• 155. Effect of methanol and DMSO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish Danio rerio

      Zampolla, Tiziana; Spikings, Emma; Zhang, Tiantian; Rawson, David M.; University of Bedfordshire (Elsevier, 2009-12)
    • 160. Effect of cryoprotectant treatment and chilling on oxidative stress in zebrafish (Danio rerio) early ovarian follicles

      Ghafari, Fataneh; Spikings, Emma; Rawson, David M.; Zhang, Tiantian; University of Bedfordshire (Elsevier, 2009-12)
    • 8th international conference on oncolytic virus therapeutics

      Guinn, Barbara-Ann; Braidwood, Lynne; Parker, Alan; Peng, Kah-Whye; Seymour, Leonard (Mary Ann Liebert, 2014-12)
      The 8th International Conference on Oncolytic Virus Therapeutics meeting was held from April 10-13, 2014, in Oxford, United Kingdom. It brought together experts in the field of oncolytics from Europe, Asia, Australasia, and the Americas and provided a unique opportunity to hear the latest research findings in oncolytic virotherapy. Presentations of recent work were delivered in an informal and intimate setting afforded by a small group of attendees and an exquisitely focused conference topic. Here we describe the oral presentations and enable the reader to share in the benefits of bringing together experts to share their findings.
    • Agrobacterium-mediated transformation and insertional mutagenesis in colletotrichum acutatum for investigating varied pathogenicity lifestyles

      Talhinhas, Pedro; Muthumeenakshi, S.; Neves-Martins, João; Oliveira, Helena; Sreenivasaprasad, Surapareddy; Technical University of Lisbon; University of Warwick (Humana Press, 2008)
      Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10(4) conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.
    • Amino acid cycling by Rhizobium leguminosarum in pea nodules

      White, James P.; Bourdes, Alex; Hosie, Arthur H.F.; Kinghorn, Seonag; Poole, Philip S. (Springer, 2005)
    • Amino-acid cycling drives nitrogen fixation in the legume–Rhizobium symbiosis

      Lodwig, Emma M.; Hosie, Arthur H.F.; Bourdes, Alex; Findlay, K.; Allaway, D.; Karunakaran, R.; Downie, J. A.; Poole, Philip S. (Nature Publishing Group, 2003)
      The biological reduction of atmospheric N2 to ammonium (nitrogen fixation) provides about 65% of the biosphere's available nitrogen. Most of this ammonium is contributed by legume–rhizobia symbioses1, which are initiated by the infection of legume hosts by bacteria (rhizobia), resulting in formation of root nodules. Within the nodules, rhizobia are found as bacteroids, which perform the nitrogen fixation: to do this, they obtain sources of carbon and energy from the plant, in the form of dicarboxylic acids2, 3. It has been thought that, in return, bacteroids simply provide the plant with ammonium. But here the authors show that a more complex amino-acid cycle is essential for symbiotic nitrogen fixation by Rhizobium in pea nodules. The plant provides amino acids to the bacteroids, enabling them to shut down their ammonium assimilation. In return, bacteroids act like plant organelles to cycle amino acids back to the plant for asparagine synthesis. The mutual dependence of this exchange prevents the symbiosis being dominated by the plant, and provides a selective pressure for the evolution of mutualism.
    • An analogue peptide from the cancer testis antigen, PASD1, induces CD8+ T cell-responses against naturally processed peptide.

      Hardwick, Nicola R.; Buchan, Sarah; Ingram, Wendy; Khan, Ghazala; Vittes, Gisella; Rice, Jason; Pulford, Karen; Mufti, Ghulam J.; Stevenson, Freda; Guinn, Barbara-Ann; et al. (Cancer Research Institute, 2013)
      We have previously identified the novel Cancer/Testis antigen PASD1 by immunoscreening a testis library with pooled acute myeloid leukemia (AML) patient sera. To develop a cytotoxic T lymphocyte (CTL)-inducing vaccine, we have now investigated the carboxy-terminal region, known to contain serological determinants, for MHC class I (HLA-A⋆0201)-binding peptides. Algorithm-selected natural peptides failed to show detectable HLA-A⋆0201 binding in T2 assays. However, anchor-modified analogue peptides showed enhanced binding, with decreased off-rates. Analogue peptide-loaded antigen-presenting cells (APCs) induced IFN-γ production by T cells from normal donors and patients. In addition, peptide-specific T cells could be expanded from cancer patients by stimulation with the PASD1 analogue peptide Pa14. For clinical application, a DNA fusion gene vaccine encoding Pa14 was designed and tested in "humanized" mice. Splenocytes from vaccinated mice showed in vitro cytotoxicity against tumour cells, either exogenously loaded with the corresponding wild-type peptide (Pw8) or expressing endogenously processed PASD1 protein. We show for the first time that a DNA vaccine encoding an altered PASD1 epitope can induce CTLs to target the natural peptide expressed by human tumour cells.
    • Analogue peptides for the immunotherapy of human acute myeloid leukemia

      Hofmann, Susanne; Mead, Andrew; Malinovskis, Aleksandrs; Hardwick, Nicola R.; Guinn, Barbara-Ann; University of Bedfordshire (Springer, 2015-10-05)
      The use of peptide vaccines, enhanced by adjuvants, has shown some efficacy in clinical trials. However, responses are often short-lived and rarely induce notable memory responses. The reason is that self-antigens have already been presented to the immune system as the tumor develops, leading to tolerance or some degree of host tumor cell destruction. To try to break tolerance against self-antigens, one of the methods employed has been to modify peptides at the anchor residues to enhance their ability to bind major histocompatibility complex molecules, extending their exposure to the T-cell receptor. These modified or analogue peptides have been investigated as stimulators of the immune system in patients with different cancers with variable but sometimes notable success. In this review we describe the background and recent developments in the use of analogue peptides for the immunotherapy of acute myeloid leukemia describing knowledge useful for the application of analogue peptide treatments for other malignancies.
    • The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells.

      St John, Justin C.; Amaral, Alexandra; Bowles, Emma J.; Oliveira, João Facucho; Lloyd, Rhiannon E.I.; Freitas, Mariana; Gray, Heather L.; Navara, Christopher S.; Oliveira, Gisela; Schatten, Gerald P.; et al. (Humana Press, 2006)
      As human embryonic stem cells (hESCs) undergo differentiation, they express genes characteristic of the lineage for which they are destined. However, fully differentiated individual cell types can be characterized by the number of mitochondria they possess and the copies of the mitochondrial genome per mitochondrion. These characteristics are indicative of a specific cell's requirement for adenosine triphosphate (ATP) and therefore cellular viability and function. Consequently, failure for an ESC to possess the full complement of mitochondria and mitochondrial DNA (mtDNA) could limit its final commitment to a particular fate. We describe a series of protocols that analyze the process of cellular mitochondrial and mtDNA differentiation during hESC differentiation. In addition, mtDNA transcription and replication are key events in cellular differentiation that require interaction between the nucleus and the mitochondrion. To this extent, we describe a series of protocols that analyze the initiation of these key events as hESCs progress from their undifferentiated state to the fully committed cell. Last, we describe real-time polymerase chain reaction protocols that allow both the identification of mtDNA copy number and determine whether mtDNA copy is uniform (homoplasmy) in its transmission or heterogeneous (heteroplasmy).
    • Anti-tumor immunity in a model of acute myeloid leukemia

      Cheuk, Adam T.C.; Wells, James W.; Chan, Lucas; Westwood, Nigel B.; Berger, Stuart A.; Yagita, Hideo; Okumura, Ko; Farzaneh, Farzin; Mufti, Ghulam J.; Guinn, Barbara-Ann; et al. (informa healthcare, 2009-03)
      Whole-cell vaccines allow the induction of anti-tumor immune responses without the need to define tumor antigens. We wished to directly compare, for the first time, the capacity of B7-1, B7-2 and 4-1BB ligand (4-1BBL) costimulatory molecules to convert murine and human acute myeloid leukemia (AML) cells into whole vaccines. 32Dc-kit is a murine myeloid cell line, which develops an AML-like disease over a protracted period, emulating human AML disease development. 32Dc-kit cells were modified to express elevated levels of B7-1, B7-2 or 4-1BBL, and each led to tumor rejection, although only mice injected with 32Dc-kit/B7-2 cells were able to reject subsequent parental tumor cell challenge. T-cell deficient nude mice were able to reject the 32Dc-kit variants, but they could not reject parental cell challenge; however, we found no evidence of cytotoxic T lymphocyte or natural killer (NK) activity ex vivo suggesting that tumor cell killing was mediated by an immune response that could not be recapitulated using purified NK or T cells as lone effectors. In human allogeneic mixed lymphocyte reactions (MLRs), we found no single costimulatory molecule was more effective, suggesting that the induction of a universal anti-tumor response will require a combination of costimulatory molecules.
    • Antitumor effects of saponin extract from Patrinia villosa (Thunb.) Juss on mice bearing U14 cervical cancer

      Zhang, Tao; Li, Qingwang; Li, Kun; Li, Yurong; Li, Jian; Wang, Guijie; Zhou, Shaobo (John Wiley and Sons, 2008-03)
    • Application of the pMHC array to characterise tumour antigen specific T cell populations in leukaemia patients at disease diagnosis

      Brooks, Suzanne E.; Bonney, Stephanie A.; Lee, Cindy; Publicover, Amy; Khan, Ghazala; Smits, Evelien L.J.; Sigurdardottir, Dagmar; Arno, Matthew; Li, Demin; Mills, Ken I.; et al. (Public Library of Science, 2015-10-22)
      Immunotherapy treatments for cancer are becoming increasingly successful, however to further improve our understanding of the T-cell recognition involved in effective responses and to encourage moves towards the development of personalised treatments for leukaemia immunotherapy, precise antigenic targets in individual patients have been identified. Cellular arrays using peptide-MHC (pMHC) tetramers allow the simultaneous detection of different antigen specific T-cell populations naturally circulating in patients and normal donors. We have developed the pMHC array to detect CD8+ T-cell populations in leukaemia patients that recognise epitopes within viral antigens (cytomegalovirus (CMV) and influenza (Flu)) and leukaemia antigens (including Per Arnt Sim domain 1 (PASD1), MelanA, Wilms’ Tumour (WT1) and tyrosinase). We show that the pMHC array is at least as sensitive as flow cytometry and has the potential to rapidly identify more than 40 specific T-cell populations in a small sample of T-cells (0.8–1.4 x 106). Fourteen of the twenty-six acute myeloid leukaemia (AML) patients analysed had T cells that recognised tumour antigen epitopes, and eight of these recognised PASD1 epitopes. Other tumour epitopes recognised were MelanA (n = 3), tyrosinase (n = 3) and WT1126-134 (n = 1). One of the seven acute lymphocytic leukaemia (ALL) patients analysed had T cells that recognised the MUC1950-958 epitope. In the future the pMHC array may be used provide point of care T-cell analyses, predict patient response to conventional therapy and direct personalised immunotherapy for patients.
    • 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)
    • Bacterial ABC transporters of amino acids

      Hosie, Arthur H.F.; Poole, Philip S. (Elsevier, 2001)
      There are two subfamilies of ABC uptake systems for amino acids in bacteria, the polar amino acid transport family and the hydrophobic amino acid transport family. We consider the general properties of these families and we examine the specific transporters. Focusing on some of the best-studied ATP binding cassette transporters we also examine the mechanism of amino acid uptake, paying particular attention to the question of bidirectionality of solute movement.
    • Binding modes of diketo-acid inhibitors of HIV-1 integrase: a comparative molecular dynamics simulation study.

      Huang, Meilan; Grant, Guy H.; Richards, W. Graham; Queens University Belfast (Elsevier, 2011-06)
      HIV-1 integrase (IN) has become an attractive target since drug resistance against HIV-1 reverse transcriptase (RT) and protease (PR) has appeared. Diketo acid (DKA) inhibitors are potent and selective inhibitors of HIV-1 IN: however the action mechanism is not well understood. Here, to study the inhibition mechanism of DKAs we performed 10 ns comparative molecular dynamics simulations on HIV-1 IN bound with three most representative DKA inhibitors: Shionogi inhibitor, S-1360 and two Merck inhibitors L-731,988 and L-708,906. Our simulations show that the acidic part of S-1360 formed salt bridge and cation-π interactions with Lys159. In addition, the catalytic Glu152 in S-1360 was pushed away from the active site to form an ion-pair interaction with Arg199. The Merck inhibitors can maintain either one or both of these ion-pair interaction features. The difference in potencies of the DKA inhibitors is thus attributed to the different binding modes at the catalytic site. Such structural information at atomic level, not only demonstrates the action modes of DKA inhibitors but also provides a novel starting point for structural-based design of HIV-1 IN inhibitors.
    • Bring the captive closer to the wild: redefining the role of ex situ conservation

      Pritchard, Diana J.; Fa, John E.; Oldfield, Sara; Harrop, Stuart R.; University of Sussex; Durrell Wildlife Conservation Trust; Imperial College London; Botanical Gardens Conservation International; University of Kent (Wiley Blackwell, 2011)
      In situ conservation is central to contemporary global biodiversity protection and is the predominant emphasis of international regulation and funding strategies. Ex situ approaches, in contrast, have been relegated to a subsidiary role and their direct contributions to conservation have been limited. We draw on a variety of sources to make the case for an enhanced role for ex situ conservation. We note the advances occurring within institutions specializing in ex situ conservation and stress that, although much remains to be done, many constraints are being addressed. We argue that the evidence of increasing extinction rates, exacerbated by climate change, challenges the wisdom of a heavy dependence on in situ strategies and necessitates increased development of ex situ approaches. A number of different techniques that enable species and their habitats to survive should now be explored. These could build on the experience of management systems that have already demonstrated the effective integration of in situ and ex situ techniques and hybrid approaches.
    • The C-4 stereochemistry of leucocyanidin substrates for anthocyanidin synthase affects product selectivity

      Turnbull, Jonathan J.; Nagle, Michael J.; Seibel, Jürgen F.; Welford, Richard W.D.; Grant, Guy H.; Schofield, Christopher J. (Elsevier, 2003)
      Anthocyanidin synthase (ANS), an iron(II) and 2-oxoglutarate (2OG) dependent oxygenase, catalyses the penultimate step in anthocyanin biosynthesis by oxidation of the 2R,3S,4S-cis-leucoanthocyanidins. It has been believed that in vivo the products of ANS are the anthocyanidins. However, in vitro studies on ANS using optically active cis- and trans-leucocyanidin substrates identified cyanidin as only a minor product; instead both quercetin and dihydroquercetin are products with the distribution being dependent on the C-4 stereochemistry of the leucocyanidin substrates.
    • Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-Kinase C2  on neurosecretory vesicles

      Wen, P.J.; Osborne, S.L.; Morrow, I.C.; Parton, R.G.; Domin, Jan; Meunier, F.A. (American Society for Cell Biology, 2008-12)