scholarly journals Active Microrheology of Intestinal Mucus in the Larval Zebrafish

2016 ◽  
Author(s):  
Michael J. Taormina ◽  
Raghuveer Parthasarathy
Keyword(s):  
Biological Fluid ◽  
Measurement Techniques ◽  
Model Organisms ◽  
Secretory Cells ◽  
Fluid Viscosity ◽  
Intestinal Microbes ◽  
Germ Free ◽  
Larval Zebrafish ◽  
Intestinal Mucus

Mucus is a complex biological fluid that plays a variety of functional roles in many physiological systems. Intestinal mucus in particular serves as a physical barrier to pathogens, a medium for the diffusion of nutrients and metabolites, and an environmental home for colonizing microbes. Its rheological properties have therefore been the subject of many investigations, thus far limited, however, to in vitro studies due to the difficulty of measurement in the natural context of the gut. This limitation especially hinders our understanding of how the gut microbiota interact with the intestinal environment, since examination of this calls not only for in vivo measurement techniques, but for techniques that can be applied to model organisms in which the microbial state of the gut can be controlled. We address this challenge by developing a method that combines magnetic microrheology, light sheet fluorescence microscopy, and microgavage of particles, applying this to the larval zebrafish, a model vertebrate. We present measurements of the viscosity of mucus within the intestinal bulb of both germ-free (devoid of intestinal microbes) and conventionally reared larval zebrafish. At the length scale probed (≈ 10 μ m), we find that mucus behaves as a Newtonian fluid, with no discernable elastic component. Surprisingly, despite known differences in the the number of secretory cells in germ-free zebrafish and their conventional counterparts, the fluid viscosity for these two groups was very similar. Our measurements provide the first in vivo measurements of intestinal mucus rheology at micron length scales in living animals, quantifying of an important biomaterial environment and highlighting the utility of active magnetic microrheology for biophysical studies.

scholarly journals Experimental models to study intestinal microbes–mucus interactions in health and disease

2019 ◽  
Vol 43 (5) ◽  
pp. 457-489 ◽  
Author(s):  
Lucie Etienne-Mesmin ◽  
Benoit Chassaing ◽  
Mickaël Desvaux ◽  
Kim De Paepe ◽  
Raphaële Gresse ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Intestinal Bacteria ◽  
Experimental Models ◽  
In Vivo Models ◽  
Intestinal Microbes ◽  
Intestinal Mucus ◽  
Health And Disease ◽  
And Function

ABSTRACTA close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

10 THE ROLE OF DIETARY L-SERINE IN THE REGULATION OF INTESTINAL MUCUS BARRIER DURING INFLAMMATION

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S42-S42
Author(s):  
Kohei Sugihara ◽  
Nobuhiko Kamada
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gut Microbiota ◽  
Control Diet ◽  
Bacterial Strains ◽  
Germ Free ◽  
Intestinal Mucus ◽  
Gnotobiotic Mice ◽  
Mucus Barrier

Abstract Background Recent accumulating evidence suggests that amino acids have crucial roles in the maintenance of intestinal homeostasis. In inflammatory bowel disease (IBD), amino acid metabolism is changed in both host and the gut microbiota. Among amino acids, L-serine plays a central role in several metabolic processes that are essential for the growth and survival of both mammalian and bacterial cells. However, the role of L-serine in intestinal homeostasis and IBD remains incompletely understood. In this study, we investigated the effect of dietary L-serine on intestinal inflammation in a murine model of colitis. Methods Specific pathogen-free (SPF) mice were fed either a control diet (amino acid-based diet) or an L-serine-deficient diet (SDD). Colitis was induced by the treatment of dextran sodium sulfate (DSS). The gut microbiome was analyzed by 16S rRNA sequencing. We also evaluate the effect of dietary L-serine in germ-free mice and gnotobiotic mice that were colonized by a consortium of non-mucolytic bacterial strains or the consortium plus mucolytic bacterial strains. Results We found that the SDD exacerbated experimental colitis in SPF mice. However, the severity of colitis in SDD-fed mice was comparable to control diet-fed mice in germ-free condition, suggesting that the gut microbiota is required for exacerbation of colitis caused by the restriction of dietary L-serine. The gut microbiome analysis revealed that dietary L-serine restriction fosters the blooms of a mucus-degrading bacterium Akkermansia muciniphila and adherent-invasive Escherichia coli in the inflamed gut. Consistent with the expansion of mucolytic bacteria, SDD-fed mice showed a loss of the intestinal mucus layer. Dysfunction of the mucus barrier resulted in increased intestinal permeability, thereby leading to bacterial translocation to the intestinal mucosa, which subsequently increased the severity of colitis. The increased intestinal permeability and subsequent bacterial translocation were observed in SDD-fed gnotobiotic mice that colonized by mucolytic bacteria. In contrast, dietary L-serine restriction did not alter intestinal barrier integrity in gnotobiotic mice that colonized only by non-mucolytic bacteria. Conclusion Our results suggest that dietary L-serine regulates the integrity of the intestinal mucus barrier during inflammation by limiting the expansion of mucus degrading bacteria.

scholarly journals Cellular dynamics of EMT: lessons from live in vivo imaging of embryonic development

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jeffrey D. Amack
Keyword(s):  
Extracellular Matrix ◽  
Embryonic Development ◽  
In Vivo Imaging ◽  
Cytoskeletal Proteins ◽  
Cell Junctions ◽  
Model Organisms ◽  
Cellular Dynamics ◽  
Mesenchymal Transition ◽  
Cell Cell

AbstractEpithelial-mesenchymal transition (EMT) refers to a process in which epithelial cells lose apical-basal polarity and loosen cell–cell junctions to take on mesenchymal cell morphologies and invasive properties that facilitate migration through extracellular matrix. EMT—and the reverse mesenchymal-epithelial transition (MET)—are evolutionarily conserved processes that are used throughout embryonic development to drive tissue morphogenesis. During adult life, EMT is activated to close wounds after injury, but also can be used by cancers to promote metastasis. EMT is controlled by several mechanisms that depend on context. In response to cell–cell signaling and/or interactions with the local environment, cells undergoing EMT make rapid changes in kinase and adaptor proteins, adhesion and extracellular matrix molecules, and gene expression. Many of these changes modulate localization, activity, or expression of cytoskeletal proteins that mediate cell shape changes and cell motility. Since cellular changes during EMT are highly dynamic and context-dependent, it is ideal to analyze this process in situ in living organisms. Embryonic development of model organisms is amenable to live time-lapse microscopy, which provides an opportunity to watch EMT as it happens. Here, with a focus on functions of the actin cytoskeleton, I review recent examples of how live in vivo imaging of embryonic development has led to new insights into mechanisms of EMT. At the same time, I highlight specific developmental processes in model embryos—gastrulation in fly and mouse embryos, and neural crest cell development in zebrafish and frog embryos—that provide in vivo platforms for visualizing cellular dynamics during EMT. In addition, I introduce Kupffer’s vesicle in the zebrafish embryo as a new model system to investigate EMT and MET. I discuss how these systems have provided insights into the dynamics of adherens junction remodeling, planar cell polarity signaling, cadherin functions, and cytoskeletal organization during EMT, which are not only important for understanding development, but also cancer progression. These findings shed light on mechanisms of actin cytoskeletal dynamics during EMT, and feature live in vivo imaging strategies that can be exploited in future work to identify new mechanisms of EMT and MET.

Photocontrolling Microtubule Dynamics with Photoswitchable Chemical Reagents

2021 ◽  
Author(s):  
Oliver Thorn-Seshold ◽  
Joyce Meiring
Keyword(s):  
3D Culture ◽  
3D Cell Culture ◽  
Relevant Information ◽  
Microtubule Dynamics ◽  
Model Organisms ◽  
Chemical Reagents ◽  
Cellular Assays ◽  
General Protein ◽  
Spatial Specificity

Microtubule dynamics can be inhibited with sub-second temporal resolution and cellular-scale spatial resolution, by using precise illuminations to optically pattern where and when photoswitchable microtubule-inhibiting chemical reagents exert their latent bioactivity. The recently-available reagents (SBTub, PST, STEpo, AzTax, PHTub) now enable researchers to use light to reversibly modulate microtubule-dependent processes in eukaryotes, in 2D and 3D cell culture as well as in vivo, across a variety of model organisms: with applications in fields from cargo transport to cell migration, cell division, and embryonic development.<br><br>However, a wide knowledge gap has remained in the literature, which has blocked further translation of these and many other classes of photopharmaceuticals. No generally-applicable procedures or workflows to establish biological assays using photopharmaceuticals have been published. Accordingly, the rate of adoption of photopharmaceutical tools in the broader chemical biology community (beyond the original chemical developers of the tools) has remained very low. Vital information about assay benchmarking for photoconversion, testing for isomer solubility, proving the retention of mechanism of action, estimating the limits of phototoxicity etc has either simply not been formalised in the literature, or has remained buried in diverse reports without being unified and codified for an audience beyond that of synthetic organic chemists.<br><br>Here we have developed a robust four-step assay establishment procedure to optimise assay parameters for achieving reliable photocontrol over microtubule dynamics, that is applicable to diverse families of photoswitchable inhibitors. This procedure also controls for these common sources of irreproducibility and includes numerous troubleshooting steps. We also collect together the relevant information for non-chemist "users" such as microscopists and biologists, to introduce the theory of small molecule photoswitching; the unique features, usage requirements, and limitations that photoswitchable chemical reagents have; and the specific performance features of the major classes of photoswitchable microtubule inhibitors that are currently available; to highlight their properties that suit them to different applications. The generally-applicable workflows that we present allow establishing cellular assays optically controlling microtubule dynamics in a temporally reversible fashion with spatial specificity down to a single selected cell within a field of view. These workflows and methods also equip the reader to tackle advanced uses of photoswitchable chemical reagents for general protein targets, in 3D culture and in vivo, and can represent an important bridge to reach the high-value biological applications that photopharmacology can promise.<br>

MIC-Drop: A platform for large-scale in vivo CRISPR screens

Science ◽  
2021 ◽  
pp. eabi8870
Author(s):  
Saba Parvez ◽  
Chelsea Herdman ◽  
Manu Beerens ◽  
Korak Chakraborti ◽  
Zachary P. Harmer ◽  
...  
Keyword(s):  
Large Scale ◽  
Cultured Cells ◽  
Cardiac Development ◽  
Droplet Microfluidics ◽  
Model Organisms ◽  
Genetic Screens ◽  
Large Numbers ◽  
And Function ◽  
Genome Scale

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we report Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we show MIC-Drop can identify small molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discover several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse-genetic screens in model organisms.

The Symptoms and Histopathology of Poisoning by Maneb in Oncopeltus fasciatus (Dallas),

1965 ◽  
Vol 97 (11) ◽  
pp. 1200-1208 ◽  
Author(s):  
R. D. McMullen
Keyword(s):  
Oxygen Consumption ◽  
Malpighian Tubules ◽  
Oncopeltus Fasciatus ◽  
Secretory Cells ◽  
Cell Nuclei ◽  
Gut Epithelium

AbstractManeb (manganous ethylene bisdithiocarhamate) applied topically to Oncopeltus fasciatus nymphs causes death after 7 to 10 days. The gross symptoms of intoxication, histopathology and effect on oxygen consumption are described. Activities such as feeding and walking are slightly reduced after 24 hours and completely inhibited after 3 to 4 days. The tissues most severely affected by the treatment are the secretory cells of the mid-gut epithelium and the cells of the Malpighian tubules. These at first show extreme vacuolization, reduction of the size of cell nuclei and finally cytolysis. Oxygen consumption in vivo is reduced by the treatment.

scholarly journals Dynamic Colon Model (DCM): A Cine-MRI Informed Biorelevant In Vitro Model of the Human Proximal Large Intestine Characterized by Positron Imaging Techniques

Pharmaceutics ◽  
2020 ◽  
Vol 12 (7) ◽  
pp. 659
Author(s):  
Konstantinos Stamatopoulos ◽  
Sharad Karandikar ◽  
Mark Goldstein ◽  
Connor O’Farrell ◽  
Luca Marciani ◽  
...  
Keyword(s):  
Particle Motion ◽  
Wall Motion ◽  
Bulk Liquid ◽  
Cine Mri ◽  
Fluid Viscosity ◽  
Positron Emission ◽  
Volume Viscosity ◽  
Viscosity Fluid

This work used in vivo MRI images of human colon wall motion to inform a biorelevant Dynamic Colon Model (DCM) to understand the interplay of wall motion, volume, viscosity, fluid, and particle motion within the colon lumen. Hydrodynamics and particle motion within the DCM were characterized using Positron Emission Tomography (PET) and Positron Emission Particle Tracking (PEPT), respectively. In vitro PET images showed that fluid of higher viscosity follows the wall motion with poor mixing, whereas good mixing was observed for a low viscosity fluid. PEPT data showed particle displacements comparable to the in vivo data. Increasing fluid viscosity favors the net forward propulsion of the tracked particles. The use of a floating particle demonstrated shorter residence times and greater velocities on the liquid surface, suggesting a surface wave that was moving faster than the bulk liquid. The DCM can provide an understanding of flow motion and behavior of particles with different buoyancy, which in turn may improve the design of drug formulations, whereby fragments of the dosage form and/or drug particles are suspended in the proximal colon.

scholarly journals A Larval Zebrafish Model for Assessing Hypoxic‐Induced In Vivo Cardiomyocyte Damage: Time Course for Induction and Cardiac Output Recovery

2020 ◽  
Vol 34 (S1) ◽  
pp. 1-1
Author(s):  
Warren Burggren ◽  
Regina Abramova ◽  
Naim Bautista ◽  
Regina Fritsche Danielson ◽  
Avi Gupta ◽  
...  
Keyword(s):  
Cardiac Output ◽  
Time Course ◽  
Zebrafish Model ◽  
Larval Zebrafish
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