• Accelerated and efficient neuronal differentiation of Sox1GFP mouse embryonic stem cells in vitro using nicotinamide

      Griffin, Sile; Pickard, Mark R.; Fricker, Rosemary; Keele University, United Kingdom (NECTAR (Network of European CNS Transplantation and Restoration) 24th Annual Meeting, 27/28th November 2014, Galway, Ireland, 2014)
      A major challenge for advancement of clinical neuronal replacement therapies is the production of high yields of purified neuronal populations of appropriate phenotype with control over proliferation to prevent tumorigenesis. We previously reported that treatment of mouse embryonic stem cell (mESC;46CSox1GFP reporter cell line) monolayer cultures with the vitamin B3 metabolite nicotinamide at the early onset of development not only increased the efficiency of neuronal generation by two-fold but also enriched the ratio of purified neurons to non-neuronal cells in culture. This study aimed to investigate if nicotinamide enhances neural induction in this model and whether it also promotes the production/differentiation of specific neuronal subtypes. To address these aims, monolayer mESC cultures were treated with nicotinamide (10 mM) for different durations and immunocytochemistry/fluorescence microscopy was performed to assess the expression of stem cell, neural progenitor (NP) and neuronal subtype markers. Morphometric analyses were also performed to assess the extent of differentiation. Nicotinamide treatment significantly decreased Oct4+ pluripotent cells and concomitantly increased GFP+ cells at day 4, suggesting enhanced neural lineage commitment. By day 14, nicotinamide treatment (from day 0-7) reduced both Oct4+ and GFP expression concomitant with enhanced expression of neuron-specific β-tubulin, indicative of accelerated neuronal differentiation. Nicotinamide selectively enhanced the production of catecholaminergic, serotonergic and GABAergic neurons and, moreover, enhanced various aspects of neuronal morphology and maturation. Collectively, these data demonstrate a direct effect of nicotinamide at the initial stages of embryonic stem cell differentiation which could be critical for rapidly andefficiently promoting neural commitment to highly enriched neuronal lineages. The strong clinical potential of nicotinamide could successfully be applied to future neural cell-based therapies including Parkinson’s and Huntington’s disease, both to eradicate proliferating cells and for a more enhanced and specific differentiation
    • Alignment of multiple glial cell populations in 3D nanofiber scaffolds: toward the development of multicellular implantable scaffolds for repair of neural injury

      Weightman, Alan P.; Jenkins, Stuart I.; Pickard, Mark R.; Chari, Divya M.; Yang, Ying; Keele University, United Kingdom (Elsevier, 2014-02)
      Non-neuronal cells of the central nervous system (CNS), termed "neuroglia," play critical roles in neural regeneration; therefore, replacement of glial populations via implantable nanofabricated devices (providing a growth-permissive niche) is a promising strategy to enhance repair. Most constructs developed to date have lacked three-dimensionality, multiple glial populations and control over spatial orientations, limiting their ability to mimic in vivo neurocytoarchitecture. We describe a facile technique to incorporate multiple glial cell populations [astrocytes, oligodendrocyte precursor cells (OPCs) and oligodendrocytes] within a three-dimensional (3D) nanofabricated construct. Highly aligned nanofibers could induce elongation of astrocytes, while OPC survival, elongation and maturation required pre-aligned astrocytes. The potential to scale-up the numbers of constituent nanofiber layers is demonstrated with astrocytes. Such complex implantable constructs with multiple glial sub-populations in defined 3D orientations could represent an effective approach to reconstruct glial circuitry in neural injury sites.
    • Anti-epileptic drugs and bone loss: phenytoin reduces pro-collagen I and alters the electrophoretic mobility of osteonectin in cultured bone cells.

      Wilson, Emma L.; Garton, Mark; Fuller, Heidi R.; RJAH Orthopaedic Hospital; RJAH Orthopaedic NHS Foundation Trust; Keele University (Elsevier, 2016-05)
      Phenytoin is an antiepileptic drug used in the management of partial and tonic-clonic seizures. In previous studies we have shown that valproate, another antiepileptic drug, reduced the amount of two key bone proteins, pro-collagen I and osteonectin (SPARC, BM-40), in both skin fibroblasts and cultured osteoblast-like cells. Here we show that phenytoin also reduces pro-collagen I production in osteoblast-like cells, but does not appear to cause a decrease in osteonectin message or protein production. Instead, a 24h exposure to a clinically relevant concentration of phenytoin resulted in a dose-dependent change in electrophoretic mobility of osteonectin, which was suggestive of a change in post-translational modification status. The perturbation of these important bone proteins could be one of the mechanisms to explain the bone loss that has been reported following long-term treatment with phenytoin.
    • Autologous chondrocyte implantation-derived synovial fluids display distinct responder and non-responder proteomic profiles

      Hulme, Charlotte H.; Wilson, Emma L.; Peffers, Mandy J.; Roberts, Sally; Simpson, Deborah M.; Richardson, James B.; Gallacher, Pete; Wright, Karina T.; Keele University; Robert Jones and Agnes Hunt Orthopaedic Hospital; University of Chester; University of Liverpool (BioMed Central, 2017-06-30)
      Background Autologous chondrocyte implantation (ACI) can be used in the treatment of focal cartilage injuries to prevent the onset of osteoarthritis (OA). However, we are yet to understand fully why some individuals do not respond well to this intervention. Identification of a reliable and accurate biomarker panel that can predict which patients are likely to respond well to ACI is needed in order to assign the patient to the most appropriate therapy. This study aimed to compare the baseline and mid-treatment proteomic profiles of synovial fluids (SFs) obtained from responders and non-responders to ACI. Methods SFs were derived from 14 ACI responders (mean Lysholm improvement of 33 (17–54)) and 13 non-responders (mean Lysholm decrease of 14 (4–46)) at the two stages of surgery (cartilage harvest and chondrocyte implantation). Label-free proteome profiling of dynamically compressed SFs was used to identify predictive markers of ACI success or failure and to investigate the biological pathways involved in the clinical response to ACI. Results Only 1 protein displayed a ≥2.0-fold differential abundance in the preclinical SF of ACI responders versus non-responders. However, there is a marked difference between these two groups with regard to their proteome shift in response to cartilage harvest, with 24 and 92 proteins showing ≥2.0-fold differential abundance between Stages I and II in responders and non-responders, respectively. Proteomic data has been uploaded to ProteomeXchange (identifier: PXD005220). We have validated two biologically relevant protein changes associated with this response, demonstrating that matrix metalloproteinase 1 was prominently elevated and S100 calcium binding protein A13 was reduced in response to cartilage harvest in non-responders. Conclusions The differential proteomic response to cartilage harvest noted in responders versus non-responders is completely novel. Our analyses suggest several pathways which appear to be altered in non-responders that are worthy of further investigation to elucidate the mechanisms of ACI failure. These protein changes highlight many putative biomarkers that may have potential for prediction of ACI treatment success.
    • Clinical Cell Therapy Guidelines for Neurorestoration (IANR/CANR 2017)

      Huang, Hongyun; Young, Wise; Chen, Lin; Feng, Shiqing; Zoubi, Ziad M. Al; Sharma, Hari Shanker; Saberi, Hooshang; Moviglia, Gustavo A.; He, Xijing; Muresanu, Dafin F.; Sharma, Alok; Otom, Ali; Andrews, Russell J.; Al-Zoubi, Adeeb; Bryukhovetskiy, Andrey S.; Chernykh, Elena R.; Domańska-Janik, Krystyna; Jafar, Emad; Johnson, W. Eustace; Li, Ying; Li, Daqing; Luan, Zuo; Mao, Gengsheng; Shetty, Ashok K.; Siniscalco, Dario; Skaper, Stephen; Sun, Tiansheng; Wang, Yunliang; Wiklund, Lars; Xue, Qun; You, Si-Wei; Zheng, Zuncheng; Dimitrijevic, Milan R.; Masri, W. S. El; Sanberg, Paul R.; Xu, Qunyuan; Luan, Guoming; Chopp, Michael; Cho, Kyoung-Suok; Zhou, Xin-Fu; Wu, Ping; Liu, Kai; Mobasheri, Hamid; Ohtori, Seiji; Tanaka, Hiroyuki; Han, Fabin; Feng, Yaping; Zhang, Shaocheng; Lu, Yingjie; Zhang, Zhicheng; Rao, Yaojian; Tang, Zhouping; Xi, Haitao; Wu, Liang; Shen, Shunji; Xue, Mengzhou; Xiang, Guanghong; Guo, Xiaoling; Yang, Xiaofeng; Hao, Yujun; Hu, Yong; Li, Jinfeng; AO, Qiang; Wang, Bin; Zhang, Zhiwen; Lu, Ming; Li, Tong (SAGE Publications, 2018-04-11)
    • Commentary: Endovascular Sealing of Abdominal Aortic Aneurysms: Do Current Data Justify Wider Use?

      Torella, Francesco; McWilliams, Richard G.; Fisher, Robert K. (SAGE Publications, 2018-04-12)
    • Conscientious objection and physician-assisted suicide: a viable option in the UK?

      Willis, Derek; email: derekwillis35@hotmail.com; George, Rob (2018-11-15)
      Conscience objection is a proposed way of ensuring that medical practitioners who object to physician-assisted suicide may avoid having to be involved in such a procedure if this is legalised. This right on the part of healthcare professionals already exists in certain circumstances. This paper examines the ethical and legal grounds for conscientious objection for medical professionals and shows how it is heavily criticised in circumstances where it is already used. The paper comes to the conclusion that as the grounds and application of conscience objection are no longer as widely accepted, its future application in any legislation can be called into question. [Abstract copyright: © Author(s) (or their employer(s)) 2018. No commercial re-use. See rights and permissions. Published by BMJ.]
    • Conserved sequence-specific lincRNA-steroid receptor interactions drive transcriptional repression and direct cell fate

      Hudson, William H.; Pickard, Mark R.; de Vera, Ian M.; Kuiper, Emily G.; Mourtada-Maarabouni, Mirna; Conn, Graeme L.; Kojetin, Douglas J.; Williams, Gwyn T.; Ortlund, Eric A.; Emory University School of Medicine; Keele University; Scripps Research Institute (Nature Publishing Group, 2014-11-07)
      The majority of the eukaryotic genome is transcribed, generating a significant number of long intergenic noncoding RNAs (lincRNAs). Although lincRNAs represent the most poorly understood product of transcription, recent work has shown lincRNAs fulfill important cellular functions. In addition to low sequence conservation, poor understanding of structural mechanisms driving lincRNA biology hinders systematic prediction of their function. Here we report the molecular requirements for the recognition of steroid receptors (SRs) by the lincRNA growth arrest-specific 5 (Gas5), which regulates steroid-mediated transcriptional regulation, growth arrest and apoptosis. We identify the functional Gas5-SR interface and generate point mutations that ablate the SR-Gas5 lincRNA interaction, altering Gas5-driven apoptosis in cancer cell lines. Further, we find that the Gas5 SR-recognition sequence is conserved among haplorhines, with its evolutionary origin as a splice acceptor site. This study demonstrates that lincRNAs can recognize protein targets in a conserved, sequence-specific manner in order to affect critical cell functions.
    • Endocytotic potential governs magnetic particle loading in dividing neural cells: studying modes of particle inheritance

      Tickle, Jacqueline A.; Jenkins, Stuart I.; Polyak, Boris; Pickard, Mark R.; Chari, Divya M.; Keele University, United Kingdom; Drexel University College of Medicine, Philadelphia, USA (Future Medicine, 2016-02)
      AIM: To achieve high and sustained magnetic particle loading in a proliferative and endocytotically active neural transplant population (astrocytes) through tailored magnetite content in polymeric iron oxide particles. MATERIALS & METHODS: MPs of varying magnetite content were applied to primary-derived rat cortical astrocytes ± static/oscillating magnetic fields to assess labeling efficiency and safety. RESULTS: Higher magnetite content particles display high but safe accumulation in astrocytes, with longer-term label retention versus lower/no magnetite content particles. Magnetic fields enhanced loading extent. Dynamic live cell imaging of dividing labeled astrocytes demonstrated that particle distribution into daughter cells is predominantly 'asymmetric'. CONCLUSION: These findings could inform protocols to achieve efficient MP loading into neural transplant cells, with significant implications for post-transplantation tracking/localization.
    • The experience of stigma in inflammatory bowel disease: an interpretive (hermeneutic) phenomenological study

      Dibley, Lesley; Norton, Christine; Whitehead, Elizabeth; University of Chester (John Wiley & Sons Ltd, 2017-12-05)
      Aim to explore experiences of stigma in people with inflammatory bowel disease. Background Diarrhoea, urgency and incontinence are common symptoms in inflammatory bowel disease. Social rules stipulate full control of bodily functions in adulthood: poor control may lead to stigmatisation, affecting patients’ adjustment to disease. Disease-related stigma is associated with poorer clinical outcomes but qualitative evidence is minimal. Design An interpretive (hermeneutic) phenomenological study of the lived experience of stigma in inflammatory bowel disease. Methods Forty community-dwelling adults with a self-reported diagnosis of inflammatory bowel disease were recruited purposively. Participants reported feeling stigmatised or not and experiencing faecal incontinence or not. Unstructured interviews took place in participants’ homes in the United Kingdom (September 2012 – May 2013). Data were analysed using Diekelmann's interpretive method. Findings Three constitutive patterns - Being in and out of control, Relationships and social Support and Mastery and mediation - reveal the experience of disease-related stigma, occurring regardless of continence status and because of name and type of disease. Stigma recedes when mastery over disease is achieved through development of resilience - influenced by humour, perspective, mental wellbeing and upbringing (childhood socialisation about bodily functions). People travel in and out of stigma, dependent on social relationships with others including clinicians and tend to feel less stigmatised over time. Conclusion Emotional control, social support and mastery over disease are key to stigma reduction. By identifying less resilient patients, clinicians can offer appropriate support, accelerating the patient's path towards disease acceptance and stigma reduction.
    • GAS5 lncRNA Modulates the Action of mTOR Inhibitors in Prostate Cancer Cells

      Yacqub-Usman, Kiren; Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (NCRI Cancer Conference 2014 Abstracts, 2014)
      Background There is a need to develop new therapies for castrate-resistant prostate cancer (CRPC) and growth arrest-specific 5 (GAS5) long non-coding RNA (lncRNA), which riborepresses androgen receptor action, may offer novel opportunities in this regard. GAS5 lncRNA expression declines as prostate cancer cells acquire castrate-resistance, and decreased GAS5 expression attenuates the responses of prostate cancer cells to apoptotic stimuli. Enhancing GAS5 lncRNA expression may therefore offer a strategy to improve the effectiveness of chemotherapeutic agents. GAS5 is a member of the 5' terminal oligopyrimidine gene family, and we have therefore examined if mTOR inhibition can enhance cellular GAS5 levels in prostate cancer cells. In addition, we have determined if GAS5 lncRNA itself is required for mTOR inhibitor action in prostate cancer cells, as recently demonstrated in lymphoid cells. Method The effects of mTOR inhibitors on GAS5 lncRNA expression and cell proliferation were determined in a range of prostate cancer cell lines. Transfection of cells with GAS5 siRNA and plasmid constructs was performed to determine the involvement of GAS5 lncRNA in mTOR inhibitor action. Results Treatment with rapamycin and rapalogues increased cellular GAS5 levels and inhibited culture growth in both androgen-dependent (LNCaP) and androgen-sensitive (22Rv1) cell lines, but not in androgen-independent (PC-3 and DU145) cells. GAS5 silencing in both LNCaP and 22Rv1 cells decreased their sensitivity to growth inhibition by mTOR inhibitors. Moreover, transfection of GAS5 lncRNA sensitized PC-3 and DU145 cells to mTOR inhibitors, resulting in inhibition of culture growth. Conclusion mTOR inhibition enhances GAS5 transcript levels in some, but not all, prostate cancer cell lines. This may in part be related to endogenous levels of GAS5 expression, which tend to be lower in prostate cancer cells representative of advanced disease, particularly since current findings demonstrate a role for GAS5 lncRNA in mTOR inhibitor action in prostate cancer cells.
    • The Hormone Response Element Mimic Sequence of GAS5 LncRNA is Sufficient to Induce Apoptosis in Breast Cancer Cell Lines

      Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (2015)
      Growth arrest-specific 5 (GAS5) encodes snoRNAs and lncRNA. The latter promotes apoptosis, but its expression is down-regulated in breast cancer. The mTOR and nonsense-mediated decay pathways together regulate GAS5 transcript levels but rapalogues fail to enhance GAS5 levels in triple-negative breast cancer cells, so that mTOR inhibitor-independent induction of GAS5 may be more productive in enhancing apoptotic responses to therapies in breast cancer. Notably, GAS5 lncRNA acts by riborepression of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM). The aim of this study was to determine if the GAS5 HREM sequence alone is sufficient to promote the apoptosis of breast cancer cells. Cells were nucleofected with a DNA oligonucleotide corresponding to the GAS5 lncRNA HREM; controls received oligonucleotides either with scrambled GAS5 sequence or with stem complementarity present but lacking the GAS5 HRE consensus. Cells were irradiated with ultraviolet-C (UV-C) light at 20 h post-transfection to induce apoptosis. The basal apoptotic rate almost doubled in MCF7 and MDA-MB-231 cells transfected with the HREM oligonucleotide compared with controls. This effect was apparent at 20 h post¬-transfection, and a corresponding decrease was observed in culture viability; clonogenic activity was also impaired. The HREM sequence also enhanced UV-C-induced apoptosis in an additive manner in both cell lines. Endogenous GAS5 lncRNA expression was unaffected by transfection of the HREM sequence. Thus the GAS5 lncRNA HREM is sufficient to induce apoptosis in breast cancer cells, including TNBC cells and this may serve as the basis for the development of novel oligonucleotide cancer therapies. Funded by the Breast Cancer Campaign.
    • The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cell lines – towards oligonucleotide therapies?

      Pickard, Mark R.; Williams, Gwyn T.; Keele University, United Kingdom (NCRI Cancer Conference 2014 Abstracts, 2014)
      Background Growth arrest-specific 5 (GAS5), a non-protein coding gene, encodes snoRNAs and lncRNA; transcript levels are controlled by the mTOR and nonsense-mediated decay pathways. GAS5 lncRNA promotes the apoptosis of breast cells, including triple-negative breast cancer (TNBC) cells, but its expression is down-regulated in breast cancer. Rapalogues enhance GAS5 levels in oestrogen receptor-positive breast cancer cells but not in TNBC cells, so that mTOR inhibitor-independent induction of GAS5 may be more productive in enhancing apoptotic responses to therapies. Notably, GAS5 lncRNA acts by riborepression of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM). The aim of this study was to determine if the GAS5 HREM sequence alone is sufficient to promote the apoptosis of breast cancer cells. Method Cells were nucleofected with a DNA oligonucleotide corresponding to the GAS5 lncRNA HREM; controls received oligonucleotides either with scrambled GAS5 sequence or retaining stem-loop structure but lacking the GAS5 HRE consensus; mock-transfected cells were also studied. Cells were irradiated with ultraviolet-C (UV-C) light at 20 h post-transfection to induce apoptosis. Culture viability and apoptosis were assessed and cellular GAS5 levels were determined by RT-qPCR. Results The basal apoptotic rate almost doubled in MCF7 and MDA-MB-231 cells transfected with the HREM oligonucleotide compared with controls. This effect was apparent at 20 h post­-transfection, and a corresponding decrease was observed in culture viability. The HREM sequence also enhanced UV-C-induced apoptosis in an additive manner in both cell lines. Endogenous GAS5 lncRNA expression was unaffected by transfection of the HREM sequence. Conclusion The GAS5 lncRNA HREM is sufficient to induce apoptosis in breast cancer cells, including TNBC cells. This study serves as an exemplar of how emerging knowledge of biologically important lncRNAs may be exploited towards the development of novel oncotherapeutic agents.
    • The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells.

      Pickard, Mark R.; Williams, Gwyn T.; Keele University (Impact Journals, 2016-02-03)
      Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and -insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies.
    • 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.
    • 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 nicotinamide on the development of neurons

      Griffin, Sile; Pickard, Mark R.; Orme, Rowan; Hawkins, Clive; Williams, Adrian; Chari, Divya M.; Fricker, Rosemary; Keele University, University Hospital of North Staffordshire NHS Trust, University of Birmingham, United Kingdom (2014-10)
      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.
    • 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.
    • In vitro cyto-toxic assessment of heavy metals and their binary mixtures on mast cell-like, rat basophilic leukemia (RBL-2H3) cells.

      ThankGod Eze, Chukwuebuka; email: thankgod.eze@fuoye.edu.ng; Michelangeli, Francesco; email: f.michelangeli@chester.ac.uk; Otitoloju, Adebayo Akeem; email: aotitoloju@unilag.edu.ng (2019-02-11)
      We investigated the cytotoxicity and mechanisms of cell death induced by salts of Cadmium (Cd ), Lead (Pb ), Arsenic (AsO ) and Chromium (Cr ) on RBL-2H3 cells (a model mast cell line). In addition, cyto-toxic effect on cell viability was assessed to reveal their nature of interaction in binary mixture. The individual cytotoxic characteristics of these metals on RBL-2H3 cell viability showed a concentration-dependent reduction of cell viability. We observed that concentration-dependent cytotoxic potency on RBL-2H3 cells of these metals range in the following order Cd >Cr >As O > Pb with LC values of 0.11 μM, 93.58 μM, 397.9 μM and 485.3 μM respectively. Additive effects were observed with Pb  + Cd , Pb  + AsO , Pb  + Cr and AsO  + Cr . The study revealed that Pb , Cd , AsO and Cr could induce significant (P < 0.01) cell death by apoptosis in RBL-2H3. Highly significant necrotic cell death was observed with Pb and Cr (P < 0.01) than Cd and AsO (P < 0.05). Overall, it can be deduced that several cell death executing pathways may be concomitantly activated on exposure to heavy metals and the predominance of one over others might depend on the type of heavy metal, concentration and the metabolic state of the cell. Eventually, binary mixtures of some of these metals showed less cytotoxicity than would be expected from their individual actions and may depend on the co-exposure of the metal ions and their modes of action. [Abstract copyright: Copyright © 2019 Elsevier Ltd. All rights reserved.]