Field Of Cell Biology Research Articles

Three-Dimensional Avian Hematopoietic Stem Cell Cultures as a Model for Studying Disease Pathogenesis.

Three-dimensional (3D) cell culture is attracting increasing attention today because it can mimic tissue environments and provide more realistic results than do conventional cell cultures. On the other hand, very little attention has been given to using 3D cell cultures in the field of avian cell biology. Although mimicking the bone marrow niche is a classic challenge of mammalian stem cell research, experiments have never been conducted in poultry on preparing in vitro the bone marrow niche. It is well known, however, that all diseases cause immunosuppression and target immune cells and their development. Hematopoietic stem cells (HSC) reside in the bone marrow and constitute a source for immune cells of lymphoid and myeloid origins. Disease prevention and control in poultry are facing new challenges, such as greater use of alternative breeding systems and expanding production of eggs and chicken meat in developing countries. Moreover, the COVID-19 pandemic will draw greater attention to the importance of disease management in poultry because poultry constitutes a rich source of zoonotic diseases. For these reasons, and because they will lead to a better understanding of disease pathogenesis, in vivo HSC niches for studying disease pathogenesis can be valuable tools for developing more effective disease prevention, diagnosis, and control. The main goal of this review is to summarize knowledge about avian hematopoietic cells, HSC niches, avian immunosuppressive diseases, and isolation of HSC, and the main part of the review is dedicated to using 3D cell cultures and their possible use for studying disease pathogenesis with practical examples. Therefore, this review can serve as a practical guide to support further preparation of 3D avian HSC niches to study the pathogenesis of avian diseases.

Robust optical flow algorithm for general single cell segmentation.

Cell segmentation is crucial to the field of cell biology, as the accurate extraction of single-cell morphology, migration, and ultimately behavior from time-lapse live cell imagery are of paramount importance to elucidate and understand basic cellular processes. In an effort to increase available segmentation tools that can perform across research groups and platforms, we introduce a novel segmentation approach centered around optical flow and show that it achieves robust segmentation of single cells by validating it on multiple cell types, phenotypes, optical modalities, and in-vitro environments with or without labels. By leveraging cell movement in time-lapse imagery as a means to distinguish cells from their background and augmenting the output with machine vision operations, our algorithm reduces the number of adjustable parameters needed for manual optimization to two. We show that this approach offers the advantage of quicker processing times compared to contemporary machine learning based methods that require manual labeling for training, and in most cases achieves higher quality segmentation as well. This algorithm is packaged within MATLAB, offering an accessible means for general cell segmentation in a time-efficient manner.

Culture of Adherent Cancer Cell Lines.

Adherent cell lines grow attached to the surface of a cell culture vessel. Due to the adherent nature of the cells, enzymes, such as trypsin, are required to lift the cells from the cell culture vessel for harvesting or subculturing. Many cancer cell lines are adherent, rendering adherent cell culture a critical experimental method in the fields of cell biology, biochemistry, and cancer research. In this chapter, we outline the protocols for culturing and maintaining adherent cells. We detail the procedures for preparing cell culture medium, thawing and reviving frozen adherent cells, subculturing adherent cells, freezing cells, and counting cells. Most notably, we outline the best techniques and practices for optimal growth of healthy adherent cells while diminishing the risk of contamination.

Emerging Frontiers in Vaccine Development: A Review of Changing Paradigm

The technology behind vaccine development varies significantly from one vaccine to another depending on the time when the vaccine was first developed. Over the years, the vaccine innovation time has significantly shortened with the advancement of knowledge in the fields of molecular and cell biology, and discoveries in the field of biotechnology. The first vaccines created were tested in a kind of trial-and-error approach which sometimes had deadly side effects. These vaccines used either living, weakened, or completely dead pathogens. The use of whole pathogen vaccines was seen to be time consuming and unpredictable because even though it would cause an immune response, it could vary from person to person, and always had the risk of pathogens returning to virulence causing sometimes fatal outcomes. The next major technology used to create vaccines was subunit vaccines which utilize purified antigens inactivated through various methods. This technology is quite prevalent among the vaccines that are currently in circulation, making them quite effective, and free from fatal side effects. The viral vector vaccine technology has been around for a few decades and utilizes knowledge of molecular genetics to the greatest extent. It uses intermediate vectors to deliver genetic instructions to trigger an immune response within the subject body. The introduction of nucleic acid vaccines is the newest technology and has come to a great deal of attention during the SARS-CoV-2 immunization efforts. The technology primarily utilizes the delivery of genetic information using messenger ribonucleic acid (mRNA) to create characteristic pathogen-specific proteins that in turn generate an immune response in the recipients.

Imaging Mitosis with Lattice LightSheet.

Mitosis is one of the most fundamental processes of life, allowing organisms to grow, develop, and evolve. Acquiring microscopic images and understanding the detailed mechanism of this process is critical in the fields of cell and developmental biology. Modern fluorescence microscopy is the standard for imaging specific molecules and proteins as they interact during this complicated process. However, researchers must take care to ensure that they are maintaining the basal cell processes during mitosis without disruption by placing the sample on a microscope. In addition, mitosis in itself is an incredibly dynamic process that requires both high-speed and high-resolution imaging (McIntosh and Hays. Biology. 5:55, 2016). The Lattice LightSheet is an advanced system, developed in the lab of Eric Betzig (Chen et al. Science. 346:1257998), that offers imaging speeds in the volumes/second while still resolving fine, intracellular structures. Here we describe how to prepare cell culture samples for ideal mitotic imaging on this cutting-edge light sheet fluorescence microscope.

Progress and Challenges in Archaeal Cell Biology.

Over the past decades there has been a growing interest in the domain of archaea. In this chapter we highlight the recent advances that have been made in studying the cell biology of archaea. We particularly focus on methods for genetic manipulation and imaging of different archaeal species and discuss the technical limitations at the often-extreme growth conditions. Several ongoing developments will help us overcoming these limitations, thereby facilitating future studies in the existing field of archaeal cell biology.

Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa.

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. This protocol was validated in: Curr Biol (2022), DOI: 10.1016/j.cub.2022.04.024.

Publication trends in nature reviews molecular cell biology: A scientometric analysis based on scopus database

Nature Review Molecular Cell Biology is one of the most prestigious journals in molecular and cellular biology. The focus of this research study is to use scientometric analysis to look at the publication trends of Nature Reviews Molecular Cell Biology. The data were collected from the Scopus database covering 20 years. The present study covers the years 2000–2019, with 3397 articles published during the study period. The study concentrates on publication trends, highly productive authors, collaborative institutions, countries, and highly cited publications. The analysis of the study revealed that 2001 witnessed the highest number of publications. Heinrichs, A. was ranked as the most productive author, contributing a total of 165 articles. The United States of America and the United Kingdom have the most international collaboration players. The maximum number of publications published in the review was 1307(38.47%) of the publication. The article “Understanding the ErbB Signaling Network by Yarden, Y., and Sliwkowski, M.X.” has the most citations and was published as a review. This scientometric study will benefit researchers in the field of molecular cell biology because it will highlight the amount of literature published during the study period and worldwide collaboration among researchers.

Analysis of Nucleoporin Function Using Inducible Degron Techniques.

Over the last decade, the use of auxin-inducible degrons (AID) to control the stability of target proteins has revolutionized the field of cell biology. AID-mediated degradation helps to overcome multiple hurdles that have been encountered in studying multisubunit protein complexes, like the nuclear pore complex (NPC), using classical biochemical and genetic methods. We have used the AID system for acute depletion of individual members of the NPC, called nucleoporins, in order to distinguish their roles both within established NPCs and during NPC assembly.Here, we describe a protocol for CRISPR/Cas9-mediated gene targeting of genes with the AID tag. As an example, we describe a step-by-step protocol for targeting of the NUP153 gene. We also provide recommendations for screening strategies and integration of the sequence encoding the Transport Inhibitor Response 1 (TIR1) protein, a E3-Ubiquitin ligase subunit necessary for AID-dependent protein degradation. In addition, we discuss applications of the NUP-AID system and functional assays for analysis of NUP-AID tagged cell lines.

Single-Molecule Clustering for Super-Resolution Optical Fluorescence Microscopy

Molecular assembly in a complex cellular environment is vital for understanding underlying biological mechanisms. Biophysical parameters (such as single-molecule cluster density, cluster-area, pairwise distance, and number of molecules per cluster) related to molecular clusters directly associate with the physiological state (healthy/diseased) of a cell. Using super-resolution imaging along with powerful clustering methods (K-means, Gaussian mixture, and point clustering), we estimated these critical biophysical parameters associated with dense and sparse molecular clusters. We investigated Hemaglutinin (HA) molecules in an Influenza type A disease model. Subsequently, clustering parameters were estimated for transfected NIH3T3 cells. Investigations on test sample (randomly generated clusters) and NIH3T3 cells (expressing Dendra2-Hemaglutinin (Dendra2-HA) photoactivable molecules) show a significant disparity among the existing clustering techniques. It is observed that a single method is inadequate for estimating all relevant biophysical parameters accurately. Thus, a multimodel approach is necessary in order to characterize molecular clusters and determine critical parameters. The proposed study involving optical system development, photoactivable sample synthesis, and advanced clustering methods may facilitate a better understanding of single molecular clusters. Potential applications are in the emerging field of cell biology, biophysics, and fluorescence imaging.

Fluorescence Microscopy-An Outline of Hardware, Biological Handling, and Fluorophore Considerations.

Fluorescence microscopy has become a critical tool for researchers to understand biological processes at the cellular level. Micrographs from fixed and live-cell imaging procedures feature in a plethora of scientific articles for the field of cell biology, but the complexities of fluorescence microscopy as an imaging tool can sometimes be overlooked or misunderstood. This review seeks to cover the three fundamental considerations when designing fluorescence microscopy experiments: (1) hardware availability; (2) amenability of biological models to fluorescence microscopy; and (3) suitability of imaging agents for intended applications. This review will help equip the reader to make judicious decisions when designing fluorescence microscopy experiments that deliver high-resolution and informative images for cell biology.

20 years of Developmental Cell: Looking back.

In our 20th anniversary year, we reflect on how the cell and developmental biology fields have changed since the publication of Developmental Cell's first few issues. In this collection of Voices, authors who published in our early issues discuss the advances that helped shape their field over the past two decades.

Contribution of Human Pluripotent Stem Cell-Based Models to Drug Discovery for Neurological Disorders.

One of the major obstacles to the identification of therapeutic interventions for central nervous system disorders has been the difficulty in studying the step-by-step progression of diseases in neuronal networks that are amenable to drug screening. Recent advances in the field of human pluripotent stem cell (PSC) biology offers the capability to create patient-specific human neurons with defined clinical profiles using reprogramming technology, which provides unprecedented opportunities for both the investigation of pathogenic mechanisms of brain disorders and the discovery of novel therapeutic strategies via drug screening. Many examples not only of the creation of human pluripotent stem cells as models of monogenic neurological disorders, but also of more challenging cases of complex multifactorial disorders now exist. Here, we review the state-of-the art brain cell types obtainable from PSCs and amenable to compound-screening formats. We then provide examples illustrating how these models contribute to the definition of new molecular or functional targets for drug discovery and to the design of novel pharmacological approaches for rare genetic disorders, as well as frequent neurodegenerative diseases and psychiatric disorders.

Multimodal on-chip nanoscopy and quantitative phase imaging reveals the nanoscale morphology of liver sinusoidal endothelial cells

Visualization of three-dimensional (3D) morphological changes in the subcellular structures of a biological specimen is a major challenge in life science. Here, we present an integrated chip-based optical nanoscopy combined with quantitative phase microscopy (QPM) to obtain 3D morphology of liver sinusoidal endothelial cells (LSEC). LSEC have unique morphology with small nanopores (50-300 nm in diameter) in the plasma membrane, called fenestrations. The fenestrations are grouped in discrete clusters, which are around 100 to 200 nm thick. Thus, imaging and quantification of fenestrations and sieve plate thickness require resolution and sensitivity of sub-100 nm along both the lateral and the axial directions, respectively. In chip-based nanoscopy, the optical waveguides are used both for hosting and illuminating the sample. The fluorescence signal is captured by an upright microscope, which is converted into a Linnik-type interferometer to sequentially acquire both superresolved images and phase information of the sample. The multimodal microscope provided an estimate of the fenestration diameter of 119 ± 53 nm and average thickness of the sieve plates of 136.6 ± 42.4 nm, assuming the constant refractive index of cell membrane to be 1.38. Further, LSEC were treated with cytochalasin B to demonstrate the possibility of precise detection in the cell height. The mean phase value of the fenestrated area in normal and treated cells was found to be 161 ± 50 mrad and 109 ± 49 mrad, respectively. The proposed multimodal technique offers nanoscale visualization of both the lateral size and the thickness map, which would be of broader interest in the fields of cell biology and bioimaging.

Rapid Encapsulation of Reconstituted Cytoskeleton Inside Giant Unilamellar Vesicles.

Giant unilamellar vesicles (GUVs) are frequently used as models of biological membranes and thus are a great tool to study membrane-related cellular processes in vitro. In recent years, encapsulation within GUVs has proven to be a helpful approach for reconstitution experiments in cell biology and related fields. It better mimics confinement conditions inside living cells, as opposed to conventional biochemical reconstitution. Methods for encapsulation inside GUVs are often not easy to implement, and success rates can differ significantly from lab to lab. One technique that has proven to be successful for encapsulating more complex protein systems is called continuous droplet interface crossing encapsulation (cDICE). Here, a cDICE-based method is presented for rapidly encapsulating cytoskeletal proteins in GUVs with high encapsulation efficiency. In this method, first, lipid-monolayer droplets are generated by emulsifying a protein solution of interest in a lipid/oil mixture. After being added into a rotating 3D-printed chamber, these lipid-monolayered droplets then pass through a second lipid monolayer at a water/oil interface inside the chamber to form GUVs that contain the protein system. This method simplifies the overall procedure of encapsulation within GUVs and speeds up the process, and thus allows us to confine and observe the dynamic evolution of network assembly inside lipid bilayer vesicles. This platform is handy for studying the mechanics of cytoskeleton-membrane interactions in confinement.

A Tale of Two Bioconjugations: pH Controlled Divergent Reactivity of Protein α-oxo-Aldehydes in Competing α-oxo-Mannich and Catalyst-Free Aldol Ligations.

Site-selective chemical methods for protein bioconjugation have revolutionized the fields of cell and chemical biology through the development of novel protein/enzyme probes bearing fluorescent, spectroscopic, or even toxic cargos. Herein, we report two new methods for the bioconjugation of α-oxo aldehyde handles within proteins using small molecule aniline and/or phenol probes. The "α-oxo-Mannich" and "catalyst-free aldol" ligations both compete for the electrophilic α-oxo aldehyde, which displays pH divergent reactivity proceeding through the "Mannich" pathway at acidic pH to afford bifunctionalized bioconjugates, and the "catalyst-free aldol" pathway at neutral pH to afford monofunctionalized bioconjugates. We explore the substrate scope and utility of both of these bioconjugations in the construction of neoglycoproteins, in the process formulating a mechanistic rationale for how both pathways intersect with each other at different reaction pH's.

Human fibroblast-like synoviocyte isolation matter: a comparison between cell isolation from synovial tissue and synovial fluid from patients with rheumatoid arthritis.

Cell culture technology has become a popular method in the field of cell biology, pharmacology, and medical researches. Primary cells represent the normal physiological condition of human cells. Fibroblasts are the most common native cells of connective tissue that play a crucial role in the entire pathogenesis of various disorders, such as rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs), which overlie the loose connective tissue of the synovial sublining, are known to be the central mediators of joint damage. The most routine approach for the isolation of FLS is an enzymatic digestion of synovial tissue. This experimental study is designed to introduce an easy, fast, and high-throughput method compared with enzymatic digestion for isolation of FLS. The synovial tissue and synovial fluid (SF) samples were collected from eight patients with RA who underwent routine knee replacement surgery. Synovial tissue was incubated with collagenase VIII enzyme, while SF was washed with a similar volume of phosphate-buffered saline. The cells were further subcultured and stored based on the standard protocols. The purity of isolated synoviocytes was confirmed using flow cytometry analysis. Isolation of FLS from SF was more successful with a faster rate, 3-5 days after culture. The morphological assessment and flow cytometry analysis confirmed the purity of SF-derived cells in passage 4. SF could be a more accessible source of FLS than synovial tissue. Obtaining primary FLS from SF is a simple, fast, and cost-effective way to have a large-scale cell during a short time.

A Review of Advanced Impedance Biosensors with Microfluidic Chips for Single-Cell Analysis.

Electrical impedance biosensors combined with microfluidic devices can be used to analyze fundamental biological processes for high-throughput analysis at the single-cell scale. These specialized analytical tools can determine the effectiveness and toxicity of drugs with high sensitivity and demonstrate biological functions on a single-cell scale. Because the various parameters of the cells can be measured depending on methods of single-cell trapping, technological development ultimately determine the efficiency and performance of the sensors. Identifying the latest trends in single-cell trapping technologies afford opportunities such as new structural design and combination with other technologies. This will lead to more advanced applications towards improving measurement sensitivity to the desired target. In this review, we examined the basic principles of impedance sensors and their applications in various biological fields. In the next step, we introduced the latest trend of microfluidic chip technology for trapping single cells and summarized the important findings on the characteristics of single cells in impedance biosensor systems that successfully trapped single cells. This is expected to be used as a leading technology in cell biology, pathology, and pharmacological fields, promoting the further understanding of complex functions and mechanisms within individual cells with numerous data sampling and accurate analysis capabilities.

Deep Learning-Based In Vitro Detection Method for Cellular Impurities in Human Cell-Processed Therapeutic Products

Automated detection of impurities is in demand for evaluating the quality and safety of human cell-processed therapeutic products in regenerative medicine. Deep learning (DL) is a powerful method for classifying and recognizing images in cell biology, diagnostic medicine, and other fields because it automatically extracts the features from complex cell morphologies. In the present study, we construct prediction models that recognize cancer-cell contamination in continuous long-term (four-day) cell cultures. After dividing the whole dataset into Early- and Late-stage cell images, we found that Late-stage images improved the DL performance. The performance was further improved by optimizing the DL hyperparameters (batch size and learning rate). These findings are first report for the implement of DL-based systems in disease cell-type classification of human cell-processed therapeutic products (hCTPs), that are expected to enable the rapid, automatic classification of induced pluripotent stem cells and other cell treatments for life-threatening or chronic diseases.

Imaging the living plant cell: From probes to quantification.

At the center of cell biology is our ability to image the cell and its various components, either in isolation or within an organism. Given its importance, biological imaging has emerged as a field of its own, which is inherently highly interdisciplinary. Indeed, biologists rely on physicists and engineers to build new microscopes and imaging techniques, chemists to develop better imaging probes, and mathematicians and computer scientists for image analysis and quantification. Live imaging collectively involves all the techniques aimed at imaging live samples. It is a rapidly evolving field, with countless new techniques, probes, and dyes being continuously developed. Some of these new methods or reagents are readily amenable to image plant samples, while others are not and require specific modifications for the plant field. Here, we review some recent advances in live imaging of plant cells. In particular, we discuss the solutions that plant biologists use to live image membrane-bound organelles, cytoskeleton components, hormones, and the mechanical properties of cells or tissues. We not only consider the imaging techniques per se, but also how the construction of new fluorescent probes and analysis pipelines are driving the field of plant cell biology.