• Identifying the cellular targets of drug action in the central nervous system following corticosteroid therapy

      Jenkins, Stuart I.; Pickard, Mark R.; Khong, Melinda; Smith, Heather L.; Mann, Carl L. A.; Emes, Richard D.; Chari, Divya M.; Keele University, University of Nottingham, University Hospital of North Staffordshire NHS Trust, United Kingdom (American Chemical Society, 2014-01-15)
      Corticosteroid (CS) therapy is used widely in the treatment of a range of pathologies, but can delay production of myelin, the insulating sheath around central nervous system nerve fibers. The cellular targets of CS action are not fully understood, that is, "direct" action on cells involved in myelin genesis [oligodendrocytes and their progenitors the oligodendrocyte precursor cells (OPCs)] versus "indirect" action on other neural cells. We evaluated the effects of the widely used CS dexamethasone (DEX) on purified OPCs and oligodendrocytes, employing complementary histological and transcriptional analyses. Histological assessments showed no DEX effects on OPC proliferation or oligodendrocyte genesis/maturation (key processes underpinning myelin genesis). Immunostaining and RT-PCR analyses show that both cell types express glucocorticoid receptor (GR; the target for DEX action), ruling out receptor expression as a causal factor in the lack of DEX-responsiveness. GRs function as ligand-activated transcription factors, so we simultaneously analyzed DEX-induced transcriptional responses using microarray analyses; these substantiated the histological findings, with limited gene expression changes in DEX-treated OPCs and oligodendrocytes. With identical treatment, microglial cells showed profound and global changes post-DEX addition; an unexpected finding was the identification of the transcription factor Olig1, a master regulator of myelination, as a DEX responsive gene in microglia. Our data indicate that CS-induced myelination delays are unlikely to be due to direct drug action on OPCs or oligodendrocytes, and may occur secondary to alterations in other neural cells, such as the immune component. To the best of our knowledge, this is the first comparative molecular and cellular analysis of CS effects in glial cells, to investigate the targets of this major class of anti-inflammatory drugs as a basis for myelination deficits.
    • The importance of clinician, patient and researcher collaborations in Alport syndrome

      Rheault, Michelle N.; Savige, Judith; Randles, Michael J.; Weinstock, André; Stepney, Melissa; Turner, Neil; Parziale, Gina; Gross, Oliver; Flinter, Frances A; Miner, Jeffrey H; et al. (Springer Nature, 2019-05-01)
      Alport syndrome (AS) is caused by mutations in the genes COL4A3, COL4A4 or COL4A5 and is characterised by progressive glomerular disease, sensorineural hearing loss and ocular defects. Occurring in less than 1:5000, AS is rare genetic disorder but still accounts for >1% of the prevalent population receiving renal replacement therapy. There is also increasing awareness about the risk of chronic kidney disease in individuals with heterozygous mutations in AS genes. The mainstay of current therapy is the use of angiotensin converting enzyme inhibitors and angiotensin receptor blockers, yet potential new therapies are now entering clinical trials. The 2017 International Workshop on Alport Syndrome in Glasgow was a preconference workshop ahead of the 50th anniversary meeting of the European Society for Pediatric Nephrology. It focussed on updates in clinical practice, genetics, basic science and also incorporated patient perspectives. More than 80 international experts including clinicians, geneticists, researchers from academia and industry, and patient representatives took part in panel discussions and breakout groups. This report summarises the workshop proceedings and the relevant contemporary literature. It highlights the unique clinician, patient and researcher collaborations achieved by regular engagement between the groups.
    • An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair

      Kohli, Nupur; Johnson, William Eustace Basil; Wright, Karina T.; Sammons, Rachel L.; Jeys, Lee; Snow, Martyn
      Aim: To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. Methods: Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. Results: A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. Conclusion: Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.
    • An in vitro spinal cord injury model to screen neuroregenerative materials

      Weightman, Alan P.; Pickard, Mark R.; Yang, Ying; Chari, Divya M.; Keele University (Elsevier, 2014-01-29)
      Implantable 'structural bridges' based on nanofabricated polymer scaffolds have great promise to aid spinal cord regeneration. Their development (optimal formulations, surface functionalizations, safety, topographical influences and degradation profiles) is heavily reliant on live animal injury models. These have several disadvantages including invasive surgical procedures, ethical issues, high animal usage, technical complexity and expense. In vitro 3-D organotypic slice arrays could offer a solution to overcome these challenges, but their utility for nanomaterials testing is undetermined. We have developed an in vitro model of spinal cord injury that replicates stereotypical cellular responses to neurological injury in vivo, viz. reactive gliosis, microglial infiltration and limited nerve fibre outgrowth. We describe a facile method to safely incorporate aligned, poly-lactic acid nanofibre meshes (±poly-lysine + laminin coating) within injury sites using a lightweight construct. Patterns of nanotopography induced outgrowth/alignment of astrocytes and neurons in the in vitro model were strikingly similar to that induced by comparable materials in related studies in vivo. This highlights the value of our model in providing biologically-relevant readouts of the regeneration-promoting capacity of synthetic bridges within the complex environment of spinal cord lesions. Our approach can serve as a prototype to develop versatile bio-screening systems to identify materials/combinatorial strategies for regenerative medicine, whilst reducing live animal experimentation.
    • Influence of Amplitude of Oscillating Magnetic Fields on Magnetic Nanoparticle-Mediated Gene Transfer to Astrocytes

      Tickle, Jacqueline A.; Jenkins, Stuart I.; Pickard, Mark R.; Chari, Divya M.; Keele University, United Kingdom (World Scientific, 2014-08-07)
      Functionalized magnetic nanoparticles (MNPs) are emerging as a major nanoplatform for regenerative neurology, particularly as transfection agents for gene delivery. Magnetic assistive technology, particularly the recent innovation of applied oscillating magnetic fields, can significantly enhance MNP-mediated gene transfer to neural cells. While transfection efficiency varies with oscillation frequency in various neural cell types, the influence of oscillation amplitude has not yet been investigated. We have addressed this issue using cortical astrocytes that were transfected using MNPs functionalized with plasmid encoding a reporter protein. Cells were exposed to a range of oscillation amplitudes (100–1000 μm), using a fixed oscillation frequency of 1 Hz. No significant differences were found in the proportions of transfected cells at the amplitudes tested, but GFP-related optical density measurements (indicative of reporter protein expression) were significantly enhanced at 200 μm. Safety data show no amplitude-dependent toxicity. Our data suggest that the amplitude of oscillating magnetic fields influences MNP-mediated transfection, and a tailored combination of amplitude and frequency may further enhance transgene expression. Systematic testing of these parameters in different neural subtypes will enable the development of a database of neuro-magnetofection protocols — an area of nanotechnology research where little information currently exists.
    • The influence of CLA on obesity, lung function, adipokines and inflammation

      Williams, John; Ireland, Elsye; Hamdallah, Hanady (University of ChesterUniversity of Chester, 2019-01-31)
      Obesity is currently widespread in the world; the epidemic and pathogenesis of the disease negatively affect several body systems including cardiovascular, endocrine and respiratory systems. Obesity influences the respiratory functions and this effect could be challenging for women, because the air way and lungs are smaller in women compared to men, as well as obesity itself exerts a negative mechanical effect on the women’s airway. Since inflammation was proposed asthe main link between obesity and lung functions, a natural supplement like conjugated linoleic acid (CLA), which has been proposed as an antiinflammatory and anti-obesity food component, could be a potential supplement that can improve the lung functions in obese women. Therefore, the aim of this thesis is to explore the effect of CLA on obesity, lung function, adipokines and inflammation. Additionally, the effect of CLA on inflammation in the current thesis was explored using novel inflammatory markers, such as adhesion molecules (CD11b and CD62L) and heat shock proteins (HSPA1A and HSPB1). Investigating the evidence about the effect of CLA supplementation on obesity in women was conducted via a systematic review with meta-analysis. The meta- analysis searched randomised control trials (RCTs) supplemented CLA mixture in form of oral capsules for less than 6 months. Two search strategies were applied, and eight eligible trials were included with 330 women. CLA significantly reduced body weight (BW; 1.2±0.26 kg, p<0.001), body mass index (BMI; 0.6 ±0.13 kg/ m², p <0.001) and total body fat (TBF; 0.76± 0.26 kg, p=0.003) when it was supplemented for short durations (6- 16 weeks). Moreover, subgroups meta-analyses were conducted which were based on obesity level, menopausal age and life style of the participants. This meta-analysis suggested a mild anti-obesity effect of CLA. However, it was not clear whether the anti-obesity effect is enough to modulate obesity-induced inflammation and lung functions. Therefore, initially a crosssectional trial was conducted to assess the direct associations between the circulating level of CLA and obesity markers, lung functions and inflammations. To the best of Knowledge, this was the first cross-sectional trial that explored these direct associations. The cross-sectional trial recruited 77 women with average age 39 years old with forced expiratory volume in one-second (FEV1) ≥70%. The level of CLA in plasma was assessed by gas chromatography; the expression of the CD markers and HSPs were assessed using flow cytometry; body composition was assessed using bioelectric impedance; and lung functions were assessed using spirometer. Interestingly, the trial revealed significant positive associations between CLA and BW (R=0.4, p<0.001), BMI (R=0.4, P<0.001) and TBF (R=0.34, P<0.001) in the overall population, and in perimenopause women. A significant inverse correlation between t10, c12-CLA and TBF was detected in overweight women (R=- 0.42, p<0.05). A significant positive association (R=0.45, P<0.04) was detected between the c9, t11-CLA and percentage peak of flow predicted (PEF %) in postmenopausal women, meanwhile t10, c12-CLA was negatively associated with peak of flow (R=-0.44, P<0.04). CLA was inversely associated with adiponectin in both obese (R=-0.55, p<0.1) and morbidly obese (R=0.48, P<0.004) women. C9, t11-CLA was positively associated with the expression of HSPA1A inside the lymphocytes in postmenopausal women (R=0.58, p=0.04). HSPB1 expression in the monocytes were associated with both c9, t11-CLA (R=0.58, p<0.05) and total CLA (R=0.71, p<0.001). The level of expression of CD11b on the pro-inflammatory monocytes (CD14++ CD16+ ) was negatively associated with CLA (R=-0.36, p<0.05). Ultimately, the study did not provide strong evidence regarding the direct relationship between CLA and obesity markers or lung functions. However, it showed a potential immunomodulatory effect of CLA on obesity-induced chronic inflammation, which subsequently could influence multiple obesity compilations. The lack of strong evidencewas primarily due to the nature of the study design (observational study). Therefore, in chapter 5 a randomised double-blind placebo control trial was conducted, for more powerful evidence based. The aim of the RCT was to look at the effect of 12-week CLA supplementation on obesity, lung function, adipokines and inflammation in obese and overweight women. The RCT recruited 56 overweight and obese women with a mean age of 42 years old, participants were randomly assigned either to receive 4.5gm/day of CLA or placebo (High Oleic Safflower oil). Participants had to attend three clinics at base line, after 6 weeks and after 12 weeks. In each clinic body composition, lung functions and inflammatory markers were assessed. The study revealed a significant 1.8% reduction in %BF in the CLA group compared to the baseline. No significant effect of CLA on the lung functions was detected, however, this study found a significant reduction in the expression of CD11b on the stimulated pro-inflammatory monocytes after 12 weeks compared to baseline in the CLA group. CLA caused a significant reduction in the expression of intracellular HSPA1A in PBMCs at week 12 compared to baseline. The results might suggest a limited anti-obesity effect of CLA, and a potential positive effect on obesity induced chronic inflammation. Ultimately, no evidence was demonstrated on the direct effect of CLA on lung functions or adipokines. The effect of CLA on adhesion molecules and HSPA1A could suggest an indirect impact on the lung function, but more research in clinically diagnosed patients with pulmonary dysfunctions could help to confirm the effect of CLA on the lung function and adipokines.
    • The influence of nicotinamide on the development of neurons

      Griffin, Sile; Pickard, Mark R.; Hawkins, Clive P.; Williams, Adrian C.; Chari, Divya M.; Fricker, Rosemary; Orme, Rowan P.; Keele University, University Hospital of North Staffordshire NHS Trust, University of Birmingham, United Kingdom (2014-09-09)
      A major challenge in translating the promise of stem cell therapies to treat a myriad of neurodegenerative disorders is to rapidly and efficiently direct pluripotent stem cells to generate differentiated neurons. The application of active vitamin metabolites known to function in embryonic development and maintenance in the adult brain such as retinoic acid (vitamin A), ascorbic acid (vitamin C) and calcitriol (vitamin D3) have proven effective in current in-vitro differentiation protocols. Therefore, in this study we investigated whether the biologically active vitamin B3 metabolite, nicotinamide could enhance the differentiation of mouse embryonic stem cells, cultured as monolayers, into mature neurons at either early or late stages of development. Interestingly, nicotinamide elicited a dose-responsive increase in the percentage of neurons when added at an early developmental stage to the cells undergoing differentiation (days 0–7). Nicotinamide (10 mM) increased the proportion of β-III tubulin positive neurons by two fold and concomitantly decreased the total number of cells in culture, measured by quantification of 4′, 6-diamidino-2-phenylindole positive cells. This effect could result from induction of cell-cycle exit and/or selective cell death in non-neural populations. Higher levels of nicotinamide (20 mM) induced cytoxicity and cell death. This study supports previous evidence that vitamins and their metabolites can efficiently direct stem cells into neurons. Current work is focusing on the effect of nicotinamide on the process of neural induction and whether nicotinamide influences the generation of particular neuronal subtypes implicated in neurodegenerative diseases, specifically focusing on midbrain dopamine neurons; towards a therapy for Parkinson's disease.
    • The influence of pH and fluid dynamics on the antibacterial efficacy of 45S5 Bioglass Short title: Antibacterial efficacy of 45S5 Bioglass

      Begum, Saima; Johnson, William Eustace Basil; Worthington, Tony; Martin, Richard; Aston University (IOP Publishing, 2016-02-02)
      In recent years, there has been considerable interest in the potential antibacterial properties that bioactive glasses may possess. However, there have been several conflicting reports on the antibacterial efficacy of 45S5 Bioglass®. Various mechanisms regarding its mode of action have been proposed, such as changes in the environmental pH, increased osmotic pressure, and 'needle-like' sharp glass debris which could potentially damage prokaryotic cell walls and thus inactivate bacteria. In this current study, a systematic investigation was undertaken on the antibacterial efficacy of 45S5 Bioglass® on Escherichia coli NCTC 10538 and Staphylococcus aureus ATCO 6538 under a range of clinically relevant scenarios including varying Bioglass® concentration, direct and indirect contact between Bioglass® and microorganisms, static and shaking incubation conditions, elevated and neutralised pH environments. The results demonstrated that, under elevated pH conditions, Bioglass® particles have no antibacterial effect on S. aureus while a concentration dependent antibacterial effect against E. coli was observed. However, the antibacterial activity ceased when the pH of the media was neutralised. The results of this current study, therefore, suggest that the mechanism of antibacterial activity of Bioglass® is associated with changes in the environmental pH; an environment that is less likely to occur in vivo due to buffering of the system.
    • Interactions between extracellular Hsp72 and blood cells

      Williams, John H. H.; Ireland, H. Elyse; Williams, Helen (University of Liverpool (University of Chester), 2010-12)
      In recent years, compelling evidence has accumulated suggesting heat shock proteins (HSPs) which are generally believed to be localised and functioning mainly within eukaryotic cells as cyto-protective molecular chaperones, are also localised in the extracellular milieu. Depending on their localisation, on the cell surface (membrance-bound or embedded), or in the peripheral circulation, extracellular HSPs may induce apoptotic cell death, or in contrast protect cells from cell damage and/or cell death when exposed to cellular stress, or may even elicit a stimulatory effect on the innate immune response including cell activiation and cytokine secretion. Hence, the localisation of intracellular and extracellular HSPs appears to be critical in determining their roles in terms of stimulating cell death, cyto-protection, or immune activiation under normal physiological conditions and following exposure to stress stimuli. This thesis describes the intracellular expression, up-regulation, and cell surface localisation of endogenous HSPs: HSP27, Hsp60, Hsp72 and Hsp90 by flow cytometry, florescence microscopy and Western blotting, under control conditions and in response to environmental stress using in vitro and ex vivo models with the intention of determining their physiological roles. The ability of extracellularly administered HSPs (Hsp70 and Hsp72) to protect cultured U937 cells in vitro or peripheral primary human leukogytes or erythrocytes ex vivo from various stress stimuli was demonstrated and was found to be dependent on surface binding and/or internalisation via scavenger receptors (SRs) or phosphatidylserine (PS), which could be blocked by receptor specific ligands. Extracellular HSPs were also shown to be able to stimulate an immune response through the induction of U937 monocyte differentiation into macrophages as evidenced through the up-regulation of the surface receptors: CD36, SR-A1 and CD91 analysed by flow cytometry. These proteins were able to stimulate TNF-x and IL-10 production and secretion by U937 macrophages, shown by ELISA, and chemotatic properties were demonstrated using Boyden chambers. The cyto-protective and immune regulatory effects of extracellular HSPs have potential therapeutic value as treatments in a wide variety of clinical situations.
    • Interactions between PP4 and PEA-15 in the regulation of cell proliferation and apoptosis of breast cancer cells

      Mohammed, Hiba N.; Pickard, Mark R.; Mourtada-Maarabouni, Mirna; Keele University, United Kingdom (NCRI Cancer Conference 2015 Abstracts, 2015)
      Background The serine/threonine protein phosphatase 4 (PP4) is recognised to regulate a variety of cellular functions. Our previous work has shown that the catalytic subunit of PP4 (PP4c) promotes cell death and inhibits proliferation in breast cancer cells, suggestive of a role of PP4c as tumour suppressor gene. Phosphoprotein enriched in astrocytes 15 (PEA-15), a member of the death effector domain protein family known to control cell survival, is reported to be regulated by PP4c. The aims of this study were to investigate the involvement of PEA-15 in mediating the effects of PP4c on breast cancer cells. Method PEA-15 phosphorylation was examined by western blot analysis on proteins extracted from MCF7 and MDA-MB-231 cells over-expressing PP4 and PP4 knock down cells. To investigate the role of PEA-15 in mediating the effects of PP4c, MCF7 and MDA-MB-231 were transfected with control (-) siRNA or with three different PEA-15 specific siRNAs. 48 h post-transfection, control cells (transfected with negative control siRNA) and cells transfected with PEA-15 siRNAs were transiently transfected with pcDNA3.1-PP4c expression construct or pcDNA3.1. Cell viability and apoptosis level were assessed post transfection. Results In MCF7 and MDA-MB-231 cells, the phosphorylation state of PEA-15 increased when PP4c expression was suppressed and decreased when PP4c was over-expressed. Over-expression of PP4c in cells transfected with (-) siRNA caused 50% reduction in viability compared to cells transfected with empty vector. Cells transfected with PEA-15 siRNAs showed a decrease in viable cell number and long term survival. However, over-expression of PP4c in these cells did not have any additional effect on the decrease in cell viability. Conclusion These observations suggest that the induction of apoptosis by over-expression of PP4c is mediated, at least in part, by the dephosphorylation of PEA-15. The interactions between PEA-15 and PP4c may therefore be critical in breast cancer tumorigenesis.
    • An Interpretive Phenomenological Analysis (IPA) of coercion towards community dwelling older adults with dementia: Findings from MYsore studies of Natal effects on Ageing and Health (MYNAH)

      Danivas, Vijay; Bharmal, Mufaddal; Keenan, Paul; Jones, Steven; Karat, Samuel C.; Kalyanaraman, Kumaran; Prince, Martin; Fall, Caroline H. D.; Krishna, Murali; University of Chester (Springer, 2016-09-29)
      Purpose Limited availability of specialist services places a considerable burden on caregivers of Persons with Dementia (PwD) in Low- and Middle-Income Countries (LMICs). There are limited qualitative data on coercive behavior towards PwD in an LMIC setting. Aim The aim of this study was to find relevant themes of the lived experience of relatives as caregivers for PwD in view of their use of coercive measures in community setting in South India. Method Primary caregivers (n = 13) of PwDs from the Mysore study of Natal effects on Ageing and Health (MYNAH) in South India were interviewed to explore the nature and impact of coercion towards community dwelling older adults with dementia. The narrative data were coded using an Interpretative Phenomenological Analysis (IPA) approach for thematic analysis and theory formation. Results Caregivers reported feeling physical and emotional burn-out, a lack of respite care, an absence of shared caregiving arrangements, limited knowledge of dementia, and a complete lack of community support services. They reported restrictions on their lives through not being able take employment, a poor social life, reduced income and job opportunities, and restricted movement that impacted on their physical and emotional well-being. Inappropriate use of sedatives, seclusion and environmental restraint, and restricted dietary intake, access to finances and participation in social events, was commonly reported methods of coercion used by caregivers towards PwD. Reasons given by caregivers for employing these coercive measures included safeguarding of the PwD and for the management of behavioral problems and physical health. Conclusion There is an urgent need for training health and social care professionals to better understand the use of coercive measures and their impact on persons with dementia in India. It is feasible to conduct qualitative research using IPA in South India.
    • Investigating the Prevalence of Anaemia in Rural Gambia, in Relation to Levels of Zinc Protoporphyrin, Haemoglobin and Haptoglobin (Phenotype and Genotype)

      Bah, Ebrima; Michelangeli, Frank (Oxford University Press (OUP), 2020-05-29)
      Abstract Objectives To find out the overlapping and correlating relationships between serum haptoglobin level, haptoglobin genotype and phenotype, blood haemoglobin level and zinc protoporphyrin (measured in washed RBCs) in association to prevalence of anaemia. It will focus on comparing all the mention components in contrast to each other. The study will also look for the frequency distribution of the major HP alleles. Methods 1278 participants were randomly selected. Blood samples collected by trained nurses. Data generation was done at the Medical research council (keneba field station) research site. Data Analysis was conducted at the university of Chester with the assistance of the computer department team. Results P = 0.000 indicating anaemia prevalence with HP 1 allele. P &amp;gt; 0.05 when ID, IDA and AI relates with HP genotype. Positive correlation between ZnPP and HP serum level, but negative between ZnPP and Hb. P = 0.000 between ZnPP and IDA. P = 0.024 between HP genotype and Hb level. P = 0.013 between HP genotype and HP serum. P = 0.100 between HP genotype and ZnPP. P = 0.000 between ZnPP and IDA. P = 0.024 between HP genotype and Hb. ZnPP shared a positive correlation with HP serum level, and a negative correlation with Hb level. The correlation significant = 0.01 level (2-tailed) P = 0.01. The correlation between HP genotype and HP serum level was significant with P = 0.013, but the correlation between HP genotype and ZnPP was not significant with P = 0.100. Conclusions HP genotype had association with anaemia prevalence and more occurrence was observed in carriers of the type ‘1’ allele. It had no association with ID, IDA and AI. HP genotype had association with HP serum level and Hb level but had no association with ZnPP level. ZnPP level was observed to have had association with HP serum level, Hb level and IDA; but had no association with ID and AI in the region. Funding Sources All the resources used in this study were from MRC Keneba (International Nutrition Group) which is supported by funds from the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreement (Hennig et al., 2015).
    • Investigating the role of heatshock on diabetic wound healing

      Contractor, Taha (University of Chester, 2017-05)
      The increasing occurrence of diabetes in the general population as a result of over nutrition and increasingly inactive lifestyle has led to an obesity epidemic which is set to grow over time. With an ever increasing obese population type 2 diabetes and cardiovascular complications are set to become the major causes of human mortality. Chronic non healing wounds are a major cause of mortality and morbidity in patients with type 2 diabetes. They are predominantly caused by macrophage dysfunction and a lack of migration of fibroblasts into the wound. This study aimed to investigate diabetic wound healing through development of an artificial scratch assay. An in vitro scratch assay developed in WS1 cells. The effect of heat shock treatments from 39°C to 45° was tested to determine if cell migration increased; however, no significant difference was seen. Mitomycin C was used to determine if wound closure occurred as a result of cell proliferation and migration or migration alone. 10μg/ml of mitomycin C inhibited cell division by 79.9% without exhibiting cytotoxicity over a 12h period. The effect of hyperglycaemia and heat shock was also tested and showed no significant difference when compared to control conditions, suggesting that fibroblast migration in vivo is hindered through other factors such as debridement or macrophage dysfunction in the wound. GLUT4 is present in insulin sensitive organs (liver, adipose and muscle) and is the major glucose transporter responsible for the clearance of glucose from the blood after a meal, thus playing a central role in glucose homeostasis. Monocytes are precursors to macrophages and can easily be isolated from whole blood. They have also been shown to express GLUT4 in response to insulin and could be used as model to assess inflammation in diabetes. A glucose uptake assay was developed in U937 cells using a fluorescent glucose analogue, 2NBDG. 2NBDG fluorescence was shown to be competitively inhibited by increasing concentrations of glucose suggesting that 2NBDG enters the cell through glucose transporters. 2NBDG uptake was also assessed at different pH and in presence of membrane fluidizers (DMSO, benzyl alcohol and phenethyl alcohol). Extremes of pH significantly reduced cell viability and only at pH 4 was 2NBDG fluorescence significantly reduced. Treatment with DMSO showed that at high concentrations (≤ 1.56%) cell viability was reduced with a concurrent reduction in 2NBDG fluorescence. The effect of benzyl alcohol and phenethyl alcohol was foundto be insignificant at the concentrations and time points tested. The presence of GLUT4 was also determined by flow cytometry and Western blotting and found to be situated in the cytoplasmic region of the cell. This study indicates that monocytes and macrophages could be a potential therapeutic target to improve diabetic wound healing as they are a source of growth factors and cytokines that can bring about resolution of inflammation and it is their dysfunction in diabetic wounds that causes poor clinical outcomes.
    • Lamin A/C dysregulation contributes to cardiac pathology in a mouse model of severe spinal muscular atrophy

      Soltic, Darija; Shorrock, Hannah K; Allardyce, Hazel; Wilson, Emma L; Holt, Ian; Synowsky, Silvia A; Shirran, Sally L; Parson, Simon H; Gillingwater, Thomas H; Fuller, HR; et al.
      Cardiac pathology is emerging as a prominent systemic feature of spinal muscular atrophy (SMA), but little is known about the underlying molecular pathways. Using quantitative proteomics analysis, we demonstrate widespread molecular defects in heart tissue from the Taiwanese mouse model of severe SMA. We identify increased levels of lamin A/C as a robust molecular phenotype in the heart of SMA mice and show that lamin A/C dysregulation is also apparent in SMA patient fibroblast cells and other tissues from SMA mice. Lamin A/C expression was regulated in vitro by knockdown of the E1 ubiquitination factor ubiquitin-like modifier activating enzyme 1, a key downstream mediator of SMN-dependent disease pathways, converging on β-catenin signaling. Increased levels of lamin A are known to increase the rigidity of nuclei, inevitably disrupting contractile activity in cardiomyocytes. The increased lamin A/C levels in the hearts of SMA mice therefore provide a likely mechanism explaining morphological and functional cardiac defects, leading to blood pooling. Therapeutic strategies directed at lamin A/C may therefore offer a new approach to target cardiac pathology in SMA.
    • The long non-coding RNA NEAT1 regulates cell survival in breast cancer cell lines

      Almnaseer, Zainab; Pickard, Mark R.; Mourtada-Maarabouni, Mirna; Keele University, United Kingdom (NCRI Cancer Conference 2015 Abstracts, 2015)
      Background Nuclear long non-coding RNAs (LncRNAs) regulate various cellular processes including the organization of nuclear sub-structures, the alteration of chromatin state, and the regulation of gene expression. Nuclear Enriched Abundant Transcript 1 (NEAT1) is a nuclear lncRNA transcribed from chromosome 11q13. Two transcripts are produced from the NEAT1 gene, 3.7-kb NEAT1_v1 and 23-kb NEAT1_v2. Both isoforms participate in the formation of the nuclear paraspeckles . NEAT1 is reported to be overexpressed in prostate cancer and a direct transcriptional target of hypoxia-inducible factor in many breast cancer cell lines. The aims of this study were to determine the effects of silencing NEAT1 on breast cancer cell survival. Method MCF7 and MDA-MB 231 cells were transfected with siRNAs to different NEAT1 sequences or NEAT1 antisense oligonucleotides (ASO). Controls received scrambled siRNA or scrambled oligonucleotide, as appropriate. In some experiments, cells were exposed to ultraviolet-C (UV-C) light post-transfection to induce apoptosis, and then culture viability and apoptosis were assessed. NEAT1 expression was evaluated by qRT-PCR TaqMan® analysis. Results In MCF7 and MDA-MB-231 cells, siRNA-mediated silencing of NEAT1 reduced basal survival and after UV-C irradiation and decreased their colony forming ability. NEAT1 ASOs were more effective in silencing NEAT1 and caused a greater reduction in cell viability. NEAT1 silencing also affected cell cycle profile by enhancing the proportion of cells in G0/G1 phase. Conclusion NEAT1 regulates the survival of Breast cells. Down regulation of NEAT1 expression decreased cell survival, proliferation and modulated cell cycle progression of breast cancer cells, indicating a link between the NEAT1 expression levels and carcinogenesis of breast cancer.
    • Long non-coding RNAs: new opportunities and old challenges in cancer therapy

      Williams, Gwyn T.; Pickard, Mark R.; Keele University; University of Chester (AME Publishing Company, 2016-09)
      No abstract - invited commentary
    • Long-term administration of the mitochondria-targeted antioxidant mitoquinone mesylate fails to attenuate age-related oxidative damage or rescue the loss of muscle mass and function associated with aging of skeletal muscle

      Nye, Gareth; Sakellariou, Giorgos; Lightfoot, Adam; Pearson, Timothy; Wells, Nicola; McArdle, Anne; Jackson, Malcolm; Giakoumaki, Ifigeneia; Griffiths, Richard; University of Liverpool (Faseb Journal, 2016-08-22)
      Age-related skeletal muscle dysfunction is the underlying cause of morbidity that affects up to half the population aged 80 and over. Considerable evidence indicates that oxidative damage and mitochondrial dysfunction contribute to the sarcopenic phenotype that occurs with aging. To examine this, we administered the mitochondria-targeted antioxidant mitoquinone mesylate {[10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl] triphenylphosphonium; 100 μM} to wild-type C57BL/6 mice for 15 wk (from 24 to 28 mo of age) and investigated the effects on age-related loss of muscle mass and function, changes in redox homeostasis, and mitochondrial organelle integrity and function. We found that mitoquinone mesylate treatment failed to prevent age-dependent loss of skeletal muscle mass associated with myofiber atrophy or alter a variety of in situ and ex vivo muscle function analyses, including maximum isometric tetanic force, decline in force after a tetanic fatiguing protocol, and single-fiber-specific force. We also found evidence that long-term mitoquinone mesylate administration did not reduce mitochondrial reactive oxygen species or induce significant changes in muscle redox homeostasis, as assessed by changes in 4-hydroxynonenal protein adducts, protein carbonyl content, protein nitration, and DNA damage determined by the content of 8-hydroxydeoxyguanosine. Mitochondrial membrane potential, abundance, and respiration assessed in permeabilized myofibers were not significantly altered in response to mitoquinone mesylate treatment. Collectively, these findings demonstrate that long-term mitochondria-targeted mitoquinone mesylate administration failed to attenuate age-related oxidative damage in skeletal muscle of old mice or provide any protective effect in the context of muscle aging
    • Low leukotriene B4 receptor 1 leads to ALOX5 downregulation at diagnosis of chronic myeloid leukemia

      Lucas, Claire; Harris, Robert; McDonald, Elizabeth; Giannoudis, Athina; Clark, Richard; University of Liverpool, Royal Liverpool University hospital, (Ferrata Storti Foundation, 2014-11-01)
      ALOX5 is implicated in chronic myeloid leukemia development in mouse leukemic stem cells, but its importance in human chronic myeloid leukemia is unknown. Functional ALOX5 was assessed using an LTB4 ELISA and ALOX5, and LTB4R1 mRNA expression was determined via a TaqMan gene expression assay. LTB4R1 and 5-LOX protein levels were assessed by cell surface flow cytometry analysis. At diagnosis ALOX5 was below normal in both blood and CD34(+) stem cells in all patients. On treatment initiation, ALOX5 levels increased in all patients except those who were destined to progress subsequently to blast crisis. LTB4 levels were increased despite low ALOX5 expression, suggesting that the arachidonic acid pathway is functioning normally up to the point of LTB4 production. However, the LTB4 receptor (BLT1) protein in newly diagnosed patients was significantly lower than after a period of treatment (P<0.0001). The low level of LTB4R1 at diagnosis explains the downregulation of ALOX5. In the absence of LTB4R1, the arachidonic acid pathway intermediates (5-HEPTE and LTA4) negatively regulate ALOX5. ALOX5 regulation is aberrant in chronic myeloid leukemia patients and may not be important for the development of the disease. Our data suggest caution when extrapolating mouse model data into human chronic myeloid leukemia.
    • Magnetic nanoparticle-mediated gene delivery to two- and three-dimensional neural stem cell cultures: magnet-assisted transfection and multifection approaches to enhance outcomes

      Pickard, Mark R.; Adams, Christopher F.; Chari, Divya M.; University of Chester; Keele University (Wiley, 2017-02-02)
      Neural stem cells (NSCs) have high translational potential in transplantation therapies for neural repair. Enhancement of their therapeutic capacity by genetic engineering is an important goal for regenerative neurology. Magnetic nanoparticles (MNPs) are major non-viral vectors for safe bioengineering of NSCs, offering critical translational benefits over viral vectors, including safety, scalability, and ease of use. This unit describes protocols for the production of suspension (neurosphere) and adherent (monolayer) murine NSC cultures. Genetic engineering of NSCs with MNPs and the application of 'magnetofection' (magnetic fields) or 'multifection' (repeat transfection) approaches to enhance gene delivery are described. Magnetofection of monolayer cultures achieves optimal transfection, but neurospheres offer key advantages for neural graft survival post-transplantation. A protocol is presented which allows the advantageous features of each approach to be combined into a single procedure for transplantation. The adaptation of these protocols for other MNP preparations is considered, with emphasis on the evaluation of procedural safety.