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1.  Mitochondrial CB1 receptor is involved in ACEA-induced protective effects on neurons and mitochondrial functions 
Scientific Reports  2015;5:12440.
Mitochondrial dysfunction contributes to cell death after cerebral ischemia/reperfusion (I/R) injury. Cannabinoid CB1 receptor is expressed in neuronal mitochondrial membranes (mtCB1R) and involved in regulating mitochondrial functions under physiological conditions. However, whether mtCB1R affords neuroprotection against I/R injury remains unknown. We used mouse models of cerebral I/R, primary cultured hippocampal neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) and Ca2+-induced injury in purified neuronal mitochondria to investigate the role of mtCB1R in neuroprotection. Our results showed selective cell-permeant CB1 receptor agonist, arachidonyl-2-chloroethylamide (ACEA), significantly up-regulated the expression of mtCB1R protein in hippocampal neurons and tissue. In vitro, ACEA restored cell viability, inhibited generation of reactive oxygen species (ROS), decreased lactate dehydrogenase (LDH) release and reduced apoptosis, improved mitochondrial function. In vivo, ACEA ameliorated neurological scores, diminished the number of TUNEL-positive neurons and decreased the expression of cleaved caspase-3. However, ACEA-induced benefits were blocked by the selective cell-permeant CB1 receptor antagonist AM251, but just partially by the selective cell-impermeant CB1 receptor antagonist hemopressin. In purified neuronal mitochondria, mtCB1R activation attenuated Ca2+-induced mitochondrial injury. In conclusion, mtCB1R is involved in ACEA-induced protective effects on neurons and mitochondrial functions, suggesting mtCB1R may be a potential novel target for the treatment of brain ischemic injury.
doi:10.1038/srep12440
PMCID: PMC4516969  PMID: 26215450
2.  Remodeling the Dendritic Spines in the Hindlimb Representation of the Sensory Cortex after Spinal Cord Hemisection in Mice 
PLoS ONE  2015;10(7):e0132077.
Spinal cord injury (SCI) can induce remodeling of multiple levels of the cerebral cortex system especially in the sensory cortex. The aim of this study was to assess, in vivo and bilaterally, the remodeling of dendritic spines in the hindlimb representation of the sensory cortex after spinal cord hemisection. Thy1-YFP transgenic mice were randomly divided into the control group and the SCI group, and the spinal vertebral plates (T11–T12) of all mice were excised. Next, the left hemisphere of the spinal cord (T12) was hemisected in the SCI group. The hindlimb representations of the sensory cortex in both groups were imaged bilaterally on the day before (0d), and three days (3d), two weeks (2w), and one month (1m) after the SCI. The rates of stable, newly formed, and eliminated spines were calculated by comparing images of individual dendritic spine in the same areas at different time points. In comparison to the control group, the rate of newly formed spines in the contralateral sensory cortex of the SCI group increased at three days and two weeks after injury. The rates of eliminated spines in the bilateral sensory cortices increased and the rate of stable spines in the bilateral cortices declined at two weeks and one month. From three days to two weeks, the stable rates of bilaterally stable spines in the SCI group decreased. In comparison to the control group and contralateral cortex in the SCI group, the re-emerging rate of eliminated spines in ipsilateral cortex of the SCI group decreased significantly. The stable rates of newly formed spines in bilateral cortices of the SCI group decreased from two weeks to one month. We found that the remodeling in the hindlimb representation of the sensory cortex after spinal cord hemisection occurred bilaterally. This remodeling included eliminating spines and forming new spines, as well as changing the reorganized regions of the brain cortex after the SCI over time. Soon after the SCI, the cortex was remodeled by increasing spine formation in the contralateral cortex. Then it was remodeled prominently by eliminating spines of bilateral cortices. Spinal cord hemisection also caused traditional stable spines to become unstable and led the eliminated spines even more hard to recur especially in the ipsilateral cortex of the SCI group. In addition, it also made the new formed spines unstable.
doi:10.1371/journal.pone.0132077
PMCID: PMC4489092  PMID: 26132157
3.  Pedican: an online gene resource for pediatric cancers with literature evidence 
Scientific Reports  2015;5:11435.
Pediatric cancer (PC), that is cancer occurring in children, is the leading cause of death among children worldwide, with an incidence of 175,000 per year. Elucidating the genetic abnormalities and underlying cellular mechanisms may provide less toxic curative treatments. Therefore, it is important to understand the pathology of pediatric cancer at the genetic, genomic and epigenetic level. To unveil the cellular complexity of PC, we have developed a database of pediatric cancers (Pedican), the first literature-based pediatric gene data resource by comprehensive literature curation and data integration. In the current release, Pedican contains 735 human genes, 88 gene fusion and 24 chromosome abnormal events curated from 2245 PubMed abstracts. Pedican provides detailed annotations for each gene, such as Entrez gene information, involved pathways, protein–protein interactions, mutations, gene expression, methylation sites, TF regulation, and post-translational modification. Additionally Pedican has a user-friendly web interface, which allows sophisticated text query, sequence searches, and browsing by highlighted literature evidence and hundreds of cancer types. Overall, our curated pediatric cancer-related gene list maps the genomic and cellular landscape for various pediatric cancers, providing a valuable resource for further experiment design. The Pedican is available at http://pedican.bioinfo-minzhao.org/.
doi:10.1038/srep11435
PMCID: PMC4466794  PMID: 26073932
4.  Sleep promotes branch-specific formation of dendritic spines after learning 
Science (New York, N.Y.)  2014;344(6188):1173-1178.
How sleep helps learning and memory remains unknown. We report in mouse motor cortex that sleep after motor learning promotes the formation of postsynaptic dendritic spines on a subset of branches of individual layer V pyramidal neurons. New spines are formed on different sets of dendritic branches in response to different learning tasks and are protected from being eliminated when multiple tasks are learned. Neurons activated during learning of a motor task are reactivated during subsequent non-rapid eye movement sleep, and disrupting this neuronal reactivation prevents branch-specific spine formation. These findings indicate that sleep has a key role in promoting learning-dependent synapse formation and maintenance on selected dendritic branches, which contribute to memory storage.
doi:10.1126/science.1249098
PMCID: PMC4447313  PMID: 24904169
5.  Discovery of Naphthyl-Fused 5-Membered Lactams as a New Class of M1 Positive Allosteric Modulators 
ACS Medicinal Chemistry Letters  2014;5(5):604-608.
Selective activation of the M1 muscarinic receptor via positive allosteric modulation represents an original approach to treat the cognitive decline in patients with Alzheimer’s disease. A series of naphthyl-fused 5-membered lactams were identified as a new class of M1 positive allosteric modulators and were found to possess good potency and in vivo efficacy.
doi:10.1021/ml500055h
PMCID: PMC4027734  PMID: 24900888
M1; muscarinic; positive allosteric modulators; Alzheimer’s disease; acetylcholine
6.  Effect of the beta-3 adrenergic receptor Trp64Arg and uncoupling protein 1–3826 A > G genotypes on lipid and apolipoprotein levels in overweight/obese and non-obese Chinese subjects 
Background
The beta-3 adrenergic receptor (β3-AR) Trp64Arg and uncoupling protein 1 (UCP1) -3826 A > G polymorphisms have been reported to be associated with obesity and/or lipid metabolism in some populations. This study examined the possible association of the β3-AR and UCP1 polymorphisms with overweight/obesity or lipid variation in a Southwest Chinese population.
Methods
A total of 418 Han Chinese (249 overweight/obese and 169 healthy control subjects) in the Chengdu area were studied using PCR-RFLP analysis. Total serum cholesterol (TC) and triglycerides (TGs) were measured using an enzymatic method. High density lipoprotein cholesterol (HDL-C) was determined after sodium phosphotungstate/magnesium chloride precipitation of low-density lipoproteins by polyvinyl sulfate. Serum apolipoproteins were quantified by radial immunodiffusion.
Results
The genotype and allele frequencies of the β3-AR Trp64Arg and UCP1 -3826 A > G polymorphisms in overweight/obese subjects exhibited no significant differences compared to the controls. However, subjects carrying the β3-AR TrpTrp genotype and UCP1 AG genotype had higher TG levels than those carrying the Arg allele and AA genotype, respectively (P < 0.05), while controls carrying the β3-AR Arg allele had significantly higher TC and apo AII concentrations than those carrying the TrpTrp genotype (P < 0.05). Additionally, subjects carrying the UCP1 AG genotype exhibited elevated apo C-II and apo C-III levels compared to those carrying the AA genotype (P < 0.05). We were unable to find an association of the UCP1 and β3-AR polymorphisms with low HDL-cholesterolemia in the overweight/obese subjects.
Conclusions
The present study provides evidence that the β3-AR Trp64Arg and UCP1 -3826 A > G polymorphisms are associated with TG levels in overweight/obese Chinese subjects and that the two polymorphisms are also associated with certain lipid and apolipoprotein variations, depending on BMI. However, these polymorphisms are not associated with overweight/obesity or low HDL-cholesterolemia in a Chinese population from the Chengdu area.
doi:10.1186/s12944-015-0029-y
PMCID: PMC4410578  PMID: 25928572
Overweight/obese; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \beta $$\end{document}β3-AR; UCP1; Gene polymorphism; PCR-RFLP
7.  Impact of Yangtze River Water Transfer on the Water Quality of the Lixia River Watershed, China 
PLoS ONE  2015;10(4):e0119720.
To improve water quality and reduce the negative impacts of sudden inputs of water pollution in the Lixia River watershed, China, a series of experimental water transfers from the Yangtze River to the Lixia River were conducted from 2 December 2006 to 7 January 2007. Water samples were collected every six days at 55 monitoring sites during this period. Eight water parameters (water temperature, pH, dissolved oxygen (DO), chemical oxygen demand (COD), potassium permanganate index (CODMn), ammonia nitrogen (NH4+-N), electrical conductivity (EC), and water transparency (WT)) were analyzed to determine changes in nutrient concentrations during water transfers. The comprehensive pollution index (Pi) and single-factor (Si) evaluation methods were applied to evaluate spatio-temporal patterns of water quality during water transfers. Water quality parameters displayed different spatial and temporal distribution patterns within the watershed. Water quality was improved significantly by the water transfers, especially for sites closer to water intake points. The degree of improvement is positively related to rates of transfer inflow and drainage outflow. The effects differed for different water quality parameters at each site and at different water transfer times. There were notable decreases in NH4+-N, DO, COD, and CODMn across the entire watershed. However, positive effects on EC and pH were not observed. It is concluded that freshwater transfers from the Yangtze River can be used as an emergency measure to flush pollutants from the Lixia River watershed. Improved understanding of the effects of water transfers on water quality can help the development and implementation of effective strategies to improve water quality within this watershed.
doi:10.1371/journal.pone.0119720
PMCID: PMC4383563  PMID: 25835525
8.  Phase 1b Study of New Posaconazole Tablet for Prevention of Invasive Fungal Infections in High-Risk Patients with Neutropenia 
Antimicrobial Agents and Chemotherapy  2014;58(10):5758-5765.
Posaconazole tablets, a new oral formulation of posaconazole, can be effective when given as antifungal prophylaxis to neutropenic patients at high risk for invasive fungal infection (e.g., those with acute myelogenous leukemia or myelodysplastic syndrome). Such effectiveness might be specifically important to patients with poor oral intake because of nausea, vomiting, or chemotherapy-associated mucositis. This was a prospective, global study in high-risk patients to characterize the pharmacokinetics and safety profile of posaconazole tablets and to identify the dose of posaconazole tablets that would provide exposure within a predefined range of exposures (steady-state average concentration [area under the concentration-time curve/24 h] of ≥500 ng/ml and ≤2,500 ng/ml in >90% of patients). The study evaluated two sequential dosing cohorts: 200 mg posaconazole once daily (n = 20) and 300 mg posaconazole once daily (n = 34) (both cohorts had a twice-daily loading dose on day 1) taken without regard to food intake during the neutropenic period for ≤28 days. The exposure target was reached (day 8) in 15 of 19 (79%) pharmacokinetic-evaluable patients taking 200 mg posaconazole once daily and in 31 of 32 (97%) patients taking 300 mg posaconazole once daily; 300 mg posaconazole once daily achieved the desired exposure target. Posaconazole tablets were generally well tolerated in high-risk neutropenic patients. (This study has been registered at ClinicalTrials.gov under registration no. NCT01777763.)
doi:10.1128/AAC.03050-14
PMCID: PMC4187965  PMID: 25049247
9.  Macrophage Migration Inhibitory Factor Interacting with Th17 Cells May Be Involved in the Pathogenesis of Autoimmune Damage in Hashimoto's Thyroiditis 
Mediators of Inflammation  2015;2015:621072.
Purpose. To explore the possible role of MIF and Th17 cells in the thyroid-specific autoimmune damage of Hashimoto's thyroiditis (HT). Material and Methods. We enrolled 40 HT patients and 30 healthy controls and divided HT patients into euthyroid subset (n = 22) and subclinical or overt hypothyroidism subset (n = 18). The percentages of Th17 cells and expressions of MIF, interleukin 17A (IL-17A) mRNA in PBMCs, as well as serum concentrations of MIF, and IL-17A, and thyroid functions, and thyroid-specific autoantibodies (TPOAb, TgAb) were detected by flow cytometry, real-time RT-PCR, ELISA, and ECLIA in all subjects. Results. MIF mRNA, IL-17A mRNA expressions and Th17 cells percentages, serum MIF, and IL-17A protein levels were all significantly higher in HT patients, even in euthyroid subgroup. Additionally, the differences became more obvious in dysfunction subgroup. Importantly, both MIF levels and Th17 cells percentage were positively correlated with serum TPOAb, TgAb, and thyrotropin (TSH) levels in HT patients. Conclusions. These data suggest that MIF and Th17 cells increased dynamically and positively correlated with the markers of thyroid autoimmune damage, which indicated that interaction between MIF and Th17 cells may participate in the pathogenesis and development of thyroid-specific autoimmunity in HT.
doi:10.1155/2015/621072
PMCID: PMC4377496  PMID: 25861163
10.  Prognostic significance of the neutrophil to lymphocyte ratio in patients with non-small cell lung cancer: a systemic review and meta-analysis 
Neutrophil to Lymphocyte Ratio (NLR) was recently demonstrated as a useful index in predicting the prognosis of Non-Small Cell Lung Cancer (NSCLC). Thus, a meta-analysis was performed to demonstrate the relationship between NLR and overall survival (OS), progress-free survival (PFS) or disease free survival (DFS) in patients with NSCLC. We searched for relevant literatures in PubMed, EMBASE and Cochrane library and pooled the eligible studies and synthesized hazard ratios (HRs) using Stata 12.0. Final analysis of NSCLC patients from 12 eligible studies was performed. Combined HR suggested that high NLR had an unfavorable effect on patients’ OS (n=1700 in 11 studies; HR= 1.43, 95% CI: 1.25-1.64; I^2=80.2%, P<0.01) and PFS (n=664 in 5 studies, HR=1.37, 95% CI: 1.07-1.74; I^2=70.8%, P=0.004). Subgroup analysis based on cutoff shown that, compared with other subgroups, the subgroup with a cutoff of 5 had a significantly poorer survival (HR=1.87, 95% CI 1.49-2.34) with less heterogeneity (I^2=21.3%, P=0.28). However, subgroup analysis based on treatment method indicated that the “surgery” subgroup seemed to have not a significant impact on survival (HR=1.32, 95% CI 0.99-1.77; I^2=80.0%, P=0.063) compared with the chemotherapy subgroup (HR=1.61, 95% CI 1.24-2.10; I^2=82.6%, P<0.01). Additionally, combined odds ratio (OR) suggested high NLR was associated inversely with response to treatment (n = 276 in 2 studies; OR = 1.73, 95% CI: 1.04-2.88; I^2=0%, P=0.40). This study suggests high NLR (especially with a cutoff of 5) seems to be associated with a worse prognosis in patients with NSCLC as well as a worse response to treatments.
PMCID: PMC4443032  PMID: 26064198
Neutrophil to lymphocyte ratio; NLR; non-small cell lung cancer; NSCLC; overall survival; progress-free survival; treatment response
11.  Unilateral posterior vertebral column resection for severe thoracolumbar kyphotic deformity caused by old compressive vertebrae fracture: a technical improvement 
Severe thoracolumbar kyphotic deformity caused by old compressive vertebrae fracture remains a big challenge for spine surgeons. When symptoms related to significant deformities cannot be adequately managed conservatively, posterior vertebral column resection (PVCR) is required, but with long operating time and severe blood loss. We develop a UPVCR technique, which is done through a unilateral approach instead of a bilateral approach, vertebral body resection advancing to cross the midline in an abrasive way from an extreme oblique orientation enable the resection of most contralateral vertebral body. In the present study, the effects of UPVCR for severe thoracolumbar kyphotic deformity were investigated. We did find that satisfactory correction of sagittal deformity, functional improvement and pain relief can be achieved by UPVCR, and it has the advantage of shortening surgery time, reducing blood loss and incidence of nerve root impingement over PVCR.
PMCID: PMC4443086  PMID: 26064252
Unilateral posterior vertebral column resection; unilateral approach; less blood loss; short operating time
12.  Systematic identification and characterization of long intergenic non-coding RNAs in fetal porcine skeletal muscle development 
Scientific Reports  2015;5:8957.
Long intergenic non-coding RNAs (lincRNAs) play important roles in many cellular processes. Here, we present the first systematic identification and characterization of lincRNAs in fetal porcine skeletal muscle. We obtained a total of 55.02 million 90-bp paired-end reads and assembled 54,550 transcripts using cufflinks. We developed a pipeline to identify 570 multi-exon lincRNAs by integrating a set of previous approaches. These putative porcine lincRNAs share many characteristics with mammalian lincRNAs, such as a relatively short length, small number of exons and low level of sequence conservation. We found that the porcine lincRNAs were preferentially located near genes mediating transcriptional regulation rather than those with developmental functions. We further experimentally analyzed the features of a conserved mouse lincRNA gene and found that isoforms 1 and 4 of this lincRNA were enriched in the cell nucleus and were associated with polycomb repressive complex 2 (PRC2). Our results provide a catalog of fetal porcine lincRNAs for further experimental investigation of the functions of these genes in the skeletal muscle developmental process.
doi:10.1038/srep08957
PMCID: PMC4354164  PMID: 25753296
13.  Identification of proteins associated with pyrethroid resistance by iTRAQ-based quantitative proteomic analysis in Culex pipiens pallens 
Parasites & Vectors  2015;8:95.
Background
Mosquito control based on chemical insecticides is considered as an important element in the current global strategies for the control of mosquito-borne diseases. Unfortunately, the development of pyrethroid resistance in important vector mosquito species jeopardizes the effectiveness of insecticide-based mosquito control. To date, the mechanisms of pyrethroid resistance are still unclear. Recent advances in proteomic techniques can facilitate to identify pyrethroid resistance-associated proteins at a large-scale for improving our understanding of resistance mechanisms, and more importantly, for seeking some genetic markers used for monitoring and predicting the development of resistance.
Methods
We performed a quantitative proteomic analysis between a deltamethrin-susceptible strain and a deltamethrin-resistant strain of laboratory population of Culex pipiens pallens using isobaric tags for relative and absolute quantitation (iTRAQ) analysis. Gene Ontology (GO) analysis was used to find the relative processes that these differentially expressed proteins were involved in. One differentially expressed protein was chosen to confirm by Western blot in the laboratory and field populations of Cx. pipiens pallens.
Results
We identified 30 differentially expressed proteins assigned into 10 different categories, including oxidoreductase activity, transporter activity, catalytic activity, structural constituent of cuticle and hypothetical proteins. GO analysis revealed that 25 proteins were sub-categorized into 35 hierarchically-structured GO classifications. Western blot results showed that CYP6AA9 as one of the up-regulated proteins was confirmed to be overexpressed in the deltamethrin-resistant strains compared with the deltamethrin-susceptible strains both in the laboratory and field populations.
Conclusions
This is the first study to use modern proteomic tools for identifying pyrethroid resistance-related proteins in Cx. pipiens. The present study brought to light many proteins that were not previously thought to be associated with pyrethroid resistance, which further expands our understanding of pyrethroid resistance mechanisms. CYP6AA9 was overexpressed in the deltamethrin-resistant strains, indicating that CYP6AA9 may be involved in pyrethroid resistance and may be used as a potential genetic marker to monitor and predict the pyrethroid resistance level of field populations.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0709-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13071-015-0709-5
PMCID: PMC4337324  PMID: 25880395
Insecticide resistance; Pyrethroids; Proteomic; iTRAQ; P450; Culex pipiens pallens
14.  Assembling single gold nanorods into large-scale highly aligned nanoarrays via vacuum-enhanced capillarity 
Nanoscale Research Letters  2014;9(1):556.
We report a simple, straightforward, and efficient approach to assemble single gold nanorods (AuNRs) into highly aligned arrays, via a unique vacuum-enhanced capillarity. The assembled AuNR arrays demonstrate both an excellently unidirectional ordering and a wonderful single-rod resolution. The key role of vacuum in this approach enables high-aspect-ratio (10 to 22) AuNR alignment and efficiently facilitates large-area alignment. Further investigation of one- and two-dimensional AuNR arrays would undoubtedly be beneficial to their potential applications.
doi:10.1186/1556-276X-9-556
PMCID: PMC4194060  PMID: 25313304
Gold; Nanorods; Assembly; Capillary; Atomic force microscopy; Nanoarray
15.  Electroacupuncture preconditioning-induced neuroprotection may be mediated by glutamate transporter type 2 
Neurochemistry international  2013;63(4):302-308.
Electroacupuncture has been shown to induce a preconditioning effect in the brain. The mechanisms for this protection are not fully elucidated. We hypothesize that this protection is mediated by excitatory amino acid transporters (EAATs) that have been shown to be neuroprotective. To test this hypothesis, two-month old male Sprague-Dawley rats and EAAT type 3 (EAAT3) knockout mice received or did not receive 30-min electroacupuncture once a day for 5 consecutive days. They were subjected to a 120-min middle cerebral arterial occlusion (MCAO) at 24 h after the last electroacupuncture. Neurological outcome was assessed 2 days after the MCAO. Brain tissues were harvested at 24 h after the last electroacupuncture for Western blotting. Rats subjected to electroacupuncture at the Baihui acupoint had smaller brain infarct volumes and better neurological deficit scores than control rats. Electroacupuncture increased EAAT type 2 (EAAT2) in the cerebral cortex, tended to increase EAAT3 in the hippocampus, and had no effect on EAAT type 1 expression. Dihydrokainate, an EAAT2 inhibitor, worsened the neurological outcome of rats with electroacupuncture pretreatment. Electroacupuncture pretreatment at the Baihui acupoint increased EAAT2 in the cerebral cortex and improved the neurological outcome of EAAT3 knockout mice. Together, our results suggest that EAAT2 may mediate the electroacupuncture preconditioning-induced neuroprotection.
doi:10.1016/j.neuint.2013.06.017
PMCID: PMC3758789  PMID: 23831620
brain; electroacupuncture; glutamate transporter; ischemia; preconditioning
16.  Timosaponin A3 induces hepatotoxicity in rats through inducing oxidative stress and down-regulating bile acid transporters 
Acta Pharmacologica Sinica  2014;35(9):1188-1198.
Aim:
To investigate the mechanisms underlying the hepatotoxicity of timosaponin A3 (TA3), a steroidal saponin from Anemarrhena asphodeloides, in rats.
Methods:
Male SD rats were administered TA3 (100 mg·kg−1·d−1, po) for 14 d, and the blood and bile samples were collected after the final administration. The viability of a sandwich configuration of cultured rat hepatocytes (SCRHs) was assessed using WST-1. Accumulation and biliary excretion index (BEI) of d8-TCA in SCRHs were determined with LC-MS/MS. RT-PCR and Western blot were used to analyze the expression of relevant genes and proteins. ROS and ATP levels, and mitochondrial membrane potential (MMP) were measured. F-actin cytoskeletal integrity was assessed under confocal microscopy.
Results:
TA3 administration in rats significantly elevated the total bile acid in serum, and decreased bile acid (BA) component concentrations in bile. TA3 inhibited the viability of the SCRHs with an IC50 value of 15.21±1.73 μmol/L. Treatment of the SCRHs with TA3 (1–10 μmol/L) for 2 and 24 h dose-dependently decreased the accumulation and BEI of d8-TCA. The TA3 treatment dose-dependently decreased the expression of BA transporters Ntcp, Bsep and Mrp2, and BA biosynthesis related Cyp7a1 in hepatocytes. Furthermore, the TA3 treatment dose-dependently increased ROS generation and HO-1 expression, decreased the ATP level and MMP, and disrupted F-actin in the SCRHs. NAC (5 mmol/L) significantly ameliorated TA3-induced effects in the SCRHs, whereas mangiferin (10–200 μg/mL) almost blocked TA3-induced ROS generation.
Conclusion:
TA3 triggers liver injury through inducing ROS generation and suppressing the expression of BA transporters. Mangiferin, an active component in Anemarrhena, may protect hepatocytes from TA3-induced hepatotoxicity.
doi:10.1038/aps.2014.65
PMCID: PMC4155534  PMID: 25087997
timosaponin A3; hepatotoxicity; cholestasis; bile acid; transporter; Cyp7a1; ROS; N-acetyl-L- cysteine; mangiferin
17.  Diagnostic Accuracy of Intracellular Mycobacterium tuberculosis Detection for Tuberculous Meningitis 
Rationale: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity.
Objectives: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis.
Methods: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6–expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients’ final diagnosis.
Measurements and Main Results: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6–6.7%), compared with 82.9% (95% confidence interval, 77.4–87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8–80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4–93.8%) and 90.0% (95% confidence interval, 75.4–96.7%), respectively.
Conclusions: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis.
doi:10.1164/rccm.201309-1686OC
PMCID: PMC3977721  PMID: 24450377
tuberculosis; central nervous system; cerebrospinal fluid; diagnosis
18.  Anandamide Protects HT22 Cells Exposed to Hydrogen Peroxide by Inhibiting CB1 Receptor-Mediated Type 2 NADPH Oxidase 
Background. Endogenous cannabinoid anandamide (AEA) protects neurons from oxidative injury in rodent models; however the mechanism of AEA-induced neuroprotection remains to be determined. Activation of neuronal NADPH oxidase 2 (Nox2) contributes to oxidative damage of the brain, and inhibition of Nox2 can attenuate cerebral oxidative stress. We aimed to determine whether the neuronal Nox2 was involved in protection mediated by AEA. Methods. The mouse hippocampal neuron cell line HT22 was exposed to hydrogen peroxide (H2O2) to mimic oxidative injury of neurons. The protective effect of AEA was assessed by measuring cell metabolic activity, apoptosis, lactate dehydrogenase (LDH) release, cellular morphology, intracellular reactive oxygen species (ROS), and antioxidant and oxidant levels and Nox2 expression. Results. HT22 cells exposed to H2O2 demonstrated morphological changes, decreased LDH release, reduced metabolic activity, increased levels of intracellular ROS and oxidized glutathione (GSSG), reduced levels of superoxide dismutase (SOD), and reduced glutathione (GSH) and increased expression of Nox2. AEA prevented these effects, a property abolished by simultaneous administration of CB1 antagonist AM251 or CB1-siRNA. Conclusion. Nox2 inhibition is involved in AEA-induced cytoprotection against oxidative stress through CB1 activation in HT22 cells.
doi:10.1155/2014/893516
PMCID: PMC4127243  PMID: 25136404
19.  Interleukin-1 beta guides the migration of cortical neurons 
Background
Proinflammatory cytokine interleukin-1beta (IL-1β) is expressed at high levels in the developing brain and declines to low constitutive levels in the adult. However, the pathophysiological function of IL-1β during brain development remains elusive. In this study, we investigated the role of IL-1β in neuronal migration.
Methods
The Boyden transwell assay was used to examine the effects of IL-1β on the migration of dissociated primary cortical neurons. To determine the role of IL-1β in neuron leading process pathfinding, we employed a growth cone turning assay. In utero electroporation combined with RNAi technology was used to examine the neuronal migration in vivo during brain development in Sprague–Dawley rats.
Results
IL-1β at concentrations ranging from 0.1 to 10 ng/mL in the lower chamber of a transwell induced a significant increase in the number of migrating neurons in a dose-dependent manner. When IL-1β was simultaneously put in both the upper and lower chambers to eliminate the gradient, no significant differences in cell migration were observed. IL-1 receptor antagonist IL-1RA dose-dependently blocked the attractive effect of IL-1β on neuronal migration. Microscopic gradients of IL-1β were created near the growth cones of isolated neurons by repetitive pulsatile application of picoliters of a IL-1β-containing solution with a micropipette. We found that growth cones exhibited a clear bias toward the source of IL-1β at the end of a one hour period in the IL-1β gradient. No significant difference was observed in the rate of neurite extension between IL-1β and controls. We electroporated specific siRNA constructs against IL-1R1 mRNA into cortical progenitors at embryonic day 16 and examined the position and distribution of transfected cells in the somatosensory cortex at postnatal day 5. We found that neurons transfected with IL-1R1-siRNA displayed a severe retardation in radial migration, with about 83% of total cells unable to arrive at the upper cortical layers.
Conclusions
Our study suggests an essential contribution of IL-1β to neuronal migration during brain development, which provides a basis to understand the physiological roles of IL-1β in the developing brain and could have significant implications for the prevention of some neurodevelopment disorders due to abnormal neuronal migration.
doi:10.1186/1742-2094-11-114
PMCID: PMC4084576  PMID: 24950657
IL-1beta; Neuronal migration; in utero electroporation; Brain development
20.  Astrocytic expression of cannabinoid type 1 receptor in rat and human sclerotic hippocampi 
Cannabinoid type 1 receptor (CB1R), which is traditionally located on axon terminals, plays an important role in the pathology of epilepsy and neurodegenerative diseases by modulating synaptic transmission. Using the pilocarpine model of chronic spontaneous recurrent seizures, which mimics the main features of mesial temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) in humans, we examined the expression of CB1R in hippocampal astrocytes of epileptic rats. Furthermore, we also examined the expression of astrocytic CB1R in the resected hippocampi from patients with medically refractory mesial TLE. Using immunofluorescent double labeling, we found increased expression of astrocytic CB1R in hippocampi of epileptic rats, whereas expression of astrocytic CB1R was not detectable in hippocampi of saline treated animals. Furthermore, CB1R was also found in some astrocytes in sclerotic hippocampi in a subset of patients with intractable mesial TLE. Detection with immune electron microscopy showed that the expression of CB1R was increased in astrocytes of epileptic rats and modest levels of CB1R were also found on the astrocytic membrane of sclerotic hippocampi. These results suggest that increased expression of astrocytic CB1R in sclerotic hippocampi might be involved in the cellular basis of the effects of cannabinoids on epilepsy.
PMCID: PMC4097232  PMID: 25031702
Epilepsy; cannabinoid type 1 receptor; hippocampal sclerosis; astrocyte; immune electron microscopy
21.  Decreased expression of miR-204 is associated with poor prognosis in patients with breast cancer 
The identification of biomarkers in breast cancer diagnosis and therapy is important in achieving early cancer diagnosis and improving patient outcomes. The aim of this study was to examine clinical significance of miR-204 expression in tissues from breast cancer patients. The relationship between miR-204 expression and clinicopathological characteristics was investigated. MiR-204 expression was significantly associated with TNM stage and metastasis. Patients with low miR-204 expression had poorer overall survival time and disease free survival time than those with high miR-204 expression. Furthermore, miR-204 expression was correlated with chemotherapeutic resistance of breast cancer patients. In conclusion, the miR-204 may be a potential diagnostic and prognostic biomarker of breast cancer.
PMCID: PMC4097245  PMID: 25031750
MiR-204; breast cancer; prognosis
22.  Honokiol Suppresses Renal Cancer Cells’ Metastasis via Dual-Blocking Epithelial-Mesenchymal Transition and Cancer Stem Cell Properties through Modulating miR-141/ZEB2 Signaling 
Molecules and Cells  2014;37(5):383-388.
Renal cell carcinoma (RCC) is associated with a high frequency of metastasis and only few therapies substantially prolong survival. Honokiol, isolated from Magnolia spp. bark, has been shown to exhibit pleiotropic anticancer effects in many cancer types. However, whether honokiol could suppress RCC metastasis has not been fully elucidated. In the present study, we found that honokiol suppressed renal cancer cells’ metastasis via dual-blocking epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. In addition, honokiol inhibited tumor growth in vivo. It was found that honokiol could up-regulate miR-141, which targeted ZEB2 and modulated ZEB2 expression. Honokiol reversed EMT and suppressed CSC properties partly through the miR-141/ZEB2 axis. Our study suggested that honokiol may be a suitable therapeutic strategy for RCC treatment.
doi:10.14348/molcells.2014.0009
PMCID: PMC4044309  PMID: 24810210
cancer stem cell; epithelial-mesenchymal transition; honokiol; microRNA; renal cell carcinoma
23.  Cancer Stem-like Cell Properties are Regulated by EGFR/AKT/β-catenin Signaling and Preferentially Inhibited by Gefitinib in Nasopharyngeal Carcinoma 
The FEBS journal  2013;280(9):10.1111/febs.12226.
We report that the EGFR pathway plays a critical role in regulating cancer stem-like cells (CSCs) in nasopharyngeal carcinoma (NPC), one of the most common malignant tumors in Southeast Asia. Effects of EGFR on maintaining CSCs are mainly mediated by AKT signaling, and β-catenin is responsible for governing CSC properties in response to EGFR/AKT activation. Significantly, CSCs are enriched by cisplatin and decreased by gefitinib in NPC xenograft models. Upon reimplantation in secondary mice, tumor cells derived from cisplatin-treated mice grew rapidly, whereas regrowth of tumor cells from gefitinib-treated mice was severely diminished. We further demonstrate that expression of EGFR correlates with expression of β-catenin and Nanog in primary tumor specimens from NPC patients. These findings provide mechanistic and preclinical evidence supporting the use of gefitinib alone or in combination with a chemotherapeutic agent in first-line therapy for patients with NPC. In addition, our results suggest that targeting β-catenin represents a rational clinical modality for patients whose tumors harbor activated EGFR or AKT.
doi:10.1111/febs.12226
PMCID: PMC3831031  PMID: 23461856
Nasopharyngeal carcinoma; Cancer stem-like cells; EGFR; β-catenin; Gefitinib
24.  A case of horizontal gene transfer from Wolbachia to Aedes albopictus C6/36 cell line 
Mobile Genetic Elements  2014;4:e28914.
Horizontal gene transfer plays an essential role in evolution and ecological adaptation, yet this phenomenon has remained controversial, particularly where it occurs between prokaryotes and eukaryotes. There are a handful of reported examples of horizontal gene transfer occurring between prokaryotes and eukaryotes in the literature, with most of these documented cases pertaining to invertebrates and endosymbionts. However, the vast majority of these horizontally transferred genes were either eventually excluded or rapidly became nonfunctional in the recipient genome. In this study, we report the discovery of a horizontal gene transfer from the endosymbiont Wolbachia in the C6/36 cell line derived from the mosquito Aedes albopictus. Moreover, we report that this horizontally transferred gene displayed high transcription level. This finding and the results of further experimentation strongly suggest this gene is functional and has been expressed and translated into a protein in the mosquito host cells.
doi:10.4161/mge.28914
PMCID: PMC4013104  PMID: 24812591
horizontal gene transfer; endosymbiont; Wolbachia; mosquito; Aedes Albopictus; C6/36 cell line
25.  Ribosomal Protein S29 Regulates Metabolic Insecticide Resistance through Binding and Degradation of CYP6N3 
PLoS ONE  2014;9(4):e94611.
Background
Many diseases are transmitted by mosquitoes, including malaria, dengue fever, yellow fever, filariasis, and West Nile fever. Chemical control plays a major role in managing mosquito-borne diseases. However, excessive and continuous application of insecticides has caused the development of insecticide resistance in many species including mosquito, and this has become the major obstacle to controlling mosquito-borne diseases. Insecticide resistance is the result of complex polygenic inheritance, and the mechanisms are not well understood. Ribosomal protein RPS29 was found to be associated with DM resistance in our previous study. In this study, we aim to further investigate the involvement of RPS29 in deltamethrin resistance.
Methodology and Principal Findings
In this study, tandem affinity purification was used to identify proteins that can interact with RPS29. Among the candidate proteins, CYP6N3, a member of the CYP450 superfamily, was identified, and binding to RPS29 was confirmed in vitro and in vivo by GST pull-down and immunofluorescence. CCK-8 assay was used to investigate the RPS29-CTP6N3 interaction in relation to DM resistance. CYP6N3 overexpression significantly enhanced DM resistance and insect cell viability, but this was reversed by RPS29 overexpression. Western blot was used to study the mechanism of interaction between RPS29 and CYP6N3. RPS29 increases CYP6N3 protein degradation through the proteasome.
Conclusions and Significance
These observations indicate that CYP6N3, a novel RPS29-interacting partner, could stimulate deltamethrin resistance in mosquito cells and RPS29 overexpression targeted CYP6N3 for proteosomal degradation, abrogating the CYP6N3-associated resistence to deltamethrin. Our findings provide a novel mechanism associated with CYP450s mediated DM resistance.
doi:10.1371/journal.pone.0094611
PMCID: PMC3984272  PMID: 24728095

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