the forty Caenorhabditis elegans ILPs coordinate diverse processes, including development, reproduction, longevity and several specific stress responses. Our systematic studies identify an ILP-based combinatorial code for these phenotypes characterized by substantial functional specificity and diversity rather than global redundancy. Notably, we show that ILPs regulate each other transcriptionally, uncovering an ILP-to-ILP regulatory network that underlies the combinatorial phenotypic coding by the ILP family. Extensive analyses of genetic interactions among ILPs reveal how their signals are integrated. A combined analysis of these functional and regulatory ILP interactions identifies local genetic circuits that act in parallel and interact by crosstalk, feedback and compensation. This organization provides emergent mechanisms for phenotypic specificity and graded regulation for the combinatorial phenotypic coding we observe. Our findings also provide insights into how large hormonal networks regulate diverse traits.
Author Summary
Insulin signaling is widely implicated in regulating diverse physiological processes ranging from metabolism to longevity across many animal species. Many animals have multiple insulin-like peptides that can regulate the activity of this signaling pathway. For example, while humans have ten, including the well-studied insulin hormone, the nematode Caenorhabditis elegans has forty such peptides. The similarity among these insulin-like peptides led to the predominant notion that widespread redundancy occurs among these peptides. Contrary to this notion, we find that the forty insulin-like peptides in the nematode C. elegans have specific and distinct effects on eight different physiological outputs that range from development, stress responses, lifespan and reproduction. Interestingly, we also find that these peptides regulate each other at the transcriptional level to form a signaling network. In addition, we observe that this network is organized into parallel circuits, whose activities are affected by compensation, feedback and crosstalk. Finally, the organization of the network helps to explain how different combinations of peptides generate specific outputs and captures the complexity of how these peptides orchestrate an animal's physiology through distinct peptide-to-peptide signaling circuits.
doi:10.1371/journal.pgen.1004225
PMCID: PMC3967928  PMID: 24675767
3.  Application of three-dimensionally area-selective atomic layer deposition for selectively coating the vertical surfaces of standing nanopillars 
Scientific Reports  2014;4:4458.
We describe a strategy for selectively coating the vertical surfaces of standing nanopillars using area-selective atomic layer deposition (ALD). Hydrophobic self-assembled monolayers (SAMs) are utilised to selectively inhibit the coating of oxides on the modified horizontal regions to ensure that only the vertical surfaces of vertical standing nanorods are coated using ALD processes. This method makes it possible to fabricate vertical nanodevices using a simple process of depositing oxide layer on a vertical surface, and can also be applied to the area-selective surface passivation of other standing structures.
doi:10.1038/srep04458
PMCID: PMC3964512  PMID: 24662775
4.  The Epidemic Characteristics and Changing Trend of Hemorrhagic Fever with Renal Syndrome in Hubei Province, China 
PLoS ONE  2014;9(3):e92700.
Background
Hemorrhagic fever with renal syndrome (HFRS) is caused by different hantaviruses within the Bunyaviridae family. HFRS is a fulminant, infectious disease that occurs worldwide and is endemic in all 31 provinces of China. Since the first HFRS case in Hubei Province was reported in 1957, the disease has spread across the province and Hubei has become one of the seriously affected areas in China with the greatest number of reported HFRS cases in the 1980's. However, the epidemic characteristics of HFRS in Hubei are still not entirely clear and long-term, systematic investigations of this epidemic area have been very limited.
Methods
The spatiotemporal distribution of HFRS was investigated using data spanning the years 1980 to 2009. The annual HFRS incidence, fatality rate and seasonal incidence between 1980 and 2009 were calculated and plotted. GIS-based spatial analyses were conducted to detect the spatial distribution and seasonal pattern of HFRS. A spatial statistical analysis, using Kulldorff's spatial scan statistic, was performed to identify clustering of HFRS.
Results
A total of 104,467 HFRS cases were reported in Hubei Province between 1980 and 2009. Incidence of and mortality due to HFRS declined after the outbreak in 1980s and HFRS cases have been sporadic in recent years. The locations and scale of disease clusters have changed during the three decades. The seasonal epidemic pattern of HFRS was characterized by the shift from the unimodal type (autumn/winter peak) to the bimodal type.
Conclusions
Socioeconomic development has great influence on the transmission of hantaviruses to humans and new epidemic characteristics have emerged in Hubei Province. It is necessary to reinforce preventative measures against HFRS according to the newly-presented seasonal variation and to intensify these efforts especially in the urban areas of Hubei Province.
doi:10.1371/journal.pone.0092700
PMCID: PMC3962441  PMID: 24658382
5.  Selective Inhibition of PI3K/Akt/mTOR Signaling Pathway Regulates Autophagy of Macrophage and Vulnerability of Atherosclerotic Plaque 
PLoS ONE  2014;9(3):e90563.
Macrophage infiltration contributes to the instability of atherosclerotic plaques. In the present study, we investigated whether selective inhibition of PI3K/Akt/mTOR signaling pathway can enhance the stability of atherosclerotic plaques by activation of macrophage autophagy. In vitro study, selective inhibitors or siRNA of PI3K/Akt/mTOR pathways were used to treat the rabbit's peritoneal primary macrophage cells. Inflammation related cytokines secreted by macrophages were measured. Ultrastructure changes of macrophages were examined by transmission electron microscope. mRNA or protein expression levels of autophagy related gene Beclin 1, protein 1 light chain 3 II dots (LC3-II) or Atg5-Atg12 conjugation were assayed by quantitative RT-PCR or Western blot. In vivo study, vulnerable plaque models were established in 40 New Zealand White rabbits and then drugs or siRNA were given for 8 weeks to inhibit the PI3K/Akt/mTOR signaling pathway. Intravascular ultrasound (IVUS) was performed to observe the plaque imaging. The ultrastructure of the abdominal aortic atherosclerosis lesions were analyzed with histopathology. RT-PCR or Western blot methods were used to measure the expression levels of corresponding autophagy related molecules. We found that macrophage autophagy was induced in the presence of Akt inhibitor, mTOR inhibitor and mTOR-siRNA in vitro study, while PI3K inhibitor had the opposite role. In vivo study, we found that macrophage autophagy increased significantly and the rabbits had lower plaque rupture incidence, lower plaque burden and decreased vulnerability index in the inhibitors or siRNA treated groups. We made a conclusion that selective inhibition of the Akt/mTOR signal pathway can reduce macrophages and stabilize the vulnerable atherosclerotic plaques by promoting macrophage autophagy.
doi:10.1371/journal.pone.0090563
PMCID: PMC3944201  PMID: 24599185
6.  MicroRNA-7a/b Protects against Cardiac Myocyte Injury in Ischemia/Reperfusion by Targeting Poly(ADP-Ribose) Polymerase 
PLoS ONE  2014;9(3):e90096.
Objectives
MicroRNA-7 (miR-7) is highly connected to cancerous cell proliferation and metastasis. It is also involved in myocardial ischemia-reperfusion (I/R) injury and is upregulated in cardiomyocyte under simulated I/R (SI/R). We aimed to investigate the role of miR-7 during myocardial I/R injury in vitro and in vivo and a possible gene target.
Methods and Results
Real-time PCR revealed that miR-7a/b expression was upregulated in H9c2 cells after SI/R. Flow cytometry showed SI/R-induced cell apoptosis was decreased with miR-7a/b mimic transfection but increased with miR-7a/b inhibitor in H9c2 cells. In a rat cardiac I/R injury model, infarct size determination and TUNEL assay revealed that miR-7a/b mimic decreased but miR-7a/b inhibitor increased cardiac infarct size and cardiomyocyte apoptosis as compared with controls. We previously identified an important gene connected with cell apoptosis -- poly(ADP-ribose) polymerase (PARP) -- as a candidate target for miR-7a/b and verified the target by luciferase reporter activity assay and western blot analysis.
Conclusions
miR-7a/b is sensitive to I/R injury and protects myocardial cells against I/R-induced apoptosis by negatively regulating PARP expression in vivo and in vitro. miR-7a/b may provide a new therapeutic approach for treatment of myocardial I/R injury. Poly(ADP-ribose) polymerase.
doi:10.1371/journal.pone.0090096
PMCID: PMC3940823  PMID: 24594984
7.  Impact of Manure Fertilization on the Abundance of Antibiotic-Resistant Bacteria and Frequency of Detection of Antibiotic Resistance Genes in Soil and on Vegetables at Harvest 
Applied and Environmental Microbiology  2013;79(18):5701-5709.
Consumption of vegetables represents a route of direct human exposure to bacteria found in soil. The present study evaluated the complement of bacteria resistant to various antibiotics on vegetables often eaten raw (tomato, cucumber, pepper, carrot, radish, lettuce) and how this might vary with growth in soil fertilized inorganically or with dairy or swine manure. Vegetables were sown into field plots immediately following fertilization and harvested when of marketable quality. Vegetable and soil samples were evaluated for viable antibiotic-resistant bacteria by plate count on Chromocult medium supplemented with antibiotics at clinical breakpoint concentrations. DNA was extracted from soil and vegetables and evaluated by PCR for the presence of 46 gene targets associated with plasmid incompatibility groups, integrons, or antibiotic resistance genes. Soil receiving manure was enriched in antibiotic-resistant bacteria and various antibiotic resistance determinants. There was no coherent corresponding increase in the abundance of antibiotic-resistant bacteria enumerated from any vegetable grown in manure-fertilized soil. Numerous antibiotic resistance determinants were detected in DNA extracted from vegetables grown in unmanured soil. A smaller number of determinants were additionally detected on vegetables grown only in manured and not in unmanured soil. Overall, consumption of raw vegetables represents a route of human exposure to antibiotic-resistant bacteria and resistance determinants naturally present in soil. However, the detection of some determinants on vegetables grown only in freshly manured soil reinforces the advisability of pretreating manure through composting or other stabilization processes or mandating offset times between manuring and harvesting vegetables for human consumption.
doi:10.1128/AEM.01682-13
PMCID: PMC3754188  PMID: 23851089
8.  Sphingosine Kinase 1 and Cancer: A Systematic Review and Meta-Analysis 
PLoS ONE  2014;9(2):e90362.
Background
Sphingosine kinase 1 (SK1) is a key regulator of the dynamic ceramide/sphingosine 1-phosphate rheostat balance and important in the pathological cancer genesis, progression, and metastasis processes. Many studies have demonstrated SK1 overexpressed in various cancers, but no meta-analysis has evaluated the relationship between SK1 and various cancers.
Methods
We retrieved relevant articles from the PubMed, EBSCO, ISI, and OVID databases. A pooled odds ratio (OR) was used to assess the associations between SK1 expression and cancer; hazard ratios (HR) were used for 5-year and overall survival. Review Manager 5.0 was used for the meta-analysis, and publication bias was evaluated with STATA 12.0 (Egger’s test).
Results
Thirty-four eligible studies (n = 4,673 patients) were identified. SK1 positivity and high expression were significantly different between cancer, non-cancer, and benign tissues. SK1 mRNA and protein expression levels were elevated in the cancer tissues, compared with the normal tissues. SK1 positivity rates differed between various cancer types (lowest [27.3%] in estrogen receptor-positive breast cancer and highest [82.2%] in tongue squamous cell carcinoma). SK1 positivity and high expression were associated with 5-year survival; the HR was 1.86 (95% confidence interval [CI], 1.18–2.94) for breast cancer, 1.58 (1.08–2.31) for gastric cancer, and 2.68 (2.10–3.44) for other cancers; the total cancer HR was 2.21 (95% CI, 1.83–2.67; P < 0.00001). The overall survival HRs were 2.09 (95% CI, 1.35–3.22), 1.56 (1.08–2.25), and 2.62 (2.05–3.35) in breast, gastric, and other cancers, respectively. The total effect HR was 2.21 (95% CI, 1.83–2.66; P < 0.00001).
Conclusions
SK1 positivity and high expression were significantly associated with cancer and a shorter 5-year and overall survival. SK1 positivity rates vary tremendously among the cancer types. It is necessary to further explore whether SK1 might be a predictive biomarker of outcomes in cancer patients.
doi:10.1371/journal.pone.0090362
PMCID: PMC3937388  PMID: 24587339
9.  Genome Re-Sequencing and Functional Analysis Places the Phytophthora sojae Avirulence Genes Avr1c and Avr1a in a Tandem Repeat at a Single Locus 
PLoS ONE  2014;9(2):e89738.
The aim of this work was to map and identify the Phytophthora sojae Avr1c gene. Progeny from a cross of P. sojae strains ACR10×P7076 were tested for virulence on plants carrying Rps1c. Results indicate that avirulence segregates as a dominant trait. We mapped the Avr1c locus by performing whole genome re-sequencing of composite libraries created from pooled samples. Sequence reads from avirulent (Pool1) and virulent (Pool2) samples were aligned to the reference genome and single nucleotide polymorphisms (SNP) were identified for each pool. High quality SNPs were filtered to select for positions where SNP frequency was close to expected values for each pool. Only three SNP positions fit all requirements, and these occurred in close proximity. Additional DNA markers were developed and scored in the F2 progeny, producing a fine genetic map that places Avr1c within the Avr1a gene cluster. Transient expression of Avr1c or Avr1a triggers cell death on Rps1c plants, but Avr1c does not trigger cell death on Rps1a plants. Sequence comparisons show that the RXLR effector genes Avr1c and Avr1a are closely related paralogs. Gain of virulence on Rps1c in P. sojae strain P7076 is achieved by gene deletion, but in most other strains this is accomplished by gene silencing. This work provides practical tools for crop breeding and diagnostics, as the Rps1c gene is widely deployed in commercial soybean cultivars.
doi:10.1371/journal.pone.0089738
PMCID: PMC3933651  PMID: 24586999
10.  Hantaan Virus Infection Induces CXCL10 Expression through TLR3, RIG-I, and MDA-5 Pathways Correlated with the Disease Severity 
Mediators of Inflammation  2014;2014:697837.
Hantaan virus (HTNV) is a major agent causing hemorrhagic fever with renal syndrome (HFRS). Although the pathogenesis of HFRS is unclear, some reports have suggested that the abundant production of proinflammatory cytokines and uncontrolled inflammatory responses may contribute to the development of HFRS. CXCL10 is one of these cytokines and is found to be involved in the pathogenesis of many virus infectious diseases. However, the role of CXCL10 in the pathogenesis of HFRS and the molecular regulation mechanism of CXCL10 in HTNV infection remain unknown. In this study, we report that CXCL10 expresses highly in the HFRS patients' sera and the elevated CXCL10 is positively correlated with the severity of HFRS. We find that HTNV, a single-strand RNA virus, can act as a double-strand RNA to activate the TLR3, RIG-I, and MDA-5 signaling pathways. Through the downstream transcription factors of these pathways, NF-κB and IRF7, which bind directly to the CXCL10's promoter, the expression of CXCL10 is increased. Our results may help to better understand the role of CXCL10 in the development of HFRS and may provide some novel insights into the immune response of HTNV infection.
doi:10.1155/2014/697837
PMCID: PMC3950924
11.  Glibenclamide Decreases ATP-Induced Intracellular Calcium Transient Elevation via Inhibiting Reactive Oxygen Species and Mitochondrial Activity in Macrophages 
PLoS ONE  2014;9(2):e89083.
Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca2+]i) handling in Raw 264.7 macrophages. In the present study, [Ca2+]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K+ channel blockers had no effect on the resting [Ca2+]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca2+]i transient elevation independent of extracellular Ca2+. The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca2+]i transient elevation, but pinacidil and other unselective K+ channel blockers had no effect. Glibenclamide also decreased the peak of [Ca2+]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca2+]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca2+]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.
doi:10.1371/journal.pone.0089083
PMCID: PMC3928368  PMID: 24558474
12.  Obstacles and alternative options for cardiac rehabilitation in Nanjing, China: an exploratory study 
Background
Coronary heart disease (CHD) is a major cause of morbidity and mortality, and cardiac rehabilitation (CR) is still not well developed in mainland China. The objective of this study is to investigate the barriers associated with those seeking cardiac rehabilitation (CR) and to explore appropriate secondary prevention modalities tailored to the needs of Chinese patients with coronary heart disease (CHD).
Methods
A consecutive series of eligible patients was recruited from the cardiac department of a teaching hospital in Nanjing, located in southeast China. Structured face-to-face interviews were conducted with 328 patients prior to hospital discharge. Patient preferences for seeking an outpatient CR program or an alternative outpatient self-choice, minimal-cost educational program were evaluated. Socio-demographic characteristics and clinical data were assessed. Additionally, patients were asked to provide the reasons affecting their choice.
Results
Overall, only 14.3% patients preferred the standard CR program. Factors associated with non-participating were female gender (odds ratios [ORs], 6.05, 95% CI, 1.30-28.19), older age (ORs, 1.11, 95% CI, 1.04-1.19, per year), less education (ORs, 8.13, 95% CI, 2.83-23.38), low income (ORs, 3.26, 95% CI, 1.24-8.54), and having either basic medical care or a lack of health insurance (ORs, 10.01, 95% CI, 3.90-25.68). The most common reason for refusing to participate in CR was that patients could not afford it. Of the remaining patients, 65.8% patients chose self-choice educational programs, especially for female (ORs, 5.84, 95% CI, 2.67-12.79), older (ORs, 1.06, 95% CI, 1.02-1.11, per year), and low income (ORs, 2.14, 95% CI, 1.12-4.10) patients. The main reasons for their preferences were their desires for more information about disease and risk factors, the low cost, feasibility, and saving time.
Conclusions
Multiple barriers, which may occur at the patient, health system, and societal levels, have prevented eligible patients from participating in CR programs. Self-choice educational programs, an alternative model incorporating more information, would strongly meet the needs of most patients. A feasible delivery format for secondary prevention should be provided for all CHD patients.
doi:10.1186/1471-2261-14-20
PMCID: PMC3930547  PMID: 24533661
Coronary heart disease; Secondary prevention; Cardiac rehabilitation; Health education
13.  Two insulin-like peptides antagonistically regulate aversive olfactory learning in C. elegans 
Neuron  2013;77(3):572-585.
SUMMARY
The insulin/insulin-like peptides (ILPs) regulate key events in physiology, including neural plasticity. However, the cellular and circuit mechanisms whereby ILPs regulate learning remain largely unknown. Here, we characterize two ILPs that play antagonistic roles in aversive olfactory learning of C. elegans. We show that the ILP ins-6 acts from ASI sensory neurons to enable learning by repressing the transcription of another ILP, ins-7, specifically in URX neurons. A high level of INS-7 from URX disrupts learning by antagonizing the insulin receptor-like homolog DAF-2 in the postsynaptic neurons RIA, which play an essential role in the neural circuit underlying olfactory learning. We also show that increasing URX-generated INS-7 and loss of INS-6, both of which abolish learning, alter RIA neuronal property. Together, our results reveal an “ILP-to-ILP” pathway that links environment-sensing neurons, ASI and URX, to the key neuron, RIA, of a network that underlies olfactory plasticity and modulates its activity.
doi:10.1016/j.neuron.2012.11.025
PMCID: PMC3569836  PMID: 23395381
14.  Loss of sorting nexin 27 contributes to excitatory synaptic dysfunction via modulation of glutamate receptor recycling in Down syndrome 
Nature medicine  2013;19(4):473-480.
Sorting nexin 27 (SNX27), a brain-enriched PDZ domain protein, regulates endocytic sorting and trafficking. Here, we show that Snx27−/− mice exhibit severe neuronal deficits in the hippocampus and cortex. While Snx27+/− mice exhibit grossly normal neuroanatomy, we find defects in synaptic function, learning and memory, and a reduction in ionotropic glutamate receptors (NMDARs and AMPARs). SNX27 interacts with these receptors through its PDZ domain, regulating their recycling to the plasma membrane. We demonstrate a concomitant reduction of SNX27 and C/EBPβ in Down syndrome brains and identify C/EBPβ as a transcription factor for SNX27. Down syndrome causes over-expression of miR-155, a chromosome 21-encoded microRNA that negatively regulates C/EBPβ, thereby reducing SNX27 and resulting in synaptic dysfunction. Up-regulating SNX27 in the hippocampus of Down syndrome mice rescues synaptic and cognitive deficits. Our identification of the role of SNX27 in synaptic function establishes a novel molecular mechanism of Down syndrome pathogenesis.
doi:10.1038/nm.3117
PMCID: PMC3911880  PMID: 23524343
15.  Quantitative screening of genes regulating tryptophan hydroxylase transcription in C. elegans using microfluidics and adaptive algorithm 
Forward genetic screen via mutagenesis is a powerful method for identifying regulatory factors in target pathways in model organisms such as the soil-dwelling free-living nematode Caenorhabditis elegans (C. elegans). Currently manual microscopy is the standard technique for conducting such screens; however, it is labor-intensive and time-consuming because screening requires imaging thousands of animals. Recently microfluidic chips have been developed to increase the throughput of some of such experiments; nonetheless, most of these chips are multilayer devices and complicated to fabricate and therefore prone to failure during fabrication and operation. In addition, most sorting decisions are made manually and the criteria used for sorting are subjective. To overcome these limitations, we developed a simple single-layer microfluidic device and an adaptive algorithm to make sorting decisions. The one-layer device greatly improves the reliability, while the quantitative analysis with adaptive algorithm allows for the identification of mutations that generate subtle changes in expression, which would have been hard to be detected by eye. The screening criterion is set based on the mutagenized population, not separate control populations measured prior to actual screening experiments, to account for stochasticity and day-to-day variations of gene expression in mutagenized worms. Moreover, during each experiment, the threshold is constantly updated to reflect the balance between maximizing sorting rate and minimizing false-positive rate. Using this system, we screened for mutants that have altered expression level of tryptophan hydroxylase, a key enzyme for serotonin synthesis in a CaMKII gain-of-function background. We found several putative mutants in this screen. Furthermore, this microfluidic system and quantitative analysis can be easily adapted to study other pathways in C. elegans.
doi:10.1039/c2ib20078c
PMCID: PMC3618955  PMID: 23168494
16.  Proteomic Profiling of Tissue-Engineered Blood Vessel Walls Constructed by Adipose-Derived Stem Cells 
Tissue Engineering. Part A  2012;19(3-4):415-425.
Adipose-derived stem cells (ASCs) can differentiate into smooth muscle cells and have been engineered into elastic small diameter blood vessel walls in vitro. However, the mechanisms involved in the development of three-dimensional (3D) vascular tissue remain poorly understood. The present study analyzed protein expression profiles of engineered blood vessel walls constructed by human ASCs using methods of two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS). These results were compared to normal arterial walls. A total of 1701±15 and 1265±26 protein spots from normal and engineered blood vessel wall extractions were detected by 2DE, respectively. A total of 20 spots with at least 2.0-fold changes in expression were identified, and 38 differently expressed proteins were identified by 2D electrophoresis and ion trap MS. These proteins were classified into seven functional categories: cellular organization, energy, signaling pathway, enzyme, anchored protein, cell apoptosis/defense, and others. These results demonstrated that 2DE, followed by ion trap MS, could be successfully utilized to characterize the proteome of vascular tissue, including tissue-engineered vessels. The method could also be employed to achieve a better understanding of differentiated smooth muscle protein expression in vitro. These results provide a basis for comparative studies of protein expression in vascular smooth muscles of different origin and could provide a better understanding of the mechanisms of action needed for constructing blood vessels that exhibit properties consistent with normal blood vessels.
doi:10.1089/ten.tea.2011.0532
PMCID: PMC3542882  PMID: 22963350
17.  TRAIL/DR5 Signaling Promotes Macrophage Foam Cell Formation by Modulating Scavenger Receptor Expression 
PLoS ONE  2014;9(1):e87059.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to have protective effects against atherosclerosis. However, whether TRAIL has any effects on expression of macrophage scavenger receptors and lipid uptake has not yet been studied. Macrophage lines RAW264.7 and THP-1, and mouse primary peritoneal macrophages, were cultured in vitro and treated with recombinant human TRAIL. Real-time PCR and western blot were performed to measure mRNA and protein expressions. Foam cell formation was assessed by internalization of acetylated and oxidized low-density lipoproteins (LDL). Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. We found that TRAIL treatment increased expression of scavenger receptor (SR)-AI and SR-BI in a time- and dose-dependent manner, and this effect was accompanied by increased foam cell formation. These effects of TRAIL were abolished by a TRAIL neutralizing antibody or in DR5 receptor-deficient macrophages. The increased LDL uptake by TRAIL was blocked by SR-AI gene silencing or the SR-AI inhibitor poly(I:C), while SR-BI blockade with BLT-1 had no effect. TRAIL-induced SR-AI expression was blocked by the inhibitor of p38 mitogen-activated protein kinase, but not by inhibitors of ERK1/2 or JNK. TRAIL also induced apoptosis in macrophages. In contrast to macrophages, TRAIL showed little effects on SR expression or apoptosis in vascular smooth muscle cells. In conclusion, our results demonstrate that TRAIL promotes macrophage lipid uptake via SR-AI upregulation through activation of the p38 pathway.
doi:10.1371/journal.pone.0087059
PMCID: PMC3899365  PMID: 24466325
18.  Dry eye management in a Sjögren’s syndrome mouse model by inhibition of p38-MAPK pathway 
Background
To investigate the therapeutic effect of p38-MAPK inhibitor, SB203580, on dry eye in a mouse model of Sjögren’s syndrome (MRL/lpr mice).
Methods
18 female BALB/c mice and 44 female MRL/lpr mice were included. Mice were randomly assigned to the control or treatment group. The expression of phospho-p38 MAPK in lacrimal glands of BALB/c mice was determined by Western blot analysis following IL-1β treatment at various time points. Different doses of SB203580 were injected into lacrimal glands of MRL/lpr mice and phenol red thread test was performed seven days post-injection. Moreover, the levels of acetylcholine and norepinephrine expression in lacrimal glands of MRL/lpr mice were measured using spectrofluoremetric assay and the histopathology of lacrimal glands was also evaluated.
Results
The expression of p-p38 MAPK in lacrimal glands of BALB/c mice gradually increased following incubation with IL-1β ex vivo. Injection of SB203580 into lacrimal glands significantly improved the results of phenol red thread test in MRL/lpr mice. In addition, the secretions of acetylcholine and norepinephrine increased significantly compared to the control group. Less lymphocytes infiltration was observed in pathological section of lacrimal glands following SB203580 injection.
Conclusions
Our results indicate that the activation of p38-MAPK pathway plays an important role in dry eye of a Sjögren’s syndrome mouse model. Inhibition of p38-MAPK pathway by SB203580 might have potential therapeutic effect on Sjögren’s syndrome associated dry eye.
Virtual slides
The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1256849631103092.
doi:10.1186/1746-1596-9-5
PMCID: PMC3976089  PMID: 24443980
Sjögren’s syndrome; Dry eye; Interleukin-1β; p38-MAPK pathway
19.  Role of Caveolin-1 in Atrial Fibrillation as an Anti-Fibrotic Signaling Molecule in Human Atrial Fibroblasts 
PLoS ONE  2014;9(1):e85144.
Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in the general population; yet, the precise mechanisms resulting in AF are not fully understood. Caveolin-1 (Cav-1), the principal structural component of caveolae organelles in cardiac fibroblasts, is involved in several cardiovascular conditions; however, the study on its function in atrium, in particular, in AF, is still lacking. This report examines the hypothesis that Cav-1 confers an anti-AF effect by mediating atrial structural remodeling through its anti-fibrotic action. We evaluated the expression of Cav-1, transforming growth factor-β1 (TGF-β1), and fibrosis in atrial specimens of 13 patients with AF and 10 subjects with sinus rhythm, and found that the expression of Cav-1 was significantly downregulated, whereas TGF-β1 level, collagens I/III contents and atrial fibrosis were markedly increased, in AF. Western blot analysis demonstrated that treatment of human atrial fibroblasts (HAFs) with TGF-β1 resulted in a concentration- and time-dependent repression of Cav-1. Downregulation of Cav-1 with siRNA increased the TGF-β1-induced activation of Smad signal pathway and collagens production in HAFs. Furthermore, incubation of HAFs with the peptides derived from Cav-1 to achieve Cav-1 gain-of-function abolished the TGF-β1-induced production of collagens I/III and decreases of MMP-2/-9 expression. Therefore it was concluded that Cav-1 is an important anti-AF signaling mediator by conferring its anti-fibrotic effects in atrium.
doi:10.1371/journal.pone.0085144
PMCID: PMC3891766  PMID: 24454806
20.  Trafficking regulation of proteins in Alzheimer’s disease 
The β-amyloid (Aβ) peptide has been postulated to be a key determinant in the pathogenesis of Alzheimer’s disease (AD). Aβ is produced through sequential cleavage of the β-amyloid precursor protein (APP) by β- and γ-secretases. APP and relevant secretases are transmembrane proteins and traffic through the secretory pathway in a highly regulated fashion. Perturbation of their intracellular trafficking may affect dynamic interactions among these proteins, thus altering Aβ generation and accelerating disease pathogenesis. Herein, we review recent progress elucidating the regulation of intracellular trafficking of these essential protein components in AD.
doi:10.1186/1750-1326-9-6
PMCID: PMC3891995  PMID: 24410826
α-secretase; Amyloid beta (A4) precursor protein; β-secretase; Beta-site APP-cleaving enzyme 1; γ-secretase; A Disintegrin and Metalloprotease 10; Alzheimer’s disease; Trafficking
21.  Correction: Saponin 1 Induces Apoptosis and Suppresses NF-κB-Mediated Survival Signaling in Glioblastoma Multiforme (GBM) 
PLoS ONE  2014;9(1):10.1371/annotation/0c3019ff-6d7b-444b-8eb5-9461f3dcd029.
doi:10.1371/annotation/0c3019ff-6d7b-444b-8eb5-9461f3dcd029
PMCID: PMC3888439
22.  Detection of Specific Antibodies against Tembusu Virus in Ducks by Use of an E Protein-Based Enzyme-Linked Immunosorbent Assay 
Journal of Clinical Microbiology  2013;51(7):2400-2402.
We developed an enzyme-linked immunosorbent assay (ELISA) using eukaryotically expressed E protein as the antigen (termed E-ELISA) to detect antibodies to tembusu virus (TMUV) in ducks. The E-ELISA did not react with antisera to other known pathogens, indicating the E protein is specific for recognizing anti-TMUV antibodies. Compared to the serum neutralization test, the specificity and sensitivity of the E-ELISA was 93.2 and 97.8%, respectively. Therefore, this E-ELISA is a sensitive and rapid method for detecting antibodies against TMUV in ducks.
doi:10.1128/JCM.00361-13
PMCID: PMC3697666  PMID: 23616462
23.  Correction: Overexpressing STAMP2 Improves Insulin Resistance in Diabetic ApoE−/−/LDLR−/− Mice via Macrophage Polarization Shift in Adipose Tissues 
PLoS ONE  2013;8(12):10.1371/annotation/39606c88-c2a1-45e7-8b2d-9b8f7df6f05f.
doi:10.1371/annotation/39606c88-c2a1-45e7-8b2d-9b8f7df6f05f
PMCID: PMC3876947
24.  Use of Advanced Treatment Technologies Among Men at Low Risk of Dying From Prostate Cancer 
JAMA : the journal of the American Medical Association  2013;309(24):10.1001/jama.2013.6882.
Importance
The use of advanced treatment technologies (ie, intensity-modulated radiotherapy [IMRT] and robotic prostatectomy) for prostate cancer is increasing. The extent to which these advanced treatment technologies have disseminated among patients at low risk of dying from prostate cancer is uncertain.
Objective
To assess the use of advanced treatment technologies, compared with prior standards (ie, traditional external beam radiation treatment [EBRT] and open radical prostatectomy) and observation, among men with a low risk of dying from prostate cancer.
Design, Setting, and Patients
Using Surveillance, Epidemiology, and End Results (SEER)-Medicare data, we identified a retrospective cohort of men diagnosed with prostate cancer between 2004 and 2009 who underwent IMRT (n=23 633), EBRT (n=3926), robotic prostatectomy (n=5881), open radical prostatectomy (n=6123), or observation (n=16 384). Follow-up data were available through December 31, 2010.
Main Outcomes and Measures
The use of advanced treatment technologies among men unlikely to die from prostate cancer, as assessed by low-risk disease (clinical stage ≤T2a, biopsy Gleason score ≤6, and prostate-specific antigen level ≤10 ng/mL), high risk of noncancer mortality (based on the predicted probability of death within 10 years in the absence of a cancer diagnosis), or both.
Results
In our cohort, the use of advanced treatment technologies increased from 32% (95% CI, 30%–33%) to 44% (95% CI, 43%–46%) among men with low-risk disease (P<.001) and from 36% (95% CI, 35%–38%) to 57% (95% CI, 55%–59%) among men with high risk of noncancer mortality (P<.001). The use of these advanced treatment technologies among men with both low-risk disease and high risk of noncancer mortality increased from 25% (95% CI, 23%–28%) to 34% (95% CI, 31%–37%) (P<.001). Among all patients diagnosed in SEER, the use of advanced treatment technologies for men unlikely to die from prostate cancer increased from 13% (95% CI, 12%–14%), or 129.2 per 1000 patients diagnosed with prostate cancer, to 24% (95% CI, 24%–25%), or 244.2 per 1000 patients diagnosed with prostate cancer (P<.001).
Conclusion and Relevance
Among men diagnosed with prostate cancer between 2004 and 2009 who had low-risk disease, high risk of noncancer mortality, or both, the use of advanced treatment technologies has increased.
doi:10.1001/jama.2013.6882
PMCID: PMC3857348  PMID: 23800935
25.  Correction: Relationship of CHA2DS2-VASc and CHADS2 Score to Left Atrial Remodeling Detected by Velocity Vector Imaging in Patients with Atrial Fibrillation 
PLoS ONE  2013;8(12):10.1371/annotation/fb95b7ed-f03b-498b-adda-2683a1b904b7.
doi:10.1371/annotation/fb95b7ed-f03b-498b-adda-2683a1b904b7
PMCID: PMC3869701

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