Microbiota analysis with next-generation 16S rDNA gene sequencing in recurrent common bile duct stones.

Background: Endoscopic retrograde cholangiopancreatography (ERCP) is the main remedy for gallstones, but the postoperative recurrence rate is high. Recent research has indicated that the biliary microbiome takes part in the pathogenesis of cholelithiasis. However, it is not yet known whether biliary microbiome dysbiosis is relevant to recurrent cholelithiasis. Methods: Thus, we investigated the bacterial communities of the biliary microbiomes of patients with recurrent common bile duct (CBD) stones and analyzed the relationship between recurrent CBD stones and biliary microbiota. The bile specimens of 5 patients with recurrent CBD stones (FF) and 45 patients with primary CBD stones (YF) were collected during the ERCP process. The microbiota was analyzed using 16S ribosomal DNA (rDNA) high-throughput sequencing. We also identified the link between recurrent CBD stones and biliary microbiota. Results: Our results showed that at the phylum level, proteobacteria and firmicutes were the main two genera groups, and proteobacteria was high in FF patients. Additionally, synergistetes were high, but Bacteroidetes and actinobacteria were low in FF patients. The microbiomes in the bile of the YF patients were more evenly distributed than those in the bile of the FF patients. We also discovered that FF patients had decreased microbial bile diversity. At the genus level, klebsiella dominated in the FF patients, while Escherichia-shigella dominated in the YF patients. Additionally, klebsiella was higher in the FF patients than the YF patients. Conclusions: The observed differences in the genera between the recurrent CBD stone FF patients and the YF patients provide novel insights into the link between biliary microbiota changes and recurrent CBD stones.

Targeted epigenomic editing ameliorates adult anxiety and excessive drinking after adolescent alcohol exposure.

Adolescent binge drinking is a major risk factor for psychiatric disorders later in life including alcohol use disorder. Adolescent alcohol exposure induces epigenetic reprogramming at the enhancer region of the activity-regulated cytoskeleton-associated protein (Arc) immediate-early gene, known as synaptic activity response element (SARE), and decreases Arc expression in the amygdala of both rodents and humans. The causal role of amygdalar epigenomic regulation at Arc SARE in adult anxiety and drinking after adolescent alcohol exposure is unknown. Here, we show that dCas9-P300 increases histone acetylation at the Arc SARE and normalizes deficits in Arc expression, leading to attenuation of adult anxiety and excessive alcohol drinking in a rat model of adolescent alcohol exposure. Conversely, dCas9-KRAB increases repressive histone methylation at the Arc SARE, decreases Arc expression, and produces anxiety and alcohol drinking in control rats. These results demonstrate that epigenomic editing in the amygdala can ameliorate adult psychopathology after adolescent alcohol exposure.

RP11-295G20.2 facilitates hepatocellular carcinoma progression via the miR-6884-3p/CCNB1 pathway.

OBJECTIVE: An increasing number of studies have indicated that long noncoding RNAs (lncRNAs) play an important role in the pathogenesis of hepatocellular carcinoma (HCC). In this study, we aimed to clarify the roles of RP11-295G20.2 in HCC progression and the underlying molecular mechanisms. RESULTS: Bioinformatics analyses based TCGA data suggested that RP11-295G20.2 was significantly upregulated in HCC tissues and increased RP11-295G20.2 expression level correlated with poor overall survival of patients with HCC. The results of RT-PCR further showed that RP11-295G20.2 was upregulated in HCC tissues and cell lines. Functionally, RP11-295G20.2 knockdown significantly inhibited the proliferation, colony formation, invasion and migration, but induced the apoptosis of HCC cells. In line with this, downregulation of RP11-295G20.2 in HCC lines markedly suppressed the tumor growth in vivo. Mechanistically, RP11-295G20.2 could upregulate CCNB1 through targeting miR-6884-3p. More importantly, our rescue experiments revealed that miR-6884-3p/CCNB1 axis was involved in RP11-295G20.2-meditated tumorigenic behaviors of HCC cells. CONCLUSIONS: RP11-295G20.2 can contribute to HCC progression at least partly via the miR-6884-3p/CCNB1 axis, suggesting that RP11-295G20.2 may be a potential target for HCC therapy. METHODS: RT-qPCR was employed to examine the expression levels of RP11-295G20.2, miR-6884-3p, and CCNB1 in HCC tissues and cell lines. CCK8 assay, transwell assay, colony formation assay and flow cytometry analysis were performed to evaluate the biological function of RP11-295G20.2 in HCC cells. The xenograft tumor assay was used to assess the effect of RP11-295G20.2 on the in vivo growth of HCC cells. The luciferase reporter assay, RIP assay and Spearman's correlation analysis were performed to explore the potential mechanisms underlying the roles of RP11-295G20.2 in HCC progression.

Estrogen Receptor α Regulates Ethanol Excitation of Ventral Tegmental Area Neurons and Binge Drinking in Female Mice.

Elevations in estrogen (17ß-estradiol, E2) are associated with increased alcohol drinking by women and experimentally in rodents. E2 alters the activity of the dopamine system, including the VTA and its projection targets, which plays an important role in binge drinking. A previous study demonstrated that, during high E2 states, VTA neurons in female mice are more sensitive to ethanol excitation. However, the mechanisms responsible for the ability of E2 to enhance ethanol sensitivity of VTA neurons have not been investigated. In this study, we used selective agonists and antagonists to examine the role of ER subtypes (ERα and ERß) in regulating the ethanol sensitivity of VTA neurons in female mice and found that ERα promotes the enhanced ethanol response of VTA neurons. We also demonstrated that enhancement of ethanol excitation requires the activity of the metabotropic glutamate receptor, mGluR1, which is known to couple with ERα at the plasma membrane. To investigate the behavioral relevance of these findings, we administered lentivirus-expressing short hairpin RNAs targeting either ERα or ERß into the VTA and found that knockdown of each receptor in the VTA reduced binge-like ethanol drinking in female, but not male, mice. Reducing ERα in the VTA had a more dramatic effect on binge-like drinking than reducing ERß, consistent with the ability of ERα to alter ethanol sensitivity of VTA neurons. These results provide important insight into sex-specific mechanisms that drive excessive alcohol drinking.SIGNIFICANCE STATEMENT Estrogen has potent effects on the dopamine system and increases the vulnerability of females to develop addiction to substances, such as alcohol. We investigated the mechanisms by which estrogen increases the response of neurons in the VTA to ethanol. We found that activation of the ERα increased the ethanol-induced excitation of VTA neurons. 17ß-Estradiol-mediated enhancement of ethanol-induced excitation required the metabotropic glutamate receptor mGluR1. We also demonstrated that ERs in the VTA regulate binge-like alcohol drinking by female, but not male, mice. The influence of ERs on binge drinking in female mice suggests that treatments for alcohol use disorder in women may need to account for this sex difference.

Astragalus Polysaccharide Promotes Adriamycin-Induced Apoptosis in Gastric Cancer Cells.

PURPOSE: Astragalus polysaccharide (APS), a common Chinese herbal compound extracted from Astragalus membranaceus, has been proposed to increase the tumour response of and stabilize chemotherapy drugs while reducing their toxicity. Here, we examined the effects of APS on apoptosis in gastric cancer (GC) cells in the presence or absence of adriamycin (0.1 µg/mL). METHODS: GC cells cultured in the presence or absence of adriamycin (0.1 µg/mL) were administered APS (50-200 µg/mL) for 24-72 h and subjected to an MTT assay to examine cell viability. Active caspase-3 expression and DNA fragmentation were assessed to evaluate apoptosis, and real-time PCR was used to analyse the expression levels of multidrug resistance (MDR1) genes and tumour suppressor genes. Western blot analysis was applied to detect cleaved caspase-3 and phosphorylated AMPK (p-AMPK). RESULTS: Cellular viability was profoundly reduced by APS, and GC cell apoptosis was strongly increased by APS in a time- and dose-dependent manner; these changes may be linked to an increase in p-AMPK levels because the AMPK inhibitor compound C blocked the effects of APS. Similarly, adriamycin-induced decreases in cellular viability and apoptosis of GC cells were enhanced by APS administration. The expression of tumour suppressor genes (SEMA3F, P21WAF1/CIP1, FBXW7), but not of MDR1, was increased by APS compared to the control, and p-AMPK levels were lower in adriamycin-resistant GC cells than in either adriamycin-sensitive GC cells or an immortalized human gastric epithelial cell line. CONCLUSION: APS induces apoptosis independently and strengthens the proapoptotic effect of adriamycin on GC cells, suggesting that APS may act as a chemotherapeutic sensitizer.

Anaplastic Lymphoma Kinase Regulates Internalization of the Dopamine D2 Receptor.

The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) expressed in regions of the brain that control motor function, cognition, and motivation. As a result, D2R is involved in the pathophysiology of disorders such as schizophrenia and drug addiction. Understanding the signaling pathways activated by D2R is crucial to finding new therapeutic targets for these disorders. D2R stimulation by its agonist, dopamine, causes desensitization and internalization of the receptor. A previous study found that inhibitors of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK) blocked D2R desensitization in neurons in the ventral tegmental area of the brain. In the present study, using a cell-based system, we investigated whether ALK regulates D2R internalization. The ALK inhibitor alectinib completely inhibited dopamine-induced D2R internalization. Since GPCRs can transactivate receptor tyrosine kinases, we also examined if D2R stimulation activated ALK signaling. ALK phosphorylation increased by almost 2-fold after dopamine treatment and ALK coimmunoprecipitated with D2R. To identify the signaling pathways downstream of ALK that might regulate D2R internalization, we used pharmacological inhibitors of proteins activated by ALK signaling. Protein kinase Cγ was activated by dopamine in an ALK-dependent manner, and a protein kinase C inhibitor completely blocked dopamine-induced D2R internalization. Taken together, these results identify ALK as a receptor tyrosine kinase transactivated by D2R that promotes its internalization, possibly through activation of protein kinase C. ALK inhibitors could be useful in enhancing D2R signaling. SIGNIFICANCE STATEMENT: Receptor internalization is a mechanism by which receptors are desensitized. In this study we found that agonist-induced internalization of the dopamine D2 receptor is regulated by the receptor tyrosine kinase ALK. ALK was also transactivated by and associated with dopamine D2 receptor. Dopamine activated protein kinase C in an ALK-dependent manner and a PKC inhibitor blocked dopamine D2 receptor internalization. These results indicate that ALK regulates dopamine D2 receptor trafficking, which has implications for psychiatric disorders involving dysregulated dopamine signaling.

Design and Synthesis of Basic Selective Estrogen Receptor Degraders for Endocrine Therapy Resistant Breast Cancer.

The clinical steroidal selective estrogen receptor (ER) degrader (SERD), fulvestrant, is effective in metastatic breast cancer, but limited by poor pharmacokinetics, prompting the development of orally bioavailable, nonsteroidal SERDs, currently in clinical trials. These trials address local breast cancer as well as peripheral metastases, but patients with brain metastases are generally excluded because of the lack of blood-brain barrier penetration. A novel family of benzothiophene SERDs with a basic amino side arm (B-SERDs) was synthesized. Proteasomal degradation of ERα was induced by B-SERDs that achieved the objectives of oral and brain bioavailability, while maintaining high affinity binding to ERα and both potency and efficacy comparable to fulvestrant in cell lines resistant to endocrine therapy or bearing ESR1 mutations. A novel 3-oxyazetidine side chain was designed, leading to 37d, a B-SERD that caused endocrine-resistant ER+ tumors to regress in a mouse orthotopic xenograft model.

Ethanol acts on KCNK13 potassium channels in the ventral tegmental area to increase firing rate and modulate binge-like drinking.

Alcohol excitation of the ventral tegmental area (VTA) is important in neurobiological processes related to the development of alcoholism. The ionotropic receptors on VTA neurons that mediate ethanol-induced excitation have not been identified. Quinidine blocks ethanol excitation of VTA neurons, and blockade of two-pore potassium channels is among the actions of quinidine. Therefore two-pore potassium channels in the VTA may be potential targets for the action of ethanol. Here, we explored whether ethanol activation of VTA neurons is mediated by the two-pore potassium channel KCNK13. Extracellular recordings of the response of VTA neurons to ethanol were performed in combination with knockdown of Kcnk13 using a short hairpin RNA (shRNA) in C57BL/6 J mice. Real-time PCR and immunohistochemistry were used to examine expression of this channel in the VTA. Finally, the role of KCNK13 in binge-like drinking was examined in the drinking in the dark test after knockdown of the channel. Kcnk13 expression in the VTA was increased by acute ethanol exposure. Ethanol-induced excitation of VTA neurons was selectively reduced by shRNA targeting Kcnk13. Importantly, knockdown of Kcnk13 in the VTA resulted in increased alcohol drinking. These results are consistent with the idea that ethanol stimulates VTA neurons at least in part by inhibiting KCNK13, a specific two-pore potassium channel, and that KCNK13 can control both VTA neuronal activity and binge drinking. KCNK13 is a novel alcohol-sensitive molecular target and may be amenable to the development of pharmacotherapies for alcoholism treatment.

Estrogen Receptor ß in the Nucleus Accumbens Regulates the Rewarding Properties of Cocaine in Female Mice.

Background: Females are more vulnerable to developing cocaine addiction compared with males, a phenomenon that may be regulated by the steroid hormone 17ß-estradiol. 17ß-Estradiol enhances cocaine reward as measured by the conditioned place preference test. It is currently not known which estrogen receptor is involved or the neuroanatomical locations in which estrogen receptors act to enhance cocaine responses. The purpose of this study was to determine if the estrogen receptors ERα and ERß regulate cocaine conditioned place preference in mice and whether they act in the nucleus accumbens, a brain region critically involved in the development of cocaine abuse. Methods: Ovariectomized mice were treated with 17ß-estradiol or agonists selective for ERα or ERß and tested for cocaine conditioned place preference and for c-fos expression in the nucleus accumbens. Female mice with intact ovaries were also tested for cocaine conditioned place preference after RNA interference-mediated knockdown of ERα or ERß in the nucleus accumbens. Results: We found that mice treated with 17ß-estradiol or an ERß agonist exhibited increased cocaine conditioned place preference, while knockdown of ERß, but not ERα, in the nucleus accumbens of females with intact ovaries abrogated cocaine conditioned place preference. Acute treatment with 17ß-estradiol or an ERß agonist induced expression of the immediate-early gene c-fos in the nucleus accumbens, whereas the ERα agonist did not. Conclusions: These data indicate that ERß in the nucleus accumbens regulates the development of cocaine conditioned place preference in female mice. 17ß-Estradiol may activate neurons in the nucleus accumbens via ERß. We speculate that this might increase the saliency of cocaine cues that predict drug reward.

Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01). VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01). Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

Repeated Binge Drinking Increases Perineuronal Nets in the Insular Cortex.

BACKGROUND: Alcohol exposure leads to changes in the extracellular matrix (ECM) in the brain, which profoundly impacts neuronal plasticity. Perineuronal nets (PNs) are specialized ECM structures that enclose subpopulations of neurons in the cortex. Adolescent exposure to alcohol induces long-lasting increases in the expression of PN components in the cortex in adult mice. However, it has not been determined whether binge alcohol exposure in young adults alters PNs. Here, we examined PNs and their core components in the insula and primary motor cortex after repeated binge-like ethanol (EtOH) consumption in adult mice. METHODS: The 4-day drinking in the dark (DID) procedure was performed in mice for 1 or 6 weeks to model binge alcohol consumption. The impact of EtOH drinking on PNs was examined by fluorescent staining of brain sections using a marker for PNs, Wisteria floribunda agglutinin (WFA). In another set of experiments, cortex was dissected and Western blots and real-time quantitative polymerase chain reaction were performed to evaluate the expression of the PN proteins aggrecan, brevican, and phosphacan. RESULTS: Binge-like EtOH drinking for 6 weeks caused a significant increase in PNs in the insula, as measured by WFA binding. Aggrecan, brevican, and phosphacan protein expression, and aggrecan mRNA expression, were also elevated in the insula after 6 weeks of EtOH drinking. In contrast, expression of PN components did not change after 1 week of DID. The increase in PNs appears to be specific to the insula, because alterations were not observed in the primary motor cortex. CONCLUSIONS: Our results provide the first evidence that insular PNs increase after long-term binge drinking. The insula mediates compulsive alcohol use. As PNs influence neuronal firing and plasticity, increased PNs in the insula after multiple binge cycles may contribute to restricted neuronal plasticity and lead to the development of compulsive alcohol use.

Ethanol activates midkine and anaplastic lymphoma kinase signaling in neuroblastoma cells and in the brain.

Alcohol engages signaling pathways in the brain. Midkine (MDK) is a neurotrophic factor that is over-expressed in the prefrontal cortex of alcoholics. MDK and one of its receptors, anaplastic lymphoma kinase (ALK), also regulate behavioral responses to ethanol in mice. The goal of this study was to determine whether MDK and ALK expression and signaling are activated by ethanol. We found that ethanol treatment of neuroblastoma cells increased MDK and ALK expression. We also assessed activation of ALK by ethanol in cells and found that ALK and ALK-dependent extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) phosphorylation increased rapidly with ethanol exposure. Similarly, treatment of cells with recombinant MDK protein increased ALK, ERK and STAT3 phosphorylation, suggesting that ethanol may utilize MDK to activate ALK signaling. In support of this, transfection of cells with MDK siRNAs attenuated ALK signaling in response to ethanol. Ethanol also activates ERK signaling in the brain. We found that inhibition of ALK or knockout of MDK attenuated ethanol-induced ERK phosphorylation in mouse amygdala. These results demonstrate that ethanol engages MDK and ALK signaling, which has important consequences for alcohol-induced neurotoxicity and the regulation of behaviors related to alcohol abuse.

Coronin 1B regulates S1P-induced human lung endothelial cell chemotaxis: role of PLD2, protein kinase C and Rac1 signal transduction.

Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.

Targeting sphingosine kinase 1 attenuates bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor ß (TGF-ß) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-ß secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-ß dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.

Sphingosine kinase 1 is required for mesothelioma cell proliferation: role of histone acetylation.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1-/- null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts. CONCLUSIONS/SIGNIFICANCE: The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium.

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

Lysophosphatidic acid receptor 1 modulates lipopolysaccharide-induced inflammation in alveolar epithelial cells and murine lungs.

Lysophosphatidic acid (LPA), a bioactive phospholipid, plays an important role in lung inflammation by inducing the release of chemokines and lipid mediators. Our previous studies have shown that LPA induces the secretion of interleukin-8 and prostaglandin E(2) in lung epithelial cells. Here, we demonstrate that LPA receptors contribute to lipopolysaccharide (LPS)-induced inflammation. Pretreatment with LPA receptor antagonist Ki16425 or downregulation of LPA receptor 1 (LPA(1)) by small-interfering RNA (siRNA) attenuated LPS-induced phosphorylation of p38 MAPK, I-κB kinase, and I-κB in MLE12 epithelial cells. In addition, the blocking of LPA(1) also suppressed LPS-induced IL-6 production. Furthermore, LPS treatment promoted interaction between LPA(1) and CD14, a LPS coreceptor, in a time- and dose-dependent manner. Disruption of lipid rafts attenuated the interaction between LPA(1) and CD14. Mice challenged with LPS increased plasma LPA levels and enhanced expression of LPA receptors in lung tissues. To further investigate the role of LPA receptors in LPS-induced inflammation, wild-type, or LPA(1)-deficient mice, or wild-type mice pretreated with Ki16425 were intratracheally challenged with LPS for 24 h. Knock down or inhibition of LPA(1) decreased LPS-induced IL-6 release in bronchoalveolar lavage (BAL) fluids and infiltration of cells into alveolar space compared with wild-type mice. However, no significant differences in total protein concentration in BAL fluids were observed. These results showed that knock down or inhibition of LPA(1) offered significant protection against LPS-induced lung inflammation but not against pulmonary leak as observed in the murine model for lung injury.

Autotaxin induces lung epithelial cell migration through lysoPLD activity-dependent and -independent pathways.

Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodelling and repair after lung injury. In the present paper we hypothesize that secreted ATX (autotaxin), which exhibits lysoPLD (lysophospholipase D) activity, stimulates lung epithelial cell migration through LPA (lysophosphatidic acid) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag overexpressed conditional medium had higher LPA levels compared with control medium and stimulated cell migration through G(αi)-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKC (protein kinase C) δ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to the LPA receptor and integrin ß4 complex on A549 cell surface. Finally, intratracheal administration of LPS (lipopolysaccharide) into the mouse airway induced ATX release and LPA production in BAL (bronchoalveolar lavage) fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodelling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated association of ATX with the epithelial cell-surface LPA receptor and integrin ß4.

Meta-analysis of letrozole versus clomiphene citrate in polycystic ovary syndrome.

The aim of this study was to systematically compare the clinical efficacy and safety of letrozole with clomiphene citrate for ovulation induction in women with polycystic ovary syndrome (PCOS). The Cochrane Central Register of Controlled Trials, PubMed, EMbase, CBMdisc and CNKI were searched for eligible randomized controlled trials (RCT) comparing letrozole with clomiphene citrate in PCOS patients. Two reviewers independently extracted information and evaluated methodological quality according to the Cochrane Handbook 5.0. Meta-analysis was performed with the fixed-effects model or random-effects model according to the heterogeneity. Six eligible RCT involving 841 patients were included. Letrozole was associated with a number of lower mature follicles per cycle (standardized mean difference (SMD) -1.41; 95% confidence intervales (CI) -1.54 to -1.28; P<0.00001) compared with clomiphene citrate. There were no significant differences in pregnancy rate (relative risk (RR) 0.97; 95% CI 0.79 to 1.18), abortion rate (RR 1.38; 95% CI 0.48 to -3.96) and multiple pregnancy rate (RR 0.34; 95% CI 0.07 to -1.72) between the two groups. The evidence from ovulation rates was not enough to support either letrozole or clomiphene citrate. In conclusion, letrozole is as effective as clomiphene citrate for ovulation induction in patients with PCOS.

Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and ß-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.