E-cadherin and APC in early-onset colorectal cancer

Colorectal cancer (CRC) is a considerable health burden being the second highest cause of cancer deaths globally. While overall cases of CRC have been declining worldwide, there has been an increase in the incidence of the disease among patients under 50 years of age. The majority of these cancers are sporadic and the increase in incidence may reflect changing lifestyle, exposing young people to more and earlier pro-oncogenic factors. An early event in the development of CRC is the loss of normal structure of the epithelium and key to this is the loss of cell to cell contact. E-cadherin, encoded by the CDH1 gene, is a membrane-bound protein whose extracellular domains bind to E-cadherin of neighbouring cells forming adherens junctions as the primary event in intercellular contact. Loss of E-cadherin leads to the breakdown of this organised epithelial structure and can lead to the development of cancer. The research detailed in this thesis looked at E-cadherin expression status and mutation of the CDH1 gene in two separate cohorts. Firstly tumours from young Pakistani patients with early-onset colorectal signet-ring cell carcinomas (SRCCs), due to their histological similarity to SRCCs found in hereditary diffuse gastric cancers where mutation of CDH1 is a common causative factor and, secondly a local New Zealand cohort of early-onset CRC cases. E-cadherin was found to be absent or weak in the colorectal SRCC samples and in a small number of cases this corresponded to germline CDH1 mutation. However, the remaining SRCC samples had low levels of CDH1 mutation despite loss of E-cadherin expression, suggesting that while E-cadherin loss is common in colorectal SRCC it is not due to CDH1 mutation in most cases. Amongst the New Zealand samples, only one tumour, the sole SRCC case in the cohort, showed loss of E-cadherin but this was not correlated with CDH1 mutation which was not a common feature of this cohort. Additionally, APC sequencing was performed on the New Zealand cohort. APC is mutated in 60-80% of sporadic CRC tumours but with reportedly lower levels in younger patients. However 72% of the New Zealand early-onset CRC cohort was found to have a mutation in APC, a higher proportion than expected. This may reflect a greater coverage of APC by the sequencing methodology employed in this study compared to previous studies with a high proportion of mutations occurring outside the commonly studied mutation cluster region of APC. Loss of heterozygosity at the APC locus was found in only three patients, all of whom had APC mutations occurring close to codon 1300, reflective of a previous studies in older-onset CRC. While environmental and lifestyle factors are widely considered to have roles in the development of sporadic CRC, there is growing evidence of the gut microbiota being a factor in colorectal carcinogenesis. One well-studied toxin-producing bacteria is Enterotoxigenic Bacteroides fragilis (ETBF) that causes cleavage of E-cadherin in colonic epithelial cells. Study of the effect of the B. fragilis toxin (BFT) on colonic epithelial cells has focused on the cell line HT29 which shows a rapid morphological change upon incubation with BFT. However, HT29 cells only have truncated forms of APC. In the last part of this study one mutated APC allele in HT29 was corrected by genome editing in order to study the effects of BFT on a cell line expressing full-length APC, and thereby to increase our understanding of the role of APC in colorectal carcinogenesis. The rapid change in morphology upon exposure to BFT in HT29 cells has been attributed to the BFT-mediated cleavage of E-cadherin. However, edited HT29 cells containing full-length APC maintained their structure after 6 hours incubation with ETBF supernatant. Moreover, fluorescent immunohistochemistry showed that cell morphology was maintained despite the cleavage of E-cadherin, suggesting that the structural integrity of the edited cells was due to some internal function of APC rather than E-cadherin cleavage. In summary, this research found that E-cadherin loss was commonly found in colorectal SRCCs but predominantly occurred independently from CDH1 mutations. Furthermore, other than in SRCCs, E-cadherin loss was not a common feature of early-onset CRC. Conversely, APC mutation was very common, with many mutations being found outside the mutation cluster region of APC, suggesting APC sequencing strategies should be targeted more widely throughout the gene. These results suggest a role for the APC protein in stabilising the cellular morphology of HT29 cells exposed to bacterial toxin, which is independent of E-cadherin. The edited HT29 cell line is likely to be a useful tool in the study of bacterial and other environmental effects on colorectal carcinogenesis

GINS motion reveals replication fork progression is remarkably uniform throughout the yeast genome

Time-resolved ChIP-chip can be utilized to monitor the genome-wide dynamics of the GINS complex, yielding quantitative information on replication fork movement.Replication forks progress at remarkably uniform rates across the genome, regardless of location.GINS progression appears to be arrested, albeit with very low frequency, at sites of highly transcribed genes.Comparison of simulation with data leads to novel biological insights regarding the dynamics of replication fork progressio

Detrital chrome spinel evidence for a Neotethyan intra-oceanic island arc collision with India in the Paleocene

Models that support a single collision scenario for India and Eurasia are incompatible with the evidence that an intra-oceanic island arc (IOIA) existed within the Neotethyan Ocean. Understanding the spatial and temporal extent of any IOIA is crucial for India-Eurasia collision studies as the entire ocean, including any intra-oceanic features, must have been consumed or emplaced prior to continental collision. Here, we review what is known about the Neotethyan IOIA and report evidence from sedimentary successions in NW India and southern Tibet to constrain when and where it was emplaced. We use detrital mineral geochemistry and supporting provenance and age data to identify the source of the sediments and compare the timing of erosion of IOIA-derived material in both regions. Detrital chrome spinels, extracted from distinct sedimentary horizons in southern Tibet (Sangdanlin) and NW India (Ladakh), exhibit similar average geochemical values (TiO\ua0=\ua00.09 and 0.24%, Cr#\ua0=\ua00.66 and 0.68 and Mg#\ua0=\ua00.45 and 0.53, respectively) and supra-subduction zone (SSZ), forearc peridotite signatures. Furthermore, they overlap with in-situ chrome spinels reported from the Spongtang Ophiolite in NW India and the Sangsang Ophiolite in southern Tibet. As with many of the ophiolitic remnants that crop out in and adjacent to the Yarlung-Tsangpo and Indus suture zones (YTSZ and ISZ respectively), the Spongtang and Sangsang ophiolites formed in an IOIA setting. Linking the source of the detrital chrome spinels to those analysed from remnant IOIA massifs in the YTSZ and ISZ is strong evidence for the emplacement of the IOIA onto the Indian margin. The timing of the IOIA collision with India is constrained by the depositional ages of the chrome spinel-bearing sediments to the end of the Paleocene (Thanetian) in southern Tibet and the Early Eocene in NW India. This indirectly provides a maximum age constraint of Late Paleocene-Early Eocene for intercontinental collision between India and Eurasia. Additionally, this study highlights the importance of targeting distinct sedimentary horizons in collision zones to find evidence for discrete tectonic events that may be obfuscated by later collisions

Virtual cultural tourism: six pillars of VCT using co-creation, value exchange and exchange value

This paper examines antecedents to the successful use of Virtual Cultural Tourism and the ways in which virtual realities can add value to Cultural Tourism offers. Success can be seen to derive from the deeper understanding of consumers’ preferences and motivations to engage with Virtual Cultural Tourism. It is also necessary to see these initiatives from the perspective of multiple stakeholders: the armchair traveller, the frequent flyer and the service provider at destinations. The latter include public sector providers such as park site managers, museum curators, interpretation and information services for tourism as well as the private sector developers

Impact of index hopping and bias towards the reference allele on accuracy of genotype calls from low-coverage sequencing

Abstract Background Inherent sources of error and bias that affect the quality of sequence data include index hopping and bias towards the reference allele. The impact of these artefacts is likely greater for low-coverage data than for high-coverage data because low-coverage data has scant information and many standard tools for processing sequence data were designed for high-coverage data. With the proliferation of cost-effective low-coverage sequencing, there is a need to understand the impact of these errors and bias on resulting genotype calls from low-coverage sequencing. Results We used a dataset of 26 pigs sequenced both at 2× with multiplexing and at 30× without multiplexing to show that index hopping and bias towards the reference allele due to alignment had little impact on genotype calls. However, pruning of alternative haplotypes supported by a number of reads below a predefined threshold, which is a default and desired step of some variant callers for removing potential sequencing errors in high-coverage data, introduced an unexpected bias towards the reference allele when applied to low-coverage sequence data. This bias reduced best-guess genotype concordance of low-coverage sequence data by 19.0 absolute percentage points. Conclusions We propose a simple pipeline to correct the preferential bias towards the reference allele that can occur during variant discovery and we recommend that users of low-coverage sequence data be wary of unexpected biases that may be produced by bioinformatic tools that were designed for high-coverage sequence data

Carbonic Anhydrase 9 Expression Increases with Vascular Endothelial Growth Factor-Targeted Therapy and Is Predictive of Outcome in Metastatic Clear Cell Renal Cancer

AbstractBackgroundThere is a lack of biomarkers to predict outcome with targeted therapy in metastatic clear cell renal cancer (mccRCC). This may be because dynamic molecular changes occur with therapy.ObjectiveTo explore if dynamic, targeted-therapy-driven molecular changes correlate with mccRCC outcome.Design, setting, and participantsMultiple frozen samples from primary tumours were taken from sunitinib-naïve (n=22) and sunitinib-treated mccRCC patients (n=23) for protein analysis. A cohort (n=86) of paired, untreated and sunitinib/pazopanib-treated mccRCC samples was used for validation. Array comparative genomic hybridisation (CGH) analysis and RNA interference (RNAi) was used to support the findings.InterventionThree cycles of sunitinib 50mg (4 wk on, 2 wk off).Outcome measurements and statistical analysisReverse phase protein arrays (training set) and immunofluorescence automated quantitative analysis (validation set) assessed protein expression.Results and limitationsDifferential expression between sunitinib-naïve and treated samples was seen in 30 of 55 proteins (p<0.05 for each). The proteins B-cell CLL/lymphoma 2 (BCL2), mutL homolog 1 (MLH1), carbonic anhydrase 9 (CA9), and mechanistic target of rapamycin (mTOR) (serine/threonine kinase) had both increased intratumoural variance and significant differential expression with therapy. The validation cohort confirmed increased CA9 expression with therapy. Multivariate analysis showed high CA9 expression after treatment was associated with longer survival (hazard ratio: 0.48; 95% confidence interval, 0.26–0.87; p=0.02). Array CGH profiles revealed sunitinib was associated with significant CA9 region loss. RNAi CA9 silencing in two cell lines inhibited the antiproliferative effects of sunitinib. Shortcomings of the study include selection of a specific protein for analysis, and the specific time points at which the treated tissue was analysed.ConclusionsCA9 levels increase with targeted therapy in mccRCC. Lower CA9 levels are associated with a poor prognosis and possible resistance, as indicated by the validation cohort.Patient summaryDrug treatment of advanced kidney cancer alters molecular markers of treatment resistance. Measuring carbonic anhydrase 9 levels may be helpful in determining which patients benefit from therapy

Sociophonetic variation in a long-term language contact situation: /l/-darkening in Welsh-English bilingual speech

This study investigates /l/-darkening in the Welsh and English speech of bilinguals in North Wales. Although it is claimed that /l/ is dark in all syllable positions in northern varieties of both languages, there have been no quantitative investigations of this feature which consider cross-linguistic phonetic differences, the differing nature of language contact between North East and North West Wales, and differences in the way both languages are acquired by speakers. The dataset of 32 Welsh-English bilinguals, aged 16-18, was stratified by speaker sex, home language, and area. Tokens of /l/ in word-initial onset and word-final coda positions were analysed acoustically. The results show cross-linguistic differences in onset position and that such differences were found to be greater in the speech of female participants and those from North West Wales. Differences were also found between Welsh-dominant and English-dominant communities. These results are discussed with reference to the influence of extra-linguistic factors on speech production and the possible social meaning associated with dark /l/

A collaborative approach to exploring the future of Cancer treatment and care in relation to Precision Medicine: A design perspective.

The Precision Medicine and the Future of Cancer project was jointly conceived by the Innovation School at Glasgow School of Art and the Institute of Cancer Sciences at the University of Glasgow. Graduating year Product Design students from the Innovation School were presented with a challenge-based project to produce a vision of the future based on current trends that relate to Precision Medicine(PM) and Cancer treatment. This project involved working closely with scientists, clinicians, patients, industry and academic professionals from Glasgow University, staff at Queen Elizabeth University Hospital and Clinical Innovation Zone, staff at Beatson West of Scotland Cancer Centre, Patient Representatives and external design experts from Studio AndThen and GOODD design consultancy. The objective of this project was to investigate, in both analytical and speculative ways, future forms and functions of cancer treatment and care in relation to Precision Medicine, to develop future scenarios and design artefacts, services, and the experiences associated with them. One of the most significant societal shifts currently taking place within the field of PM is the transformation around what it means to be a patient and a professional working within this context. The public’s role is developing beyond once-passive patients into stakeholders valued within the medical industry and healthcare sector for their participation in clinical trials, and contribution towards policy-making and decision-making committees. This new dynamic is changing the traditional patient-doctor relationship and challenging the hegemony of medical practice at an institutional level. The impetus for this shift is relentless technological acceleration and increased scientific research, in particular driven by advances in PM. This project asked students to consider what will happen in a cancer landscape ten years from now, where PM has evolved to the extent that new forms of medical practice, cancer treatment and care transform how we interact with each other, with professionals and the world around us. The brief gave students the opportunity to reflect on the underlying complexities regarding the future of health, technological acceleration, post-capitalism and human agency, to envision a future world context, develop it as an experiential exhibit, and produce the designed products, services and experiences for the people who might live and work within it. The project was divided into two sections: The first was a collaborative stage where groups of students were assigned a specific area of focus from Social, Technological, Economic, Ethical, Educational, Political, Legal, Ecological [STEEEPLE]. These groups focused on researching and exploring their specific lenses and gathering as much information and understanding while working with external experts to further their knowledge. This group stage culminated in an exhibition of the collaborative understanding of what the future could look like in 10 years from now, after exploring the possible consequences of current actions. The second stage saw students explore their individual response to the world that had been defined in the first stage. Each student had their own response to the research by iteratively creating a design outcome that was appropriate to the subject matter. This culminated in each student having created a design product/service/experience relating to the future scenario. A full report (Project Process Journal [PPJ]) is included within the repository of each student which breaks down their process of designing and the outcome they have designed. The project aims to tackle the emerging possibilities where medical professionals and design can collaborate, to create a future where forms of medical practice are more preventative and are more appropriate for an aging population now and into the future. The deposited materials are arranged as follows: Readme files - two readme files relate to stage one and stage two of the project as outlined above. Overview poster - gives a visual overview of the structure and timeline of the project. Data folders - the data folders for stage one of the project are named for the lens through which each group viewed possible futures. The data folders for stage two of the project are named for the individual students who conducted the work