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1.  RNA-Based Fluorescent Biosensors for Live Cell Imaging of Second Messenger Cyclic di-AMP 
Cyclic di-AMP (cdiA) is a second messenger predicted to be widespread in Gram-positive bacteria, some Gram-negative bacteria, and Archaea. In the human pathogen Listeria monocytogenes, cdiA is an essential molecule that regulates metabolic function and cell wall homeostasis, and decreased levels of cdiA result in increased antibiotic susceptibility. We have generated fluorescent biosensors for cdiA through fusion of the Spinach2 aptamer to ligand-binding domains of cdiA riboswitches. The biosensor was used to visualize intracellular cdiA levels in live L. monocytogenes strains and to determine the catalytic domain of the phosphodiesterase PdeA. Furthermore, a flow cytometry assay based on this biosensor was used to screen for diadenylate cyclase activity and confirmed the enzymatic activity of DisA-like proteins from Clostridium difficile and Methanocaldococcus jannaschii. Thus, we have expanded the development of RNA-based biosensors for in vivo metabolite imaging in Gram-positive bacteria and have validated the first dinucleotide cyclase from Archaea.
Graphical abstract
PMCID: PMC4521591  PMID: 25965978
2.  Differences in Copper Absorption and Accumulation between Copper-Exclusion and Copper-Enrichment Plants: A Comparison of Structure and Physiological Responses 
PLoS ONE  2015;10(7):e0133424.
Differences in copper (Cu) absorption and transport, physiological responses and structural characteristics between two types of Cu-resistant plants, Oenothera glazioviana (Cu-exclusion type) and Elsholtzia haichowensis (Cu-enrichment type), were investigated in the present study. The results indicated the following: (1) After 50 μM Cu treatment, the Cu ratio in the xylem vessels of E. haichowensis increased by 60%. A Cu adsorption experiment indicated that O. glazioviana exhibited greater resistance to Cu, and Cu absorption and the shoot/root ratio of Cu were significantly lower in O. glazioviana than in E. haichowensis. (2) An analysis of the endogenous abscisic acid (ABA) variance and exogenous ABA treatment demonstrated that the ABA levels of both plants did not differ; exogenous ABA treatment clearly reduced Cu accumulation in both plants. (3) The leaf stomatal density of O. glazioviana was significantly less than that of E. haichowensis. Guard cells in E. haichowensis plants were covered with a thick cuticle layer, the epidermal hair was more numerous and longer, and the number of xylem conduits in the root was small. (4) The transpiration rate and the stomatal conductance of O. glazioviana were both significantly lower than those of E. haichowensis, regardless of whether the plants were treated with Cu. Taken together, these results indicate that the differences in the structural characteristics between these two plant species, particularly in the characteristics related to plant transpiration, are important factors that govern whether plants acquire or exclude Cu.
PMCID: PMC4514476  PMID: 26207743
3.  Computational Prediction of One-Step Synthesis of Seven-membered Fused Rings by (5+2) Cycloaddition Utilising Cycloalkenes 
Scientific Reports  2015;5:12272.
The (5+2) cycloaddition reaction utilising cycloalkenes is rare, although it is one of the most efficient methods of constructing seven-membered fused rings because of its high atom- and step-economy. In this study, we used quantum mechanical calculations to predict the plausibility of using the Rh-catalysed intermolecular (5+2) cycloaddition of 3-acyloxy-1,4-enynes and cycloalkenes to produce fused seven-membered carbocycles. The calculation results suggest a convenient, highly efficient and energetically practical approach. Strained cycloalkenes, such as cyclopropene, have been predicted to be active, and the desired bicyclic product should be favoured, accompanied by the formation of byproducts from rearrangement reactions. The energy barriers of the alkene insertion step were analysed by the distortion/interaction model to disclose the origins of the different reactivities of cycloalkenes with different ring sizes.
PMCID: PMC4510518  PMID: 26198317
4.  Dynamic structural evolution of supported palladium–ceria core–shell catalysts revealed by in situ electron microscopy 
Nature Communications  2015;6:7778.
The exceptional activity for methane combustion of modular palladium–ceria core–shell subunits on silicon-functionalized alumina that was recently reported has created renewed interest in the potential of core–shell structures as catalysts. Here we report on our use of advanced ex situ and in situ electron microscopy with atomic resolution to show that the modular palladium–ceria core–shell subunits undergo structural evolution over a wide temperature range. In situ observations performed in an atmospheric gas cell within this temperature range provide real-time evidence that the palladium and ceria nanoparticle constituents of the palladium–ceria core–shell participate in a dynamical process that leads to the formation of an unanticipated structure comprised of an intimate mixture of palladium, cerium, silicon and oxygen, with very high dispersion. This finding may open new perspectives about the origin of the activity of this catalyst.
There is currently renewed interest in the use of core–shell catalysts for methane combustion. Here, the authors perform an ex situ and in situ electron microscopy study to probe the structural evolution of palladium–cerium dioxide catalytic core–shell subunits over a wide temperature range.
PMCID: PMC4510970  PMID: 26160065
5.  Minimally invasive unilateral versus bilateral technique in performing single-segment pedicle screw fixation and lumbar interbody fusion 
The minimally invasive transforaminal lumbar interbody fusion procedure with percutaneous pedicle screws was adopted in clinical practice, but the choice between a unilateral pedicle screw (UPS) or bilateral pedicle screw (BPS) fixation after lumbar fusion remains controversial. The purpose of the present retrospective study was to compare the clinical outcomes and radiological results of unilateral and bilateral pedicle screw fixations.
The retrospective study recruited seventy-eight patients with a single-level pedicle screw fixation and lumbar interbody fusion at L4–L5 or L5–S1 from January 2010 to January 2013. The patients were treated with MIS TLIF with BPS fixation, and since May 2012, all patients were treated with UPS fixation. The perioperative outcomes including operative time, blood loss, hospital-stay length, and complication rates were accessed. Radiological outcomes regarding fusion were determined with the Bridwell grading system. Clinical outcomes were evaluated with the Oswestry Disability Index (ODI) and visual analog scale (VAS) during the mean follow-up of 2 years.
According to perioperative assessments, the operative time was significantly shorter for group UPS (84.7 ± 6.4 min) than for group BPS (103.6 ± 10.6 min; p < 0.0001), and similar results were found with regard to the mean blood loss (UPS, 96.3 ± 17.5; BPS, 137.4 ± 32.9, p < 0.0001). With regard to the hospital-stay period, though the UPS group seems shorter, there is no statistical significance (UPS, 10.0 ± 2.1; BPS, 10.4 ± 2.4, p = 0.428). There were four in the BPS group and six in the UPS group defined as unfused at 6 months pest-operative, but at 12 months post-surgery, all patients achieved solid fusion. Regarding clinical outcomes, the VAS and ODI scores were significantly lower in the UPS group than the BPS group at 7 days post-surgery, but there was no difference at 1 month post-surgery and during the later follow-up.
There was no difference between the UPS and BPS flexion techniques about the clinical outcomes at 24 months post-surgery. However, because the UPS involves a shorter surgical time, less blood loss, faster pain relief, and faster functional recovery, UPS might be more suitable in performing single-segment pedicle screw fixation and lumbar interbody fusion.
PMCID: PMC4504127  PMID: 26179281
Minimally invasive transforaminal lumbar interbody fusion; Unilateral pedicle screw; Bilateral pedicle screw
6.  β-2 Adrenergic receptor gene polymorphism and response to propranolol in cirrhosis 
AIM: To evaluate the association of β-2 adrenergic receptor (β2-AR) gene polymorphism with response of variceal pressure to propranolol in cirrhosis.
METHODS: Sixty-four non-related cirrhotic patients participated in this study and accepted variceal pressure measurement before and after propranolol administration. Polymorphism of the β2-AR gene was determined by directly sequencing of the polymerase chain reaction products from the DNA samples that were prepared from the patients.
RESULTS: The prevalence of Gly16-Glu/Gln27 and Arg16-Gln27 homozygotes, and compound heterozygotes was 29.7%, 10.9%, and 59.4%, respectively. Patients with cirrhosis with Gly16-Glu/Gln27 homozygotes had a greater decrease of variceal pressure after propranolol administration than those with Arg16-Gln27 homozygotes or with compound heterozygotes (22.4% ± 2.1%, 13.1% ± 2.7% and 12.5% ± 3.1%, respectively, P < 0.01).
CONCLUSION: The variceal pressure response to propranolol was associated with polymorphism of β2-AR gene. Patients with the Gly16-Glu/Gln27 homozygotes probably benefit from propranolol therapy.
PMCID: PMC4476880  PMID: 26109805
Variceal bleeding; β2-adrenergic receptor; Propranolol; Variceal pressure; Homozygotes
7.  Ectopic Expression of a Glycine soja myo-Inositol Oxygenase Gene (GsMIOX1a) in Arabidopsis Enhances Tolerance to Alkaline Stress 
PLoS ONE  2015;10(6):e0129998.
Myo-inositol participates in various aspects of plant physiology, and myo-inositol oxygenase is the key enzyme of the myo-inositol oxygenation pathway. Previous studies indicated that myo-inositol oxygenase may play a role in plant responses to abiotic stresses. In this study, we focused on the functional characterization of GsMIOX1a, a remarkable alkaline stress-responsive gene of Glycine soja 07256, based on RNA-seq data. Using quantitative real-time PCR, we demonstrated that GsMIOX1a is rapidly induced by alkaline stress and expressed predominantly in flowers. We also elucidated the positive function of GsMIOX1a in the alkaline response in the wild type, atmiox1 mutant as well as GsMIOX1a-overexpressing Arabidopsis. We determined that atmiox1 mutant decreased Arabidopsis tolerance to alkaline stress, whereas GsMIOX1a overexpression increased tolerance. Moreover, the expression levels of some alkaline stress-responsive and inducible marker genes, including H+-Ppase, NADP-ME, KIN1 and RD29B, were also up-regulated in GsMIOX1a overexpression lines compared with the wild type and atmiox1 mutant. Together, these results suggest that the GsMIOX1a gene positively regulates plant tolerance to alkaline stress. This is the first report to demonstrate that ectopic expression of myo-inositol oxygenase improves alkaline tolerance in plants.
PMCID: PMC4474918  PMID: 26091094
8.  In Situ OH Generation from O2− and H2O2 Plays a Critical Role in Plasma-Induced Cell Death 
PLoS ONE  2015;10(6):e0128205.
Reactive oxygen and nitrogen species produced by cold atmospheric plasma (CAP) are considered to be the most important species for biomedical applications, including cancer treatment. However, it is not known which species exert the greatest biological effects, and the nature of their interactions with tumor cells remains ill-defined. These questions were addressed in the present study by exposing human mesenchymal stromal and LP-1 cells to reactive oxygen and nitrogen species produced by CAP and evaluating cell viability. Superoxide anion (O2−) and hydrogen peroxide (H2O2) were the two major species present in plasma, but their respective concentrations were not sufficient to cause cell death when used in isolation; however, in the presence of iron, both species enhanced the cell death-inducing effects of plasma. We propose that iron containing proteins in cells catalyze O2− and H2O2 into the highly reactive OH radical that can induce cell death. The results demonstrate how reactive species are transferred to liquid and converted into the OH radical to mediate cytotoxicity and provide mechanistic insight into the molecular mechanisms underlying tumor cell death by plasma treatment.
PMCID: PMC4457797  PMID: 26046915
9.  Autoregulatory Mechanism for Dynactin Control of Processive and Diffusive Dynein Transport 
Nature cell biology  2014;16(12):1192-1201.
Dynactin is the longest known cytoplasmic dynein regulator, with roles in dynein recruitment to subcellular cargo and in stimulating processive dynein movement. The latter function was thought to involve the N-terminal microtubule binding region of the major dynactin polypeptide p150Glued, though recent results disputed this. To understand how dynactin regulates dynein we generated recombinant fragments of the N-terminal half of p150Glued. We find that the dynein-binding coiled-coil α-helical domain CC1B is sufficient to stimulate dynein processivity, which it accomplishes by increasing average dynein step size and forward step frequency, while decreasing lateral stepping and microtubule detachment. In contrast, the immediate upstream coiled-coil domain, CC1A, activates a novel diffusive dynein state. CC1A interacts physically with CC1B and interferes with its effect on dynein processivity. We also identify a role for the N-terminal portion of p150Glued in coordinating these activities. Our results reveal an unexpected form of long-range allosteric control of dynein motor function by internal p150Glued sequences, and evidence for p150Glued auto regulation.
PMCID: PMC4250405  PMID: 25419851
10.  BRCC3 acts as a prognostic marker in nasopharyngeal carcinoma patients treated with radiotherapy and mediates radiation resistance in vitro 
BRCC3 has been found to be aberrantly expressed in breast tumors and involved in DNA damage response. The contribution of BRCC3 to nasopharyngeal carcinoma prognosis and radiosensitivity is still unclear.
Immunohistochemical analysis of BRCC3 was carried out in 100 nasopharyngeal carcinoma tissues, and the protein level was correlated to patient survival. BRCC3 expression of nasopharyngeal carcinoma cell lines was determined by Western-blotting and real-time PCR. Additionally, the effects of BRCC3 knockdown on nasopharyngeal carcinoma cell clongenic survival, DNA damage repair, and cell cycle distribution after irradiation was assessed.
The BRCC3 protein level was inversely correlated with nasopharyngeal carcinoma patient overall survival (P < 0.001) and 3-year loco-regional relapse-free survival (P = 0.034). Multivariate analysis demonstrated that BRCC3 expression was an independent prognostic factor (P = 0.010). The expression of BRCC3 was much higher in radioresistant nasopharyngeal carcinoma cells than in radiosensitive cells. Knockdown of BRCC3 increased the cell survival fraction, attenuated DNA damage repair and resulted in G2/M cell cycle arrest in radioresistant NPC cells.
High BRCC3 expression in nasopharyngeal carcinoma patients is associated with poor survival. BRCC3 knockdown could abate the radioresistance in nasopharyngeal carcinoma cells. These findings suggest the utility of BRCC3 as a prognostic biomarker and novel target for nasopharyngeal carcinoma.
PMCID: PMC4511524  PMID: 26024915
BRCC3; Nasopharyngeal carcinoma; Prognostic marker; Radioresistance
11.  Residual disease and risk factors in patients with high-grade cervical intraepithelial neoplasia and positive margins after initial conization 
The purpose of this study was to determine the clinicopathologic predictors of residual disease in patients with high-grade cervical intraepithelial neoplasia (CIN) and margin involvement after initial conization.
Data from 145 patients who underwent subsequent surgery for high-grade CIN with positive margins were retrospectively analyzed.
After subsequent surgery, residual disease was diagnosed in 47 (34.2%) patients, of whom five had invasive cervical carcinoma, 31 had CIN 3, nine had CIN 2, and two had CIN 1. Multivariate analysis revealed that only age ≥35 years (P=0.033), major abnormal cytology (P=0.002), and pre-cone high-risk human papillomavirus load ≥300 relative light units (P=0.011) were significant factors associated with residual disease.
Age ≥35 years, major abnormal cytology, and pre-cone high-risk human papillomavirus load ≥300 relative light units were the only significant factors predicting post-cone residual disease. Appropriate application of these predictive factors may avoid delayed treatment and overtreatment.
PMCID: PMC4445701  PMID: 26056463
cervical intraepithelial neoplasia; residual disease; conization; positive margin; predictor
12.  Circulating microRNAs are promising novel biomarkers for drug-resistant epilepsy 
Scientific Reports  2015;5:10201.
MicroRNAs (miRNAs) open up a new field for molecular diagnosis for cancer and other diseases based on their stability in serum. However, the role of circulating miRNAs in plasma/serum in epilepsy diagnosis is still unclear. The aim of this study was to evaluate whether miRNAs can be used as biomarkers for drug-resistant epilepsy. We measured the differences in serum miRNA levels between 30 drug-resistant patients and 30 drug-responsive epilepsy patients in discovery and training phases using Illumina HiSeq2000 sequencing followed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assays. The selected miRNAs were then validated in 77 drug-resistant epilepsy patients, 81 drug-responsive epilepsy patients and 85 healthy controls by qRT-PCR. We found that circulating miRNAs are differentially expressed between drug-resistant group and drug-responsive group. MiR-194-5p, -301a-3p, -30b-5p, -342-5p and -4446-3p were significantly deregulated in drug-resistant group compared to drug-responsive group and control group. Among these 5 miRNAs, miR-301a-3p had the best diagnostic value for drug-resistant epilepsy with 80.5% sensitivity and 81.2% specificity, and was negatively associated with seizure severity. These provide the rationale for further confirmation studies in larger prospective cohorts and in other ethnics.
PMCID: PMC4435024  PMID: 25984652
13.  Biological Significance of Photoreceptor Photocycle Length: VIVID Photocycle Governs the Dynamic VIVID-White Collar Complex Pool Mediating Photo-adaptation and Response to Changes in Light Intensity 
PLoS Genetics  2015;11(5):e1005215.
Most organisms on earth sense light through the use of chromophore-bearing photoreceptive proteins with distinct and characteristic photocycle lengths, yet the biological significance of this adduct decay length is neither understood nor has been tested. In the filamentous fungus Neurospora crassa VIVID (VVD) is a critical player in the process of photoadaptation, the attenuation of light-induced responses and the ability to maintain photosensitivity in response to changing light intensities. Detailed in vitro analysis of the photochemistry of the blue light sensing, FAD binding, LOV domain of VVD has revealed residues around the site of photo-adduct formation that influence the stability of the adduct state (light state), that is, altering the photocycle length. We have examined the biological significance of VVD photocycle length to photoadaptation and report that a double substitution mutant (vvdI74VI85V), previously shown to have a very fast light to dark state reversion in vitro, shows significantly reduced interaction with the White Collar Complex (WCC) resulting in a substantial photoadaptation defect. This reduced interaction impacts photoreceptor transcription factor WHITE COLLAR-1 (WC-1) protein stability when N. crassa is exposed to light: The fast-reverting mutant VVD is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation as assayed both by attenuation of gene expression and the ability to respond to increasing light intensity. Additionally, transcription of the clock gene frequency (frq) is sensitive to changing light intensity in a wild-type strain but not in the fast photo-reversion mutant indicating that the establishment of this dynamic VVD-WCC pool is essential in general photobiology and circadian biology. Thus, VVD photocycle length appears sculpted to establish a VVD-WCC reservoir of sufficient size to sustain photoadaptation while maintaining sensitivity to changing light intensity. The great diversity in photocycle kinetics among photoreceptors may be viewed as reflecting adaptive responses to specific and salient tasks required by organisms to respond to different photic environments.
Author Summary
Sensing light from the environment using a variety of photoreceptors is of great adaptive significance for most eukaryotes. A key feature of photoreceptors is the photocycle length, the time taken to decay from the initial signaling light state back to the receptive dark state; however, the significance of photocycle length, or adduct decay length, has not been tested in a biological setting. The photocycle length is determined by the chemical environment of the active site where a photon absorbing chromophore forms an adduct with a conserved amino acid. There is clear evidence of evolutionary selection for a particular photocycle length even between photoreceptors containing the same prototypic light-sensing domains suggesting functional relevance. Using defined in vitro mutations that change the photocycle length of the VIVID (VVD) protein over 4 orders of magnitude we were able to ascribe a pivotal role of the native photochemistry of the protein in its function as a photoreceptor in the light and circadian biology of Neurospora crassa. This study links in vitro photochemical studies with in vivo function and provides evidence that the true evolutionary and functional significance of native photochemistry of photoreceptors can be enhanced by studying photocycle mutants in their native systems.
PMCID: PMC4433212  PMID: 25978382
14.  Berberine reverses abnormal expression of L-type pyruvate kinase by DNA demethylation and histone acetylation in the livers of the non-alcoholic fatty disease rat 
Berberine (BBR) can potentially be used as a drug against non-alcoholic fatty liver disease (NAFLD) and diabetes. Our previous study found that BBR could change the pattern of DNA methylation. But the mechanisms underlying berberine are still far from completely understood. In this study, the function of L-PK in cell metabolism was explored, and high-fat-diet induced SD rats NAFLD models were created. The NAFLD rats were randomly grouped to be oral administration with BBR at a dosage of 200 mg/kg daily. Then DNA methylation and histone acetylation around the L-type Pyruvate Kinase (L-PK) gene were examined. In the results, we found that L-PK had a positive effect on cell proliferation, glucose utilization and triglyceride metabolism. However, the expression of L-PK was reduced in the livers of NAFLD rats, in accord with the decrease of DNA hypermethylation and histone deacetylation in the regulatory regions of L-PK. Notably, BBR treatment can restore the expression of L-PK by the demethylation of L-PK promoter and the increase in acetylation levels of histone H3 and H4 around L-PK, which indicated that BBR may be a potential drug for epigenetic-included diseases.
PMCID: PMC4509242  PMID: 26221297
Berberine; L-type pyruvate kinase; non-alcoholic fatty liver disease; DNA methylation; histone acetylation
15.  Involvement of enhancer of zeste homolog 2 in cisplatin-resistance in ovarian cancer cells by interacting with several genes 
Molecular Medicine Reports  2015;12(2):2503-2510.
In the present study, gene expression profiles of cisplatin-sensitive ovarian cancer (OC) cells were compared with those of cisplatin-resistant OC cells to identify key genes and pathways contributing to cisplatin resistance in ovarian cancer cells. The GSE15372 gene expression data set was downloaded from Gene Expression Omnibus, and included five biological replicates of cisplatin-sensitive OC cells and five biological replicates of cisplatin-resistant OC cells. Differentially expressed genes (DEGs) were screened using the limma package in R, based on the cut-off values of P<0.05 and |log2 (fold change)|>1. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis and Gene Ontology enrichment analysis were performed on the DEGs using the Database for Annotation, Visualization and Integration Discovery. The protein-protein interaction (PPI) network was constructed for the DEGs using STRING, and sub-networks were analyzed by Clustering with Overlapping Neighborhood Expansion. A total of 556 DEGs were identified in the cisplatin-sensitive OC cells, of which 246 were upregulated and 310 were downregulated. Functional enrichment analysis revealed metabolism-associated pathways, DNA replication and cell cycle were significantly enriched in the downregulated genes, while cell growth and differentiation, response to stimulus, and apoptosis were significantly enriched in the upregulated genes. A PPI network, including 342 nodes was constructed for the DEGs and four subnetworks were extracted from the entire network. A total of 34 DEGs interacting with enhancer of zeste homolog 2 (EZH2) were identified, which were associated with DNA replication, pyrimidine metabolism and cell cycle. In conclusion, a number of key genes and pathways associated with the cisplatin-resistance of OC were revealed, particularly EZH2. These findings assist in the development of therapy for OC.
PMCID: PMC4464270  PMID: 25955318
ovarian cancer; gene expression data; differentially expressed genes; functional enrichment analysis; protein-protein interaction network; EZH2
17.  Intermittent fasting attenuates increases in neurogenesis after ischemia and reperfusion and improves recovery 
Intermittent fasting (IF) is neuroprotective across a range of insults, but the question of whether extending the interval between meals alters neurogenesis after ischemia remains unexplored. We therefore measured cell proliferation, cell death, and neurogenesis after transient middle cerebral artery occlusion (MCAO) or sham surgery (SHAM) in mice fed ad libitum (AL) or maintained on IF for 3 months. IF was associated with twofold reductions in circulating levels of the adipocyte cytokine leptin in intact mice, but also prevented further reductions in leptin after MCAO. IF/MCAO mice also exhibit infarct volumes that were less than half those of AL/MCAO mice. We observed a 30% increase in basal cell proliferation in the hippocampus and subventricular zone (SVZ) in IF/SHAM, relative to AL/SHAM mice. However, cell proliferation after MCAO was limited in IF mice, which showed twofold increases in cell proliferation relative to IF/SHAM, whereas AL/MCAO mice exhibit fivefold increases relative to AL/SHAM. Attenuation of stroke-induced neurogenesis was correlated with reductions in cell death, with AL/MCAO mice exhibiting twice the number of dying cells relative to IF/MCAO mice. These observations indicate that IF protects against neurological damage in ischemic stroke, with circulating leptin as one possible mediator.
PMCID: PMC4013772  PMID: 24549184
caloric restriction; hippocampus; intermittent fasting; neurogenesis; stroke; subventricular zone
We report that PGE2 promotes Smad2-Smad4 complex formation and this phenomenon could be blocked by DIDS, an anion transporter inhibitor. Our data suggest that PGE2 had no effects on Smad2 phosphorylation, suggesting that PGE2-mediated Smad2-Smad4 complex formation is independent of TGF-β signaling and that PGE2 induced Smad2 modification which is different from TGF-β-mediated phosphorylation. We demonstrate that in primary human glomerular mesangial cells PGE2 caused modification of Smad2 as detected by Smad2N antibody, raised against a peptide near the N-terminus of Smad2. We hypothesize that Smad2 protein is post-translationaly modified by PGE2. Direct evidence of Smad2 modification by PGE2 was achieved by avidin pulldown assay which showed that endogenous Smad2 and recombinant Smad2 protein were attached by biotin-labeled PGE2. Taken together, our results provided evidence that post-translational modification of Smad2 could be a mechanism for the action of PGE2 in the pathogenesis of human pathologies.
PMCID: PMC4036222  PMID: 24613014
19.  The Acceptability of Incorporating a Youth Smoking Prevention Intervention in the Pediatric Emergency Department 
The pediatric emergency department (PED) is under-utilized as a setting in which to provide tobacco prevention interventions for at-risk children. We sought to determine the acceptability and feasibility of incorporating a brief, parental tobacco prevention intervention to 520 parents during the PED visit. Mean age (SD) of parents and children was 38.6 (7.1) and 11.5 (1.1), respectively; 47% of children were female; 45% were African American; 36% of parents had an annual income less than $25,000; 28.8% of parents were current smokers. Over 90% of parents said the intervention provided “useful” and “easy to understand” information and 97% of practitioners said it did not “interfere with clinical care.” Given the high prevalence of parental smoking in the PED, there is a high likelihood that their children will initiate smoking in the future. Thus, the use of the PED as a venue to providing tobacco prevention interventions warrants further evaluation.
PMCID: PMC4166505  PMID: 24858886
Tobacco; adolescent smoking; emergency department; parenting; prevention and control
20.  MiR-320a contributes to atherogenesis by augmenting multiple risk factors and down-regulating SRF 
Atherosclerosis progress is regulated by a variety of factors. Here, we show that miR-320a, an intergenic miRNA, is markedly elevated in the peripheral blood of coronary heart disease patients and high-risk patients. Microarray analysis and qRT-PCR assays showed that circulating miRNA-320a was highly expressed in coronary artery disease patients. In vivo study showed that overexpression of miR-320a resulted in significant increase in levels of plasma lipid (total cholesterol, Triglyceride and low-density lipoprotein) and serum inflammatory cytokines (IL-6, MCP-1, sICAM, pSelectin, TNF-α and fibrinogen). In ApoE−/− mice, miR-320a expression attenuates endothelium cell function and promotes atherogenesis. Bioinformatics analysis identified serum response factor as a potential target for miR-320a, which was validated by luciferase reporter activity assay and western-blot in vitro and in vivo. Moreover, miR-320a expression inhibits human-derived endothelium cell proliferation and induces apoptosis. We also found that SP1 transcriptionally up-regulates hsa-miR-320a expression. Our observations indicate that miR-320a is a key regulator contributing to multiple aspects of atherogenesis.
PMCID: PMC4420600  PMID: 25728840
atherosclerosis; miRNA; SRF; endothelium
21.  A Sensorless Predictive Current Controlled Boost Converter by Using an EKF with Load Variation Effect Elimination Function 
Sensors (Basel, Switzerland)  2015;15(5):9986-10003.
To realize accurate current control for a boost converter, a precise measurement of the inductor current is required to achieve high resolution current regulating. Current sensors are widely used to measure the inductor current. However, the current sensors and their processing circuits significantly contribute extra hardware cost, delay and noise to the system. They can also harm the system reliability. Therefore, current sensorless control techniques can bring cost effective and reliable solutions for various boost converter applications. According to the derived accurate model, which contains a number of parasitics, the boost converter is a nonlinear system. An Extended Kalman Filter (EKF) is proposed for inductor current estimation and output voltage filtering. With this approach, the system can have the same advantages as sensored current control mode. To implement EKF, the load value is necessary. However, the load may vary from time to time. This can lead to errors of current estimation and filtered output voltage. To solve this issue, a load variation elimination effect elimination (LVEE) module is added. In addition, a predictive average current controller is used to regulate the current. Compared with conventional voltage controlled system, the transient response is greatly improved since it only takes two switching cycles for the current to reach its reference. Finally, experimental results are presented to verify the stable operation and output tracking capability for large-signal transients of the proposed algorithm.
PMCID: PMC4481978  PMID: 25928061
boost converter; sensorless predictive current control; extended Kalman filter; load variation effect elimination
22.  Gamma secretase inhibitor enhances sensitivity to doxorubicin in MDA-MB-231 cells 
Deregulated expression of molecular of the Notch signaling pathway is observed in malignant tumor. Notch signaling pathway is activated by a series of catalytic cleavage of the Notch receptor by gamma secretase. Gamma secretase inhibitor (GSI) have demonstrated clinical activity in patients with solid tumor. Triple negative breast cancer (TNBC) is related to poor prognosis and a high probability of lung and brain metastases. As first line therapy for TNBC, doxorubicin is partially effective in TNBC control. An understanding of the mechanisms for enhancing sensitivity to doxorubicin would be significant for future drug development. We hypothesized that a combination of cytotoxic chemotherapy doxorubicin to inhibit cell proliferation, together with GSI, would result in more effective outcome than either monotherapy alone. We treated MDA-MB-231 cell lines with doxorubicin and evaluated the monotherapy efficacy and in combination with GSI in both vitro and vivo. GSI-induced proliferation inhibition and apoptosis was achieved with an induction of PTEN and pro-apoptotic protein Bax expression and suppression of Notch-1, HES-1, CyclinD1 and anti-apoptotic protein Bcl-2. These results indicate that MDA-MB-231 cells are susceptible to a GSI, whether alone or in combination with doxorubicin, are correlated with changing of some surrogate marker. This study demonstrates practicability of combined use of GSI and doxorubicin, and together these results encourage new therapeutic method in triple negative breast cancer.
PMCID: PMC4503001  PMID: 26191129
Notch-1; doxorubicin; triple negative breast cancer
23.  Elevation of Soluble Guanylate Cyclase Suppresses Proliferation and Survival of Human Breast Cancer Cells 
PLoS ONE  2015;10(4):e0125518.
Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and β1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCβ1 mRNAs. However, levels of sGCβ1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCβ1 in MDA-MB-231 cells but only sGCβ1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCβ1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCβ1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.
PMCID: PMC4416047  PMID: 25928539
24.  Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion 
Oncotarget  2015;6(12):10473-10486.
The neuronal membrane protein sortilin has been reported in a few cancer cell lines, but its expression and impact in human tumors is unclear. In this study, sortilin was analyzed by immunohistochemistry in a series of 318 clinically annotated breast cancers and 53 normal breast tissues. Sortilin was detected in epithelial cells, with increased levels in cancers, as compared to normal tissues (p = 0.0088). It was found in 79% of invasive ductal carcinomas and 54% of invasive lobular carcinomas (p < 0.0001). There was an association between sortilin expression and lymph node involvement (p = 0.0093), suggesting a relationship with metastatic potential. In cell culture, sortilin levels were higher in cancer cell lines compared to non-tumorigenic breast epithelial cells and siRNA knockdown of sortilin inhibited cancer cell adhesion, while proliferation and apoptosis were not affected. Breast cancer cell migration and invasion were also inhibited by sortilin knockdown, with a decrease in focal adhesion kinase and SRC phosphorylation. In conclusion, sortilin participates in breast tumor aggressiveness and may constitute a new therapeutic target against tumor cell invasion.
PMCID: PMC4496368  PMID: 25871389
breast cancer; sortilin; protein expression; cell adhesion; cell invasion
25.  Proteomic Analysis of Copper-Binding Proteins in Excess Copper-Stressed Roots of Two Rice (Oryza sativa L.) Varieties with Different Cu Tolerances 
PLoS ONE  2015;10(4):e0125367.
To better understand the mechanisms involved in the heavy metal stress response and tolerance in plants, a proteomic approach was used to investigate the differences in Cu-binding protein expression in Cu-tolerant and Cu-sensitive rice varieties. Cu-binding proteins from Cu-treated rice roots were separated using a new IMAC method in which an IDA-sepharose column was applied prior to the Cu-IMAC column to remove metal ions from protein samples. More than 300 protein spots were reproducibly detected in the 2D gel. Thirty-five protein spots exhibited changes greater than 1.5-fold in intensity compared to the control. Twenty-four proteins contained one or more of nine putative metal-binding motifs reported by Smith et al., and 19 proteins (spots) contained one to three of the top six motifs reported by Kung et al. The intensities of seven protein spots were increased in the Cu-tolerant variety B1139 compared to the Cu-sensitive variety B1195 (p<0.05) and six protein spots were markedly up-regulated in B1139, but not detectable in B1195. Four protein spots were significantly up-regulated in B1139, but unchanged in B1195 under Cu stress. In contrast, two protein spots were significantly down-regulated in B1195, but unchanged in B1139. These Cu-responsive proteins included those involved in antioxidant defense and detoxification (spots 5, 16, 21, 22, 28, 29 and 33), pathogenesis (spots 5, 16, 21, 22, 28, 29 and 33), regulation of gene transcription (spots 8 and 34), amino acid synthesis (spots 8 and 34), protein synthesis, modification, transport and degradation (spots 1, 2, 4, 10, 15, 19, 30, 31, 32 and 35), cell wall synthesis (spot 14), molecular signaling (spot 3), and salt stress (spots 7, 9 and 27); together with other proteins, such as a putative glyoxylate induced protein, proteins containing dimeric alpha-beta barrel domains, and adenosine kinase-like proteins. Our results suggest that these proteins, together with related physiological processes, play an important role in the detoxification of excess Cu and in maintaining cellular homeostasis.
PMCID: PMC4412397  PMID: 25919452

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