Georgina E. Menzies

A New Class of Safe Oligosaccharide Polymer Therapy To Modify the Mucus Barrier of Chronic Respiratory Disease

The host- and bacteria-derived extracellular polysaccharide coating of the lung is a considerable challenge in chronic respiratory disease and is a powerful barrier to effective drug delivery. A low molecular weight 12-15-mer alginate oligosaccharide (OligoG CF-5/20), derived from plant biopolymers, was shown to modulate the polyanionic components of this coating. Molecular modeling and Fourier transform infrared spectroscopy demonstrated binding between OligoG CF-5/20 and respiratory mucins. Ex vivo studies showed binding induced alterations in mucin surface charge and porosity of the three-dimensional mucin networks in cystic fibrosis (CF) sputum. Human studies showed that OligoG CF-5/20 is safe for inhalation in CF patients with effective lung deposition and modifies the viscoelasticity of CF-sputum. OligoG CF-5/20 is the first inhaled polymer therapy, represents a novel mechanism of action and therapeutic approach for the treatment of chronic respiratory disease, and is currently in Phase IIb clinical trials for the treatment of CF.

Base damage, local sequence context and TP53 mutation hotspots: a molecular dynamics study of benzo[a]pyrene induced DNA distortion and mutability

The mutational pattern for the TP53 tumour suppressor gene in lung tumours differs to other cancer types by having a higher frequency of G:C>T:A transversions. The aetiology of this differing mutation pattern is still unknown. Benzo[a]pyrene,diol epoxide (BPDE) is a potent cigarette smoke carcinogen that forms guanine adducts at TP53 CpG mutation hotspot sites including codons 157, 158, 245, 248 and 273. We performed molecular modelling of BPDE-adducted TP53 duplex sequences to determine the degree of local distortion caused by adducts which could influence the ability of nucleotide excision repair. We show that BPDE adducted codon 157 has greater structural distortion than other TP53 G:C>T:A hotspot sites and that sequence context more distal to adjacent bases must influence local distortion. Using TP53 trinucleotide mutation signatures for lung cancer in smokers and non-smokers we further show that codons 157 and 273 have the highest mutation probability in smokers. Combining this information with adduct structural data we predict that G:C>T:A mutations at codon 157 in lung tumours of smokers are predominantly caused by BPDE. Our results provide insight into how different DNA sequence contexts show variability in DNA distortion at mutagen adduct sites that could compromise DNA repair at well characterized cancer related mutation hotspots.

Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors

The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways, that yield a cytokine output in response to pathogenic stimuli, have been postulated to link infection at the maternal–fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll‐like receptors (TLR‐3, −7, −8 and −9), and retinoic acid‐inducible gene 1 (RIG‐I)‐like receptors [RIG‐I, melanoma differentiation‐associated protein 5 (MDA5) and Laboratory of Genetics and Physiology 2(LGP2)] in human term gestation‐associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)‐6 and/or IL‐8 production in response to specific agonists for TLR‐3 (Poly(I:C); low and high molecular weight), TLR‐7 (imiquimod), TLR‐8 (ssRNA40) and RIG‐I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR‐9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL‐8 response, while the choriodecidua and amnion generate a more similar IL‐6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR‐3, TLR‐7, TLR‐8 and RIG‐1/MDA5 is a broad feature of human term gestation‐associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes.

Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides

Acquisition of a mucoid phenotype by Pseudomonas sp. in the lungs of cystic fibrosis (CF) patients, with subsequent over-production of extracellular polymeric substance (EPS), plays an important role in mediating the persistence of multi-drug resistant (MDR) infections. The ability of a low molecular weight (Mn = 3200 g mol−1) alginate oligomer (OligoG CF-5/20) to modify biofilm structure of mucoid Pseudomonas aeruginosa (NH57388A) was studied in vitro using scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) with Texas Red (TxRd®)-labelled OligoG and EPS histochemical staining. Structural changes in treated biofilms were quantified using COMSTAT image-analysis software of CLSM z-stack images, and nanoparticle diffusion. Interactions between the oligomers, Ca2+ and DNA were studied using molecular dynamics (MD) simulations, Fourier transform infrared spectroscopy (FTIR) and isothermal titration calorimetry (ITC). Imaging demonstrated that OligoG treatment (≥0.5%) inhibited biofilm formation, revealing a significant reduction in both biomass and biofilm height (P < 0.05). TxRd®-labelled oligomers readily diffused into established (24 h) biofilms. OligoG treatment (≥2%) induced alterations in the EPS of established biofilms; significantly reducing the structural quantities of EPS polysaccharides, and extracellular (e)DNA (P < 0.05) with a corresponding increase in nanoparticle diffusion (P < 0.05) and antibiotic efficacy against established biofilms. ITC demonstrated an absence of rapid complex formation between DNA and OligoG and confirmed the interactions of OligoG with Ca2+ evident in FTIR and MD modelling. The ability of OligoG to diffuse into biofilms, potentiate antibiotic activity, disrupt DNA-Ca2+-DNA bridges and biofilm EPS matrix highlights its potential for the treatment of biofilm-related infections.Drug-resistant biofilms: Marine algal molecules could helpSmall carbohydrate molecules derived from marine algae show potential for inhibiting biofilm formation in multi-drug resistant infections. A research team led by Lydia Powell at Cardiff University, UK, investigated the action of carbohydrates called alginate oligosaccharides, composed of a small number of linked sugar molecules. The oligosaccharides modified and disrupted the structure of cultured biofilms of Pseudomonas aeruginosa, the cause of many serious drug resistant infections. This effect significantly inhibited the formation and maintenance of the biofilm state, which is known to be a crucial factor allowing the bacteria to resist drug treatment. Antibiotics proved more effective following the oligosaccharide intervention. The researchers uncovered key molecular details involved in the ability of the oligosaccharides to diffuse into and disrupt biofilms. The therapeutic potential of these small carbohydrates is currently being investigated in clinical trials.

NEXT GENERATION EXOME SEQUENCING IN A LARGE SAMPLE OF ALZHEIMER’S PATIENTS

P2-112 NEXT GENERATION EXOME SEQUENCING IN A LARGE SAMPLE OF ALZHEIMER’S PATIENTS Detelina Grozeva, Aura Frizzati, Rebecca Sims, Taniesha Morgan, Rachel Raybould, Elliott Rees, Elisa Majounie, Nicola Denning, Alun Meggy, Rachel Marshall, Bartholomew Beaumont, William Nash, Chloe Davies, Joanne Morgan, Ganna Leonenko, Georgina E. Menzies, Nandini Badarinarayan, Valentina EscottPrice, Julie Williams, MRC Centre for Neuropsychiatric Genetics and Genomics, Institute of Psychological Medicine and Clinical Neurosciences, Cardiff University, Cardiff, United Kingdom; U.K. Dementia Research Institute at Cardiff, Cardiff, United Kingdom. Contact e-mail: grozevaD@cardiff.ac.uk