• Targeted long-read sequencing of a locus under long-term balancing selection in Capsella

      Bachmann, J.A.; Tedder, Andrew; Laenen, B.; Steige, K.A.; Slotte, T. (2018-04)
      Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7×10−5. A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach.
    • Targeting HOX transcription factors in prostate cancer

      Morgan, Richard; Boxall, A.; Harrington, K.J.; Simpson, G.R.; Michael, A.; Pandha, H.S. (2014-12)
      Background: The HOX genes are a family of transcription factors that help to determine cell and tissue identity during early development, and which are also over-expressed in a number of malignancies where they have been shown to promote cell proliferation and survival. The purpose of this study was to evaluate the expression of HOX genes in prostate cancer and to establish whether prostate cancer cells are sensitive to killing by HXR9, an inhibitor of HOX function. Methods: HOX function was inhibited using the HXR9 peptide. HOX gene expression was assessed by RNA extraction from cells or tissues followed by quantitative PCR, and siRNA was used to block the expression of the HOX target gene, cFos. In vivo modelling involved a mouse flank tumour induced by inoculation with LNCaP cells. Results: In this study we show that the expression of HOX genes in prostate tumours is greatly increased with respect to normal prostate tissue. Targeting the interaction between HOX proteins and their PBX cofactor induces apoptosis in the prostate cancer derived cell lines PC3, DU145 and LNCaP, through a mechanism that involves a rapid increase in the expression of cFos, an oncogenic transcription factor. Furthermore, disrupting HOX/PBX binding using the HXR9 antagonist blocks the growth of LNCaP tumours in a xenograft model over an extended period. Conclusion: Many HOX genes are highly over-expressed in prostate cancer, and prostate cancer cells are sensitive to killing by HXR9 both in vitro and in vivo. The HOX genes are therefore a potential therapeutic target in prostate cancer.
    • Targeting HOX-PBX interactions causes death in oral potentially malignant and squamous carcinoma cells but not normal oral keratinocytes

      Platais, C.; Radhakrishnan, R.; Ebensberger, S.N.; Morgan, Richard; Lambert, D.W.; Hunter, K.D. (2018-07)
      Background: High HOX gene expression has been described in many cancers, including oral squamous cell carcinoma and the functional roles of these genes are gradually being understood. The pattern of overexpression suggests that inhibition may be useful therapeutically. Inhibition of HOX protein binding to PBX cofactors by the use of synthetic peptides, such as HXR9, results in apoptosis in multiple cancers. Methods: Activity of the HOX-PBX inhibiting peptide HXR9 was tested in immortalised normal oral (NOK), potentially-malignant (PMOL) and squamous cell carcinoma (OSCC) cells, compared to the inactive peptide CXR9. Cytotoxicity was assessed by LDH assay. Expression of PBX1/2 and c-Fos was assessed by qPCR and western blotting. Apoptosis was assessed by Annexin-V assay. Results: PMOL and OSCC cells expressed PBX1/2. HOX-PBX inhibition by HXR9 caused death of PMOL and OSCC cells, but not NOKs. HXR9 treatment resulted in apoptosis and increased expression of c-Fos in some cells, whereas CXR9 did not. A correlation was observed between HOX expression and resistance to HXR9. Conclusion: Inhibition of HOX-PBX interactions causes selective apoptosis of OSCC/PMOL, indicating selective toxicity that may be useful clinically.
    • Targeting HOX/PBX dimers in cancer

      Morgan, Richard; El-Tanani, Mohamed; Hunter, K.D.; Harrington, K.J.; Pandha, H.S. (2017-03-07)
      The HOX and PBX gene families encode transcription factors that have key roles in establishing the identity of cells and tissues in early development. Over the last 20 years it has become apparent that they are also dysregulated in a wide range of solid and haematological malignancies and have a predominantly pro-oncogenic function. A key mode of transcriptional regulation by HOX and PBX proteins is through their interaction as a heterodimer or larger complex that enhances their binding affinity and specificity for DNA, and there is growing evidence that this interaction is a potential therapeutic target in malignancies that include prostate, breast, renal, ovarian and lung cancer, melanoma, myeloma, and acute myeloid leukaemia. This review summarizes the roles of HOX and PBX genes in cancer and assesses the therapeutic potential of HOX/PBX dimer inhibition, including the availability of biomarkers for its application in precision medicine.
    • Targeting of Hypoxia in AQ4N-treated Tumour Xenografts by MALDI-Ion Mobility Separation-Mass Spectrometry Imaging

      Djidja, M-C.; Francese, S.; Claude, E.; Loadman, Paul M.; Sutton, Chris W.; Shnyder, Steven D.; Cooper, Patricia A.; Patterson, Laurence H.; Carolan, V.A.; Clench, M.R. (2013-04-01)
      Hypoxia is a common feature observed in solid tumours. It is a target of interest in oncology as it has been found to be closely associated with tumour progression, metastasis and aggressiveness and confers resistance to a variety of chemotherapeutic agents as well as radiotherapy. AQ4N, also known as banoxatrone or 1,4-bis-[2-(dimethylamino-Noxide) ethyl]amino-5,8-dihydroxyanthracene-9,10-dione is a very promising bioreductive prodrug. This paper, describes an application of MALDI-MSI combined with ion mobility separation and an "on-tissue" bottom up proteomic strategy to obtain proteomic data from AQ4N dosed tumour xenograft tissue sections. These data are then correlated with the drug distribution determined also using MALDI-ion mobility separation-mass spectrometry imaging (MALDI-IMS-MSI). PCA-DA and OPLS-DA have been used to compare treated and untreated xenografts and of note is the marked increase in expression of Histone H3.
    • Targeting the mesolithic: Interdisciplinary approaches to archaeological prospection inthe Brown Bank area, southern North Sea

      Missiaen, T.; Fitch, Simon; Muru, Merle; Harding, Rachel; Fraser, Andy; De Clercq, M.; Garcia Moreno, David; Versteeg, W.; Gaffney, Vincent L. (2020)
      This paper describes some results of the research undertaken over the Brown Bank area during recent (2018/2019) geoarchaeological surveys in the North Sea which included seismic imaging, shallow (vibro)coring and dredging. It examines the benefits of simultaneous high-resolution (0.5 – 1m) and ultra-high-resolution (10 – 20cm) seismic survey techniques and a staged approach to resolving the submerged Holocene landscape in the highest possible detail for the purpose of targeted prospecting for archaeological material from the Mesolithic landscape of Doggerland. The materials recovered from such surveys offer significantly greater information due to an enhanced understanding of the context in which they were recovered. The importance of this information cannot be understated archaeologically, as few locations on land provide the opportunity to recover archaeological finds in situ within preserved landscapes. Moreover, it allows offshore areas of potential human activity to be prospected with some certainty of success.
    • Tattoo ink nanoparticles in skin tissue and fibroblasts

      Grant, Colin A.; Twigg, Peter C.; Baker, Richard; Tobin, Desmond J. (2015-05-20)
      Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.
    • Tea phenols in bulk and nanoparticle form modify DNA damage in human lymphocytes from colon cancer patients and healthy individuals treated in vitro with platinum-based chemotherapeutic drugs

      Alotaibi, Amal; Bhatnagar, P.; Najafzadeh, Mojgan; Gupta, K.C.; Anderson, Diana (2013)
      Tea catechin epigallocatechin-3-gallate (EGCG) and other polyphenols, such as theaflavins (TFs), are increasingly proving useful as chemopreventives in a number of human cancers. They can also affect normal cells. The polyphenols in tea are known to have antioxidant properties that can quench free radical species, and pro-oxidant activities that appear to be responsible for the induction of apoptosis in tumor cells. The bioavailability of these natural compounds is an important factor that determines their efficacy. Nanoparticle (NP)-mediated delivery techniques of EGCG and TFs have been found to improve their bioavailability to a level that could benefit their effectiveness as chemopreventives. AIM: The present study was conducted to compare the effects of TFs and EGCG, when used in the bulk form and in the polymer (poly[lactic-co-glycolic acid])-based NP form, in oxaliplatin- and satraplatin-treated lymphocytes as surrogate cells from colorectal cancer patients and healthy volunteers. NPs were examined for their size distribution, surface morphology, entrapment efficiency and release profile. Lymphocytes were treated in the Comet assay with oxaliplatin and satraplatin, washed and treated with bulk or NP forms of tea phenols, washed and then treated with hydrogen peroxide to determine single-strand breaks after crosslinking. The results of DNA damage measurements by the Comet assay revealed opposite trends in bulk and NP forms of TFs, as well as EGCG. Both the compounds in the bulk form produced statistically significant concentration-dependent reductions in DNA damage in oxaliplatin- or satraplatin-treated lymphocytes. In contrast, when used in the NP form both TFs and EGCG, although initially causing a reduction, produced a concentration-dependent statistically significant increase in DNA damage in the lymphocytes. These observations support the notion that TFs and EGCG act as both antioxidants and pro-oxidants, depending on the form in which they are administered under the conditions of investigation.
    • Team-based learning in pharmacy: The faculty experience

      Tweddell, Simon; Clark, D.; Nelson, M. (2016)
      Aim To assess faculty perceptions and experiences when implementing team-based learning (TBL) across a pharmacy curriculum. Study design A total of 19 faculty members participated in a series of individual semi-structured interviews that allowed freedom of discussion within a structured framework of inquiry. Data were transcribed, coded using NVivo, and analyzed to establish common themes. Participant quotations were chosen to reinforce the themes and give a voice to the participants. Findings and discussion The benefits of TBL were perceived to be enhanced student engagement, peer learning, increased faculty enjoyment of teaching, and student development of transferable skills. Challenges included increased initial workload, writing effective application exercises, and facilitating learner-centered classes. TBL may be useful in optimizing course content to ensure outcomes and activities focus on important concepts. Peer learning appears to benefit student learning. TBL may help equip students with valuable transferable skills. TBL requires an initial upfront investment in faculty development and time to prepare resources. A student-centered approach to learning may be daunting for faculty and require new skill sets. Conclusions Faculty described their support for TBL concluding that the pedagogical benefits of engaging students in active learning, the development of transferable skills for the workplace, and the personal satisfaction felt after a TBL class, outweigh the initial challenges of transitioning to TBL.
    • Team-based Learning: Engaging learners and creating team accountability

      de Vries, J.; Tweddell, Simon; McCarter, Rebecca (2018-06)
      Team-based Learning (TBL) is a new teaching strategy that may take small group learning to a new level of effectiveness. TBL shifts the focus from content delivery by teachers to the application of course content by student teams. Teams work on authentic problems, make collaborative decisions, and develop problem-solving skills required in their future workplace. Prior to redesigning the MPharm programme according to TBL principles, several pilots were set up to research how students responded to this new way of teaching. One pilot focussed on the introduction of TBL as a phenomena and aimed to find out if and how TBL engaged students, how students were held accountable by their teams, and more importantly how that affected their lifeworld. Ashworth’s lifeworld contingencies provided the theoretical framework as it ranges from students’ selfhood, embodiment and social interactions to their ability to carry out tasks they are committed to and regard as essential (Ashworth, 2003).
    • Technical note: reliability of Suchey-Brooks and Buckberry-Chamberlain methods on 3D visualizations from CT and laser scans.

      Villa, C.; Buckberry, Jo; Cattaneo, C.; Lynnerup, N. (2013)
      Previous studies have reported that the ageing method of Suchey-Brooks (pubic bone) and some of the features applied by Lovejoy et al. and Buckberry-Chamberlain (auricular surface) can be confidently performed on 3D visualizations from CT-scans. In this study, seven observers applied the Suchey-Brooks and the Buckberry-Chamberlain methods on 3D visualizations based on CT-scans and, for the first time, on 3D visualizations from laser scans. We examined how the bone features can be evaluated on 3D visualizations and whether the different modalities (direct observations of bones, 3D visualization from CT-scan and from laser scans) are alike to different observers. We found the best inter-observer agreement for the bones versus 3D visualizations, with the highest values for the auricular surface. Between the 3D modalities, less variability was obtained for the 3D laser visualizations. Fair inter-observer agreement was obtained in the evaluation of the pubic bone in all modalities. In 3D visualizations of the auricular surfaces, transverse organization and apical changes could be evaluated, although with high inter-observer variability; micro-, macroporosity and surface texture were very difficult to score. In conclusion, these methods were developed for dry bones, where they perform best. The Suchey-Brooks method can be applied on 3D visualizations from CT or laser, but with less accuracy than on dry bone. The Buckberry-Chamberlain method should be modified before application on 3D visualizations. Future investigation should focus on a different approach and different features: 3D laser scans could be analyzed with mathematical approaches and sub-surface features should be explored on CT-scans
    • Techniques for identifying the age and sex of children at death

      Buckberry, Jo (2018-05)
      The skeletal remains of infants and children are a poignant reminder of the perilous nature of childhood in the past, yet they offer valuable insight into the life histories of individuals and into the health of populations. Many osteoarchaeological and bioarchaeological analyses are dependent on two vital pieces of information: the age-at-death and sex of the individual(s) under study. This chapter will outline how age-at-death and sex can be estimated from the skeletal remains and dental development of non-adults, and how these are easier or more difficult to determine than for adults, and will discuss the complexities and controversies surrounding different methods.
    • Technological Analysis of the World’s Earliest Shamanic Costume: A Multi-Scalar, Experimental Study of a Red Deer Headdress from the Early Holocene Site of Star Carr, North Yorkshire, UK

      Little, A.; Elliott, B.; Conneller, C.; Pomstra, D.; Evans, Adrian A.; Fitton, L.C.; Holland, Andrew D.; Davis, R.; Kershaw, Rachael; O'Connor, Sonia A.; et al. (2016-04-13)
      Shamanic belief systems represent the first form of religious practice visible within the global archaeological record. Here we report on the earliest known evidence of shamanic costume: modified red deer crania headdresses from the Early Holocene site of Star Carr (c. 11 kya). More than 90% of the examples from prehistoric Europe come from this one site, establishing it as a place of outstanding shamanistic/cosmological significance. Our work, involving a programme of experimental replication, analysis of macroscopic traces, organic residue analysis and 3D image acquisition, metrology and visualisation, represents the first attempt to understand the manufacturing processes used to create these artefacts. The results produced were unexpected—rather than being carefully crafted objects, elements of their production can only be described as expedient.
    • Technological, Refitting and Microwear of the Stone Artefact Assemblage

      Pope, M.; Davis, R.; Evans, Adrian A. (SpoilHeap, 2020-08)
    • Temperature-dependent structure and dynamics of highly-branched poly(N -isopropylacrylamide) in aqueous solution

      Al-Baradi, A.M.; Rimmer, Stephen; Carter, S.R.; de Silva, J.P.; King, S.M.; Maccarini, M.; Farago, B.; Noirez, L.; Geoghegan, M. (2018-02)
      Small-angle neutron scattering (SANS) and neutron spin-echo (NSE) have been used to investigate the temperature-dependent solution behaviour of highly-branched poly(N-isopropylacrylamide) (HB-PNIPAM). SANS experiments have shown that water is a good solvent for both HB-PNIPAM and a linear PNIPAM control at low temperatures where the small angle scattering is described by a single correlation length model. Increasing the temperature leads to a gradual collapse of HB-PNIPAM until above the lower critical solution temperature (LCST), at which point aggregation occurs, forming disperse spherical particles of up to 60 nm in diameter, independent of the degree of branching. However, SANS from linear PNIPAM above the LCST is described by a model that combines particulate structure and a contribution from solvated chains. NSE was used to study the internal and translational solution dynamics of HB-PNIPAM chains below the LCST. Internal HB-PNIPAM dynamics is described well by the Rouse model for non-entangled chains.
    • Temporal characteristics of L and M-cone isolating steady-state ERGs

      Kommanapalli, Deepika; Murray, I.J.; Kremers, Jan; Parry, Neil R.A.; McKeefry, Declan J. (2014-04)
      Cone isolating stimuli were used to assess the temporal frequency response characteristics of L- and M-cone electroretinograms (ERGs) in nine trichromatic and four dichromatic human observers. The stimuli comprised sinusoidal temporal modulations varying from 5 to 100 Hz. ERGs were recorded using corneal fiber electrodes and subjected to fast Fourier transform analysis. At low temporal frequencies (<10  Hz<10  Hz) the L- and M-cone ERGs had similar amplitude and exhibited minimal differences in apparent latency. At higher flicker rates (>20  Hz>20  Hz) L-cone ERGs had greater amplitudes and shorter apparent latencies than the M-cone responses. These differences between the L- and M-cone ERGs are consistent with their mediation by chromatic and luminance postreceptoral processing pathways at low and high temporal frequencies, respectively.
    • The temporal dynamics of Arc expression regulate cognitive flexibility

      Wall, M.J.; Collins, D.R.; Chery, S.L.; Allen, Z.D.; Pastuzyn, E.D.; George, A.J.; Nikolova, V.D.; Moy, S.S.; Philpot, B.D.; Shepherd, J.D.; et al. (2018-06)
      Neuronal activity regulates the transcription and translation of the immediate-early gene Arc/Arg3.1, a key mediator of synaptic plasticity. Proteasomedependent degradation of Arc tightly limits its temporal expression, yet the significance of this regulation remains unknown. We disrupted the temporal control of Arc degradation by creating an Arc knockin mouse (ArcKR) where the predominant Arc ubiquitination sites were mutated. ArcKR mice had intact spatial learning but showed specific deficits in selecting an optimal strategy during reversal learning. This cognitive inflexibility was coupled to changes in Arc mRNA and protein expression resulting in a reduced threshold to induce mGluR-LTD and enhanced mGluR-LTD amplitude. These findings show that the abnormal persistence of Arc protein limits the dynamic range of Arc signaling pathways specifically during reversal learning. Our work illuminates how the precise temporal control of activity-dependent molecules, such as Arc, regulates synaptic plasticity and is crucial for cognition.
    • Temporal estimation in prediction motion tasks is biased by a moving destination

      Flavell, Jonathan; Barrett, Brendan T.; Buckley, John G.; Harris, J.M.; Scally, Andy J.; Beebe, Nathan B.; Cruickshank, Alice G.; Bennett, S.J. (2018-02)
      An ability to predict the time-to-contact (TTC) of moving objects that become momentarily hidden is advantageous in everyday life and could be particularly so in fast-ball sports. Prediction motion (PM) experiments have sought to test this ability using tasks where a disappearing target moves towards a stationary destination. Here, we developed two novel versions of the PM task in which the destination either moved away from (Chase) or towards (Attract) the moving target. The target and destination moved with different speeds such that collision occurred 750, 1000 or 1250ms after target occlusion. To determine if domain-specific experience conveys an advantage in PM tasks, we compared the performance of different sporting groups ranging from internationally competing athletes to non-sporting controls. There was no difference in performance between sporting groups and non-sporting controls but there were significant and independent effects on response error by target speed, destination speed and occlusion period. We simulated these findings using a revised version of the linear TTC model of response timing for PM tasks (Yakimoff et al. 1987, 1993) in which retinal input from the moving destination biases the internal representation of the occluded target. This revision closely reproduced the observed patterns of response error and thus describes a means by which the brain might estimate TTC when the target and destination are in motion.
    • Temporal evaluation of methionine synthase and related metabolites in the MAC15A mouse adenocarcinoma animal mode.l

      Blackburn, Alison; Bibby, Michael C.; Lucock, M.D.; Nicolaou, Anna (2004)
      Methionine dependence is unique to cancer cells and defined as the inability to grow in a methionine-deprived environment even if supplemented with the metabolic precursor homocysteine. Cobalamin-dependent methionine synthase (MS) catalyses the formation of methionine and tetrahydrofolate from homocysteine and methyltetrahydrofolate, thus linking the methionine and folate pathways. The apparent altered methionine metabolism in methionine-dependent cancer cells suggests a role for MS, although results to date are conflicting. We have analysed key metabolites of the MS-associated transmethylation, transsulphuration and folate pathways of the methionine-dependent MAC15A tumour model as a function of tumour progression over a 10-day period. MS activity increased 2-fold from day I to day 10. Cysteine, homocysteine, S-adenosylmethionine and S-adenosylhomocysteine levels in tumour cytosolic fractions decreased as a function of tumour progression. Plasma cysteine levels also decreased, whilst the distribution of folates in erythrocytes was altered, with a maximum increase in methyltetrahydrofolate observed by day 5. The increasing MS activity and decreasing cysteine levels suggest an increasing methionine requirement by the tumour, whilst the induction of enzyme activity indicates that MS is not defective in the methionine-dependent MAC15A tumour. The decrease in tumour S-adenosylmethionine and S-adenosylhomocysteine levels suggests that methionine is required for some function other than cellular methylation, e.g., incorporation into protein. Overall, the results support a theory of methionine conservation in response to tumour growth, where the methionine-dependent MAC15A tumour has a higher than normal methionine requirement.