Resource and animal use implications of the proposed REACH information requirements for endocrine disruptor assessment.

Revised information requirements for endocrine disruptor (ED) assessment of chemicals under the European Union's Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation have been proposed. Implementation will substantially increase demands for new data to inform ED assessment. This article evaluates the potential animal use and financial resource associated with two proposed ED policy options, and highlights areas where further clarification is warranted. This evaluation demonstrates that studies potentially conducted to meet the proposed requirements could use tens of millions of animals, and that the approach is unlikely to be feasible in practice. Given the challenges with implementing either policy option and the need to minimise the reliance on animal testing, further consideration and clarification is needed on several aspects prior to implementation of the requirements. This includes how testing will be prioritised in a proportionate approach; how to harness new approach methodologies to waive higher-tier animal testing; and need for provision of clear guidance particularly in applying weight-of-evidence approaches. There is now a clear opportunity for the European Commission to lead the way in developing a robust and transparent ED assessment process for industrial chemicals which fully implements replacement, refinement, and reduction of the use of animals (the 3Rs).

Understanding Medical Standards for Entrance Into Military Service and Disqualifying Dermatologic Conditions.

The military is a desirable career field for many young adults; however, the Department of Defense (DoD) requires its members to maintain a level of health necessary to meet the physical demands of military duties and be able to deploy to austere environments. The strict standards are designed to protect the health of the individual and maximize mission success. Standards for entrance into the US Armed Forces, called accession standards, are codified in DoD Instruction 6130.03, Volume 1 and include a section dedicated to skin and soft tissue conditions. This document lists medical conditions that do not meet the standard due to current and prior diagnoses and is regularly updated by a board using the best available scientific evidence. Applicants who do not meet the physical and medical standards can be considered for a medical waiver, although not guaranteed. Generally, retention standards differ for those already serving in the military and will not be addressed here. The focus of this article is to inform the general dermatologic community that these standards exist, to discuss specific dermatologic conditions that are disqualifying at the current time, and to provide resources for the dermatologist or primary care physician to access current information.

Effects of a senior practicum course on nursing students' confidence in speaking up for patient safety.

As patient advocates, nurses are responsible for speaking up against unsafe practices. Nursing students must develop the confidence to speak up for patient safety so that they can hold themselves, as well as their peers and coworkers, accountable for patients' well-being. The purpose of this study was to examine the effects of a senior practicum course on confidence for speaking up for patient safety in nursing students. Confidence in speaking up for patient safety was measured with the Health Professional Education in Patient Safety Survey. The study showed a significant increase in nursing students' confidence after the senior practicum course, but there was no significant change in students' confidence in questioning someone of authority.

The effects of dexamethasone on cigarette smoke induced gene expression changes in COPD macrophages.

Chronic obstructive pulmonary disease (COPD) is a smoking related inflammatory airway disease in which macrophages play a key role. Previously we have shown that cigarette smoke extract (CSE) causes suppression of macrophage inflammatory mediators, with the exception of IL-8. We now investigate the effects of dexamethasone on these gene expression changes. Monocyte derived macrophages (MDMs) were cultured with CSE and dexamethasone. Microarray analysis was used to assess inflammatory mediator regulation, with qPCR and ELISA also performed for selected cytokines. The major effect of CSE was down-regulation of inflammatory genes (11 probe sets). For CSE regulated genes (n=13), the median fold change with CSE alone was -2.84 and with dexamethasone alone was -2.97. Both treatments combined caused the greatest suppression of gene expression; -4.47. qPCR also showed that IL-1beta, GM-CSF and IL-6 mRNA levels were significantly reduced by CSE and further suppressed by dexamethasone. qPCR and ELISA showed that IL-8 levels were increased by CSE, with suppression by dexamethasone. We show that CSE suppressed the expression of some inflammatory genes whilst up-regulating IL-8. Dexamethasone further suppressed gene expression when combined with CSE. The combined effect of GC and CSE causes suppression of the macrophage innate immune response.

Evolution of the gene lineage encoding the carbon dioxide receptor in insects.

A heterodimer of the insect chemoreceptors Gr21a and Gr63a has been shown to be the carbon dioxide receptor in Drosophila melanogaster (Meigen) (Diptera: Drosophilidae). Comparison of the genes encoding these two proteins across the 12 available drosophilid fly genomes allows refined definition of their N-termini. These genes are highly conserved, along with a paralog of Gr21a, in the Anopheles gambiae, Aedes aegypti, and Culex pipiens mosquitoes, as well as in the silk moth Bombyx mori and the red flour beetle Tribolium castaneum. In the latter four species we name these three proteins Gr1, Gr2, and Gr3. Intron evolution within this distinctive three gene lineage is considerable, with at least 13 inferred gains and 39 losses. Surprisingly, this entire ancient gene lineage is absent from all other available more basal insect and related arthropod genomes, specifically the honey bee, parasitoid wasp, human louse, pea aphid, waterflea, and blacklegged tick genomes. At least two of these species can detect carbon dioxide, suggesting that they evolved other means to do so.

Evolution of the sugar receptors in insects.

BACKGROUND: Perception of sugars is an invaluable ability for insects which often derive quickly accessible energy from these molecules. A distinctive subfamily of eight proteins within the gustatory receptor (Gr) family has been identified as sugar receptors (SRs) in Drosophila melanogaster (Gr5a, Gr61a, and Gr64a-f). We examined the evolution of these SRs within the 12 available Drosophila genome sequences, as well as three mosquito, two moth, and beetle, bee, and wasp genome sequences. RESULTS: While most Drosophila species retain all eight genes, we find that the three Drosophila subgenus species have lost Gr64d, while D. grimshawi and the D. pseudoobscura/persimilis sibling species have also lost Gr5a function. The entire Gr64 gene complex was also duplicated in the D. grimshawi lineage, but only one potentially functional copy of each gene has been retained. The numbers of SRs range from two in the hymenopterans Apis mellifera and Nasonia vitripennis to 16 in the beetle Tribolium castaneum. An unusual aspect is the evolution of a novel exon from intronic sequence in an expanded set of four SRs in Bombyx mori (BmGr5-8), which appears to be the first example of such exonization in insects. Twelve intron gains and 63 losses are inferred within the SR family. CONCLUSION: Examination of the SRs in these fly, mosquito, moth, beetle, and hymenopteran genome sequences reveals that they appear to have originated independently from single ancestral genes within the dipteran and coleopteran lineages, and two genes in the lepidopteran and hymenopteran lineages. The origin of the insect SRs will eventually be illuminated by additional basal insect and arthropod genome sequences.

Inhibition of lipopolysaccharide-stimulated chronic obstructive pulmonary disease macrophage inflammatory gene expression by dexamethasone and the p38 mitogen-activated protein kinase inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d] pyrimidin-2-yl]amino}ethyl)guanidine (SB706504).

p38 mitogen-activated protein kinase (MAPK) signaling is known to be increased in chronic obstructive pulmonary disease (COPD) macrophages. We have studied the effects of the p38 MAPK inhibitor N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]-pyrimidin-2-yl]amino}ethyl)guanidine (SB706504) and dexamethasone on COPD macrophage inflammatory gene expression and protein secretion. We also studied the effects of combined SB706504 and dexamethasone treatment. Lipopolysaccharide (LPS)-stimulated monocyte derived macrophages (MDMs) and alveolar macrophages (AMs) were cultured with dexamethasone and/or SB706504. MDMs were used for gene array and protein studies, whereas tumor necrosis factor (TNF) alpha protein production was measured from AMs. SB706504 caused transcriptional inhibition of a range of cytokines and chemokines in COPD MDMs. The use of SB706504 combined with dexamethasone caused greater suppression of gene expression (-8.90) compared with SB706504 alone (-2.04) or dexamethasone (-3.39). Twenty-three genes were insensitive to the effects of both drugs, including interleukin (IL)-1beta, IL-18, and chemokine (CC motif) ligand (CCL) 5. In addition, the chromosome 4 chemokine cluster members, CXCL1, CXCL2, CXCL3, and CXCL8, were all glucocorticoid-resistant. SB706504 significantly inhibited LPS-stimulated TNFalpha production from COPD and smoker AMs, with near-maximal suppression caused by combination treatment with dexamethasone. We conclude that SB706504 targets a subset of inflammatory macrophage genes and when used with dexamethasone causes effective suppression of these genes. SB706504 and dexamethasone had no effect on the transcription of a subset of LPS-regulated genes, including IL-1beta, IL-18, and CCL5, which are all known to be involved in the pathogenesis of COPD.

Cigarette smoke extract induced cytokine and chemokine gene expression changes in COPD macrophages.

Macrophages are key inflammatory cells in chronic obstructive pulmonary disease (COPD). The transcriptional regulation of inflammatory signalling pathways by cigarette smoke (CS) in COPD macrophages is not well understood. We have studied the effects of acute CS exposure on COPD macrophage cytokine, chemokine and signal transduction gene expression profiles. Monocyte derived macrophages (MDMs) from whole blood from patients with COPD (n=6) were stimulated with 1%, 10% and 25% CS extract (CSE) for 6h for microarray and quantitative polymerase chain reaction (Q-PCR) analysis. We observed a CSE dose dependant increase in the numbers of significantly regulated genes; 24, 340 and 627 genes at 1%, 10% and 25% CSE, respectively. IL-8 mRNA levels were up-regulated by 10% CSE (2.25-fold increase, 95% CI 1.28-4.00). In contrast a range of other cytokines and chemokines were down-regulated at both 10% and 25% CSE, including IL-1beta, -6, -10 and -18, chemokine ligands CCL-2, -3, -4, -5, -8, -15, -20 and CXCL-1, -2 and -10. Q-PCR and microarray data were highly correlated (r=0.95, p=0.0001). NF-kappaB component p50 and IkappaBalpha expression were suppressed by CSE, while there was up-regulation of the AP-1 components c-Jun, FOSL1 and FOSL2. Acute CSE exposure decreased macrophage inflammatory gene expression, with the exception of increased IL-8. There was diverse regulation of key inflammatory signal pathway genes. The effects of acute CS exposure appear to encompass both up-regulation of chemotaxis mechanisms through IL-8, but also down-regulation of innate immunity.

The Gr family of candidate gustatory and olfactory receptors in the yellow-fever mosquito Aedes aegypti.

The gustatory receptor (Gr) protein family contains most of the diversity in the insect chemoreceptor superfamily, including within it not only taste receptors but select olfactory receptors as well. Manual annotation of the Gr family in the genome sequence of the yellow-fever mosquito, Aedes aegypti, yielded a total of 114 potential proteins encoded by 79 genes. In the sequenced genome, 23 of these genes and protein isoforms are pseudogenic, leaving 91 putatively functional Grs. Comparison with our previously published set of 76 Grs encoded by 52 genes in the distantly related Anopheles gambiae mosquito revealed 13 new AgGrs encoded by 8 genes. Phylogenetic analysis reveals the conservation of carbon dioxide, sugar, and several orphan receptors in these 2 mosquitoes and Drosophila flies. On the other hand, most of these Grs are unique to mosquitoes and many are specific to the Aedes or Anopheles lineages, indicating their involvement in mosquito-specific aspects of both gustatory and olfactory perception. In particular, most instances of alternative splicing in orthologous loci appear to have evolved after the culicine-anopheline split +/-150 million years ago.

G protein-coupled receptors in Anopheles gambiae.

We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.