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.     

The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane

Cytochrome P450scc (CYP11A1) is a protein attached to the inner surface of the inner mitochondrial membrane that uses cholesterol from the membrane phase as its substrate for the first step in steroid hormone synthesis. We investigated the mechanism by which CYP11A1 interacts with the membrane. Hydrophobicity profiles of CYP11A1 and two other mitochondrial cytochromes P450, plus a model structure of CYP11A1 using CYP2C5 as template, suggest that CYP11A1 has a monotopic association with the membrane which may involve the A' helix and the F-G loop. Deletion of the A' helix reduced the proportion of expressed CYP11A1 associated with the bacterial membrane fraction, indicating a role for the A' helix in membrane binding. However, introduction of a cysteine residue in this helix at position 24 (L24C) and subsequent labelling with the fluorescent probe N'-(7-nitrobenz-2-oxal,3-diazol-4-yl)ethylenediamine (NBD) failed to show a membrane localisation. Cysteine mutagenesis and fluorescent labelling of other residues appearing on the distal surface of the CYP11A1 model revealed that V212C and L219C have enhanced fluorescence and a blue shift following association of the mutant CYP11A1 with phospholipid vesicles. This indicates that these residues, which are located in the F-G loop, become localised to a more hydrophobic environment following membrane binding. Analysis of the quenching of tryptophan residues in CYP11A1 by acrylamide indicates that at least one and probably two tryptophans are involved in membrane binding. We conclude that CYP11A1 has a monotopic association with the membrane that is mediated, at least in part, by the F-G loop region.     

Beta-scission of side-chain alkoxyl radicals on peptides and proteins results in the loss of side-chains as aldehydes and ketones

Exposure of proteins to radicals in the presence of O(2) results in side-chain oxidation and backbone fragmentation; the interrelationship between these processes is not fully understood. Recently, initial attack on Ala side-chains was shown to give alpha-carbon radicals (and hence backbone cleavage) and formaldehyde, via the formation and subsequent beta-scission, of C-3 alkoxyl radicals. We now show that this side-chain to backbone damage transfer, is a general mechanism for aliphatic side-chains. Oxidation of Val, Leu, and Asp residues by HO(*)/O(2) results in the release of a family of carbonyls (including formaldehyde, acetone, isobutyraldehyde, and glyoxylic acid) via the formation, and subsequent beta-scission of alkoxyl radicals. The concentration of these products increases with the HO(*) flux. The release of multiple carbonyls confirms the occurrence of oxidation at C-3 and C-4 for Val, and these sites, plus C-5, for Leu. The detection of glyoxylic acid and CO(2)(-*) from Asp demonstrates the occurrence of competing beta-scission processes for the Asp C-3 alkoxyl radical. The yield of hydroperoxides and released carbonyls account for 10-145% of the initial HO(*). The greater than 100% yields confirm the occurrence of chain reactions in peptide/protein oxidation, with more than one residue being damaged per initiating radical.     

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.     

Protein--protein interactions in the eubacterial replisome

Replication of genomic DNA is a universal process that proceeds in distinct stages, from initiation to elongation and finally to termination. Each stage involves multiple stable or transient interactions between protein subunits with functions that are more or less conserved in all organisms. In Escherichia coli, initiation of bidirectional replication at the origin (oriC) occurs through the concerted actions of the DnaA replication initiator protein, the hexameric DnaB helicase, the DnaC?helicase loading partner and the DnaG primase, leading to establishment of two replication forks. Elongation of RNA primers at each fork proceeds simultaneously on both strands by actions of the multimeric replicase, DNA polymerase III holoenzyme. The fork that arrives first in the terminus region is halted by its encounter with a correctly-oriented complex of the Tus replication terminator protein bound at one of several Ter sites, where it is trapped until the other fork arrives. We summarize current understanding of interactions among the various proteins that act in the different stages of replication of the chromosome of E. coli, and make some comparisons with the analogous proteins in Bacillus subtilis and the coliphages T4 and T7.