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1.  Mechanical Stretch-Induced Activation of ROS/RNS Signaling in Striated Muscle 
Antioxidants & Redox Signaling  2014;20(6):929-936.
Significance: Mechanical activation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) occurs in striated muscle and affects Ca2+ signaling and contractile function. ROS/RNS signaling is tightly controlled, spatially compartmentalized, and source specific. Recent Advances: Here, we review the evidence that within the contracting myocyte, the trans-membrane protein NADPH oxidase 2 (Nox2) is the primary source of ROS generated during contraction. We also review a newly characterized signaling cascade in cardiac and skeletal muscle in which the microtubule network acts as a mechanotransduction element that activates Nox2-dependent ROS generation during mechanical stretch, a pathway termed X-ROS signaling. Critical Issues: In the heart, X-ROS acts locally and affects the sarcoplasmic reticulum (SR) Ca2+ release channels (ryanodine receptors) and tunes Ca2+ signaling during physiological behavior, but excessive X-ROS can promote Ca2+-dependent arrhythmias in pathology. In skeletal muscle, X-ROS sensitizes Ca2+-permeable sarcolemmal “transient receptor potential” channels, a pathway that is critical for sustaining SR load during repetitive contractions, but when in excess, it is maladaptive in diseases such as Duchenne Musclar dystrophy. Future Directions: New advances in ROS/RNS detection as well as molecular manipulation of signaling pathways will provide critical new mechanistic insights into the details of X-ROS signaling. These efforts will undoubtedly reveal new avenues for therapeutic intervention in the numerous diseases of striated muscle in which altered mechanoactivation of ROS/RNS production has been identified. Antioxid. Redox Signal. 20, 929–936.
PMCID: PMC3924793  PMID: 23971496
2.  Sleep deprivation impairs recall of social transmission of food preference in rats 
Nature and Science of Sleep  2014;6:129-135.
Evidence indicates that sleep plays an important role in learning and memory, and disruption of sleep especially seems to interfere with hippocampal memory processes. Social transmission of food preference (STFP), a natural test of paired associative learning, has been shown to be dependent on the hippocampus. While social transmission of food preference is not a novel task, it has not been used to examine the role of sleep in memory consolidation. Male Sprague-Dawley rats were randomly divided into three groups: cage control; sleep-deprived; and device control. Demonstrator rats were given powdered food mixed with a target spice. Test rats then interacted with demonstrator rats before being given a two choice test of powered food with the target spice or a novel spice. Sleep-deprived rats were then placed in an automated device that prevented sleep for 24 hours. After sleep deprivation, animals were given a preference test again to determine memory for the target spice at both 24 hours and 72 hours. Polysomnography was used to validate the method of sleep deprivation. During immediate preference testing, rats demonstrated a clear preference for the food containing the target spice. Rats that experienced 24 hours of sleep deprivation following the initial testing indicated a significant reduction in the recall of the target spice at 24 and 72 hours. The cage control and device animals maintained their preference for food containing the target spice. Therefore, the loss of sleep interfered with memory consolidation for food preference learned via social transmission.
PMCID: PMC4226519  PMID: 25395874
hippocampus; learning; consolidation
3.  Should we offer routine hepatitis C antibody testing in men who have sex with men? 
Journal of the International AIDS Society  2014;17(4Suppl 3):19591.
There has been a significant rise in the number of HIV positive men who have sex with men (MSM) co-infected with hepatitis C (HCV). Most infections are thought to occur through high risk sexual practices, exacerbated by drug use. Previous data has suggested no need for routine screening in HIV negative MSM. We looked at HCV antibody testing and HCV risk assessment in all MSM clinic attenders as part of a Public Health England initiative.
Materials and Methods
Routine HCV antibody testing was offered to all MSM attending a large inner city sexual health clinic from April to June 2014. Patients were asked to fill in a questionnaire assessing HCV risk. Demographic data, HIV status and STI results were collected and compared.
We collected 471 HCV risk assessment questionnaires during the eight-week period. The median age was 34 (range 18–71) and 403 (85.6%) were White British. Ten (2.1%) patients were known to be HCV positive, of which 3 were on treatment and 5 (1.1%) had cleared HCV. One hundred and forty-nine (31.6%) patients were HIV negative, 254 (53.9%) were HIV positive and 68 (14.5%) had unknown HIV status at time of clinic visit. In the last three months 151 (32.1%) reported unprotected receptive anal intercourse, 58 (12.3%) reported group sex, 11 (2.3%) reported receptive fisting and 32 (6.8%) reported more than 10 partners. Eighty-seven (18.5%) patients had shared notes/straws to snort drugs and 29 (6.2%) reported injecting drugs or slamming. One hundred and forty-two (30.0%) patients reported recreational drug use in the last 12 months, with cocaine, methadrone and ketamine most popular. One hundred and fifteen (24.4%) patients reported sex under the influence of recreational drugs. There were no statistical differences between HIV positive and HIV negative patients in their risk, sexual behaviour and drug use. STI screens were performed on 269 patients with nine (3.3%) new HIV diagnoses, four (1.5%) early syphilis, and 28 (10.4%) rectal gonorrhoea infections. There were three (1.1%) new HCV diagnoses, and one (33.3%) was in an HIV negative patient.
Our results show increased risk behaviour for both HIV positive and HIV negative MSM. There are a high number of patients using party drugs, participating in group sex and not using condoms, leading to high rates of new STI diagnoses. With similar rates of risk we believe HCV testing and risk assessment should be considered in all MSM regardless of HIV status.
PMCID: PMC4224777  PMID: 25394096
4.  Experience of acute hepatitis C and HIV co-infection in an inner city clinic in the UK 
Journal of the International AIDS Society  2014;17(4Suppl 3):19639.
Acute hepatitis C infection (HCV) is increasing in the HIV-infected population, particularly among men who have sex with men (MSM). Patients co-infected with HCV and HIV progress more rapidly to liver cirrhosis and are at higher risk of hepatocellular carcinoma. We looked at our management of acute HCV to assess treatment outcome.
Materials and Methods
We performed a retrospective and prospective case note review of HIV-HCV co-infected patients attending a large inner city sexual health clinic from 2006-to date. Acute HCV infections (less than six months) were identified and data was collected on demographics, transmission and treatment outcomes. Treatment regime was 48 weeks of weight-based ribavirin and pegylated interferon α2a.
Sixty-seven acute HCV infections were identified among 142 co-infected patients, all of whom were male and 66 (98.5%) were MSM. Median age at diagnosis was 37 (range 20–59) and 58 (86.6%) were White British. Sixty patients (89.6%) were genotype 1, 3 (4.5%) were genotype 4 and 2 (3.0%) were genotype 2/3. A further 2 (3.0%) were re-infections. A peak in new HCV diagnoses was seen in 2013 with 17 (25.4%). Route of transmission was sexual in all cases with 13 (19.4%) also injecting drugs, pointing to mixed transmission routes. Nine (69.2%) of these occurred in 2013. Nine (13.4%) patients cleared HCV themselves. Of the 58 who didn't clear HCV, 12 (20.7%) were lost to follow up/transferred care, 4 (6.9%) declined treatment awaiting newer agents, and 10 (17.2%) are waiting to start. A total of 32 patients started treatment. Six (18.8%) patients are currently on treatment and three (9.4%) await a final sustained virological response (SVR) test. Six out of twenty-four (25.0%) stopped treatment due to lack of response and 1 stopped due to side effects. Fifteen (62.5%) achieved SVR and 2 (8.3%) failed to achieve SVR. Eight out of ten (80.0%) patients who had an early virological response (EVR) achieved SVR.
Our data shows good treatment outcomes for acute HCV infection in HIV patients with an SVR rate of 62.5%. We've seen a steady increase in acute HCV infection, particularly in MSM injecting party drugs. Changing risk behaviours, particularly a rise in chem sex parties and club drug use, along with more anonymous partners and disclosure issues create difficulties in managing the HCV epidemic. More education is needed to raise awareness of HCV transmission and disclosure in our MSM population.
PMCID: PMC4224802  PMID: 25394143
5.  Subfractionation, characterization and in-depth proteomic analysis of glomerular membrane vesicles in human urine 
Kidney international  2013;85(5):1225-1237.
Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.
PMCID: PMC4008663  PMID: 24196483
Exosome; proteomics; podocyte; glomerular disease; Integrin; actin cytoskeleton; Rho GDI. Q-Exactive mass spectrometer
7.  Insulinotropic treatments exacerbate metabolic syndrome in mice lacking MeCP2 function 
Human Molecular Genetics  2013;22(13):2626-2633.
Rett syndrome (RTT), an X-linked postnatal disorder, results from mutations in Methyl CpG-binding protein 2 (MECP2). Survival and breathing in Mecp2NULL/Y animals are improved by an N-terminal tripeptide of insulin-like growth factor I (IGF-I) treatment. We determined that Mecp2NULL/Y animals also have a metabolic syndrome and investigated whether IGF-I treatment might improve this phenotype. Mecp2NULL/Y mice were treated with a full-length IGF-I modified with the addition of polyethylene glycol (PEG-IGF-I), which improves pharmacological properties. Low-dose PEG-IGF-I treatment slightly improved lifespan and heart rate in Mecp2NULL/Y mice; however, high-dose PEG-IGF-I decreased lifespan. To determine whether insulinotropic off-target effects of PEG-IGF-I caused the detrimental effect, we treated Mecp2NULL/Y mice with insulin, which also decreased lifespan. Thus, the clinical benefit of IGF-I treatment in RTT may critically depend on the dose used, and caution should be taken when initiating clinical trials with these compounds because the beneficial therapeutic window is narrow.
PMCID: PMC3674803  PMID: 23462290
8.  Recovery of altered neuromuscular junction morphology and muscle function in mdx mice after injury 
Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease in which weakness, increased susceptibility to muscle injury, and inadequate repair underlie the pathology. While most attention has focused within the muscle fiber, we recently demonstrated significant alterations in the neuromuscular junction (NMJ) morphology and resulting neuromuscular transmission failure (NTF) 24 h after injury in mdx mice (murine model for DMD). Here we determine the contribution of NMJ morphology and NTF to the recovery of muscle contractile function post-injury. NMJ morphology and NTF rates were assessed day 0 (immediately after injury) and days 1, 7, 14 and 21 after quadriceps injury. Eccentric injury of the quadriceps resulted in a significant loss of maximal torque in both WT (39 ± 6 %) and mdx (76 ± 8 %) with a full recovery in WT by day 7 and in mdx by day 21. Post-injury alterations in NMJ morphology and NTF were found only in mdx, were limited to days 0 and 1, and were independent of changes in MuSK or AChR expression. Such early changes at the NMJ after injury are consistent with mechanical disruption rather than newly forming NMJs. Furthermore, we show that the dense microtubule network that underlies the NMJ is significantly reduced and disorganized in mdx compared to WT. These structural changes at the NMJ may play a role in the increased NMJ disruption and the exaggerated loss of nerve-evoked muscle force seen after injury to dystrophic muscles.
Electronic supplementary material
The online version of this article (doi:10.1007/s00018-014-1663-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4282693  PMID: 24947322
mdx; NMJ; Muscular dystrophy; Eccentric injury; MuSK; Microtubules
9.  Purification of Exosome-like Vesicles from Urine 
Methods in enzymology  2013;524:225-241.
Urinary exosome-like vesicles (ELVs), 20–200nm membrane bound particles shed by renal epithelium, are emerging as an important source of protein, mRNA, and miRNA biomarkers to monitor renal disease. However, purification of ELVs is compromised by the presence of large amounts of the urinary protein Tamm-Horsfall Protein (THP). THP molecules oligomerize into long, double-helical strands several microns long. These linear assemblies form a 3-dimensional gel which traps and sequesters ELVs in any centrifugation based protocol. Here we present a purification protocol that separates ELVs from THP and divides urinary ELVs into three distinct populations.
PMCID: PMC4028690  PMID: 23498743
10.  Endothelial outgrowth cells shift macrophage phenotype and improve kidney viability in swine renal artery stenosis 
Endothelial outgrowth cells (EOC) decrease inflammation and improve endothelial repair. Inflammation aggravates kidney injury in renal artery stenosis (RAS), and may account for its persistence upon revascularization. We hypothesized that EOC would decrease inflammatory (M1) macrophages and improve renal recovery in RAS.
Approach and Results
Pigs with 10 weeks of RAS were studied 4 weeks after percutaneous transluminal renal angioplasty (PTRA+stenting) or sham, with or without adjunct intra-renal delivery of autologous EOC (10×10^6), and compared to similarly-treated normal controls (n=7 each). Single-kidney function, microvascular and tissue remodeling, inflammation, oxidative stress, and fibrosis were evaluated. Four weeks after PTRA, EOC engrafted in injected RAS-kidneys. Stenotic-kidney glomerular filtration rate was restored in RAS+EOC, RAS+PTRA, and RAS+PTRA+EOC pigs, while stenotic-kidney blood flow and angiogenesis were improved and fibrosis attenuated only in EOC-treated pigs. Furthermore, EOC increased cell proliferation and decreased the ratio of M1 (inflammatory)/M2 (reparative) macrophages, as well as circulating levels and stenotic-kidney release of inflammatory cytokines. Cultured-EOC released microvesicles in-vitro and induced phenotypic switch (M1-to-M2) in cultured monocytes, which was inhibited by VEGF blockade. Finally, a single intra-renal injection of rhVEGF (0.05 μg/kg) in 7 additional RAS pigs also restored M1/M2 ratio 4 weeks later.
Intra-renal infusion of EOC after PTRA induced a VEGF-mediated attenuation in macrophages inflammatory phenotype, preserved microvascular architecture and function, and decreased inflammation and fibrosis in the stenotic kidney, suggesting a novel mechanism and therapeutic potential for adjunctive EOC delivery in experimental RAS to improve PTRA outcomes.
PMCID: PMC3644980  PMID: 23430615
renal artery stenosis; progenitor cells; kidney hypertension; revascularization; macrophages
11.  X-ROS signaling in heart and skeletal muscle: stretch-dependent local ROS regulates [Ca2+]i 
X-ROS signaling is a novel redox signaling pathway that links mechanical stress to changes in [Ca2+]i. This pathway is activated rapidly and locally within a muscle cell under physiological conditions, but can also contribute to Ca2+-dependent arrhythmia in heart and to the dystrophic phenotype in heart and skeletal muscle. Upon physiologic cellular stretch, microtubules serve as mechanotransducers to activate NADPH oxidase 2 in the transverse tubules and sarcolemmal membranes to produce reactive oxygen species (ROS). In heart, the ROS acts locally to activate ryanodine receptor Ca2+ release channels in the junctional sarcoplasmic reticulum, increasing the Ca2+ spark rate and “tuning” excitation-contraction coupling. In skeletal muscle, where Ca2+ sparks are not normally observed, the X-ROS signaling process is muted. However in muscular dystrophies, such as Duchenne Muscular Dystrophy and dysferlinopathy, X-ROS signaling operates at a high level and contributes to myopathy. Importantly, Ca2+ permeable stretch-activated channels are activated by X-ROS and contribute to skeletal muscle pathology. Here we review X-ROS signaling and mechanotransduction in striated muscle, and highlight important questions to drive future work on stretch-dependent signaling. We conclude that X-ROS provides an exciting mechanism for the mechanical control of redox and Ca2+ signaling, but much work is needed to establish its contribution to physiologic and pathophysiologic processes in diverse cell systems.
PMCID: PMC3951390  PMID: 23220288
12.  X-ROS signalling is enhanced and graded by cyclic cardiomyocyte stretch 
Cardiovascular Research  2013;98(2):307-314.
A sustained, single stretch of a cardiomyocyte activates a transient production of reactive oxygen species by membrane-located NADPH oxidase 2 (Nox2). This NoX2-dependent ROS (X-ROS) tunes cardiac Ca2+ signalling by reversibly sensitizing sarcoplasmic reticulum Ca2+ release channels. In contrast to static length changes, working heart cells are cyclically stretched and shortened in the living animal. Additionally, this stretch cycle is constantly varied by changes in the pre-load and heart rate. Thus, the objective of this study was (i) to characterize X-ROS signalling during stretch-shortening cycles and (ii) to evaluate how the amplitude (pre-load) and frequency (heart rate) of cell stretch affects X-ROS and Ca2+ signalling.
Methods and results
Single adult rat cardiomyocytes were attached to MyoTak™-coated micro-rods and stretched, while ROS production and Ca2+ signals were monitored optically. Although a sustained stretch led to only a transient burst of ROS, cyclic stretch-shortening cycles led to a steady-state elevation of ROS production. Importantly, this new redox state was graded by both the amplitude of stretch (3–15%) and cycle frequency (1–4 Hz). Elevated ROS production enhanced Ca2+ signalling sensitivity as measured by the Ca2+ spark rate.
The steady-state level of ROS production in a cardiomyocyte is graded by the amplitude and frequency of cell stretch. Thus, mechanical changes that depend on the pre-load and heart rate regulate a dynamic redox balance that tunes cellular Ca2+ signalling.
PMCID: PMC3633162  PMID: 23524301
Stretch; ROS; NADPH oxidase; Calcium sparks; Nox2
13.  Familial Alzheimer’s disease modelling using induced pluripotent stem cell technology 
World Journal of Stem Cells  2014;6(2):239-247.
Alzheimer’s disease (AD) is a progressive neurodegenerative disease in which patients exhibit gradual loss of memory that impairs their ability to learn or carry out daily tasks. Diagnosis of AD is difficult, particularly in early stages of the disease, and largely consists of cognitive assessments, with only one in four patients being correctly diagnosed. Development of novel therapeutics for the treatment of AD has proved to be a lengthy, costly and relatively unproductive process with attrition rates of > 90%. As a result, there are no cures for AD and few treatment options available for patients. Therefore, there is a pressing need for drug discovery platforms that can accurately and reproducibly mimic the AD phenotype and be amenable to high content screening applications. Here, we discuss the use of induced pluripotent stem cells (iPSCs), which can be derived from adult cells, as a method of recapitulation of AD phenotype in vitro. We assess their potential use in high content screening assays and the barriers that exist to realising their full potential in predictive efficacy, toxicology and disease modelling. At present, a number of limitations need to be addressed before the use of iPSC technology can be fully realised in AD therapeutic applications. However, whilst the use of AD-derived iPSCs in drug discovery remains a fledgling field, it is one with immense potential that is likely to reach fruition within the next few years.
PMCID: PMC3999781  PMID: 24772250
Human induced pluripotent stem cells; Alzheimer’s disease; Neurodegenerative diseases; High-throughput screening assays; Cholinergic neurons; Drug discovery; Stratified medicine
14.  Human MX2 is an interferon-induced post-entry inhibitor of HIV-1 infection 
Nature  2013;502(7472):10.1038/nature12542.
Animal cells harbour multiple innate effector mechanisms that inhibit virus replication. For the pathogenic retrovirus human immunodeficiency virus type-1 (HIV-1), these include widely expressed restriction factors1 such as APOBEC3 proteins2, TRIM5α3, tetherin/BST24,5 and SAMHD16,7, as well as additional factors that are stimulated by type-1 interferon (IFN)8,9,10,11,12,13,14. Here, we employ both ectopic expression and gene silencing experiments to define the human dynamin-like, IFN-induced guanosine triphosphatase (GTPase), myxovirus resistance 2 (MX2 or MxB) protein, as a potent inhibitor of HIV-1 infection and as a major effector of IFNα-mediated resistance to HIV-1 infection. MX2 suppresses infection by all HIV-1 strains tested, has similar to modest effects on divergent simian immunodeficiency viruses (SIVs), and does not inhibit other retroviruses such as murine leukaemia virus (MLV). The capsid (CA) region of the viral Gag protein dictates susceptibility to MX2, and the block to infection occurs at a late post-entry step with the nuclear accumulation and chromosomal integration of nascent viral cDNA both being suppressed. Finally, human MX1 (or MxA), a closely related protein that has long been recognised as a broadly acting inhibitor of RNA/DNA viruses, including the orthomyxovirus influenza A virus15,16, does not affect HIV-1,whereas MX2 is ineffective against influenza virus. MX2 is therefore a cell-autonomous, anti-HIV-1 resistance factor whose purposeful mobilisation may represent a new therapeutic approach for the treatment of HIV/AIDS.
PMCID: PMC3808269  PMID: 24048477
15.  Dysferlin at transverse tubules regulates Ca2+ homeostasis in skeletal muscle 
The class of muscular dystrophies linked to the genetic ablation or mutation of dysferlin, including Limb Girdle Muscular Dystrophy 2B (LGMD2B) and Miyoshi Myopathy (MM), are late-onset degenerative diseases. In lieu of a genetic cure, treatments to prevent or slow the progression of dysferlinopathy are of the utmost importance. Recent advances in the study of dysferlinopathy have highlighted the necessity for the maintenance of calcium handling in altering or slowing the progression of muscular degeneration resulting from the loss of dysferlin. This review highlights new evidence for a role for dysferlin at the transverse (t-) tubule of striated muscle, where it is involved in maintaining t-tubule structure and function.
PMCID: PMC3944681  PMID: 24639655
muscular dystrophy; calcium; excitation-contraction coupling; myopathy
16.  Genetic silencing of Nrf2 enhances X-ROS in dysferlin-deficient muscle 
Oxidative stress is a critical disease modifier in the muscular dystrophies. Recently, we discovered a pathway by which mechanical stretch activates NADPH Oxidase 2 (Nox2) dependent ROS generation (X-ROS). Our work in dystrophic skeletal muscle revealed that X-ROS is excessive in dystrophin-deficient (mdx) skeletal muscle and contributes to muscle injury susceptibility, a hallmark of the dystrophic process. We also observed widespread alterations in the expression of genes associated with the X-ROS pathway and redox homeostasis in muscles from both Duchenne muscular dystrophy patients and mdx mice. As nuclear factor erythroid 2-related factor 2 (Nrf2) plays an essential role in the transcriptional regulation of genes involved in redox homeostasis, we hypothesized that Nrf2 deficiency may contribute to enhanced X-ROS signaling by reducing redox buffering. To directly test the effect of diminished Nrf2 activity, Nrf2 was genetically silenced in the A/J model of dysferlinopathy—a model with a mild histopathologic and functional phenotype. Nrf2-deficient A/J mice exhibited significant muscle-specific functional deficits, histopathologic abnormalities, and dramatically enhanced X-ROS compared to control A/J and WT mice, both with functional Nrf2. Having identified that reduced Nrf2 activity is a negative disease modifier, we propose that strategies targeting Nrf2 activation may address the generalized reduction in redox homeostasis to halt or slow dystrophic progression.
PMCID: PMC3928547  PMID: 24600403
Nrf2; X-ROS; ROS; dysferlin; dystrophy
17.  Mitochondrial oxygen consumption deficits in skeletal muscle isolated from an Alzheimer’s disease-relevant murine model 
BMC Neuroscience  2014;15:24.
Age is considered a primary risk factor for neurodegenerative diseases including Alzheimer’s disease (AD). It is also now well understood that mitochondrial function declines with age. Mitochondrial deficits have been previously assessed in brain from both human autopsy tissue and disease-relevant transgenic mice. Recently it has been recognized that abnormalities of muscle may be an intrinsic aspect of AD and might contribute to the pathophysiology. However, deficits in mitochondrial function have yet to be clearly assessed in tissues outside the central nervous system (CNS). In the present study, we utilized a well-characterized AD-relevant transgenic mouse strain to assess mitochondrial respiratory deficits in both brain and muscle. In addition to mitochondrial function, we assessed levels of transgene-derived amyloid precursor protein (APP) in homogenates isolated from brain and muscle of these AD-relevant animals.
We now demonstrate that skeletal muscles isolated from these animals have differential levels of mutant full-length APP depending on muscle type. Additionally, isolated muscle fibers from young transgenic mice (3 months) have significantly decreased maximal mitochondrial oxygen consumption capacity compared to non-transgenic, age-matched mice, with similar deficits to those previously described in brain.
This is the first study to directly examine mitochondrial function in skeletal muscle from an AD-relevant transgenic murine model. As with brain, these deficits in muscle are an early event, occurring prior to appearance of amyloid plaques.
PMCID: PMC3930757  PMID: 24524276
Alzheimer’s disease; Mitochondria; Amyloid plaque; Neurodegeneration; Muscle
18.  Female Mecp2+/− mice display robust behavioral deficits on two different genetic backgrounds providing a framework for pre-clinical studies 
Human Molecular Genetics  2012;22(1):96-109.
Rett syndrome (RTT) is an X-linked neurological disorder caused by mutations in the gene encoding the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2). Typical RTT primarily affects girls and is characterized by a brief period of apparently normal development followed by the loss of purposeful hand skills and language, the onset of anxiety, hand stereotypies, autistic features, seizures and autonomic dysfunction. Mecp2 mouse models have extensively been studied to demonstrate the functional link between MeCP2 dysfunction and RTT pathogenesis. However, the majority of studies have focused primarily on the molecular and behavioral consequences of the complete absence of MeCP2 in male mice. Studies of female Mecp2+/− mice have been limited because of potential phenotypic variability due to X chromosome inactivation effects. To determine whether reproducible and reliable phenotypes can be detected Mecp2+/− mice, we analyzed Mecp2+/− mice of two different F1 hybrid isogenic backgrounds and at young and old ages using several neurobehavioral and physiological assays. Here, we report a multitude of phenotypes in female Mecp2+/− mice, some presenting as early as 5 weeks of life. We demonstrate that Mecp2+/− mice recapitulate several aspects of typical RTT and show that mosaic expression of MeCP2 does not preclude the use of female mice in behavioral and molecular studies. Importantly, we uncover several behavioral abnormalities that are present in two genetic backgrounds and report on phenotypes that are unique to one background. These findings provide a framework for pre-clinical studies aimed at improving the constellation of phenotypes in a mouse model of RTT.
PMCID: PMC3522402  PMID: 23026749
19.  Practical management of patients on apixaban: a consensus guide 
Thrombosis Journal  2013;11:27.
Atrial fibrillation (AF) is a common tachyarrhythmia in Australia, with a prevalence over 10% in older patients. AF is the leading preventable cause of ischaemic stroke, and strokes due to AF have a higher mortality and morbidity. Stroke prevention is therefore a key management strategy for AF patients, in addition to rate and rhythm control. Anticoagulation with warfarin has been an enduring gold standard for stroke prevention in NVAF patients. In Australia, three novel oral anticoagulants (NOACs), apixaban, dabigatran and rivaroxaban are now approved and reimbursed for stroke prevention in patients with non-valvular AF (NVAF). International European Cardiology guidelines now recommend either a NOAC or warfarin for NVAF patients with a CHA2DS2-VASc score ≥2, unless contraindicated. Apixaban is a direct factor Xa inhibitor with a 12-hour half-life and 25% renal excretion that was found in a large trial of NVAF patients to be superior to warfarin in preventing stroke or systemic embolism. In this trial population, apixaban also resulted in less bleeding and a lower mortality rate than warfarin.
Clinical experience with apixaban outside of clinical trials has been limited, and there is currently little evidence to guide the management of bleeding or invasive procedures in patients taking apixaban. The relevant currently available animal and ex vivo human data were collected, analyzed and summarized.
This multi-disciplinary consensus statement has been written to serve as a guide for healthcare practitioners prescribing apixaban in Australia, with a focus on acute and emergency management.
The predictable pharmacokinetics and minimal drug interactions of apixaban should allow for safe anticoagulation in the majority of patients, including temporary interruption for elective procedures. In the absence of published data, patients actively bleeding on apixaban should receive standard supportive treatment. Quantitative assays of apixaban level such as chromogenic anti-Xa assays are becoming available but their utility is unproven in this setting. Specific antidotes for novel anticoagulants, including apixaban, are in clinical development.
PMCID: PMC3904756  PMID: 24380488
Apixaban; Novel oral anticoagulants; Bleeding; Perioperative management
20.  Dramatic Variability of the Carbonate System at a Temperate Coastal Ocean Site (Beaufort, North Carolina, USA) Is Regulated by Physical and Biogeochemical Processes on Multiple Timescales 
PLoS ONE  2013;8(12):e85117.
Increasing atmospheric carbon dioxide (CO2) from anthropogenic sources is acidifying marine environments resulting in potentially dramatic consequences for the physical, chemical and biological functioning of these ecosystems. If current trends continue, mean ocean pH is expected to decrease by ~0.2 units over the next ~50 years. Yet, there is also substantial temporal variability in pH and other carbon system parameters in the ocean resulting in regions that already experience change that exceeds long-term projected trends in pH. This points to short-term dynamics as an important layer of complexity on top of long-term trends. Thus, in order to predict future climate change impacts, there is a critical need to characterize the natural range and dynamics of the marine carbonate system and the mechanisms responsible for observed variability. Here, we present pH and dissolved inorganic carbon (DIC) at time intervals spanning 1 hour to >1 year from a dynamic, coastal, temperate marine system (Beaufort Inlet, Beaufort NC USA) to characterize the carbonate system at multiple time scales. Daily and seasonal variation of the carbonate system is largely driven by temperature, alkalinity and the balance between primary production and respiration, but high frequency change (hours to days) is further influenced by water mass movement (e.g. tides) and stochastic events (e.g. storms). Both annual (~0.3 units) and diurnal (~0.1 units) variability in coastal ocean acidity are similar in magnitude to 50 year projections of ocean acidity associated with increasing atmospheric CO2. The environmental variables driving these changes highlight the importance of characterizing the complete carbonate system rather than just pH. Short-term dynamics of ocean carbon parameters may already exert significant pressure on some coastal marine ecosystems with implications for ecology, biogeochemistry and evolution and this shorter term variability layers additive effects and complexity, including extreme values, on top of long-term trends in ocean acidification.
PMCID: PMC3866137  PMID: 24358377
21.  Polyploidy in myelofibrosis: analysis by cytogenetic and SNP array indicates association with advancing disease 
Myelofibrosis occurs as primary myelofibrosis or as a late occurrence in the evolution of essential thrombocythaemia and polycythaemia vera. It is the rarest of the three classic myeloproliferative neoplasms (MPN). Polyploidy has only rarely been reported in MPN despite the prominent involvement of abnormal megakaryocytes. The use of peripheral blood samples containing increased numbers of haematopoietic progenitors has improved the output from cytogenetic studies in myelofibrosis and together with the use of single nucleotide polymorphism arrays (SNPa) has contributed to an improved knowledge regarding the diverse genetic landscape of this rare disease.
Cytogenetic studies performed on a consecutive cohort of 42 patients with primary or post ET/PV myelofibrosis showed an abnormal karyotype in 24 cases and of these, nine showed a polyploid clone. Six of the nine cases showed a tetraploid (4n) subclone, whereas three showed mixed polyploid subclones with both tetraploid and octoploid (4n/8n) cell lines. The abnormal clone evolved from a near diploid karyotype at the initial investigation to a tetraploid karyotype in follow-up cytogenetic analysis in four cases. In total, six of the nine polyploid cases showed gain of 1q material. The remaining three cases showed polyploid metaphases, but with no detectable structural karyotypic rearrangements. Three of the nine cases showed chromosome abnormalities of 6p, either at diagnosis or later acquired. SNPa analysis on eight polyploid cases showed additional changes not previously recognised by karyotype analysis alone, including recurring changes involving 9p, 14q, 17q and 22q. Except for gain of 1q, SNPa findings from the polyploid group compared to eight non-polyploid cases with myelofibrosis found no significant differences in the type of abnormality detected.
The study showed the use of peripheral blood samples to be suitable for standard karyotyping evaluation and DNA based studies. The overall profile of abnormalities found were comparable with that of post-MPN acute myeloid leukaemia or secondary myelodysplastic syndrome and cases in the polyploidy group were associated with features of high risk disease. The above represents the first documented series of polyploid karyotypes in myelofibrosis and shows a high representation of gain of 1q.
PMCID: PMC3906908  PMID: 24341401
Gain of 1q; Myelofibrosis; Tetraploidy; Polyploidy; SNP array
22.  Microtubules Underlie Dysfunction in Duchenne Muscular Dystrophy 
Science signaling  2012;5(236):10.1126/scisignal.2002829.
Duchenne muscular dystrophy (DMD) is a fatal X-linked degenerative muscle disease caused by the absence of the microtubule-associated protein dystrophin, which results in a disorganized and denser microtubule cytoskeleton. In addition, mechanotransduction-dependent activation of calcium (Ca2+) and reactive oxygen species (ROS) signaling underpins muscle degeneration in DMD. We show that in muscle from adult mdx mice, a model of DMD, a brief physiologic stretch elicited microtubule-dependent activation of NADPH (reduced-form nicotinamide adenine dinucleotide phosphate) oxidase–dependent production of ROS, termed X-ROS. Further, X-ROS amplified Ca2+ influx through stretch-activated channels in mdx muscle. Consistent with the importance of the microtubules to the dysfunction in mdx muscle, muscle cells with dense microtubule structure, such as those from adult mdx mice or from young wild-type mice treated with Taxol, showed increased X-ROS production and Ca2+ influx, whereas cells with a less dense microtubule network, such as young mdx or adult mdx muscle treated with colchicine or nocodazole, showed little ROS production or Ca2+ influx. In vivo treatments that disrupted the microtubule network or inhibited NADPH oxidase 2 reduced contraction-induced injury in adult mdx mice. Furthermore, transcriptome analysis identified increased expression of X-ROS–related genes in human DMD skeletal muscle. Together, these data show that microtubules are the proximate element responsible for the dysfunction in Ca2+ and ROS signaling in DMD and could be effective therapeutic targets for intervention.
PMCID: PMC3835660  PMID: 22871609
23.  Breathing challenges in Rett Syndrome: Lessons learned from humans and animal models✩,✩✩ 
Respiratory physiology & neurobiology  2013;189(2):10.1016/j.resp.2013.06.022.
Breathing disturbances are a major challenge in Rett Syndrome (RTT). These disturbances are more pronounced during wakefulness; but irregular breathing occurs also during sleep. During the day patients can exhibit alternating bouts of hypoventilation and irregular hyperventilation. But there is significant individual variability in severity, onset, duration and type of breathing disturbances. Research in mouse models of RTT suggests that different areas in the ventrolateral medulla and pons give rise to different aspects of this breathing disorder. Pre-clinical experiments in mouse models that target different neuromodulatory and neurotransmitter receptors and MeCP2 function within glia cells can partly reverse breathing abnormalities. The success in animal models raises optimism that one day it will be possible to control or potentially cure the devastating symptoms also in human patients with RTT.
PMCID: PMC3812329  PMID: 23816600
Pre-Boetzinger Complex; Mecp2; dysautonomia
24.  Elevated Paracellular Glucose Flux across Cystic Fibrosis Airway Epithelial Monolayers Is an Important Factor for Pseudomonas aeruginosa Growth 
PLoS ONE  2013;8(10):e76283.
People with cystic fibrosis (CF) who develop related diabetes (CFRD) have accelerated pulmonary decline, increased infection with antibiotic-resistant Pseudomonas aeruginosa and increased pulmonary exacerbations. We have previously shown that glucose concentrations are elevated in airway surface liquid (ASL) of people with CF, particularly in those with CFRD. We therefore explored the hypotheses that glucose homeostasis is altered in CF airway epithelia and that elevation of glucose flux into ASL drives increased bacterial growth, with an effect over and above other cystic fibrosis transmembrane conductance regulator (CFTR)-related ASL abnormalities. The aim of this study was to compare the mechanisms governing airway glucose homeostasis in CF and non-CF primary human bronchial epithelial (HBE) monolayers, under normal conditions and in the presence of Ps. aeruginosa filtrate. HBE-bacterial co-cultures were performed in the presence of 5 mM or 15 mM basolateral glucose to investigate how changes in blood glucose, such as those seen in CFRD, affects luminal Ps. aeruginosa growth. Calu-3 cell monolayers were used to evaluate the potential importance of glucose on Ps. aeruginosa growth, in comparison to other hallmarks of the CF ASL, namely mucus hyperviscosity and impaired CFTR-dependent fluid secretions. We show that elevation of basolateral glucose promotes the apical growth of Ps. aeruginosa on CF airway epithelial monolayers more than non-CF monolayers. Ps. aeruginosa secretions elicited more glucose flux across CF airway epithelial monolayers compared to non-CF monolayers which we propose increases glucose availability in ASL for bacterial growth. In addition, elevating basolateral glucose increased Ps. aeruginosa growth over and above any CFTR-dependent effects and the presence or absence of mucus in Calu-3 airway epithelia-bacteria co-cultures. Together these studies highlight the importance of glucose as an additional factor in promoting Ps. aeruginosa growth and respiratory infection in CF disease.
PMCID: PMC3790714  PMID: 24124542
25.  Surface Glycosylation Profiles of Urine Extracellular Vesicles 
PLoS ONE  2013;8(9):e74801.
Urinary extracellular vesicles (uEVs) are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.
PMCID: PMC3777961  PMID: 24069349

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