Publications by authors named "Pasan Fernando"

24 Publications

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The spliceosome inhibitors isoginkgetin and pladienolide B induce ATF3-dependent cell death.

PLoS One 2020 28;15(12):e0224953. Epub 2020 Dec 28.

Department of Biology, Carleton University, Ottawa, ON, Canada.

The spliceosome assembles on pre-mRNA in a stepwise manner through five successive pre-spliceosome complexes. The spliceosome functions to remove introns from pre-mRNAs to generate mature mRNAs that encode functional proteins. Many small molecule inhibitors of the spliceosome have been identified and they are cytotoxic. However, little is known about genetic determinants of cell sensitivity. Activating transcription factor 3 (ATF3) is a transcription factor that can stimulate apoptotic cell death in response to a variety of cellular stresses. Here, we used a genetic approach to determine if ATF3 was important in determining the sensitivity of mouse embryonic fibroblasts (MEFs) to two splicing inhibitors: pladienolide B (PB) and isoginkgetin (IGG), that target different pre-spliceosome complexes. Both compounds led to increased ATF3 expression and apoptosis in control MEFs while ATF3 null cells were significantly protected from the cytotoxic effects of these drugs. Similarly, ATF3 was induced in response to IGG and PB in the two human tumour cell lines tested while knockdown of ATF3 protected cells from both drugs. Taken together, ATF3 appears to contribute to the cytotoxicity elicited by these spliceosome inhibitors in both murine and human cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0224953PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7769279PMC
January 2021

Integration of anatomy ontology data with protein-protein interaction networks improves the candidate gene prediction accuracy for anatomical entities.

BMC Bioinformatics 2020 Oct 7;21(1):442. Epub 2020 Oct 7.

Division of Biostatistics and Computational Biology, College of Dentistry, University of Iowa, Iowa City, IA, USA.

Background: Identification of genes responsible for anatomical entities is a major requirement in many fields including developmental biology, medicine, and agriculture. Current wet lab techniques used for this purpose, such as gene knockout, are high in resource and time consumption. Protein-protein interaction (PPI) networks are frequently used to predict disease genes for humans and gene candidates for molecular functions, but they are rarely used to predict genes for anatomical entities. Moreover, PPI networks suffer from network quality issues, which can be a limitation for their usage in predicting candidate genes. Therefore, we developed an integrative framework to improve the candidate gene prediction accuracy for anatomical entities by combining existing experimental knowledge about gene-anatomical entity relationships with PPI networks using anatomy ontology annotations. We hypothesized that this integration improves the quality of the PPI networks by reducing the number of false positive and false negative interactions and is better optimized to predict candidate genes for anatomical entities. We used existing Uberon anatomical entity annotations for zebrafish and mouse genes to construct gene networks by calculating semantic similarity between the genes. These anatomy-based gene networks were semantic networks, as they were constructed based on the anatomy ontology annotations that were obtained from the experimental data in the literature. We integrated these anatomy-based gene networks with mouse and zebrafish PPI networks retrieved from the STRING database and compared the performance of their network-based candidate gene predictions.

Results: According to evaluations of candidate gene prediction performance tested under four different semantic similarity calculation methods (Lin, Resnik, Schlicker, and Wang), the integrated networks, which were semantically improved PPI networks, showed better performances by having higher area under the curve values for receiver operating characteristic and precision-recall curves than PPI networks for both zebrafish and mouse.

Conclusion: Integration of existing experimental knowledge about gene-anatomical entity relationships with PPI networks via anatomy ontology improved the candidate gene prediction accuracy and optimized them for predicting candidate genes for anatomical entities.
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http://dx.doi.org/10.1186/s12859-020-03773-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7542696PMC
October 2020

Automated Integration of Trees and Traits: A Case Study Using Paired Fin Loss Across Teleost Fishes.

Syst Biol 2018 07;67(4):559-575

Department of Biology, University of South Dakota, 414 East Clark St., Vermillion, SD 57069, USA.

Data synthesis required for large-scale macroevolutionary studies is challenging with the current tools available for integration. Using a classic question regarding the frequency of paired fin loss in teleost fishes as a case study, we sought to create automated methods to facilitate the integration of broad-scale trait data with a sizable species-level phylogeny. Similar to the evolutionary pattern previously described for limbs, pelvic and pectoral fin reduction and loss are thought to have occurred independently multiple times in the evolution of fishes. We developed a bioinformatics pipeline to identify the presence and absence of pectoral and pelvic fins of 12,582 species. To do this, we integrated a synthetic morphological supermatrix of phenotypic data for the pectoral and pelvic fins for teleost fishes from the Phenoscape Knowledgebase (two presence/absence characters for 3047 taxa) with a species-level tree for teleost fishes from the Open Tree of Life project (38,419 species). The integration method detailed herein harnessed a new combined approach by utilizing data based on ontological inference, as well as phylogenetic propagation, to reduce overall data loss. Using inference enabled by ontology-based annotations, missing data were reduced from 98.0% to 85.9%, and further reduced to 34.8% by phylogenetic data propagation. These methods allowed us to extend the data to an additional 11,293 species for a total of 12,582 species with trait data. The pectoral fin appears to have been independently lost in a minimum of 19 lineages and the pelvic fin in 48. Though interpretation is limited by lack of phylogenetic resolution at the species level, it appears that following loss, both pectoral and pelvic fins were regained several (3) to many (14) times respectively. Focused investigation into putative regains of the pectoral fin, all within one clade (Anguilliformes), showed that the pectoral fin was regained at least twice following loss. Overall, this study points to specific teleost clades where strategic phylogenetic resolution and genetic investigation will be necessary to understand the pattern and frequency of pectoral fin reversals.
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http://dx.doi.org/10.1093/sysbio/syx098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005059PMC
July 2018

Cardiotrophin 1 stimulates beneficial myogenic and vascular remodeling of the heart.

Cell Res 2017 Oct 8;27(10):1195-1215. Epub 2017 Aug 8.

Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa Hospital, Ottawa, Ontario K1H 8L6, Canada.

The post-natal heart adapts to stress and overload through hypertrophic growth, a process that may be pathologic or beneficial (physiologic hypertrophy). Physiologic hypertrophy improves cardiac performance in both healthy and diseased individuals, yet the mechanisms that propagate this favorable adaptation remain poorly defined. We identify the cytokine cardiotrophin 1 (CT1) as a factor capable of recapitulating the key features of physiologic growth of the heart including transient and reversible hypertrophy of the myocardium, and stimulation of cardiomyocyte-derived angiogenic signals leading to increased vascularity. The capacity of CT1 to induce physiologic hypertrophy originates from a CK2-mediated restraining of caspase activation, preventing the transition to unrestrained pathologic growth. Exogenous CT1 protein delivery attenuated pathology and restored contractile function in a severe model of right heart failure, suggesting a novel treatment option for this intractable cardiac disease.
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http://dx.doi.org/10.1038/cr.2017.87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5630684PMC
October 2017

Acute and subacute toxicity studies of CMICE-013, a novel iodinated rotenone-based myocardial perfusion tracer, in Sprague Dawley rats and Gottingen minipigs.

Regul Toxicol Pharmacol 2016 Oct 10;80:195-209. Epub 2016 May 10.

Canadian Molecular Imaging Center of Excellence (CMICE), University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON K1Y 4W7, Canada; Division of Cardiology, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON K1Y 4W7, Canada.

Purpose: Extensive acute and subacute toxicities studies are required to evaluate the toxicological profile of the novel cardiac perfusion imaging tracer (123)I-CMICE-013 to support applications for clinical trials.

Methods: Sprague-Dawley rats and Gottingen minipigs received injections of non-radioactive 127I-CMICE-013 at two dosage levels of 1 and 5 μg/kg, and vehicle buffer as control. In the acute toxicity studies, each animal was injected on two occasions 24 h apart and then underwent a 14-day recovery period; in the subacute study, animals received daily injections for 14 days continuously. The health status and mortality of test animals were monitored daily and body weight, food consumption, physiological and biochemical parameters were measured at various time points during the study. Animals were euthanized at the end of the studies and dissected for pathologic examination of organs and tissues.

Results: The acute and subacute administrations of injections of the non-radioactive CMICE-013 in rats and minipigs were well tolerated. Little to no dosing-related adverse effects were observed in animal body and organ weights, hematology, coagulation, clinical chemistry, urinalysis, ophthalmoscopy, electrocardiograms, heart rates, blood pressure, macroscopic and microscopic examination of the preserved animal tissues including the brain.

Conclusion: The lack of adverse effects from acute and subacute dosing suggest that the CMICE-013 injection solution has a reasonable safety margin within the designed concentration range to be utilized in imaging applications. The dosage level of 5 μg/kg was considered the no adverse effect level for both rats and minipigs based on our acute and subacute studies.
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http://dx.doi.org/10.1016/j.yrtph.2016.05.011DOI Listing
October 2016

Flow-Dependent Uptake of ¹²³I-CMICE-013, a Novel SPECT Perfusion Agent, Compared with Standard Tracers.

J Nucl Med 2015 May 3;56(5):764-70. Epub 2015 Apr 3.

Division of Cardiology, University of Ottawa Heart Institute, Ottawa, Ontario, Canada Canadian Molecular Imaging Center of Excellence (CMICE), University of Ottawa Heart Institute, Ottawa, Ontario, Canada; and.

Unlabelled: Rotenone derivatives have shown promise in myocardial perfusion imaging (MPI). CMICE-013 is a novel (123)I-labeled rotenone derivative developed for SPECT MPI. The objective of this study was to assess the image quality of CMICE-013 and compare its uptake with tetrofosmin, sestamibi, and (201)Tl in vivo in a porcine model of stress-induced myocardial ischemia.

Methods: Microspheres were injected simultaneously with the radiotracer injections at rest and stress to measure blood flow. Mimicking a 1-d tetrofosmin protocol, stress imaging used 3 times as much activity and occurred 1 h after the rest injection. SPECT images were obtained at both rest and stress. After imaging, the heart was sectioned into 44-50 pieces. In each heart sample, the tracer uptake was measured in a γ counter. The images were aligned, and the decay-corrected ratio of the signals at rest and stress was used to separate the well-counter signal into rest and stress components. The uptake at rest and stress was compared with microsphere flow measurements.

Results: The CMICE-013 images showed good contrast between the heart and surrounding organs, with heart-to-liver and heart-to-lung uptake ratios similar to those of the standard tracers. Uptake of CMICE-013 was 1.5% of the injected dose at rest and increased more rapidly with increased blood flow than did the standard SPECT tracers. The percentage injected dose of CMICE-013 taken up by the heart was greater (P < 0.05) than (201)Tl, tetrofosmin, or sestamibi at flows greater than 1.5 mL/min/g.

Conclusion: CMICE-013 is a promising new SPECT MPI agent.
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http://dx.doi.org/10.2967/jnumed.114.151563DOI Listing
May 2015

N-[(11)C]-methyl-hydroxyfasudil is a potential biomarker of cardiac hypertrophy.

Nucl Med Biol 2015 Feb 30;42(2):192-7. Epub 2014 Sep 30.

Division of Cardiology and Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5; Canadian Molecular Imaging Center of Excellence (CMICE), University of Ottawa Heart Institute, Ottawa, ON K1Y 4W7; Nordion Inc., Ottawa, ON, K2K 1X8. Electronic address:

Introduction: Pathologic cardiac hypertrophy is one of the leading causes of sudden death from cardiac disease and involves a complex network of bio-signaling mechanisms. To date, the clinical detection and pathologic progression of hypertrophy remains elusive. Here we tested whether imaging Rho kinase activity would serve an accurate proxy for detecting hypertrophy. Specifically, we examine the use of the N-[(11)C]-methylated derivative of hydroxyfasudil, a Rho kinase inhibitor, as a biomarker for accurate identification of cardiomyocyte hypertrophy.

Methods: Both transformed and primary neonatal cardiomyocytes were treated with isoproterenol to induce β-adrenergic receptor stimulation and hypertrophy. Phenotypic hypertrophy was verified using cytochemical evaluation of cell and nuclear size. Western blot and activity assays were used to detect ERK 1/2 mTOR and Rho kinase activation. N-[(11)C]-methyl-hydroxyfasudil binding was verified using in vitro binding assays with isoproterenol stimulated cells.

Results: Isoproterenol induced a rapid and distinct activation of ERK 1/2, mTOR and Rho kinase with negligible cytotoxicity. Subsequent expansion in cell and nuclear size that is typically associated with hypertrophy was also observed. Enhanced retention of N-[(11)C]-methyl-hydroxyfasudil observed after ISO-induced Rho kinase activation in hypertrophic cells was prevented by pre-treatment with unlabeled hydroxyfasudil.

Conclusions: N-[(11)C]-methyl-hydroxyfasudil is able to measure increased Rho kinase activity via specific binding in hypertrophied cardiomyocytes and demonstrates the potential for molecular imaging of altered Rho kinase activity in diseases such as cardiac hypertrophy.
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http://dx.doi.org/10.1016/j.nucmedbio.2014.09.008DOI Listing
February 2015

Characterization of the four isomers of (123)I-CMICE-013: a potential SPECT myocardial perfusion imaging agent.

Bioorg Med Chem 2014 Apr 4;22(7):2033-44. Epub 2014 Mar 4.

Division of Cardiology, University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON K1Y 4W7, Canada; Canadian Molecular Imaging Center of Excellence (CMICE), Nordion Lab/University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON K1Y 4W7, Canada.

Unlabelled: Myocardial perfusion imaging (MPI) with single photon emission computed tomography (SPECT) is widely used in the assessment of coronary artery disease (CAD). We have developed (123)I-CMICE-013 based on rotenone, a mitochondrial complex I (MC-1) inhibitor, as a promising new MPI agent. Our synthesis results in a mixture of four species of (123)I-CMICE-013 A, B, C, D. In this study, we separated the four species and evaluated their biodistribution and imaging properties. The cold analogs (127)I-CMICE-013 A, B, C, D were isolated and characterized and their chemical structures proposed.

Methods: (123)I-CMICE-013 was synthesized by radiolabeling rotenone with Na(123)I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60°C for 45min, and the four species were separated by RP-HPLC. The cold analogs (127)I-CMICE-013 A, B, C and D were isolated with a similar procedure and characterized by NMR and mass spectrometry. Biodistribution and microSPECT imaging studies were carried out on normal rats.

Results: We propose the mechanism of the rotenone iodination and the structures of the four species. First, I(+) forms an intermediate three-membered ring with 6' and 7' carbons. Second, the lone electron pair of the water molecule attacks the 6' or 7'-carbon, following by the formation of 6'-OH, and 7'-I bonds as in major products C and D, or 6'-I and 7'-OH bonds as in minor products A and B. The weaker 6'-I bond in the intermediate prompts the nucleophilic attachment of water at the favorable 6'-carbon to generate C and D. MicroSPECT images of (123)I-CMICE-013 A, B, C, D in rats showed clear visualization of myocardium and little interference from lung and liver. The imaging time activity curves and biodistribution data showed complex profiles for the four isomers, which is not expected from the structure activity relationship theory.

Conclusion: (123/127)I-CMICE-013 A and B are constitutional isomers with C and D, while A and C are diastereomers of B and D, respectively. Overall, the biological characteristics of the four species are not correlated perfectly with their molecular structures.
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http://dx.doi.org/10.1016/j.bmc.2014.02.052DOI Listing
April 2014

Biodistribution and radiodosimetry of a novel myocardial perfusion tracer 123I-CMICE-013 in healthy rats.

EJNMMI Res 2014 Mar 13;4(1):16. Epub 2014 Mar 13.

Nordion Inc, 447 March Road, Ottawa, ON K2K 1X8, Canada.

Background: 123I-CMICE-013 is a novel radiotracer previously reported to have promising characteristics for single-photon emission computed tomography (SPECT) myocardial perfusion imaging. We evaluated the biokinetics and radiodosimetry of this rotenone-like 123I-labeled tracer in a microSPECT imaging-based study.

Methods: 37 to 111 MBq of 123I-CMICE-013 was synthesized and administered intravenously to 14 healthy rats. Images were acquired with a microSPECT/CT camera at various time intervals and reconstructed to allow activity quantification in the tissues of interest. Radiation dosage resulted from the injection of 123I-CMICE-013 was estimated base on the biodistribution data. Tissue uptake values from image analysis were verified by gamma-counting dissected organs ex vivo.

Results: The heart/stomach and heart/intestine uptake ratios peaked shortly after the injection of 123I-CMICE-013, meanwhile the heart/liver ratio reached 2 as early as at 23 min post-injection. Little activity was observed in the lung and overnight clearance was significant in most of the measured tissues. The radiation dosimetry analysis based on the time-activity curves provided an estimate of the effective human dose of 6.99E-03 mSv/MBq using ICRP 60 and 7.15E-03 mSv/MBq using ICRP 103, which is comparable to the popular myocardium perfusion imaging (MPI) agents such as 99mTc-tetrofosmin and 99mTc-sestamibi, as well as other 123I-based radiotracers.

Conclusions: 123I-CMICE-013 demonstrated desirable characteristics in its biokinetic and radiodosimetric profiles, supporting its potential application as a novel myocardial perfusion imaging agent.
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http://dx.doi.org/10.1186/2191-219X-4-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3995622PMC
March 2014

Ketoacidosis is not always due to diabetes.

BMJ Case Rep 2014 Feb 25;2014. Epub 2014 Feb 25.

Acute Medicine, Endocrinology & Diabetes, Stepping Hill Hospital, Stockport, UK.

Chronic alcoholism is a frequently unrecognised cause of ketoacidosis. Most patients with alcoholic ketoacidosis present with normal or low glucose, but this condition can present with hyperglycaemia. This can lead to misdiagnosis of diabetes ketoacidosis and, therefore, inappropriate treatment with insulin. We describe a 37-year-old Caucasian woman with chronic pancreatitis secondary to excess alcohol consumption, admitted with abdominal pain and vomiting, fulfilling the criteria for diabetes ketoacidosis. She was treated according to diabetes ketoacidosis protocol and experienced a hypoglycaemic attack within an hour of initiation of insulin. On review of her history, she was found to have three similar episodes over the past 12 months. Alcoholic ketoacidosis can present with hyperglycaemia due to relative deficiency of insulin and relative surplus in counter-regulatory stress hormones including glucagon. Awareness of the syndrome with a detailed history helps to differentiate alcohol ketoacidosis from diabetes ketoacidosis and prevent iatrogenic hypoglycaemia.
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http://dx.doi.org/10.1136/bcr-2013-203263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3939410PMC
February 2014

Toxicological evaluation of a rotenone derivative in rodents for clinical myocardial perfusion imaging.

Cardiovasc Toxicol 2014 Jun;14(2):170-82

Nordion, 447 March Road, Ottawa, ON, K2K 1X8, Canada,

Myocardial perfusion scintigraphy is a valuable clinical tool for assessing coronary blood flow deficits in patients. We recently synthesized a new iodinated compound ((123)I-CMICE-013) based on rotenone and showed that it has excellent performance as a radiotracer for myocardial perfusion imaging. Here, we describe the cellular toxicity and subacute toxicity of CMICE-013 in rats. Cultured hepatocytes displayed sensitivity to rotenone but not CMICE-013 at equimolar concentrations. Following i.v. injection of CMICE-013 for 14 days, body weight, ambulation, behavior, grooming, guarding (abdominal, muscular), pale conjunctivae, and food intake were observed. Biochemical, hematological, and histopathological changes in tissues (heart, liver, kidney, spleen, lung, and brain) and echocardiography at pre- and post-dosing were also examined. All animals responded well to the daily injections of CMICE-013 and showed no mortality or adverse reactions with respect to the parameters above. Subacute i.v. injections at high- (5 μg/kg) and low (1 μg/kg)-dose levels did not result in any significant changes to either biochemical or hematological parameters and no detectable changes in histopathology compared to the vehicle or untreated animals. Echocardiographic analyses, including the measurements of cardiac function and anatomy (wall thickness, left atrial size, and left ventricular mass), were not different at pre- versus post-dose measures and were not different compared to the vehicle or untreated animals. Our observations in small animals reveal that CMICE-013 induces minimal toxicity when delivered intravenously for 14 days.
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http://dx.doi.org/10.1007/s12012-013-9241-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4032473PMC
June 2014

Intrinsic-mediated caspase activation is essential for cardiomyocyte hypertrophy.

Proc Natl Acad Sci U S A 2013 Oct 7;110(43):E4079-87. Epub 2013 Oct 7.

Ottawa Hospital Research Institute, Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital, Ottawa, ON, Canada K1H 8L6.

Cardiomyocyte hypertrophy is the cellular response that mediates pathologic enlargement of the heart. This maladaptation is also characterized by cell behaviors that are typically associated with apoptosis, including cytoskeletal reorganization and disassembly, altered nuclear morphology, and enhanced protein synthesis/translation. Here, we investigated the requirement of apoptotic caspase pathways in mediating cardiomyocyte hypertrophy. Cardiomyocytes treated with hypertrophy agonists displayed rapid and transient activation of the intrinsic-mediated cell death pathway, characterized by elevated levels of caspase 9, followed by caspase 3 protease activity. Disruption of the intrinsic cell death pathway at multiple junctures led to a significant inhibition of cardiomyocyte hypertrophy during agonist stimulation, with a corresponding reduction in the expression of known hypertrophic markers (atrial natriuretic peptide) and transcription factor activity [myocyte enhancer factor-2, nuclear factor kappa B (NF-κB)]. Similarly, in vivo attenuation of caspase activity via adenoviral expression of the biologic effector caspase inhibitor p35 blunted cardiomyocyte hypertrophy in response to agonist stimulation. Treatment of cardiomyocytes with procaspase 3 activating compound 1, a small-molecule activator of caspase 3, resulted in a robust induction of the hypertrophy response in the absence of any agonist stimulation. These results suggest that caspase-dependent signaling is necessary and sufficient to promote cardiomyocyte hypertrophy. These results also confirm that cell death signal pathways behave as active remodeling agents in cardiomyocytes, independent of inducing an apoptosis response.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808644PMC
http://dx.doi.org/10.1073/pnas.1315587110DOI Listing
October 2013

Synthesis and characterization of 123I-CMICE-013: a potential SPECT myocardial perfusion imaging agent.

Bioorg Med Chem 2013 Jun 6;21(11):2903-11. Epub 2013 Apr 6.

Nordion Inc., 447 March Road, Ottawa, ON, Canada K2K 1X8.

Unlabelled: Coronary artery disease (CAD) is a major cause of death in Canada and the United States. Single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is a useful diagnostic test in the management of patients with CAD. The widely used SPECT MPI agents, (99m)Tc sestamibi and (99m)Tc tetrofosmin, exhibit less than ideal pharmacokinetic properties with decreasing uptake with higher flows. (123)I has a similar energy as (99m)Tc, an ideal half life, and is readily available from cyclotrons. The objective of this study was to develop an (123)I labeled MPI agent based on rotenone, a mitochondrial complex I inhibitor, as an alternative to currently available SPECT MPI agents.

Methods: (123)I-CMICE-013 was synthesized by radiolabeling rotenone with (123)I in trifluoroacetic acid (TFA) with iodogen as the oxidizing agent at 60 °C for 45 min, followed by RP-HPLC purification. The product was formulated in 5% EtOH in 10 mM NaOAc pH 6.5. The inactive analog (127)I-CMICE-013 was isolated and characterized by NMR and mass spectrometry, and the structure determined. Micro-SPECT imaging studies were carried out in normal and infarcted rats. Biodistribution studies were performed in normal rats at 2 h (n=6) and 24 h (n=8) post injection (p.i.).

Results: (123)I-CMICE-013 was isolated with >95% radiochemical purity and high specific activity (14.8-111 GBq/μmol; 400-3000 mCi/μmol). Structural analysis showed that rotenone was iodinated at 7'-position, with removal of the 6',7'-double bond, and addition of a hydroxy group at 6'-position. MicroSPECT images in normal rats demonstrated homogeneous and sustained myocardial uptake with minimal interference from lung and liver. Absent myocardial perfusion was clearly identified in rats with permanent left coronary artery ligation and ischemia-reperfusion injury. In vivo biodistribution studies in normal rats at 2 h p.i. showed significant myocardial uptake (2.01±0.48%ID/g) and high heart to liver (2.98±0.93), heart to lung (4.11±1.04) and heart to blood (8.37±3.97) ratios. At 24 h p.i., the majority of (123)I-CMICE-013 was cleared from tissues, and a significant amount of tracer was found in the thyroid, indicating in vivo deiodination of the tracer.

Conclusion: (123)I-CMICE-013 is a promising new radiotracer for SPECT MPI with high myocardial uptake, very good target to background ratios and favorable biodistribution characteristics.
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http://dx.doi.org/10.1016/j.bmc.2013.03.080DOI Listing
June 2013

MicroRNA-133 controls brown adipose determination in skeletal muscle satellite cells by targeting Prdm16.

Cell Metab 2013 Feb;17(2):210-24

Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON K1H 8L6, Canada.

Brown adipose tissue (BAT) is an energy-dispensing thermogenic tissue that plays an important role in balancing energy metabolism. Lineage-tracing experiments indicate that brown adipocytes are derived from myogenic progenitors during embryonic development. However, adult skeletal muscle stem cells (satellite cells) have long been considered uniformly determined toward the myogenic lineage. Here, we report that adult satellite cells give rise to brown adipocytes and that microRNA-133 regulates the choice between myogenic and brown adipose determination by targeting the 3'UTR of Prdm16. Antagonism of microRNA-133 during muscle regeneration increases uncoupled respiration, glucose uptake, and thermogenesis in local treated muscle and augments whole-body energy expenditure, improves glucose tolerance, and impedes the development of diet-induced obesity. Finally, we demonstrate that miR-133 levels are downregulated in mice exposed to cold, resulting in de novo generation of satellite cell-derived brown adipocytes. Therefore, microRNA-133 represents an important therapeutic target for the treatment of obesity.
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http://dx.doi.org/10.1016/j.cmet.2013.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641657PMC
February 2013

Evaluation of bifunctional chelates for the development of gallium-based radiopharmaceuticals.

Bioconjug Chem 2010 Mar 22;21(3):531-6. Epub 2010 Feb 22.

MDS Nordion, 4004 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 2A3, Ottawa Heart Institute, Ottawa, Ontario, Canada, MDS Nordion, Kanata, Ontario, Canada, and Macrocyclics, Dallas, Texas.

Ga radioisotopes, including the generator-produced positron-emitting isotope (68)Ga (t1/2 = 68 min), are of increasing interest for the development of new radiopharmaceuticals. Bifunctional chelates (BFCs) that can be efficiently radiolabeled with Ga to yield complexes with good in vivo stability are needed. To this end, we undertook a systematic comparison of four BFCs containing different chelating moieties: two novel BFCs, p-NO2-Bn-Oxo (1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) and p-NO2-Bn-PCTA (3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid), and two more commonly used BFCs, p-NO2-Bn-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) and p-NO2-Bn-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Each BFC was compared with respect to radiolabeling conditions, radiochemical yield, stability, and in vivo clearance properties. p-NO2-Bn-PCTA, p-NO2-Bn-Oxo, and p-NO2-Bn-NOTA were all more efficiently radiolabeled with Ga compared to p-NO2-Bn-DOTA. p-NO2-Bn-DOTA required longer reaction time, higher concentrations of BFC, or heating to obtain equivalent radiochemical yields. Better stability was observed for p-NO2-Bn-NOTA and p-NO2-Bn-PCTA compared to p-NO2-Bn-DOTA and p-NO2-Bn-Oxo, especially with respect to transmetalation to transferrin. Ga-radiolabled p-NO2-Bn-Oxo was found to be kinetically labile and therefore unstable in vivo. Ga-radiolabeled p-NO2-Bn-NOTA and p-NO2-Bn-PCTA were relatively inert, while Ga-radiolabeled p-NO2-Bn-DOTA had intermediate stability, losing >20% of Ga in less than one hour when incubated with apo-transferrin. Similar stability differences were seen when incubating at pH 2. In vivo PET imaging and biodistribution studies in mice showed that (68)Ga-radiolabeled p-NO2-Bn-PCTA, p-NO2-Bn-NOTA, and p-NO2-Bn-DOTA all cleared through the kidneys. While there was no statistical difference in the biodistribution results of (68)Ga-radiolabeled p-NO2-Bn-PCTA and p-NO2-Bn-DOTA, (68)Ga-radiolabeled p-NO2-Bn-NOTA cleared more rapidly from blood and muscle tissue but retained at up to 5 times higher activity in the kidneys.
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http://dx.doi.org/10.1021/bc900443aDOI Listing
March 2010

Bin1 SRC homology 3 domain acts as a scaffold for myofiber sarcomere assembly.

J Biol Chem 2009 Oct 26;284(40):27674-86. Epub 2009 Jul 26.

The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa, Ontario K1H 8L6, Canada.

In skeletal muscle development, the genes and regulatory factors that govern the specification of myocytes are well described. Despite this knowledge, the mechanisms that regulate the coordinated assembly of myofiber proteins into the functional contractile unit or sarcomere remain undefined. Here we explored the hypothesis that modular domain proteins such as Bin1 coordinate protein interactions to promote sarcomere formation. We demonstrate that Bin1 facilitates sarcomere organization through protein-protein interactions as mediated by the Src homology 3 (SH3) domain. We observed a profound disorder in myofiber size and structural organization in a murine model expressing the Bin1 SH3 region. In addition, satellite cell-derived myogenesis was limited despite the accumulation of skeletal muscle-specific proteins. Our experiments revealed that the Bin1 SH3 domain formed transient protein complexes with both actin and myosin filaments and the pro-myogenic kinase Cdk5. Bin1 also associated with a Cdk5 phosphorylation domain of titin. Collectively, these observations suggest that Bin1 displays protein scaffold-like properties and binds with sarcomeric factors important in directing sarcomere protein assembly and myofiber maturation.
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http://dx.doi.org/10.1074/jbc.M109.029538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785696PMC
October 2009

Cardiotrophin-1 maintains the undifferentiated state in skeletal myoblasts.

J Biol Chem 2009 Jul 12;284(29):19679-93. Epub 2009 May 12.

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

Skeletal myogenesis is potently regulated by the extracellular milieu of growth factors and cytokines. We observed that cardiotrophin-1 (CT-1), a member of the interleukin-6 (IL-6) family of cytokines, is a potent regulator of skeletal muscle differentiation. The normal up-regulation of myogenic marker genes, myosin heavy chain (MyHC), myogenic regulatory factors (MRFs), and myocyte enhancer factor 2s (MEF2s) were inhibited by CT-1 treatment. CT-1 also represses myogenin (MyoG) promoter activation. CT-1 activated two signaling pathways: signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase kinase (MEK), a component of the extracellular signal-regulated MAPK (ERK) pathway. In view of the known connection between CT-1 and STAT3 activation, we surprisingly found that pharmacological blockade of STAT3 activity had no effect on the inhibition of myogenesis by CT-1 suggesting that STAT3 signaling is dispensable for myogenic repression. Conversely, MEK inhibition potently reversed the inhibition of myotube formation and attenuated the repression of MRF transcriptional activity mediated by CT-1. Taken together, these data indicate that CT-1 represses skeletal myogenesis through interference with MRF activity by activation of MEK/ERK signaling. In agreement with these in vitro observations, exogenous systemic expression of CT-1 mediated by adenoviral vector delivery increased the number of myonuclei in normal post-natal mouse skeletal muscle and also delayed skeletal muscle regeneration induced by cardiotoxin injection. The expression pattern of CT-1 in embryonic and post-natal skeletal muscle and in vivo effects of CT-1 on myogenesis implicate CT-1 in the maintenance of the undifferentiated state in muscle progenitor cells.
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http://dx.doi.org/10.1074/jbc.M109.017319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740593PMC
July 2009

Is caspase-dependent apoptosis only cell differentiation taken to the extreme?

FASEB J 2007 Jan 8;21(1):8-17. Epub 2006 Nov 8.

Ottawa Health Research Institute, Molecular Medicine Program, Ottawa Hospital, General Campus, Ottawa ON, K1H 8L6, Canada.

The benefits of apoptosis for a multicellular organism are obvious and fit the current dogma that the maintenance and viability of such organisms are dependent on the selective elimination of unneeded or deleterious cell types. However, self destruction at the level of the individual cell defies the most basic precepts of biology (sustaining life). If apoptosis is viewed through this construct then one question becomes paramount, i.e., why would an individual cell and its progeny develop, retain, or evolve a mechanism the sole purpose of which is to eliminate itself? In consideration of such a paradox, it is reasonable to postulate that prospective apoptotic pathways coevolved with and or were co-opted from another basic cell function(s) that did not involve the death of the cell per se. In the following article, we present the hypothesis that the conserved biochemical pathways of apoptosis are integral components of terminal cell differentiation and it is the time of engagement and activity level of these pathways that ultimately determines the choice between cell death or cell maturation.
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http://dx.doi.org/10.1096/fj.06-5912hypDOI Listing
January 2007

Neural stem cell differentiation is dependent upon endogenous caspase 3 activity.

FASEB J 2005 Oct 15;19(12):1671-3. Epub 2005 Aug 15.

Ottawa Health Research Institute, Molecular Medicine Program, Ottawa, Ontario, Canada.

Caspase proteases have become the focal point for the development and application of anti-apoptotic therapies in a variety of central nervous system diseases. However, this approach is based on the premise that caspase function is limited to invoking cell death signals. Here, we show that caspase-3 activity is elevated in nonapoptotic differentiating neuronal cell populations. Moreover, peptide inhibition of protease activity effectively inhibits the differentiation process in a cultured neurosphere model. These results implicate caspase-3 activation as a conserved feature of neuronal differentiation and suggest that targeted inhibition of this protease in neural cell populations may have unintended consequences.
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http://dx.doi.org/10.1096/fj.04-2981fjeDOI Listing
October 2005

Active kinase proteome screening reveals novel signal complexity in cardiomyopathy.

Mol Cell Proteomics 2005 May 18;4(5):673-82. Epub 2005 Feb 18.

Molecular Medicine Program, Ottawa Health Research Institute, Ottawa Hospital, Ottawa, Ontario K1H 8L6, Canada.

Recent advances in the characterization of the phosphoproteome have been limited to measuring phosphorylation statuses, which imply but do not measure protein kinase activity directly. As such, the ability to screen, compare, and define multiple protein enzymatic activities across divergent samples remains a daunting challenge in proteomics. Here, we describe a gel-based kinase assay coupled to MS identification as an approach to map global kinase activity and assign pathway architecture to specified biologic contexts. We demonstrate the utility of this method as a platform for the comparison of proteomes based on differences in both kinase activities and for use in the de novo substrate identification for individual kinases. This approach allowed us to map the signal perturbations in the post-natal heart that were associated with activation of a myopathic cascade as mediated by the mitogen-activated protein kinase MKK6 and established the novel observation that MKK6 promotes the development of cardiomyopathy through multiple substrate interactions.
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http://dx.doi.org/10.1074/mcp.M400200-MCP200DOI Listing
May 2005

Phosphorylation-dependent structural alterations in the small hsp30 chaperone are associated with cellular recovery.

Exp Cell Res 2003 Jun;286(2):175-85

Ottawa Health Research Institute, Ottawa General Hospital, Center for Molecular Medicine, Ottawa, Ontario, Canada K1H 8L6.

Small heat shock proteins (hsps) act as molecular chaperones by preventing the thermal aggregation and unfolding of cellular protein; however, the manner by which cells regulate chaperone activity remains unclear. In the present study, we examined the role of phosphorylation on the chaperone function of the Xenopus small hsp30. Both heat stress and sodium arsenite treatment in A6 cells resulted in a rapid activation of p38alpha and MAPKAPK-2. Surprisingly, the association of MAPKAPK-2 with hsp30 and its subsequent phosphorylation were more prevalent during recovery after heat stress. Treatment of A6 cells with SB203580, an inhibitor of the p38 MAP kinase pathway, resulted in a loss of hsp30 phosphorylation. Phosphorylation resulted in the formation of smaller multimeric hsp30 complexes and resulted in a significant loss of secondary structure. Consequently the phosphorylation-induced structural changes severely compromised the ability of hsp30 to prevent the heat-induced aggregation of citrate synthase and luciferase in vitro. We confirmed that the loss of chaperone activity was coincident with an attenuated binding of phosphorylated hsp30 with target proteins. Our data suggest that phosphorylation may be necessary to regulate the post-heat stress molecular chaperone activity of hsp30.
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http://dx.doi.org/10.1016/s0014-4827(03)00067-3DOI Listing
June 2003

Caspase 3 activity is required for skeletal muscle differentiation.

Proc Natl Acad Sci U S A 2002 Aug 12;99(17):11025-30. Epub 2002 Aug 12.

Ottawa Health Research Institute, Molecular Medicine Program, Ottawa General Hospital, Ottawa, ON, Canada K1H 8L6.

The cellular alterations associated with skeletal muscle differentiation share a high degree of similarity with key phenotypic changes usually ascribed to apoptosis. For example, actin fiber disassembly/reorganization is a conserved feature of both apoptosis and differentiating myoblasts and the conserved muscle contractile protein, myosin light chain kinase, is required for the apoptotic feature of membrane blebbing. As such, these observations suggest that the induction of differentiation and apoptosis in the myogenic lineage may use overlapping cellular mechanisms. Here, we report that skeletal muscle differentiation depends on the activity of the key apoptotic protease, caspase 3. Peptide inhibition of caspase 3 activity or homologous deletion of caspase 3 leads to dramatic reduction in both myotube/myofiber formation and expression of muscle-specific proteins. Subsequently, we have identified Mammalian Sterile Twenty-like kinase as a crucial caspase 3 effector in this cellular process. Mammalian Sterile Twenty-like kinase is cleavage-activated by caspase 3, and restoration of this truncated kinase in caspase 3 null myoblasts restores the differentiation phenotype. Taken together, these results confirm a unique and unanticipated role for a caspase 3-mediated signal cascade in the promotion of myogenesis.
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http://dx.doi.org/10.1073/pnas.162172899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC123204PMC
August 2002

Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state.

Cell Stress Chaperones 2002 Jan;7(1):6-16

Department of Biology, University of Waterloo, Ontario, Canada.

In this study we characterized the chaperone functions of Xenopus recombinant Hsp30C and Hsp30D by using an in vitro rabbit reticulocyte lysate (RRL) refolding assay system as well as a novel in vivo Xenopus oocyte microinjection assay. Whereas heat- or chemically denaturated luciferase (LUC) did not regain significant enzyme activity when added to RRL or microinjected into Xenopus oocytes, compared with native LUC, denaturation of LUC in the presence of Hsp30C resulted in a reactivation of enzyme activity up to 80-100%. Recombinant Hsp30D, which differs from Hsp30C by 19 amino acids, was not as effective as its isoform in preventing LUC aggregation or maintaining it in a folding-competent state. Removal of the first 17 amino acids from the N-terminal region of Hsp30C had little effect on its ability to maintain LUC in a folding-competent state. However, deletion of the last 25 residues from the C-terminal end dramatically reduced Hsp30C chaperone activity. Coimmunoprecipitation and immunoblot analyses revealed that Hsp30C remained associated with heat-denatured LUC during incubation in reticulocyte lysate and that the C-terminal mutant exhibited reduced affinity for unfolded LUC. Finally, we found that Hsc70 present in RRL interacted only with heat-denatured LUC bound to Hsp30C. These findings demonstrate that Xenopus Hsp30 can maintain denatured target protein in a folding-competent state and that the C-terminal end is involved in this function.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC514803PMC
http://dx.doi.org/10.1379/1466-1268(2002)007<0006:xshsph>2.0.co;2DOI Listing
January 2002