Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber.

RATIONALE: Analysis of steroids from precious blubber biopsies obtained from marine mammals, especially endangered species, can provide valuable information on their endocrine status. Challenges with currently used ELISA methodology include lack of absolute quantitation and incompatibility with multiple steroids analysis due to limited biopsy mass. Development of a sensitive, accurate analytical method for this purpose is critical. METHODS: A nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) method was validated for sensitive, specific and quantitative analysis of three steroid hormones, without derivatization, extracted from 50 mg blubber samples. Data was acquired with an LTQ XL ion trap mass spectrometer in positive ion mode, using single reaction monitoring. All three steroids were analyzed in a single run. Cholic acid was used as a surrogate internal standard for quantitation due to its steroidal structure and lack of measurable endogenous levels in blubber. RESULTS: The lowest limits of quantitation for progesterone, testosterone, and hydrocortisone were significantly improved compared to previous studies using conventional LC/MS/MS. The lowest limit of detection was 7 fg/µL using a 1 µL injection volume. Calibration curves for steroid quantification showed good linearity (r2 >0.99) between 14 and 3620 fg/µL, and accuracy was <20% for interday and <10% for intraday. After validation, the method was successfully applied to quantification of steroids in gray whale blubber samples. CONCLUSIONS: The nanoLC/MS/MS method is more sensitive than traditional LC/MS/MS for steroid analysis. It is also compatible with other important biopsy analyses due to its small blubber mass requirement. This will benefit the reproductive and stress assessments for all marine mammals, particularly endangered populations. Copyright © 2017 John Wiley & Sons, Ltd.

Associations between tissue visfatin/nicotinamide, phosphoribosyltransferase (Nampt), retinol binding protein-4, and vaspin concentrations and insulin resistance in morbidly obese subjects.

Visfatin/Nampt, vaspin, and retinol binding protein-4 (RBP-4) play an important role in insulin resistance. The objectives of this study were to measure visfatin/Nampt, vaspin, and RBP-4 concentrations in blood, liver, muscle, subcutaneous, omental, and mesenteric adipose tissues in morbidly obese subjects and investigate their relationship to insulin resistance. Blood and tissue samples were collected from 38 morbidly obese subjects during Roux-en-Y surgery. Insulin resistance biomarkers were measured using standard kits. Visfatin/Nampt, vaspin, and RBP-4 gene expression levels in tissues were measured using real-time PCR. Their protein concentrations in blood and tissues were measured using ELISA kits. Diabetic subjects had significantly higher homeostasis model of assessment-insulin resistance and age and lower blood HDL-cholesterol concentrations than nondiabetic and prediabetic subjects. Diabetic and prediabetic subjects had significantly higher blood concentrations of visfatin/Nampt and vaspin than nondiabetic subjects. Liver RBP-4 concentrations were positively associated with blood glucose concentrations. Blood insulin resistance biomarker levels were positively associated with visfatin/Nampt concentrations in omental adipose tissue and liver, and vaspin concentrations in mesenteric adipose tissue. In conclusion, the correlations of visfatin/Nampt, vaspin, and RBP-4 with insulin resistance are tissue dependent.

Browning white adipose tissue using adipose stromal cell-targeted resveratrol-loaded nanoparticles for combating obesity.

Enhancing thermogenic energy expenditure via promoting the browning of white adipose tissue (WAT) is a potential therapeutic strategy to manage energy imbalance and the consequent comorbidities associated with excess body weight. Adverse effects and toxicities of currently available methods to induce browning of WAT have retarded exploration of this promising therapeutic approach. Targeted delivery of browning agents to adipose stromal cells (ASCs) in subcutaneous WAT to induce differentiation into beige adipocytes may overcome these barriers. Herein, we report for the first time, ASC-targeted delivery of trans-resveratrol (R), a representative agent, using ligand-coated R-encapsulated nanoparticles (L-Rnano) that selectively bind to glycanation site-deficient decorin receptors on ASCs. After biweekly intravenous administration of L-Rnano to obese C57BL/6 J mice for 5 weeks targeted R delivery significantly induced ASCs differentiation into beige adipocytes, which subsequently resulted in 40% decrease in fat mass, accompanied by improved glucose homeostasis and decreased inflammation. Our results suggest that the ASC-targeted nanoparticle delivery of browning agents could be a transformative technology in combating obesity and its comorbidities with high efficacy and low toxicity.

Placentation in watersnakes I: Placental histology and development in North American Nerodia (Colubridae: Natricinae).

As part of a broad survey of placental structure, function, and evolution in reptilian sauropsids paraffin-section histology was used to study microscopic anatomy of the uterus and fetal membranes of three species of North American watersnakes (Nerodia: Colubridae). The pre-ovulatory uterus is poorly vascularized with inactive shell glands. These shell glands are activated during vitellogenesis but regress during pregnancy. Two placentas develop through apposition of the uterine lining to the chorioallantois and the yolk sac omphalopleure. Fetal and maternal components of the chorioallantoic placenta are progressively vascularized during development. Their epithelia are attenuated, but (contrary to a previous report), epithelia of neither the uterus nor the chorion are eroded. The fetal portion of the yolk sac placenta is an omphalallantois, formed of avascular omphalopleure, isolated yolk mass, and allantois. This placenta is progressively replaced by chorioallantoic placenta during mid- to late-development through depletion of the isolated yolk mass. The chorioallantoic placenta is anatomically specialized for maternal-fetal gas exchange, and its expansion during development reflects the growing needs of the fetus for gas exchange. The yolk sac placenta is morphologically unsuited for gas exchange, but may serve other functions in maternal-fetal exchange.

Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone.

Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure.

A novel subtilisin-like serine protease of Batrachochytrium dendrobatidis is induced by thyroid hormone and degrades antimicrobial peptides.

Batrachochytrium dendrobatidis (B. dendrobatidis), a chytrid fungus, is one of the major contributors to the global amphibian decline. The fungus infects both tadpoles and adult amphibians. Tadpoles are infected in their keratinized mouthparts, and infected adults exhibit hyperkeratosis and loss of righting reflex. Infections of adults may result in death from cardiac arrest in susceptible species. Thyroid hormone plays a key role in amphibian metamorphosis. The occurrence of B. dendrobatidis in tadpoles during metamorphosis may result in exposure of the fungus to host morphogens including TH. This exposure may induce gene expression in the fungus contributing to invasion and colonization of the host. Here, we demonstrate movement of fungal zoospores toward TH. Additionally, expression of a subtilisin-like serine protease is up-regulated in B. dendrobatidis cells exposed to TH. A gene encoding this protease was cloned from B. dendrobatidis and expressed in Escherichia coli. The protein was partially purified and characterized. The similarity between subtilases of human dermatophytes and the B. dendrobatidis subtilisin-like serine protease suggests the importance of this enzyme in B. dendrobatidis pathogenicity. Cleavage of frog skin antimicrobial peptides (AMPs) by this B. dendrobatidis subtilisin-like serine protease suggests a role for this enzyme in fungal survival and colonization.

Histology of abortive egg sites in the uterus of a viviparous, placentotrophic lizard, the skink Chalcides chalcides.

Although abortive uterine eggs are often assumed to be resorbed by females of the viviparous skink Chalcides chalcides, little microscopic evidence of resorption of such eggs is available. Oviducts from pregnant female C. chalcides in which egg resorption was inferred were examined histologically to seek a morphological basis for resorption. Uterine histology at the site of abortive eggs was very similar to that of lizards in early pregnancy. The uterine epithelium consisted of a monolayer of pseudostratifed columnar cells that showed no evidence of yolk phagocytosis. The uterine lamina propria exhibited shell glands and modest vascularity, typical of early gestation, and contained neither yolk droplets nor accumulating leukocytes. Unattenuated regions of the lamina propria contained occasional macrophages and mast cells, some of the latter of which were undergoing degranulation. The abortive eggs often were collapsed with ruptured shell membranes, and some were undergoing extrusion from the incubation chambers down the oviduct. In eggs that had begun developing, extraembryonic ectoderm and endoderm were atypical in location, and had failed to enclose yolk leaking from the eggs. Oviducts sampled from later in the reproductive season were reproductively inactive, and showed no trace of abortive eggs or egg components. We postulate that abortive eggs are extruded from the oviduct by pregnant females under conditions of physiological stress, as a means of enhancing future reproductive effort. J. Morphol. 235:97-108, 1998. © 1998 Wiley-Liss, Inc.

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis.

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.