FindFoci: a focus detection algorithm with automated parameter training that closely matches human assignments, reduces human inconsistencies and increases speed of analysis

Accurate and reproducible quantification of the accumulation of proteins into foci in cells is essential for data interpretation and for biological inferences. To improve reproducibility, much emphasis has been placed on the preparation of samples, but less attention has been given to reporting and standardizing the quantification of foci. The current standard to quantitate foci in open-source software is to manually determine a range of parameters based on the outcome of one or a few representative images and then apply the parameter combination to the analysis of a larger dataset. Here, we demonstrate the power and utility of using machine learning to train a new algorithm (FindFoci) to determine optimal parameters. FindFoci closely matches human assignments and allows rapid automated exploration of parameter space. Thus, individuals can train the algorithm to mirror their own assignments and then automate focus counting using the same parameters across a large number of images. Using the training algorithm to match human assignments of foci, we demonstrate that applying an optimal parameter combination from a single image is not broadly applicable to analysis of other images scored by the same experimenter or by other experimenters. Our analysis thus reveals wide variation in human assignment of foci and their quantification. To overcome this, we developed training on multiple images, which reduces the inconsistency of using a single or a few images to set parameters for focus detection. FindFoci is provided as an open-source plugin for ImageJ

Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway

This article has been made available through the Brunel Open Access Publishing Fund - Copyright @ 2009 Orton et al.BACKGROUND: The Epidermal Growth Factor Receptor (EGFR) activated Extracellular-signal Regulated Kinase (ERK) pathway is a critical cell signalling pathway that relays the signal for a cell to proliferate from the plasma membrane to the nucleus. Deregulation of the EGFR/ERK pathway due to alterations affecting the expression or function of a number of pathway components has long been associated with numerous forms of cancer. Under normal conditions, Epidermal Growth Factor (EGF) stimulates a rapid but transient activation of ERK as the signal is rapidly shutdown. Whereas, under cancerous mutation conditions the ERK signal cannot be shutdown and is sustained resulting in the constitutive activation of ERK and continual cell proliferation. In this study, we have used computational modelling techniques to investigate what effects various cancerous alterations have on the signalling flow through the ERK pathway. RESULTS: We have generated a new model of the EGFR activated ERK pathway, which was verified by our own experimental data. We then altered our model to represent various cancerous situations such as Ras, B-Raf and EGFR mutations, as well as EGFR overexpression. Analysis of the models showed that different cancerous situations resulted in different signalling patterns through the ERK pathway, especially when compared to the normal EGF signal pattern. Our model predicts that cancerous EGFR mutation and overexpression signals almost exclusively via the Rap1 pathway, predicting that this pathway is the best target for drugs. Furthermore, our model also highlights the importance of receptor degradation in normal and cancerous EGFR signalling, and suggests that receptor degradation is a key difference between the signalling from the EGF and Nerve Growth Factor (NGF) receptors. CONCLUSION: Our results suggest that different routes to ERK activation are being utilised in different cancerous situations which therefore has interesting implications for drug selection strategies. We also conducted a comparison of the critical differences between signalling from different growth factor receptors (namely EGFR, mutated EGFR, NGF, and Insulin) with our results suggesting the difference between the systems are large scale and can be attributed to the presence/absence of entire pathways rather than subtle difference in individual rate constants between the systems.This work was funded by the Department of Trade and Industry (DTI), under their Bioscience Beacon project programme. AG was funded by an industrial PhD studentship from Scottish Enterprise and Cyclacel

Holographic Brownian Motion in Magnetic Environments

Using the gauge/gravity correspondence, we study the dynamics of a heavy quark in two strongly-coupled systems at finite temperature: Super-Yang-Mills in the presence of a magnetic field and non-commutative Super-Yang-Mills. In the former, our results agree qualitatively with the expected behavior from weakly-coupled theories. In the latter, we propose a Langevin equation that accounts for the effects of non-commutativity and we find new interesting features. The equation resembles the structure of Brownian motion in the presence of a magnetic field and implies that the fluctuations along non-commutative directions are correlated. Moreover, our results show that the viscosity is smaller than the commutative case and that the diffusion properties of the quark are unaffected by non-commutativity. Finally, we compute the random force autocorrelator and verify that the fluctuation-dissipation theorem holds in the presence of non-commutativity.Comment: 34 pages. v2: typos corrected. v3: title and abstract slightly modified in order to better reflect the contents of the paper; footnote 3 and one reference were also added; version accepted for publication in JHE

Trends in health services utilization, medication use, and health conditions among older adults: a 2-year retrospective chart review in a primary care practice

<p>Abstract</p> <p>Background</p> <p>Population aging poses significant challenges to primary care providers and healthcare policy makers. Primary care reform can alleviate the pressures, but these initiatives require clinical benchmarks and evidence regarding utilization patterns. The objectives of this study is to measure older patients' use of health services, number of health conditions, and use of medications at the level of a primary care practice, and to investigate age- and gender-related utilization trends.</p> <p>Methods</p> <p>A cross-sectional chart audit over a 2-year study period was conducted in the academic family practice clinic of Sunnybrook Health Sciences Centre in Toronto, Ontario, Canada. All patients 65 years and older (n = 2450) were included. Main outcome measures included the number of family physician visits, specialist visits, emergency room visits, surgical admissions, diagnostic test days, inpatient hospital admissions, health conditions, and medications.</p> <p>Results</p> <p>Older patients (80-84 and 85+ age-group) had significantly more family physician visits (average of 4.4 visits per person per year), emergency room visits (average of 0.22 ER visits per year per patient), diagnostic days (average of 5.1 test days per person per year), health conditions (average of 7.7 per patient), and medications average of 8.2 medications per person). Gender differences were also observed: females had significantly more family physician visits and number of medications, while men had more specialist visits, emergency room visits, and surgical admissions. There were no gender differences for inpatient hospital admissions and number of health conditions. With the exception of the 85+ age group, we found greater intra-group variability with advancing age.</p> <p>Conclusion</p> <p>The data present a map of greater interaction with and dependency on the health care system with advancing age. The magnitudes are substantial and indicate high demands on patients and families, on professional health care providers, and on the health care system itself. There is the need to create and evaluate innovative models of care of multiple chronic conditions in the late life course.</p

Early development of spasticity following stroke: a prospective, observational trial

This study followed a cohort of 103 patients at median 6 days, 6 and 16 weeks after stroke and recorded muscle tone, pain, paresis, Barthel Index and quality of life score (EQ-5D) to identify risk-factors for development of spasticity. 24.5% of stroke victims developed an increase of muscle tone within 2 weeks after stroke. Patients with spasticity had significantly higher incidences of pain and nursing home placement and lower Barthel and EQ-5D scores than patients with normal muscle tone. Early predictive factors for presence of severe spasticity [modified Ashworth scale score (MAS) ≥3] at final follow-up were moderate increase in muscle tone at baseline and/or first follow-up (MAS = 2), low Barthel Index at baseline, hemispasticity, involvement of more than two joints at first follow-up, and paresis at any assessment point. The study helps to identify patients at highest risk for permanent and severe spasticity, and advocates for early treatment in this group

Star forming dwarf galaxies

Star forming dwarf galaxies (SFDGs) have a high gas content and low metallicities, reminiscent of the basic entities in hierarchical galaxy formation scenarios. In the young universe they probably also played a major role in the cosmic reionization. Their abundant presence in the local volume and their youthful character make them ideal objects for detailed studies of the initial stellar mass function (IMF), fundamental star formation processes and its feedback to the interstellar medium. Occasionally we witness SFDGs involved in extreme starbursts, giving rise to strongly elevated production of super star clusters and global superwinds, mechanisms yet to be explored in more detail. SFDGs is the initial state of all dwarf galaxies and the relation to the environment provides us with a key to how different types of dwarf galaxies are emerging. In this review we will put the emphasis on the exotic starburst phase, as it seems less important for present day galaxy evolution but perhaps fundamental in the initial phase of galaxy formation.Comment: To appear in JENAM Symposium "Dwarf Galaxies: Keys to Galaxy Formation and Evolution", P. Papaderos, G. Hensler, S. Recchi (eds.). Lisbon, September 2010, Springer Verlag, in pres

Comparative study of in situ N2 rotational Raman spectroscopy methods for probing energy thermalisation processes during spin-exchange optical pumping

Spin-exchange optical pumping (SEOP) has been widely used to produce enhancements in nuclear spin polarisation for hyperpolarised noble gases. However, some key fundamental physical processes underlying SEOP remain poorly understood, particularly in regards to how pump laser energy absorbed during SEOP is thermalised, distributed and dissipated. This study uses in situ ultra-low frequency Raman spectroscopy to probe rotational temperatures of nitrogen buffer gas during optical pumping under conditions of high resonant laser flux and binary Xe/N2 gas mixtures. We compare two methods of collecting the Raman scattering signal from the SEOP cell: a conventional orthogonal arrangement combining intrinsic spatial filtering with the utilisation of the internal baffles of the Raman spectrometer, eliminating probe laser light and Rayleigh scattering, versus a new in-line modular design that uses ultra-narrowband notch filters to remove such unwanted contributions. We report a ~23-fold improvement in detection sensitivity using the in-line module, which leads to faster data acquisition and more accurate real-time monitoring of energy transport processes during optical pumping. The utility of this approach is demonstrated via measurements of the local internal gas temperature (which can greatly exceed the externally measured temperature) as a function of incident laser power and position within the cell

Molecular characterization of partial-open reading frames 1a and 2 of the human astroviruses in South Korea

Human astroviruses (HAstVs) are among the major causes of gastroenteritis in South Korea. In this study, the partial regions of the open reading frame (ORF) 1a and ORF2 genes of HAstVs from gastroenteritis patients in nine hospitals were sequenced, and the molecular characterization of the viruses was revealed. 89 partial nucleotide sequences of ORF1a and 88 partial nucleotide sequences of ORF2 were amplified from 120 stool specimens. Phylogenetic analysis showed that most of the nucleotide sequences of ORF1a and ORF2 were grouped with HAstV type 1 but had evolutionary genetic distance compared with the reference sequences, such as the HAstV-1 prototype, Dresden strain, and Oxford strain. According to the phylogenetic analysis, some nucleotide sequences including SE0506041, SE0506043, and SE0506058, showed the discrepancy of the genotypes, but there was no proof of recombination among the HAstV types. In conclusion, this study showed that the dominant HAstV isolated from the Seoul metropolitan area in 2004-2005 was HAstV type 1, and that Korean HAstV-1 had the genetic distance in evolution compared with the reference sequences of HAstVs. Lots of nucleotide sequences of the ORF1a and ORF2 genes of HAstV will be useful for studying for the control and prevention of HAstV gastroenteritis in South Korea

A Vaccine against Nicotine for Smoking Cessation: A Randomized Controlled Trial

BACKGROUND: Tobacco dependence is the leading cause of preventable death and disabilities worldwide and nicotine is the main substance responsible for the addiction to tobacco. A vaccine against nicotine was tested in a 6-month randomized, double blind phase II smoking cessation study in 341 smokers with a subsequent 6-month follow-up period. METHODOLOGY/PRINCIPAL FINDINGS: 229 subjects were randomized to receive five intramuscular injections of the nicotine vaccine and 112 to receive placebo at monthly intervals. All subjects received individual behavioral smoking cessation counseling. The vaccine was safe, generally well tolerated and highly immunogenic, inducing a 100% antibody responder rate after the first injection. Point prevalence of abstinence at month 2 showed a statistically significant difference between subjects treated with Nicotine-Qbeta (47.2%) and placebo (35.1%) (P = 0.036), but continuous abstinence between months 2 and 6 was not significantly different. However, in subgroup analysis of the per-protocol population, the third of subjects with highest antibody levels showed higher continuous abstinence from month 2 until month 6 (56.6%) than placebo treated participants (31.3%) (OR 2.9; P = 0.004) while medium and low antibody levels did not increase abstinence rates. After 12 month, the difference in continuous abstinence rate between subjects on placebo and those with high antibody response was maintained (difference 20.2%, P = 0.012). CONCLUSIONS: Whereas Nicotine-Qbeta did not significantly increase continuous abstinence rates in the intention-to-treat population, subgroup analyses of the per-protocol population suggest that such a vaccination against nicotine can significantly increase continuous abstinence rates in smokers when sufficiently high antibody levels are achieved. Immunotherapy might open a new avenue to the treatment of nicotine addiction. TRIAL REGISTRATION: Swiss Medical Registry 2003DR2327; ClinicalTrials.gov NCT00369616