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1.  Predicting response and survival in chemotherapy-treated triple-negative breast cancer 
British Journal of Cancer  2014;111(8):1532-1541.
In this study, we evaluated the ability of gene expression profiles to predict chemotherapy response and survival in triple-negative breast cancer (TNBC).
Gene expression and clinical–pathological data were evaluated in five independent cohorts, including three randomised clinical trials for a total of 1055 patients with TNBC, basal-like disease (BLBC) or both. Previously defined intrinsic molecular subtype and a proliferation signature were determined and tested. Each signature was tested using multivariable logistic regression models (for pCR (pathological complete response)) and Cox models (for survival). Within TNBC, interactions between each signature and the basal-like subtype (vs other subtypes) for predicting either pCR or survival were investigated.
Within TNBC, all intrinsic subtypes were identified but BLBC predominated (55–81%). Significant associations between genomic signatures and response and survival after chemotherapy were only identified within BLBC and not within TNBC as a whole. In particular, high expression of a previously identified proliferation signature, or low expression of the luminal A signature, was found independently associated with pCR and improved survival following chemotherapy across different cohorts. Significant interaction tests were only obtained between each signature and the BLBC subtype for prediction of chemotherapy response or survival.
The proliferation signature predicts response and improved survival after chemotherapy, but only within BLBC. This highlights the clinical implications of TNBC heterogeneity, and suggests that future clinical trials focused on this phenotypic subtype should consider stratifying patients as having BLBC or not.
PMCID: PMC4200088  PMID: 25101563
breast cancer; genomics; subtypes; intrinsic; basal like; chemotherapy; neoadjuvant
2.  Insulin signaling in the aging of healthy and proteotoxically stressed mechanosensory neurons 
Frontiers in Genetics  2014;5:212.
Insulin signaling is central to cellular metabolism and organismal aging. However, the role of insulin signaling in natural and proteotoxically stressed aging neurons has yet to be fully described. We studied aging of Caenorbaditis elegans mechanosensory neurons expressing a neurotoxic expanded polyglutamine transgene (polyQ128), or lacking this proteotoxicity stressor (polyQ0), under conditions in which the insulin signaling pathway was disrupted by RNA interference (RNAi). We describe specific changes in lifespan, mechanosensory neuronal morphologies, and mechansensory function following RNAi treatment targeting the insulin signaling pathway. Overall, we confirmed that transcription factor DAF-16 is neuroprotective in the proteotoxically stressed model, though not strikingly in the naturally aging model. Decreased insulin signaling through daf-2 RNAi improved mechanosensory function in both models and decreased protein aggregation load in polyQ128, yet showed opposing effects on accumulation of neuronal aberrations in both strains. Decreased daf-2 signaling slightly enhanced mechanosensation while greatly enhancing branching of the mechanosensory neuron axons and dendrites in polyQ0 animals, suggesting that branching is an adaptive response in natural aging. These effects in polyQ0 did not appear to involve DAF-16, suggesting the existence of a non-canonical DAF-2 pathway for the modulation of morphological adaptation. However, in polyQ128 animals, decreased daf-2 signaling significantly enhanced mechanosensation while decreasing neuronal aberrations. Unlike other interventions that reduce the strength of insulin signaling, daf-2 RNAi dramatically redistributed large polyQ128 aggregates to the cell body, away from neuronal processes. Our results suggest that insulin signaling strength can differentially affect specific neurons aging naturally or under proteotoxic stress.
PMCID: PMC4107846  PMID: 25101108
neuronal aging; insulin signaling; proteotoxicity; Huntington's Disease; Caenorhabditis elegans
3.  Fishing for causes and cures of motor neuron disorders 
Disease Models & Mechanisms  2014;7(7):799-809.
Motor neuron disorders (MNDs) are a clinically heterogeneous group of neurological diseases characterized by progressive degeneration of motor neurons, and share some common pathological pathways. Despite remarkable advances in our understanding of these diseases, no curative treatment for MNDs exists. To better understand the pathogenesis of MNDs and to help develop new treatments, the establishment of animal models that can be studied efficiently and thoroughly is paramount. The zebrafish (Danio rerio) is increasingly becoming a valuable model for studying human diseases and in screening for potential therapeutics. In this Review, we highlight recent progress in using zebrafish to study the pathology of the most common MNDs: spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). These studies indicate the power of zebrafish as a model to study the consequences of disease-related genes, because zebrafish homologues of human genes have conserved functions with respect to the aetiology of MNDs. Zebrafish also complement other animal models for the study of pathological mechanisms of MNDs and are particularly advantageous for the screening of compounds with therapeutic potential. We present an overview of their potential usefulness in MND drug discovery, which is just beginning and holds much promise for future therapeutic development.
PMCID: PMC4073270  PMID: 24973750
ALS; HSP; SMA; Zebrafish; Drug discovery; Motor neuron disorders
4.  The Wnt Receptor Ryk Reduces Neuronal and Cell Survival Capacity by Repressing FOXO Activity During the Early Phases of Mutant Huntingtin Pathogenicity 
PLoS Biology  2014;12(6):e1001895.
A study of Huntington's disease reveals that neurons might fail to cope with maintaining their function during the pre-symptomatic, pathogenic phases of HD, possibly due to the early repression of key longevity-promoting transcription factors by abnormal developmental signaling.
The Wnt receptor Ryk is an evolutionary-conserved protein important during neuronal differentiation through several mechanisms, including γ-secretase cleavage and nuclear translocation of its intracellular domain (Ryk-ICD). Although the Wnt pathway may be neuroprotective, the role of Ryk in neurodegenerative disease remains unknown. We found that Ryk is up-regulated in neurons expressing mutant huntingtin (HTT) in several models of Huntington's disease (HD). Further investigation in Caenorhabditis elegans and mouse striatal cell models of HD provided a model in which the early-stage increase of Ryk promotes neuronal dysfunction by repressing the neuroprotective activity of the longevity-promoting factor FOXO through a noncanonical mechanism that implicates the Ryk-ICD fragment and its binding to the FOXO co-factor β-catenin. The Ryk-ICD fragment suppressed neuroprotection by lin-18/Ryk loss-of-function in expanded-polyQ nematodes, repressed FOXO transcriptional activity, and abolished β-catenin protection of mutant htt striatal cells against cell death vulnerability. Additionally, Ryk-ICD was increased in the nucleus of mutant htt cells, and reducing γ-secretase PS1 levels compensated for the cytotoxicity of full-length Ryk in these cells. These findings reveal that the Ryk-ICD pathway may impair FOXO protective activity in mutant polyglutamine neurons, suggesting that neurons are unable to efficiently maintain function and resist disease from the earliest phases of the pathogenic process in HD.
Author Summary
Neuronal cell decline in neurodegenerative disease can be caused by inherited mutations and involves neuronal dysfunction followed by neuronal death. The ability of neurons to cope with the chronic stress induced by mutant protein expression may determine the course of their decline and eventual demise. Although the pathophysiological importance of these stress responses has been previously shown, very little is known about the signaling networks that regulate neuronal homeostasis during the early presymptomatic—but pathogenic—phases of a neurodegenerative disorder such as Huntington's disease (HD). In particular, it remains unclear whether neuronal differentiation factors regulate stress response pathways during neurodegenerative disease and how this might impact the overall capacity of neurons to cope with stress and maintain their function. Here, we show that the Wnt receptor Ryk, a protein known to be important for neurogenesis, is increased in different animal models of HD, before or during the early phases of the disease process. Interestingly, increased levels of Ryk repress activity of the FOXO proteins—a family of transcription factors that play a role in cell survival/longevity and in neuronal homeostasis and protection. Ryk represses FOXO protective activity, possibly directly, through its intracellular domain, a product of γ-secretase–mediated cleavage previously implicated in the birth of new cortical neurons. This highlights the regulation of HD neuron survival by a Ryk-dependent pathway that is distinct from canonical Wnt/Ryk signaling. From our findings, we postulate that neurons are unable to develop an efficient FOXO-mediated survival response during the very early, pathogenic phases of HD.
PMCID: PMC4068980  PMID: 24960609
5.  Deciphering genetic interactions between ALS genes using C. elegans 
Worm  2014;3:e29047.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder causing selective death of motor neurons in which it is speculated that 10% of cases have a familial history. In the past 20 years, many genes causative for ALS have been discovered, but the link between them and their roles in neurodegeneration remain unknown. The identification of genes associated with both ALS and frontotemporal dementia (FTD), along with the observation of patients affected by both diseases, have suggested that they are part of the same neurodegenerative spectrum. Investigating possible genetic interactions among ALS/FTD genes could help understand the role of these genes in neurodegeneration. To pursue this goal, our group has developed several ALS models to study potential genetic interactions. More recently, we characterized the deletion mutant alfa-1, the ortholog of C9ORF72, to evaluate the potential genetic interactions between C9ORF72/alfa-1 and other ALS genes. Here, we discuss the genetic interactions identified in our models and how some of these proteins may also be linked to other neurodegenerative disorders.
PMCID: PMC4165534  PMID: 25254150
ALS; C9ORF72; FUS; PGRN; SOD1; TDP-43; neurodegeneration
6.  Heritable Transmission of Stress Resistance by High Dietary Glucose in Caenorhabditis elegans 
PLoS Genetics  2014;10(5):e1004346.
Glucose is a major energy source and is a key regulator of metabolism but excessive dietary glucose is linked to several disorders including type 2 diabetes, obesity and cardiac dysfunction. Dietary intake greatly influences organismal survival but whether the effects of nutritional status are transmitted to the offspring is an unresolved question. Here we show that exposing Caenorhabditis elegans to high glucose concentrations in the parental generation leads to opposing negative effects on fecundity, while having protective effects against cellular stress in the descendent progeny. The transgenerational inheritance of glucose-mediated phenotypes is dependent on the insulin/IGF-like signalling pathway and components of the histone H3 lysine 4 trimethylase complex are essential for transmission of inherited phenotypes. Thus dietary over-consumption phenotypes are heritable with profound effects on the health and survival of descendants.
Author Summary
Nutritional state has major effects on health and longevity, and investigations into the mechanisms of dietary restriction have taken the lion's share of recent genetic discoveries. We used Caenorhabditis elegans to investigate the role of diet on nematode physiology and report the surprising finding that exposure to high glucose at one generational time point has heritable effects in descendent progeny. Glucose promotes resistance against cellular stress and neurodegeneration in parental and descendent progeny, while reducing lifespan only in the parental generation. Furthermore, we found that glucose mediated protection is dependent on well-known metabolic and stress response genes. Numerous strategies have evolved to ensure reproductive success in the face of changing and challenging environments. It is believed that extended lifespan phenotypes observed under dietary restriction conditions maximize an organism's survival until environmental conditions improve allowing for reproduction. We discovered a novel diet-influenced reproductive advantage; animals subjected to high dietary glucose are resistant to protein damaging stress, and this resistance is transmitted to their progeny. The trade-off for stress-resistant progeny is decreased lifespan and fecundity in the parental strain suggesting that this strategy may be adaptive under nutrient rich conditions.
PMCID: PMC4006733  PMID: 24785260
7.  Worming forward: amyotrophic lateral sclerosis toxicity mechanisms and genetic interactions in Caenorhabditis elegans 
Neurodegenerative diseases share pathogenic mechanisms at the cellular level including protein misfolding, excitotoxicity and altered RNA homeostasis among others. Recent advances have shown that the genetic causes underlying these pathologies overlap, hinting at the existence of a genetic network for neurodegeneration. This is perhaps best illustrated by the recent discoveries of causative mutations for amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD). Once thought to be distinct entities, it is now recognized that these diseases exist along a genetic spectrum. With this wealth of discoveries comes the need to develop new genetic models of ALS and FTD to investigate not only pathogenic mechanisms linked to causative mutations, but to uncover potential genetic interactions that may point to new therapeutic targets. Given the conservation of many disease genes across evolution, Caenorhabditis elegans is an ideal system to investigate genetic interactions amongst these genes. Here we review the use of C. elegans to model ALS and investigate a putative genetic network for ALS/FTD that may extend to other neurological disorders.
PMCID: PMC4029022  PMID: 24860590
C. elegans; ALS (Amyotrophic lateral sclerosis); TDP-43; FUS; C9orf72; SOD1; genetic networks; motor neuron disease
8.  Medical students’ experiences learning intimate physical examination skills: a qualitative study 
BMC Medical Education  2014;14:39.
Intimate physical examination skills are essential skills for any medical graduate to have mastered to an appropriate level for the safety of his or her future patients. Medical schools are entrusted with the complex task of teaching and assessing these skills for their students. The objectives of this study were to explore a range of medical students’ experiences of learning intimate physical examination skills and to explore their perceptions of factors which impede or promote the learning of these skills.
Individual semi-structured interviews (N = 16) were conducted with medical students in years two to five from the University of Newcastle, as part of a larger research project investigating how medical students develop their attitudes to gender and health. This was a self-selected sample of the entire cohort who were all invited to participate. A thematic analysis of the transcribed data was performed.
Students reported differing levels of discomfort with their learning experiences in the area of intimate physical examination and differing beliefs about the helpfulness of these experiences. The factors associated with levels of discomfort and the helpfulness of the experience for learning were: satisfaction with teaching techniques, dealing with an uncomfortable situation and perceived individual characteristics in both the patients and the students. The examination causing the greatest reported discomfort was the female pelvic examination by male students.
Student discomfort with the experience of learning intimate physical examination skills may be common and has ongoing repercussions for students and patients. Recommendations are made of ways to modify teaching technique to more closely match students’ perceived needs.
PMCID: PMC3943277  PMID: 24575827
Medical student; Intimate physical examination; Learning experiences; Teaching techniques
9.  Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in C. elegans 
PLoS ONE  2013;8(12):e83450.
An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.
PMCID: PMC3861484  PMID: 24349511
10.  Integration of β-catenin, Sirtuin and FOXO Signaling Protects from Mutant Huntingtin Toxicity 
One of the current challenges of neurodegenerative disease research is to determine whether signaling pathways that are essential to cellular homeostasis might contribute to neuronal survival and modulate the pathogenic process in human disease. In C. elegans, sir-2.1/SIRT1 overexpression protects neurons from the early phases of expanded polyglutamine (polyQ) toxicity, and this protection requires the lonfity-promoting factor daf-16/FOXO. Here, we show this neuroprotective effect also requires the DAF-16/FOXO partner bar-1/β-catenin and putative DAF-16 regulated gene, ucp-4, the sole mitochondrial uncoupling protein (UCP) in nematodes. These results fit with a previously-proposed mechanism in which the β-catenin, FOXO and SIRT1 proteins may together regulate gene expression and cell survival. Knockdown of β-catenin enhanced the vulnerability to cell death of mutant-huntingtin striatal cells derived from the HdhQ111 knock-in mice. In addition, this effect was compensated by SIRT1 overexpression and accompanied by the modulation of neuronal UCP expression levels, further highlighting a cross-talk between β-catenin and SIRT1 in the modulation of mutant polyQ cytoxicity. Taken together, these results suggest that integration of β-catenin, sirtuin and FOXO signaling protects from the early phases of mutant huntingtin toxicity.
PMCID: PMC3780431  PMID: 22956852
11.  Single-Neuron Sequencing Analysis of L1 Retrotransposition and Somatic Mutation in the Human Brain 
Cell  2012;151(3):483-496.
A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Since recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of 3 normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.
PMCID: PMC3567441  PMID: 23101622
12.  Baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) 
McMurray, John J.V. | Anand, Inder S. | Diaz, Rafael | Maggioni, Aldo P. | O'Connor, Christopher | Pfeffer, Marc A. | Solomon, Scott D. | Tendera, Michal | van Veldhuisen, Dirk J. | Albizem, Moetaz | Cheng, Sunfa | Scarlata, Debra | Swedberg, Karl | Young, James B. | Amuchastegui, M. | Belziti, C. | Bluguermann, J. | Caccavo, M. | Cartasegna, L. | Colque, R. | Cuneo, C. | Fernandez, A. | Gabito, A. | Goicochea, R. | Gonzalez, M. | Gorosito, V. | Grinfeld, L. | Hominal, M. | Kevorkian, R. | Litvak Bruno, M. | Llanos, J. | Mackinnon, I. | Manuale, O. | Marzetti, E. | Nul, D. | Perna, E. | Riccitelli, M. | Sanchez, A. | Santos, D. | Schygiel, P. | Toblli, J. | Vogel, D. | Aggarwal, A. | Amerena, J. | De Looze, F. | Fletcher, P. | Hare, D. | Ireland, M. | Krum, H. | Lattimore, J. | Marwick, T. | Sindone, A. | Thompson, P. | Waites, J. | Altenberger, J. | Ebner, C. | Lenz, K. | Pacher, R. | Poelzl, G. | Charlier, F. | de Ceuninck, M. | De Keulenaer, G. | Dendale, P. | Maréchal, 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L. | Senni, M. | Tavazzi, L. | Erglis, A. | Jasinkevica, I. | Kakurina, N. | Veze, I. | Volans, E. | Bagdonas, A. | Berukstis, E. | Celutkiene, J. | Dambrauskaite, A. | Jarasuniene, D. | Luksiene, D. | Rudys, A. | Sakalyte, G. | Sliaziene, S. | Aguilar-Romero, R. | Cardona-Muñoz, E. | Castro-Jimenez, J. | Chavez-Herrera, J. | Chuquiure Valenzuela, E. | De la Pena, G. | Herrera, E. | Leiva-Pons, J. | Lopez Alvarado, A. | Mendez Machado, G. | Ramos-Lopez, G. | Basart, D. | Buijs, E. | Cornel, J. | de Leeuw, M. | Dijkgraaf, R. | Dunselman, P. | Freericks, M. | Hamraoui, K. | Lenderlink, T. | Linssen, G. | Lodewick, P. | Lodewijks, C. | Lok, D. | Nierop, P. | Ronner, E. | Somsen, A. | van Dantzig, J. | van der Burgh, P. | van Kempen, L. | van Vlies, B. | Voors, A. | Wardeh, A. | Willems, F. | Dickstein, K. | Gundersen, T. | Hole, T. | Thalamus, J. | Westheim, A. | Dabrowski, M. | Gorski, J. | Korewicki, J. | Kuc, K. | Miekus, P. | Musial, W. | Niegowska, J. | Piotrowski, W. | Podolec, P. | Polonski, L. | Ponikowski, P. | Rynkiewicz, A. | Szelemej, R. | Trusz-Gluza, M. | Ujda, M. | Wojciechowski, D | Wysokinski, A. | Camacho, A. | Fonseca, C. | Monteiro, P. | Apetrei, E. | Bruckner, I. | Carasca, E. | Coman, I. | Datcu, M. | Dragulescu, S. | Ionescu, P. | Iordachescu-Petica, D. | Manitiu, I. | Popa, V. | Pop-Moldovan, A. | Radoi, M. | Stamate, S. | Tomescu, M. | Vita, I. | Aroutiounov, G. | Ballyuzek, M. | Bart, B. | Churina, S. | Glezer, M. | Goloshchekin, B. | Ivleva, A. | Kobalava, Z. | Kostenko, V. | Lopatin, Y. | Martynov, A. | Orlov, V. | Semernin, E. | Shogenov, Z. | Sidorenko, B. | Skvortsov, A. | Storzhakov, G. | Sulimov, V. | Talibov, O. | Tereshenko, S. | Tsyrline, V. | Zadionchenko, V. | Zateyshchikov, D. | Dzupina, A. | Hranai, M. | Kmec, J. | Micko, K. | Murin, J. | Pella, D. | Sojka, G. | Spisak, V. | Vahala, P. | Vinanska, D. | Badat, A. | Bayat, J. | Dawood, S. | Delport, E. | Ellis, G. | Garda, R. | Klug, E. | Mabin, T. | Naidoo, D. | Pretorius, M. | Ranjith, N. | Van Zyl, L. | Weich, H. | Anguita, M. | Berrazueta, J. | Bruguera i Cortada, J. | de Teresa, E. | Gómez Sánchez, M. | González Juanatey, J. | Gonzalez-Maqueda, I. | Jordana, R. | Lupon, J. | Manzano, L. | Pascual Figal, D. | Pulpón, L. | Recio, J. | Ridocci Soriano, F. | Rodríguez Lambert, J. | Roig Minguell, E. | Roig Minguell, E. | Romero, J. | Valdovinos, P. | Klintberg, L. | Kronvall, T. | Lycksell, M. | Morner, S. | Rydberg, E. | Swedberg, K. | Timberg, I. | Wikstrom, G. | Moccetti, T.4 | Ashok, J. | Banerjee, P. | Carr-White, G. | Cleland, J. | Connolly, E. | Francis, M. | Greenbaum, R. | Kadr, H. | Lindsay, S. | McMurray, J. | Megarry, S. | Memon, A. | Murdoch, D. | Senior, R. | Squire, I. | Tan, L. | Witte, K. | Adams, K. | Adamson, P. | Adler, A. | Altschul, L. | Altschuller, A. | Amirani, H. | Anand, I. | Andreou, C. | Ansari, M. | Antonishen, M. | Banchs, H. | Banerjee, S. | Banish, D. | Bank, A. | Barbagelata, A. | Barnard, D. | Bellinger, R. | Benn, A. | Berk, M. | Berry, B. | Bethala, V. | Bilazarian, S. | Bisognano, J. | Bleyer, F. | Blum, M. | Boehmer, J. | Bouchard, A. | Boyle, A. | Bozkurt, B. | Brown, C. | Burlew, B. | Burnham, K. | Butler, J. | Call, J. | Cambier, P. | Cappola, T. | Carlson, R. | Chandler, B. | Chandra, R. | Chandraratna, P. | Chernick, R. | Colan, D. | Colfer, H. | Colucci, W. | Connelly, T. | Costantini, O. | Dadkhah, S. | Dauber, I. | Davis, J. | Davis, S. | Denning, S. | Drazner, M. | Dunlap, S. | Egbujiobi, L. | Elkayam, U. | Elliott, J. | El-Shahawy, M. | Essandoh, L. | Ewald, G. | Fang, J. | Farhoud, H. | Felker, G. | Fernandez, J. | Festin, R. | Fishbein, G. | Florea, V. | Flores, E. | Floro, J. | Gabris, M. | Garg, M. | Gatewood, R. | Geller, M. | Ghali, J. | Ghumman, W. | Gibbs, G. | Gillespie, E. | Gilmore, R. | Gogia, H. | Goldberg, L. | Gradus-Pizlo, I. | Grainger, T. | Gudmundsson, G. | Gunawardena, D. | Gupta, D. | Hack, T. | Hall, S. | Hamroff, G. | Hankins, S. | Hanna, M. | Hargrove, J. | Haught, W. | Hauptman, P. | Hazelrigg, M. | Herzog, C. | Heywood, J. | Hill, T. | Hilton, T. | Hirsch, H. | Hunter, J. | Ibrahim, H. | Imburgia, M. | Iteld, B. | Jackson, B. | Jaffrani, N. | Jain, D. | Jain, A. | James, M. | Jimenez, J. | Johnson, E. | Kale, P. | Kaneshige, A. | Kapadia, S. | Karia, D. | Karlsberg, R. | Katholi, R. | Kerut, E. | Khoury, W. | Kipperman, R. | Klapholz, M. | Kosinski, E. | Kozinn, M. | Kraus, D. | Krueger, S. | Krum, H. | Kumar, S. | Lader, E. | Lee, C. | Levy, W. | Lewis, E. | Light-McGroary, K. | Loh, I. | Lombardi, W. | Machado, C. | Maislos, F. | Mancini, D. | Markus, T. | Mather, P. | McCants, K. | McGrew, F. | McLaurin, B. | McMillan, E. | McNamara, D. | Meyer, T. | Meymandi, S. | Miller, A. | Minami, E. | Modi, M. | Mody, F. | Mohanty, P. | Moscoso, R. | Moskowitz, R. | Moustafa, M. | Mullen, M. | Naz, T. | Noonan, T. | O'Brien, T. | Oellerich, W. | Oren, R. | Pamboukian, S. | Pereira, N. | Pitt, W. | Porter, C. | Prabhu, S. | Promisloff, 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European Journal of Heart Failure  2013;15(3):334-341.
This report describes the baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) which is testing the hypothesis that anaemia correction with darbepoetin alfa will reduce the composite endpoint of death from any cause or hospital admission for worsening heart failure, and improve other outcomes.
Methods and results
Key demographic, clinical, and laboratory findings, along with baseline treatment, are reported and compared with those of patients in other recent clinical trials in heart failure. Compared with other recent trials, RED-HF enrolled more elderly [mean age 70 (SD 11.4) years], female (41%), and black (9%) patients. RED-HF patients more often had diabetes (46%) and renal impairment (72% had an estimated glomerular filtration rate <60 mL/min/1.73 m2). Patients in RED-HF had heart failure of longer duration [5.3 (5.4) years], worse NYHA class (35% II, 63% III, and 2% IV), and more signs of congestion. Mean EF was 30% (6.8%). RED-HF patients were well treated at randomization, and pharmacological therapy at baseline was broadly similar to that of other recent trials, taking account of study-specific inclusion/exclusion criteria. Median (interquartile range) haemoglobin at baseline was 112 (106–117) g/L.
The anaemic patients enrolled in RED-HF were older, moderately to markedly symptomatic, and had extensive co-morbidity.
PMCID: PMC3576902  PMID: 23329651
Heart failure; Anaemia
13.  A Transcriptional Regulatory Role of the THAP11–HCF-1 Complex in Colon Cancer Cell Function 
Molecular and Cellular Biology  2012;32(9):1654-1670.
The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes, including transcriptional regulation, but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown. In this work, we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohistochemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases. Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation, and profiling of gene expression in these cells identified a novel gene set composed of 80 differentially expressed genes, 70% of which were derepressed by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor 1) and recruit HCF-1 to target promoters. Importantly, THAP11-mediated gene regulation and its chromatin association require HCF-1, while HCF-1 recruitment at these genes requires THAP11. Collectively, these data provide the first characterization of THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11–HCF-1 complex may be an important transcriptional and cell growth regulator in human colon cancer.
PMCID: PMC3347244  PMID: 22371484
14.  A pilot study to evaluate assisted freehand ultrasound elasticity imaging in the sizing of early breast cancer: a comparison of B-mode and AFUSON elasticity ultrasound with histopathology measurements 
The British Journal of Radiology  2011;84(1007):1011-1019.
This pilot study investigates the role of assisted-freehand ultrasound (AFUSON) elasticity imaging of the breast in assessing the contour, size and area of 23 early breast cancers by making comparison of AFUSON with the equivalent B-mode ultrasound images and gold standard histopathology slides.
The B-mode, AFUSON and digitised histopathology slides of three early breast cancers were compared for contour, size and area with histopathology scans. AFUSON features that corresponded to areas of known malignant change on the histopathology slides were regarded as diagnostic. These diagnostic criteria were then applied to the B-mode and AFUSON elasticity images of all 23 breast cancers in the pilot study without having the availability of the histopathology scans for reference. Corresponding diameters were measured and the results were compared with the equivalent measurements on the scans of the histology slides. The results were tabulated in histogram form. Diagnostic confidence levels were evaluated.
Size dimension accuracy increased from 66% using B-mode alone to 82% using combined B-mode and AFUSON elasticity images. Tumour area accuracy was also increased. A small number of cases had a striking visual similarity of shape on AFUSON elasticity scans and histopathology slides.
In spite of the shortfalls in this study, AFUSON elasticity imaging was capable of acquiring some high-quality images that showed strong correlation between AFUSON elasticity and scans of histology slides. Further studies will be carried out to refine the technique and determine if it has a role in the diagnosis and management of breast cancer.
PMCID: PMC3473697  PMID: 21632651
15.  Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence 
The inactivation of ERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism of in vitro resistance to azole antifungal drugs in the human pathogen Candida albicans. ERG3 inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified a C. albicans clinical isolate (VSY2) with high-level resistance to azole drugs in vitro and an absence of ergosterol but normal filamentation. Sequencing of ERG3 in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming that ERG3 inactivation was the mechanism of azole resistance. Additionally, the replacement of both ERG3 alleles by erg3-1 in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinical ERG3 mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole both in vitro and in vivo, the ERG3-derived mutant of SC5314 was resistant only in vitro and was less virulent than the wild type. This suggests that VSY2 compensated for the in vivo fitness defect of ERG3 inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation of ERG3 does not necessarily affect filamentation and virulence.
PMCID: PMC3318373  PMID: 22252807
16.  Equine Stomachs Harbor an Abundant and Diverse Mucosal Microbiota 
Little is known about the gastric mucosal microbiota in healthy horses, and its role in gastric disease has not been critically examined. The present study used a combination of 16S rRNA bacterial tag-encoded pyrosequencing (bTEFAP) and fluorescence in situ hybridization (FISH) to characterize the composition and spatial distribution of selected gastric mucosal microbiota of healthy horses. Biopsy specimens of the squamous, glandular, antral, and any ulcerated mucosa were obtained from 6 healthy horses by gastroscopy and from 3 horses immediately postmortem. Pyrosequencing was performed on biopsy specimens from 6 of the horses and yielded 53,920 reads in total, with 631 to 4,345 reads in each region per horse. The microbiome segregated into two distinct clusters comprised of horses that were stabled, fed hay, and sampled at postmortem (cluster 1) and horses that were pastured on grass, fed hay, and biopsied gastroscopically after a 12-h fast (cluster 2). The types of bacteria obtained from different anatomic regions clustered by horse rather than region. The dominant bacteria in cluster 1 were Firmicutes (>83% reads/sample), mainly Streptococcus spp., Lactobacillus spp. and, Sarcina spp. Cluster 2 was more diverse, with predominantly Proteobacteria, Bacteroidetes, and Firmicutes, consisting of Actinobacillus spp. Moraxella spp., Prevotella spp., and Porphyromonas spp. Helicobacter sp. sequences were not identified in any of 53,920 reads. FISH (n = 9) revealed bacteria throughout the stomach in close apposition to the mucosa, with significantly more Streptococcus spp. present in the glandular region of the stomach. The equine stomach harbors an abundant and diverse mucosal microbiota that varies by individual.
PMCID: PMC3318809  PMID: 22307294
17.  Methylene Blue Protects against TDP-43 and FUS Neuronal Toxicity in C. elegans and D. rerio 
PLoS ONE  2012;7(7):e42117.
The DNA/RNA-binding proteins TDP-43 and FUS are found in protein aggregates in a growing number of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and related dementia, but little is known about the neurotoxic mechanisms. We have generated Caenorhabditis elegans and zebrafish animal models expressing mutant human TDP-43 (A315T or G348C) or FUS (S57Δ or R521H) that reflect certain aspects of ALS including motor neuron degeneration, axonal deficits, and progressive paralysis. To explore the potential of our humanized transgenic C. elegans and zebrafish in identifying chemical suppressors of mutant TDP-43 and FUS neuronal toxicity, we tested three compounds with potential neuroprotective properties: lithium chloride, methylene blue and riluzole. We identified methylene blue as a potent suppressor of TDP-43 and FUS toxicity in both our models. Our results indicate that methylene blue can rescue toxic phenotypes associated with mutant TDP-43 and FUS including neuronal dysfunction and oxidative stress.
PMCID: PMC3407135  PMID: 22848727
18.  TDP-1/TDP-43 Regulates Stress Signaling and Age-Dependent Proteotoxicity in Caenorhabditis elegans 
PLoS Genetics  2012;8(7):e1002806.
TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS–associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16–dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
Author Summary
TAR DNA Binding Protein 43 (TDP-43) is implicated in several human age-dependent neurodegenerative disorders, but until now little was known about TDP-43's role in the aging process. Here we used the nematode Caenorhabditis elegans to study the role of the TDP-43 orthologue tdp-1 in aging and neurodegeneration. In this study we discovered that tdp-1 is a stress-responsive gene acting within the Insulin/IGF signaling pathway to regulate lifespan and the response to oxidative stress. We found that, although worms missing tdp-1 were stress-sensitive, elevated expression of tdp-1 was toxic. We asked if tdp-1 also responded to the stress caused by toxic proteins found in Amyotrophic Lateral Sclerosis (ALS). Using worm models for ALS, we discovered that mutant TDP-43 generated oxidative stress and induced tdp-1 expression with negative consequences on neuronal function and lifespan. Consistently, removing tdp-1 rescued toxicity in our worm ALS models. tdp-1's role in the cellular stress response likely reflects an ancient adaptation to deal with unfavorable environmental conditions that is inappropriately activated and maintained by genetic mutations leading to proteotoxic and oxidative stress. We predict that similar mechanisms may exist in humans, helping explain the involvement of TDP-43 in a growing number of neurodegenerative disorders.
PMCID: PMC3390363  PMID: 22792076
19.  PAM50 assay and the three-gene model for identifying the major and clinically relevant molecular subtypes of breast cancer 
It has recently been proposed that a three-gene model (SCMGENE) that measures ESR1, ERBB2, and AURKA identifies the major breast cancer intrinsic subtypes and provides robust discrimination for clinical use in a manner very similar to a 50-gene subtype predictor (PAM50). However, the clinical relevance of both predictors was not fully explored, which is needed given that a ~30 % discordance rate between these two predictors was observed. Using the same datasets and subtype calls provided by Haibe-Kains and colleagues, we compared the SCMGENE assignments and the research-based PAM50 assignments in terms of their ability to (1) predict patient outcome, (2) predict pathological complete response (pCR) after anthracycline/taxane-based chemotherapy, and (3) capture the main biological diversity displayed by all genes from a microarray. In terms of survival predictions, both assays provided independent prognostic information from each other and beyond the data provided by standard clinical–pathological variables; however, the amount of prognostic information was found to be significantly greater with the PAM50 assay than the SCMGENE assay. In terms of chemotherapy response, the PAM50 assay was the only assay to provide independent predictive information of pCR in multivariate models. Finally, compared to the SCMGENE predictor, the PAM50 assay explained a significantly greater amount of gene expression diversity as captured by the two main principal components of the breast cancer microarray data. Our results show that classification of the major and clinically relevant molecular subtypes of breast cancer are best captured using larger gene panels.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-012-2143-0) contains supplementary material, which is available to authorized users.
PMCID: PMC3413822  PMID: 22752290
Breast cancer; Microarrays; PAM50; Prognosis; Gene expression
20.  Study protocol: The registrar clinical encounters in training (ReCEnT) study 
BMC Family Practice  2012;13:50.
Patient encounters are the core learning activity of Australian general practice (family practice) training. Exposure to patient demographics and presentations may vary from one general practice registrar (vocational trainee) to another. This can affect comprehensiveness of training. Currently, there is no mechanism to systematically capture the content of GP registrar consultations. The aim of the Registrar Clinical Encounters in Training (ReCEnT) study is to document longitudinally the nature and associations of consultation-based clinical and educational experiences of general practice registrars.
This is an ongoing prospective multi-site cohort study of general practice registrars’ consultations, entailing paper-based recording of consultation data. The study setting is general practices affiliated with three geographically-based Australian general practice regional training providers. Registrars record details of 60 consecutive consultations. Data collected includes registrar demographics, details of the consultation, patient demographics, reasons for encounter and problems managed. Problems managed are coded with the International Classification of Primary Care (second edition) classification system. Additionally, registrars record educational factors related to the encounter. The study will follow the clinical exposure of each registrar six-monthly over the 18 months to two years (full-time equivalent) of their general practice training program.
The study will provide data on a range of factors (patient, registrar and consultation factors). This data will be used to inform a range of educational decisions as well as being used to answer educational research questions. We plan to use ReCEnT as a formative assessment tool for registrars and help identify and address educational needs. The study will facilitate program evaluation by the participating training providers and thus improve articulation of educational programs with practice experience. From the research point of view it will address an evidence gap – the in-practice clinical and educational experience of general practice trainees, determinants of these experiences, and the determinants of registrars’ patterns of practice (for example, prescribing practice) over the course of their training.
PMCID: PMC3507666  PMID: 22672139
21.  Concordance among gene expression-based predictors for ER-positive breast cancer treated with adjuvant tamoxifen 
Annals of Oncology  2012;23(11):2866-2873.
ER-positive (ER+ ) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. In this study, we evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER+ tumors treated with adjuvant tamoxifen only.
Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (OncotypeDX), Mammaprint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapse-free survival (DRFS) was estimated by Kaplan–Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance.
All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, OncotypeDX, Mammaprint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%–100%).
Most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative disease who might be considered suitable candidates for adjuvant endocrine therapy alone.
PMCID: PMC3477878  PMID: 22532584
breast cancer; genomics; luminal; mammaprint; oncotype; PAM50
22.  Large-scale functional RNAi screen in C. elegans identifies genes that regulate the dysfunction of mutant polyglutamine neurons 
BMC Genomics  2012;13:91.
A central goal in Huntington's disease (HD) research is to identify and prioritize candidate targets for neuroprotective intervention, which requires genome-scale information on the modifiers of early-stage neuron injury in HD.
Here, we performed a large-scale RNA interference screen in C. elegans strains that express N-terminal huntingtin (htt) in touch receptor neurons. These neurons control the response to light touch. Their function is strongly impaired by expanded polyglutamines (128Q) as shown by the nearly complete loss of touch response in adult animals, providing an in vivo model in which to manipulate the early phases of expanded-polyQ neurotoxicity. In total, 6034 genes were examined, revealing 662 gene inactivations that either reduce or aggravate defective touch response in 128Q animals. Several genes were previously implicated in HD or neurodegenerative disease, suggesting that this screen has effectively identified candidate targets for HD. Network-based analysis emphasized a subset of high-confidence modifier genes in pathways of interest in HD including metabolic, neurodevelopmental and pro-survival pathways. Finally, 49 modifiers of 128Q-neuron dysfunction that are dysregulated in the striatum of either R/2 or CHL2 HD mice, or both, were identified.
Collectively, these results highlight the relevance to HD pathogenesis, providing novel information on the potential therapeutic targets for neuroprotection in HD.
PMCID: PMC3331833  PMID: 22413862
23.  National Athletic Trainers' Association Position Statement: Prevention of Pediatric Overuse Injuries 
Journal of Athletic Training  2011;46(2):206-220.
To provide certified athletic trainers, physicians, and other health care professionals with recommendations on best practices for the prevention of overuse sports injuries in pediatric athletes (aged 6–18 years).
Participation in sports by the pediatric population has grown tremendously over the years. Although the health benefits of participation in competitive and recreational athletic events are numerous, one adverse consequence is sport-related injury. Overuse or repetitive trauma injuries represent approximately 50% of all pediatric sport-related injuries. It is speculated that more than half of these injuries may be preventable with simple approaches.
Recommendations are provided based on current evidence regarding pediatric injury surveillance, identification of risk factors for injury, preparticipation physical examinations, proper supervision and education (coaching and medical), sport alterations, training and conditioning programs, and delayed specialization.
PMCID: PMC3070508  PMID: 21391806
adolescents; children; chronic injuries; microtrauma; growth; development
24.  Mutant TDP-43 and FUS Cause Age-Dependent Paralysis and Neurodegeneration in C. elegans 
PLoS ONE  2012;7(2):e31321.
Mutations in the DNA/RNA binding proteins TDP-43 and FUS are associated with Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration. Intracellular accumulations of wild type TDP-43 and FUS are observed in a growing number of late-onset diseases suggesting that TDP-43 and FUS proteinopathies may contribute to multiple neurodegenerative diseases. To better understand the mechanisms of TDP-43 and FUS toxicity we have created transgenic Caenorhabditis elegans strains that express full-length, untagged human TDP-43 and FUS in the worm's GABAergic motor neurons. Transgenic worms expressing mutant TDP-43 and FUS display adult-onset, age-dependent loss of motility, progressive paralysis and neuronal degeneration that is distinct from wild type alleles. Additionally, mutant TDP-43 and FUS proteins are highly insoluble while wild type proteins remain soluble suggesting that protein misfolding may contribute to toxicity. Populations of mutant TDP-43 and FUS transgenics grown on solid media become paralyzed over 7 to 12 days. We have developed a liquid culture assay where the paralysis phenotype evolves over several hours. We introduce C. elegans transgenics for mutant TDP-43 and FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.
PMCID: PMC3283630  PMID: 22363618
25.  Molecular Epidemiology and Phylogeny Reveal Complex Spatial Dynamics in Areas Where Canine Parvovirus Is Endemic ▿† 
Journal of Virology  2011;85(15):7892-7899.
Canine parvovirus type 2 (CPV-2) is a severe enteric pathogen of dogs, causing high mortality in unvaccinated dogs. After emerging, CPV-2 spread rapidly worldwide. However, there is now some evidence to suggest that international transmission appears to be more restricted. In order to investigate the transmission and evolution of CPV-2 both nationally and in relation to the global situation, we have used a long-range PCR to amplify and sequence the full VP2 gene of 150 canine parvoviruses obtained from a large cross-sectional sample of dogs presenting with severe diarrhea to veterinarians in the United Kingdom, over a 2-year period. Among these 150 strains, 50 different DNA sequence types (S) were identified, and apart from one case, all appeared unique to the United Kingdom. Phylogenetic analysis provided clear evidence for spatial clustering at the international level and for the first time also at the national level, with the geographical range of some sequence types appearing to be highly restricted within the United Kingdom. Evolution of the VP2 gene in this data set was associated with a lack of positive selection. In addition, the majority of predicted amino acid sequences were identical to those found elsewhere in the world, suggesting that CPV VP2 has evolved a highly fit conformation. Based on typing systems using key amino acid mutations, 43% of viruses were CPV-2a, and 57% CPV-2b, with no type 2 or 2c found. However, phylogenetic analysis suggested complex antigenic evolution of this virus, with both type 2a and 2b viruses appearing polyphyletic. As such, typing based on specific amino acid mutations may not reflect the true epidemiology of this virus. The geographical restriction that we observed both within the United Kingdom and between the United Kingdom and other countries, together with the lack of CPV-2c in this population, strongly suggests the spread of CPV within its population may be heterogeneously subject to limiting factors. This cross-sectional study of national and global CPV phylogeographic segregation reveals a substantially more complex epidemic structure than previously described.
PMCID: PMC3147911  PMID: 21593180

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