Biochemistry, Biophysics, and Structural Biology Commons^™

Ultrasound 96 Probe Device Protocol For Cancer Cell Treatment, Aisling Field, Brijesh K. Tiwari, James F. Curtin, Julie R M Mondala, Janith Wanigasekara

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Ultrasound is a sound wave with frequencies ranging between 20 kHz and 20 MHz. Ultrasound is able to temporarily and repeatedly open the BBB safely and enhance chemotherapeutic delivery without adverse effects. This novel technique in drug delivery benefits from the powerful ability of ultrasound to produce cavitation activity. Cavitation is the generation and activity of gas-filled bubbles in a medium exposed to ultrasound. As the pressure wave passes through the media, gas bubbles expand at low pressure and contract at high pressure. This leads to oscillation which produces a circulating fluid flow known as microstreaming around the bubble with …

Plasma Induced Reactive Oxygen Species-Dependent Cytotoxicity In Glioblastoma 3d Tumourspheres, Janith Wanigasekara, Carlos Barcia, Patrick J. Cullen, Brijesh Tiwari, James F. Curtin

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The aim of this study was to determine the effects of a pin‐to‐plate cold atmospheric plasma (CAP) on U‐251 MG three‐dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species(ROS) plays a major role, while H2O2plays a partial role in CAP‐induced cytotoxicity in tumour-spheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid …

Converging Technologies: Targeting The Hallmarks Of Cancer Using Ultrasound And Microbubbles, Janith Wanigasekara, Andressa Maria Aguiar De Carvalho, Patrick J. Cullen, Brijesh Tiwari, James F. Curtin

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Various complex biological effects occur when ultrasonic compression waves travel through biological material. The myriad of biological outcomes instigated by ultrasound are evident when viewed through the lens of the hallmarks of cancer. Herein, we summarise the therapeutic potential of ultrasound, enhanced by microbubbles, for the treatment of cancer.

Optimisation Of Estrogen Receptor Subtype-Selectivity Of A 4-Aryl-4h-Chromene Scaffold Previously Identified By Virtual Screening, Miriam Carr, Andrew Knox, Daniel Nevin, Niamh O'Boyle, Shu Wang, Billy Egan, Thomas Mccabe, Brendan Twamley, Daniela Zisterer, David Lloyd, Mary Meegan

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4-Aryl-4H-Chromene derivatives have been previously shown to exhibit anti-proliferative, apoptotic and anti-angiogenic activity in a variety of tumor models in vitro and in vivo generally via activation of caspases through inhibition of tubulin polymerisation. We have previously identified by Virtual Screening (VS) a 4-aryl-4H-chromene scaffold, of which two examples were shown to bind Estrogen Receptor α and β with low nanomolar affinity and <20-fold selectivity for α over β and low micromolar anti-proliferative activity in the MCF-7 cell line. Thus, using the 4-aryl-4H-chromene scaffold as a starting point, a series of compounds with a range of basic arylethers at C-4 and modifications at the C3-ester substituent of the benzopyran ring were synthesised, producing some potent ER antagonists in the MCF-7 cell line which were highly selective for ERα (compound 35; 350-fold selectivity) or ERβ (compound 42; 170-fold selectivity).

Urban Aerobiological Risk Mapping Of Ornamental Trees Using A New Index Based On Lidar And Kriging: A Case Study Of Plane Trees, Raúl Pecero-Casimiro, Santiago Fernández-Rodríguez, Rafael Tormo-Molina, Alejandro Monroy-Colín, Inmaculada Silva-Palacios, Juan Pedro Cortés-Pérez, Ángela Gonzalo-Garijo, J. M. Maya-Manzano

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Ornamental trees bring benefits for human health, including reducing urban pollution. However, some species, such as plane trees (Platanus sp.), produce allergenic pollen. Consequently, urban maps are a valuable tool for allergic patients and allergists, but they often fail to include variables that contribute to the “building downwash effect”, such as the width and shape of streets and the height of buildings. Other factors that directly influence pollen dispersion (slopes and other geographical features) also have not traditionally been discussed. The LiDAR (Laser Imaging Detection and Ranging) technique enables one to consider these variables with high accuracy. This work proposes …

Investigating The Role Of Gold Nanoparticle Shape And Size In Their Toxicities To Fungi, Kangze Liu, Zhonglei He, Hugh Byrne, James Curtin, Furong Tian Technological University Dublin

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Gold nanoparticles (GNPs) are increasingly being used in a wide range of applications, and such they are being released in greater quantities into the environment. Consequently, the environmental effects of GNPs, especially toxicities to living organisms, have drawn great attention. However, their toxicological characteristics still remain unclear. Fungi, as the decomposers of the ecosystem, interact directly with the environment and critically control the overall health of the biosphere. Thus, their sensitivity to GNP toxicity is particularly important. The aim of this study was to evaluate the role of GNP shape and size in their toxicities to fungi, which could help …

Raman Spectral Analysis For Rapid Screening Of Dengue Infection, Tahir Mahmood, Haq Nawaz, A. Ditta, M.I. Majeed, M.A. Hanif, N. Rashid, H.N. Bhatti, H.F. Nargis, M. Saleem, Franck Bonnier, Hugh Byrne

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Infection with the dengue virus is currently clinically detected according to different biomarkers in human blood plasma, commonly measured by enzyme linked immunosorbent assays, including non-structural proteins (Ns1), immunoglobulin M (IgM) and immunoglobulin G (IgG). However, there is little or no mutual correlation between the biomarkers, as demonstrated in this study by a comparison of their levels in samples from 17 patients. As an alternative, the label free, rapid screening technique, Raman spectroscopy has been used for the characterisation/diagnosis of healthy and dengue infected human blood plasma samples. In dengue positive samples, changes in specific Raman spectral bands associated with …

Clinical Applications Of Infrared And Raman Spectroscopy: State Of Play And Future Challenges, Matthew Baker, Hugh Byrne, John Chalmers, Peter Gardner, Royston Goodacre, Alex Henderson, Sergei Kazarian, Francis Martin, Julian Moger, Nick Stone, Josep Sule-Suso

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Vibrational spectroscopies, based on Infrared absorption and/or Raman scattering provide a detailed fingerprint of a material, based on the chemical content. Diagnostic and prognostic tools based on these technologies have the potential to revolutionise our clinical systems leading to improved patient outcome, more efficient public services and significant economic savings. However, despite these strong drivers, there are many fundamental scientific and technological challenges which have limited the implementation of this technology in the clinical arena, although recent years have seen significant progress in addressing these challenges. This review examines (i) the state of the art of clinical applications of infrared …

Toxicological Assessment Of Nanomaterials: The Role Of In Vitro Raman Microspectroscopic Analysis, Esen Efeoglu, Marcus Maher, Alan Casey, Hugh Byrne

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The acceleration of nanomaterials research has brought about increased demands for rapid analysis of their bioactivity, in a multi-parametric fashion, to minimise the gap between potential applications and knowledge of their toxicological properties. The potential of Raman microspectroscopy for the analysis of biological systems with the aid of multivariate analysis techniques has been demonstrated. In this study, an overview of recent efforts towards establishing a ‘label-free high content nanotoxicological assessment technique’ using Raman microspectroscopy is presented. The current state of the art for cellular toxicity assessment and the potential of Raman microspectroscopy are discussed, and the spectral markers of the …

Isolation And Spectroscopic Characterization Of Zn(Ii), Cu(Ii), And Pd(Ii) Complexes Of 1,3,4-Thiadiazole-Derived Ligand, Dariusz Karcz, Arkadiusz Matwijczuk, Bozena Boron, Bernadette Creaven, Leszek Fiedor, Andrzej Niewiadomy, Mariusz Gagos

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A series of complexes incorporating Zn(II), Cu(II), and Pd(II) ions, and 4-(5-heptyl-1,3,4-thiadiazol-2-yl)benzene-1,3-diol (L1) as model ligand, was synthesized in order to examine the nature of potential interactions between biologically active ligands, 1,3,4-thiadiazoles and metal ions with proven biological relevance. The structures of the compounds isolated were characterized using a number of spectroscopic methods including IR, Uv–vis, AAS, steady state and time-resolved fluorescence (TRF). The results obtained suggest that the L1-Zn(II) and L1-Pd(II) complexes consist of one molecule of L1 and one acetate ion acting as ligands, while the L1-Cu(II) complex adapts a 2:1 (L1: metal) stoichiometry. The coordination of L1 …

Piperlongumine (Piplartine) And Analogues: Antiproliferative Microtubule-Destabilising Agents, Mary J. Meegan, Seema M. Nathwani, Brendan Twamley, Daniela M. Zisterer, Niamh O'Boyle

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Piperlongumine (piplartine, 1) is a small molecule alkaloid that is receiving intense interest due to its antiproliferative and anticancer activities. We investigated the effects of 1 on tubulin and microtubules. Using both an isolated tubulin assay, and a combination of sedimentation and Western blotting, we demonstrated that 1 is a tubulin-destabilising agent. This result was confirmed by immunofluorescence and confocal microscopy, which showed that microtubules in MCF-7 breast cancer cells were depolymerised when treated with 1. We synthesised a number of analogues of 1 to explore structure-activity relationships. Compound 13 had the best cytotoxic profile of this series, …

Synthesis And Biochemical Evaluation Of 3-Phenoxy-1,4-Diarylazetidin-2-Ones As Tubulin-Targeting Antitumor Agents, Thomas F. Greene, Shu Wang, Lisa M. Greene, Seema M. Nathwani, Jade K. Pollock, Azizah M. Malebari, Thomas Mccabe, Brendan Twamley, Niamh O'Boyle, Daniela M. Zisterer, Mary J. Meegan

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Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl ‘A’ ring and 4-phenyl ‘B’ ring for potent antiproliferative activity, and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC₅₀ values of 38 nM and 19 nM respectively in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular …

Artemisinin Production By Plant Hairy Root Cultures In Gas- And Liquid-Phase Bioreactors, Nivedita Patra, Ashok K. Srivastava

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Key message

Alternative biotechnological protocol for large-scale artemisinin production was established. It featured enhanced growth and artemisinin production by cultivation of hairy roots in nutrient mist bioreactor (NMB) coupled with novel cultivation strategies.

Artemisinin is used for the treatment of cerebral malaria. Presently, its main source is from seasonal plant Artemisia annua. This study featured investigation of growth and artemisinin production by A. annua hairy roots (induced by Agrobacterium rhizogenes-mediated genetic transformation of explants) in three bioreactor configurations—bubble column reactor, NMB and modified NMB particularly to establish their suitability for commercial production. It was observed that cultivation of …

Β-Lactam Estrogen Receptor Antagonists And A Dual-Targeting Estrogen Receptor/Tubulin Ligand, Niamh O'Boyle, Jade K. Pollock, Miriam Carr, Andrew Js Knox, Seema M. Nathwani, Shu Wang, Laura Caboni, Daniela M. Zisterer, Mary Meegan

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Twelve novel β-lactams were synthesised and their antiproliferative effects and binding affinity for the predominant isoforms of the estrogen receptor (ER), ERα and ERβ, were determined. β-Lactams 23 and 26 had the strongest binding affinities for ERα (IC₅₀ values: 40 and 8 nM respectively) and ERβ (IC₅₀ values: 19 and 15 nM). β-Lactam 26 was the most potent in antiproliferative assays using MCF-7 breast cancer cells, and further biochemical analysis showed that it caused accumulation of cells in G₂/M phase (mitotic blockade) and depolymerisation of tubulin in MCF-7 cells. Compound 26 also induced apoptosis and downregulation …

Raman Spectroscopic Analysis Of Human Skin Tissue Sections Ex-Vivo: Evaluation Of The Effects Of Tissue Processing And Dewaxing, Syed Mehmood Ali, Franck Bonnier, Ali Tfayli, Helen Lambkin, Kathleen Flynn, Vincent Mcdonagh, Claragh Healy, Thomas Lee, Fiona Lyng, Hugh Byrne

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Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections, and the effects of tissue processing. Both hand and thigh sections of human cadavers were analysed in their unprocessed and formalin fixed paraffin processed (FFPP) and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum, intermediate underlying epithelium and dermal layers for sections from both anatomical sites. The stratum corneum is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that …

Synthesis And Biochemical Activities Of Antiproliferative Amino Acid And Phosphate Derivatives Of Microtubule-Disrupting Beta-Lactam Combretastatins, Niamh M. O'Boyle, Lisa M. Greene, Niall O. Keely, Shu Wang, Tadhg S. Cotter, Daniela M. Zisterer, Mary J. Meegan

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The synthesis and biochemical activities of novel water-soluble β-lactam analogues of combretastatin A-4 are described. The first series of compounds investigated, β-lactam phosphate esters 7a, 8a and 9a, exhibited potent antiproliferative activity and caused microtubule disruption in human breast carcinoma-derived MCF-7 cells. They did not inhibit tubulin polymerisation in vitro, indicating that biotransformation was necessary for their antiproliferative and tubulin binding effects in MCF-7 cells. The second series of compounds, β-lactam amino acid amides (including 10k and 11l) displayed potent antiproliferative activity in MCF-7 cells, disrupted microtubules in MCF-7 cells and also inhibited the polymerisation of …

Novel Cis-Restricted Β-Lactam Combretastatin A-4 Analogues Display Anti-Vascular And Anti-Metastatic Properties In Vitro, Seema M. Nathwani, Lisa M. Greene, Linda Hughes, Miriam Carr, Niamh O'Boyle, Susan Mcdonnell, Mary J. Meegan, Daniela M. Zisterer

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No abstract provided.

Electric Field Standing Wave Effects In Ft-Ir Transflection Spectra Of Biological Tissue Sections: Simulated Models Of Experimental Variability, Tomasz P. Wrobel, Barbara Wajnchold, Hugh Byrne, Malgorzata Baranska

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The so-called electric field standing wave effect (EFSW) has recently been demonstrated to significantly distort FT-IR spectra acquired in a transflection mode, both experimentally and in simulated models, bringing into question the appropriateness of the technique for sample characterization, particularly in the field of spectroscopy of biological materials. The predicted effects are most notable in the regime where the sample thickness is comparable to the source wavelength. In this work, the model is extended to sample thicknesses more representative of biological tissue sections and to include typical experimental factors which are demonstrated to reduce the predicted effects. These include integration …

Quantitative Reagent-Free Detection Of Fibrinogen Levels In Human Blood Plasma Using Raman Spectroscopy, Kelvin Poon, Fiona Lyng, Peter Knief, Orla L. Howe, Aidan Meade, James Curtin, Hugh Byrne, Joseph Vaughan

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Fibrinogen assays are commonly used as part of clinical screening tests to investigate haemorrhagic states, for detection of disseminated intravascular coagulation and as a predictor of a variety of cardiovascular events. The Clauss assay, which measures thrombin clotting time, is the most commonly used method for measuring fibrinogen levels. Nevertheless, inconsistencies are present in inter-manufacturer reagent sources, calibration standards and methodologies. Automated coagulation analysers, which measure changes in optical density during the prothrombin time (PT-Fg), have found use in many hospitals. However, the PT-Fg method is found to give falsely elevated values due to varying choices of calibrants, reagents and …

Lead Identification Of Β-Lactam And Related Imine Inhibitors Of The Molecular Caperone Heat Shock Protein 90, Niamh O'Boyle, Andrew Js Knox, Trevor P. Price, D. Clive Williams, Daniela M. Zisterer, David G. Lloyd, Mary J. Meegan

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Heat shock protein 90 is an emerging target for oncology therapeutics. Inhibitors of this molecular chaperone, which is responsible for the maintenance of a number of oncogenic proteins, have shown promise in clinical trials and represent a new and exciting area in the treatment of cancer. Heat shock protein 90 inhibitors have huge structural diversity, and here we present the identification of inhibitors based on β-lactam and imine templates. β-Lactam 5 and imines 12 and 18 exhibit binding to heat shock protein 90-α with IC₅₀ values of 5.6 μM, 14.5 μM and 22.1 μM respectively. The binding affinity displayed …

Synthesis, Biochemical And Molecular Modelling Studies Of Antiproliferative Azetidinones Causing Microtubule Disruption And Mitotic Catastrophe, Niamh O'Boyle, Miriam Carr, Lisa M. Greene, Niall O. Keely, Andrew Js Knox, Thomas Mccabe, David G. Lloyd, Daniela M. Zisterer, Mary J. Meegan

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The structure-activity relationships of antiproliferative β-lactams, focusing on modifications at the 4-position of the β-lactam ring, is described. Synthesis of this series of compounds was achieved utilizing the Staudinger and Reformatsky reactions. The antiproliferative activity was assessed in MCF-7 cells, where the 4-(4-ethoxy)phenyl substituted compound 26 displayed the most potent activity with an IC₅₀ value of 0.22 μM. The mechanism of action was demonstrated to be by inhibition of tubulin. Cell exposure to combretastatin A-4 and 26 led to arrest of MCF-7 cells in the G2/M phase of the cell cycle and induction of apoptosis. Additionally, mitotic catastrophe for …

Genetic Connections Between Neurological Disorders And Cholesterol Metabolism, Ingemar Bjorkhem, Valerio Leoni, Steve Meaney

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Cholesterol is an essential component of both the peripheral and central nervous systems of mammals. Over the last decade, evidence has accumulated that disturbances in cholesterol metabolism are associated with the development of various neurological conditions. In addition to genetically defined defects in cholesterol synthesis, which will be covered in another review in this Thematic Series, defects in cholesterol metabolism (cerebrotendinous xanthomatosis) and intracellular transport (Niemann Pick Syndrome) lead to neurological disease. A subform of hereditary spastic paresis (type SPG5) and Huntington's disease are neurological diseases with mutations in genes that are of importance for cholesterol metabolism. Neurodegeneration is generally …

Hmgb1 Mediates Endogenous Tlr2 Activation And Brain Tumor Regression, James Curtin, Naiyou Liu, Marianela Candolfi, Weidong Xiong, Hikmat Assi, Kader Yagiz, Matthew Edwards, Kathrin Michelsen, Kurt Kroeger, Chunyan Liu, Akm Ghulam Muhammad, Mary Clark, Moshe Arditi, Begonya Comin-Anduix, Antoni Ribas, Pedro Lowenstein, Maria Castro

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BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling …

Treg Depletion Inhibits Efficacy Of Cancer Immunotherapy: Implications For Clinical Trials., James Curtin, Marianela Candolfi, Tamer Fakhouri, Chunyan Liu, Anderson Alden, Matthew Edwards, Pedro Lowenstein, Maria Castro

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BACKGROUND: Regulatory T lymphocytes (Treg) infiltrate human glioblastoma (GBM); are involved in tumor progression and correlate with tumor grade. Transient elimination of Tregs using CD25 depleting antibodies (PC61) has been found to mediate GBM regression in preclinical models of brain tumors. Clinical trials that combine Treg depletion with tumor vaccination are underway to determine whether transient Treg depletion can enhance anti-tumor immune responses and improve long term survival in cancer patients. FINDINGS: Using a syngeneic intracrabial glioblastoma (GBM) mouse model we show that systemic depletion of Tregs 15 days after tumor implantation using PC61 resulted in a decrease in Tregs …

Novel Functions Of Acyl-Coa Thioesterases And Acyltransferases As Auxiliary Enzymes In Peroxisomal Lipid Metabolism., Mary Hunt, Stefan Alexson

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Peroxisomes are single membrane bound organelles present in almost all eukaryotic cells, and to date have been shown to contain approximately 60 identified enzymes involved in various metabolic pathways, including the oxidation of a variety of lipids. These lipids include very long-chain fatty acids, methyl branched fatty acids, prostaglandins, bile acid precursors, and xenobiotics that are either β-oxidized or α-oxidized in peroxisomes. The recent identification of several acyl-CoA thioesterases and acyltransferases in peroxisomes has revealed their various functions in acting as auxiliary enzymes in α- and β-oxidation in this organelle. To date, 9 functional acyl-CoA thioesterases and acyltransferases have been …

Identification Of Fatty Acid Oxidation Disorder Patients With Lowered Acyl-Coa Thioesterase Activity In Human Skin Fibroblasts, Mary Hunt, Jos Ruiter, Petra Mooyer, Carlo W T Van Roermond, Rob Ofman, Lodewig Ijlst, Ronald J A Wanders

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Background: Acyl-CoA thioesterases are enzymes that hydrolyze acyl-CoAs to the free fatty acid and coenzyme A (CoASH). These enzymes have been identified in several cellular compartments and are thought to regulate intracellular levels of acyl-CoAs, free fatty acids and CoASH. However, to date no patients deficient in acyl-CoA thioesterases have been identified. Design: Acyl-CoA thioesterase activity was measured in human skin fibroblasts. Western blot analysis was used to determine Type-II acyl-CoA thioesterase protein levels in patients. Results: Activity was found in human fibroblasts with all saturated acyl-CoAs from C4:0- to C18:0-CoA, with highest activity detected with lauroyl-CoA and myristoyl-CoA (C12:0 …