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1.  The Phosphodiesterase-4 (PDE4) Inhibitor Rolipram Decreases Ethanol Seeking and Consumption in Alcohol-preferring Fawn-Hooded Rats 
Background
Alcohol dependence is a complex psychiatric disorder demanding development of novel pharmacotherapies. Since the cyclic AMP (cAMP) signaling cascade has been implicated in mediating behavioral responses to alcohol, key components in this cascade may serve as potential treatment targets. Phosphodiesterase 4 (PDE4), an enzyme that specifically catalyzes the hydrolysis of cAMP, represents as a key point in regulating intracellular cAMP levels. Thus, it was of interest to determine whether PDE4 was involved in the regulation of alcohol use and abuse.
Methods
Male Fawn-Hooded (FH/Wjd) rats were tested for 5% (v/v) ethanol and 10% (w/v) sucrose operant oral self-administration following treatment with the selective PDE4 inhibitor rolipram (0.0125, 0.025, or 0.05 mg/kg, s.c.); rolipram at higher doses (0.05, 0.1, and 0.2 mg/kg, s.c.) was tested to determine its impact on the intake of ethanol, sucrose, or water using the two-bottle choice drinking paradigm. Subsequent open-field testing was performed to evaluate the influence of higher doses of rolipram on locomotor activity.
Results
Acute administration of rolipram dose-dependently reduced operant self-administration of 5% ethanol, but had no effect on 10% sucrose responding. Time-course assessment revealed significant decreases in ethanol consumption after rolipram (0.1, 0.2 mg/kg) treatment in continuous- and intermittent-access to ethanol at 5% or 10%, respectively. Moreover, chronic rolipram treatment time-dependently decreased 5% ethanol consumption and preference during treatment days and after the termination of rolipram administration. Rolipram at the highest doses (0.1 and 0.2 mg/kg) did decrease locomotor activity, but the effect lasted only 10 and 20 min, respectively, which did not likely alter long-term ethanol drinking.
Conclusions
These results suggest that PDE4 plays a role in alcohol seeking and consumption behavior. Drugs interfering with PDE4 may be a potential pharmacotherapy for alcohol dependence.
doi:10.1111/j.1530-0277.2012.01845.x
PMCID: PMC4335658  PMID: 22671516
Cyclic AMP Signaling; Phosphodiesterase-4 (PDE4); Rolipram; FH/Wjd Rat; Ethanol Intake
2.  Expanding Chemical Diversity of Conotoxins: Peptoid-Peptide Chimeras of the Sodium Channel Blocker µ-KIIIA and its Selenopeptide Analogues 
The µ-conotoxin KIIIA is a three disulfide-bridged blocker of voltage-gated sodium channels (VGSCs). The Lys7 residue in KIIIA is an attractive target for manipulating the selectivity and efficacy of this peptide. Here, we report the design and chemical synthesis of µ-conopeptoid analogues (peptomers) in which we replaced Lys7 with peptoid monomers of increasing side-chain size: N-methylglycine, N-butylglycine and N-octylglycine. In the first series of analogues, the peptide core contained all three disulfide bridges; whereas in the second series, a disulfide-depleted selenoconopeptide core was used to simplify oxidative folding. The analogues were tested for functional activity in blocking the Nav1.2 subtype of mammalian VGSCs exogenously expressed in Xenopus oocytes. All six analogues were active, with the N-methylglycine analogue, [Sar7]KIIIA, the most potent in blocking the channels while favoring lower efficacy. Our findings demonstrate that the use of N-substituted Gly residues in conotoxins show promise as a tool to optimize their pharmacological properties as potential analgesic drug leads.
doi:10.1016/j.ejmech.2013.04.041
PMCID: PMC4332556  PMID: 23707919
µ-conotoxins; KIIIA, peptomers; selenocysteine; diselenide; sodium channels; electrophysiology
3.  Comparison of single versus repeated methamphetamine injection induced behavioral sensitization in mice 
Neuroscience letters  2013;560:103-106.
Repeated exposure to drugs of abuse produces a persistent behavioral sensitization to stimulants, which is often used to study drug-associated behavioral plasticity. Interestingly, even a single exposure to some drugs of abuse is sufficient to elicit long-lasting behavioral sensitization. However, few studies have directly compared the magnitude of sensitization between single versus repeated drug treatments. This study examined the magnitude and duration of single methamphetamine (METH) injection-induced behavioral sensitization and compared it to the more typical repeated drug injection-induced sensitization in mice. Different groups of mice were injected with METH (0.5, 1.0, 2.0 mg/kg, i.p.) only once or daily for 7 consecutive days. A challenge dose of METH (1.0 mg/kg, i.p.) was tested 7 days later. The time-course of a single METH injection-induced behavioral sensitization was assessed where METH (2.0 mg/kg, i.p.) was injected and a challenge dose of METH (1.0 mg/kg, i.p.) was tested after different drug-free periods. Single METH injection produced similar magnitude of behavioral sensitization as compared to repeated injection. Such a sensitized locomotor response peaked 8 days after METH injection and lasted for at least 21 days. This long lasting behavioral alteration induced by single METH injection suggests the value of future studies to explore the underlying neural mechanisms, particularly in comparison to those underlying repeated METH-induced sensitization.
doi:10.1016/j.neulet.2013.12.024
PMCID: PMC4017776  PMID: 24361545
Methamphetamine; Behavioural sensitization; Single-dose injection; Repeated-dose injection
4.  In Vivo and In Vitro Metabolites from the Main Diester and Monoester Diterpenoid Alkaloids in a Traditional Chinese Herb, the Aconitum Species 
Diester diterpenoid alkaloids (DDAs), such as aconitine (AC), mesaconitine (MA), and hypaconitine (HA), are both pharmacologically active compounds and toxic ingredients in a traditional Chinese herb, the Aconitum species. Many DDA metabolism studies have been performed to explore mechanisms for reducing toxicity in these compounds and in Aconitum species extracts for safe clinical administration. In this review, we summarize recent progress on the metabolism of toxic AC, MA, and HA and corresponding monoester diterpenoid alkaloids (MDAs) in the gastrointestinal tract and liver in different animal species and humans in vivo and/or in vitro, where these alkaloids are primarily metabolized by cytochrome P450 enzymes, carboxylesterases, and intestinal bacteria, which produces phase I metabolites, ester hydrolysed products, and lipoalkaloids. Furthermore, we classify metabolites detected in the blood and urine, where the aforementioned metabolites are absorbed and excreted. Less toxic MDAs and nontoxic alcohol amines are the primary DDA metabolites detected in the blood. Most other DDAs metabolites produced in the intestine and liver detected in the urine have not been reported in the blood. We propose an explanation for this nonconformity. Finally, taking AC, for instance, we generalize a process of toxicity reduction in the body after oral AC administration for the first time.
doi:10.1155/2015/252434
PMCID: PMC4332761
5.  Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription 
Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3 week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth.
Highlights
•Cardiomyocyte cell cycling is under redox control in vivo.•Nox4 is a potential source of ROS regulating this process.•Redox activation of ERK1/2 promotes cyclin D2 transcription via c-myc activation.•Promotion of proliferation via increased cyclin D2 may be useful therapeutically.
doi:10.1016/j.yjmcc.2014.10.017
PMCID: PMC4312357  PMID: 25450615
BrdU, bromodeoxyuridine; AdNox4, adenoviral Nox4; AdβGal, adenoviral β-galactosidase; PEG, polyethylene glycol; Wt, wild type; Tg, transgenic; NRC, neonatal rat cardiomyocyte; Cardiomyocyte proliferation; ERK1/2; Nox4; Cyclin D2; Redox signalling; c-Myc
6.  Cooperative Interaction of trp Melastatin Channel TRPM2 with its Splice Variant TRPM2-S is Essential for Endothelial Cell Apoptosis 
Circulation research  2013;114(3):469-479.
Rationale:
Oxidants generated by activated endothelial cells are known to induce apoptosis, a pathogenic feature of vascular injury and inflammation from multiple etiologies. The melastatin-family transient receptor potential 2 (TRPM2) channel is an oxidant-sensitive Ca2+ permeable channel implicated in mediating apoptosis; however, the mechanisms of gating of the supra-normal Ca2+ influx required for initiating of apoptosis are not understood.
Objective:
Here we addressed the role TRPM2 and its interaction with the short splice variant TRPM2-S in mediating the Ca2+ entry burst required for induction of endothelial cell apoptosis.
Methods and Results:
We observed that TRPM2-S was basally associated with TRPM2 in the endothelial plasmalemma and this interaction functioned to constitutively suppress TRPM2-dependent Ca2+ gating. ROS production in endothelial cells or directly applying ROS induced PKCα activation and phosphorylation of TRPM2 at Ser 39. This in turn stimulated a large entry of Ca2+ and activated the apoptosis pathway. A similar TRPM2-dependent endothelial apoptosis mechanism was seen in intact vessels. The PKCα-activated phospho-switch opened the TRPM2 channel to allow large Ca2+ influx by releasing TRPM2-S inhibition of TRPM2, which in turn activated caspase-3 and cleaved the caspase substrate poly(ADP-ribose) polymerase.
Conclusions:
Here we describs a fundamental mechanism by which activation of the trp super-family TRPM2 channel induces apoptosis of endothelial cells. The signaling mechanism involves ROS-induced PKCα activation resulting in phosphorylation of TRPM2-S that allows enhanced TRPM2-mediated gating of Ca2+ and activation of the apoptosis program. Strategies aimed at preventing the uncoupling of TRPM2-S from TRPM2 and subsequent Ca2+ gating during oxidative stress may mitigate endothelial apoptosis and its consequences in mediating vascular injury and inflammation.
doi:10.1161/CIRCRESAHA.114.302414
PMCID: PMC3978731  PMID: 24337049
Apoptosis; oxidant-activated cation channel; endothelium; vascular permeability; inflammation; oxidant signaling
7.  Recharge and Groundwater Use in the North China Plain for Six Irrigated Crops for an Eleven Year Period 
PLoS ONE  2015;10(1):e0115269.
Water tables are dropping by approximately one meter annually throughout the North China Plain mainly due to water withdrawals for irrigating winter wheat year after year. In order to examine whether the drawdown can be reduced we calculate the net water use for an 11 year field experiment from 2003 to 2013 where six irrigated crops (winter wheat, summer maize, cotton, peanuts, sweet potato, ryegrass) were grown in different crop rotations in the North China Plain. As part of this experiment moisture contents were measured each at 20 cm intervals in the top 1.8 m. Recharge and net water use were calculated based on these moisture measurement. Results showed that winter wheat and ryegrass had the least recharge with an average of 27 mm/year and 39 mm/year, respectively; cotton had the most recharge with an average of 211 mm/year) followed by peanuts with 118 mm/year, sweet potato with 76 mm/year, and summer maize with 44 mm/year. Recharge depended on the amount of irrigation water pumped from the aquifer and was therefore a poor indicator of future groundwater decline. Instead net water use (recharge minus irrigation) was found to be a good indicator for the decline of the water table. The smallest amount of net (ground water) used was cotton with an average of 14 mm/year, followed by peanut with 32 mm/year, summer maize with 71 mm/year, sweet potato with 74 mm/year. Winter wheat and ryegrass had the greatest net water use with the average of 198 mm/year and 111 mm/year, respectively. Our calculations showed that any single crop would use less water than the prevalent winter wheat summer maize rotation. This growing one crop instead of two will reduce the decline of groundwater and in some rain rich years increase the ground water level, but will result in less income for the farmers.
doi:10.1371/journal.pone.0115269
PMCID: PMC4308074  PMID: 25625765
8.  In vivo Molecular Imaging and Radionuclide (131I) Therapy of Human Nasopharyngeal Carcinoma Cells Transfected with a Lentivirus Expressing Sodium Iodide Symporter 
PLoS ONE  2015;10(1):e0116531.
Introduction
Despite recent improvements in the survival rates for nasopharyngeal carcinoma (NPC), novel treatment strategies are required to improve distant metastasis-free survival. The sodium iodine symporter (NIS) gene has been applied for in vivo imaging and cancer therapy. In this study, we examined the potential of NIS gene therapy as a therapeutic approach in NPC by performing non-invasive imaging using 125I and 131I therapy in vivo.
Methods
We constructed a lentiviral vector expressing NIS and enhanced green fluorescent protein (EGFP) under the control of the human elongation factor-1α (EF1α) promoter, and stably transfected the vector into CNE-2Z NPC cells to create CNE-2Z-NIS cells. CNE-2Z and CNE-2Z-NIS tumor xenografts were established in nude mice; 125I uptake, accumulation and efflux were measured using micro-SPECT/CT imaging; the therapeutic effects of treatment with 131I were assessed over 25 days by measuring tumor volume and immunohistochemical staining of the excised tumors.
Results
qPCR, immunofluorescence and Western blotting confirmed that CNE-2Z-NIS cells expressed high levels of NIS mRNA and protein. CNE-2Z-NIS cells and xenografts took up and accumulated significantly more 125I than CNE-2Z cells and xenografts. In vitro, 131I significantly reduced the clonogenic survival of CNE-2Z-NIS cells. In vivo, 131I effectively inhibited the growth of CNE-2Z-NIS xenografts. At the end of 131I therapy, CNE-2Z-NIS xenograft tumor cells expressed higher levels of NIS and caspase-3 and lower levels of Ki-67.
Conclusion
Lentiviruses effectively delivered and mediated long-lasting expression of NIS in CNE-2Z cells which enabled uptake and accumulation of radioisotopes and provided a significant therapeutic effect in an in vivo model of NPC. NIS-mediated radioiodine treatment merits further investigation as a potentially effective, low toxicity therapeutic strategy for NPC.
doi:10.1371/journal.pone.0116531
PMCID: PMC4306548  PMID: 25621996
9.  Effect of Exposure to Atmospheric Ultrafine Particles on Production of Free Fatty Acids and Lipid Metabolites in the Mouse Small Intestine 
Background: Exposure to ambient ultrafine particulate matter (UFP) is a well-recognized risk factor for cardiovascular and respiratory diseases. However, little is known about the effects of air pollution on gastrointestinal disorders.
Objective: We sought to assess whether exposure to ambient UFP (diameter < 180 nm) increased free fatty acids and lipid metabolites in the mouse small intestine.
Methods: Ldlr-null mice were exposed to filtered air (FA) or UFP collected at an urban Los Angeles, California, site that was heavily affected by vehicular emissions; the exposure was carried out for 10 weeks in the presence or absence of D-4F, an apolipoprotein A-I mimetic peptide with antioxidant and anti-inflammation properties on a high-fat or normal chow diet.
Results: Compared with FA, exposure to UFP significantly increased intestinal hydroxyeicosatetraenoic acids (HETEs), including 15-HETE, 12-HETE, 5-HETE, as well as hydroxyoctadecadienoic acids (HODEs), including 13-HODE and 9-HODE. Arachidonic acid (AA) and prostaglandin D2 (PGD2) as well as some of the lysophosphatidic acids (LPA) in the small intestine were also increased in response to UFP exposure. Administration of D-4F significantly reduced UFP-mediated increase in HETEs, HODEs, AA, PGD2, and LPA. Although exposure to UFP further led to shortened villus length accompanied by prominent macrophage and neutrophil infiltration into the intestinal villi, administration of D-4F mitigated macrophage infiltration.
Conclusions: Exposure to UFP promotes lipid metabolism, villus shortening, and inflammatory responses in mouse small intestine, whereas administration of D-4F attenuated these effects. Our findings provide a basis to further assess the mechanisms underlying UFP-mediated lipid metabolism in the digestive system with clinical relevance to gut homeostasis and diseases.
Citation: Li R, Navab K, Hough G, Daher N, Zhang M, Mittelstein D, Lee K, Pakbin P, Saffari A, Bhetraratana M, Sulaiman D, Beebe T, Wu L, Jen N, Wine E, Tseng CH, Araujo JA, Fogelman A, Sioutas C, Navab M, Hsiai TK. 2015. Effect of exposure to atmospheric ultrafine particles on production of free fatty acids and lipid metabolites in the mouse small intestine. Environ Health Perspect 123:34–41; http://dx.doi.org/10.1289/ehp.1307036
doi:10.1289/ehp.1307036
PMCID: PMC4286268  PMID: 25170928
10.  Development of a poly (ether urethane) system for the controlled release of two novel anti-biofilm agents based on gallium or zinc and its efficacy to prevent bacterial biofilm formation 
Traditional antibiotic therapy to control medical device-based infections typically fails to clear biofilm infections and may even promote the evolution of antibiotic resistant species. We report here the development of two novel antibiofilm agents; gallium (Ga) or zinc (Zn) complexed with protoporphyrin IX (PP) or mesoprotoporphyrin IX (MP) that are both highly effective in negating suspended bacterial growth and biofilm formation. These chelated gallium or zinc complexes act as iron siderophore analogs, surplanting the natural iron uptake of most bacteria. Poly (ether urethane) (PEU; Biospan®) polymer films were fabricated for the controlled sustained release of the Ga- or Zn-complexes, using an incorporated pore-forming agent, poly (ethylene glycol) (PEG). An optimum formulation containing 8% PEG (MW=1450) in the PEU polymer effectively sustained drug release for at least 3 months. All drug-loaded PEU films exhibited in vitro ≥ 90% reduction of Gram-positive (Staphylococcus epidermidis) and Gram-negative (Pseudomonas aeruginosa) bacteria in both suspended and biofilm culture versus the negative control PEU films releasing nothing. Cytotoxicity and endotoxin evaluation demonstrated no adverse responses to the Ga- or Zn-complex releasing PEU films. Finally, in vivo studies further substantiate the anti-biofilm efficacy of the PEU films releasing Ga- or Zn- complexes.
doi:10.1016/j.jconrel.2013.10.005
PMCID: PMC3858484  PMID: 24140747
Anti-biofilm biomaterials; interrupting iron metabolism, gallium and zinc siderophores, poly (ether urethane); drug release; Staphylococcus epidermidis; Pseudomonas aeruginosa
11.  Overexpression of CD90 (Thy-1) in Pancreatic Adenocarcinoma Present in the Tumor Microenvironment 
PLoS ONE  2014;9(12):e115507.
CD90 (Thy-1) plays important roles in oncogenesis and shows potential as a candidate marker for cancer stem cells (CSCs) in various malignancies. Herein, we investigated the expression of CD90 in pancreatic adenocarcinoma (PDAC), with a comparison to normal pancreas and non-malignant pancreatic disease, by immunohistochemical (IHC) analysis of tissue microarrays containing 183 clinical tissue specimens. Statistical analysis was performed to evaluate the correlation between CD90 expression and the major clinicopathological factors after adjustment of age and gender. The IHC data showed that CD90 was significantly overexpressed in PDAC and its metastatic cancers as compared to chronic pancreatitis and benign islet tumors, while it was negative in normal pancreas and 82.7% of adjacent normal pancreas tissues. The abundant CD90 expression was predominantly present in PDAC stroma, such as fibroblasts and vascular endothelial cells, which could serve as a promising marker to distinguish pancreatic adenocarcinoma from normal pancreas and non-malignant pancreatic diseases. Double immunostaining of CD90 with CD24, a CSC marker for PDAC, showed that there was little overlap between these two markers. However, CD90+ fibroblast cells were clustered around CD24+ malignant ducts, suggesting that CD90 may be involved in the tumor-stroma interactions and promote pancreatic cancer development. Furthermore, CD90 mostly overlapped with α-smooth muscle actin (αSMA, a marker of activated pancreatic stellate cells (PSCs)) in PDAC stroma, which demonstrated that CD90+ stromal cells consist largely of activated PSCs. Double immunostaining of CD90 and a vascular endothelial cell marker CD31 demonstrated that CD90 expression on vascular endothelial cells was significantly increased in PDACs as compared to normal pancreas and non-malignant pancreatic diseases. Our findings suggest that CD90 could serve as a promising marker for pancreatic adenocarcinoma where desmoplastic stroma plays an important role in tumor growth and angiogenesis.
doi:10.1371/journal.pone.0115507
PMCID: PMC4275230  PMID: 25536077
12.  Optimal contouring of seminal vesicle for definitive radiotherapy of localized prostate cancer: comparison between EORTC prostate cancer radiotherapy guideline, RTOG0815 protocol and actual anatomy 
Background
Intermediate- to-high-risk prostate cancer can locally invade seminal vesicle (SV). It is recommended that anatomic proximal 1-cm to 2-cm SV be included in the clinical target volume (CTV) for definitive radiotherapy based on pathology studies. However, it remains unclear whether the pathology indicated SV extent is included into the CTV defined by current guidelines. The purpose of this study is to compare the volume of proximal SV included in CTV defined by EORTC prostate cancer radiotherapy guideline and RTOG0815 protocol with the actual anatomic volume.
Methods
Radiotherapy planning CT images from 114 patients with intermediate- (36.8%) or high-risk (63.2%) prostate cancer were reconstructed with 1-mm-thick sections. The starting and ending points of SV and the cross sections of SV at 1-cm and 2-cm from the starting point were determined using 3D-view. Maximum (D1H, D2H) and minimum (D1L, D2L) vertical distance from these cross sections to the starting point were measured. Then, CTV of proximal SV defined by actual anatomy, EORTC guideline and RTOG0815 protocol were contoured and compared (paired t test).
Results
Median length of D1H, D1L, D2H and D2L was 10.8 mm, 2.1 mm, 17.6 mm and 8.8 mm (95th percentile: 13.5mm, 5.0mm, 21.5mm and 13.5mm, respectively). For intermediate-risk patients, the proximal 1-cm SV CTV defined by EORTC guideline and RTOG0815 protocol inadequately included the anatomic proximal 1-cm SV in 62.3% (71/114) and 71.0% (81/114) cases, respectively. While for high-risk patients, the proximal 2-cm SV CTV defined by EORTC guideline inadequately included the anatomic proximal 2-cm SV in 17.5% (20/114) cases.
Conclusions
SV involvement indicated by pathology studies was not completely included in the CTV defined by current guidelines. Delineation of proximal 1.4 cm and 2.2 cm SV in axial plane may be adequate to include the anatomic proximal 1-cm and 2-cm SV. However, part of SV may be over-contoured.
doi:10.1186/s13014-014-0288-1
PMCID: PMC4299806  PMID: 25526901
Prostate cancer; Radiotherapy; Seminal vesicle; Target delineation; CT reconstruction
13.  Molecular Cloning, Characterization and Positively Selected Sites of the Glutathione S-Transferase Family from Locusta migratoria 
PLoS ONE  2014;9(12):e114776.
Glutathione S-transferases (GSTs) are multifunctional enzymes that are involved in the metabolism of endogenous and exogenous compounds and are related to insecticide resistance. The purpose of this study was to provide new information on the molecular characteristics and the positive selection of locust GSTs. Based on the transcriptome database, we sequenced 28 cytosolic GSTs and 4 microsomal GSTs from the migratory locust (Locusta migratoria). We assigned the 28 cytosolic GSTs into 6 classes—sigma, epsilon, delta, theta, omega and zeta, and the 4 microsomal GSTs into 2 subclasses—insect and MGST3. The tissue- and stage-expression patterns of the GSTs differed at the mRNA level. Further, the substrate specificities and kinetic constants of the cytosolic GSTs differed markedly at the protein level. The results of likelihood ratio tests provided strong evidence for positive selection in the delta class. The result of Bayes Empirical Bayes analysis identified 4 amino acid sites in the delta class as positive selection sites. These sites were located on the protein surface. Our findings will facilitate the elucidation of the molecular characteristics and evolutionary aspects of insect GST superfamily.
doi:10.1371/journal.pone.0114776
PMCID: PMC4259467  PMID: 25486043
14.  Heterologous Expression of Xylanase Enzymes in Lipogenic Yeast Yarrowia lipolytica 
PLoS ONE  2014;9(12):e111443.
To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.
doi:10.1371/journal.pone.0111443
PMCID: PMC4251831  PMID: 25462572
15.  Associations of Diabetes Mellitus, Insulin, Leptin, and Ghrelin With Gastroesophageal Reflux and Barrett's Esophagus 
Gastroenterology  2013;145(6):1237-44.e1-5.
Background & Aims
Insulin and leptin have proliferative and anti-apoptotic effects. Ghrelin promotes gastric emptying and secretion of growth hormone and inhibits inflammation. We assessed whether diabetes mellitus and serum levels of insulin, leptin, and ghrelin are associated with gastroesophageal reflux disease (GERD) and Barrett's esophagus.
Methods
We conducted a case-control study in 822 men undergoing colorectal cancer screening who were recruited to also undergo upper endoscopy. We identified 70 with Barrett's esophagus; 80 additional men with Barrett's esophagus were recruited shortly after their clinical diagnoses. Serum levels of insulin, leptin, and ghrelin were assayed in all 104 fasting men with Barrett's esophagus without diabetes and 271 without diabetes or Barrett's esophagus. Logistic regression was used to estimate the effects of diabetes and levels of insulin, leptin, and ghrelin on GERD and Barrett's esophagus.
Results
Among men with GERD, diabetes was inversely associated with Barrett's esophagus (adjusted odds ratio [OR] = 0.383; 95% confidence interval [CI]: 0.179–0.821). Among nondiabetics, hyperinsulinemia was positively associated with Barrett's esophagus, but the association was attenuated by adjustment for leptin and ghrelin. Leptin was positively associated with Barrett's esophagus, adjusting for obesity, GERD, and levels of insulin and ghrelin (OR for 3rd vs 1st tertile = 3.25; 95% CI: 1.29–8.17); this association was stronger in men with GERD (P = .01 for OR heterogeneity). Ghrelin was positively associated with Barrett's esophagus (OR for an increment of 400 pg/mL = 1.39; 95% CI: 1.09–1.76), but inversely associated with GERD (OR for 3rd vs 1st tertile = 0.364; 95% CI: 0.195–0.680).
Conclusions
Based on a case-control study, leptin was associated with Barrett's esophagus, particularly in men with GERD. Serum insulin level was associated with Barrett's esophagus, but might be mediated by leptin. Serum ghrelin was inversely associated with GERD, as hypothesized, but positively associated with Barrett's esophagus, contrary to our hypothesis. Additional studies are needed in men and women to replicate these findings.
doi:10.1053/j.gastro.2013.08.052
PMCID: PMC3914630  PMID: 23999171
Insulin; Leptin; Ghrelin; Gastroesophageal Reflux
16.  Advances in the study of HLA-restricted epitope vaccines 
Human Vaccines & Immunotherapeutics  2013;9(12):2566-2577.
Vaccination is a proven strategy for protection from disease. An ideal vaccine would include antigens that elicit a safe and effective protective immune response. HLA-restricted epitope vaccines, which include T-lymphocyte epitopes restricted by HLA alleles, represent a new and promising immunization approach. In recent years, research in HLA-restricted epitope vaccines for the treatment of tumors and for the prevention of viral, bacterial, and parasite-induced infectious diseases have achieved substantial progress. Approaches for the improvement of the immunogenicity of epitope vaccines include (1) improving the accuracy of the methods used for the prediction of epitopes, (2) making use of additional HLA-restricted CD8+ T-cell epitopes, (3) the inclusion of specific CD4+ T-cell epitopes, (4) adding B-cell epitopes to the vaccine construction, (5) finding more effective adjuvants and delivery systems, (6) using immunogenic carrier proteins, and (7) using multiple proteins as epitopes sources. In this manuscript, we review recent research into HLA-restricted epitope vaccines.
doi:10.4161/hv.26088
PMCID: PMC4162067  PMID: 23955319
HLA restriction; epitope vaccine; adjuvant; MHC; bioinformatic
17.  Characterization of Nestin-positive stem Leydig cells as a potential source for the treatment of testicular Leydig cell dysfunction 
Cell Research  2014;24(12):1466-1485.
The ability to identify and isolate lineage-specific stem cells from adult tissues could facilitate cell replacement therapy. Leydig cells (LCs) are the primary source of androgen in the mammalian testis, and the prospective identification of stem Leydig cells (SLCs) may offer new opportunities for treating testosterone deficiency. Here, in a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, we observed Nes-GFP+ cells located in the testicular interstitial compartment where SLCs normally reside. We showed that these Nes-GFP+ cells expressed LIFR and PDGFR-α, but not LC lineage markers. We further observed that these cells were capable of clonogenic self-renewal and extensive proliferation in vitro and could differentiate into neural or mesenchymal cell lineages, as well as LCs, with the ability to produce testosterone, under defined conditions. Moreover, when transplanted into the testes of LC-disrupted or aging models, the Nes-GFP+ cells colonized the interstitium and partially increased testosterone production, and then accelerated meiotic and post-meiotic germ cell recovery. In addition, we further demonstrated that CD51 might be a putative cell surface marker for SLCs, similar with Nestin. Taken together, these results suggest that Nes-GFP+ cells from the testis have the characteristics of SLCs, and our study would shed new light on developing stem cell replacement therapy for testosterone deficiency.
doi:10.1038/cr.2014.149
PMCID: PMC4260348  PMID: 25418539
stem Leydig cell; Nestin; self-renewal; multipotency; Leydig cell dysfunction
18.  The Dynamics of Microcystis Genotypes and Microcystin Production and Associations with Environmental Factors during Blooms in Lake Chaohu, China 
Toxins  2014;6(12):3238-3257.
Lake Chaohu, which is a large, shallow, hypertrophic freshwater lake in southeastern China, has been experiencing lake-wide toxic Microcystis blooms in recent decades. To illuminate the relationships between microcystin (MC) production, the genotypic composition of the Microcystis community and environmental factors, water samples and associated environmental data were collected from June to October 2012 within Lake Chaohu. The Microcystis genotypes and MC concentrations were quantified using quantitative real-time PCR (qPCR) and HPLC, respectively. The results showed that the abundances of Microcystis genotypes and MC concentrations varied on spatial and temporal scales. Microcystis exists as a mixed population of toxic and non-toxic genotypes, and the proportion of toxic Microcystis genotypes ranged from 9.43% to 87.98%. Both Pearson correlation and stepwise multiple regressions demonstrated that throughout the entire lake, the abundances of total and toxic Microcystis and MC concentrations showed significant positive correlation with the total phosphorus and water temperature, suggesting that increases in temperature together with the phosphorus concentrations may promote more frequent toxic Microcystis blooms and higher concentrations of MC. Whereas, dissolved inorganic carbon (DIC) was negatively correlated with the abundances of total and toxic Microcystis and MC concentrations, indicating that rising DIC concentrations may suppress toxic Microcystis abundance and reduce the MC concentrations in the future. Therefore, our results highlight the fact that future eutrophication and global climate change can affect the dynamics of toxic Microcystis blooms and hence change the MC levels in freshwater.
doi:10.3390/toxins6123238
PMCID: PMC4280532  PMID: 25474494
Microcystis; microcystin; 16S rDNA; mcyD; qPCR; environmental factors; Lake Chaohu
19.  Design and Experiment of FBG-Based Icing Monitoring on Overhead Transmission Lines with an Improvement Trial for Windy Weather 
Sensors (Basel, Switzerland)  2014;14(12):23954-23969.
A scheme for monitoring icing on overhead transmission lines with fiber Bragg grating (FBG) strain sensors is designed and evaluated both theoretically and experimentally. The influences of temperature and wind are considered. The results of field experiments using simulated ice loading on windless days indicate that the scheme is capable of monitoring the icing thickness within 0–30 mm with an accuracy of ±1 mm, a load cell error of 0.0308v, a repeatability error of 0.3328v and a hysteresis error is 0.026%. To improve the measurement during windy weather, a correction factor is added to the effective gravity acceleration, and the absolute FBG strain is replaced by its statistical average.
doi:10.3390/s141223954
PMCID: PMC4299095  PMID: 25615733
overhead power transmission line; ice monitoring; fiber Bragg grating (FBG); strain sensor
20.  Inhibition of mTORC1 renders cardiac protection against lipopolysaccharide 
Sepsis-induced cardiac dysfunction is a severe clinical problem. It is evident that rapamycin can protect heart from pathological injuries. However, there are no data demonstrating rapamycin reverse cardiac dysfunction induced by sepsis. In this study, Lipopolysaccharide (LPS) was administrated to mice and H9c2 cells. After treatment, we further determined cardiac function by echocardiography, ANP, BNP and inflammatory markers by qPCR and apoptosis by TUNEL staining. Moreover, mTORC1 signaling pathway and Akt activity were measured by Western blots. We found that rapamycin attenuated cardiac dysfunction, increase in ANP and BNP as well as apoptosis induced by LPS both in mice and in H9c2 cells. Unexpectedly, LPS did not significantly affect the mRNA levels of TNF-α and IL-6. Furthermore, rapamycin further reduced the decrease in mTORC1 signaling and Akt activity induced by LPS. In conclusion, rapamycin can protect heart from LPS induced damages by inhibition mTORC1 signaling and elevation of Akt activity.
PMCID: PMC4313975
LPS; cardiac dysfunction; rapamycin; mTORC1
21.  The differential susceptibilities of MCF-7 and MDA-MB-231 cells to the cytotoxic effects of curcumin are associated with the PI3K/Akt-SKP2-Cip/Kips pathway 
Cancer Cell International  2014;14(1):126.
Background
The mechanism underlying the differential cytotoxicity of curcumin in various cancer types, however, remains largely unclear. The aims of this study is to examine the concentration- and time-related effects of curcumin on two different breast cancer cells, MCF-7 and MDA-MB-231, and investigated the functional changes induced by curcumin treatment, as well as their relationship to the PI3K/Akt-SKP2-Cip/Kips pathway.
Methods
First, WST-1 and clonogenic assay were performed to determine the cytotoxicity of curcumin in MCF-7 and MDA-MB-231 cells. Then, the expression of CDK interacting protein/Kinase inhibitory protein (Cip/Kips) members (p27, p21 and p57) and S-phase kinase-associated protein-2 (SKP2) was investigated by QRT PCR and Western Blotting. Curcumin’s effect on PI3K (phosphatidylinositol 3-kinase) /Akt and its substrates Foxo1 and Foxo3a were then studied by Western Blotting. Small interfering RNAs (siRNAs) targeting SKP2 was used to explore the relationship between SKP2 and Cip/Kips members. Finally, WST-1 assay was tested to explore the concomitant treatment with curcumin and the inhibition of PKB or SKP2 signaling on curcumin sensitivity in MCF-7 and MDA-MB-231 cells.
Results
We demonstrated MCF-7 and MDA-MB-231 cells exhibited differential responses to curcumin by WST-1 and clonogenic assay (MDA-MB-231 cells was sensitive, and MCF-7 cells was resistant), which were found to be related to the differential curcumin-mediated regulation of SKP2-Cip/Kips (p21 and p27 but not p57) signaling. The differential cellular responses were further linked to the converse effects of curcumin on PI3K/Akt and its substrates Foxo1 and Foxo3a. Importantly, PI3K inhibitor wortmannin could counteract both curcumin-induced phosphorylation of Akt and up-regulation of SKP2 in MCF-7 cells. Subsequent WST-1 assay demonstrated concomitant treatment with curcumin and wortmannin or SKP2 siRNA not only further augmented curcumin sensitivity in MDA-MB-231 cells but also overcame curcumin resistance in MCF-7 cells.
Conclusions
Our study established PI3K/Akt-SKP2-Cip/Kips signaling pathway is involved in the mechanism of action of curcumin and revealed that the discrepant modulation of this pathway by curcumin is responsible for the differential susceptibilities of these two cell types to curcumin.
doi:10.1186/s12935-014-0126-4
PMCID: PMC4272549  PMID: 25530715
Curcumin sensitivity; Breast cancer; PI3K; SKP2; FOXO; Cip/Kips
22.  Phosphatidylinositol 3-kinase and COPII generate LC3 lipidation vesicles from the ER-Golgi intermediate compartment 
eLife  null;3:e04135.
Formation of the autophagosome requires significant membrane input from cellular organelles. However, no direct evidence has been developed to link autophagic factors and the mobilization of membranes to generate the phagophore. Previously, we established a cell-free LC3 lipidation reaction to identify the ER-Golgi intermediate compartment (ERGIC) as a membrane source for LC3 lipidation, a key step of autophagosome biogenesis (Ge et al., eLife 2013; 2:e00947). We now report that starvation activation of autophagic phosphotidylinositol-3 kinase (PI3K) induces the generation of small vesicles active in LC3 lipidation. Subcellular fractionation studies identified the ERGIC as the donor membrane in the generation of small lipidation-active vesicles. COPII proteins are recruited to the ERGIC membrane in starved cells, dependent on active PI3K. We conclude that starvation activates the autophagic PI3K, which in turn induces the recruitment of COPII to the ERGIC to bud LC3 lipidation-active vesicles as one potential membrane source of the autophagosome.
DOI: http://dx.doi.org/10.7554/eLife.04135.001
doi:10.7554/eLife.04135
PMCID: PMC4270069  PMID: 25432021
autophagy; autophagosome; COPII; ER-Golgi intermediate compartment; LC3 lipidation; Phosphatidylinositol 3-kinase; human; mouse
23.  Role of Moesin in Renal Fibrosis 
PLoS ONE  2014;9(11):e112936.
Background
Renal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.
Method
Human proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.
Result
HK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.
Conclusion
We find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin.
doi:10.1371/journal.pone.0112936
PMCID: PMC4236084  PMID: 25406076
24.  The Ratio of Circulating Regulatory T Cells (Tregs)/Th17 Cells Is Associated with Acute Allograft Rejection in Liver Transplantation 
PLoS ONE  2014;9(11):e112135.
CD4+CD25+FoxP3+ regulatory T cells (Tregs) and Th17 cells are known to be involved in the alloreactive responses in organ transplantation, but little is known about the relationship between Tregs and Th17 cells in the context of liver alloresponse. Here, we investigated whether the circulating Tregs/Th17 ratio is associated with acute allograft rejection in liver transplantation. In present study, thirty-eight patients who received liver transplant were enrolled. The patients were divided into two groups: acute allograft rejection group (Gr-AR) (n = 16) and stable allograft liver function group (Gr-SF) (n = 22). The frequencies of circulating Tregs and circulating Th17 cells, as well as Tregs/Th17 ratio were determined using flow cytometry. The association between Tregs/Th17 ratio and acute allograft rejection was then analyzed. Our results showed that the frequency of circulating Tregs was significantly decreased, whereas the frequency of circulating Th17 cells was significantly increased in liver allograft recipients who developed acute rejection. Tregs/Th17 ratio had a negative correlation with liver damage indices and the score of rejection activity index (RAI) after liver transplantation. In addition, the percentages of CTLA-4+, HLA-DR+, Ki67+, and IL-10+ Tregs were higher in Gr-SF group than in Gr-AR group. Our results suggested that the ratio of circulating Tregs/Th17 cells is associated with acute allograft rejection, thus the ratio may serve as an alternative marker for the diagnosis of acute rejection.
doi:10.1371/journal.pone.0112135
PMCID: PMC4221545  PMID: 25372875
25.  Liver myofibroblasts from hepatitis B related liver failure patients may regulate natural killer cell function via PGE2 
Background
Natural killer (NK) cells are abundant in the liver and constitute a major innate immune component that contributes to immune-mediated liver injury. However, few studies have investigated the phenotypes and functions of NK cells involved in hepatitis B related liver failure (LF), and the precise mechanism underlying NK cell regulation is not fully understood.
Methods
We detected the percentage and function of peripheral NK cells both in hepatitis B related LF patients and healthy volunteers by flow cytometry and isolated the liver myofibroblasts (LMFs) from hepatitis B related LF livers. To determine the possible effects of LMFs on NK cells, mixed cell cultures were established in vitro.
Results
We found a down-regulated percentage of peripheral NK cells in hepatitis B related LF patients, and their NK cells also displayed decreased activated natural cytotoxicity receptors (NCRs) and cytokine production. In a co-culture model, LMFs sharply attenuated IL-2-induced NK cell triggering receptors, cytotoxicity, and cytokine production. The inhibitory effect of LMFs on NK cells correlated with their ability to produce prostaglandin (PG) E2.
Conclusion
These data suggest that LMFs may protect against immune-mediated liver injury in hepatitis B related LF patients by inhibiting NK cell function via PGE2.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-014-0308-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12967-014-0308-9
PMCID: PMC4232720  PMID: 25367326
Liver myofibroblasts; Natural killer cell; Immune-mediated liver injury; Liver failure; Hepatitis B

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