Role of cytoskeleton in morphological changes of blood platelets

Platelets are an important component of blood that help maintain haemostasis. They are derived from large bone marrow residents, called megakaryocytes, which release tubular processes into the blood stream that fragment and eventually form 2 to 3 μm sized enucleate discoid platelets. Activation of platelets is caused by factors released into the bloodstream upon endothelial damage, to which they respond by undergoing a distinct order of shape transition, from discoid, to spheroid, to dendritic, and finally an extended morphology, required to form a clot. Despite the simple architecture, platelets are able to drastically alter their morphology owing to the repertoire of cytoskeletal proteins they express. Microtubules, which form a bundle running along the platelet periphery, are known to be important for maintaining the resting discoid morphology of a platelet, while actin is heavily implicated in the later stages that require adhesion. During the first step of platelet activation, the microtubule marginal band undergoes coiling, while the platelet changes from a disc to a sphere shape. Both actin and microtubules are implicated in this process but the mechanics of the process are not clearly understood. The following project has been carried out to explore the role of cytoskeletal mechanics in triggering activation of a platelet. A combination of experimental and analysis techniques was used to quantitatively assess mechanical properties of the system in a resting state, as well as during activation, by direct measurement of the marginal band morphology. The structure and composition of the marginal band was analyzed using electron tomography, which provided detailed information on individual microtubules. Super-resolution microscopy was also used to visualize the overall morphology and composition of the marginal band. With this data we could infer the mechanical properties of the resting marginal band in fixed platelets. To analyze the dynamics of coiling process, live cell fluorescence microscopy was used in combination with a microfluidic system. With this setup, changes in the marginal band shape could be followed in response to treatment with agonists or inhibitors that affect the cytoskeleton, that is, a process analogous to mechanical perturbations of the platelet. A large population of platelets was also analyzed to infer the intrinsic variations mechanical properties of the marginal band. The Cytosim software was used to set up simulation of marginal band coiling. By using a multifaceted approach, we were able to get novel insight into the mechanics of marginal band coiling. Firstly, we showed that length distribution of microtubules in a set of resting platelet marginal bands follows an exponential distribution. The sum of all polymerized microtubule length was found to be 101.84µm ±12.63 per platelet. The typical distance between two microtubules was found to be 30nm in a tightly packed marginal band. Secondly, by measuring the dynamics of coiling, we could infer that the marginal band behaves like a visco-elastic ring upon activation with ADP. This response was found to be dependent on actin, while thrombin activation elicited a response that manifested in an actin independent manner. Analysis of large population of platelets showed that the tubulin intensity scales as a power of five to the platelet radius, indicating a possible enrichment of tubulin in platelets. Finally, our data suggests that platelets with longer marginal bands have a higher propensity to coil. Although some of our results need to be followed up with further investigations, this study provides an experimental and analysis framework that allows us to quantitatively analyze platelet cytoskeleton morphology with an aim to understand the mechanics of platelet activation in healthy and disease states

Guidelines for Urban Regeneration in India through Parameters of Landscape Transformation

This paper is a study of the residential urban regeneration programs in the city of Delhi. This research aims to prepare a landscape-driven holistic vision for urban regeneration by examining and evaluating elements of landscape planning through quantitative parameters such as mobility, building heights and other built-form regulations, blue-green networks, and qualitative aspects of open spaces such as identity. In Delhi, the government has initiated an urban regeneration program called the General Pool Residential Accommodation (GPRA[1]) that uses existing residential plots for re-densification. An undertaking of such magnitude warranted a nuanced policy intervention for urban re-generation. However, it was found that the current research gap lay in a deficit of parameters to study the existing planning norms of redevelopment through the lens of landscape urban values. The paper begins by surveying, analyzing, and assessing the prevailing redevelopment patterns of completed, under-construction, and future GPRA redevelopment projects. At the same time, case studies of landscape-driven evaluations of previous GPRA projects were analysed to establish benchmarks of landscape parameters. Based on this assessment, a set of “Efficiency Factors” were derived pertaining to each quantitative parameter and an “Assessment Index” was generated for qualitative parameters. The paper concludes with a catalogue of benchmarking criteria to be used as a guiding framework for future resilient and sustainable urban regeneration interventions in India

miTuner - a kit for microRNA based gene expression tuning in mammalian cells

The purpose of this RFC is to introduce a modular expression tuning kit for use in mammalian cells. The kit enables the regulation of the gene expression of any gene of interest (GOI) based on synthetic microRNAs, endogenous microRNAs or a combination of both

MIFA: Metadata, Incentives, Formats, and Accessibility guidelines to improve the reuse of AI datasets for bioimage analysis

Artificial Intelligence methods are powerful tools for biological image analysis and processing. High-quality annotated images are key to training and developing new methods, but access to such data is often hindered by the lack of standards for sharing datasets. We brought together community experts in a workshop to develop guidelines to improve the reuse of bioimages and annotations for AI applications. These include standards on data formats, metadata, data presentation and sharing, and incentives to generate new datasets. We are positive that the MIFA (Metadata, Incentives, Formats, and Accessibility) recommendations will accelerate the development of AI tools for bioimage analysis by facilitating access to high quality training data.Comment: 16 pages, 3 figure

miMeasure – a standard for miRNA binding site characterization in mammalian cells

This RFC proposes a standard for the quantitative characterization of miRNA binding sites (miRNA-BS) in mammalian cells. The miMeasure standard introduces a ready-to-use standard measurement plasmid (pSMB_miMeasure, BBa_K337049) enabling rapid experimental characterization of any miRNA-BS of choice. We recommend a new standard unit, RKDU (relative knock-down unit) to describe the knock-down efficiency of a miRNA-BS in a specific cell type. pSMB_miMeasure allows for an easy and fast measurement of RKDU while providing effective normalization against variance stemming from differences in transfection efficiency and from other sources

Community-developed checklists for publishing images and image analysis

Images document scientific discoveries and are prevalent in modern biomedical research. Microscopy imaging in particular is currently undergoing rapid technological advancements. However for scientists wishing to publish the obtained images and image analyses results, there are to date no unified guidelines. Consequently, microscopy images and image data in publications may be unclear or difficult to interpret. Here we present community-developed checklists for preparing light microscopy images and image analysis for publications. These checklists offer authors, readers, and publishers key recommendations for image formatting and annotation, color selection, data availability, and for reporting image analysis workflows. The goal of our guidelines is to increase the clarity and reproducibility of image figures and thereby heighten the quality of microscopy data is in publications.Comment: 28 pages, 8 Figures, 3 Supplmentary Figures, Manuscript, Essential recommendations for publication of microscopy image dat

Balance of microtubule stiffness and cortical tension determines the size of blood cells with marginal band across species

International audienceThe fast bloodstream of animals is associated with large shear stresses. To withstand these conditions, blood cells have evolved a special morphology and a specific internal architecture to maintain their integrity over several weeks. For instance, nonmammalian red blood cells, mammalian erythroblasts, and platelets have a peripheral ring of microtubules, called the marginal band, that flattens the overall cell morphology by pushing on the cell cortex. In this work, we model how the shape of these cells stems from the balance between marginal band rigidity and cortical tension. We predict that the diameter of the cell scales with the total microtubule polymer and verify the predicted law across a wide range of species. Our analysis also shows that the combination of the marginal band rigidity and cortical tension increases the ability of the cell to withstand forces without deformation. Finally, we model the marginal band coiling that occurs during the disk-to-sphere transition observed, for instance, at the onset of blood platelet activation. We show that when cortical tension increases faster than cross-linkers can unbind, the marginal band will coil, whereas if the tension increases more slowly, the marginal band may shorten as microtubules slide relative to each other

Open Imaging Data Sharing in EOSC: COVID-19 as Demonstrator

This Science Project (SP) brings together three different domains of life sciences with the aim to create reproducible workflows, tools and web-services for data visualization. This SP focuses in building resources for handling data from bioimaging, structural and bio-chemical studies. The Euro-Bioimaging will implement a community standard cloud compatible open image data format and data submission workflow for high-throughput screening data. Whereas, Instruct-ERIC will develop a user-friendly web-service to access to multi-dimensional structural and imaging data. Lastly, EU-OpenScreen/Fraunhofer ITMP will create reproducible workflow for generating Knowledge Graphs that represent phenotype-chemotype of diseases. While these resources are being developed, the collaborators will also simultaneously harmonize the resources right from the beginning to enable FAIR data principles. This SP uses COVID-19 as a demonstrator, however the resources will be generalized for any disease of interest

Amifostine Analog, DRDE-30, Attenuates Bleomycin-Induced Pulmonary Fibrosis in Mice

Bleomycin (BLM) is an effective curative option in the management of several malignancies including pleural effusions; but pulmonary toxicity, comprising of pneumonitis and fibrosis, poses challenge in its use as a front-line chemotherapeutic. Although Amifostine has been found to protect lungs from the toxic effects of radiation and BLM, its application is limited due to associated toxicity and unfavorable route of administration. Therefore, there is a need for selective, potent, and safe anti-fibrotic drugs. The current study was undertaken to assess the protective effects of DRDE-30, an analog of Amifostine, on BLM-induced lung injury in C57BL/6 mice. Whole body micro- computed tomography (CT) was used to non-invasively observe tissue damage, while broncheo-alveolar lavage fluid (BALF) and lung tissues were assessed for oxidative damage, inflammation and fibrosis. Changes in the lung density revealed by micro-CT suggested protection against BLM-induced lung injury by DRDE-30, which correlated well with changes in lung morphology and histopathology. DRDE-30 significantly blunted BLM-induced oxidative stress, inflammation and fibrosis in the lungs evidenced by reduced oxidative damage, endothelial barrier dysfunction, Myeloperoxidase (MPO) activity, pro-inflammatory cytokine release and protection of tissue architecture, that could be linked to enhanced anti-oxidant defense system and suppression of redox-sensitive pro-inflammatory signaling cascades. DRDE-30 decreased the BLM-induced augmentation in BALF TGF-β and lung hydroxyproline levels, as well as reduced the expression of the mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the suppression of epithelial to mesenchymal transition (EMT) as one of its anti-fibrotic effects. The results demonstrate that the Amifostine analog, DRDE-30, ameliorates the oxidative injury and lung fibrosis induced by BLM and strengthen its potential use as an adjuvant in alleviating the side effects of BLM

BatchConvert: A command-line tool for parallelised conversion of image collections into the standard bioimage file formats OME-TIFF and OME-Zarr

File formats incompatibility has become a major obstacle in biological imaging, complicating downstream processes such as image processing and analysis. One way to address this challenge is to convert the acquired image data into standard image file formats. Here we introduce BatchConvert, a command line tool for parallelised conversion of image collections into OME-TIFF or OME-Zarr using the workflow management system Nextflow. BatchConvert offers functionalities such as remote input-output support, optional execution on Slurm clusters and pattern-based filtering of input files. Conversion can be coupled to image concatenation, allowing selected images to be merged along specified dimensions. Support for remote locations includes an option to submit the output data to S3-compatible object stores or public archives such as BioImage Archive. Overall, BatchConvert is a flexible tool for researchers who are routinely managing and analysing large multidimensional image data that is either locally or remotely stored