• Sonic Hedgehog regulates thymic epithelial cell differentiation

      Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa; University College London; et al. (Elsevier, 2016-01)
      Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.
    • Haemophilia A and cardiovascular morbidity in a female SHAM syndrome carrier due to skewed X chromosome inactivation

      Janczar, Szymon; Kosinska, Joanna; Ploski, Rafal; Pastorczak, Agata; Wegner, Olga; Zalewska-Szewczyk, Beata; Paige, Adam J.W.; Borowiec, Maciej; Mlynarski, Wojciech; Medical University of Lodz; et al. (Elsevier, 2016-01)
      We have recently described a severe haemophilia A and moyamoya (SHAM) syndrome caused by Xq28 deletions encompassing F8 and the BRCC3 familial moyamoya gene. The phenotype includes haemophilia A, moyamoya angiopathy, dysmorphia and hypertension. The genetic analysis of the family of our SHAM patient demonstrated carrier state in proband's mother and sister. The patient's mother is apparently well, whereas his currently 18-years-old sister presents with mild haemophilia A, coarctation of the aorta, hypertension, and ventricular arrhythmia. We performed X chromosome inactivation assay based on HpaII methylation analysis of a polymorphic short tandem repeat (STR) in the X linked AR (androgen receptor) gene and used quantitative real-time RT PCR to measure the expression of genes from the deleted region in proband's family members. We found an extremely skewed X chromosome inactivation pattern in the female members of the family leading to preferential inactivation of the X chromosome without Xq28 deletion in patient's sister. We demonstrated differential expression of the genes from the deleted region in four members of the family, that tightly correlates with the clinical features. In conclusion, we show that the haematologic and cardiovascular morbidity and the discrepancy between patient's sister and mother despite the same genetic lesion are due to skewed X chromosome inactivation leading to clinically relevant differential expression of SHAM syndrome genes. This report highlights the role for BRCC3 in cardiovascular physiology and disease, and demonstrates that in some complex hereditary syndromes full diagnostics may require the examination of both genetic and epigenetic events.
    • Preserved global histone H4 acetylation linked to ETV6-RUNX1 fusion and PAX5 deletions is associated with favorable outcome in pediatric B-cell progenitor acute lymphoblastic leukemia.

      Janczar, Karolina; Janczar, Szymon; Pastorczak, Agata; Mycko, K.; Paige, Adam J.W.; Zalewska-Szewczyk, Beata; Wagrowska-Danilewicz, M.; Danilewicz, Marian; Mlynarski, Wojciech; Medical University of Lodz; et al. (Elsevier, 2015-10-20)
      Epigenetic dysregulation is a hallmark of cancer executed by a number of complex processes the most important of which converge on DNA methylation and histone protein modifications. Epigenetic marks are potentially reversible and thus promising drug targets. In the setting of acute lymphoblastic leukemia (ALL) they have been associated with clinicopathological features including risk of relapse or molecular subgroups of the disease. Here, using immunocytochemistry of bone marrow smears from diagnosis, we studied global histone H4 acetylation, whose loss was previously linked to treatment failure in adults with ALL, in pediatric patients. We demonstrate that preserved global histone H4 acetylation is significantly associated with favorable outcome (RFS, EFS, OS) in children with B cell progenitor (BCP) ALL, recapitulating the findings from adult populations. Further, for the first time we demonstrate differential histone H4 acetylation in molecular subclasses of BCP-ALL including cases with ETV6-RUNX1 fusion gene or PAX5 deletion or deletions in genes linked to B cell development. We conclude global histone H4 acetylation is a prognostic marker and a potential therapeutic target in ALL.
    • A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation

      Sahni, Hemant; Ross, Susan; Barbarulo, Alessandro; Solanki, Anisha; Lau, Ching-In; Furmanski, Anna L.; Saldaña, José Ignacio; Ono, Masahiro; Hubank, Mike; Barenco, Martino; et al. (Impact Journals, 2015-09-20)
      Developing thymocytes require pre-TCR signalling to differentiate from CD4-CD8- double negative to CD4+CD8+ double positive cell. Here we followed the transcriptional response to pre-TCR signalling in a synchronised population of differentiating double negative thymocytes. This time series analysis revealed a complex transcriptional response, in which thousands of genes were up and down-regulated before changes in cell surface phenotype were detected. Genome-wide measurement of RNA degradation of individual genes showed great heterogeneity in the rate of degradation between different genes. We therefore used time course expression and degradation data and a genome wide transcriptional modelling (GWTM) strategy to model the transcriptional response of genes up-regulated on pre-TCR signal transduction. This analysis revealed five major temporally distinct transcriptional activities that up regulate transcription through time, whereas down-regulation of expression occurred in three waves. Our model thus placed known regulators in a temporal perspective, and in addition identified novel candidate regulators of thymocyte differentiation.
    • Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos

      Desai, Kunjan; Spikings, Emma; Zhang, Tiantian (Elsevier, 2015-08)
      Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6h chilling in methanol (0.2-, 0.5- and 1M). Subsequent experiments in embryos that had been chilled for 3h in 1M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3h in the presence of 1M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol.
    • Gli2, hedgehog and TCR signalling

      Furmanski, Anna L.; Crompton, Tessa (Impact Journals, 2015-07)
    • Degradation of mitochondrial DNA in cryoprotectant-treated hard coral (Echinopora spp.) oocytes

      Tsai, Sujune; Chen, Jiann-Chu; Spikings, Emma; Li, Jan-Jung; Lin, Chiahsin; Mingdao University; National Taiwan Ocean University; University of Bedfordshire; Nationall Museum Marine Biology & Aquarium, Taiwan; National Dong Hwa University (Informa Healthcare, 2015-06)
      A critical step for successful cryopreservation is to determine the optimal cryoprotectant treatment that can provide protective effects against cryoinjury during freezing and with minimal toxicity. Most cryoprotectants have chemical and osmotic effects when used at high concentrations. Cryoprotectants can damage coral mitochondrial distributions and membrane potentials, which results in reduced ATP production. As mitochondrial DNA (mtDNA) encodes for components of the electron transport chain (ETC) and plays a critical role in ATP synthesis capacity, we determined the effects of cryoprotectants on mtDNA in hard coral (Echinopora spp.) oocytes using quantitative real-time PCR. Our results showed that an insult from a cryoprotectant may be compensated for by the genetic defense mechanisms of these cells. Methanol was found to have the least effect on coral oocytes with regard to their energy status. A single oocyte without cryoprotectant treatment produced an average of 4,220,645 ± 169,990 mtDNA copies, which was greater than that in mammals. However, relatively lower mtDNA copy numbers (<2,000,000) were observed when oocytes were treated with dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG), or glycerol at a concentration of 3 M for 20 min. These results provide direct evidence that hard coral (Echinopora spp.) oocytes are extremely susceptible to cryoprotectants and support the concerns with regard to the adverse effects of cryoprotectants.
    • Short-term chilled storage of Zebrafish (Danio rerio) embryos in cryoprotectant as an alternative to cryopreservation

      Desai, Kunjan; Spikings, Emma; Zhang, Tiantian; Georgia Regents University; University of Bedfordshire; Bournemouth University (Mary Ann Liebert, 2015-02)
      As zebrafish embryos have never been cryopreserved, we developed a protocol to store zebrafish embryos (50% epiboly-5.3 hour post fertilization) for up to 18 h at 0°C. Initial experiments to optimize the cryoprotectant (CPA) solution demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH+0.1 M sucrose (56 ± 5%) compared with other combinations of methanol (0.2-0.5 M) and sucrose (0.05-0.1 M). This combination of CPAs that protects against chilling injury was further tested to assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH+0.1 sucrose compared with non-chilled controls, however the expression of both sox2 and sox3 proteins was unaffected. Significant decreases in sox2 protein expression were, however, observed in embryos that had been chilled without CPAs and these embryos also had lower hatching rates than those chilled with the optimal CPA solution. We, therefore, conclude that the CPA combination of 1 M MeOH+0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish embryos for up to 18 h without compromising transcriptional response.
    • Are there systemic comorbidities in haemophilia unrelated to bleeding and transfusion-transmitted infections?

      Janczar, Szymon; Wegner, O.; Kostrzewska, M.; Stolarska, M.; Paige, Adam J.W.; Mlynarski, Wojciech; Hematology and Diabetology Medical University of Lodz; University of Bedfordshire (Wiley, 2015-01)
    • The transcriptional activator Gli2 modulates T-cell receptor signalling through attenuation of AP-1 and NFκB activity

      Furmanski, Anna L.; Barbarulo, Alessandro; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Saldaña, José Ignacio; D’Acquisto, Fulvio; Crompton, Tessa; University College London; QMUL; et al. (The Company of Biologists Ltd, 2015)
      Different tissues contain diverse and dynamic cellular niches, providing distinct signals to tissue-resident or migratory infiltrating immune cells. Hedgehog (Hh) proteins are secreted inter-cellular signalling molecules, which are essential during development and are important in cancer, post-natal tissue homeostasis and repair. Hh signalling mediated by the Hh-responsive transcription factor Gli2 also has multiple roles in T-lymphocyte development and differentiation.Here, we investigate the function of Gli2 in T-cell signalling and activation. Gene transcription driven by the Gli2 transcriptional activator isoform (Gli2A) attenuated T-cell activation and proliferation following T-cell receptor (TCR) stimulation. Expression of Gli2A in T-cells altered gene expression profiles, impaired the TCR-induced Ca2+ flux and nuclear expression of NFAT2, suppressed upregulation of molecules essential for activation, and attenuated signalling pathways upstream of the AP-1 and NFκB complexes, leading to reduced activation of these important transcription factors. Inhibition of physiological Hh-dependent transcription increased NFκB activity upon TCR ligation. These data are important for nderstanding the molecular mechanisms of immunomodulation, particularly in tissues where Hh proteins or other Gli-activating ligands such as TGFβ are upregulated, including during inflammation, tissue damage and repair, and in tumour microenvironments.
    • Ultrastructural observations of the early and late stages of gorgonian coral (Junceella juncea) oocytes

      Tsai, Sujune; Jhuang, Yating; Spikings, Emma; Sung, Ping-Jyun; Lin, Chiahsin; Mingdao University; National Dong Hwa University; University of Bedfordshire (Elsevier, 2014-08)
      The developmental oogenesis of gorgonian coral was investigated at the histological level. The objective of this study was to examine and improve the understanding of Junceella juncea oogenesis using ultrastructural methods, such as histological sectioning and transmission electron microscopy. At least three types of yolk materials were observed in this study: yolk body, lipid granules and cortical alveoli. Some of the complex yolk materials were encompassed by concentric or arched layers of smooth and rough endoplasmic reticulum and the Golgi complex in early stage oocytes. Different types of vesicles were found in both early and late stage oocytes and some granules could be seen inside the empty vesicles. This may be a possible method for elaborating complex yolk materials. Homogeneous yolks from different types of inclusions were abundant and the autosynthesis of yolk may be a major mechanism in J. juncea oocytes. This is the first report of the ultrastructural observation of oogenesis in gorgonian coral species using transmission electron microscopy. Our study obtained relatively detailed information at the ultrastructural level, and it provides an overview of the oocyte ultrastucture of the gorgonian coral J. juncea.
    • Pre-hybridisation: an efficient way of suppressing endogenous biotin-binding activity inherent to biotin–streptavidin detection system

      Ahmed, Raju; Spikings, Emma; Zhou, Shaobo; Thompsett, Andrew; Zhang, Tiantian; University of Bedfordshire; University of East London; Bournemouth University (Elsevier, 2014-04)
      Endogenous biotin or biotinylated protein binding activity is a major drawback to biotin-avidin/streptavidin detection system. The avidin/streptavidin conjugate used to detect the complex of the biotinylated secondary antibody and the primary antibody binds to endogenous biotin or biotinylated proteins leading to non-specific signals. In Western blot, the endogenous biotin or biotinylated protein binding activity is usually manifested in the form of ~72kDa, ~75kDa and ~150kDa protein bands, which often mask the signals of interest. To overcome this problem, a method based on prior hybridisation of the biotinylated secondary antibody and the streptavidin conjugate was developed. The method was tested alongside the conventional biotin-streptavidin method on proteins extracted from zebrafish (Danio rerio) embryos. Results showed that the newly developed method efficiently suppresses the endogenous biotin or biotinylated protein binding activity inherent to the biotin-streptavidin detection system.
    • The role of caspases in Parkinson’s Disease pathogenesis: a brief look at the mitochondrial pathway

      Chaudhry, Zahara Latif; Ahmed, Bushra Y.; University of Bedfordshire (Austin Publishing Group, 2014)
      Parkinson’s disease (PD) is a neurodegenerative disorder characterised by tremor, rigidity, Bradykinesia and reduced facial expression. Development of PD is considered to be the result of deficiency of the neurotransmitter dopamine, which is due to death of Dopamine-Containing Neurons (DCNs) that produce dopamine in the pars compacta region of the substantia nigra. Although the concentrated efforts of the scientific community over the last decades, the etiology of the death of DCN is yet to be understood. Oxidative stress has been considered as one of the causes of defects in the mitochondria leading to the dopaminergic cell damage [1]. Levodopa therapy is a well-known treatment for the symptoms of PD, however long term use of L-dopa causes side effects including further enhancement of oxidative stress [2]. The elevated levels of Reactive Oxidative Species (ROS) such as hydrogen peroxide, superoxide and hydroxyl ions, induce stimulation to the Permeability Transition Pore (mPTP) of the mitochondria leading to the collapse of the mitochondrial membrane potential and the release of cytochrome C. Furthermore, increased ROS activity promote nitric oxide binding to superoxide producing peroxynitrate enhancing oxidative and nitrosative stress, which results in DNA damage, chromosomal mutations, lipid peroxidation and enzyme defects [3]. Mutation of E3 ligase caused by peroxynitrate damage leads to impairment of ubiquitin-proteasome system, resulting in high levels of defective proteins, which accumulate in the Endoplasmic Reticulum (ER) promoting ER stress and ultimately cell death. Moreover, the apoptotic neuron triggers injury signals that activate microglia and promote release of cytokines such as interleukins-6 and -8. Subsequently, interleukins trigger Caspase activation along with inducible NO synthase, which further elevates formation of nitric oxide. Exposure to excessive reactive nitrite species along with enhanced production of ROS and peroxynitrate lead to dysfunction of complex-IV and complex-I activities of the mitochondria and promote mitochondrialmediated apoptosis through Caspase activation [4,5].
    • Tissue-derived hedgehog proteins modulate Th differentiation and disease

      Furmanski, Anna L.; Saldaña, José Ignacio; Ono, Masahiro; Sahni, Hemant; Paschalidis, Nikolaos; D'Acquisto, Fulvio; Crompton, Tessa; University College London (American Association for Immunologists, 2013-02-13)
      Genome-wide association studies of complex immune-mediated diseases have indicated that many genetic factors, each with individual low risk, contribute to overall disease. It is therefore timely and important to characterize how immune responses may be subtly modified by tissue context. In this article, we explore the role of tissue-derived molecules in influencing the function of T cells, which, owing to their migratory nature, come into contact with many different microenvironments through their lifespan. Hedgehog (Hh) proteins act as secreted morphogens, providing concentration-dependent positional and temporal cell-fate specification in solid tissues. Hh signaling is required for embryogenesis and is important in postnatal tissue renewal and in malignancy. However, the function of Hh in dynamic, fluid systems, such as in mammalian immunity, is largely unknown. In this article, we show that Hh-dependent transcription in T cells promoted Th2 transcriptional programs and differentiation, exacerbating allergic disease. Of interest, expression of Sonic Hh increased in lung epithelial cells following the induction of allergic disease, and lung T cells upregulated Hh target gene expression, indicating that T cells respond to locally secreted Hh ligands in vivo. We show that Il4, the key Th2 cytokine, is a novel transcriptional target of Hh signals in T cells, providing one mechanism for the role of Hh in Th differentiation. We propose that Hh, secreted from inflamed, remodeling, or malignant tissue, can modulate local T cell function. Our data present an unexpected and novel role for tissue-derived morphogens in the regulation of fluid immune responses, with implications for allergy and tumor responses, suggesting new uses for anti-Hh therapeutics.