Automatic segmentation of focal adhesions from mouse embryonic fibroblasts

This work describes an automatic algorithm for the segmentation and quantification of focal adhesions from mouse embryonic fibroblasts. The main challenges solved by this algorithm are: the variability of the intensity of the focal adhesions, the detection of an outer ring, which distinguishes the cell periphery responsible for the cell migration, and the quantification of the characteristics of the focal adhesions. The algorithm detects maximal regions through gradients and uses a region-growing algorithm limited by intensity-based edges. The outer ring is calculated based on the average radial intensity from an extended centroid of the cell. Finally, traditional morphological characteristics are obtained to distinguish between two groups of cells. Two of the measurements employed showed statistical difference between two groups of cells

Time consistent expected mean-variance in multistage stochastic quadratic optimization: a model and a matheuristic

In this paper, we present a multistage time consistent Expected Conditional Risk Measure for minimizing a linear combination of the expected mean and the expected variance, so-called Expected Mean-Variance. The model is formulated as a multistage stochastic mixed-integer quadratic programming problem combining risk-sensitive cost and scenario analysis approaches. The proposed problem is solved by a matheuristic based on the Branch-and-Fix Coordination method. The multistage scenario cluster primal decomposition framework is extended to deal with large-scale quadratic optimization by means of stage-wise reformulation techniques. A specific case study in risk-sensitive production planning is used to illustrate that a remarkable decrease in the expected variance (risk cost) is obtained. A competitive behavior on the part of our methodology in terms of solution quality and computation time is shown when comparing with plain use of CPLEX in 150 benchmark instances, ranging up to 711,845 constraints and 193,000 binary variables.project MTM2015-65317-P (MINECO/FEDER/EU); BERC 2014-2017; IT-928-16; and by the University of the Basque Country UPV/EHU; BCAM Severo Ochoa excellence accreditation Grant SEV-2013-0323; BERC 2014-201

Astrocytes and Inflammatory Processes in Alzheimer’s Disease

A significant increase in inflammation has been shown to be a crucial factor in the progression of the Alzheimer’s disease (AD). Moreover, inflammatory signals are already present in mild cognitive impairment (MCI) patients before they develop AD. The amyloid hypothesis argues that in AD, there is an increase in oxidative stress caused by the accumulation of β-amyloid (Aβ) and that its elimination should be a priority. Also, hyperphosphorylation of the protein TAU occurs, which is characteristic of this disease. In AD oxidative stress processes occur and also inflammation. The basal chronic inflammation produces a cascade of cellular, such as astrocytes and microglial cells, and molecular processes in AD patients. We here have tried to explore the action of the inflammatory process and its implication in the neurodegenerative process of the AD. We can see that the role of Aβ is only one component that gives rise to inflammation, probably mediated by activation of microglia and astrocytes with the goal of getting rid of these brain waste products. In fact, it is related to a greater degree with the progression of the disease and worsening of the symptoms with the increase of phosphorylated TAU in different parts of the brain

Measuring Red Blood Cell Velocity with a Keyhole Tracking Algorithm

A tracking algorithm is proposed to measure the velocity of red blood cells traveling through microvessels of tumors growing in skin flaps implanted on mice. The tracking is based on a keyhole model that describes the probable movement of a segmented cell between contiguous frames in a video sequence. When a history of movements exists, past, present and a predicted landing position define two regions of probability with a keyhole shape. This keyhole is used to de- termine if cells in contiguous frames should be linked to form tracks. Pre-processing segments cells from background and post-processing joins tracks and discards links that could have been formed due to noise or uncertainty. The algorithm pre- sents several advantages over traditional methods such as kymographs or particle image velocimetry: manual interven- tion is restricted to the thresholding, several vessels can be analyzed simultaneously, algorithm is robust to noise and a wealth of statistical measures can be obtained. Two tumors with different geometries were analyzed; average velocities were 211±136 [μm/s] (mean±std) with a range 15.9-797 [μm/s], and 89±62 [μm/s] with a range 5.5-300 [μm/s] respec- tively, which are consistent with previous results in the litera- ture

A parallel Branch-and-Fix Coordination based matheuristic algorithm for solving large sized multistage stochastic mixed 0-1 problems

A parallel matheuristic algorithm is presented as a spin-off from the exact Branch-and-Fix Coordination (BFC) algorithm for solving multistage stochastic mixed 0-1 problems. Some steps to guarantee the solution’s optimality are relaxed in the BFC algorithm, such that an incomplete backward branching scheme is considered for solving large sized problems. Additionally, a new branching criterion is considered, based on dynamically-guided and stage-wise ordering schemes, such that fewer Twin Node Families are expected to be visited during the execution of the so-called H-DBFC algorithm. The inner parallelization IH-DBFC of the new approach, allows to solve in parallel scenario clusters MIP submodels at different steps of the algorithm. The outer parallel version, OH-DBFC, considers independent paths and allows iterative incumbent solution values exchanges to obtain tighter bounds of the solution value of the original problem. A broad computational experience is reported for assessing the quality of the matheuristic solution for large sized instances. The instances dimensions that are considered are up to two orders of magnitude larger than in some other works that we are aware of. The optimality gap of the H-DBFC solution value versus the one obtained by a state-of-the-artMIP solver is very small, if any. The new approach frequently outperforms it in terms of solution’s quality and computing time. A comparison with our Stochastic Dynamic Programming algorithm is also reported. The use of parallel computing provides, on one hand, a perspective for solving very large sized instances and, on the other hand, an expected large reduction in elapsed time.MTM2015-65317-P, MTM2015-63710-P, IT928-16; UFI BETS 2011; IZO-SGI SGIke

A Framework for Providing Research Applications as a Service Using the IOME Toolkit

This paper presents a unique, multi-purpose toolkit, enabling researchers to easily develop modelling and analysis applications, which can be run as web services and accessed interactively. The development kit is based on a protocol that uses an XML markup called the "Interactive Object Management Environment Markup Language" (IOME ML). The paper describes the IOME ML and its development kit. We illustrate the capabilities of IOME with two case studies the first case study is based on a medical image processing application (CAIMAN: CAncer IMage ANalysis), offering image analysis tools for life scientists. For the second case study, the Pi-Phi collaboration have developed an inverse imaging method for ‘lensless’ microscopy a demonstrator is introduced for the Pi-Phi project. For both case studies the application is wrapped as a web service and accessed through a web browser. The paper concludes with a review of further developments, including refinements to the mark up language and the development of a service factory, enabling a more scalable service provision model through the dynamic invocation of published simulations as IOME web service applications

Signaling interactions between RhoGTPase and cAMP/cGMP influence endothelial responses to the vascular disrupting agent combretastatin A4 phosphate

Background: Combretastatin A4 phosphate (CA4P) is a tumour vascular disrupting agent (VDA) that targets endothelial microtubules, triggering remodelling of the actin cytoskeleton, contractility and disruption of VE-cadherin junctions through RhoGTPase/ROCK-dependent pathways. These events lead to a rise in endothelial monolayer permeability. A rise in permeability is considered crucial for vascular shutdown elicited by CA4P in vivo. CA4P also inhibits endothelial migration and induces mitotic arrest and apoptosis, so potentially it could also target tumour angiogenesis. Method: In this study, the nature of signalling interactions between Rho/ROCK and cAMP/cGMP and their influence on cytoskeletal and functional responses of endothelial cells to CA4P were investigated. Results: Several cAMP/cGMP analogues inhibited CA4P-induced Rho/ROCK activation and prevented actin remodeling, disruption of cell-to-cell junctions and permeability rise in endothelial monolayers. cAMP inhibits Rho by either protein kinase A (PKA)-dependent mechanisms or via activation of Epac1/Rap1. O-Me-cAMP, an analogue that selectively activates Epac1/Rap1 abolished activation of Rho/ROCK by CA4P while selective PKA activator 6-Bnz-cAMP only partially inhibited Rho/ROCK activation and actin remodelling by CA4P. Inhibitors of PKA did not alter endothelial responses to CA4P in the presence of cAMP analogues suggesting that cAMP acts primarily via Epac1/Rap1 to inhibit Rho/CA4P interactions. CA-4-P also inhibited endothelial migration and abolished lamellipodia at the leading edge of migrating cells in injured monolayers. Rho inhibitor C3 exoenzyme and ROCK inhibitor Y27632 as well as cAMP analogues re-established cell movement and formation of lamellipodia in wounded monolayers exposed to CA-4-P, suggesting that inhibitory effects on migration were mediated via Rho/ROCK. Conclusion: Deciphering molecular pathways that modulate endothelial responses to VDAs is important for further targeting. Our data demonstrate that interactions between cGMP/cAMP and Rho influence both the vascular disrupting and anti-angiogenic activities of CA-4-P and point to cAMP/cGMP as potential targets for improving VDA activity. Acknowledgements Funded by Cancer Research U

Tracking neutrophils in zebrafish: the use of synthetic data sets

In this work we present a series of data sets that model the behaviour of neutrophils as observed with a confocal microscope. The data sets describe important characteristics of the migration of neutrophils such as collisions and path tortuosity as well as different levels of background noise. Neutrophil trajectories were manually defined, and Gaussian shapes similar to those of real data sets were assigned to each position of a neutrophil. The availability of synthetic data sets such as the ones proposed here, together with appropriate gold standards will benefit those wanting to test the robustness and accuracy of segmentation and tracking algorithms

Exploring neutrophil behaviour in a zebrafish model of inflammation through the generation of novel parameters using MatLab algorithms

Purpose/Objective: Tracking of immune cells is key to understanding their behaviour during inflammation. Current software available for tracking of immune cells is limited. The aim of this study was to develop a MatLab package of segmentation and tracking algorithms to apply in the tracking of neutrophils, fluorescently labelled in our zebrafish model [1]. The development of algorithms in MatLab allows us to explore parameters not available in other software packages such as directionality of neutrophil movement and neutrophil behaviour in- side and outside of a wound region. Materials and methods: Tail fin transection was performed on Tg(mpx:GFP) zebrafish (3 dpf) which were imaged on a spinning disk confocal from 1 h post injury (hpi) to 7 hpi. Images were exported from VolocityTM and analysed using MatLab m-files written for the tracking of immune cells. This is a fully automated analysis, after the user defines the initial thresholds based on fluorescent intensity of the images. Results: Neutrophils from injured embryos had a lower meandering ratio and a greater speed than neutrophils tracked in uninjured embryos (meandering ratio 0.24 ± 0.03 versus 0.42 ± 0.05, P = 0.003; speed 4.03 ± 0.32 versus 1.31 ± 0.21 pixels/frame, P 0.05). In injured embryos with a defined wound region, the oriented velocity towards the wound was 0.31 ± 0.24 pixels/frame. Once within the wound region, the oriented velocity of neutrophil tracks was -0.39 ± 0.32 pixels/frame; indicating that while the neutrophils travel at a similar speed, they are now travelling away from the wound. The ‘in wound ratio’ was 0.91 ± 0.04, indicating that once neutrophils enter the wound region they tend to stay, in the timeframe studied. The ‘leave wound ratio’ was 0.37 ± 0.03, a measure of the rate at which neutrophils move away from the site of injury once they have entered the wound region. Conclusions: Using these algorithms, we can analyse the behaviour of immune cells in a more detailed way. In addition to previously available parameters such as meandering ratio and speed, more complex parameters such as velocity towards or away from a wound region and a measurement of how neutrophils behave while in a wound region are available. Combining this novel tracking technology with established assays in our laboratory will enable the further dissection of neutrophil fate following an inflammatory stimulus

Microflow of fluorescently labelled red blood cells in tumours expressing single isoforms of VEGF and their response to VEGF-R tyrosine kinase inhibition

This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.In this work we studied the functional differences between the microcirculation of murine tumours that only express single isoforms of vascular endothelial growth factor-A (VEGF), VEGF120 and VEGF188, and the effect of VEGF receptor tyrosine kinase (VEGF-R TK) inhibition on their functional response to the vascular disrupting agent, combretastatin A-4 phosphate (CA-4-P). We used measurement of fluorescentlylabelled red blood cell (RBC) velocities in tumour microvessels to study this functional response. RBC velocity for control VEGF120-expressing tumours was over 50% slower than for control VEGF188-expressing tumours, which may be due to the immature and haemorrhagic vasculature of the VEGF120 tumour. After chronic treatment with a VEGF-R tyrosine kinase inhibitor, SU5416, RBC velocities in VEGF120 tumours were significantly increased compared to control VEGF120 tumours, and similar to velocities in both VEGF188 treatment groups. Control and SU5416 treated VEGF188 tumours were not different from each other. Treatment of VEGF120 tumours with SU5416 reduced their vascular response to CA-4-P to a similar level to the VEGF188 tumours. Differential expression of VEGF isoforms not only affected vascular function in untreated tumours but also impacted on response to a vascular disrupting drug, CA-4-P, alone and in combination with an anti-angiogenic approach involving VEGF-R TK inhibition. Analysis of RBC velocities is a useful tool in measuring functional responses to vascular targeted treatments.This study is funded by the Cancer Research UK