An improved liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of dexmedetomidine concentrations in samples of human plasma

Dexmedetomidine (DMET) is a sedative, analgesic and anxiolytic with minimum adverse respiratory effects. An LC-MS/MS bioanalytical method has been developed and validated to accurately measure DMET concentrations in samples of human plasma. The method overcomes difficulties in the extraction and quantification of DMET due to the fact that it binds strongly to glass and plastic tubes, as well as solid phase extraction (SPE) cartridges. Human plasma (50 μL) was mixed with the internal standard (IS) (DMET-d4) solution (100 μL) and 0.1% formic acid (50 μL) and extracted using Oasis HLB 1 CC (30 mg) solid phase extraction (SPE) cartridges (Waters®). The glass tubes were coated with bovine serum albumin (BSA) 0.5% (20 μL) before eluting DMET and the IS. After evaporation under nitrogen at room temperature, the analytes were reconstituted in 20% acetonitrile in 0.1% formic acid in water and transferred to silanized glass vials. An electrospray ionisation (ESI) mass spectrometry method in positive mode was created and the precursor/product transitions (m/z) were 201.1 → 95.0 (DMET) and 204.9 → 99.0 (IS). The method was robust and fully validated based on the 2012 EMEA guideline for bioanalytical method validation in the concentration range of 0.5-20 ng/mL. Using this assay, we showed that DMET binds strongly to Extracorporeal Membrane Oxygenation (ECMO) circuits, consistent with expectations for small lipophilic compounds

Complications following Bacille Calmette-Guérin Vaccination in Children under the Age of 18 Months: A Multi-center Study

Background: The BCG vaccine, used since 1921 to prevent tuberculosis (TB), considered the world's most widely used vaccine. This study aimed to investigate the frequency and the type of complications associated with Bacillus Calmette-Guérin (BCG) vaccination in Iranian children. Materials and Methods This cross-sectional study conducted for 6months among children aged up to 18 months who presented to primary health care centers in Tehran (under supervision of all three Medical Universities of Tehran) for their routine vaccinations. All children had received BCG vaccination at birth. We investigated the occurrence of BCG complications through history taking from the parents and physical examination by the study physician. Complications categorized into four major groups: local, regional, remote, and generalized and the rate of occurrence compared between the two genders. Results: Finally, 14,095 children enrolled during the study period; 574 patients (4%) had experienced at least one complication following BCG vaccination. The most common complications were local side effects observed in 304 children (2.1%); followed by lymph node involvement detected in 270 children (1.9%). Lymph node involvement was more common in males (

Content and Feedback Analysis of YouTube Videos: Football Clubs and Fans as Brand Communities

The use of Web 2.0 tools has been transforming the interaction between companies and their clients, especially for those that are selling emotional products. Consumers are generating and sharing contents concerning their favourite products on the web. Even if this process has been widely acknowledged, only a few studies have been specifically devoted to the analysis of both the contents and the feedback the consumers receive from other users. This article analyzes the online presence of sport brands through contents that are generated by sport clubs (official contents) and their fans (User Generated Content, UGC) on YouTube. After a description and classification of video contents, it examines the factors that influence the performance of the videos in terms of passive (videos views) and active behaviour (any kinds of interaction with videos) among the viewers. In order to carry out this analysis, 125 YouTube channels were considered thereby accounting for a total of 375 videos. Results show that official contents are those preferred by the users/consumers and that if the video displays a passive/purely informative content, the chance of getting an active behaviour from the users tends to decrease. These findings may help companies manage their online presence, creating awareness about contents and information that should be spread and shared on the web

Down syndrome-recent progress and future prospects

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and is associated with a number of deleterious phenotypes, including learning disability, heart defects, early-onset Alzheimer's disease and childhood leukaemia. Individuals with DS are affected by these phenotypes to a variable extent; understanding the cause of this variation is a key challenge. Here, we review recent research progress in DS, both in patients and relevant animal models. In particular, we highlight exciting advances in therapy to improve cognitive function in people with DS and the significant developments in understanding the gene content of Hsa21. Moreover, we discuss future research directions in light of new technologies. In particular, the use of chromosome engineering to generate new trisomic mouse models and large-scale studies of genotype-phenotype relationships in patients are likely to significantly contribute to the future understanding of DS

Disruption of Nrf2, a Key Inducer of Antioxidant Defenses, Attenuates ApoE-Mediated Atherosclerosis in Mice

Background: Oxidative stress and inflammation are two critical factors that drive the formation of plaques in atherosclerosis. Nrf2 is a redox-sensitive transcription factor that upregulates a battery of antioxidative genes and cytoprotective enzymes that constitute the cellular response to oxidative stress. Our previous studies have shown that disruption of Nrf2 in mice (Nrf2-/-) causes increased susceptibility to pulmonary emphysema, asthma and sepsis due to increased oxidative stress and inflammation. Here we have tested the hypothesis that disruption of Nrf2 in mice causes increased atherosclerosis. Principal Findings: To investigate the role of Nrf2 in the development of atherosclerosis, we crossed Nrf2-/- mice with apoliporotein E-deficient (ApoE-/- mice. ApoE-/- and ApoE-/- Nrf2-/- mice were fed an atherogenic diet for 20 weeks, and plaque area was assessed in the aortas. Surprisingly, ApoE-/- Nrf2-/- mice exhibited significantly smaller plaque area than ApoE-/- controls (11.5% vs 29.5%). This decrease in plaque area observed in ApoE-/- Nrf2-/- mice was associated with a significant decrease in uptake of modified low density lipoproteins (AcLDL) by isolated macrophages from ApoE-/- Nrf2-/- mice. Furthermore, atherosclerotic plaques and isolated macrophages from ApoE-/- Nrf2-/- mice exhibited decreased expression of the scavenger receptor CD36. Conclusions: Nrf2 is pro-atherogenic in mice, despite its antioxidative function. The net pro-atherogenic effect of Nrf2 may be mediated via positive regulation of CD36. Our data demonstrates that the potential effects of Nrf2-targeted therapies on cardiovascular disease need to be investigated.9 page(s

RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism

Mechanism of Chemical Activation of Nrf2

NF-E2 related factor-2 (Nrf2) promotes the transcription of many cytoprotective genes and is a major drug target for prevention of cancer and other diseases. Indeed, the cancer-preventive activities of several well-known chemical agents were shown to depend on Nrf2 activation. It is well known that chemopreventive Nrf2 activators stabilize Nrf2 by blocking its ubiquitination, but previous studies have indicated that this process occurs exclusively in the cytoplasm. Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and orchestrates Nrf2 ubiquitination, and it has been a widely-held view that inhibition of Nrf2 ubiquitination by chemopreventive agents results from the dissociation of Nrf2 from its repressor Keap1. Here, we show that while the activation of Nrf2 by prototypical chemical activators, including 5,6-dihydrocyclopenta-1,2-dithiole-3-thione (CPDT) and sulforaphane (SF), results solely from inhibition of its ubiquitination, such inhibition occurs predominantly in the nucleus. Moreover, the Nrf2 activators promote Nrf2 association with Keap1, rather than disassociation, which appears to result from inhibition of Nrf2 phosphorylation at Ser40. Available evidence suggests the Nrf2 activators may block Nrf2 ubiquitination by altering Keap1 conformation via reaction with the thiols of specific Keap1 cysteines. We further show that while the inhibitory effects of CPDT and SF on Nrf2 ubiquitination depend entirely on Keap1, Nrf2 is also degraded by a Keap1-independent mechanism. These findings provide significant new insight about Nrf2 activation and suggest that exogenous chemical activators of Nrf2 enter the nucleus to exert most of their inhibitory impact on Nrf2 ubiquitination and degradation

Nrf2, a PPARγ Alternative Pathway to Promote CD36 Expression on Inflammatory Macrophages: Implication for Malaria

CD36 is the major receptor mediating nonopsonic phagocytosis of Plasmodium falciparum-parasitized erythrocytes by macrophages. Its expression on macrophages is mainly controlled by the nuclear receptor PPARγ. Here, we demonstrate that inflammatory processes negatively regulate CD36 expression on human and murine macrophages, and hence decrease Plasmodium clearance directly favoring the worsening of malaria infection. This CD36 downregulation in inflammatory conditions is associated with a failure in the expression and activation of PPARγ. Interestingly, using siRNA mediating knock down of Nrf2 in macrophages or Nrf2- and PPARγ-deficient macrophages, we establish that in inflammatory conditions, the Nrf2 transcription factor controls CD36 expression independently of PPARγ. In these conditions, Nrf2 activators, but not PPARγ ligands, enhance CD36 expression and CD36-mediated Plasmodium phagocytosis. These results were confirmed in human macrophages and in vivo where only Nrf2 activators improve the outcome of severe malaria. Collectively, this report highlights that the Nrf2 transcription factor could be an alternative target to PPARγ in the control of severe malaria through parasite clearance