How much information can be obtained from tracking the position of the leading edge in a scratch assay?

Moving cell fronts are an essential feature of wound healing, development and disease. The rate at which a cell front moves is driven, in part, by the cell motility, quantified in terms of the cell diffusivity $D$, and the cell proliferation rate �$\lambda$. Scratch assays are a commonly-reported procedure used to investigate the motion of cell fronts where an initial cell monolayer is scratched and the motion of the front is monitored over a short period of time, often less than 24 hours. The simplest way of quantifying a scratch assay is to monitor the progression of the leading edge. Leading edge data is very convenient since, unlike other methods, it is nondestructive and does not require labeling, tracking or counting individual cells amongst the population. In this work we study short time leading edge data in a scratch assay using a discrete mathematical model and automated image analysis with the aim of investigating whether such data allows us to reliably identify $D$ and $\lambda$�. Using a naıve calibration approach where we simply scan the relevant region of the ($D$;$\lambda$�) parameter space, we show that there are many choices of $D$ and $\lambda$� for which our model produces indistinguishable short time leading edge data. Therefore, without due care, it is impossible to estimate $D$ and $\lambda$� from this kind of data. To address this, we present a modified approach accounting for the fact that cell motility occurs over a much shorter time scale than proliferation. Using this information we divide the duration of the experiment into two periods, and we estimate $D$ using data from the first period, while we estimate �$\lambda$ using data from the second period. We confirm the accuracy of our approach using in silico data and a new set of in vitro data, which shows that our method recovers estimates of $D$ and $\lamdba$� that are consistent with previously-reported values except that that our approach is fast, inexpensive, nondestructive and avoids the need for cell labeling and cell counting.

A multi-genome analysis approach enables tracking of the invasion of a single Russian wheat aphid (Diuraphis noxia) clone throughout the New World

This study investigated the population genetics, demographic history and pathway of invasion of the Russian wheat aphid (RWA) from its native range in Central Asia, the Middle East and Europe to South Africa and the Americas. We screened microsatellite markers, mitochondrial DNA and endosymbiont genes in 504 RWA clones from nineteen populations worldwide. Following pathway analyses of microsatellite and endosymbiont data, we postulate that Turkey and Syria were the most likely sources of invasion to Kenya and South Africa, respectively. Furthermore, we found that one clone transferred between South Africa and the Americas was most likely responsible for the New World invasion. Finally, endosymbiont DNA was found to be a high resolution population genetic marker, extremely useful for studies of invasion over a relatively short evolutionary history time frame. This study has provided valuable insights into the factors that may have facilitated the recent global invasion by this damaging pest.

Visible and near infrared spectroscopy of hayabusa reentry using semi-autonomous tracking

Aground-based tracking camera and coaligned slitless spectrograph were used to measure the spectral signature of visible radiation emitted from the Hayabusa capsule as it entered into the Earth’s atmosphere in June 2010. Good quality spectra were obtained, which showed the presence of radiation from the heat shield of the vehicle and the shock-heated air in front of the vehicle. An analysis of the blackbody nature of the radiation concluded that the peak average temperature of the surface was about (3100± 100)K. Line spectra from oxygen and nitrogen atoms were used to infer a peak average shock-heated gas temperature of around((7000±400))K.

Challenges for implementation of a new model of collaborative design management: Analyzing the impact of human factor

The ineffectiveness of current design processes has been well studied and has resulted in widespread calls for the evolution and development of new management processes. Even following the advent of BIM, we continue to move from one stage to another without necessarily having resolved all the issues. CAD design technology, if well handled, could have significantly raised the level of quality and efficiency of current processes, but in practice this was not fully realized. Therefore, technology alone can´t solve all the problems and the advent of BIM could result in a similar bottleneck. For a precise definition of the problem to be solved we should start by understanding what are the main current bottlenecks that have yet to be overcome by either new technologies or management processes, and the impact of human behaviour-related issues which impact the adoption and utilization of new technologies. The fragmented and dispersed nature of the AEC sector, and the huge number of small organizations that comprise it, are a major limiting factor. Several authors have addressed this issue and more recently IDDS has been defined as the highest level of achievement. However, what is written on IDDS shows an extremely ideal situation on a state to be achieved; it shows a holistic utopian proposition with the intent to create the research agenda to move towards that state. Key to IDDS is the framing of a new management model which should address the problems associated with key aspects: technology, processes, policies and people. One of the primary areas to be further studied is the process of collaborative work and understanding, together with the development of proposals to overcome the many cultural barriers that currently exist and impede the advance of new management methods. The purpose of this paper is to define and delimit problems to be solved so that it is possible to implement a new management model for a collaborative design process.

Robust speed tracking control of synchronous motors using immersion and invariance

This paper presents a novel control strategy for velocity tracking of Permanent Magnet Synchronous Machines (PMSM). The model of the machine is considered within the port-Hamiltonian framework and a control is designed using concepts of immersion and invariance (I&I) recently developed in the literature. The proposed controller ensures internal stability and output regulation, and it forces integral action on non-passive outputs.

Manoeuvring control of fully-actuated marine vehicles : a Port-Hamiltonian system approach to tracking

This paper presents a novel control strategy for trajectory tracking of marine vehicles manoeuvring at low speed. The model of the marine vehicle is formulated as a Port-Hamiltonian system, and the tracking controller is designed using energy shaping and damping assignment. The controller guarantees global asymptotic stability and includes integral action for output variables with relative degree greater than one.

Assessment of gene expression of intracellular calcium channels, pumps and exchangers with epidermal growth factor-induced epithelial-mesenchymal transition in a breast cancer cell line

Background Epithelial-mesenchymal transition (EMT) is a process implicated in cancer metastasis that involves the conversion of epithelial cells to a more mesenchymal and invasive cell phenotype. In breast cancer cells EMT is associated with altered store-operated calcium influx and changes in calcium signalling mediated by activation of cell surface purinergic receptors. In this study, we investigated whether MDA-MB-468 breast cancer cells induced to undergo EMT exhibit changes in mRNA levels of calcium channels, pumps and exchangers located on intracellular calcium storing organelles, including the Golgi, mitochondria and endoplasmic reticulum (ER). Methods Epidermal growth factor (EGF) was used to induce EMT in MDA-MB-468 breast cancer cells. Serum-deprived cells were treated with EGF (50 ng/mL) for 12 h and gene expression was assessed using quantitative RT-PCR. Results and conclusions These data reveal no significant alterations in mRNA levels of the Golgi calcium pump secretory pathway calcium ATPases (SPCA1 and SPCA2), or the mitochondrial calcium uniporter (MCU) or Na+/Ca2+ exchanger (NCLX). However, EGF-induced EMT was associated with significant alterations in mRNA levels of specific ER calcium channels and pumps, including (sarco)-endoplasmic reticulum calcium ATPases (SERCAs), and inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RYR) calcium channel isoforms. The most prominent change in gene expression between the epithelial and mesenchymal-like states was RYR2, which was enriched 45-fold in EGF-treated MDA-MB-468 cells. These findings indicate that EGF-induced EMT in breast cancer cells may be associated with major alterations in ER calcium homeostasis.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population

Background Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. Results Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals. A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia. The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. Conclusions The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

An R2R3 MYB transcription factor associated with regulation of the anthocyanin biosynthetic pathway in Rosaceae

Background The control of plant anthocyanin accumulation is via transcriptional regulation of the genes encoding the biosynthetic enzymes. A key activator appears to be an R2R3 MYB transcription factor. In apple fruit, skin anthocyanin levels are controlled by a gene called MYBA or MYB1, while the gene determining fruit flesh and foliage anthocyanin has been termed MYB10. In order to further understand tissue-specific anthocyanin regulation we have isolated orthologous MYB genes from all the commercially important rosaceous species. Results We use gene specific primers to show that the three MYB activators of apple anthocyanin (MYB10/MYB1/MYBA) are likely alleles of each other. MYB transcription factors, with high sequence identity to the apple gene were isolated from across the rosaceous family (e.g. apples, pears, plums, cherries, peaches, raspberries, rose, strawberry). Key identifying amino acid residues were found in both the DNA-binding and C-terminal domains of these MYBs. The expression of these MYB10 genes correlates with fruit and flower anthocyanin levels. Their function was tested in tobacco and strawberry. In tobacco, these MYBs were shown to induce the anthocyanin pathway when co-expressed with bHLHs, while over-expression of strawberry and apple genes in the crop of origin elevates anthocyanins. Conclusions This family-wide study of rosaceous R2R3 MYBs provides insight into the evolution of this plant trait. It has implications for the development of new coloured fruit and flowers, as well as aiding the understanding of temporal-spatial colour change.

A diode for correction-less small field output factor measurements

Introduction Due to their high spatial resolution diodes are often used for small field relative output factor measurements. However, a field size specific correction factor [1] is required and corrects for diode detector over-response at small field sizes. A recent Monte Carlo based study has shown that it is possible to design a diode detector that produces measured relative output factors that are equivalent to those in water. This is accomplished by introducing an air gap at the upstream end of the diode [2]. The aim of this study was to physically construct this diode by placing an ‘air cap’ on the end of a commercially available diode (the PTW 60016 electron diode). The output factors subsequently measured with the new diode design were compared to current benchmark small field output factor measurements. Methods A water-tight ‘cap’ was constructed so that it could be placed over the upstream end of the diode. The cap was able to be offset from the end of the diode, thus creating an air gap. The air gap width was the same as the diode width (7 mm) and the thickness of the air gap could be varied. Output factor measurements were made using square field sizes of side length from 5 to 50 mm, using a 6 MV photon beam. The set of output factor measurements were repeated with the air gap thickness set to 0, 0.5, 1.0 and 1.5 mm. The optimal air gap thickness was found in a similar manner to that proposed by Charles et al. [2]. An IBA stereotactic field diode, corrected using Monte Carlo calculated kq,clin,kq,msr values [3] was used as the gold standard. Results The optimal air thickness required for the PTW 60016 electron diode was 1.0 mm. This was close to the Monte Carlo predicted value of 1.15 mm2. The sensitivity of the new diode design was independent of field size (kq,clin,kq,msr = 1.000 at all field sizes) to within 1 %. Discussion and conclusions The work of Charles et al. [2] has been proven experimentally. An existing commercial diode has been converted into a correction-less small field diode by the simple addition of an ‘air cap’. The method of applying a cap to create the new diode leads to the diode being dual purpose, as without the cap it is still an unmodified electron diode.

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

Object tracking via non-Euclidean geometry : a Grassmann approach

A robust visual tracking system requires an object appearance model that is able to handle occlusion, pose, and illumination variations in the video stream. This can be difficult to accomplish when the model is trained using only a single image. In this paper, we first propose a tracking approach based on affine subspaces (constructed from several images) which are able to accommodate the abovementioned variations. We use affine subspaces not only to represent the object, but also the candidate areas that the object may occupy. We furthermore propose a novel approach to measure affine subspace-to-subspace distance via the use of non-Euclidean geometry of Grassmann manifolds. The tracking problem is then considered as an inference task in a Markov Chain Monte Carlo framework via particle filtering. Quantitative evaluation on challenging video sequences indicates that the proposed approach obtains considerably better performance than several recent state-of-the-art methods such as Tracking-Learning-Detection and MILtrack.

Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis : role of fibroblast growth factor receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.