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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]

G protein‐coupled receptors (GPCRs) comprise the largest group of membrane receptors in eukaryotic genomes and collectively they regulate nearly all cellular processes. Despite the widely recognized importance of this class of proteins, many GPCRs remain understudied. G protein‐coupled receptor 27 (Gpr27) is an orphan GPCR that displays high conservation during vertebrate evolution. Although, GPR27 is known to be expressed in tissues that regulate metabolism including the pancreas, skeletal muscle, and adipose tissue, its functions are poorly characterized. Therefore, to investigate the potential roles of Gpr27 in energy metabolism, we generated a whole body gpr27 knockout zebrafish line. Loss of gpr27 potentiated the elevation in glucose levels induced by pharmacological or nutritional perturbations. We next leveraged a mass spectrometry metabolite profiling platform to identify other potential metabolic functions of Gpr27. Notably, genetic deletion of gpr27 elevated medium‐chain acylcarnitines, in particular C6‐hexanoylcarnitine, C8‐octanoylcarnitine, C9‐nonanoylcarnitine, and C10‐decanoylcarnitine, lipid species known to be associated with insulin resistance in humans. Concordantly, gpr27 deletion in zebrafish abrogated insulin‐dependent Akt phosphorylation and glucose utilization. Finally, loss of gpr27 increased the expression of key enzymes in carnitine shuttle complex, in particular the homolog to the brain‐specific isoform of CPT1C which functions as a hypothalamic energy senor. In summary, our findings shed light on the biochemical functions of Gpr27 by illuminating its role in lipid metabolism, insulin signaling, and glucose homeostasis. [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]

Blood circulation is dependent on heart valves to direct blood flow through the heart and great vessels. Valve development relies on epithelial to mesenchymal transition (EMT), a central feature of embryonic development and metastatic cancer. Abnormal EMT and remodeling contribute to the etiology of several congenital heart defects. Leptin and its receptor were detected in the mouse embryonic heart. Using an ex vivo model of cardiac EMT, the inhibition of leptin results in a signal transducer and activator of transcription 3 and Snail/vascular endothelial cadherin-independent decrease in EMT and migration. Our data suggest that an Akt signaling pathway underlies the observed phenotype. Furthermore, loss of leptin phenocopied the functional inhibition of αvβ3 integrin receptor and resulted in decreased αvβ3 integrin and matrix metalloprotease 2, suggesting that the leptin signaling pathway is involved in adhesion and migration processes. This study adds leptin to the repertoire of factors that mediate EMT and, for the first time, demonstrates a role for the interleukin 6 family in embryonic EMT. [ABSTRACT FROM AUTHOR]

Abstract: Nitric oxide (NO) participates in a diverse array of biological functions in mammalian organ systems. Depending on the biochemical environment, the production of NO may result in cytoprotection or cytotoxicity. The paradoxical actions of NO arise from the complexities generated by the redox milieu, NO concentration/bioavailability, and tissue/cell context, which ultimately result in the wide range of regulatory roles observed. Additionally, in physiological versus pathological states, NO often displays diametrically opposing affects in several organ systems. Here, we will discuss the roles of NO during reproduction, organ system development, in particular, the cardiovascular system, and its potential implications in diabetes-induced fetal defects. [Copyright &y& Elsevier]

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]

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.

Studies the biological action of the hormone leptin as an angiogenic factor. Information on leptin; Expression of the receptor of leptin, OB-Rb; Angiogenic activity of leptin revealed by in vitro and in vivo; Findings on vascular endothelium.

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]

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]

Summary Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning. [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]

The article presents a study on the role of embryonic inhibition of topoisomerase IIa (Top2a) in the development of sociality in zebrafish. Topics discussed include the development of an automated assay system called Fishbook to assess the social behavior in zebrafish, the identification of fluoroquinolones as inhibitors of social development, and the down-regulation of autism risk genes by Top2a depletion.

The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-γ and IFN-α treatments revealed dramatic changes in binding locations between the two treatments. The IFN-α induction revealed nonconserved STAT1 occupancy at IFN-γ-induced sites, as well as novel sites of STAT1 binding not evident in IFN-γ-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states. [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]

Abstract: Objectives:: The study of isolated microvascular endothelial cells from mice has long been impeded due to the many difficulties encountered in isolating and culturing these cells. We focused on developing a method to isolate microvascular endothelial cells from the skin fragments of newborn mice. We also aimed at establishing optimal culture conditions to sustain the growth of these cells. Methods and results:: Isolation of murine dermal microvascular endothelial cells (mDMEC) from P3 newborn mice was based first on enzymatic separation of the skin epidermal layer from the dermis using dispase and then on disaggregating dermal cellular elements using collagenase. The cells obtained from the dermis were subjected to a continuous density gradient centrifugation. Cells situated between densities 1.033 and 1.047 were then cultured on collagen IV-coated culture flasks using optimized growth culture conditions. Cells were characterized by endothelial appearance and by the presence and genetic expression of endothelial markers like CD31, NOS3, VEGFR-2 and Tie-2. Uptake of acetylated low-density lipoprotein (Ac-LDL) was used as a functional assay. Conclusions:: The methodology described herein for isolation and culture of murine microvascular endothelium offers a distinctive advantage for those using mouse models to study endothelial cell biology. [Copyright &y& Elsevier]

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.