Medicine and Health Sciences Commons^™

Gene Expression Pattern Characterises Development Of Multiple Sclerosis, Lotti Tajouri, Ekua Brenu, Kevin Ashton, Don Staines, Sonja Marshall-Gradisnik

Kevin Ashton

No abstract provided.

Dna Barcoding Of Freshwater Fishes And The Development Of A Quantitative Qpcr Assay For The Species-Specific Detection And Quantification Of Fish Larvae From Plankton Samples, William Loh, P Bond, Kevin Ashton, David Roberts, I.R. Tibbetts

Kevin Ashton

The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0·52 ± 0·10 and 23·8 ± 2·20% respectively, indicating that barcodes can …

High-Throughput Sequencing Of Plasma Microrna In Chronic Fatigue Syndrome/Myalgic Encephalomyelitis, Ekua Brenu, Kevin Ashton, Jana Batovska, Don Staines, Sonja Marshall-Gradisnik

Kevin Ashton

Background: MicroRNAs (miRNAs) are known to regulate many biological processes and their dysregulation has been associated with a variety of diseases including Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME). The recent discovery of stable and reproducible miRNA in plasma has raised the possibility that circulating miRNAs may serve as novel diagnostic markers. The objective of this study was to determine the role of plasma miRNA in CFS/ME. Results: Using Illumina high-throughput sequencing we identified 19 miRNAs that were differentially expressed in the plasma of CFS/ME patients in comparison to non-fatigued controls. Following RT-qPCR analysis, we were able to confirm the significant up-regulation …

Heat Shock Proteins And Regulatory T Cells, Ekua Brenu, Don Staines, Lotti Tajouri, Teilah Huth, Kevin Ashton, Sonja Marshall-Gradisnik

Kevin Ashton

Heat shock proteins (HSPs) are important molecules required for ideal protein function. Extensive research on the functional properties of HSPs indicates that HSPs may be implicated in a wide range of physiological functions including immune function. In the immune system, HSPs are involved in cell proliferation, differentiation, cytokine release, and apoptosis. Therefore, the ability of the immune system, in particular immune cells, to function optimally and in unison with other physiological systems is in part dependent on signaling transduction processes, including bidirectional communication with HSPs. Regulatory T cells (Tregs) are important T cells with suppressive functions and impairments in their …

Natural Killer Cells In Patients With Severe Chronic Fatigue Syndrome, Ekua Brenu, Sharni Hardcastle, Gunn Atkinson, Mieke Van Driel, Sanne Kreijkamp-Kaspers, Kevin Ashton, Don Staines, Sonja Marshall-Gradisnik

Kevin Ashton

Maintenance of health and physiological homeostasis is a synergistic process involving tight regulation of proteins, transcription factors and other molecular processes. The immune system consists of innate and adaptive immune cells that are required to sustain immunity. The presence of pathogens and tumour cells activates innate immune cells, in particular Natural Killer (NK) cells. Stochastic expression of NK receptors activates either inhibitory or activating signals and results in cytokine production and activation of pathways that result in apoptosis of target cells. Thus, NK cells are a necessary component of the immunological process and aberrations in their functional processes, including equivocal …

Chronic Fatigue Syndrome/Myalgic Encephalomyelitis And Parallels With Autoimmune Disorders, Ekua Brenu, Lotti Tajouri, Kevin Ashton, Don Staines, Sonja Marshall-Gradisnik

Kevin Ashton

Autoimmune disorders are known to affect a substantial number of people worldwide and in some cases may be fatal. They occur in the presence of unregulated inflammatory responses including failure in self-tolerance. Some unexplained disorders with immune compromises may demonstrate certain characteristics that suggest an autoimmune disorderincluding Chronic Fatigue syndrome/Myalgic Encephalomyelitis (CFS/ME). CFS/ME remains an unsolved disorder with multiple symptoms and no single causative factor. Thesesymptoms may include but are not limited to incapacitating fatigue, weakened short term memory or attentiveness, sore throat, tender cervical or axillary lymph nodes, muscle pain, severe headaches, impaired sleep and postexertional malaise. To date …

Assessment Of Dna Degradation And The Genotyping Success Of Highly Degraded Samples, Sheree Hughes-Stamm, Kevin Ashton, Angela Van Daal

Kevin Ashton

DNA becomes progressively more fragmented as biological tissue degrades resulting in decreasing ability to gain a complete DNA profile. Successful identification of samples exhibiting very high levels of DNA degradation may be complicated by presenting in minute quantities. The industry standard method for human DNA identification utilising short tandem repeats (STR) may produce partial or no DNA profile with such samples. We report a comparative study of genotyping using STRs, mini-STRs and single nucleotide polymorphisms (SNPs) with template at different levels of degradation in varying amounts. Two methods of assessing quantity and quality of a DNA sample prior to genotyping …

Elasmobranch Qpcr Reference Genes: A Case Study Of Hypoxia Preconditioned Epaulette Sharks, Kalle T. Rytkönen, Gillian M. C. Renshaw, Kevin J. Ashton, Grant Williams-Pritchard, Erica H. Leder, Mikko Nikinmaa

Kevin Ashton

Background: Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined the suitability of 9 reference candidates from various functional categories after a single hypoxic insult or after hypoxia preconditioning in epaulette shark (Hemiscyllium ocellatum).Results: Epaulette sharks were caught and exposed to hypoxia. Tissues were collected from 10 controls, 10 individuals …

Unique Transcriptional Profile Of Sustained Ligand-Activated Preconditioning In Pre- And Post-Ischemic Myocardium, Kevin Ashton, Amanda Tupicoff, Grant Williams-Pritchard, Can Kiessling, Louise See Hoe, John Headrick, Jason Peart

Kevin Ashton

Background: Opioidergic SLP (sustained ligand-activated preconditioning) induced by 3–5 days of opioid receptor (OR) agonism induces persistent protection against ischemia-reperfusion (I-R) injury in young and aged hearts, and is mechanistically distinct from conventional preconditioning responses. We thus applied unbiased gene-array interrogation to identify molecular effects of SLP in pre- and post-ischemic myocardium. Methodology/Principal Findings: Male C57Bl/6 mice were implanted with 75 mg morphine or placebo pellets for 5 days. Resultant SLP did not modify cardiac function, and markedly reduced dysfunction and injury in perfused hearts subjected to 25 min ischemia/45 min reperfusion. Microarray analysis identified 14 up- and 86 down-regulated …

Cytotoxic Lymphocyte Micrornas As Prospective Biomarkers For Chronic Fatigue Syndrome/Myalgic Encephalomyelitis, Ekua Brenu, Kevin Ashton, Mieke Van Driel, Donald Staines, Daniel Peterson, Gunn Atkinson, Sonia Marshall-Gradisnika

Kevin Ashton

Background: Immune dysfunction associated with a disease often has a molecular basis. A novel group of molecules known as microRNAs (miRNAs) have been associated with suppression of translational processes involved in cellular development and proliferation, protein secretion, apoptosis, immune function and inflammatory processes. MicroRNAs may be implicated in Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME), where immune function is impaired. The objective of this study was to determine the association between miRNAs in cytotoxic cells and CFS/ME. Methods: Natural Killer (NK) and CD8+T cells were preferentially isolated from peripheral blood mononuclear cells from all participants (CFS/ME, n = 28; mean age = …

Voluntary Running In Mice Beneficially Modulates Myocardial Ischemic Tolerance, Signaling Kinases, And Gene Expression Patterns, Boris Budiono, Louise See Hoe, Jason Peart, Surendran Sabapathy, Kevin Ashton, Luke Haseler, John Headrick

Kevin Ashton

Exercise triggers hormesis, conditioning hearts against damaging consequences of subsequent ischemia-reperfusion (I/R). We test whether “low-stress” voluntary activity modifies I/R tolerance and molecular determinants of cardiac survival. Male C57BL/6 mice were provided 7-day access to locked (7SED) or rotating (7EX) running-wheels before analysis of cardiac prosurvival (Akt, ERK 1/2) and prodeath (GSK3β) kinases, transcriptomic adaptations, and functional tolerance of isolated hearts to 25-min ischemia/45-min reperfusion. Over 7 days, 7EX mice increased running from 2.1 ± 0.2 to 5.3 ± 0.3 km/day (mean speed 38 ± 2 m/min), with activity improving myocardial I/R tolerance: 7SED hearts recovered 43 ± 3% of …

Selective Effects Of Intrinsic A2aar Activity On Cardiac And Coronary Injuries With Lps Challenge, Melissa Reichelt, Kevin Ashton, S Mustafa, B Tang, Catherine Ledent, Xing Tan, John Headrick, R Morrison

Kevin Ashton

We assessed the impact of A2A adenosine receptor (A2AAR) knockout (KO) on LPS-triggered cardiovascular injuries, inflammation, gene expression and mortality. LPS precipitated cardiac injury, with 7-fold elevations in serum cardiac troponin I (cTnI) and 25–35% reductions in ventricular contractility. Coronary dysfunction was evident as a 20% reduction in reactive hyperaemic flows. A2AAR KO augmented cTnI release 3-fold without modifying ventricular dysfunction. Coronary effects of LPS and A2AAR KO were identical, and LPS no longer modified hyperaemia in A2AAR KO hearts. Effects of A2AAR activity were largely independent of shifts in acute phase reactants (CRP, haptoglobin) and circulating cytokines. Thus, up …

Cardiovascular Adenosine Receptors: Expression, Actions And Interactions, John Headrick, Kevin Ashton, Roselyn Rose'meyer, Jason Peart

Kevin Ashton

Intra- and extracellular adenosine levels rise in response to physiological stimuli and with metabolic/energetic perturbations, inflammatory challenge and tissue injury. Extracellular adenosine engages members of the G-protein coupled adenosine receptor (AR) family to mediate generally beneficial acute and adaptive responses within all constituent cells of the heart. In this way the four AR sub-types—A1, A2A, A2B, and A3Rs—regulate myocardial contraction, heart rate and conduction, adrenergic control, coronary vascular tone, cardiac and vascular growth, inflammatory–vascular cell interactions, and cellular stress-resistance, injury and death. The AR sub-types exert both distinct and overlapping effects, and may interact in mediating these cardiovascular responses. The …

Longitudinal Investigation Of Natural Killer Cells And Cytokines In Chronic Fatigue Syndrome/Myalgic Encephalomyelitis, Ekua Brenu, Mieke Van Driel, Donald Staines, Kevin Ashton, Sharni Hardcastle, James Keane, Lotti Tajouri, Daniel Peterson, Sandra Ramos, Sonya Marshall-Gradisnik

Kevin Ashton

Background: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is an etiologically unexplained disorder characterised by irregularities in various aspects of the immunological function. Presently, it is unknown whether these immunological changes remain consistent over time. This study investigates Natural Killer (NK) cell cytotoxic activity, NK cell subsets (CD56brightCD16- and CD56dimCD16+) and cytokines, over the course of a12 month period in patients with CFS/ME. Methods: The participants in the study comprised 65 (47.2 ± 11.5 years) CFS/ME participants and 21 (45.2 ±9.3 years) non-fatigued controls. Flow cytometry protocols were used to assess NK subsets and NK cytotoxic activity at various time points that …

The Adenosine A2a Receptor — Myocardial Protectant And Coronary Target In Endotoxemia, Melissa Reichelt, Kevin Ashton, Xing Tan, S Mustafa, Catherine Ledent, Lea Delbridge, Polly Hofmann, John Headrick, R Morrison

Kevin Ashton

Background: Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A2A adenosine receptor (A2AAR) activity during lipopolysaccharide (LPS)-induced inflammation.

Methods: We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A2AARs (A2AAR KO).

Results: Cardiac injury was evident in LPS-treated WTs, with ~ 7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A2AARs increased LPS-provoked cTnI release at 24 h by …

Immunological Abnormalities As Potential Biomarkers In Chronic Fatigue Syndrome/Myalgic Encephalomyelitis, Ekua Brenu, Mieke Van Driel, Don Staines, Kevin Ashton, Sandra Ramos, James Keane, Nancy Kilmas, Sonya Marshall-Gradisnik

Kevin Ashton

Background: Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) is characterized by severe prolonged fatigue, and decreases in cognition and other physiological functions, resulting in severe loss of quality of life, difficult clinical management and high costs to the health care system. To date there is no proven pathomechanism to satisfactorily explain this disorder. Studies have identified abnormalities in immune function but these data are inconsistent. We investigated the profile of markers of immune function (including novel markers) in CFS/ME patients.

Methods: We included 95 CFS/ME patients and 50 healthy controls. All participants were assessed on natural killer (NK) and CD8+T cell cytotoxic …

Immune And Hemorheological Changes In Chronic Fatigue Syndrome, Ekua W. Brenu, Donald R. Staines, Oguz K. Baskurt, Kevin J. Ashton, Sandra B. Ramos, Rhys M. Christy, Sonya M. Marshall-Gradisnik

Kevin Ashton

Background - Chronic Fatigue Syndrome (CFS) is a multifactorial disorder that affects various physiological systems including immune and neurological systems. The immune system has been substantially examined in CFS with equivocal results, however, little is known about the role of neutrophils and natural killer (NK) phenotypes in the pathomechanism of this disorder. Additionally the role of erythrocyte rheological characteristics in CFS has not been fully expounded. The objective of this present study was to determine deficiencies in lymphocyte function and erythrocyte rheology in CFS patients. Methods - Flow cytometric measurements were performed for neutrophil function, lymphocyte numbers, NK phenotypes (CD56dimCD16+ …

Modulation Of Ischaemic Contracture In Mouse Hearts: A 'Supraphysiological' Response To Adenosine, Melissa Reichelt, Laura Willems, Jason Peart, Kevin Ashton, G Matherne, Michael Blackburn, John Headrick

Kevin Ashton

While inhibition of ischaemic contracture was one of the first documented cardioprotective actions of exogenously applied adenosine, it is not known whether this is a normal function of endogenous adenosine generated during ischaemic stress. Additionally, the relevance of delayed contracture to postischaemic outcome is unclear. We tested the ability of endogenous versus exogenous adenosine to modify contracture (and postischaemic outcomes) in C57/Bl6 mouse hearts. During ischaemia, untreated hearts developed peak contracture (PC) of 85 ± 5 mmHg at 8.9 ± 0.8 min, with time to reach 20 mmHg (time to onset of contracture; TOC) of 4.4 ± 0.3 min. Adenosine …

Quantitative (Real-Time) Rt-Pcr In Cardiovascular Research, Kevin Ashton, John Headrick

Kevin Ashton

Quantitative (real-time) PCR (qPCR) represents a highly sensitive, sequence-specific, and reproducible technique for the gel-free detection and quantitation of nucleic acids. Owing to its large dynamic range and throughput, this approach has become the chosen method for rapid quantification of mRNA levels in biological samples. The sensitivity of this method permits the reliable detection of low concentrations of initial template, while delivering a linear range of up to 10 orders of magnitude in copy number. This chapter details the basic methodology behind key components of a qPCR experiment, including sample preparation, fluorescent chemistries, primer/probe design, and data analysis applicable to …