Journal articles on the topic 'Time-lapse biological experiment'
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Štys, Dalibor, Jan Urban, Jan Vaněk, and Petr Císař. "Analysis of biological time-lapse microscopic experiment from the point of view of the information theory." Micron 42, no. 4 (June 2011): 360–65. http://dx.doi.org/10.1016/j.micron.2010.01.012.
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Štys, Dalibor, Jan Urban, Jan Vaněk, and Petr Císař. "Analysis of biological time-lapse microscopic experiment from the point of view of the information theory [Micron Vol. 41 Issue 5]." Micron 41, no. 5 (July 2010): 478. http://dx.doi.org/10.1016/s0968-4328(10)00110-1.
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Yoshioka, Akira, Akira Shimizu, Hiroyuki Oguma, Nao Kumada, Keita Fukasawa, Shoma Jingu, and Taku Kadoya. "Development of a camera trap for perching dragonflies: a new tool for freshwater environmental assessment." PeerJ 8 (September 18, 2020): e9681. http://dx.doi.org/10.7717/peerj.9681.
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Although dragonflies are excellent environmental indicators for monitoring terrestrial water ecosystems, automatic monitoring techniques using digital tools are limited. We designed a novel camera trapping system with an original dragonfly detector based on the hypothesis that perching dragonflies can be automatically detected using inexpensive and energy-saving photosensors built in a perch-like structure. A trial version of the camera trap was developed and evaluated in a case study targeting red dragonflies (Sympetrum spp.) in Japan. During an approximately 2-month period, the detector successfully detected Sympetrum dragonflies while using extremely low power consumption (less than 5 mW). Furthermore, a short-term field experiment using time-lapse cameras for validation at three locations indicated that the detection accuracy was sufficient for practical applications. The frequency of false positive detection ranged from 17 to 51 over an approximately 2-day period. The detection sensitivities were 0.67 and 1.0 at two locations, where a time-lapse camera confirmed that Sympetrum dragonflies perched on the trap more than once. However, the correspondence between the detection frequency by the camera trap and the abundance of Sympetrum dragonflies determined by field observations conducted in parallel was low when the dragonfly density was relatively high. Despite the potential for improvements in our camera trap and its application to the quantitative monitoring of dragonflies, the low cost and low power consumption of the detector make it a promising tool.
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Zhu, Xueqing, Cristina Alonso, Makram T. Suidan, Hongwen Cao, Byung J. Kim, and Byung R. Kim. "The effect of liquid phase on VOC removal in trickle-bed biofilters." Water Science and Technology 38, no. 3 (August 1, 1998): 315–22. http://dx.doi.org/10.2166/wst.1998.0226.
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The objective of this work was to analyze the effect of water content on the removal of volatile organic compounds (VOCs) in gas phase trickle-bed biofilters. Previous studies revealed that excessive accumulation of biomass in the reactor had a negative effect on contaminant removal efficiency. To solve this problem, periodic backwash was used to remove excess biomass and maintain an effective operation. Results showed that the ether removal efficiency dropped immediately after backwashing and gradually improved to reach the optimal value within about 24 hours. The initial drop in performance can be attributed to biological effects, such as loss of active biomass and the necessity for an adaptation period for the microbial culture. However, mass transfer limitations due to the water retained in the reactor may also be important. To investigate the effect of water content on the biofilter performance, an experiment evaluated the effect of draining after backwashing was conducted. Biofilters were allowed to drain for different periods of time before restarting the reactors. A longer lapse time in draining before the restart resulted in better ether removal efficiency. The improvement of biofilter performance after backwashing was explained with a combination of biological and physical effects. A mathematical model was then used to simulate the performance of the biofilter under these conditions and to support the conclusions obtained. The mathematical model considered a three-phase system (biofilm, water, and gas phase), dunamic processes, non-uniform bacterial population, and one limiting substrate.
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Nolan, Peter, James Pinto, Javier González-Rocha, Anders Jensen, Christina Vezzi, Sean Bailey, Gijs de Boer, et al. "Coordinated Unmanned Aircraft System (UAS) and Ground-Based Weather Measurements to Predict Lagrangian Coherent Structures (LCSs)." Sensors 18, no. 12 (December 15, 2018): 4448. http://dx.doi.org/10.3390/s18124448.
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Concentrations of airborne chemical and biological agents from a hazardous release are not spread uniformly. Instead, there are regions of higher concentration, in part due to local atmospheric flow conditions which can attract agents. We equipped a ground station and two rotary-wing unmanned aircraft systems (UASs) with ultrasonic anemometers. Flights reported here were conducted 10 to 15 m above ground level (AGL) at the Leach Airfield in the San Luis Valley, Colorado as part of the Lower Atmospheric Process Studies at Elevation—a Remotely-Piloted Aircraft Team Experiment (LAPSE-RATE) campaign in 2018. The ultrasonic anemometers were used to collect simultaneous measurements of wind speed, wind direction, and temperature in a fixed triangle pattern; each sensor was located at one apex of a triangle with ∼100 to 200 m on each side, depending on the experiment. A WRF-LES model was used to determine the wind field across the sampling domain. Data from the ground-based sensors and the two UASs were used to detect attracting regions (also known as Lagrangian Coherent Structures, or LCSs), which have the potential to transport high concentrations of agents. This unique framework for detection of high concentration regions is based on estimates of the horizontal wind gradient tensor. To our knowledge, our work represents the first direct measurement of an LCS indicator in the atmosphere using a team of sensors. Our ultimate goal is to use environmental data from swarms of sensors to drive transport models of hazardous agents that can lead to real-time proper decisions regarding rapid emergency responses. The integration of real-time data from unmanned assets, advanced mathematical techniques for transport analysis, and predictive models can help assist in emergency response decisions in the future.
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Hadjidemetriou, Stathis, Rania Hadjisavva, Andri Christodoulou, Ismini Papageorgiou, Ioanna Panayiotou, and Paris Skourides. "Spatiotemporal Identification of Cell Divisions Using Symmetry Properties in Time-Lapse Phase Contrast Microscopy." Symmetry 14, no. 9 (August 30, 2022): 1802. http://dx.doi.org/10.3390/sym14091802.
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A variety of biological and pharmaceutical studies, such as for anti-cancer drugs, require the quantification of cell responses over long periods of time. This is performed with time-lapse video microscopy that gives a long sequence of frames. For this purpose, phase contrast imaging is commonly used since it is minimally invasive. The cell responses of interest in this study are the mitotic cell divisions. Their manual measurements are tedious, subjective, and restrictive. This study introduces an automated method for these measurements. The method starts with preprocessing for restoration and reconstruction of the phase contrast time-lapse sequences. The data are first restored from intensity non-uniformities. Subsequently, the circular symmetry of the contour of the mitotic cells in phase contrast images is used by applying a Circle Hough Transform (CHT) to reconstruct the entire cells. The CHT is also enhanced with the ability to “vote” exclusively towards the center of curvature. The CHT image sequence is then registered for misplacements between successive frames. The sequence is subsequently processed to detect cell centroids in individual frames and use them as starting points to form spatiotemporal trajectories of cells along the positive as well as along the negative time directions, that is, anti-causally. The connectivities of different trajectories enhanced by the symmetry of the trajectories of the daughter cells provide as topological by-products the events of cell divisions together with the corresponding entries into mitoses as well as exits from cytokineses. The experiments use several experimental video sequences from three different cell lines with many cells undergoing mitoses and divisions. The quantitative validations of the results of the processing demonstrate the high performance and efficiency of the method.
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Ghafari, Mehran, Justin Clark, Hao-Bo Guo, Ruofan Yu, Yu Sun, Weiwei Dang, and Hong Qin. "Complementary performances of convolutional and capsule neural networks on classifying microfluidic images of dividing yeast cells." PLOS ONE 16, no. 3 (March 17, 2021): e0246988. http://dx.doi.org/10.1371/journal.pone.0246988.
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Microfluidic-based assays have become effective high-throughput approaches to examining replicative aging of budding yeast cells. Deep learning may offer an efficient way to analyze a large number of images collected from microfluidic experiments. Here, we compare three deep learning architectures to classify microfluidic time-lapse images of dividing yeast cells into categories that represent different stages in the yeast replicative aging process. We found that convolutional neural networks outperformed capsule networks in terms of accuracy, precision, and recall. The capsule networks had the most robust performance in detecting one specific category of cell images. An ensemble of three best-fitted single-architecture models achieves the highest overall accuracy, precision, and recall due to complementary performances. In addition, extending classification classes and data augmentation of the training dataset can improve the predictions of the biological categories in our study. This work lays a useful framework for sophisticated deep-learning processing of microfluidic-based assays of yeast replicative aging.
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Yoo, Woo Sik, Jung Gon Kim, Kitaek Kang, and Yeongsik Yoo. "Development of Static and Dynamic Colorimetric Analysis Techniques Using Image Sensors and Novel Image Processing Software for Chemical, Biological and Medical Applications." Technologies 11, no. 1 (January 28, 2023): 23. http://dx.doi.org/10.3390/technologies11010023.
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Colorimetric sensing techniques for point(s), linear and areal array(s) were developed using image sensors and novel image processing software for chemical, biological and medical applications. Monitoring and recording of colorimetric information on one or more specimens can be carried out by specially designed image processing software. The colorimetric information on real-time monitoring and recorded images or video clips can be analyzed for point(s), line(s) and area(s) of interest for manual and automatic data collection. Ex situ and in situ colorimetric data can be used as signals for process control, process optimization, safety and security alarms, and inputs for machine learning, including artificial intelligence. As an analytical example, video clips of chromatographic experiments using different colored inks on filter papers dipped in water and randomly blinking light-emitting-diode-based decorative lights were used. The colorimetric information on points, lines and areas, with different sizes from the video clips, were extracted and analyzed as a function of time. The video analysis results were both visualized as time-lapse images and RGB (red, green, blue) color/intensity graphs as a function of time. As a demonstration of the developed colorimetric analysis technique, the colorimetric information was expressed as static and time-series combinations of RGB intensity, HSV (hue, saturation and value) and CIE L*a*b* values. Both static and dynamic colorimetric analysis of photographs and/or video files from image sensors were successfully demonstrated using a novel image processing software.
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Laketa, Vibor, Jeremy C. Simpson, Stephanie Bechtel, Stefan Wiemann, and Rainer Pepperkok. "High-Content Microscopy Identifies New Neurite Outgrowth Regulators." Molecular Biology of the Cell 18, no. 1 (January 2007): 242–52. http://dx.doi.org/10.1091/mbc.e06-08-0666.
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Neurons, with their long axons and elaborate dendritic arbour, establish the complex circuitry that is essential for the proper functioning of the nervous system. Whereas a catalogue of structural, molecular, and functional differences between axons and dendrites is accumulating, the mechanisms involved in early events of neuronal differentiation, such as neurite initiation and elongation, are less well understood, mainly because the key molecules involved remain elusive. Here we describe the establishment and application of a microscopy-based approach designed to identify novel proteins involved in neurite initiation and/or elongation. We identified 21 proteins that affected neurite outgrowth when ectopically expressed in cells. Complementary time-lapse microscopy allowed us to discriminate between early and late effector proteins. Localization experiments with GFP-tagged proteins in fixed and living cells revealed a further 14 proteins that associated with neurite tips either early or late during neurite outgrowth. Coexpression experiments of the new effector proteins provide a first glimpse on a possible functional relationship of these proteins during neurite outgrowth. Altogether, we demonstrate the potential of the systematic microscope-based screening approaches described here to tackle the complex biological process of neurite outgrowth regulation.
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D’Agostino, Antonella, Anna Virginia Adriana Pirozzi, Rosario Finamore, Fabrizia Grieco, Massimiliano Minale, and Chiara Schiraldi. "Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing." Molecules 25, no. 3 (January 21, 2020): 431. http://dx.doi.org/10.3390/molecules25030431.
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Background: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. Methods: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose–response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. Results: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. Conclusions: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment.
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Neelam, Srujana, Audrey Lee, Michael A. Lane, Ceasar Udave, Howard G. Levine, and Ye Zhang. "Module to Support Real-Time Microscopic Imaging of Living Organisms on Ground-Based Microgravity Analogs." Applied Sciences 11, no. 7 (April 1, 2021): 3122. http://dx.doi.org/10.3390/app11073122.
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Since opportunities for spaceflight experiments are scarce, ground-based microgravity simulation devices (MSDs) offer accessible and economical alternatives for gravitational biology studies. Among the MSDs, the random positioning machine (RPM) provides simulated microgravity conditions on the ground by randomizing rotating biological samples in two axes to distribute the Earth’s gravity vector in all directions over time. Real-time microscopy and image acquisition during microgravity simulation are of particular interest to enable the study of how basic cell functions, such as division, migration, and proliferation, progress under altered gravity conditions. However, these capabilities have been difficult to implement due to the constantly moving frames of the RPM as well as mechanical noise. Therefore, we developed an image acquisition module that can be mounted on an RPM to capture live images over time while the specimen is in the simulated microgravity (SMG) environment. This module integrates a digital microscope with a magnification range of 20× to 700×, a high-speed data transmission adaptor for the wireless streaming of time-lapse images, and a backlight illuminator to view the sample under brightfield and darkfield modes. With this module, we successfully demonstrated the real-time imaging of human cells cultured on an RPM in brightfield, lasting up to 80 h, and also visualized them in green fluorescent channel. This module was successful in monitoring cell morphology and in quantifying the rate of cell division, cell migration, and wound healing in SMG. It can be easily modified to study the response of other biological specimens to SMG.
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Csepregi, Rita, Viktória Temesfői, Sourav Das, Ágnes Alberti, Csenge Anna Tóth, Róbert Herczeg, Nóra Papp, and Tamás Kőszegi. "Cytotoxic, Antimicrobial, Antioxidant Properties and Effects on Cell Migration of Phenolic Compounds of Selected Transylvanian Medicinal Plants." Antioxidants 9, no. 2 (February 18, 2020): 166. http://dx.doi.org/10.3390/antiox9020166.
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Medicinal plants are widely used in folk medicine but quite often their composition and biological effects are hardly known. Our study aimed to analyze the composition, cytotoxicity, antimicrobial, antioxidant activity and cellular migration effects of Anthyllis vulneraria, Fuchsia magellanica, Fuchsia triphylla and Lysimachia nummularia used in the Romanian ethnomedicine for wounds. Liquid chromatography with mass spectrometry (LC-MS/MS) was used to analyze 50% (v/v) ethanolic and aqueous extracts of the plants’ leaves. Antimicrobial activities were estimated with a standard microdilution method. The antioxidant properties were evaluated by validated chemical cell-free and biological cell-based assays. Cytotoxic effects were performed on mouse fibroblasts and human keratinocytes with a plate reader-based method assessing intracellular adenosine triphosphate (ATP), nucleic acid and protein contents and also by a flow cytometer-based assay detecting apoptotic–necrotic cell populations. Cell migration to cover cell-free areas was visualized by time-lapse phase-contrast microscopy using standard culture inserts. Fuchsia species showed the strongest cytotoxicity and the highest antioxidant and antimicrobial activity. However, their ethanolic extracts facilitated cell migration, most probably due to their various phenolic acid, flavonoid and anthocyanin derivatives. Our data might serve as a basis for further animal experiments to explore the complex action of Fuchsia species in wound healing assays.
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Zhang, Hong, Zhihui Lyu, Yongqiang Fan, Christopher R. Evans, Karl W. Barber, Kinshuk Banerjee, Oleg A. Igoshin, Jesse Rinehart, and Jiqiang Ling. "Metabolic stress promotes stop-codon readthrough and phenotypic heterogeneity." Proceedings of the National Academy of Sciences 117, no. 36 (August 24, 2020): 22167–72. http://dx.doi.org/10.1073/pnas.2013543117.
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Accurate protein synthesis is a tightly controlled biological process with multiple quality control steps safeguarded by aminoacyl-transfer RNA (tRNA) synthetases and the ribosome. Reduced translational accuracy leads to various physiological changes in both prokaryotes and eukaryotes. Termination of translation is signaled by stop codons and catalyzed by release factors. Occasionally, stop codons can be suppressed by near-cognate aminoacyl-tRNAs, resulting in protein variants with extended C termini. We have recently shown that stop-codon readthrough is heterogeneous among single bacterial cells. However, little is known about how environmental factors affect the level and heterogeneity of stop-codon readthrough. In this study, we have combined dual-fluorescence reporters, mass spectrometry, mathematical modeling, and single-cell approaches to demonstrate that a metabolic stress caused by excess carbon substantially increases both the level and heterogeneity of stop-codon readthrough. Excess carbon leads to accumulation of acid metabolites, which lower the pH and the activity of release factors to promote readthrough. Furthermore, our time-lapse microscopy experiments show that single cells with high readthrough levels are more adapted to severe acid stress conditions and are more sensitive to an aminoglycoside antibiotic. Our work thus reveals a metabolic stress that promotes translational heterogeneity and phenotypic diversity.
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Skopalík, Josef, Jiří Sekora, Martin Pešl, Markéta Bébarová, Olga Švecová, Tomáš Parák, Vratislav Čmiel, Ivo Provaznik, Edita Jeklová, and Josef Mašek. "UPGRADE OF THE LANGENDORFF APPARATUS USING THE INFRARED THERMO-CONTROL SYSTEM AND AN INTELLIGENT HEATER." Lékař a technika - Clinician and Technology 50, no. 4 (December 31, 2020): 137–41. http://dx.doi.org/10.14311/ctj.2020.4.03.
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Biological experiments involving isolated organs and tissues demand precise temperature monitoring and regulation. An automatic temperature control system was proposed and optimised on real isolated swine hearts and the prototype is described in this work. The traditional Langendorff apparatus consists of a heart holder, a reservoir of perfusion solution flowing to aortic cannula and a heating bath allowing passive heat transfer to the reservoir of perfusion solution. The commercial infrared camera FLIR T62101 was added to this basic set-up and used for very precise monitoring of the temperature kinetic of the organ and connected with an electronic feedback loop, which allowed real-time and precise regulation of heat transfer from the heating bath to the perfusion solution and in turn indirectly to the heart tissue. This provides real time control and active regulation of the myocardial tissue temperature. The infrared camera was tested in several modes and several variants of detection were optimised for ideal measurement of the region of interest of the ex vivo organ. The kinetics of the temperature changes and temperature stability of the tissue were recorded and calibrated by external electronic thermometers (type Pt100, inserted in tissue). The time lapse from the hang-up of the hypo termed organ (30 °C) until optimal warming (37 °C) was less than eight minutes in the final instrument prototype. The final stability of the 37 °C tissue temperature was approved; the temperature fluctuation of left ventricle tissue was characterised as 36.8 ± 0.5 °C. This upgraded traditional instrument could be used in specific preclinical and clinical transplantation and analytical projects in future.
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Yao, Yao, Ihor Smal, Ilya Grigoriev, Anna Akhmanova, and Erik Meijering. "Deep-learning method for data association in particle tracking." Bioinformatics 36, no. 19 (July 6, 2020): 4935–41. http://dx.doi.org/10.1093/bioinformatics/btaa597.
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Abstract Motivation Biological studies of dynamic processes in living cells often require accurate particle tracking as a first step toward quantitative analysis. Although many particle tracking methods have been developed for this purpose, they are typically based on prior assumptions about the particle dynamics, and/or they involve careful tuning of various algorithm parameters by the user for each application. This may make existing methods difficult to apply by non-expert users and to a broader range of tracking problems. Recent advances in deep-learning techniques hold great promise in eliminating these disadvantages, as they can learn how to optimally track particles from example data. Results Here, we present a deep-learning-based method for the data association stage of particle tracking. The proposed method uses convolutional neural networks and long short-term memory networks to extract relevant dynamics features and predict the motion of a particle and the cost of linking detected particles from one time point to the next. Comprehensive evaluations on datasets from the particle tracking challenge demonstrate the competitiveness of the proposed deep-learning method compared to the state of the art. Additional tests on real-time-lapse fluorescence microscopy images of various types of intracellular particles show the method performs comparably with human experts. Availability and implementation The software code implementing the proposed method as well as a description of how to obtain the test data used in the presented experiments will be available for non-commercial purposes from https://github.com/yoyohoho0221/pt_linking. Supplementary information Supplementary data are available at Bioinformatics online.
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Watkins, Catherine L., Dirk Schmaljohann, Shiroh Futaki, and Arwyn T. Jones. "Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide." Biochemical Journal 420, no. 2 (May 13, 2009): 179–91. http://dx.doi.org/10.1042/bj20090042.
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The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 μM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 °C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-β-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 °C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 °C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-β-cyclodextrin–cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.
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Nath, D., P. M. Slocombe, A. Webster, P. E. Stephens, A. J. Docherty, and G. Murphy. "Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1)integrin, leading to a marked induction of fibroblast cell motility." Journal of Cell Science 113, no. 12 (June 15, 2000): 2319–28. http://dx.doi.org/10.1242/jcs.113.12.2319.
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The ADAMs (A Disintegrin and Metalloprotease Domains) are a family of membrane-anchored proteins that play a role in fertilisation, myoblast fusion and ectodomain shedding of cell surface proteins. Meltrin gamma (ADAM-9) is a widely expressed member of this family and is involved in the shedding of heparin binding epidermal growth factor. Here we report that meltrin gamma can function as a cell adhesion molecule via its disintegrin domain. Using solid-phase binding assays and antibody inhibition experiments, we demonstrate that a murine meltrin gamma-Fc (Mel gamma -Fc) fusion protein binds to the integrin alpha(6)beta(1) on the surface of fibroblast cell lines, HT1080 and Wehi 164 in a specific manner. Since alpha(6)beta(1) is important for the motility of several cell types on laminin, cell migration studies using time-lapse video microscopy were performed. Cells adhering to Mel gamma-Fc displayed a rounded morphology and a marked increase (eight- to tenfold) in their motility compared to that on laminin. Furthermore, the p160 ROCK kinase inhibitor Y-27632 specifically reduced the migration of cells on meltrin gamma but had no effect on migration of cells on laminin, whilst the general tyrosine phoshorylation inhibitor, genistein, inhibited cell migration on both substrates. These results together suggest that meltrin gamma may play a role in regulating the motility of cells by binding to alpha(6)beta(1) integrin and this may be important during a variety of biological and pathological processes.
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Markham, John F., Cameron J. Wellard, Edwin D. Hawkins, Ken R. Duffy, and Philip D. Hodgkin. "A minimum of two distinct heritable factors are required to explain correlation structures in proliferating lymphocytes." Journal of The Royal Society Interface 7, no. 48 (January 6, 2010): 1049–59. http://dx.doi.org/10.1098/rsif.2009.0488.
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During the adaptive immune response, lymphocyte populations undergo a characteristic three-phase process: expansion through a series of cell divisions; cessation of expansion; and, finally, most of the accumulated lymphocytes die by apoptosis. The data used, thus far, to inform understanding of these processes, both in vitro and in vivo , are taken from flow cytometry experiments. One significant drawback of flow cytometry is that individual cells cannot be tracked, so that it is not possible to investigate interdependencies in the fate of cells within a family tree. This deficit in experimental information has recently been overcome by Hawkins et al . (Hawkins et al . 2009 Proc. Natl Acad. Sci. USA 106 , 13 457–13 462 ( doi:10.1073/pnas.0905629106 )), who reported on time-lapse microscopy experiments in which B-cells were stimulated through the TLR-9 receptor. Cells stimulated in this way do not aggregate, so that data regarding family trees can be recorded. In this article, we further investigate the Hawkins et al . data. Our conclusions are striking: in order to explain the familial correlation structure in division times, death times and propensity to divide, a minimum of two distinct heritable factors are necessary. As the data show that two distinct factors are necessary, we develop a stochastic model that has two heritable factors and demonstrate that it can reproduce the key features of the data. This model shows that two heritable factors are sufficient. These deductions have a clear impact upon biological understanding of the adaptive immune response. They also necessitate changes to the fundamental premises behind the tools developed by statisticians to draw deductions from flow cytometry data. Finally, they affect the mathematical modelling paradigms that are used to study these systems, as these are widely developed based on assumptions of cellular independence that are not accurate.
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Montano, Elena, Maria Vivo, Andrea Maria Guarino, Orsola di Martino, Blanda Di Luccia, Viola Calabrò, Sergio Caserta, and Alessandra Pollice. "Colloidal Silver Induces Cytoskeleton Reorganization and E-Cadherin Recruitment at Cell-Cell Contacts in HaCaT Cells." Pharmaceuticals 12, no. 2 (May 15, 2019): 72. http://dx.doi.org/10.3390/ph12020072.
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Up until the first half of the 20th century, silver found significant employment in medical applications, particularly in the healing of open wounds, thanks to its antibacterial and antifungal properties. Wound repair is a complex and dynamic biological process regulated by several pathways that cooperate to restore tissue integrity and homeostasis. To facilitate healing, injuries need to be promptly treated. Recently, the interest in alternatives to antibiotics has been raised given the widespread phenomenon of antibiotic resistance. Among these alternatives, the use of silver appears to be a valid option, so a resurgence in its use has been recently observed. In particular, in contrast to ionic silver, colloidal silver, a suspension of metallic silver particles, shows antibacterial activity displaying less or no toxicity. However, the human health risks associated with exposure to silver nanoparticles (NP) appear to be conflicted, and some studies have suggested that it could be toxic in different cellular contexts. These potentially harmful effects of silver NP depend on various parameters including NP size, which commonly range from 1 to 100 nm. In this study, we analyzed the effect of a colloidal silver preparation composed of very small and homogeneous nanoparticles of 0.62 nm size, smaller than those previously tested. We found no adverse effect on the cell proliferation of HaCaT cells, even at high NP concentration. Time-lapse microscopy and indirect immunofluorescence experiments demonstrated that this preparation of colloidal silver strongly increased cell migration, re-modeled the cytoskeleton, and caused recruitment of E-cadherin at cell-cell junctions of human cultured keratinocytes.
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Aust, G., M. Loeffler, I. Hanisch, M. Wobus, E. Wandel, and J. Galle. "Linking altered cell motility and proteolytic activity to tumor invasion—An active role of CD97 in tumor progression." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 10103. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.10103.
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10103 Background: Tumor cells at the invasion front of several carcinomas differ in their molecule pattern from cells in central tumor regions. As recently shown by us, this includes the cell surface receptor CD97 (Am J Pathol 2002,161:1657–67). Here, we link related differences in cell biological and biomechanical properties to the characteristics of tumor invasion. We combine in vitro and in vivo experiments with computer simulations of tumor progression and analyze the particular role of CD97 in this process. Methods: We compared the cDNA pattern of clones with adjustable expression of normal or C- terminal truncated CD97 using microarrays and confirmed the results at the protein level. Clonal cell motility was analyzed by time-lapse video microscopy. The scid mouse model was used to monitor tumor growth in vivo. Additionally, we introduce a novel class of individual cell-based computer models of tumor invasion into stroma. The approach enables us to analyze the impact of different cellular alterations on the organization and dynamics of the tumor invasion front and we can study several assumptions about the origin of these alterations. Results: CD97 overexpression stimulates single cell motility and increases proteolytic activity and IL-8 secretion in vitro and promotes growth of tumors in scid mice. In contrast, tumor cells overexpressing truncated CD97 show lower proteolytic activity, impaired in vitro motility and in vivo tumor growth. By computer simulation studies we demonstrate that the observed effects induced by CD97 can strongly increase the invasion capacity of tumors. Furthermore, they can cause a specific morphology of the invasion front which is known to correlate with poor prognosis. Thus, as a consequence of our computer simulations and findings in vitro and in vivo, we suggest that CD97 plays an active role in the propagation of de-differentiated carcinomas. Conclusions: Our combined experimental and theoretical computer analysis provides a novel insight in how variations of individual cell properties can be linked to different patterns of tumor cell invasion. Our results suggest that proteolytic activity at the tumor front in conjunction with elevated and directed cell motility are key steps to aggressive tumor invasion. No significant financial relationships to disclose.
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Zhadan, Peter M., and Marina A. Vaschenko. "Long-term study of behaviors of two cohabiting sea urchin species, Mesocentrotus nudus and Strongylocentrotus intermedius, under conditions of high food quantity and predation risk in situ." PeerJ 7 (November 22, 2019): e8087. http://dx.doi.org/10.7717/peerj.8087.
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Background In the predator–sea urchin–macrophyte trophic cascade, the ecological effect of sea urchins as grazers depends both on their density and the changes in foraging activity, which are influenced by various disturbing factors. However, the complete duration of the alarm reactions of echinoids has not been studied until now. Here, we tested a hypothesis that two cohabiting sea urchins, Mesocentrotus nudus and Strongylocentrotus intermedius, which differ morphologically, might display different behavioral responses to high hydrodynamic activity and predation. Methods We used continuous time-lapse video recording to clarify behavioral patterns of M. nudus and S. intermedius in presence of a large quantity of food (the kelp Saccharina japonica) but under different weather conditions and different types of predation threat: (1) calm weather conditions, (2) stormy weather conditions, (3) predation risk associated with the presence of several sea star species and (4) predation risk associated with an alarm stimulus (crushed conspecifics or heterospecifics). Three separate video recording experiments (134 days in total) were conducted under field conditions. Video recording analysis was performed to determine the number of specimens of each sea urchin species in the cameras’ field of view, size of sea urchins’ groups, movement patterns and the duration of the alarm responses of both sea urchin species. Results We showed that in the presence of kelp, M. nudus and S. intermedius exhibited both similar and different behavioral responses to hydrodynamics and predation threat. Under calm weather, movement patterns of both echinoids were similar but M. nudus exhibited the higher locomotion speed and distance traveled. Furthermore, S. intermedius but not M. nudus tended to group near the food substrate. The stormy weather caused a sharp decrease in movement activity followed by escape response in both echinoids. Six starfish species failed to predate on healthy sea urchins of either species and only a few attacks on ailing S. intermedius specimens were successful. The alarm response of S. intermedius lasted approximately 90 h and 20 h for starfish attacks on ailing conspecifics and for simulated attacks (crushed conspecifics or heterospecifics), respectively and involved several phases: (1) flight response, (2) grouping close to the food, (3) leaving the food and (4) return to the food. Phase three was the more pronounced in a case of starfish attack. M. nudus only responded to crushed conspecifics and exhibited no grouping behavior but displayed fast escape (during 4 h) and prolonged (up to 19 days) avoidance of the food source. This outcome is the longest alarm response reported for sea urchins. Discussion The most interesting finding is that two cohabiting sea urchin species, M. nudus and S. intermedius, display different alarm responses to predation threat. Both alarm responses are interpreted as defensive adaptations against visual predators.
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Dander, Erica, Federica Portale, Daniela Silvestri, Giulia Cricrì, Silvia Bresolin, Stefania Gaspari, Barbara Russo, et al. "Activin A, a Potential Key Factor of the Malignant Bone Marrow Niche, Enhances B-Cell Precursor-Acute Lymphoblastic Leukemic Cell Migratory and Invasive Properties." Blood 132, Supplement 1 (November 29, 2018): 1296. http://dx.doi.org/10.1182/blood-2018-99-116668.
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Abstract The bone marrow (BM) represents a peculiar microenvironment characterized by a high concentration of growth factors and cytokines necessary for hematopoiesis, that make it a sanctuary for leukemic cell homing, survival and proliferation. B-cell precursor-Acute Lymphoblastic Leukemia (BCP-ALL) reprogram the BM stroma to create a leukemia-supporting and chemoprotective niche. Strategies to modulate the microenvironment could offer new approaches for anti-leukemia therapies. We identified ActivinA, a TGF-β family member, with a well-described promoting role in several solid malignancies, as a new potentially targetable leukemia-favoring factor. ActivinA resulted overexpressed in the BM plasma of 108 BCP-ALL pediatric patients compared to 44 Healthy Donors (HDs), as evaluated by ELISA assay. Upon in vitro culture, primary BCP-ALL cells significantly increased ActivinA secretion by mesenchymal stromal cells (MSCs) derived from HD BM. Interestingly this effect was achieved both by cell-to-cell contact-mediated mechanisms (direct contact) and by soluble factor release (transwell-mediated co-culture). Interestingly, MSCs isolated from the BM of leukemic patients showed an intrinsic ability to secrete higher amounts of ActivinA (mean=983.6±362.9 pg/mL), compared to their normal counterpart (mean=218.5±31.99 pg/mL). In addition, we demonstrated that the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α are increased in the leukemic BM and that they were able to synergize with leukemic blasts in inducing ActivinA release by MSCs (>100 fold increase compared to basal production). Both type I and type II Activin receptors were found to be expressed by leukemic cells as demonstrated by flow cytometry analysis of ALK4, ACVR2A and ACVR2B receptors and western blot analysis of ALK2. Of note, ActivinA exposure could increase the mRNA expression levels of type I receptors ALK2 and ALK4, thus suggesting a possible self-reinforcing mechanism. Gene expression analysis of ActivinA-treated BCP-ALL cells showed that this protein was able to significantly influence motility-associated molecular pathways. Accordingly, time lapse microscopy analyses revealed that ActivinA significantly increased random motility of leukemic cells (p<0.0001). In addition, ActivinA was able to almost double the migration of BCP-ALL primary cells in response to CXCL12, as demonstrated by in vitro chemotaxis assays. The specificity of the observed effect was demonstrated by using the ALK4 specific inhibitor SB431542. CXCL12 reduction is one of the typical microenvironmental alterations occurring in the leukemic BM. In line with literature data, we showed 6-fold decrese of CXCL12 levels in the BM of BCP-ALL patients compared to HDs (p<0.0001). Dose-responses chemotaxis experiments revealed that ActivinA was able to sensitize leukemic cells to suboptimal CXCL12 concentrations. On the other site, ActivinA exerted an opposite effect on CD34+ cells isolated both from HD cord blood or BM. In detail, in HD-CD34+ cells ActivinA severely impaired CXCL12-induced migration (p<0.0001). This opposite effect could be explained by ActivinA ability to increase free cytosolic calcium only in leukemic cells, both basally and after addition of CXCL12 (flow cytometry analysis of Fluo-4 NW stained cells). Of note, calcium levels of HD-CD34+ cells resulted unaffected or even decreased (in 2 out of 3 experiments performed) by ActivinA treatment. Since calcium is critically involved in boosting cytoskeleton dynamics, we analysed, by flow cytometry, actin polymerization in phalloidin stained BCP-ALL cells. Interestingly, ActivinA treatment of BCP-ALL cells significantly increased the rate of conversion of globular into filamentous actin, which is a prerequisite of site-directed migration, as soon as CXCL12 was added. Moreover, ActivinA resulted a leukemia-promoting factor: protein treatment significantly increased the in vitro migration of BCP-ALL cells through Matrigel-coated transwells in response to CXCL12, thus stimulating leukemic cell invasiveness. Overall, ActivinA resulted a key factor conferring a migratory advantage to leukemic cells over healthy hematopoiesis within the leukemic niche. Indeed, our in vitro findings provide the biological rationale for designing novel therapeutical approaches targeting the leukemia-stroma interplay. Disclosures Locatelli: bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.
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Kim, Diane N. H., Alexander A. Lim, and Michael A. Teitell. "Rapid, label-free classification of tumor-reactive T cell killing with quantitative phase microscopy and machine learning." Scientific Reports 11, no. 1 (September 30, 2021). http://dx.doi.org/10.1038/s41598-021-98567-8.
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AbstractQuantitative phase microscopy (QPM) enables studies of living biological systems without exogenous labels. To increase the utility of QPM, machine-learning methods have been adapted to extract additional information from the quantitative phase data. Previous QPM approaches focused on fluid flow systems or time-lapse images that provide high throughput data for cells at single time points, or of time-lapse images that require delayed post-experiment analyses, respectively. To date, QPM studies have not imaged specific cells over time with rapid, concurrent analyses during image acquisition. In order to study biological phenomena or cellular interactions over time, efficient time-dependent methods that automatically and rapidly identify events of interest are desirable. Here, we present an approach that combines QPM and machine learning to identify tumor-reactive T cell killing of adherent cancer cells rapidly, which could be used for identifying and isolating novel T cells and/or their T cell receptors for studies in cancer immunotherapy. We demonstrate the utility of this method by machine learning model training and validation studies using one melanoma-cognate T cell receptor model system, followed by high classification accuracy in identifying T cell killing in an additional, independent melanoma-cognate T cell receptor model system. This general approach could be useful for studying additional biological systems under label-free conditions over extended periods of examination.
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Barber, P. R., S. M. Ameer-Beg, J. Gilbey, L. M. Carlin, M. Keppler, T. C. Ng, and B. Vojnovic. "Multiphoton time-domain fluorescence lifetime imaging microscopy: practical application to protein–protein interactions using global analysis." Journal of The Royal Society Interface 6, suppl_1 (December 9, 2008). http://dx.doi.org/10.1098/rsif.2008.0451.focus.
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Förster resonance energy transfer (FRET) detected via fluorescence lifetime imaging microscopy (FLIM) and global analysis provide a way in which protein–protein interactions may be spatially localized and quantified within biological cells. The FRET efficiency and proportion of interacting molecules have been determined using bi-exponential fitting to time-domain FLIM data from a multiphoton time-correlated single-photon counting microscope system. The analysis has been made more robust to noise and significantly faster using global fitting, allowing higher spatial resolutions and/or lower acquisition times. Data have been simulated, as well as acquired from cell experiments, and the accuracy of a modified Levenberg–Marquardt fitting technique has been explored. Multi-image global analysis has been used to follow the epidermal growth factor-induced activation of Cdc42 in a short-image-interval time-lapse FLIM/FRET experiment. Our implementation offers practical analysis and time-resolved-image manipulation, which have been targeted towards providing fast execution, robustness to low photon counts, quantitative results and amenability to automation and batch processing.
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Balomenos, Athanasios D., Victoria Stefanou, and Elias S. Manolakos. "Analytics and visualization tools to characterize single-cell stochasticity using bacterial single-cell movie cytometry data." BMC Bioinformatics 22, no. 1 (October 29, 2021). http://dx.doi.org/10.1186/s12859-021-04409-9.
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Abstract Background Time-lapse microscopy live-cell imaging is essential for studying the evolution of bacterial communities at single-cell resolution. It allows capturing detailed information about the morphology, gene expression, and spatial characteristics of individual cells at every time instance of the imaging experiment. The image analysis of bacterial "single-cell movies" (videos) generates big data in the form of multidimensional time series of measured bacterial attributes. If properly analyzed, these datasets can help us decipher the bacterial communities' growth dynamics and identify the sources and potential functional role of intra- and inter-subpopulation heterogeneity. Recent research has highlighted the importance of investigating the role of biological "noise" in gene regulation, cell growth, cell division, etc. Single-cell analytics of complex single-cell movie datasets, capturing the interaction of multiple micro-colonies with thousands of cells, can shed light on essential phenomena for human health, such as the competition of pathogens and benign microbiome cells, the emergence of dormant cells (“persisters”), the formation of biofilms under different stress conditions, etc. However, highly accurate and automated bacterial bioimage analysis and single-cell analytics methods remain elusive, even though they are required before we can routinely exploit the plethora of data that single-cell movies generate. Results We present visualization and single-cell analytics using R (ViSCAR), a set of methods and corresponding functions, to visually explore and correlate single-cell attributes generated from the image processing of complex bacterial single-cell movies. They can be used to model and visualize the spatiotemporal evolution of attributes at different levels of the microbial community organization (i.e., cell population, colony, generation, etc.), to discover possible epigenetic information transfer across cell generations, infer mathematical and statistical models describing various stochastic phenomena (e.g., cell growth, cell division), and even identify and auto-correct errors introduced unavoidably during the bioimage analysis of a dense movie with thousands of overcrowded cells in the microscope's field of view. Conclusions ViSCAR empowers researchers to capture and characterize the stochasticity, uncover the mechanisms leading to cellular phenotypes of interest, and decipher a large heterogeneous microbial communities' dynamic behavior. ViSCAR source code is available from GitLab at https://gitlab.com/ManolakosLab/viscar.
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Li, Xinyang, Yixin Li, Yiliang Zhou, Jiamin Wu, Zhifeng Zhao, Jiaqi Fan, Fei Deng, et al. "Real-time denoising enables high-sensitivity fluorescence time-lapse imaging beyond the shot-noise limit." Nature Biotechnology, September 26, 2022. http://dx.doi.org/10.1038/s41587-022-01450-8.
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AbstractA fundamental challenge in fluorescence microscopy is the photon shot noise arising from the inevitable stochasticity of photon detection. Noise increases measurement uncertainty and limits imaging resolution, speed and sensitivity. To achieve high-sensitivity fluorescence imaging beyond the shot-noise limit, we present DeepCAD-RT, a self-supervised deep learning method for real-time noise suppression. Based on our previous framework DeepCAD, we reduced the number of network parameters by 94%, memory consumption by 27-fold and processing time by a factor of 20, allowing real-time processing on a two-photon microscope. A high imaging signal-to-noise ratio can be acquired with tenfold fewer photons than in standard imaging approaches. We demonstrate the utility of DeepCAD-RT in a series of photon-limited experiments, including in vivo calcium imaging of mice, zebrafish larva and fruit flies, recording of three-dimensional (3D) migration of neutrophils after acute brain injury and imaging of 3D dynamics of cortical ATP release. DeepCAD-RT will facilitate the morphological and functional interrogation of biological dynamics with a minimal photon budget.
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Zhu, Yanming, and Erik Meijering. "Automatic improvement of deep learning-based cell segmentation in time-lapse microscopy by neural architecture search." Bioinformatics, July 30, 2021. http://dx.doi.org/10.1093/bioinformatics/btab556.
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Abstract Motivation Live cell segmentation is a crucial step in biological image analysis and is also a challenging task because time-lapse microscopy cell sequences usually exhibit complex spatial structures and complicated temporal behaviors. In recent years, numerous deep learning-based methods have been proposed to tackle this task and obtained promising results. However, designing a network with excellent performance requires professional knowledge and expertise and is very time-consuming and labor-intensive. Recently emerged neural architecture search (NAS) methods hold great promise in eliminating these disadvantages, because they can automatically search an optimal network for the task. Results We propose a novel NAS-based solution for deep learning-based cell segmentation in time-lapse microscopy images. Different from current NAS methods, we propose (i) jointly searching non-repeatable micro architectures to construct the macro network for exploring greater NAS potential and better performance and (ii) defining a specific search space suitable for the live cell segmentation task, including the incorporation of a convolutional long short-term memory network for exploring the temporal information in time-lapse sequences. Comprehensive evaluations on the 2D datasets from the cell tracking challenge demonstrate the competitiveness of the proposed method compared to the state of the art. The experimental results show that the method is capable of achieving more consistent top performance across all ten datasets than the other challenge methods. Availabilityand implementation The executable files of the proposed method as well as configurations for each dataset used in the presented experiments will be available for non-commercial purposes from https://github.com/291498346/nas_cellseg. Supplementary information Supplementary data are available at Bioinformatics online.
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Arts, Marloes, Ihor Smal, Maarten W. Paul, Claire Wyman, and Erik Meijering. "Particle Mobility Analysis Using Deep Learning and the Moment Scaling Spectrum." Scientific Reports 9, no. 1 (November 20, 2019). http://dx.doi.org/10.1038/s41598-019-53663-8.
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AbstractQuantitative analysis of dynamic processes in living cells using time-lapse microscopy requires not only accurate tracking of every particle in the images, but also reliable extraction of biologically relevant parameters from the resulting trajectories. Whereas many methods exist to perform the tracking task, there is still a lack of robust solutions for subsequent parameter extraction and analysis. Here a novel method is presented to address this need. It uses for the first time a deep learning approach to segment single particle trajectories into consistent tracklets (trajectory segments that exhibit one type of motion) and then performs moment scaling spectrum analysis of the tracklets to estimate the number of mobility classes and their associated parameters, providing rich fundamental knowledge about the behavior of the particles under study. Experiments on in-house datasets as well as publicly available particle tracking data for a wide range of proteins with different dynamic behavior demonstrate the broad applicability of the method.
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Cheng, Ryan P., Puneet Dang, Alemji A. Taku, Yoon Ji Moon, Vi Pham, Xiaohe Sun, Ethan Zhao, and Jonathan A. Raper. "Loss of Neuropilin2a/b or Sema3fa alters olfactory sensory axon dynamics and protoglomerular targeting." Neural Development 17, no. 1 (January 3, 2022). http://dx.doi.org/10.1186/s13064-021-00157-x.
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Abstract Background Olfactory Sensory Neuron (OSN) axons project from the zebrafish olfactory epithelium to reproducible intermediate target locations in the olfactory bulb called protoglomeruli at early stages in development. Two classes of OSNs expressing either OMP or TRPC2 exclusively target distinct, complementary protoglomeruli. Using RNAseq, we identified axon guidance receptors nrp2a and nrp2b, and their ligand sema3fa, as potential guidance factors that are differentially expressed between these two classes of OSNs. Methods To investigate their role in OSN axon guidance, we assessed the protoglomerular targeting fidelity of OSNs labeled by OMP:RFP and TRPC2:Venus transgenes in nrp2a, nrp2b, or sema3fa mutants. We used double mutant and genetic interaction experiments to interrogate the relationship between the three genes. We used live time-lapse imaging to compare the dynamic behaviors of OSN growth cones during protoglomerular targeting in heterozygous and mutant larvae. Results The fidelity of protoglomerular targeting of TRPC2-class OSNs is degraded in nrp2a, nrp2b, or sema3fa mutants, as axons misproject into OMP-specific protoglomeruli and other ectopic locations in the bulb. These misprojections are further enhanced in nrp2a;nrp2b double mutants suggesting that nrp2s work at least partially in parallel in the same guidance process. Results from genetic interaction experiments are consistent with sema3fa acting in the same biological pathway as both nrp2a and nrp2b. Live time-lapse imaging was used to examine the dynamic behavior of TRPC2-class growth cones in nrp2a mutants compared to heterozygous siblings. Some TRPC2-class growth cones ectopically enter the dorsal-medial region of the bulb in both groups, but in fully mutant embryos, they are less likely to correct the error through retraction. The same result was observed when TRPC2-class growth cone behavior was compared between sema3fa heterozygous and sema3fa mutant larvae. Conclusions Our results suggest that nrp2a and nrp2b expressed in TRPC2-class OSNs help prevent their mixing with axon projections in OMP-specific protoglomeruli, and further, that sema3fa helps to exclude TRPC2-class axons by repulsion from the dorsal-medial bulb.
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Campbell, Ian. "Bothering Myself." M/C Journal 16, no. 6 (December 6, 2013). http://dx.doi.org/10.5204/mcj.748.
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Figure 1: photos by Ian Campbell Figure 2: photos by Ian Cambell Figure 3: photos by Ian Campbell Figure 4: photos by Ian Campbell Bothering Myself Bothering Myself is an ongoing experiment that originated in 2004. The latest incarnation of the project was presented at the MacKenzie Art Gallery, Regina, Saskatchewan, Canada in 2012. The work is presented as a series of single channel videos. The project involves a custom-built electronic/mechanical system that is designed to wake up a subject (the artist) and record the results on video. Typical mechanical interventions include being sprayed with water, having objects (shoes, paper, clothing) land on the subject’s head, being hit with a large Styrofoam hammer, and other absurdist performance actions. Project Themes Portraiture Bothering Myself is a way of approaching the body at rest as a method of uncovering the possibilities of consciousness. One of the main questions at stake was: How can one capture the moment of consciousness? It was decided to locate this question within the idea of the cinematic but also that of portraiture. The subject is alone and at the mercy of the technological system employed to trigger consciousness. Vulnerability was heightened through the use of the "medium" of sleep in order to present the human being at the centre of a system that is not under their control. There is a tension in that the body is both cooperating and at odds with the activity of being awoken. The aesthetic of the system, the visual parts that appear in the bedroom, are meant to be functional. The wires, lighting stands, computer peripherals, cameras and mechanical interventions that make up the visual landscape of the piece are there as props for an installation. This language of new media installation and objectivity of showing the means of production is meant to carry both a context of the art gallery and a scientific experiment. The "Absent" Body The artist is both developer and subject of the project. This forced the investigation of several methods of working with a subject where there was no in site access. While programming the system and positioning the elements (cameras, lights, interventions) educated guesses had to be made as to where the body would be at any one time. The physical gesturing that happens during the night makes any precise positioning impossible. Experimentation with means of tracking the body was attempted (in order to guide the camera). Eventually it was found that employing multiple wide-angle cameras close to the locus of activity was an acceptable alternative. When the video was produced for the final installation, the use of split screen was employed to show two angles of the intervention simultaneously. Duplication like this underlines the idea of "coverage;" a term in film that is used to describe capturing successive takes for the purpose of continuity editing. Although these techniques are used for practical reasons, the hope is that they would add up to a feeling of expansiveness of the visual record. That this project is capturing more than the sum of the parts. That it might capture a slice of any number of things that it is investigating: transference of emotion, empathetic response to physical discomfort, interrogation of the point of consciousness. The Invasive Another aspect of working with the absent was making more intimate interventions on the body. As the project turned from an absurdist expanded cinema project to a faux science experiment, the project became concerned with getting closer to the biological. One phenomenon that seemed like an intriguing subject was R.E.M. (rapid eye movement). This involuntary reflex accompanies 20-25% of the sleep period of typical human subjects and is concurrent with vivid dream states near wakefulness. A kind of electronic "sleep mask" was designed that was a microcosm of my system as a whole. It contained a camera, a small light source, computer controller and video recorder. The camera and light source were positioned directly over the eye. Recordings were made of a series of time-lapse videos of an entire night's worth of eye movements. In the end the video proved unsatisfying enough that the immense discomfort of wearing this sleep mask was not worth inflicting on the subject. This dead end in the project points to the limits of physical invasiveness when working with the body. When the goal of the art is to maintain an emotional connection to the audience, you must use elements that are familiar (i.e. the language of film). In the end, abandoning this R.E.M. monitoring avoided the reduction of the body to its constituent parts and maintained the primacy of more traditional cinematic language. Cinema Originally the project was conceptualized as a kind of "expanded cinema" performance piece. The artist's body is an actor in a mechanical film set. By completely controlling the movie-making apparatus (multiple cameras providing multiple angles, automated controllable lighting) the participation of human and machine could be contrasted. The human subject plays the role of the organic, changeable biological/social actor imbued with agency. The machine plays the role of the inflexible mechanistic logical system that repeats itself without deviation. The tension between what it means to be human and the function of the machine is what provides the serious message underneath the humour and absurdity of the situation. Extending the Nervous System For this project the idea of augmentation ties into the artistic project of "making a film." These short fragments or visual modules of Bothering Myself are the product of a technological system designed to make art on the cusp of consciousness. Much like the tools of media art making are extensions of the senses in a McLuhan-esque sense, the camera is to the eye as the computer network is to the nervous system. The communication signals that allow the director to engage with the tools of the film set are at play on a micro level in Bothering Myself. Instead of relying on sets of skills ranged across numerous crew members: from fine motor skills to adjust the focus of a camera to the cognitive activity at work in editing, the system at play in Bothering Myself is a hybrid of the pre-programmed and the immediate. By combining these two aspects of production, a record is created that allows for a single individual to create an impossibility without technology; to view oneself at the moment of consciousness. This is the promise and fear of technological augmentation and what makes it both exhilarating to work with and frightening to contemplate.
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Fuchino, Katsuya, Klas Flärdh, Paul Dyson, and Nora Ausmees. "Cell-Biological Studies of Osmotic Shock Response in Streptomyces spp." Journal of Bacteriology 199, no. 1 (October 17, 2016). http://dx.doi.org/10.1128/jb.00465-16.
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ABSTRACT Most bacteria are likely to face osmotic challenges, but there is yet much to learn about how such environmental changes affect the architecture of bacterial cells. Here, we report a cell-biological study in model organisms of the genus Streptomyces, which are actinobacteria that grow in a highly polarized fashion to form branching hyphae. The characteristic apical growth of Streptomyces hyphae is orchestrated by protein assemblies, called polarisomes, which contain coiled-coil proteins DivIVA and Scy, and recruit cell wall synthesis complexes and the stress-bearing cytoskeleton of FilP to the tip regions of the hyphae. We monitored cell growth and cell-architectural changes by time-lapse microscopy in osmotic upshift experiments. Hyperosmotic shock caused arrest of growth, loss of turgor, and hypercondensation of chromosomes. The recovery period was protracted, presumably due to the dehydrated state of the cytoplasm, before hyphae could restore their turgor and start to grow again. In most hyphae, this regrowth did not take place at the original hyphal tips. Instead, cell polarity was reprogrammed, and polarisomes were redistributed to new sites, leading to the emergence of multiple lateral branches from which growth occurred. Factors known to regulate the branching pattern of Streptomyces hyphae, such as the serine/threonine kinase AfsK and Scy, were not involved in reprogramming of cell polarity, indicating that different mechanisms may act under different environmental conditions to control hyphal branching. Our observations of hyphal morphology during the stress response indicate that turgor and sufficient hydration of cytoplasm are required for Streptomyces tip growth. IMPORTANCE Polar growth is an intricate manner of growth for accomplishing a complicated morphology, employed by a wide range of organisms across the kingdoms of life. The tip extension of Streptomyces hyphae is one of the most pronounced examples of polar growth among bacteria. The expansion of the cell wall by tip extension is thought to be facilitated by the turgor pressure, but it was unknown how external osmotic change influences Streptomyces tip growth. We report here that severe hyperosmotic stress causes cessation of growth, followed by reprogramming of cell polarity and rearrangement of growth zones to promote lateral hyphal branching. This phenomenon may represent a strategy of hyphal organisms to avoid osmotic stress encountered by the growing hyphal tip.
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Ebert, Karolin, Gwen Zwingenberger, Elena Barbaria, Simone Keller, Corinna Heck, Rouven Arnold, Vanessa Hollerieth, et al. "Determining the effects of trastuzumab, cetuximab and afatinib by phosphoprotein, gene expression and phenotypic analysis in gastric cancer cell lines." BMC Cancer 20, no. 1 (October 28, 2020). http://dx.doi.org/10.1186/s12885-020-07540-7.
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Abstract Background Gastric cancer is the fifth most frequently diagnosed cancer and the third leading cause of cancer death worldwide. The molecular mechanisms of action for anti-HER-family drugs in gastric cancer cells are incompletely understood. We compared the molecular effects of trastuzumab and the other HER-family targeting drugs cetuximab and afatinib on phosphoprotein and gene expression level to gain insights into the regulated pathways. Moreover, we intended to identify genes involved in phenotypic effects of anti-HER therapies. Methods A time-resolved analysis of downstream intracellular kinases following EGF, cetuximab, trastuzumab and afatinib treatment was performed by Luminex analysis in the gastric cancer cell lines Hs746T, MKN1, MKN7 and NCI-N87. The changes in gene expression after treatment of the gastric cancer cell lines with EGF, cetuximab, trastuzumab or afatinib for 4 or 24 h were analyzed by RNA sequencing. Significantly enriched pathways and gene ontology terms were identified by functional enrichment analysis. Furthermore, effects of trastuzumab and afatinib on cell motility and apoptosis were analyzed by time-lapse microscopy and western blot for cleaved caspase 3. Results The Luminex analysis of kinase activity revealed no effects of trastuzumab, while alterations of AKT1, MAPK3, MEK1 and p70S6K1 activations were observed under cetuximab and afatinib treatment. On gene expression level, cetuximab mainly affected the signaling pathways, whereas afatinib had an effect on both signaling and cell cycle pathways. In contrast, trastuzumab had little effects on gene expression. Afatinib reduced average speed in MKN1 and MKN7 cells and induced apoptosis in NCI-N87 cells. Following treatment with afatinib, a list of 14 genes that might be involved in the decrease of cell motility and a list of 44 genes that might have a potential role in induction of apoptosis was suggested. The importance of one of these genes (HBEGF) as regulator of motility was confirmed by knockdown experiments. Conclusions Taken together, we described the different molecular effects of trastuzumab, cetuximab and afatinib on kinase activity and gene expression. The phenotypic changes following afatinib treatment were reflected by altered biological functions indicated by overrepresentation of gene ontology terms. The importance of identified genes for cell motility was validated in case of HBEGF.
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Cui, Guoxin, Yi Jin Liew, Migle K. Konciute, Ye Zhan, Shiou-Han Hung, Jana Thistle, Lucia Gastoldi, Sebastian Schmidt-Roach, Job Dekker, and Manuel Aranda. "Nutritional control regulates symbiont proliferation and life history in coral-dinoflagellate symbiosis." BMC Biology 20, no. 1 (May 13, 2022). http://dx.doi.org/10.1186/s12915-022-01306-2.
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Abstract Background The coral-Symbiodiniaceae symbiosis is fundamental for the coral reef ecosystem. Corals provide various inorganic nutrients to their algal symbionts in exchange for the photosynthates to meet their metabolic demands. When becoming symbionts, Symbiodiniaceae cells show a reduced proliferation rate and a different life history. While it is generally believed that the animal hosts play critical roles in regulating these processes, far less is known about the molecular underpinnings that allow the corals to induce the changes in their symbionts. Results We tested symbiont cell proliferation and life stage changes in vitro in response to different nutrient-limiting conditions to determine the key nutrients and to compare the respective symbiont transcriptomic profiles to cells in hospite. We then examined the effects of nutrient repletion on symbiont proliferation in coral hosts and quantified life stage transitions in vitro using time-lapse confocal imaging. Here, we show that symbionts in hospite share gene expression and pathway activation profiles with free-living cells under nitrogen-limited conditions, strongly suggesting that symbiont proliferation in symbiosis is limited by nitrogen availability. Conclusions We demonstrate that nitrogen limitation not only suppresses cell proliferation but also life stage transition to maintain symbionts in the immobile coccoid stage. Nutrient repletion experiments in corals further confirmed that nitrogen availability is the major factor limiting symbiont density in hospite. Our study emphasizes the importance of nitrogen in coral-algae interactions and, more importantly, sheds light on the crucial role of nitrogen in symbiont life history regulation.
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Basak, Arpan Kumar, Mohamadreza Mirzaei, Kazimierz Strzałka, and Kenji Yamada. "Texture feature extraction from microscope images enables a robust estimation of ER body phenotype in Arabidopsis." Plant Methods 17, no. 1 (October 26, 2021). http://dx.doi.org/10.1186/s13007-021-00810-w.
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Abstract Background Cellular components are controlled by genetic and physiological factors that define their shape and size. However, quantitively capturing the morphological characteristics and movement of cellular organelles from micrograph images is challenging, because the analysis deals with complexities of images that frequently lead to inaccuracy in the estimation of the features. Here we show a unique quantitative method to overcome biases and inaccuracy of biological samples from confocal micrographs. Results We generated 2D images of cell walls and spindle-shaped cellular organelles, namely ER bodies, with a maximum contrast projection of 3D confocal fluorescent microscope images. The projected images were further processed and segmented by adaptive thresholding of the fluorescent levels in the cell walls. Micrographs are composed of pixels, which have information on position and intensity. From the pixel information we calculated three types of features (spatial, intensity and Haralick) in ER bodies corresponding to segmented cells. The spatial features include basic information on shape, e.g., surface area and perimeter. The intensity features include information on mean, standard deviation and quantile of fluorescence intensities within an ER body. Haralick features describe the texture features, which can be calculated mathematically from the interrelationship between the pixel information. Together these parameters were subjected to multivariate analysis to estimate the morphological diversity. Additionally, we calculated the displacement of the ER bodies using the positional information in time-lapse images. We captured similar morphological diversity and movement within ER body phenotypes in several microscopy experiments performed in different settings and scanned under different objectives. We then described differences in morphology and movement of ER bodies between A. thaliana wild type and mutants deficient in ER body-related genes. Conclusions The findings unexpectedly revealed multiple genetic factors that are involved in the shape and size of ER bodies in A. thaliana. This is the first report showing morphological characteristics in addition to the movement of cellular components and it quantitatively summarises plant phenotypic differences even in plants that show similar cellular components. The estimation of morphological diversity was independent of the cell staining method and the objective lens used in the microscopy. Hence, our study enables a robust estimation of plant phenotypes by recognizing small differences in complex cell organelle shapes and their movement, which is beneficial in a comprehensive analysis of the molecular mechanism for cell organelle formation that is independent of technical variations.