Heterogeneity in melanoma and the microenvironment

This thesis addresses the signalling pathways and gene networks associated with melanoma cell motility in vivo and heterogeneity in tumour-associated vasculature. Melanoma metastasis begins with the acquisition of motility in the primary tumour, however, there is little characterisation of signalling pathways or microenvironments associated with melanoma motility in vivo. Intra-vital imaging of clonal signalling reporter cell-lines showed heterogeneous cell behaviour in vivo and increased Notch, TGF-β and SRF dependent transcription in motile cells. Further investigations into Notch signalling in melanoma metastasis showed that Notch promotes metastasis through cell-autonomous and non-cell autonomous mechanisms. The reporter cell-lines were used to enrich for the motile melanoma cell population and genes up-regulated in this population were identified. Many cell-cycle regulators, including regulators of cytokinesis, were up-regulated in the B16 F2 melanoma population enriched for motile cells. Bioinformatic analysis indicated that EZH2, a histone methyltransferase in the Polycomb repressor 2 complex, can regulate the genes associated with motile cells and EZH2 depletion decreased melanoma cell motility in vitro and metastasis in vivo. EZH2 was also shown to regulate cell morphology and the actin cytoskeleton through regulating activation of a subset of ERM proteins. Due to association between melanoma metastasis and differentiation, the role of EZH2 in melanoma differentiation state was investigated. EZH2 depletion did not significantly affect expression of melanoma differentiation markers but increased Oca2 mRNA expression leading to increased pigment. This thesis also investigates heterogeneity in the tumour microenvironment. Morphologically and dynamically distinct regions of the tumour vasculature were identified by intra-vital imaging and quantitative image analysis, and these regions could be followed over time using imaging windows. The tumour vasculature showed a heterogeneous response to the anti-angiogenic drug, Sunitinib, possibly due to microenvironmental factors. Aspects of vascular morphology and dynamics correlated with melanoma motility and may be used to describe microenvironments favourable for motility

A bifunctional kinase-phosphatase in bacterial chemotaxis.

addresses: Oxford Centre for Integrative Systems Biology and Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.notes: PMCID: PMC2587623types: Journal Article; Research Support, Non-U.S. Gov'tCopyright © 2008, The National Academy of SciencesPhosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, CheA(3), is not only a phosphodonor for the principal CheY protein, CheY(6), but is also is a specific phosphatase for CheY(6)-P. This phosphatase activity accelerates CheY(6)-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. CheA(3) possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of CheA(3) is phosphorylated by CheA(4), and it subsequently acts as a phosphodonor for the response regulators. A CheA(3) mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of CheA(3) is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, CheA(3) lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases

Long-term high frequency measurements of ethane, benzene and methyl chloride at Ragged Point, Barbados: Identification of long-range transport events

Abstract Here we present high frequency long-term observations of ethane, benzene and methyl chloride from the AGAGE Ragged Point, Barbados, monitoring station made using a custom built GC-MS system. Our analysis focuses on the first three years of data (2005–2007) and on the interpretation of periodic episodes of high concentrations of these compounds. We focus specifically on an exemplar episode during September 2007 to assess if these measurements are impacted by long-range transport of biomass burning and biogenic emissions. We use the Lagrangian Particle Dispersion model, NAME, run forwards and backwards in time to identify transport of air masses from the North East of Brazil during these events. To assess whether biomass burning was the cause we used hot spots detected using the MODIS instrument to act as point sources for simulating the release of biomass burning plumes. Excellent agreement for the arrival time of the simulated biomass burning plumes and the observations of enhancements in the trace gases indicates that biomass burning strongly influenced these measurements. These modelling data were then used to determine the emissions required to match the observations and compared with bottom up estimates based on burnt area and literature emission factors. Good agreement was found between the two techniques highlight the important role of biomass burning. The modelling constrained by in situ observations suggests that the emission factors were representative of their known upper limits, with the in situ data suggesting slightly greater emissions of ethane than the literature emission factors account for. Further analysis was performed concluding only a small role for biogenic emissions of methyl chloride from South America impacting measurements at Ragged Point. These results highlight the importance of long-term high frequency measurements of NMHC and ODS and highlight how these data can be used to determine sources of emissions 1000’s km away.ATA would like to thank the GWR and the Met Office for the PhD funding that was useful in the initiation of this research and the Herchel Smith foundation for a Postdoctoral fellowship, and NCAS for funding. The operation of the AGAGE station at Ragged Point is supported by the National Aeronautic and Space Administration (NASA, USA) (grants NNX07AE89G and NNX11AF17G to MIT) and the National Oceanic and Atmospheric Administration (NOAA, USA) (contract RA133R09CN0062).This is the final version of the article. It first appeared from BioOne via http://dx.doi.org/10.12952/journal.elementa.00006

'Can you recommend any good STI apps?' A review of content, accuracy and comprehensiveness of current mobile medical applications for STIs and related genital infections.

OBJECTIVE: Seeking sexual health information online is common, and provision of mobile medical applications (apps) for STIs is increasing. Young people, inherently at higher risk of STIs, are avid users of technology, and apps could be appealing sources of information. We undertook a comprehensive review of content and accuracy of apps for people seeking information about STIs. METHODS: Search of Google Play and iTunes stores using general and specific search terms for apps regarding STIs and genital infections (except HIV), testing, diagnosis and management, 10 September 2014 to 16 September 2014. We assessed eligible apps against (1) 19 modified Health on The Net (HON) Foundation principles; and (2) comprehensiveness and accuracy of information on STIs/genital infections, and their diagnosis and management, compared with corresponding National Health Service STI information webpage content. RESULTS: 144/6642 apps were eligible. 57 were excluded after downloading. 87 were analysed. Only 29% of apps met ≥6 HON criteria. Content was highly variable: 34/87 (39%) covered one or two infections; 40 (46%) covered multiple STIs; 5 (6%) focused on accessing STI testing. 13 (15%) were fully, 46 (53%) mostly and 28 (32%) partially accurate. 25 (29%) contained ≥1 piece of potentially harmful information. Apps available on both iOS and Android were more accurate than single-platform apps. Only one app provided fully accurate and comprehensive information on chlamydia. CONCLUSIONS: Marked variation in content, quality and accuracy of available apps combined with the nearly one-third containing potentially harmful information risks undermining potential benefits of an e-Health approach to sexual health and well-being

Stochasticity in the miR-9/Hes1 oscillatory network can account for clonal heterogeneity in the timing of differentiation.

Recent studies suggest that cells make stochastic choices with respect to differentiation or division. However, the molecular mechanism underlying such stochasticity is unknown. We previously proposed that the timing of vertebrate neuronal differentiation is regulated by molecular oscillations of a transcriptional repressor, HES1, tuned by a post-transcriptional repressor, miR-9. Here, we computationally model the effects of intrinsic noise on the Hes1/miR-9 oscillator as a consequence of low molecular numbers of interacting species, determined experimentally. We report that increased stochasticity spreads the timing of differentiation in a population, such that initially equivalent cells differentiate over a period of time. Surprisingly, inherent stochasticity also increases the robustness of the progenitor state and lessens the impact of unequal, random distribution of molecules at cell division on the temporal spread of differentiation at the population level. This advantageous use of biological noise contrasts with the view that noise needs to be counteracted

The frequency of genes encoding three putative group B streptococcal virulence factors among invasive and colonizing isolates

BACKGROUND: Group B Streptococcus (GBS) causes severe infections in very young infants and invasive disease in pregnant women and adults with underlying medical conditions. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. Three proteins, Rib encoded by rib, and alpha and beta C proteins encoded by bca and bac, respectively, have been suggested as potential vaccine candidates for GBS. It is not known, however, whether these genes occur more frequently in invasive versus colonizing GBS strains. METHODS: We screened 162 invasive and 338 colonizing GBS strains from different collections using dot blot hybridization to assess the frequency of bca, bac and rib. All strains were defined by serotyping for capsular type, and frequency differences were tested using the Chi square test. RESULTS: Genes encoding the beta C protein (bac) and Rib (rib) occurred at similar frequencies among invasive and colonizing isolates, bac (20% vs. 23%), and rib (28% vs. 20%), while the alpha (bca) C protein was more frequently found in colonizing strains (46%) vs, invasive (29%). Invasive strains were associated with specific serotype/gene combinations. CONCLUSION: Novel virulence factors must be identified to better understand GBS disease

The application of multiplex PCR to detect seven different DNA targets in group B streptococci

Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection

Expression, Localization, and Phosphorylation of Akt1 in Benign and Malignant Thyroid Lesions

The serine/threonine protein kinase Akt is a key molecule in the phosphatidyl inositol 3-kinase pathway that is often overactivated in human cancers. Three Akt isoforms (Akt1, Akt2, Akt3) have been identified in human cells and they show different distribution and have non-redundant functions. The aim of this study was to determine whether the expression, phosphorylation, and localization of Akt1 isoform in human thyroid malignant lesions are different from those in benign lesions. Nuclear and cytoplasmic fractions were isolated from tissue samples and Western blot method was used to detect Akt1 presence in both cellular fractions. Akt1 expression was also assessed by ELISA method. To estimate Akt1 phosphorylation, kinase was immunoprecipitated from cell lysates and tested with anti-phospho-Akt antibodies. The Akt1 expression in majority of thyroid cancer samples was significantly higher than in benign lesions (p < 0.05). Akt1 both in differentiated cancers (follicular and papillary) and benign lesions was localized mainly in cytoplasmic fraction. In two of three anaplastic cancer samples Akt1 was predominantly localized in nucleus. The ratio of phosphorylated Akt1 to total Akt1 was lower in cancers than in non-neoplastic lesions and adenomas. Thus, although Akt1 seems to be overexpressed in thyroid neoplasms, its high phosphorylation is not characteristic for thyroid cancers

Membranes by the Numbers

Many of the most important processes in cells take place on and across membranes. With the rise of an impressive array of powerful quantitative methods for characterizing these membranes, it is an opportune time to reflect on the structure and function of membranes from the point of view of biological numeracy. To that end, in this article, I review the quantitative parameters that characterize the mechanical, electrical and transport properties of membranes and carry out a number of corresponding order of magnitude estimates that help us understand the values of those parameters.Comment: 27 pages, 12 figure

Machine Learning Classification of Females Susceptibility to Visceral Fat Associated Diseases

The problem of classifying subjects into risk categories is a common challenge in medical research. Machine Learning (ML) methods are widely used in the areas of risk prediction and classification. The primary objective of these algorithms is to predict dichotomous responses (e.g. healthy/at risk) based on several features. Similarly to statistical inference models, also ML models are subject to the common problem of class imbalance. Therefore, they are affected by the majority class increasing the false-negative rate. In this paper, we built and evaluated eighteen ML models classifying approximately 4300 female participants from the UK Biobank into three categorical risk statuses based on responses for the discretised visceral adipose tissue values from magnetic resonance imaging. We also examined the effect of sampling techniques on classification modelling when dealing with class imbalance. Results showed that the use of sampling techniques had a significant impact. They not only drove an improvement in predicting patients risk status but also facilitated an increase in the information contained within each variable. Based on domain experts criteria, the three best models for classification were finally identified. These encouraging results will guide further developments of classification models for predicting visceral adipose tissue without the need for a costly scan