Extracellular Fluid Flow Research Articles

Nodal flow transfers polycystin to determine mouse left-right asymmetry

Left-dominant [Ca2+]i elevation on the left margin of the ventral node furnishes the initial laterality of mouse embryos. It depends on extracellular leftward fluid flow (nodal flow), fibroblast growth factor receptor (FGFR)/sonic hedgehog (Shh) signaling, and the PKD1L1 polycystin subunit, of which interrelationship is still elusive. Here, we show that leftward nodal flow directs PKD1L1-containing fibrous strands and facilitates Nodal-mediated [Ca2+]i elevation on the left margin. We generate KikGR-PKD1L1 knockin mice in order to monitor protein dynamics with a photoconvertible fluorescence protein tag. By imaging those embryos, we have identified fragile meshwork being gradually transferred leftward involving pleiomorphic extracellular events. A portion of the meshwork finally bridges over the left nodal crown cells in an FGFR/Shh-dependent manner. As PKD1L1 N-term is predominantly associated with Nodal on the left margin and that PKD1L1/PKD2 overexpression significantly augments cellular Nodal sensitivity, we propose that leftward transfer of polycystin-containing fibrous strands determines left-right asymmetry in developing embryos.

Molecular Dynamics Simulation from Symmetry Breaking Changing to Asymmetrical Phospholipid Membranes Due to Variable Capacitors during Resonance with Helical Proteins

Biological symmetry breaking is a mechanism in biosystems that is necessary for human survival, and depends on chemical physics concepts at both microscopic and macroscopic scales. In this work, we present a few mechanisms of the signaling phenomenon that have been studied in various tissues of human origin. We exhibit that anatomical asymmetry in the structure of a membrane can produce a flow of extracellular fluid. Furthermore, we exhibit that membrane asymmetry is a misbalance in the composition of the aqueous phases and interaction forces with the protein trans-membrane. Various biological membranes such as DPPC, DMPC, DLPC, and so on, have considerable electrostatic voltages that extend across the phosphor lipids bilayer. For studying these phenomena, we modeled DPPC, DMPC, and DLPC lipid bilayers with a net charge misbalance across the phospholipids. Because asymmetric membranes create the shifted voltages among the various aqueous tissues, this effect makes the charge misbalances cause a voltage of 1.3 V across the DPPC bilayer and 0.8 V across the DMPC bilayer. This subject exhibits the importance of membrane structures on electrostatic potential gradients. Finally, we exhibited that a quantum effect was created in small parts of the cell’s thickness due to the symmetry breaking of asymmetrical phospholipid bilayers.

Blood-Brain barrier dysfunction: reduced flow of Extracellular Fluid - no leakage

Blood-Brain barrier dysfunction: reduced flow of Extracellular Fluid - no leakage

Plutonium Systemic Biokinetic Model for Rats

A baseline compartmental model (relative to modeling decorporation) of the distribution and retention of plutonium (Pu) in the rat for a systemic intake is derived. The model is derived from data obtained from a study designed to evaluate the behavior of plutonium in the first 28 days after incorporation. The model is based on a recently published model of americium (Am) in rats, which incorporated a pharmacokinetic (PK)-front-end modeling approach, which was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. In the americium model, the approach was "cell-membrane limited," meaning that rapid diffusion of americium occurred throughout all the extracellular fluids (i.e., the blood plasma and interstitial fluids), while back-end rates representing transport into and out of the cells were determined empirically. However, this approach was inconsistent with the plutonium dataset. A good fit to the data is obtained by incorporating aspects of the Durbin et al. model structure, with plutonium in plasma separated into "free" and "bound" components. Free plutonium uses a cell-membrane-limited front end as for americium. Bound plutonium uses a capillary-wall-limited front end, where transfer rates from blood plasma into the interstitial fluids are relatively slow, and must be determined either empirically or from a priori knowledge. As in the Durbin et al. model, both free and bound plutonium are available for deposition in bone. In addition, our model has some bound plutonium associated with uptake to the gastrointestinal (GI) tract. Uncertainties in transfer rates were investigated using Markov Chain Monte Carlo (MCMC). It is anticipated that this model structure of plutonium will also be useful in interpreting comparable data from decorporation studies done in experimental animals.

Fluidic Flow Enhances the Differentiation of Placental Trophoblast-Like 3D Tissue from hiPSCs in a Perfused Macrofluidic Device.

The human placenta serves as a multifunctional organ to maintain the proper development of a fetus. However, our knowledge of the human placenta is limited due to the lack of appropriate experimental models. In this work, we created an in vitro placental trophoblast-like model via self-organization of human induced pluripotent stem cells (hiPSCs) in a perfused 3D culture macrofluidic device. This device allowed cell seeding, in situ trophoblast lineage differentiation, and formation of trophoblast-like tissues from hiPSCs in a biomimetic microenvironment. It incorporated extracellular matrix (ECM) and fluid flow in a single device. After trophoblast lineage differentiation, we were able to generate the 3D clusters with major cell types of the human placenta, including trophoblast progenitor cytotrophoblasts (CTBs), differentiated subtypes, syncytiotrophoblasts (STBs), and extravillous trophoblasts (EVTs) under long-term 3D culture (∼23 days). Moreover, the formed tissues exhibited enhanced expressions of CTB-, STB-, and EVT-related markers at the level of genes and proteins under a dynamic culture compared with static conditions. RNA-seq analysis revealed the higher expression of trophoblast-specific genes in 3D tissues, indicating the essential role of fluid flow to promote the trophoblast differentiation of hiPSCs. The established placental 3D model combined a bioengineering strategy with developmental principles, providing a promising platform for the study of placental biology in a biomimetic microenvironment in health and disease.

Transverse Magnetic Field Effect on Extracellular Fluid Flow along with a Semi-Infinite Vertical Rotating Porous Plate

The primary intention of this practice is to numerically investigate the extracellular fluid (ECF) flow for unsteady 2-dimenstional case along with a porous vertical plate with the appearance of a transverse magnetic field in a rotating system. The dimensional basic equations have been non-dimensionalized by necessary dimensionless variables. The EFDM has been practiced to solve the dimensionless equations. The numerical data have been evaluated by FORTRAN software version 6.6a. For a perfect conductivity, Magnetic Diffusivity Number values have been taken between 5 to 15 in the induction formula. For proper exactness, stability and convergence tests have been performed. For initial time , the outputs have been illustrated for the primary, secondary and angular velocity, primary and secondary induced magnetic field, temperature field with shear stresses for and -direction, couple stress for -direction, and -direction current densities and Nusselt number. Finally, the outcomes of different parameters are discussed separately and pictorial graphically by MATLAB R2018a.The findings of this research may be used to control cell temperature, measurement of extracellular fluid motion, and so on.

Extracellular Fluid Flow Induces Shallow Quiescence Through Physical and Biochemical Cues.

The balance between cell quiescence and proliferation is fundamental to tissue physiology and homeostasis. Recent studies have shown that quiescence is not a passive and homogeneous state but actively maintained and heterogeneous. These cellular characteristics associated with quiescence were observed primarily in cultured cells under a static medium. However, cells in vivo face different microenvironmental conditions, particularly, under interstitial fluid flows distributed through extracellular matrices. Interstitial fluid flow exerts shear stress on cells and matrix strain, and results in continuous replacement of extracellular factors. In this study, we analyzed individual cells under varying fluid flow rates in microfluidic devices. We found quiescence characteristics previously identified under conventional static medium, including serum signal-dependant quiescence entry and exit and time-dependant quiescence deepening, are also present under continuous fluid flow. Furthermore, increasing the flow rate drives cells to shallower quiescence and become more likely to reenter the cell cycle upon growth stimulation. This effect is due to flow-induced physical and biochemical cues. Specifically, increasing shear stress or extracellular factor replacement individually, without altering other parameters, results in shallow quiescence. We show our experimental results can be quantitatively explained by a mathematical model connecting extracellular fluid flow to an Rb-E2f bistable switch that regulates the quiescence-to-proliferation transition. Our findings uncover a previously unappreciated mechanism that likely underlies the heterogeneous responses of quiescent cells for tissue repair and regeneration in different physiological tissue microenvironments.

Xenopus to the rescue: A model to validate and characterize candidate ciliopathy genes.

Cilia are present on most vertebrate cells and play a central role in development, growth, and homeostasis. Thus, cilia dysfunction can manifest into an array of diseases, collectively termed ciliopathies, affecting millions of lives worldwide. Yet, our understanding of the gene regulatory networks that control cilia assembly and functions remain incomplete. With the advances in next-generation sequencing technologies, we can now rapidly predict pathogenic variants from hundreds of ciliopathy patients. While the pace of candidate gene discovery is exciting, most of these genes have never been previously implicated in cilia assembly or function. This makes assigning the disease causality difficult. This review discusses how Xenopus, a genetically tractable and high-throughput vertebrate model, has played a central role in identifying, validating, and characterizing candidate ciliopathy genes. The review is focused on multiciliated cells (MCCs) and diseases associated with MCC dysfunction. MCCs harbor multiple motile cilia on their apical surface to generate extracellular fluid flow inside the airway, the brain ventricles, and the oviduct. In Xenopus, these cells are external and present on the embryonic epidermal epithelia, facilitating candidate genes analysis in MCC development in vivo. The ability to introduce patient variants to study their effects on disease progression makes Xenopus a powerful model to improve our understanding of the underlying disease mechanisms and explain the patient phenotype.

Chemically‐Induced Cross‐Linking of Peptidic Fibrils for Scaffolding Polymeric Particles and Macrophages

EAK16-II (EAK) is a self-assembling peptide (SAP) that forms β-sheets and β-fibrils through ionic-complementary interactions at physiological ionic strengths. The soft materials can be injected in vivo, creating depots of drugs and cells for rendering pharmacological and biological actions. The scope of the applications of EAK is sought to extend to tissues through which the flow of extracellular fluid tends to be limited. In such anatomical locales the rate and extent of the fibrilization are limited insofar as drug delivery and cellular scaffolding would be impeded. A method is generated utilizing a carbodiimide cross-linker by which EAK fibrils are pre-assembled yet remain injectable soft materials. It is hypothesized that the resulting de novo covalent linkages enhance the stacking of the β-sheet bilayers, thereby increasing the lengths of the fibrils and the extent of their cross-linking, as evidenced in Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy, scanning electron microscopy, and atomic force microscopy analyses. The cross-linked EAK (clEAK) retains polymeric microspheres with an average diameter of 1µm. Macrophages admixed with clEAK remain viable and do not produce the inflammatory mediator interleukin-1β. These results indicate that clEAK should be investigated further as a platform for delivering particles and cells in vivo.

Structure of a microtubule-bound axonemal dynein

Axonemal dyneins are tethered to doublet microtubules inside cilia to drive ciliary beating, a process critical for cellular motility and extracellular fluid flow. Axonemal dyneins are evolutionarily and biochemically distinct from cytoplasmic dyneins that transport cargo, and the mechanisms regulating their localization and function are poorly understood. Here, we report a single-particle cryo-EM reconstruction of a three-headed axonemal dynein natively bound to doublet microtubules isolated from cilia. The slanted conformation of the axonemal dynein causes interaction of its motor domains with the neighboring dynein complex. Our structure shows how a heterotrimeric docking complex specifically localizes the linear array of axonemal dyneins to the doublet microtubule by directly interacting with the heavy chains. Our structural analysis establishes the arrangement of conserved heavy, intermediate and light chain subunits, and provides a framework to understand the roles of individual subunits and the interactions between dyneins during ciliary waveform generation.

Hall Effect on ECF Flow along a Rotating Infinite Porous Plate in the Presence of Transverse Magnetic Field

Unsteady extracellular fluid (ECF) flow along with a rotating infinite vertical porous plate in the presence of a transverse magnetic field has been studied numerically. The dimensional governing equations have been non-dimensionalized by useful dimensionless variables. The explicit finite difference method has been used to solve dimensionless equations. The numerical results have been calculated by studio developer FORTRAN 6.6a and MATLAB 2018a. For perfect conducting case, Magnetic Diffusivity Parameter 5 ≤ Pm ≤ 15 has been taken in induction equation. For good accuracy, stability and convergence analysis have been analyzed. Mesh Sensitivity test, steady-state solution test, and code validation test have been performed. For time step τ = 1, the numerical results have been found for the primary velocity, secondary velocity, angular velocity, primary induced magnetic field, secondary induced magnetic field, temperature as well as shear stresses along x and z direction, couple stress along z direction, current densities along x and z direction and the Nusselt number. Finally, the effects of various parameters have been separately discussed and illustrated graphically.

Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating.

Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial spoke (RS) protein complexes and the microtubule central pair (CP). Despite their importance for ciliary motility across eukaryotes, the molecular function of the RSs is unknown. Here, we reconstituted the Chlamydomonas reinhardtii RS head that abuts the CP and determined its structure using single-particle cryo-EM to 3.1 Å resolution, revealing a flat, negatively-charged surface supported by a rigid core of tightly intertwined proteins. Mutations in this core, corresponding to those involved in human ciliopathies, compromised stability of the recombinant complex, providing a molecular basis for disease. Partially reversing the negative charge on the RS surface impaired motility in C. reinhardtii. We propose that the RS head architecture is well-suited for mechanoregulation of ciliary beating through physical collisions with the CP.

Characterisation of centriole biogenesis during multiciliation in planarians.

Dense multicilia in protozoa and metazoa generate a strong force important for locomotion and extracellular fluid flow. During ciliogenesis, multiciliated cells produce hundreds of centrioles to serve as basal bodies through various pathways including deuterosome-dependent (DD), hyper-activated mother centriole-dependent (MCD) and basal bodydependent (BBD) pathways. The centrosome-free planarian Schmidtea mediterranea is widely used for regeneration studies because its neoblasts are capable of regenerating any body part after injury. However, it is currently unclear how the flatworms generate massive centrioles for multiciliated cells in the pharynx and body epidermis when their cells are initially centriole-free. In this study, we investigate the progress of centriole amplification during the pharynx regeneration. We observe that the planarian pharyngeal epithelial cells generate their centrioles asynchronously through a de novo pathway. Most of the de novo centrioles are formed individually, whereas the remaining ones are assembled in pairs, possibly by sharing a cartwheel, or in small clusters lacking a nucleation center. Further RNAi experiments show that the known key factors of centriole duplication, including Cep152, Plk4 and Sas6, are crucial for the centriole amplification. Our study demonstrates the distinct process of massive centriole biogenesis in S. mediterranea and helps to understand the diversity of centriole biogenesis during evolution.

Rsph4a is essential for the triplet radial spoke head assembly of the mouse motile cilia.

Motile cilia/flagella are essential for swimming and generating extracellular fluid flow in eukaryotes. Motile cilia harbor a 9+2 arrangement consisting of nine doublet microtubules with dynein arms at the periphery and a pair of singlet microtubules at the center (central pair). In the central system, the radial spoke has a T-shaped architecture and regulates the motility and motion pattern of cilia. Recent cryoelectron tomography data reveal three types of radial spokes (RS1, RS2, and RS3) in the 96 nm axoneme repeat unit; however, the molecular composition of the third radial spoke, RS3 is unknown. In human pathology, it is well known mutation of the radial spoke head-related genes causes primary ciliary dyskinesia (PCD) including respiratory defect and infertility. Here, we describe the role of the primary ciliary dyskinesia protein Rsph4a in the mouse motile cilia. Cryoelectron tomography reveals that the mouse trachea cilia harbor three types of radial spoke as with the other vertebrates and that all triplet spoke heads are lacking in the trachea cilia of Rsph4a-deficient mice. Furthermore, observation of ciliary movement and immunofluorescence analysis indicates that Rsph4a contributes to the generation of the planar beating of motile cilia by building the distal architecture of radial spokes in the trachea, the ependymal tissues, and the oviduct. Although detailed mechanism of RSs assembly remains unknown, our results suggest Rsph4a is a generic component of radial spoke heads, and could explain the severe phenotype of human PCD patients with RSPH4A mutation.

Americium Systemic Biokinetic Model for Rats.

In this work, a baseline compartmental model of the distribution and retention of americium in the rat for a systemic intake was derived. The model was derived from data obtained from a study designed to evaluate the behavior of americium in the first 28 days after incorporation. A pharmacokinetic (PK)-front-end modeling approach was used to specify transfer to and from the extracellular fluids (ECF) in the various tissues in terms of vascular flow and volumes of ECF. Back-end rates representing transport into and out of the cells were determined empirically. Uncertainties in transfer rates were investigated using Markov chain Monte Carlo (MCMC). The combination of PK-front-end model and the back-end model structure used allowed for extrapolation to the earliest times with small uncertainty. This approach clearly demonstrated the rapid transfer of material from ECF to liver and bone. This model provides a baseline for modeling the action of decorporation agents, such as DTPA.

A multiphase theory for spreading microbial swarms and films.

Bacterial swarming and biofilm formation are collective multicellular phenomena through which diverse microbial species colonize and spread over water-permeable tissue. During both modes of surface translocation, fluid uptake and transport play a key role in shaping the overall morphology and spreading dynamics. Here we develop a generalized two-phase thin-film model that couples bacterial growth, extracellular matrix swelling, fluid flow, and nutrient transport to describe the expansion of both highly motile bacterial swarms, and sessile bacterial biofilms. We show that swarm expansion corresponds to steady-state solutions in a nutrient-rich, capillarity dominated regime. In contrast, biofilm colony growth is described by transient solutions associated with a nutrient-limited, extracellular polymer stress driven limit. We apply our unified framework to explain a range of recent experimental observations of steady and unsteady expansion of microbial swarms and biofilms. Our results demonstrate how the physics of flow and transport in slender geometries serve to constrain biological organization in microbial communities.

CTENO64 is Required for Coordinated Paddling of Ciliary Comb Plate in Ctenophores

Motile cilia are oscillating nanomachinery that are essential for cell locomotion and the generation of extracellular fluid flow. They typically comprise an axoneme with a 9+2 microtubule structure with dynein arms on each microtubule doublet that generate force by sliding between adjacent microtubules. These structures are efficient for motility and have therefore been conserved in cellular-level aquatic microflow throughout eukaryotic evolution. Diversity in the ciliary structures is seen in the basic microtubule arrangements but also in the bundling of multiple cilia, such as compound cilia. Studies on the structural diversity of cilia shed light on the mechanisms underlying ciliary formation and the biological adaptation to different aquatic environments. Ctenophores (comb jellies) have eight rows of iridescent comb plates that facilitate locomotion. A comb plate consists of tens of thousands of cilia to form a “puddle-like” ciliary plate with lengths of up to 1 mm. It exhibits structural color by light diffraction and is reported to have properties similar to photonic crystals. Here we identified a protein specific to the construction of comb plates (CTENO64). This protein is specifically present in ctenophore species and is localized on the compartmenting lamella (CL), which is the structure connecting adjacent cilia on the comb plates. Knockdown of the CTENO64 gene caused loss or misformation of CLs, resulting in aberrant waveforms in the comb plates. Our findings provide the initial characterization of a protein involved in the bundling of cilia into giant compound ciliary comb plates.

Nutrient-limited growth with non-linear cell diffusion as a mechanism for floral pattern formation in yeast biofilms

Previous experiments have shown that mature yeast mat biofilms develop a floral morphology, characterised by the formation of petal-like structures. In this work, we investigate the hypothesis that nutrient-limited growth is the mechanism by which these floral patterns form. To do this, we use a combination of experiments and mathematical analysis. In mat formation experiments of the yeast species Saccharomyces cerevisiae, we observe that mats expand radially at a roughly constant speed, and eventually undergo a transition from circular to floral morphology. To determine the extent to which nutrient-limited growth can explain these features, we adopt a previously proposed mathematical model for yeast growth. The model consists of a coupled system of reaction–diffusion equations for the yeast cell density and nutrient concentration, with a non-linear, degenerate diffusion term for cell spread. Using geometric singular perturbation theory and numerics, we show that the model admits travelling wave solutions in one dimension, which enables us to infer the diffusion ratio from experimental data. We then use a linear stability analysis to show that two-dimensional planar travelling wave solutions for feasible experimental parameters are linearly unstable to non-planar perturbations. This provides a potential mechanism by which petals can form, and allows us to predict the characteristic petal width. There is good agreement between these predictions, numerical solutions to the model, and experimental data. We therefore conclude that the non-linear cell diffusion mechanism provides a possible explanation for pattern formation in yeast mat biofilms, without the need to invoke other mechanisms such as flow of extracellular fluid, cell adhesion, or changes to cellular shape or behaviour.

Classifying Heterotaxy Syndrome

Heterotaxy syndrome is a complex set of abnormalities related to abnormal left–right axis patterning. Although rare (occurring in just over 1 in 10 000 live births1), congenital heart defects associated with heterotaxy syndrome carry a disproportionate burden of morbidity and mortality.2,3 Despite numerous investigations on the genetic underpinning, epidemiology, anatomic pathology, diagnostic imaging, and management, agreement regarding the best method to classify patients with heterotaxy continues to elude our field.4 Specifically, a growing body of experience has noted that the 2 most common methods of classifying patients with heterotaxy—based on the status of the spleen or on the morphology of the atrial appendages—is frequently incongruent with the expected pattern of cardiac and noncardiac anomalies,5 is difficult to ascertain clinically, and may not be helpful in guiding clinical management.2–4 See Article by Yim et al In this issue of Circulation: Cardiovascular Imaging , Yim et al6 provide further evidence that the prevailing classification schemes used in heterotaxy syndrome are flawed. In a retrospective analysis of noninvasive imaging data from 114 patients with heterotaxy, they noted that the classic pattern of isomerism was breached in 1 of every 5 patients. Furthermore, they documented the limited clinical utility of relying on atrial appendage morphology or the status of the spleen and …

Cilia-related protein SPEF2 regulates osteoblast differentiation

Sperm flagellar protein 2 (SPEF2) is essential for motile cilia, and lack of SPEF2 function causes male infertility and primary ciliary dyskinesia. Cilia are pointing out from the cell surface and are involved in signal transduction from extracellular matrix, fluid flow and motility. It has been shown that cilia and cilia-related genes play essential role in commitment and differentiation of chondrocytes and osteoblasts during bone formation. Here we show that SPEF2 is expressed in bone and cartilage. The analysis of a Spef2 knockout (KO) mouse model revealed hydrocephalus, growth retardation and death prior to five weeks of age. To further elucidate the causes of growth retardation we analyzed the bone structure and possible effects of SPEF2 depletion on bone formation. In Spef2 KO mice, long bones (tibia and femur) were shorter compared to wild type, and X-ray analysis revealed reduced bone mineral content. Furthermore, we showed that the in vitro differentiation of osteoblasts isolated from Spef2 KO animals was compromised. In conclusion, this study reveals a novel function for SPEF2 in bone formation through regulation of osteoblast differentiation and bone growth.