Leptin signaling promotes blood vessel formation in the Xenopus tail during the embryo-larval transition.

The signals that regulate peripheral blood vessel formation during development are still under investigation. The hormone leptin promotes blood vessel formation, adipose tissue establishment and expansion, tumor growth, and wound healing, but the underlying mechanisms for these actions are currently unknown. We investigated whether leptin promotes angiogenesis in the developing tail fin using embryonic transgenic xflk-1:GFP Xenopus laevis, which express a green fluorescent protein on vascular endothelial cells to mark blood vessels. We found that leptin protein is expressed in endothelial cells of developing blood vessels and that leptin treatment via injection increased phosphorylated STAT3 signaling, which is indicative of leptin activation of its receptor, in blood vessels of the larval tail fin. Leptin administration via media increased vessel length, branching, and reconnection with the cardinal vein, while decreased leptin signaling via immunoneutralization had an opposing effect on vessel development. We also observed disorganization of major vessels and microvessels of the tail fin and muscle when leptin signaling was decreased. Reduced leptin signaling lowered mRNA expression of cenpk, gpx1, and mmp9, markers for cell proliferation, antioxidation, and extracellular matrix remodeling/cell migration, respectively, in the developing tail, providing insight into three possible mechanisms underlying leptin's promotion of angiogenesis. Together these results illustrate that leptin levels are correlated with embryonic angiogenesis and that leptin coordinates multiple aspects of blood vessel growth and development, showing that leptin is an important morphogen during embryonic development.

Phosphorylation of serine residues S252, S268/S269, and S879 in p120 catenin activates migration of presomitic mesoderm in gastrulating zebrafish embryos.

BACKGROUND: Cadherin-associated protein p120 catenin regulates cell adhesion and migration in cell cultures and is required for axial elongation in embryos. Its roles in adhesion and cell migration are regulated by phosphorylation. We determined the effects of phosphorylation of six serine and three threonine residues in p120 catenin during zebrafish (Danio rerio) embryogenesis. RESULTS: We knocked down endogenous p120 catenin-δ1 with an antisense RNA-splice-site morpholino (Sp-MO) causing defects in axis elongation. These defects were rescued by co-injections of mRNAs for wildtype mouse p120 catenin-δ1-3A or various mutated forms. Several mRNAs containing serine or threonine codons singly or doubly mutated to phosphomimetic glutamic acid rescued, and some nonphosphorylatable mutants did not. CONCLUSIONS: We discovered that phosphorylation of serine residue S252 or S879 is required for convergent extension of zebrafish embryos, since rescue occurred only when these residues were mutated to glutamic acid. In addition, the phosphorylation of either S268 or S269 is required, not both, consistent with the presence of only a single one of these residues in two isoforms of zebrafish and Xenopus laevis. In summary, phosphorylation of multiple serine and threonine residues of p120 catenin activates migration of presomitic mesoderm of zebrafish embryos facilitating elongation of the dorsal axis.

Detection of sensitivity and vestigiality of presumptive tests for swabbed blood stains.

It is a common practice in forensic casework to use presumptive tests for blood stains before DNA extraction and testing. Stains are usually swabbed and then the swabs are sent for analysis. The Kastle-Meyer (KM) and Leucomalachite green (LMG) presumptive tests for blood are widely used, and their sensitivities have been thoroughly tested in the literature in solution and directly on stains, but not on swabbed stains to mimic casework. In this study, the sensitivity of the KM and LMG tests was tested on eight blood dilutions on cotton fabric and ceramic tile that were stained and subsequently swabbed. Both tests showed sensitivity up to 1:5000, which is slightly lower than reported values in solution or directly on stain but still highly effective in most cases. Stains were also cleaned with common agents, then swabbed and re-tested. Stained ceramic tiles cleaned with soap/water or bleach gave mixed positive and negative results for the 1:10 dilution, presumably due to variance in how thoroughly each investigator cleaned the stain, and other dilutions were undetectable after cleaning. The LMG test gave false positives for bleach cleaned stains, due to reagent reactivity with bleach. Surprisingly, blood was detectible up to the 1:100 dilution with both tests on stained cotton fabric that was cleaned in a washing machine with detergent and dried. Ultimately the KM and LMG presumptive tests remain effective tools for swabbed blood stains, and their practicality for cleaned stains is dependent on material containing the stain, cleaning agent and processing.

Trans-ovo permethrin exposure affects growth, brain morphology and cardiac development in quail.

Permethrin is a commonly used, highly effective pesticide in poultry agriculture, and has recently been trialed in conservation efforts to protect Galápagos finch hatchlings from an invasive ectoparasite. Although permethrin is considered safe for adults, pesticides can have health consequences when animals are exposed during early life stages. The few studies that have examined permethrin's effects in embryonic chicks and rats have shown hydrocephaly, anencephaly, reduced cellular energy conversion, and disruption of developing heart muscle. To test whether trans-ovo exposure of permethrin affects early development in birds, we exposed Japanese quail (Coturnix japonica) eggs to cotton treated with 1% permethrin that was incorporated into nests in two amounts (0.2, 0.8 g), each with a paired untreated cotton control group. When measured on incubation Day 15, we found permethrin-treated developing birds were smaller and showed signs of microcephaly, although mortality rates were the same. Despite no difference in heart mass, ventricular tissue was less compact, cardiac arteries were reduced and heart rates were slower in permethrin-treated birds. Differences in heart development were also observed at 5 days of incubation, indicating that abnormalities are present from early in cardiac development. Future studies are needed to examine permethrin's effects on developmental pathways and to determine if these effects persist after hatching to affect offspring health. This study provides evidence that permethrin can cross the eggshell to cause non-lethal but adverse effects on embryonic development, and studies should look beyond hatching when monitoring the efficacy of permethrin on wild bird populations.

Bromophenolics from the Red Alga Polysiphonia decipiens.

The isolation and the structure determination of a new bromophenolic compound, polysiphonol (10), as well as five previously reported compounds, (4-8), from the red alga Polysiphonia decipiens is reported. In addition, the absolute configuration of the natural product rhodomelol (8) could be unequivocally confirmed for the first time, and on biosynthetic grounds, the absolute configuration of polysiphonol (10) was tentatively suggested. Compounds 4-8 were evaluated for their antibacterial activity against both Gram-positive and Gram-negative bacteria, but none of the compounds showed any appreciable activity.

Diet- and salinity-induced modifications of the gut microbiota are associated with differential physiological responses to ranavirus infection in Rana sylvatica.

Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Evolutionary conservation of leptin effects on wound healing in vertebrates: Implications for veterinary medicine.

In mammals, the cytokine hormone leptin promotes wound healing by increasing inflammation, cellular recruitment, angiogenic regrowth, and re-epithelialization; however, it is not known whether leptin has conserved actions on wound healing in other vertebrates. Here, we tested the hypothesis that leptin promotes both the quality and speed of wound healing in the South African clawed frog, Xenopus laevis. First, fluorescent immunohistochemistry using a polyclonal antibody specific to Xenopus leptin showed that in juvenile dorsal skin, leptin protein is expressed in the dorsal epidermal layer, as well in blood vessel endothelial cells and sensory nerves that run along the base of the dermis. Injection of recombinant Xenopus leptin (rXleptin) stimulates phosphorylated STAT3 (pSTAT3), indicative of leptin-activated JAK/STAT signaling in the epidermis. Similar to mammals, leptin protein expression increases at the wound site after injury of the epidermis. We then cultured "punch-in-a-punch" full-thickness dorsal skin explants in three doses of rXleptin (0, 10, and 100 ng/ml) and showed that leptin treatment doubled the rate of wound closure after 48 h relative to skin punches cultured without leptin. Food restriction prior to wound explant culture reduced the amount of wound closure, but leptin injection prior to euthanasia rescued closure to similar control levels. Leptin treatment also significantly reduced bacterial infection of these epidermal punches by 48 h in culture. This study shows that leptin is likely an endogenous promoter of wound healing in amphibians. Leptin-based therapies have the potential to expedite healing and reduce the incidence of secondary infections without toxicity issues, the threat of antibiotic resistance, or environmental antibiotic contamination. The conservation of leptin's actions on wound healing also suggests that it may have similar veterinary applications for other exotic species.