Identification of nitroxide radioprotectors.

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.     

Operation Crayweed: Ecological and sociocultural aspects of restoring Sydney’s underwater forests

Operation Crayweed focuses on the restoration of underwater forests that disappeared from the coastline of Sydney, Australia’s largest city, 40 years previously. We show how a combination of science, hands‐on restoration, community engagement and art has helped the project to reach its goals as well as raise awareness about the importance of underwater kelp forests that are experiencing global decline.     

Genetic deciphering of the antagonistic activities of the melanin-concentrating hormone and melanocortin pathways in skin pigmentation

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte–stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers.     

Clozapine-Induced Mitochondria Alterations and Inflammation in Brain and Insulin-Responsive Cells

Background

Metabolic syndrome (MetS) is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS) and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined.

Methodology/Principal Findings

Cultured mouse myoblasts (C2C12), adipocytes (3T3-L1), hepatocytes (FL-83B), and monocytes (RAW 264.7) were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line.

Conclusions/Significance

Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated with the development of these diseases in the general population.     

Plasmid and Host Strain Characteristics of Escherichia coli Resistant to Extended-Spectrum Cephalosporins in the Norwegian Broiler Production

Escherichia coli resistant to extended-spectrum cephalosporins have been detected in the Norwegian broiler production, despite the fact that antimicrobial agents are rarely used. The genetic mechanism responsible for cephalosporin resistance is mainly attributed to the presence of the bla_CMY-2 gene encoding a plasmid-mediated AmpC-beta-lactamase (pAmpC). The aim of this study was to characterize and compare bla_CMY-2 containing Escherichia coli isolated from the intestinal flora of broilers and retail chicken meat (fillets) to identify possible successful clones and/or resistance plasmids widespread in the Norwegian broiler production. Methods used included PCR based phylotyping, conjugation experiments, plasmid replicon typing, pulsed-field gel electrophoresis, multiple locus variable-number tandem-repeats analysis and whole genome sequencing. The nucleotide sequence of an IncK plasmid carrying bla_CMY-2 was determined. Intestinal isolates displayed a higher degree of genetic diversity than meat isolates. A cluster of genetically related isolates belonging to ST38, phylogroup D, carrying bla_CMY-2 containing IncK plasmids was identified. Furthermore, genes encoding plasmid stability systems (relBE/stbDE and pndAC) were identified on the IncK plasmid. Single nucleotide polymorphism (SNP) analysis of a subset of isolates confirmed a close genetic relationship within the two most prevalent STs. The IncK plasmids within these two STs also shared a high degree of similarity. Cephalosporin-resistant E. coli with the same genetic characteristics have been identified in the broiler production in other European countries, and the IncK plasmid characterized in this study showed close homology to a plasmid isolated from retail chicken meat in the Netherlands. The results indicate that both clonal expansion and horizontal transfer of bla_CMY-2 containing plasmids contribute to dissemination of cephalosporin resistant E. coli in the broiler production. The presence of plasmid stability systems may explain why the IncK plasmid containing bla_CMY-2 is maintained and disseminated in the Norwegian broiler production in absence of selection pressure from the use of antimicrobial agents.     

Associative learning in immature lacewings (Ceraeochrysa cubana)

It is known that many social insects and arthropod predators and parasitoids can learn the association between a resource and volatile cues. Although there are various studies on the effect of experience in immature arthropods on behavior later in adult life, not much is known about the effects of such experiences on immature behavior. This was investigated here in the lacewing Ceraeochrysa cubana (Hagen) (Neuroptera: Chrysopidae). Whereas adults of this lacewing feed on plant‐provided food and honeydew, larvae are voracious polyphagous predators of several insect pests, and therefore important for biological control. Hence, studying the foraging behavior and the effects of learning in immatures of this species is important. We exposed immatures to the volatile methyl salicylate (MeSA), which was either associated with food or with the absence of food. Subsequently, their response to this volatile was tested in an olfactometer. Immatures that had experienced the association of MeSA with food were attracted to it and immatures that were exposed to MeSA during food deprivation were repelled. Subsequently, predator immatures that had experienced the association between MeSA and food were released on a plant without food and were found to use this volatile in locating patches with food. In contrast, larvae without such experience were found equally on food patches with and without the volatile. We conclude that these immature predators are capable of learning the association between volatiles and food, or the absence of food, and use this during foraging.     

Vaccination of macaques with long-standing SIVmac251 infection lowers the viral set point after cessation of antiretroviral therapy

A cohort of rhesus macaques with long-standing SIVmac251 infection (> or =5 mo) was treated with continuous antiretroviral therapy (ART). A group of eight macaques was vaccinated with or without simultaneous administration of low dose IL-2 with the highly attenuated poxvirus vector (NYVAC) vaccine candidate expressing the SIVmac structural gag-pol-env (gpe) genes and a novel chimeric fusion protein derived from the rev-tat-nef (rtn) regulatory genes. Control groups consisted of mock-vaccinated macaques or animals treated only with IL-2. Vaccination significantly expanded both virus-specific CD4(+) and CD8(+) T cell responses, and IL-2 further increased the vaccine-induced response to an immunodominant Gag epitope. Following antiretroviral treatment interruption, the viral set point was significantly lower in vaccinated than in control macaques for at least 4 consecutive mo, and viral containment was inversely correlated with vaccine-induced, virus-specific CD4(+) and CD8(+) T cell responses. These data provide the proof of concept that therapeutic vaccination before cessation of ART may be a feasible approach in the clinical management of HIV-1 infection.     

Endostatin inhibits microvessel formation in the ex vivo rat aortic ring angiogenesis assay

Endostatin has demonstrated potent antiangiogenic and antitumor activity in mouse models. We have investigated the ex vivo rat aortic ring assay and a human vein model to assess the biological activity of murine and human endostatin. Rat aortic rings were exposed to recombinant murine endostatin (Spodoptera frugipera; Calbiochem, San Diego, CA) or recombinant human endostatin (Pichia pastoris; EntreMed, Rockville, MD). After 5 days, murine endostatin (500 microgram/ml) demonstrated inhibition of microvessel outgrowth with dose-dependent effects (down to 16 microgram/ml). No significant inhibition was observed with human endostatin in the rat assay. Human endostatin at 250 and 500 microgram/ml inhibited outgrowths from human saphenous vein rings after a 14-day incubation. Electron microscopy assessed the formation of basal lamina, confirming that the microvessels were progenitors of patent vessels. Immunostaining for Factor VIII or CD34 demonstrated that the microvessel cells were endothelial. BrdU incorporation assays supported the presence of proliferating endothelial cells, correlating with neovascularization from the aortic wall. We conclude that the rat aortic ring assay confirms the antiangiogenic activity of murine but not human endostatin, suggesting that the model may have species specificity. However, the human form shows biological activity against human vascular tissue.     

Improvement in multiple dimensions of fatigue in patients with fibromyalgia treated with duloxetine: secondary analysis of a randomized,placebo-controlled trial

Introduction  

Fatigue is one of the most disabling symptoms associated with fibromyalgia that greatly impacts quality of life. Fatigue was assessed as a secondary objective in a 2-phase, 24-week study in outpatients with American College of Rheumatology-defined fibromyalgia.