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Purines are the building blocks of DNA/RNA, energy substrates, and cofactors. Purine metabolites, including ATP, GTP, NADH, and coenzyme A, are essential molecules in diverse biological processes such as energy metabolism, signal transduction, and enzyme activity. When purine levels increase, excess purines are either recycled to synthesize purine metabolites or catabolized to the end product uric acid. Purine catabolism increases during states of low oxygen tension (hypoxia and ischemia), but this metabolic pathway is incompletely understood in the context of histotoxic hypoxia (i.e., inhibition of oxygen utilization despite normal oxygen tension). In rabbits exposed to cyanide—a classical histotoxic hypoxia agent—we demonstrated significant increases in several concordant metabolites in the purine catabolic pathway (including plasma levels of uric acid, xanthosine, xanthine, hypoxanthine, and inosine) via mass spectrometry-based metabolite profiling. Pharmacological inhibition of the purine catabolic pathway with oxypurinol mitigated the deleterious effects of cyanide on skeletal muscle cytochrome c oxidase redox state, measured by non-invasive diffuse optical spectroscopy. Finally, plasma uric acid levels correlated strongly with those of lactic acid, an established clinical biomarker of cyanide exposure, in addition to a tissue biomarker of cyanide exposure (skeletal muscle cytochrome c oxidase redox state). Cumulatively, these findings not only shed light on the in vivo role(s) of cyanide but also have implications in the field of medical countermeasure (MCM) development. [ABSTRACT FROM AUTHOR]

Cyanide represents a persistent threat for accidental or malicious misuse due to easy conversion into a toxic gas and access to large quantities through several industries. The high safety index of hydroxocobalamin is a cornerstone quality as a cyanide scavenger. Unfortunately, intravenous infusion of hydroxocobalamin limits the utility in a mass casualty setting. We previously reported platinum(II) [Pt(II)] complexes with trans-directing sulfur ligands as an efficacious alternative to hydroxocobalamin when delivered by a bolus intramuscular (IM) injection in mice and rabbits. Thus, to enable Pt(II) as an alternative to hydroxocobalamin, a high safety factor is needed. The objective is to maintain efficacy and mitigate the risk of nephrotoxicity. Platinum amino acid complexes with the ability to form 5- or 6-membered rings and possessing either carboxylates or carboxamides are evaluated in vitro for cyanide scavenging. In vivo efficacy wa s evaluated in the zebrafish and mice cyanide exposure models. In addition, Pt(II) complex toxicity and pharmacokinetics were evaluated in a cyanide naive Sprague Dawley model. Doses for toxicity are escalated to 5× from the efficacious dose in mice using a body surface area adjustment. The results show the carboxamide ligands display a time and pH dependence on cyanide scavenging in vitro and efficacy in vivo. Additionally, exchanging the carboxylate for carboxamide showed reduced indications of renal injury. A pharmacokinetic analysis of the larger bidentate complexes displayed rapid absorption by IM administration and having similar plasma exposure. These findings point to the importance of pH and ligand structures for methionine carboxamide complexes with Pt(II). [ABSTRACT FROM AUTHOR]

Throughout a vertebrate organism's lifespan, skeletal muscle mass and function progressively decline. This age-related condition is termed sarcopenia. In humans, sarcopenia is associated with risk of falling, cardiovascular disease, and all-cause mortality. As the world population ages, projected to reach 2 billion older adults worldwide in 2050, the economic burden on the healthcare system is also projected to increase considerably. Currently, there are no pharmacological treatments for sarcopenia, and given the long-term nature of aging studies, high-throughput chemical screens are impractical in mammalian models. Zebrafish is a promising, up-and-coming vertebrate model in the field of sarcopenia that could fill this gap. Here, we developed a surface electrical impedance myography (sEIM) platform to assess skeletal muscle health, quantitatively and noninvasively, in adult zebrafish (young, aged, and genetic mutant animals). In aged zebrafish (~85% lifespan) as compared to young zebrafish (~20% lifespan), sEIM parameters (2 kHz phase angle, 2 kHz reactance, and 2 kHz resistance) robustly detected muscle atrophy (p < 0.000001, q = 0.000002; p = 0.000004, q = 0.000006; p = 0.000867, q = 0.000683, respectively). Moreover, these same measurements exhibited strong correlations with an established morphometric parameter of muscle atrophy (myofiber cross-sectional area), as determined by histological-based morphometric analysis (r = 0.831, p = 2 × 10−12; r = 0.6959, p = 2 × 10−8; and r = 0.7220; p = 4 × 10−9, respectively). Finally, the genetic deletion of gpr27, an orphan G-protein coupled receptor (GPCR), exacerbated the atrophy of skeletal muscle in aged animals, as evidenced by both sEIM and histology. In conclusion, the data here show that surface EIM techniques can effectively discriminate between healthy young and sarcopenic aged muscle as well as the advanced atrophied muscle in the gpr27 KO animals. Moreover, these studies show how EIM values correlate with cell size across the animals, making it potentially possible to utilize sEIM as a "virtual biopsy" in zebrafish to noninvasively assess myofiber atrophy, a valuable measure for muscle and gerontology research. [ABSTRACT FROM AUTHOR]

Age-related deficits in skeletal muscle function, termed sarcopenia, are due to loss of muscle mass and changes in the intrinsic mechanisms underlying contraction. Sarcopenia is associated with falls, functional decline, and mortality. Electrical impedance myography (EIM)—a minimally invasive, rapid electrophysiological tool—can be applied to animals and humans to monitor muscle health, thereby serving as a biomarker in both preclinical and clinical studies. EIM has been successfully employed in several species; however, the application of EIM to the assessment of zebrafish—a model organism amenable to high-throughput experimentation—has not been reported. Here, we demonstrated differences in EIM measures between the skeletal muscles of young (6 months of age) and aged (33 months of age) zebrafish. For example, EIM phase angle and reactance at 2 kHz showed significantly decreased phase angle (5.3 ± 2.1 versus 10.7 ± 1.5°; p = 0.001) and reactance (89.0 ± 3.9 versus 172.2 ± 54.8 ohms; p = 0.007) in aged versus young animals. Total muscle area, in addition to other morphometric features, was also strongly correlated to EIM 2 kHz phase angle across both groups (r = 0.7133, p = 0.01). Moreover, there was a strong correlation between 2 kHz phase angle and established metrics of zebrafish swimming performance, including turn angle, angular velocity, and lateral motion (r = 0.7253, r = 0.7308, r = 0.7857, respectively, p < 0.01 for all). In addition, the technique was shown to have high reproducibility between repeated measurements with a mean percentage difference of 5.34 ± 1.17% for phase angle. These relationships were also confirmed in a separate replication cohort. Together, these findings establish EIM as a fast, sensitive method for quantifying zebrafish muscle function and quality. Moreover, identifying the abnormalities in the bioelectrical properties of sarcopenic zebrafish provides new opportunities to evaluate potential therapeutics for age-related neuromuscular disorders and to interrogate the disease mechanisms of muscle degeneration. [ABSTRACT FROM AUTHOR]

Cyanide—a fast-acting poison—is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (p  < .01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide. [ABSTRACT FROM AUTHOR]

Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation. [ABSTRACT FROM AUTHOR]

Objective: As there is significant heterogeneity in the weight loss response to pharmacotherapy, one of the most important clinical questions in obesity medicine is how to predict an individual's response to pharmacotherapy. The present study examines patterns of weight loss among overweight and obese women who demonstrated early robust response to twice daily exenatide treatment compared to those treated with hypocaloric diet and matched placebo injections. Methods: We randomized 182 women (BMI 25-48 kg/m2) to treatment with exenatide alone or matched placebo injections plus hypocaloric diet. In both treatment groups, women who demonstrated ≥ 5% weight loss at 12 weeks were characterized as high responders and those who lost ≥10% of body weight were classified as super responders. Our primary outcome was long-term change in body weight among early high responders to either treatment. An exploratory metabolomic analysis was also performed. Results: We observed individual variability in weight loss with both exenatide and hypocaloric diet plus placebo injections. There was a trend toward a higher percentage of subjects who achieved ≥ 5% weight loss with exenatide compared to diet (56% of those treated with exenatide, 76% of those treated with diet, p = 0.05) but no significant difference in those who achieved ≥ 10% weight loss (23% of individuals treated with exenatide and 36% of those treated with diet, p = 0.55). In both treatment groups, higher weight loss at 3 months of treatment predicted super responder status (diet p=0.0098, exenatide p=0.0080). Both treatment groups also demonstrated similar peak weight loss during the study period. We observed lower cysteine concentrations in the exenatide responder group (0.81 vs 0.48 p < 0.0001) and a trend toward higher levels of serotonin, aminoisobutyric acid, anandamide, and sarcosine in the exenatide super responder group. Conclusion: In a population of early high responders, longer term weight loss with exenatide treatment is similar to that achieved with a hypocaloric diet. Clinical Trial Registration: www.clinicaltrialsgov , identifier NCT01590433. [ABSTRACT FROM AUTHOR]

Cyanide is a highly toxic industrial chemical that is widely used by manufactures. Smoke inhalation during household fires is the most common source of cyanide poisoning while additional risks to civilians include industrial accidents and terrorist attacks. Despite the risks to large numbers of individuals, an antidote capable of administration at scale adequate for a mass casualty, prehospital scenario does not yet exist. Previously, we demonstrated that intravenous cisplatin analogues accelerate recovery from cyanide poisoning in mice and rabbits. Of the dozens of platinum‐based organometallic complexes tested, hexachloroplatinate (HCP) emerged as a promising lead compound, exhibiting strong affinity for cyanide and efficacy across model systems. Here, we show HCP is an antidote to lethal cyanide exposure and is importantly effective when delivered intramuscularly. The pharmacokinetic profile of HCP exhibited bioavailability in the systemic circulation 2.5 minutes post‐treatment and subsequent renal clearance of HCP‐cyanide. HCP restored parameters of cellular physiology including cytochrome c oxidase redox state and TCA cycle metabolism. We next validated these findings in a large animal model (swine). Finally, preclinical safety studies in mice revealed minimal toxicity. Cumulatively, these findings demonstrate that HCP is a promising lead compound for development of an intramuscular injectable cyanide antidote for mass casualty scenarios. [ABSTRACT FROM AUTHOR]

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning. [ABSTRACT FROM AUTHOR]

Summary Virtually all organisms seek to maximize fitness by matching fuel availability with energy expenditure. In vertebrates, glucose homeostasis is central to this process, with glucose levels finely tuned to match changing energy requirements. To discover new pathways regulating glucose levels in vivo, we performed a large-scale chemical screen in live zebrafish and identified the small molecule alexidine as a potent glucose-lowering agent. We found that alexidine inhibits the PTEN-like mitochondrial phosphatase PTPMT1 and that other pharmacological and genetic means of inactivating PTPMT1 also decrease glucose levels in zebrafish. Mutation of ptpmt1 eliminates the effect of alexidine, further confirming it as the glucose-lowering target of alexidine. We then identified succinate dehydrogenase (SDH) as a substrate of PTPMT1. Inactivation of PTPMT1 causes hyperphosphorylation and activation of SDH, providing a possible mechanism by which PTPMT1 coordinates glucose homeostasis. Therefore, PTPMT1 appears to be an important regulator of SDH phosphorylation status and glucose concentration. [ABSTRACT FROM AUTHOR]

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity. [ABSTRACT FROM AUTHOR]

Background: Cardiovascular development is vital for embryonic survival and growth. Early gestation embryo loss or malformation has been linked to yolk sac vasculopathy and congenital heart defects (CHDs). However, the molecular pathways that underlie these structural defects in humans remain largely unknown hindering the development of molecular-based diagnostic tools and novel therapies. Methodology/Principal Findings: Murine embryos were exposed to high glucose, a condition known to induce cardiovascular defects in both animal models and humans. We further employed a mass spectrometry-based proteomics approach to identify proteins differentially expressed in embryos with defects from those with normal cardiovascular development. The proteins detected by mass spectrometry (WNT16, ST14, Pcsk1, Jumonji, Morca2a, TRPC5, and others) were validated by Western blotting and immunoflorescent staining of the yolk sac and heart. The proteins within the proteomic dataset clustered to adhesion/migration, differentiation, transport, and insulin signaling pathways. A functional role for several proteins (WNT16, ADAM15 and NOGO-A/B) was demonstrated in an ex vivo model of heart development. Additionally, a successful application of a cluster of protein biomarkers (WNT16, ST14 and Pcsk1) as a prenatal screen for CHDs was confirmed in a study of human amniotic fluid (AF) samples from women carrying normal fetuses and those with CHDs. Conclusions/Significance: The novel finding that WNT16, ST14 and Pcsk1 protein levels increase in fetuses with CHDs suggests that these proteins may play a role in the etiology of human CHDs. The information gained through this bed-side to bench translational approach contributes to a more complete understanding of the protein pathways dysregulated during cardiovascular development and provides novel avenues for diagnostic and therapeutic interventions, beneficial to fetuses at risk for CHDs. [ABSTRACT FROM AUTHOR]

Nitric oxide (NO) has been demonstrated to mediate events during ovulation, pregnancy, blastocyst invasion and preimplantation embryogenesis. However, less is known about the role of NO during postimplantation development. Therefore, in this study, we explored the effects of NO during vascular development of the murine yolk sac, which begins shortly after implantation. Establishment of the vitelline circulation is crucial for normal embryonic growth and development. Moreover, functional inactivation of the endodermal layer of the yolk sac by environmental insults or genetic manipulations during this period leads to embryonic defects/lethality, as this stature is vital for transport, metabolism and induction of vascular development. In this study, we describe the temporally/ spatially regulated distribution of nitric oxide synthase (NOS) isoforms during the three stages of yolk sac vascular development (blood island formation, primary capillary plexus formation and vessel maturation/remodeling) and found NOS expression patterns were diametrically opposed. To pharmacologically manipulate vascular development, an established in vitro system of whole murine embryo culture was employed. During blood island formation, the endoderm produced NO and inhibition of NO (L-NMMA) at this stage resulted in developmental arrest at the primary plexus stage and vasculopathy. Furthermore, administration of a NO donor did not cause abnormal vascular development; however, exogenous NO correlated with increased eNOS and decreased iNOS protein levels. Additionally, a known environmental insult (high glucose) that produces reactive oxygen species (ROS) and induces vasculopathy also altered eNOS/iNOS distribution and induced NO production during yolk sac vascular development. However, administration of a NO donor rescued the high glucose induced vasculopathy, restored the eNOS/iNOS distribution and decreased ROS production. These data suggest that NO acts as an endoderm-derived factor... [ABSTRACT FROM AUTHOR]

The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa164 cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect. [ABSTRACT FROM AUTHOR]

Examines the corticotropin-releasing factor receptor 2 (CRFR2) in tonic suppressor of vascularization. Regulation of angiogenesis by means of balance between activators and inhibitors; Mechanism responsible for CRFR2 inhibition of angiogenesis; Effects of ligand activation on cell proliferation.

Studies the role of the cardiac myocyte as a mediator of paracrine signaling in the heart. Generation of cmVEGF-Null mice; Ventricular thinning, dilation and diminished contractile function in cmVEGF-Null hearts.

Describes an optimized methodology for induction of tube formation by cultured human umbilical-vein endothelial cells within 3D gels and for inosculation of preformed networks of cultured cells with the circulatory system of a host. Formation of vascular construct; Immunohistochemistry; Electron microscopy.

This study was performed to investigate the role of leptin in wound healing. The specific aims were to determine whether leptin is present at the wound site and whether this might reflect up-regulation of leptin expression as a consequence of active synthesis in response to the tissue insult. Leptin is a hypoxia-inducible cytokine functionally related to the IL-6 cytokine family. As a well-documented angiogenic molecule, leptin may mediate wound neovascularization and have additional effects in cells involved in the healing process, including fibroblasts, macrophages, and keratinocytes. Wound resident cells actively engage in acute synthesis of leptin within the first 4 h after injury, which is maintained throughout the various phases of the healing process. Treatment of wounds with neutralizing anti-leptin antibodies severely disrupts a variety of morphological parameters of wound healing such as wound contraction, re-epithelialization, and matrix density. The increase observed in leptin synthesis within the wound results in a transient elevation in circulating leptin, arising directly from the wound bed. This report demonstrates for the first time that leptin synthesis occurs rapidly in wound ischemic tissue and that the presence of leptin is necessary for normal healing progression to occur. [ABSTRACT FROM AUTHOR]