Aims

Hypoxia causes protein kinase C epsilon (PKCɛ) gene repression in foetal hearts, resulting in heightened cardiac susceptibility to ischaemic injury in offspring. We tested the hypothesis that hypoxia inducible factor 1 (HIF-1) and/or reactive oxygen species (ROS) mediate hypoxia-induced PKCɛ gene repression.

Methods and results

Hypoxia induced in vivo to pregnant rats, ex vivo to isolated foetal rat hearts, and in vitro in the rat embryonic ventricular myocyte cell line H9c2 resulted in a comparable decrease in PKCɛ protein and mRNA abundance in foetal hearts and H9c2 cells, which was associated with a significant increase in CpG methylation of the SP1-binding sites at the PKCɛ promoter. In H9c2 cells and foetal hearts, hypoxia caused nuclear accumulation of HIF-1α, which was inhibited by 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole and 2-methoxy estradiol. The HIF-1α inhibitors had no significant effect on hypoxia-induced PKCɛ mRNA repression. Hypoxia produced a time-dependent increase in ROS production in H9c2 cells and foetal hearts that was blocked by ROS scavengers N-acetyl-cysteine or tempol. In accordance, N-acetyl-cysteine and tempol, but not apocynin, inhibited the hypoxic effect and restored PKCɛ protein and mRNA expression to the control values in foetal hearts and H9c2 cells. The ROS scavengers blocked hypoxia-induced CpG methylation of the SP1-binding sites, restored SP1 binding to the PKCɛ promoter, and abrogated the hypoxia-induced increase in the susceptibility of the heart to ischaemic injury in offspring.

Conclusions

The results demonstrate that hypoxia induces epigenetic repression of the PKCɛ gene through a NADPH oxidase-independent ROS-mediated pathway in the foetal heart, leading to heightened heart vulnerability to ischaemic injury in offspring.

Autosomal Dominant Polycystic Kidney Disease (ADPKD; MIM ID's 173900, 601313, 613095) leads to end-stage kidney disease, caused by mutations in PKD1 or PKD2. Inactivation of Pkd1 before or after P13 in mice results in distinct early- or late-onset disease. Using a mouse model of ADPKD carrying floxed Pkd1 alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that Pkd1-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4α as a likely network node. These results are further supported by a meta-analysis of 1,114 published gene expression arrays in Pkd1 wild-type tissues. These analyses also predict that metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that Pkd1 cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating Hnf4α and Pkd1. The Pkd1/Hnf4α double mutants have significantly more cystic kidneys, thus indicating that metabolic pathways could play a role in Pkd1-cystogenesis.

Author Summary

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common genetic cause of polycystic kidney disease and is responsible for 4.6% of the end-stage renal disease (ESRD) cases in the United States. It is most often caused by mutation in the PKD1 gene. To understand this disease, we made a mouse model in which we could delete the Pkd1 gene and study the animal as its kidney becomes cystic. Using this model, we had previously found that the maturation status of the animal determines whether cysts form within days or within months, and we had narrowed down this switch to a two-day interval. In the current study, we used the rapid cyst-forming model to analyze the expression pattern of thousands of genes in mutant and control kidneys, and metabolites excreted in the urine. Our results identify a number of genes that may be involved in cyst formation and suggest that metabolic changes may play a role in ADPKD and could alter disease progression. These analyses also predict that metabolic pathways are key elements in normal postnatal kidney maturation.

There has been limited analysis of the effects of hepatocellular carcinoma (HCC) on liver metabolism and circulating endogenous metabolites. Here we report the findings of a plasma metabolomic investigation of HCC patients using ultraperformance liquid chromatography-electrospray ionization-quadrupole mass spectrometry (UPLC-ESI-QTOFMS), random forests machine learning algorithm and multivariate data analysis. Control subjects included healthy individuals as well as patients with liver cirrhosis or acute myeloid leukemia. We found that HCC was associated with increased plasma levels of glycodeoxycholate, deoxycholate 3-sulfate and bilirubin. Accurate mass measurement also indicated upregulation of biliverdin and the fetal bile acids 7α-hydroxy-3-oxochol-4-en-24-oic acid and 3-oxochol-4,6-dien-24-oic acid in HCC patients. A quantitative lipid profiling of patient plasma was also performed using ultra-performance liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (UPLC-ESI-TQMS). Using this method we found that that HCC was associated also with reduced levels of lysophosphocholines (LPC) and in 4/20 patients with increased levels of lysophosphatidic acid (LPA(16:0)), where it correlated with plasma α-fetoprotein levels. Interestingly, when fatty acids were quantitatively profiled by gas chromatography-mass spectrometry (GCMS), we found that lignoceric acid (24:0) and nervonic acid (24:1) were virtually absent from HCC plasma. Overall, this investigation illustrates the power of the new discovery technologies represented in the UPLC-ESI-QTOFMS platform combined with the targeted, quantitative platforms of UPLC-ESI-TQMS and GCMS for conducting metabolomic investigations that can engender new insights into cancer pathobiology.

Rationale

Pulmonary arterial hypertension (PAH) is an incurable disease associated with viral infections and connective tissue diseases. The relationship between inflammation and disease pathogenesis in these disorders remains poorly understood.

Objective

To determine whether immune dysregulation due to absent T cell populations directly contributes to the development of PAH.

Methods and Results

Vascular endothelial growth factor receptor 2 (VEGFR2) blockade induced significant pulmonary endothelial apoptosis in T-cell deficient rats but not in immune-reconstituted (IR) rats. T cell-lymphopenia in association with VEGFR2 blockade resulted in periarteriolar inflammation with macrophages, and B cells even prior to vascular remodeling and elevated pulmonary pressures. IR prevented early inflammation and attenuated PAH development. IR with either CD8 T cells alone or with CD4-depleted spleen cells was ineffective in preventing PAH whereas CD4-depleting immunocompetent euthymic animals increased PAH susceptibility. IR with either CD4+CD25hi or CD4+CD25- T cell subsets prior to vascular injury attenuated the development of PAH. Immune reconstitution limited perivascular inflammation and endothelial apoptosis in rat lungs in association with increased FoxP3+-, IL-10- and TGF-β– expressing CD4 cells, and upregulation of pulmonary bone morphogenetic protein receptor type 2 (BMPR2)-expressing cells, a receptor that activates endothelial cell survival pathways.

Conclusions

PAH may arise when regulatory T cell (Treg) activity fails to control endothelial injury. These studies suggest that regulatory T cells normally function to limit vascular injury and may protect against the development of PAH.

Data-intensive research depends on tools that manage multi-dimensional, heterogeneous data sets. We have built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices, and tables. OMERO’s design and flexibility have enabled its use for light microscopy, high content screening, electron microscopy, and even non-image genotype data. OMERO is open source software and available at http://openmicroscopy.org.

The role of the β2AR (β2 adrenergic receptor) after stroke is unclear as pharmacological manipulations of the β2AR have produced contradictory results. We previously showed that mice deficient in the β2AR (β2KO) had smaller infarcts compared with WT (wild-type) mice (FVB) after MCAO (middle cerebral artery occlusion), a model of stroke. To elucidate mechanisms of this neuroprotection, we evaluated changes in gene expression using microarrays comparing differences before and after MCAO, and differences between genotypes. Genes associated with inflammation and cell deaths were enriched after MCAO in both genotypes, and we identified several genes not previously shown to increase following ischaemia (Ccl9, Gem and Prg4). In addition to networks that were similar between genotypes, one network with a central core of GPCR (G-protein-coupled receptor) and including biological functions such as carbohydrate metabolism, small molecule biochemistry and inflammation was identified in FVB mice but not in β2KO mice. Analysis of differences between genotypes revealed 11 genes differentially expressed by genotype both before and after ischaemia. We demonstrate greater Glo1 protein levels and lower Pmaip/Noxa mRNA levels in β2KO mice in both sham and MCAO conditions. As both genes are implicated in NF-κB (nuclear factor κB) signalling, we measured p65 activity and TNFα (tumour necrosis factor α) levels 24 h after MCAO. MCAO-induced p65 activation and post-ischaemic TNFα production were both greater in FVB compared with β2KO mice. These results suggest that loss of β2AR signalling results in a neuroprotective phenotype in part due to decreased NF-κB signalling, decreased inflammation and decreased apoptotic signalling in the brain.

Since the development and prognosis of alcohol-induced liver disease (ALD) vary significantly with genetic background, identification of a genetic background-independent noninvasive ALD biomarker would significantly improve screening and diagnosis. This study explored the effect of genetic background on the ALD-associated urinary metabolome using the Ppara-null mouse model on two different backgrounds, C57BL/6 (B6) and 129/SvJ (129S), along with their wild-type counterparts. Reversed-phase gradient UPLC-ESI-QTOF-MS analysis revealed that urinary excretion of a number of metabolites, such as, ethylsulfate, 4-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid sulfate, adipic acid, pimelic acid, xanthurenic acid, and taurine were background-dependent. Elevation of ethyl-β-D-glucuronide and N-acetylglycine was found to be common signature of the metabolomic response to alcohol exposure in wild-type as well as in Ppara-null mice of both strains. However, increased excretion of indole-3-lactic acid and phenyllactic acid was found to be a conserved feature exclusively associated with the alcohol-treated Ppara-null mouse on both backgrounds that develop liver pathologies similar to the early stages of human ALD. These markers reflected the biochemical events associated with early stages of ALD pathogenesis. The results suggest that indole-3-lactic acid and phenyllactic acid are potential candidates for conserved and pathology-specific high-throughput noninvasive biomarkers for early stages of ALD.

Lung tumors from smokers as well as lung tumors from mice exposed to tobacco carcinogens such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), often carry mutations in K-ras, which activates downstream-signaling pathways such as PI3K/AKT/mTOR pathway. Mice with genetic deletion of one of three isoforms of AKT were used to investigate the role of AKT in mutant K-ras-induced lung tumorigenesis in mice. Although deletion of Akt1 or Akt2 decreased NNK-induced lung tumor formation by 90%, deletion of Akt2 failed to decrease lung tumorigenesis in two other mouse models driven by mutant K-ras. Genetic mapping showed that Akt2 was tightly linked to the cytochrome P450 Cyp2a locus on chromosome 7. Consequently, targeted deletion of Akt2 created linkage to a strain-specific Cyp2a5 polymorphism that decreased activation of NNK in vitro. Mice with this Cyp2a5 polymorphism had decreased NNK-induced DNA adduct formation in vivo and decreased NNK-induced lung tumorigenesis. These studies support human epidemiological studies linking CYP2A polymorphisms with lung cancer risk in humans and highlight the need to confirm phenotypes of genetically engineered mice in multiple mouse strains.

Lithocholic acid (LCA) is an endogenous compound associated with hepatic toxicity during cholestasis. LCA exposure in mice resulted in decreased serum lysophosphatidylcholine (LPC) and sphingomyelin levels due to elevated lysophosphatidylcholine acyltransferase (LPCAT) and sphingomyelin phosphodiesterase (SMPD) expression. Global metabolome analysis indicated significant decreases in serum palmitoyl-, stearoyl-, oleoyl- and linoleoyl-LPC levels after LCA exposure. LCA treatment also resulted in decreased serum sphingomyelin levels and increased hepatic ceramide levels, and induction of LPCAT and SMPD mRNAs. Transforming growth factor-β TGF-β) induced Lpcat2/4 and Smpd3 gene expression in primary hepatocytes and the induction was diminished by pretreatment with the SMAD3 inhibitor SIS3. Furthermore, alteration of the LPC metabolites and Lpcat1/2/4 and Smpd3 expression was attenuated in LCA-treated farnesoid X receptor-null mice that are resistant to LCA-induced intrahepatic cholestasis. This study revealed that LCA induced disruption of phospholipid/sphingolipid homeostasis through TGF-β signaling and that serum LPC is a biomarker for biliary injury.

Radiation metabolomics has aided in the identification of a number of biomarkers in cells and mice by ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-ESI-QTOFMS) and in rats by gas chromatography-coupled mass spectrometry (GCMS). These markers have been shown to be both dose- and time-dependent. Here UPLC-ESI-QTOFMS was used to analyze rat urine samples taken from 12 rats over 7 days; they were either sham-irradiated or γ-irradiated with 3 Gy after 4 days of metabolic cage acclimatization. Using multivariate data analysis, nine urinary biomarkers of γ radiation in rats were identified, including a novel mammalian metabolite, N-acetyltaurine. These upregulated urinary biomarkers were confirmed through tandem mass spectrometry and comparisons with authentic standards. They include thymidine, 2′-deoxyuridine, 2′deoxyxanthosine, N1-acetylspermidine, N-acetylglucosamine/galactosamine-6-sulfate, N-acetyltaurine, N-hexanoylglycine, taurine and, tentatively, isethionic acid. Of these metabolites, 2′-deoxyuridine and thymidine were previously identified in the rat by GCMS (observed as uridine and thymine) and in the mouse by UPLC-ESI-QTOFMS. 2′Deoxyxanthosine, taurine and N-hexanoylglycine were also seen in the mouse by UPLC-ESI-QTOFMS. These are now unequivocal cross-species biomarkers for ionizing radiation exposure. Downregulated biomarkers were shown to be related to food deprivation and starvation mechanisms. The UPLC-ESI-QTOFMS approach has aided in the advance for finding common biomarkers of ionizing radiation exposure.

Ifosfamide (IF) and cyclophosphamide (CP) are common chemotherapeutic agents. Interestingly, while the two drugs are isomers, only IF treatment is known to cause nephrotoxicity and neurotoxicity. Therefore, it was anticipated that a comparison of IF and CP drug metabolites in the mouse would reveal reasons for this selective toxicity. Drug metabolites were profiled by ultra-performance liquid chromatography-linked electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOFMS), and the results analyzed by multivariate data analysis. Of the total 23 drug metabolites identified by UPLC-ESI-QTOFMS for both IF and CP, five were found to be novel. Ifosfamide preferentially underwent N-dechloroethylation, the pathway yielding 2-chloroacetaldehyde, while cyclophosphamide preferentially underwent ring-opening, the pathway yielding acrolein (AC). Additionally, S-carboxymethylcysteine and thiodiglycolic acid, two downstream IF and CP metabolites, were produced similarly in both IF- and CP-treated mice. This may suggest that other metabolites, perhaps precursors of thiodiglycolic acid, may be responsible for IF encephalopathy and nephropathy.

Metabolomic analysis will provide the next large set of clues to further our understanding of human health and disease. A recent study has elucidated the significant differences in the metabolomes of adipocytes, serum and an adipocyte cell line after activation of two nuclear receptors, peroxisome proliferator activated receptor β/δ (PPARβ/δ) and PPARγ. These findings hold great promise for explaining fundamental differences in the mechanisms of PPAR agonists and for identifying targets for the treatment of diabetes.

See related research article: http://genomebiology.com/2011/12/8/R75

Alcohol-induced liver disease (ALD) is a leading cause of non-accident-related deaths in the United States. Although liver damage caused by ALD is reversible when discovered at the earlier stages, current risk assessment tools are relatively non-specific. Identification of an early specific signature of ALD would aid in therapeutic intervention and recovery. In this study the metabolic changes associated with alcohol-induced liver disease were examined using alcohol-fed male Ppara-null mouse as a model of ALD. Principal components analysis of the mass spectrometry-based urinary metabolic profile showed that alcohol-treated wild-type and Ppara-null mice could be distinguished from control animals without information on history of alcohol consumption. The urinary excretion of ethyl-sulfate, ethyl-β-D-glucuronide, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetic acid sulfate was elevated and that of the 2-hydroxyphenylacetic acid, adipic acid, and pimelic acid was depleted during alcohol treatment in both wild-type and the Ppara-null mice albeit to different extents. However, indole-3-lactic acid was exclusively elevated by alcohol exposure in Ppara-null mice. The elevation of indole-3-lactic acid is mechanistically related to the molecular events associated with development of ALD in alcohol-treated Ppara-null mice. This study demonstrated the ability of metabolomics approach to identify early, noninvasive biomarkers of ALD pathogenesis in Ppara-null mouse model.

Rationale

Epidemiological studies demonstrate a clear association of adverse intrauterine environment with an increased risk of ischemic heart disease in adulthood. Hypoxia is a common stress to the fetus, and results in decreased protein kinase C epsilon (PKCε) expression in the heart and increased cardiac vulnerability to ischemia and reperfusion injury in adult offspring in rats.

Objectives

The present study tested the hypothesis that fetal hypoxia-induced methylation of CpG dinucleotides at the PKCε promoter is repressive and contributes to PKCε gene repression in the heart of adult offspring.

Methods and Results

Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in significant decreases in PKCε protein and mRNA in fetal hearts. Similar results were obtained in ex vivo hypoxic treatment of isolated fetal hearts and rat embryonic ventricular myocyte cell line H9c2. Increased methylation of PKCε promoter at SP1 binding sites, −346 and −268, were demonstrated in both fetal hearts of maternal hypoxia and H9c2 cells treated with 1% O2 for 24 hours. Whereas hypoxia had no significant effect on the binding affinity of SP1 to the unmethylated sites in H9c2 cells, hearts of fetuses and adult offspring, methylation of both SP1 sites reduced SP1 binding. The addition of 5-aza-2’-deoxycytidine blocked the hypoxia-induced increase in methylation of both SP1 binding sites and restored PKCε mRNA and protein to the control levels. In hearts of both fetuses and adult offspring, hypoxia-induced methylation of SP1 sites was significantly greater in males than in females, and decreased PKCε mRNA was seen only in males. In fetal hearts, there was significantly higher abundance of estrogen receptor α (ERα ) and β (ERβ ) isoforms in females than in males. Both ERα and ERβ interacted with the SP1 binding sites in the fetal heart, which may explain the gender differences in SP1 methylation in the fetal heart. Additionally, selective activation of PKCε restored the hypoxia-induced cardiac vulnerability to ischemic injury in offspring.

Conclusion

The findings demonstrate a direct effect of hypoxia on epigenetic modification of DNA methylation and programming of cardiac PKCε gene repression in a sex-dependent manner, linking fetal hypoxia and pathophysiological consequences in the hearts of adult offspring.

The combination of advanced ultra-performance liquid chromatography coupled with mass spectrometry, chemometrics, and genetically modified mice provide an attractive raft of technologies with which to examine the metabolism of xenobiotics. Here a reexamination of the metabolism of the food mutagen PhIP (2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine), the suspect carcinogen areca alkaloids (arecoline, arecaidine, and arecoline 1-oxide), the hormone supplement melatonin, and the metabolism of the experimental cancer therapeutic agent aminoflavone is presented. In all cases, the metabolic maps of the xenobiotics were considerably enlarged, providing new insights into their toxicology. The inclusion of transgenic mice permitted unequivocal attribution of individual and often novel metabolic pathways to particular enzymes. Lastly, a future perspective for xenobiotic metabolomics is discussed and its impact on the metabolome is described. The studies reviewed here are not specific to the mouse, and can be adapted to study xenobiotic metabolism in any animal species, including Man. The view through the metabolometer is unique and visualizes a metabolic space that contains both established and unknown metabolites of a xenobiotic thereby enhancing knowledge of their modes of toxic action.

Technology to enable rapid screening for radiation exposure has been identified as an important need, and, as a part of a NIH / NIAD effort in this direction, metabolomic biomarkers for radiation exposure have been identified in a recent series of papers. To reduce the time necessary to detect and measure these biomarkers, differential mobility spectrometry – mass spectrometry (DMS-MS) systems have been developed and tested. Differential mobility ion filters preselect specific ions and also suppress chemical noise created in typical atmospheric-pressure ionization sources (ESI, MALDI, and others). Differential-mobility-based ion selection is based on the field dependence of ion mobility, which, in turn, depends on ion characteristics that include conformation, charge distribution, molecular polarizability, and other properties, and on the transport gas composition which can be modified to enhance resolution. DMS-MS is able to resolve small-molecule biomarkers from nearly-isobaric interferences, and suppresses chemical noise generated in the ion source and in the mass spectrometer, improving selectivity and quantitative accuracy. Our planar DMS design is rapid, operating in a few milliseconds, and analyzes ions before fragmentation. Depending on MS inlet conditions, DMS-selected ions can be dissociated in the MS inlet expansion, before mass analysis, providing a capability similar to MS/MS with simpler instrumentation. This report presents selected DMS-MS experimental results, including resolution of complex test mixtures of isobaric compounds, separation of charge states, separation of isobaric biomarkers (citrate and isocitrate), and separation of nearly-isobaric biomarker anions in direct analysis of a bio-fluid sample from the radiation-treated group of a mouse-model study. These uses of DMS combined with moderate resolution MS instrumentation indicate the feasibility of field-deployable instrumentation for biomarker evaluation.

Abstract

Numerous studies characterizing the function of glutathione peroxidase 4 (GPx4) have demonstrated that this selenoenzyme is protective against oxidative stress. Herein, we characterized the function of this protein by targeting GPx4 downregulation using RNA interference. Partial knockdown of GPx4 levels resulted in growth retardation and morphological changes. Surprisingly, GPx4 knockdown cells showed virtually unchanged levels of intracellular ROS, yet highly increased levels of oxidized lipid by-products. GPx1, another glutathione peroxidase and a major cellular peroxide scavenging enzyme, did not rescue GPx4-deficient cells and did not reduce lipid peroxide levels. The data established an essential role of GPx4 in protecting cells against lipid hydroperoxide damage, yet a limited role as a general antioxidant enzyme. As oxidized lipid hydroperoxides are a characteristic of neurodegenerative diseases, we analyzed brain tissues of mice suffering from a model of Alzheimer's disease and found that oxidized lipid by-products were enriched, and expression of both GPx4 and guanine-rich sequence-binding factor, which is known to control GPx4 synthesis, was downregulated. Brain tissue from an Alzheimer's diseased human also manifested enhanced levels of one of the oxidized lipid by-products, 4-hydroxynonenal. These data suggest a role of GPx4 in neurodegenerative diseases through its function in removal of lipid hydroperoxides. Antioxid. Redox Signal. 12, 819–827.

Data sharing is important in the biological sciences to prevent duplication of effort, to promote scientific integrity, and to facilitate and disseminate scientific discovery. Sharing requires centralized repositories, and submission to and utility of these resources require common data formats. This is particularly challenging for multidimensional microscopy image data, which are acquired from a variety of platforms with a myriad of proprietary file formats (PFFs). In this paper, we describe an open standard format that we have developed for microscopy image data. We call on the community to use open image data standards and to insist that all imaging platforms support these file formats. This will build the foundation for an open image data repository.

Excessive use of broad-spectrum antibiotics in hospitals has led to the emergence of highly resistant strains of Pseudomonas aeruginosa. To reduce the selection pressure for resistance, it is important to determine the antibiotic susceptibility pattern of bacteria so that hospital patients can be treated with more narrow-spectrum and target-specific antibiotics. This study describes the development of a technique for detecting point muations in the fluoroquinolone resistance-determining region of the gyrA and parC genes as well as the efflux regulatory genes mexR, mexZ and mexOZ that are associated with fluoroquinolone and aminoglycoside resistance. The assay is based on a short DNA sequencing method using multiplex-fast polymerase chain reaction (PCR) and Pyrosequencing™ for amplification and sequencing of the selected genes. Fifty-nine clinical isolates of P. aeruginosa were examined for mutations in the abovementioned genes. Mutations related to antibiotic resistance were detected in codons 83 and 87 of gyrA and codon 126 of the mexR regulatory gene. Results of this study suggest Pyrosequencing™ as a substitute for traditional methods as it provides a rapid and reliable technique for determinating the antibiotic resistance pattern of a given bacterial strain in <1 h.

Activation of the peroxisome proliferator-activated receptor α (PPARα) is associated with increased fatty acid catabolism and is commonly targeted for the treatment of hyperlipidemia. To identify latent, endogenous biomarkers of PPARα activation and hence increased fatty acid β-oxidation, healthy human volunteers were given fenofibrate orally for 2 weeks and their urine profiled by UPLC-QTOFMS. Biomarkers identified by the machine learning algorithm random forests included significant depletion by day 14 of both pantothenic acid (>5-fold) and acetylcarnitine (>20-fold), observations that are consistent with known targets of PPARα including pantothenate kinase and genes encoding proteins involved in the transport and synthesis of acylcarnitines. It was also concluded that serum cholesterol (-12.7%), triglycerides (-25.6%), uric acid (-34.7%), together with urinary propylcarnitine (>10-fold), isobutyrylcarnitine (>2.5-fold), (S)-(+)-2-methylbutyrylcarnitine (5-fold), and isovalerylcarnitine (>5-fold) were all reduced by day 14. Specificity of these biomarkers as indicators of PPARα activation was demonstrated using the Ppara-null mouse. Urinary pantothenic acid and acylcarnitines may prove useful indicators of PPARα-induced fatty acid β-oxidation in humans. This study illustrates the utility of a pharmacometabolomic approach to understand drug effects on lipid metabolism in both human populations and in inbred mouse models.

Radiation metabolomics employing mass spectral technologies represents a plausible means of high-throughput minimally invasive radiation biodosimetry. A simplified metabolomics protocol is described that employs ubiquitous gas chromatography-mass spectrometry and open source software including random forests machine learning algorithm to uncover latent biomarkers of 3 Gy γ radiation in rats. Urine was collected from six male Wistar rats and six sham-irradiated controls for 7 days, 4 prior to irradiation and 3 after irradiation. Water and food consumption, urine volume, body weight, and sodium, potassium, calcium, chloride, phosphate and urea excretion showed major effects from exposure to γ radiation. The metabolomics protocol uncovered several urinary metabolites that were significantly up-regulated (glyoxylate, threonate, thymine, uracil, p-cresol) and down-regulated (citrate, 2-oxoglutarate, adipate, pimelate, suberate, azelaate) as a result of radiation exposure. Thymine and uracil were shown to derive largely from thymidine and 2′-deoxyuridine, which are known radiation biomarkers in the mouse. The radiation metabolomic phenotype in rats appeared to derive from oxidative stress and effects on kidney function. Gas chromatography-mass spectrometry is a promising platform on which to develop the field of radiation metabolomics further and to assist in the design of instrumentation for use in detecting biological consequences of environmental radiation release.

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal γ-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2′-deoxyuridine, 2′-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2′-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8–12 h but returning to baseline by 36 h after exposure. It is proposed that 2′-deoxyuridine and 2′-deoxyxanthosine arise as a result of γ irradiation by nitrosative deamination of 2′-deoxycytidine and 2′-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

β1- and β2-adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling, and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here, we demonstrate that β1AR forms a signaling complex with a cAMP-specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β-arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner, and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β-arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1- and β2-adrenoceptor signaling.

Apelin and its cognate G protein–coupled receptor APJ constitute a signaling pathway with a positive inotropic effect on cardiac function and a vasodepressor function in the systemic circulation. The apelin-APJ pathway appears to have opposing physiological roles to the renin-angiotensin system. Here we investigated whether the apelin-APJ pathway can directly antagonize vascular disease-related Ang II actions. In ApoE-KO mice, exogenous Ang II induced atherosclerosis and abdominal aortic aneurysm formation; we found that coinfusion of apelin abrogated these effects. Similarly, apelin treatment rescued Ang II–mediated increases in neointimal formation and vascular remodeling in a vein graft model. NO has previously been implicated in the vasodepressor function of apelin; we found that apelin treatment increased NO bioavailability in ApoE-KO mice. Furthermore, infusion of an NO synthase inhibitor blocked the apelin-mediated decrease in atherosclerosis and aneurysm formation. In rat primary aortic smooth muscle cells, apelin inhibited Ang II–mediated transcriptional regulation of multiple targets as measured by reporter assays. In addition, we demonstrated by coimmunoprecipitation and fluorescence resonance energy transfer analysis that the Ang II and apelin receptors interacted physically. Taken together, these findings indicate that apelin signaling can block Ang II actions in vascular disease by increasing NO production and inhibiting Ang II cellular signaling.