Study Of Cell Physiology Research Articles

Quantitative measurements of reactive oxygen species partitioning in electron transfer flavoenzyme magnetic field sensing.

Biological magnetic field sensing that gives rise to physiological responses is of considerable importance in quantum biology. The radical pair mechanism (RPM) is a fundamental quantum process that can explain some of the observed biological magnetic effects. In magnetically sensitive radical pair (RP) reactions, coherent spin dynamics between singlet and triplet pairs are modulated by weak magnetic fields. The resulting singlet and triplet reaction products lead to distinct biological signaling channels and cellular outcomes. A prevalent RP in biology is between flavin semiquinone and superoxide (O2 •-) in the biological activation of molecular oxygen. This RP can result in a partitioning of reactive oxygen species (ROS) products to form either O2 •- or hydrogen peroxide (H2O2). Here, we examine magnetic sensing of recombinant human electron transfer flavoenzyme (ETF) reoxidation by selectively measuring O2 •- and H2O2 product distributions. ROS partitioning was observed between two static magnetic fields at 20nT and 50μT, with a 13% decrease in H2O2 singlet products and a 10% increase in O2 •- triplet products relative to 50µT. RPM product yields were calculated for a realistic flavin/superoxide RP across the range of static magnetic fields, in agreement with experimental results. For a triplet born RP, the RPM also predicts about three times more O2 •- than H2O2, with experimental results exhibiting about four time more O2 •- produced by ETF. The method presented here illustrates the potential of a novel magnetic flavoprotein biological sensor that is directly linked to mitochondria bioenergetics and can be used as a target to study cell physiology.

A general method for the development of multicolor biosensors with large dynamic ranges

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.

Efficient calculation of fluid-induced wall shear stress within tissue engineering scaffolds by an empirical model

Mechanical stimulation, such as fluid-induced wall shear stress (WSS), is known that can influence the cellular behaviours. Therefore, in some tissue engineering experiments in vitro, mechanical stimulation is applied via bioreactors to the cells in cell culturing to study cell physiology and pathology. In 3D cell culturing, porous scaffolds are used for housing the cells. It is known that the scaffold porous geometries can influence the scaffold permeability and internal WSS in a bioreactor (such as perfusion bioreactor). To calculate the WSS generated on cells within scaffolds, usually computational fluid dynamics (CFD) simulation is needed. However, the limitations of the computational method for WSS calculation are: (i) the high time cost of the CFD simulation (in particular for the highly irregular geometries); (ii) accessibility to the CFD model for some cell culturing experimentalists due to the knowledge gap. To address these limitations, this study aims to develop an empirical model for calculating the WSS based on scaffold permeability. This model can allow the tissue engineers to efficiently calculate the WSS generated within the scaffold and/or determine the bioreactor loading without performing the computational simulations.

Messenger RNA in lipid nanoparticles rescues HEK 293 cells from lipid-induced mitochondrial dysfunction as studied by real time pulse chase NMR, RTPC-NMR, spectroscopy

Analytical tools to study cell physiology are critical for optimizing drug-host interactions. Real time pulse chase NMR spectroscopy, RTPC-NMR, was introduced to monitor the kinetics of metabolite production in HEK 293T cells treated with COVID-19 vaccine-like lipid nanoparticles, LNPs, with and without mRNA. Kinetic flux parameters were resolved for the incorporation of isotopic label into metabolites and clearance of labeled metabolites from the cells. Changes in the characteristic times for alanine production implicated mitochondrial dysfunction as a consequence of treating the cells with lipid nanoparticles, LNPs. Mitochondrial dysfunction was largely abated by inclusion of mRNA in the LNPs, the presence of which increased the size and uniformity of the LNPs. The methodology is applicable to all cultured cells.

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Recent Advances in Single-Cell Metabolomics Based on Mass Spectrometry

Engineering a dynamic three-dimensional cell culturing microenvironment using a ‘sandwich’ structure-liked microfluidic device with 3D printing scaffold

Most of in vivo tissue cells reside in 3D extracellular matrix (ECM) with fluid flow. To better study cell physiology and pathophysiology, there has been an increasing need in the development of methods for culturing cells in in vivo like microenvironments with a number of strategies currently being investigated including hydrogels, spheroids, tissue scaffolds and very promising microfluidic systems. In this paper, a ‘sandwich’ structure-liked microfluidic device integrated with a 3D printing scaffold is proposed for three-dimensional and dynamic cell culture. The device consists of three layers, i.e. upper layer, scaffold layer and bottom layer. The upper layer is used for introducing cells and fixing scaffold, the scaffold layer mimicking ECM is used for providing 3D attachment areas, and the bottom layer mimicking blood vessels is used for supplying dynamic medium for cells. Thermally assisted electrohydrodynamic jet (TAEJ) printing technology and microfabrication technology are combined to fabricate the device. The flow field in the chamber of device is evaluated by numerical simulation and particle tracking technology to investigate the effects of scaffold on fluid microenvironment. The cell culturing processes are presented by the flow behaviors of inks with different colors. The densities and viabilities of HeLa cells are evaluated and compared after 72 h of culturing in the microfluidic devices and 48-well plate. The dose-dependent cell responses to doxorubicin hydrochloride (DOX) are observed after 24 h treatment at different concentrations. These experimental results, including the evaluation of cell proliferation and in vitro cytotoxicity assessment of DOX in the devices and plate, demonstrate that the presented microfluidic device has good biocompatibility and feasibility, which have great potential in providing native microenvironments for in vitro cell studies, tissue engineering and drug screening for tumor therapy.

An Optimized Direct Lysis Gene Expression Microplate Assay and Applications for Disease, Differentiation, and Pharmacological Cell-Based Studies.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

Robust engraftment of fetal nonhuman primate CD34-positive cells in immune-deficient mice.

Nonhuman primates (NHPs) represent one of the most important models for preclinical studies of novel biomedical interventions. In contrast with small animal models, however, widespread utilization of NHPs is restricted by cost, logistics, and availability. Therefore, we sought to develop a translational primatized mouse model, akin to a humanized mouse, to allow for high-throughput in vivo experimentation leveraged to inform large animal immunology-based studies. We found that adult rhesus macaque mobilized blood (AMb) CD34+-enriched hematopoietic stem and progenitor cells (HSPCs) engrafted at low but persistent levels in immune-deficient mice harboring transgenes for human (NHP cross-reactive) GM-CSF and IL3, but did not in mice with wild-type murine cytokines lacking NHP cross-reactivity. To enhance engraftment, fetal liver-derived HSPCs were selected as the infusion product based on an increased CD34hi fraction compared with AMb and bone marrow. Coupled with cotransplantation of rhesus fetal thymic fragments beneath the mouse kidney capsule, fetal liver-derived HSPC infusion in cytokine-transgenic mice yielded robust multilineage lymphohematopoietic engraftment. The emergent immune system recapitulated that of the fetal monkey, with similar relative frequencies of lymphocyte, granulocyte, and monocyte subsets within the thymic, secondary lymphoid, and peripheral compartments. Importantly, while exhibiting a predominantly naïve phenotype, in vitro functional assays demonstrated robust cellular activation in response to nonspecific and allogenic stimuli. This primatized mouse represents a viable and translatable model for the study of hematopoietic stem cell physiology, immune development, and functional immunology in NHPs. Summary Sentence: Engraftment of rhesus macaque hematopoietic tissues in immune-deficient mice yields a robust BLT/NeoThy-type primatized mouse model for studying nonhuman primate hematopoiesis and immune function in vivo.

Optogenetics Illuminates Applications in Microbial Engineering.

Optogenetics has been used in a variety of microbial engineering applications, such as chemical and protein production, studies of cell physiology, and engineered microbe-host interactions. These diverse applications benefit from the precise spatiotemporal control that light affords, as well as its tunability, reversibility, and orthogonality. This combination of unique capabilities has enabled a surge of studies in recent years investigating complex biological systems with completely new approaches. We briefly describe the optogenetic tools that have been developed for microbial engineering, emphasizing the scientific advancements that they have enabled. In particular, we focus on the unique benefits and applications of implementing optogenetic control, from bacterial therapeutics to cybergenetics. Finally, we discuss future research directions, with special attention given to the development of orthogonal multichromatic controls. With an abundance of advantages offered by optogenetics, the future is bright in microbial engineering.

K+ -independent Kir blockade by external Cs+ and Ba2.

Cations such as Cs+ and Ba2+ are known to block K+ currents by entering an open channel and binding to the selectivity filter, where they obstruct the pore and block diffusion of the permeant ion. This obstruction is voltage‐ and K+‐dependent and is relieved by the trans permeant ion flux. The present patch‐clamp study on Xenopus muscle cells shows that, unlike the voltage‐activated K+ (Kv) channels, blockade of the inward rectifier K+ (Kir) channels by external foreign cations results from the combination of pore obstruction with a new and independent mechanism. This new blockade is independent of the K+ concentrations and flux and acts indiscriminately on both the outward and the inward Kir components. External Cs+ and Ba2+ compete for this blockade with free access to common channel sites. These features suggest that the blocking cations do not need to enter the channel for this new mechanism, and should bind to the extracellular side of the channel. When K+ fluxes are flowing outward, the pore obstruction is relieved for both Kir and Kv currents, and the K+‐independent blockade here described is responsible for a selective Kir inhibition, justifying the use of these external cations as tools in cell physiology studies.

Approach for patch‐clamping using an upright microscope with z‐axis movable stage

We describe an approach for studying the physiology of single live cells using the conceptionally novel upright microscope/patch-clamp configuration. Electrophysiology experiments typically require a microscope with the fixed stage position and the motion control of the microscope objective. Here, we demonstrate that a microscope with a z-axis movable stage and a fixed objective can also be efficiently used in combination with the patch-clamp technique. We define a set of underlying principles governing the operation of this microscope/patch-clamp configuration and demonstrate its performance in practice using cultured astrocytes, microglia, and oligodendrocytes. Experimental results show that our custom configuration provides stable recordings, has a high success rate of the whole-cell patch-clamp trials, can be effectively applied to study cellular physiology of glial cells, and provides comparable performance and usability to the commercially available systems. Our system can be easily replicated or adapted to suit the needs of the research groups and can be cost-effective in reducing the investments in purchasing additional equipment. We provide step-by-step instructions on implementing an upright microscope with z-axis movable stage as a routine workhorse for patch-clamping. RESEARCH HIGHLIGHTS: Approach for efficient patch-clamping using an upright microscope with a z-axis movable stage. Routine study of live cell physiology. Custom microscope/patch-clamp configuration that is cost-effective and overcomes equipment limitations.

Polyatomic Liquid Oxygen (PLO®): A new methodology for the production in aqueous solution of reactive oxygen and nitrogen species (RONS) to be applied in medical treatments

Free radicals play a pivotal role in cell physiology as “gaseous messengers/transmitters.” The radical superoxide (O2·−) and H2O2 molecules are called Reactive Oxygen Species (ROS); nitric oxide and peroxynitrite are named Reactive Nitrogen Species (RNS). All these species constitute an integrated cellular signaling system. ROS and RNS act on cell proliferation, differentiation, migration, and apoptosis, thus becoming potential anticancer drugs. Because of their chemical instability and short half-life, they cannot be used directly. In this work, we describe an original methodology to produce an aqueous mixture of reactive oxygen and nitrogen species (RONS) in which the gas transmitter molecules derived from the dioxygen and nitrogen oxide have sufficient chemical stability, suitable for in vitro studies of cell physiology. This technique is based on the generation of an electron beam obtained through an inverse sputtering electron device. The result is a gaseous mixture of allotropes of both oxygen and nitrogen in trace amounts, later dissolved in an aqueous phase. This mixture is defined either with the acronym OPL® (Ossigeno Poliatomico Liquido) or PLO® (Polyatomic Liquid Oxygen) or OPL-RONS®. We report herein the chemical characterization of PLO. The stability of PLO makes it suitable for in vivo studies and medical applications.

Arabidopsis root growth and development under metal exposure presented in an adverse outcome pathway framework

Due to human activities, soils become more and more polluted with metals, which imposes risks for human health and wildlife welfare. As most of the metals end up in the food chain through accumulation in plants, we need to establish science‐based environmental criteria and risk management policies. To meet these necessities, a thorough understanding is required of how these metals accumulate in and affect plants. Many studies have been conducted towards this aim, but strikingly, only a few entries can be found in ecotoxicological databases, especially on Arabidopsis thaliana, which serves as a model species for plant (cell) physiology and genetic studies. As experimental conditions seem to vary considerably throughout literature, extrapolation or comparison of data is rather difficult or should be approached with caution. Furthermore, metal‐polluted soils often contain more than one metal, yet limited studies investigated the impact of metal mixtures on plants. This review aims to compile all data concerning root system architecture under Cu, Cd and Zn stress, in single or multi‐metal exposure in A. thaliana, and link it to metal‐induced responses at different biological levels. Global incorporation into an adverse outcome pathway framework is presented.

Computational Cell Physiology: With Examples in Python by Stephen M. Baylor

Computational Cell Physiology: With Examples in Python introduces key cellular mechanisms that cover a wide range of cell physiology topics, particularly associated with excitable cells such as nerve, muscle, and endocrine cells. The book is written by Dr. Stephen M. Baylor, Professor Emeritus of Physiology at the University of Pennsylvania, whose expertise lies in excitation-contraction coupling in vertebrate skeletal muscle and the role of the calcium ion as an intracellular chemical messenger. What makes this book stand out from the rest is that it uses a relatively easy-to-learn programming language, Python, to describe and computationally simulate the mechanisms in question from a quantitative perspective. By doing so, the author exemplifies how a quantitative approach can be of service to studying the underlying cellular mechanisms. Prospective readers, especially those with some programming skills who want to pursue research in cell biology, will find this book extremely valuable. More importantly, as the author points out, the book is a concrete resource for nurturing one's quantitative thinking habits.The book explains the principles of diffusion, electrophysiology, and mass action and how these components come into play in describing biophysical mechanisms underlying various cellular processes, such as the generation of action potentials and muscle contraction. Chapters 1 and 2 cover the general background of cell physiology. Needless to say, it is not feasible to include all the aspects of the field in just 2 chapters. However, the chapters discuss topics that are pertinent to the contents in later chapters. More background materials are elaborated at the beginnings of later chapters, which helps the reader grasp each chapter's main subject. In some chapters, the author describes experimental techniques used to study the electrical properties of cells, such as the patch-clamp and voltage-clamp techniques.Many readers, especially those with limited exposure to experimental settings, will appreciate these descriptions. The cellular mechanisms elaborated in the later chapters are well supported by figures that show experimental recordings and schematics of the relevant cellular units.Chapters 3 and 4 are entirely dedicated to introducing Python to the readers. Chapter 3 provides simple Python scripts that generate graphs of 1-dimensional and 2-dimensional diffusion profiles. The author clarifies that the purpose of theses scripts was not to intimidate the readers but to illustrate the elegance of Python scripts in executing calculations and generating graphs. Each script is well commented and followed by extensive line-by-line explanations, Program Notes, that inform the readers about the role and components of each line. Likewise, the Python scripts presented in the succeeding chapters have detailed, line-by-line descriptions to facilitate understanding the code and quantitative aspects of the cellular mechanisms. Such documentation is tremendously useful for the reader to explore the code setup and simulate cellular processes in different parameter regimes. Chapter 4 includes a summary of the essential elements of Python, including the types of variables, statements, and syntaxes, along with some shortcuts.Although the chapter provides relevant information about the programming language, to follow the examples and exercises discussed in the later chapters, it may be useful to read a few introductory books on Python to gain some background knowledge.Having a GitHub repository with Binder or Google Colaboratory with all the examples and exercises in the book as Jupyter notebooks would be great supplementary material. To follow the examples, the readers need to install Python and necessary libraries on their working machine, which may not be allowed if they do not have administrator privileges. One of the advantages of having a GitHub repository or Google Colaboratory is that the readers can access and execute the example notebooks interactively without having to install the required software and Python libraries on their local machines. It also provides a solution to dealing with different versions of Python libraries, because the notebooks can be saved with the user-defined working environment and its dependencies. Moreover, the readers can build a user community in which they can share their programming experiences and help each other better understand the content of the book. Putting all the materials together and setting up a web page could be an exciting summer project for an undergraduate student.Computational Cell Physiology: With Examples in Python has a balanced combination of content from biology, cell physiology, mathematics, and programming that synergistically support each other. One of the strengths of this book is that it has substantial documentation on the Python script examples, which provides all the necessary programming information to understand the code, while also explaining how the cellular mechanism in question is incorporated. A suitable audience for this book would be undergraduate and graduate students studying cell physiology who want to develop their programming and quantitative skills.

Expression of a Malassezia Codon Optimized mCherry Fluorescent Protein in a Bicistronic Vector.

The use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labeling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a model Malassezia species, the haploid strain M. furfur CBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies, Malassezia cellular localization, and co-expression of genes required for targeted mutagenesis.

Illuminating Cellular Biochemistry: Fluorogenic Chemogenetic Biosensors for Biological Imaging

Cellular activity is defined by the precise spatiotemporal regulation of various components, such as ions, small molecules, or proteins. Studying cell physiology consequently requires the optical recording of these processes, notably by using fluorescent biosensors. The recent development of various fluorogenic systems greatly expanded the palette of reporters to be included in these sensors design. Fluorogenic reporters consist of a protein or RNA tag that can complex either an endogenous or a synthetic fluorogenic dye (so-called fluorogen). The intrinsic nature of these tags, along with the high tunability of their cognate chromophore provide interesting features such as far-red to near-infrared emission, oxygen independence, or unprecedented color versatility. These engineered photoreceptors, self-labelling proteins, or noncovalent aptamers and protein tags were rapidly identified as promising reporters to observe biological events. This Minireview focuses on the new perspectives they offer to design unique and innovative biosensors, thus pushing the boundaries of cellular imaging.

Facilitating gene editing in potato: a Single-Nucleotide Polymorphism (SNP) map of the Solanum tuberosum L. cv. Desiree genome

Genome editing is a powerful tool for plant functional genomics allowing for multiallelic targeted mutagenesis. The recent development of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) systems for gene editing in plants allows for simple, cost-effective introduction of site-specific double-stranded DNA breaks. The nuclear genomes of a homozygous doubled-monoploid potato clone (DM) and a heterozygous diploid clone (RH) have been sequenced in 2011. However, common potato cultivars display a highly heterozygous autotetraploid genome thus complicating target design for tetra-allelic gene editing. Here, we report on the SNP physical map of the widely used Solanum tuberosum L. cv. Desiree and on the position of the diverse indels providing an essential tool for target design in genome editing approaches. We used this tool for designing a specific gRNA and successfully knocking-out a newly discovered starch synthase gene (SS6) in potato. Resequencing data are publicly available at the Sequence Read Archive of the NCBI (accession number: PRJNA507597) and will represent a valuable resource for functional genomic studies of various metabolic pathways, cell and plant physiology as well as high-throughput reverse genetics in potato.

Kinetic Analysis of the Motility of Giant Virus-Infected Amoebae Using Phase-Contrast Microscopic Images.

Tracking cell motility is a useful tool for the study of cell physiology and microbiology. Although phase-contrast microscopy is commonly used, the existence of optical artifacts called “halo” and “shade-off” have inhibited image analysis of moving cells. Here we show kinetic image analysis of Acanthamoeba motility using a newly developed computer program named “Phase-contrast-based Kinetic Analysis Algorithm for Amoebae (PKA3),” which revealed giant-virus-infected amoebae-specific motilities and aggregation profiles using time-lapse phase-contrast microscopic images. This program quantitatively detected the time-dependent, sequential changes in cellular number, size, shape, and direction and distance of cell motility. This method expands the potential of kinetic analysis of cultured cells using versatile phase-contrast images. Furthermore, this program could be a useful tool for investigating detailed kinetic mechanisms of cell motility, not only in virus-infected amoebae but also in other cells, including cancer cells, immune response cells, and neurons.

Using Pancreas Tissue Slices for the Study of Islet Physiology.

Studies on islet of Langerhans physiology are crucial to understand the role of the endocrine pancreas in diabetes pathogenesis and the development of new therapeutic approaches. However, so far most research addressing islet of Langerhans biology relies on islets obtained via enzymatic isolation from the pancreas, which is known to cause mechanical and chemical stress, thus having a major impact on islet cell physiology. To circumvent the limitations of islet isolation, we have pioneered a platform for the study of islet physiology using the pancreas tissue slice technique. This approach allows to explore the detailed three-dimensional morphology of intact pancreatic tissue at a cellular level and to investigate islet cell function under near-physiological conditions. The described procedure is less damaging and faster than alternative approaches and particularly advantageous for studying infiltrated and structurally damaged islets. Furthermore, pancreas tissue slices have proven valuable for acute studies of endocrine as well as exocrine cell physiology in their conserved natural environment. We here provide a detailed protocol for the preparation of mouse pancreas tissue slices, the assessment of slice viability, and the study of pancreas cell physiology by hormone secretion and immunofluorescence staining.

Transplantation of Islets of Langerhans into the Anterior Chamber of the Eye for Longitudinal In Vivo Imaging.

Noninvasive in vivo imaging techniques are attractive tools to longitudinally study various aspects of islet of Langerhans physiology and pathophysiology. Unfortunately, most imaging modalities currently applicable for clinical use do not allow the comprehensive investigation of islet cell biology due to limitations in resolution and/or sensitivity, while high-resolution imaging technologies like laser scanning microscopy (LSM) lack the penetration depth to assess islets of Langerhans within the pancreas. Significant progress in this area was made by the combination of LSM with the anterior chamber of the mouse eye platform, utilizing the cornea as a natural body window to study cell physiology of transplanted islets of Langerhans. We here describe the transplantation and longitudinal in vivo imaging of islets of Langerhans in the anterior chamber of the mouse eye as a versatile tool to study different features of islet physiology in health and disease.