Migration Tracks Research Articles

Extended live-tracking and quantitative characterization of wound healing and cell migration with SiR-Hoechst

Cell migration is essential to many life processes, including immune response, tissue repair, and cancer progression. A reliable quantitative characterization of the cell migration can therefore aid in the high throughput screening of drug efficacy in wound healing and cancer treatments. In this work, we report what we believe is the first use of SiR-Hoechst for extended live tracking and automated analysis of cell migration and wound healing. We showed through rigorous statistical comparisons that this far-red label does not affect migratory behavior. We observed excellent automated tracking of random cell migration, in which the motility parameters (speed, displacement, path length, directionality ratio, persistence time, and direction autocorrelation) obtained closely match those obtained from manual tracking. We also present an analysis framework to characterize the healing of a scratch wound from the perspective of single cells. The use of SiR-Hoechst is advantageous for the crowded environments in wound healing assays because as long as cell nuclei do not overlap, continuous tracking can be maintained even if there is cell-cell contact. In this paper, we report wound recovery based on the number of cells migrating into the wound over time, normalized by the initial cell count prior to the infliction of the wound. This normalized cell count approach is impervious to operator bias during the arbitration of wound edges and is also robust against variability that arises due to differences in the cell density of different samples. Additional wound healing characteristics were also defined based on the evolution of cell speed and directionality during healing. Not unexpected, the wound healing cells exhibited much higher tendency to maintain the same migratory direction in comparison to the randomly migrating cells. The use of SiR-Hoechst thus greatly simplified the automation of single cell and whole population analysis with high spatial and temporal resolution over extended periods of time.

Migratory connectivity and philopatry of cownose rays Rhinoptera bonasus along the Atlantic coast, USA

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 602:197-211 (2018) - DOI: https://doi.org/10.3354/meps12686 Migratory connectivity and philopatry of cownose rays Rhinoptera bonasus along the Atlantic coast, USA Matthew B. Ogburn1,*, Charles W. Bangley1, Robert Aguilar1, Robert A. Fisher2, Mary Carla Curran3, Sarah Fae Webb3, Anson H. Hines1 1Smithsonian Environmental Research Center, 647 Contees Wharf Road, Edgewater, MD 21037, USA 2Virginia Institute of Marine Science, College of William and Mary, PO Box 1346, Gloucester Point, Virginia 23062, USA 3Savannah State University, Box 20467, Savannah, GA 31404, USA *Corresponding author: ogburnm@si.edu ABSTRACT: Migratory species link spatially separated ecosystems, and understanding their migrations is critical for conservation and management. The cownose ray Rhinoptera bonasus is a large-bodied batoid ray implicated in shellfish declines along the US Atlantic coast, but its migrations and habitat use remain poorly understood. We used passive acoustic telemetry to track tagged adult female (N = 27) and male (N = 9) rays released during summer and fall 2014-2016 in Maryland, Virginia, and Georgia. Twenty-three tags provided data for more than 1 yr. Individuals from all tagging locations overwintered in the same region offshore of Cape Canaveral, Florida, then returned in summer to the estuaries where tagging took place. Hidden Markov modeling identified 3 behavioral states (Resident, Ranging, Migratory), with ray movements generally classified as non-migratory (Resident and Ranging behavioral states) in summer and winter, and migratory (Migratory behavioral state) in spring and fall. Linear discriminant analysis suggested strong philopatry to tagging locations. This study provides the first full annual migration tracks for cownose rays along the US Atlantic coast, indicating that they migrate between summer pupping and mating habitats in estuaries south of Long Island, New York, and shared overwintering habitats off the east coast of Florida near Cape Canaveral. Our results highlight the value of national-scale networks of acoustic telemetry arrays for identifying migratory patterns of highly mobile marine species. KEY WORDS: Migration · Connectivity · Behavior · Acoustic telemetry · Philopatry · Fisheries management · Conservation · Rhinoptera bonasus Full text in pdf format Supplementary material PreviousNextCite this article as: Ogburn MB, Bangley CW, Aguilar R, Fisher RA, Curran MC, Webb SF, Hines AH (2018) Migratory connectivity and philopatry of cownose rays Rhinoptera bonasus along the Atlantic coast, USA. Mar Ecol Prog Ser 602:197-211. https://doi.org/10.3354/meps12686 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 602. Online publication date: August 23, 2018 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2018 Inter-Research.

Multicell migration tracking within angiogenic networks by deep learning-based segmentation and augmented Bayesian filtering.

.Cell migration is a key feature for living organisms. Image analysis tools are useful in studying cell migration in three-dimensional (3-D) in vitro environments. We consider angiogenic vessels formed in 3-D microfluidic devices (MFDs) and develop an image analysis system to extract cell behaviors from experimental phase-contrast microscopy image sequences. The proposed system initializes tracks with the end-point confocal nuclei coordinates. We apply convolutional neural networks to detect cell candidates and combine backward Kalman filtering with multiple hypothesis tracking to link the cell candidates at each time step. These hypotheses incorporate prior knowledge on vessel formation and cell proliferation rates. The association accuracy reaches 86.4% for the proposed algorithm, indicating that the proposed system is able to associate cells more accurately than existing approaches. Cell culture experiments in 3-D MFDs have shown considerable promise for improving biology research. The proposed system is expected to be a useful quantitative tool for potential microscopy problems of MFDs.

Microfluidic system for modelling 3D tumour invasion into surrounding stroma and drug screening

Tumour invasion into the surrounding stroma is a critical step in metastasis, and it is necessary to clarify the role of microenvironmental factors in tumour invasion. We present a microfluidic system that simulated and controlled multi-factors of the tumour microenvironment for three-dimensional (3D) assessment of tumour invasion into the stroma. The simultaneous, precise and continuous arrangement of two 3D matrices was visualised to observe the migration of cancer cell populations or single cells by transfecting cells with a fluorescent protein. A vascular endothelial layer was formed to simulate transendothelial transport of nutrients, and its endothelial barrier function was verified by the diffusion of 70 kDa fluorescein isothiocyanate (FITC)-Dextran in 3D matrices. Through high-throughput cell migration tracking observation and statistic evaluation, we clarified that cell density of the tumour directly determined its invasiveness. The results suggested that increased secretion of IL-6 among both cancer cells (MDA-MB-231) and noncancerous cells (MCF-10A or HDF-n) after co-culture contributes to cancer cell invasiveness, and this was verified by an IL-6 inhibitor assay. Finally, the drug efficacy of paclitaxel was reflected as changes in cancer cell migration ability, viability, and morphology. Together, our microfluidic devices could be a useful tool to study the mechanism of tumour invasion into the stroma and to screen anti-metastatic drugs.

Chapter 6 – Mobility, migration and transnational connectivity

Chapter 6 – Mobility, migration and transnational connectivity

Self‐Assembled pH‐Sensitive Fluoromagnetic Nanotubes as Archetype System for Multimodal Imaging of Brain Cancer

AbstractFluoromagnetic systems are recognized as an emerging class of materials with great potential in the biomedical field. Here, it is shown how to fabricate fluoromagnetic nanotubes that can serve as multimodal probes for the imaging and targeting of brain cancer. An ionic self‐assembly strategy is used to functionalize the surface of synthetic chrysotile nanotubes with pH‐sensitive fluorescent chromophores and ferromagnetic nanoparticles. The acquired magnetic properties permit their use as contrast agent for magnetic resonance imaging, and enable the tracking of tumor cell migration and infiltration responsible for metastatic growth and disease recurrence. Their organic component, changing its fluorescence attitude as a function of local pH, targets the cancer distinctive acidity, and allows localizing and monitoring the tumor occurrence and progression by mapping the acidic spatial distribution within biopsy tissues. The fluoromagnetic properties of nanotubes are preserved from the in vitro to the in vivo condition and they show the ability to migrate across the blood brain barrier, thus spontaneously reaching the brain tumor after injection. The simplicity of the synthesis route of these geomimetic nanomaterials combined with their demonstrated affinity with the in vivo condition strongly highlights their potential for developing effective functional materials for multimodal theranostics of brain cancer.

Distinct roles for phosphoinositide 3-kinases \u03b3 and \u03b4 in malignant B cell migration

The PI 3-kinases (PI3K) are essential mediators of chemokine receptor signaling necessary for migration of chronic lymphocytic leukemia (CLL) cells and their interaction with tissue-resident stromal cells. While the PI3Kδ-specific inhibitor idelalisib shows efficacy in treatment of CLL and other B cell malignancies, the function of PI3Kγ has not been extensively studied in B cells. Here, we assess whether PI3Kγ has non-redundant functions in CLL migration and adhesion to stromal cells. We observed that pharmaceutical PI3Kγ inhibition with CZC24832 significantly impaired CLL cell migration, while dual PI3Kδ/γ inhibitor duvelisib had a greater impact than single isoform-selective inhibitors. Knockdown of PI3Kγ reduced migration of CLL cells and cell lines. Expression of the PI3Kγ subunits increased in CLL cells in response to CD40L/IL-4, whereas BCR cross-linking had no effect. Overexpression of PI3Kγ subunits enhanced cell migration in response to SDF1α/CXCL12, with the strongest effect observed within ZAP70 + CLL samples. Microscopic tracking of cell migration within chemokine gradients revealed that PI3Kγ functions in gradient sensing and impacts cell morphology and F-actin polarization. PI3Kγ inhibition also reduced CLL adhesion to stromal cells to a similar extent as idelalisib. These findings provide the first evidence that PI3Kγ has unique functions in malignant B cells.

Examination of Clock and Adcyap1 gene variation in a neotropical migratory passerine.

Complex behavioral traits, such as those making up a migratory phenotype, are regulated by multiple environmental factors and multiple genes. We investigated possible relationships between microsatellite variation at two candidate genes implicated in the control of migratory behavior, Clock and Adcyap1, and several aspects of migratory life-history and evolutionary divergence in the Painted Bunting (Passerina ciris), a species that shows wide variation in migratory and molting strategies across a disjunct distribution. We focused on Clock and Adcyap1 microsatellite variation across three Painted Bunting populations in Oklahoma, Louisiana, and North Carolina, and for the Oklahoma breeding population we used published migration tracking data on adult males to explore phenotypic variation in individual migratory behavior. We found no correlation between microsatellite allele size within either Clock and Adcyap1 relative to the initiation or duration of fall migration in adult males breeding in Oklahoma. We also show the lack of significant correlations with aspects of the migratory phenotype for the Louisiana population. Our research highlights the limitations of studying microsatellite allelic mutations that are of undetermined functional influence relative to complex behavioral phenotypes.

Automated Tracking of Cell Migration with Rapid Data Analysis.

Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time-consuming and labor-intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image-processing experience or familiarity with particle-tracking approaches. Using this GUI, users can interactively determine a minimum of four parameters to identify fluorescently labeled cells and automate acquisition of cell trajectories. Additional features allow for batch processing of numerous time-lapse images, curation of unwanted tracks, and subsequent statistical analysis of tracked cells. Statistical outputs allow users to evaluate migratory phenotypes, including cell speed, distance, displacement, and persistence, as well as measures of directional movement, such as forward migration index (FMI) and angular displacement. © 2017 by John Wiley & Sons, Inc.

Optimization of Dendritic Cell-Mediated Cytotoxic T-Cell Activation by Tracking of Dendritic Cell Migration Using Reporter Gene Imaging.

The aim of this study is to optimize the dendritic cell (DC)-mediated T-cell activation using reporter gene imaging and flow cytometric analysis in living mice. A murine dendritic cell line (DC2.4) co-expressing effluc and Thy1.1 genes were established by transfection with retroviral vectors. Thy1.1 positive cells were sorted by magnetic bead separation system (DC2.4/effluc). Cell proliferation assay and phenotype analysis to determine the effects of gene transduction on the function of dendritic cells between parental DC2.4 and DC2.4/effluc were performed. To optimize the DC-mediated immune response by cell number or frequency, different cell numbers (5×105, 1×106, and 2×106DC2.4/effluc) or different frequencies of DC2.4/effluc (first, second, and third injections) were injected in the right footpad of mice. The migration of the DC2.4/effluc into the draining popliteal lymph node of mice was monitored by bioluminescence imaging (BLI). Flow cytometric analysis was performed with splenocytes to determine the cytotoxic T-cell population after injection of DC2.4/effluc. Parental DC2.4 and DC2.4/effluc exhibit no significant differences in their proliferation and phenotype. BLI signals were observed in the draining popliteal lymph node at day 1 after injection of DC2.4/effluc in 1×106 and 2×106 cells-injected groups. The highest BLI signal intensity was detected in 2×106 cells-injected mice. On day 11, the BLI signal was detected in only 2×106 cell-injected group but not in other groups. Optimized cell numbers (2×106) were injected in three animal groups with a different frequency (first, second, and third injection groups). The BLI signal was detected at day 1 and maintained until day 7 in the first injection group, but there is low signal intensity in the second and the third injection groups. Although the expression levels of Thy1.1 gene in the first injection group were very high, there reveals no expression of Thy1.1 gene in the second and the third injection groups. The number of tumor-specific CD8+ T-cells in the spleen significantly increased, as the number of DC injections increases. Successful optimization of DC-mediated cytotoxic T-cell activation in living mice using reporter gene imaging and flow cytometric analysis was achieved. The optimization of DC-mediated cytotoxic T-cell activation could be applied for the future DC-based immunotherapy.

A Cell Migration Tracking Tool Supports Coupling of Tissue Rotation to Elongation

Cell migration is indispensable to morphogenesis and homeostasis. Live imaging allows mechanistic insights, but long-term observation can alter normal biology, and tools to track movements invivo without perturbation are lacking. We develop here a tool called M-TRAIL (matrix-labeling technique for real-time and inferred location), which reveals migration histories in fixed tissues. Using clones that overexpress GFP-tagged extracellular matrix (ECM) components, motility trajectories are mapped based on durable traces deposited onto basement membrane. We applied M-TRAIL to Drosophila follicle rotation, comparing invivo and exvivo migratory dynamics. The rate, trajectory, and cessation of rotation in wild-type (WT) follicles measured invivo and exvivo were identical, as was rotation failure in fat2 mutants. However, follicles carrying intracellularly truncated Fat2, previously reported to lack rotation exvivo, in fact rotate invivo at a reduced speed, thus revalidating the hypothesis that rotation is required for tissue elongation. The M-TRAIL approach could be applied to track and quantitate invivo cell motility in other tissues and organisms.

Single Cell Phenotyping Reveals Heterogeneity Among Hematopoietic Stem Cells Following Infection.

The hematopoietic stem cell (HSC) niche provides essential microenvironmental cues for the production and maintenance of HSCs within the bone marrow. During inflammation, hematopoietic dynamics are perturbed, but it is not known whether changes to the HSC-niche interaction occur as a result. We visualize HSCs directly in vivo, enabling detailed analysis of the 3D niche dynamics and migration patterns in murine bone marrow following Trichinella spiralis infection. Spatial statistical analysis of these HSC trajectories reveals two distinct modes of HSC behavior: (a) a pattern of revisiting previously explored space and (b) a pattern of exploring new space. Whereas HSCs from control donors predominantly follow pattern (a), those from infected mice adopt both strategies. Using detailed computational analyses of cell migration tracks and life-history theory, we show that the increased motility of HSCs following infection can, perhaps counterintuitively, enable mice to cope better in deteriorating HSC-niche microenvironments following infection. Stem Cells 2017;35:2292-2304.

Summer and fall movement of cownose ray, Rhinoptera bonasus, along the east coast of United States observed with pop-up satellite tags

Cownose ray, Rhinoptera bonasus, is a common elasmobranch species along the southeast United States coast that recently has received negative attention. These rays can consume considerable amounts of commercial shellfish raising concerns regarding their control and need for effective management. However, limited information is known regarding their population abundance and migration patterns. We addressed the latter by reviewing 25 tagged cownose rays in Chesapeake Bay with pop-up satellite archival tags (PSATs) to study their movement patterns during summer and fall and identify wintering grounds. Eleven tags provided useful data on temperature, depth, light level and/or end locations. The migration tracks were deciphered through geolocation based on light levels, sea surface temperatures and depth constraints. PSAT end locations indicated southern wintering grounds in the coastal waters of central Florida. Female rays migrated out of Chesapeake Bay at the end of September to early October and continued their southerly migration to Florida. Male rays exited the bay in July and migrated northward based on their estimated movement tracks. The male rays appeared to have a second summer feeding ground off the coast of southern New England. In the fall, males migrated south from New England to the same wintering grounds as the females. No diel differences in habitat use were detected; however, males tended to occupy a wider depth and temperature range compared to females. Information on the movement patterns and habitat use for cownose rays will assist in determining more effective recreational and commercial management plans.

Abstract 909: Tracking of migration using digital holography to monitor drug-induced phenotypic shifts in breast cancer cells

Abstract This study aims to investigate the migration behaviour of individual JIMT-1 breast cancer cells during the cell cycle. Mechanisms behind cell migration are important to unravel in the context of cancer relapses caused by distant metastases. JIMT-1 cells were cultivated in normoxia or hypoxia and treated with 0.5 µM of the cancer stem cell inhibiting compound salinomycin. The cells were imaged in a digital holography microscope placed in a regular cell culturing CO2 incubator (normoxia) or in a hypoxia chamber (1% O2). In digital holography, a laser beam is split into two beams - the reference beam and the object beam. The object beam is directed through the sample, where the difference in refractive index between air, the cell medium, and the cell body induces a phase shift in the laser beam. After having passed the object, the beam is merged with the reference beam giving an interference pattern, which is used to construct a three-dimensional image of the cells in a software program. The laser is non-phototoxic and therefor it can be used for long-term studies. In our study, images were acquired every 5th minute during a 72-hour incubation period. After acquisition, the individual cells in the images were tracked throughout the time lapse. Cell movement is described by three parameters: motility, which is the length of the total cell movement path, migration, which is the shortest distance from the start to the end position, and migration directness, which is migration divided by motility. When cells were exposed to hypoxia and/or treated with salinomycin, the cell cycle time increased compared to non-treated cells in normoxia. When the cell cycle time increased, motility increased as well. However, migration directness decreased despite increase in motility implying increased local movement of cells treated with salinomycin compared to control cells. We have previously shown that salinomycin-treated JIMT-1 cells obtain an epithelia phenotype (Huang et al., 2016). Thus, we decided to investigate a breast cancer cell line with epithelial traits i.e, the MCF-7 cells. The MCF-7 cells behaved like the JIMT-1 cells treated with salinomycin i.e. the motility was high and the migration directness was low. Thus, we suggest that cells with mesenchymal and epithelial traits can be distinguished using digital holographic imaging based on motility and migration directness and that drug-induced phenotypic shifts can be studied using this technique.

Abstract 5814: Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration

Abstract In extravasation cancer cells and vascular niche are involved in a tight cross-talk which has been defined as the rate-regulating event for metastases establishment [1]. Recent, animal studies support the hypothesis that metastatic deficiency lies in focal adhesion complex alterations, however, it still needs to be elucidated which are the specific regulators of each event composing extravasation. Focal adhesion proteins Talin-1 (TLN-1) and Focal Adhesion Kinase (FAK) are up-regulated in breast cancer. Both targets due to their structural and functional role, dramatically influence cancer mechanotransduction leading to endothelial junction disruption, critical in extravasation process [2]. Here, we generated ad hoc engineered models for each extravasation step allowing single cell behavior analyses through high resolution real time imaging in a reliable and quantitative way in a physiological environment. Through this novel approach we analyzed the effect of TLN-1 and FAK and their genetic and chemical inhibition in breast cancer extravasation. The 3D-microfluidic vasculature displayed maturation markers and physiological permeability (1.5±0.76*10-6cm/s) and allowed cancer cell injection through the hollow vessels. Western blot confirmed TLN-1 and FAK knock down (KD) in MDA-MB231. Both targets significantly affected morphology and proliferation (p<0.001). We demonstrated the involvement of both targets in adhesion to the endothelium (p<0.001). Trans endothelial migration (TEM) was decreased in TLN-1 KD (p<0.05) and in FAK KD (P<0.01). Both TLN-1 and FAK KD cells were trapped into the vessels and were not able to extend pseudopodia. Cancer migration tracking in 3D matrix was statistically lower in both KD (p<0.001). The calculated ratios of adhesion/TEM and invasion/TEM were lower in TLN-1 compared to FAK KD, demonstrating that TLN-1 plays a major role in adhesion to endothelium and early invasion while FAK was identified as the main driver in TEM. Additive (TLN-1 and FAK) KD did not show significant difference respect to the target that mostly affected adhesion and invasion demonstrating TLN-1 role in these events. Additive KD was statistically lower compared to TLN-1 KD (P<0.05), supporting FAK crucial role in TEM. Inhibition of TLN-1 and FAK phosphorylation revealed that actin polymerization and pseudopodia formation (required for extravasation) were dependent by their structural role and were independent by their phosphorylation. However, the inhibition of FAK activity showed FAK phosphorylation as the key driver of TEM mechanism (p<0.01). Concluding, our results supported by in vivo data (p<0.05) showed that TLN-1 and FAK inhibition may represent novel strategies challenging mechanisms leading to metastatic establishment. 1. Cell,2011.147(2):p.275-92; 2. Proc Natl Acad Sci USA,2015.112(1):p.214-9 Citation Format: Mara Gilardi, Simone Bersini, Rosa Maria Moresco, Marco Vanoni, Roger D. Kamm, Matteo Moretti. Engineered microfluidic 3D human microvasculature identifies Talin-1-dependent adhesion and FAK activation as the key promoter of cancer cell trans-endothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5814. doi:10.1158/1538-7445.AM2017-5814

Horizontal movement and emigration of juvenile spotted halibut Verasper variegatus released in a shallow brackish lagoon: Matsukawa-ura, northeastern Japan, revealed by acoustic telemetry

To elucidate the post-release movement and emigration of juvenile spotted halibut Verasper variegatus, ten hatchery-reared age-0 individuals (10 months old, 17.0–19.7 cm total length) surgically implanted with acoustic transmitters were released near the center of Matsukawa-ura, a shallow brackish lagoon, on 25 November 2009 (water temperature 13.8 °C). They were monitored by ten acoustic receivers for 5 months. Of ten individuals, eight left the release site during December–March. Three of these emigrated to the outer ocean on 17 and 29 January and 30 March 2010, respectively. Juveniles probably started wintering migration in December and January when water temperatures decreased considerably in the lagoon (mean 9.5 and 6.0 °C, respectively). They stopped their migration in the coldest month, February (mean 5.7 °C), and restarted it in March when water temperatures frequently exceeded a plausible threshold for movement (≥6 °C). Statistical analyses revealed that the fish started migration significantly more frequently at nighttime. The migration track of an individual recorded from 11 to 30 March showed gradual nocturnal movements and a slow migration speed (estimated maximum speed 2.2 m/min). Our results revealed that water temperature primarily governed the seasonal timing of nocturnal migratory movements of juvenile spotted halibut.

SiMA: A simplified migration assay for analyzing neutrophil migration.

In lung inflammation, neutrophils are the first leukocytes migrating to an inflammatory site, eliminating pathogens by multiple mechanisms. The term "migration" describes several stages of neutrophil movement to reach the site of inflammation, of which the passage of the interstitium and basal membrane of the airway are necessary to reach the site of bronchial inflammation. Currently, several methods exist (e.g., Boyden Chamber, under-agarose assay, or microfluidic systems) to assess neutrophil mobility. However, these methods do not allow for parameterization on single cell level, that is, the individual neutrophil pathway analysis is still considered challenging. This study sought to develop a simplified yet flexible method to monitor and quantify neutrophil chemotaxis by utilizing commercially available tissue culture hardware, simple video microscopic equipment and highly standardized tracking. A chemotaxis 3D µ-slide (IBIDI) was used with different chemoattractants [interleukin-8 (IL-8), fMLP, and Leukotriene B4 (LTB4 )] to attract neutrophils in different matrices like Fibronectin (FN) or human placental matrix. Migration was recorded for 60 min using phase contrast microscopy with an EVOS® FL Cell Imaging System. The images were normalized and texture based image segmentation was used to generate neutrophil trajectories. Based on these spatio-temporal information a comprehensive parameter set is extracted from each time series describing the neutrophils motility, including velocity and directness and neutrophil chemotaxis. To characterize the latter one, a sector analysis was employed enabling the quantification of the neutrophils response to the chemoattractant. Using this hard- and software framework we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications (Prednisolone). Additionally, a comparison of four asthmatic and three non-asthmatic patients gives a first hint to the capability of SiMA assay in the context of migration based diagnostics. Using SiMA we were able to identify typical migration profiles of the chemoattractants IL-8, fMLP, and LTB4 , the effect of the matrices FN versus HEM as well as the response to different medications, that is, Prednisolone induced a change of direction of migrating neutrophils in FN but no such effect was observed in human placental matrix. In addition, neutrophils of asthmatic individuals showed an increased proportion of cells migrating toward the vehicle. With the SiMA platform we presented a simplified but yet flexible platform for cost-effective tracking and quantification of neutrophil migration. The introduced method is based on a simple microscopic video stage, standardized, commercially available, µ-fluidic migration chambers and automated image analysis, and track validation software. © 2017 International Society for Advancement of Cytometry.

Repeated migration of a juvenile European Nightjar Caprimulgus europaeus

Most of our current knowledge on migration strategies in juvenile light-weight birds originates from laboratory studies, displacement experiments and partial migration tracks. Here we report on the first recording of two consecutive migration cycles of one such light-weight migrant, the European Nightjar Caprimulgus europaeus. This bird visited the same stopover zones and wintering area in both years, but with earlier arrival times and less time spent at stopover zones in the second year. This unique dataset shows that adult nightjars seem to possess the capability to perform some form of navigation towards areas which are established during the juvenile migration.

Migration tracking is a mess.

Monitoring technologies only add to the political noise over managing mobility, warns Huub Dijstelbloem.

Organoid technologies meet genome engineering.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.