Characterisation of the p53-Mediated Cellular Responses Evoked in Primary Mouse Cells Following Exposure to Ultraviolet Radiation

Exposure to ultraviolet (UV) light can cause significant damage to mammalian cells and, although the spectrum of damage produced varies with the wavelength of UV, all parts of the UV spectrum are recognised as being detrimental to human health. Characterising the cellular response to different wavelengths of UV therefore remains an important aim so that risks and their moderation can be evaluated, in particular in relation to the initiation of skin cancer. The p53 tumour suppressor protein is central to the cellular response that protects the genome from damage by external agents such as UV, thus reducing the risk of tumorigenesis. In response to a variety of DNA damaging agents including UV light, wild-type p53 plays a role in mediating cell-cycle arrest, facilitating apoptosis and stimulating repair processes, all of which prevent the propagation of potentially mutagenic defects. In this study we examined the induction of p53 protein and its influence on the survival of primary mouse fibroblasts exposed to different wavelengths of UV light. UVC was found to elevate p53 protein and its sequence specific DNA binding capacity. Unexpectedly, UVA treatment failed to induce p53 protein accumulation or sequence specific DNA binding. Despite this, UVA exposure of wild-type cells induced a p53 dependent G1 cell cycle arrest followed by a wave of p53 dependent apoptosis, peaking 12 hours post-insult. Thus, it is demonstrated that the elements of the p53 cellular response evoked by exposure to UV radiation are wavelength dependent. Furthermore, the interrelationship between various endpoints is complex and not easily predictable. This has important implications not only for understanding the mode of action of p53 but also for the use of molecular endpoints in quantifying exposure to different wavelengths of UV in the context of human health protection

Connect, converse, collaborate: Encountering belonging and forging resilience through creative practice

This paper is a dialogue between two colleagues who teach drama and performance in Higher Education. Our work here has developed across a series of formal, semi-structured and informal discussions about our experiences of teaching and supporting students within the Drama and Performance department at University of South Wales. Instantly we connected on our commitment to prioritising student needs and our intentions to co-construct reflexive learning spaces. Within the disciplines of drama and performance, we (Allinson and Crews) see practice, collaboration and dialogue as equally important and core to all learning environments and encounters. Because of this we continually question how to hold a space for students through focusing on individual needs and difference, whilst simultaneously attempting to find connection through shared intentions and practices. Acknowledging individual and collective anxiety in learning environments is important because, left unchecked, these individual anxieties risk generating collective frustration, resistance to the creative process and fatigue. Openly discussing and agreeing on how to create spaces and structures for feeling heard and seen fosters belonging and in turn resilience, both in ourselves and our students. Here we propose that working within creative practices and exploring dynamic ways of holding space for ourselves and for students generates repeated experiences of successful encounters that build resourcefulness and resilience. This allows educators and students to collectively and mindfully encounter future situations and engage with them transformatively

The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks

The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5′-hydroxyl phosphorylation and 3′-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3′-phosphate and 5′-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5′-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3′-phosphate, but it need not be present in the same strand break as the 5′-hydroxyl. PNK preferentially binds 3′-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain

Characterisation of unusual DNA glycosylases from Trichomonas vaginalis

Trichomonas vaginalis is a parasitic protozoan responsible for 170 million new infections each year and is the most prevalent non-viral sexually transmitted infection worldwide according to The World Health Organisation. Research on the parasites pathogenicity and lifestyle has been limited until the publishing of the parasites’ genome in 2007 and an emergence of studies documenting links between T. vaginalis infection and a number of adverse health outcomes, including an increased risk of HIV acquisition and cervical cancer. Research into DNA repair pathways within this organism have also been limited. In this research I describe two DNA 3-methyl adenine glycosylase homologues (TvAAG1 and TvAAG2) characterised via biochemical methods. In addition to the creation of an atlas of DNA repair proteins and enzymes present in T. vaginalis, and a close evolutionary relative, Tritrichomonas foetus via bioinformatical analysis. Biochemical analysis of TvAAG1 reveals it as the first bifunctional AAG type enzyme to be described. Bioinformatic analysis suggests TvAAG1 and TvAAG2 are most likely the result of horizontal gene transfer between an ancestor of both T. vaginalis and T. foetus and a donor belonging to the Bacteroidetes phylum. The DNA repair pathways of both parasites are reduced compared to humans. The parasites have the main components for base excision repair, mismatch repair and homologous recombination repair but not non-homologous end joining. In light of reports of antibiotic resistance further research should be done on the parasites DNA repair pathway to identify potential novel drug targets for treatment of T. vaginalis infection

Platinum trimethyl bipyridyl thiolates – new, tunable, red- to near IR emitting luminophores for bioimaging applications

Synthetic, spectroscopic, computational and biological imaging studies of platinum trimethyl bipyridyl thiolate complexes of the general formula [PtMe3(bpy)SR] reveal these to be easily accessed, tunable bioimaging agents which feature an unusual σ–π* Inter-Ligand Charge Transfer (ILCT) transition, and in some cases emit into the Near infra-red (NIR)

Electron Beam-Treated Enzymatically Mineralized Gelatin Hydrogels for Bone Tissue Engineering

Biological hydrogels are highly promising materials for bone tissue engineering (BTE) due to their high biocompatibility and biomimetic characteristics. However, for advanced and customized BTE, precise tools for material stabilization and tuning material properties are desired while optimal mineralisation must be ensured. Therefore, reagent-free crosslinking techniques such as high energy electron beam treatment promise effective material modifications without formation of cytotoxic by-products. In the case of the hydrogel gelatin, electron beam crosslinking further induces thermal stability enabling biomedical application at physiological temperatures. In the case of enzymatic mineralisation, induced by Alkaline Phosphatase (ALP) and mediated by Calcium Glycerophosphate (CaGP), it is necessary to investigate if electron beam treatment before mineralisation has an influence on the enzymatic activity and thus affects the mineralisation process. The presented study investigates electron beam-treated gelatin hydrogels with previously incorporated ALP and successive mineralisation via incubation in a medium containing CaGP. It could be shown that electron beam treatment optimally maintains enzymatic activity of ALP which allows mineralisation. Furthermore, the precise tuning of material properties such as increasing compressive modulus is possible. This study characterizes the mineralised hydrogels in terms of mineral formation and demonstrates the formation of CaP in dependence of ALP concentration and electron dose. Furthermore, investigations of uniaxial compression stability indicate increased compression moduli for mineralised electron beam-treated gelatin hydrogels. In summary, electron beam-treated mineralized gelatin hydrogels reveal good cytocompatibility for MG-63 osteoblast like cells indicating a high potential for BTE applications

Limiting Pseudomonas aeruginosa Biofilm Formation Using Cold Atmospheric Pressure Plasma

We investigate the ability to disrupt and limit growth biofilms of Pseudomonas aeruginosa using application of cold atmospheric pressure (CAP) plasma. The effect of the bio-film's exposure to a helium (CAP) jet was assessed at varying time points during biofilm maturation. Results showed that the amount of time during biofilm growth that CAP pressure was applied has a crucial role on the ability of biofilms to mature and recover after CAP exposure. Intervention during the early stages of biofilm formation (0-8 h) results in a 4-5-log reduction in viable bacterial cells (measured at 24 h of incubation) relative to untreated biofilms. However, CAP treatment of biofilm at 12 h and above only results in a 2-log reduction in viable cells. This has potentially important implications for future clinical application of CAP to treat infected wounds

Photochemical Oxidation of Pt(IV)Me3(1,2-diimine) Thiolates to Luminescent Pt(IV) Sulfinates

We report the formation of dinuclear complexes from, and photochemical oxidation of, (CH3)3-Pt(IV)(N^N) (N^N = 1,2-diimine derivatives) complexes of thiophenolate ligands to the analogous sulfinates (CH3)3Pt(N^N)(SO2Ph) and structural, spectroscopic, and theoretical studies of the latter revealing tunable photophysics depending upon the 1,2-diimine ligands. Electron-rich thiolate and conjugated 1,2-diimines encourage formation of thiolate-bridged dinuclear complexes; smaller 1,2-diimines or electron-poor thiolates favor mononuclear complexes. Photooxidation of the thiolate ligand yields hitherto unreported Pt(IV)-SO2R complexes, promoted by electron-deficient thiolates such as 4-nitrothiophenol, which exclusively forms the sulfinate complex. Such complexes exhibit expected absorptions due to π-π* ligand transitions of the 1,2-diimines mixed with spin-allowed singlet MLCT (d-π*) at relatively high energy (270–290 nm), as well as unexpected broad, lower energy absorptions between 360 and 490 nm. DFT data indicate that these low energy absorption bands result from excitation of Pt–S and Pt–C σ-bonding electrons to π* orbitals on sulfinate and 1,2-diimine, the latter of which gives rise to emission in the visible range