Cell Systems Biology Research Articles

High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

EGG: Accuracy Estimation of Individual Multimeric Protein Models Using Deep Energy-Based Models and Graph Neural Networks.

Reliable and accurate methods of estimating the accuracy of predicted protein models are vital to understanding their respective utility. Discerning how the quaternary structure conforms can significantly improve our collective understanding of cell biology, systems biology, disease formation, and disease treatment. Accurately determining the quality of multimeric protein models is still computationally challenging, as the space of possible conformations is significantly larger when proteins form in complex with one another. Here, we present EGG (energy and graph-based architectures) to assess the accuracy of predicted multimeric protein models. We implemented message-passing and transformer layers to infer the overall fold and interface accuracy scores of predicted multimeric protein models. When evaluated with CASP15 targets, our methods achieved promising results against single model predictors: fourth and third place for determining the highest-quality model when estimating overall fold accuracy and overall interface accuracy, respectively, and first place for determining the top three highest quality models when estimating both overall fold accuracy and overall interface accuracy.

Engineering fibronectin-templated multi-component fibrillar extracellular matrices to modulate tissue-specific cell response

Cells assemble fibronectin, the major extracellular matrix (ECM) protein, into fibrillar matrices, which serve as 3D architectural scaffolds to provide, together with other ECM proteins tissue-specific environments. Although recent approaches enable to bioengineer 3D fibrillar fibronectin matrices in vitro, it remains elusive how fibronectin can be co-assembled with other ECM proteins into complex 3D fibrillar matrices that recapitulate tissue-specific compositions and cellular responses. Here, we introduce the engineering of fibrillar fibronectin-templated 3D matrices that can be complemented with other ECM proteins, including vitronectin, collagen, and laminin to resemble ECM architectures observed in vivo. For the co-assembly of different ECM proteins, we employed their innate fibrillogenic mechanisms including shear forces, pH-dependent electrostatic interactions, or specific binding domains. Through recapitulating various tissue-specific ECM compositions and morphologies, the large scale multi-composite 3D fibrillar ECM matrices can guide fibroblast adhesion, 3D fibroblast tissue formation, or tissue morphogenesis of epithelial cells. In other examples, we customize multi-composite 3D fibrillar matrices to support the growth of signal propagating neuronal networks and of human brain organoids. We envision that these 3D fibrillar ECM matrices can be tailored in scale and composition to modulate tissue-specific responses across various biological length scales and systems, and thus to advance manyfold studies of cell biological systems.

An open-source FACS automation system for high-throughput cell biology.

Recent advances in gene editing are enabling the engineering of cells with an unprecedented level of scale. To capitalize on this opportunity, new methods are needed to accelerate the different steps required to manufacture and handle engineered cells. Here, we describe the development of an integrated software and hardware platform to automate Fluorescence-Activated Cell Sorting (FACS), a central step for the selection of cells displaying desired molecular attributes. Sorting large numbers of samples is laborious, and, to date, no automated system exists to sequentially manage FACS samples, likely owing to the need to tailor sorting conditions ("gating") to each individual sample. Our platform is built around a commercial instrument and integrates the handling and transfer of samples to and from the instrument, autonomous control of the instrument's software, and the algorithmic generation of sorting gates, resulting in walkaway functionality. Automation eliminates operator errors, standardizes gating conditions by eliminating operator-to-operator variations, and reduces hands-on labor by 93%. Moreover, our strategy for automating the operation of a commercial instrument control software in the absence of an Application Program Interface (API) exemplifies a universal solution for other instruments that lack an API. Our software and hardware designs are fully open-source and include step-by-step build documentation to contribute to a growing open ecosystem of tools for high-throughput cell biology.

Monitoring the oxidative function of hydroxytyrosol and potential interactions with glutathione produced by human cells

Hydroxytyrosol (HT) has outstanding antioxidant properties, however, the lack of information regarding its potential interactions with biological systems prevents the direct application of HT in the field of medicine. In particular, the interaction of HT with antioxidant defense systems based on tripeptides containing thiols (glutathione, GSH) remains elusive and requires further investigation. This study investigated the electrochemical behavior of HT in Britton-Robinson buffer solution (B-R) with and without GSH using bulk electrolysis (BE) and cyclic voltammetry (CV). The reaction pathways of HT and oxidation products were identified by combining ultrahigh-performance liquid chromatography and mass spectrometry (UHPLC-MS). The findings of the study indicate that the phenolic ring of HT experiences a quasi-reversible oxidation process. This process is associated with the oxidation of the catechol moiety through the transfer of 2e−/2H+ in the presence of existing GSH. As a result of this oxidation, a quinone is produced. The quinone molecule can undergo reduction, leading to the regeneration of the HT molecule. Alternatively, it can undergo further modification by interacting with GSH, resulting in the formation of mono-GSH-HT, bi-GSH-HT, and bi-GSH-HT trimer conjugates. Using density functional theory (DFT), the ratio of disulfide, mono-GSH-HT, bi-GSH-HT, and bi-GSH-HT trimer formation energies (Ef) was determined to be 4.5:1.8:1.1:1.4 which are consistent with UHPLC-MS measurement. Accordingly, the formation of the disulfide form of GSH and parent HT is preferred over the formation of other conjugates. The higher values of electrophilicity index (ω) for the HT trimer and quinone suggest a remarkable ability to interact with cells in biological systems.

π -Extended BTD derivatives: synthesis, photophysical properties, and applications in biological systemsimagingfor discriminating living and dead cells.

Benzothiadiazole (BTD) is one of the popular compounds for organic electronic devices, organic synthesis, and fluorescent probes. Here, benzothiadiazole derivatives were successfully synthesized by heck coupling reaction, which exhibited the the advantages of simplicity and efficiency, wild substrate csope, easily available substrates and high yield. The fluorescent probe BTDAr-PEG targeting lipid droplets was successfully prepared by the nucleophilic substitution reaction between the heck coupling reaction product 3h and Amino polyethylene glycol monomethyl ether (Mn=2000). BTDAr-PEG exhibited the advantages of high selectivity, good stability and pH resistance. The use of PEG as a substrate gave BTDAr-PEG good biocompatibility. It was worth mentioning that BTDAr-PEG could not only track LDs in cells under different physiological conditions, but also distinguish between living and dead cells in biological systems.

Single-cell transcriptomics of pathological pregnancies

Pregnancy is a complicated process involving various anatomical and physiological changes to provide a suitable environment for fetal development, to meet the increased metabolic demands, and to prepare for labor. While most pregnancies and births are uneventful, all pregnancies are at risk of complications. No less than 15% of all pregnant women develop a potentially lifethreatening complication or may also experience an unpleasant outcome. Conventional sequencing has been widely used in this field for diagnoses and treatment planning. However, it lacks the precise resolution to identify transcriptomic variation between the nearby cell types. Single-cell RNA sequencing is a novel approach that through single-cell isolation, transcript capturing, and development and sequencing of expression libraries allows the assessments of fundamental biological properties of cell populations and biological systems at unprecedented resolution. The single-cell sequencing technology is a powerful tool for investigating cell distribution and cell-to-cell relationships in human reproduction. Since 2016, the technique has contributed to the discovery of many transcriptomic atlases of human embryos, placenta, decidua, and endometrium and the identification of various crucial regulatory pathways in fetal development. Here, we briefly describe the application of single-cell RNA sequencing in the studies of physiological processes and pathological mechanisms of pregnancy.

Virtual cell modeling and simulation software.

Virtual Cell (VCell, http://vcell.org) is free open-source software for modeling cell biological systems. Biology-based interface is designed for researchers with no programming/scripting experience: they enter reactions and reaction rules in an intuitive graphical way, and VCell automatically creates the math for you. Models and simulations can be accessed from anywhere using the VCell database; models can be shared among collaborators or made publicly available. Simulations can be run locally (on a user's computer) or using our server - a job can be sent to a server and results can be retrieved any time later. VCell provides a variety of simulation frameworks. The same reaction network can be simulated using deterministic (compartmental ODE) and/or stochastic reactions (SSA solvers) simulators. By adding a geometry for compartments and diffusion for species, the same reaction network can be simulated as a reaction-diffusion-advection PDE with support for 2D kinematics and/or using spatial stochastic (reaction-diffusion with Smoldyn, http://smoldyn.org). Geometries from 2D or 3D microscope images or from idealized analytical expressions can be used, and membrane flux, lateral membrane diffusion and electrophysiology could be incorporated. If elements of the model are defined by reaction rules (using a novel graphical user interface for defining, visualizing, and verifying rule-based models), the model can be simulated using BioNetGen (http://bionetgen.org) engine and/or network-free agent-based simulations. The VCell models can be exchanged with other tools using standard formats such as SBML and BNGL, as well as exported as MatLab, SED-ML, and COMBINE archive. The latest developments include better support of SBML features, the possibility to fully annotate the models, view models online, interact with ImageJ, and send simulations to runBiosimulations (https://run.biosimulations.org) service for online simulations and visualization with modified parameters. (Supported by NIGMS grant R24 GM137787)

Development and Evaluation of a Low‐Cost LEGO 3D Bioprinter: From Building‐Blocks to Building Blocks of Life

AbstractThe development of low‐cost accessible technologies for rapid prototyping of mechanical components has democratised engineering tools for hobbyists and researchers alike. The development of analogous approaches to fabrication of soft‐matter, and biologically compatible materials containing living cells, is anticipated to be similarly enabling across multiple fields of biological research. LEGO toy construction bricks represent low‐cost, precision engineered, and versatile construction materials for rapid prototyping. This study demonstrates construction of a benchtop LEGO 3D bioprinter for additive layer manufacture of a 3D structure containing viable human skin cells within a hydrogel scaffold. 3D bioprinted structures are formed from the deposition of microfluidically generated bio‐ink droplets containing live keratinocyte skin cells, representing components toward an artificial skin model. Fluid flow rates and printer speed, together with bio‐ink gelation rate, determine droplet packing arrangement in the bioprinted structures. The printing of 3D structures containing multiple bio‐inks is demonstrated and live cells are imaged in the resulting bioprints. Fluid delivery can be achieved using LEGO pumps and readily available, or home‐3D‐printed, microfluidic components, therefore avoiding the need for any specialist microfluidic hardware. Build instructions are described to enable easy uptake, modification and improvement by other laboratories, as well provide an accessible platform for learning and education. Affordable, accessible, and easy to use tools for 3D bioprinting are anticipated to open opportunities for a greater number of research labs to work with 3D cell culture and bio‐printed materials, with bioprinting expected to assist in better understanding of disease, contribute to tissue engineering and repair, and enable personalised medicine through the printing of cultured patient cells. The presented approach is not only an easily accessible laboratory tool for bioprinting, but also provides a learning system for mechanical construction, robotics, coding, microfluidics and cell biology, making it a versatile platform for research, education, and science engagement.

Evaluation of Salivary Sialic Acid and Salivary Alpha-L-Fucosidase Levels in Patients with Oral Submucous Fibrosis- A Cross-Sectional Study

Background: Saliva, a non-invasive diagnostic tool, is used to evaluate sialic acid and Alpha-L-Fucosidase in OSMF. Sialylation and fucosylation are glycosylation changes that mediate vital biological functions. By stabilizing glycoproteins and cellular membranes, sialic acid contributes to the identification and interaction of cells in biological systems. Alpha-L-Fucosidase is an enzyme found in lysosomes and responsible for catalyzing the hydrolytic cleavage of terminal fucose residue. Aim: To evaluate and compare salivary Sialic acid and salivary Alpha-L-fucosidase levels in patients with OSMF with age and gender-matched healthy controls and different stages of OSMF. Materials and Methods: The study included 30 Patients with clinically diagnosed OSMF and 30 age and gender-matched healthy controls. Unstimulated whole saliva samples were collected by the spit method. A saliva sample was used to estimate salivary sialic acid and salivary Alpha-L-glucosidase (ELISA) method. Results: Salivary Sialic acid and salivary Alpha-L-fucosidase levels were significantly higher (P < 0.001***) in patients with OSMF compared to healthy controls, and the levels gradually increased with an increase in the stages of the OSMF. Conclusion: Salivary Sialic acid and salivary Alpha-L-fucosidase in patients with OSMF can be a reliable biomarker for early detection and progression and have prognostic value.

Motility induced phase separation of deformable cells.

Using a multi-phase field model, we examine how particle deformability, which is a proxy for cell stiffness, affects motility induced phase separation (MIPS). We show that purely repulsive deformable, i.e., squishy, cells phase separate more effectively than their rigid counterparts. This can be understood as due to the fact that deformability increases the effective duration of collisions. In addition, the dense regions become increasingly disordered as deformability increases. Our results contextualize the applicability of MIPS to biological systems and have implications for how cells in biological systems may self-organize.

Making a chink in their armor: Current and next-generation antimicrobial strategies against the bacterial cell envelope.

Gram-negative bacteria are uniquely equipped to defeat antibiotics. Their outermost layer, the cell envelope, is a natural permeability barrier that contains an array of resistance proteins capable of neutralizing most existing antimicrobials. As a result, its presence creates a major obstacle for the treatment of resistant infections and for the development of new antibiotics. Despite this seemingly impenetrable armor, in-depth understanding of the cell envelope, including structural, functional and systems biology insights, has promoted efforts to target it that can ultimately lead to the generation of new antibacterial therapies. In this article, we broadly overview the biology of the cell envelope and highlight attempts and successes in generating inhibitors that impair its function or biogenesis. We argue that the very structure that has hampered antibiotic discovery for decades has untapped potential for the design of novel next-generation therapeutics against bacterial pathogens.

Bringing Cell Subpopulation Discovery on a Cloud-HPC Using rCASC and StreamFlow.

The idea behind novel single-cell RNA sequencing (scRNA-seq) pipelines is to isolate single cells through microfluidic approaches and generate sequencing libraries in which the transcripts are tagged to track their cell of origin. Modern scRNA-seq platforms are capable of analyzing up to many thousands of cells in each run. Then, combined with massive high-throughput sequencing producing billions of reads, scRNA-seq allows the assessment of fundamental biological properties of cell populations and biological systems at unprecedented resolution.In this chapter, we describe how cell subpopulation discovery algorithms, integrated into rCASC, could be efficiently executed on cloud-HPC infrastructure. To achieve this task, we focus on the StreamFlow framework which provides container-native runtime support for scientific workflows in cloud/HPC environments.

Immune response in retinal degenerative diseases - Time to rethink?

Retinal degeneration comprises a group of diseases whereby either the retinal neurons or the neurovascular unit degenerates leading to the loss of visual function. Although the initial cause varies in different conditions, inflammation is known to play an important role in disease pathogenesis. Recent advances in molecular and cell biology and systems biology have yielded unexpected findings, including the heterogeneity of immune cells in the degenerative retina, bidirectional neuron-microglia cross talk, and links to the gut microbiome. Here we discuss the immune response in retinal degenerative conditions, taking into account both regional (retinal) and systemic factors. We propose to classify retinal degeneration into dry and wet forms based on whether the blood-retinal barrier (BRB) is breached and fluid is accumulated in retinal parenchyma. The dry form has a relatively intact BRB and is characterised by progressive retinal thinning. Immune response to degenerative insults is dominated by the retinal defence system, which remains to be regulated by neurons. In contrast, the wet form has retinal oedema due to BRB damaged. Inflammation is executed by infiltrating immune cells as well as the retinal defence system. The gut microbiome will have easy access to the retina in wet retinal degeneration and may affect significantly retinal immune response.

Role of NS2 specific RNA binding and phosphorylation in liquid-liquid phase separation and virus assembly.

Liquid-liquid phase separation (LLPS) has assumed a prominent role in biological cell systems, where it underpins the formation of subcellular compartments necessary for cell function. We investigated the underlying mechanism of LLPS in virus infected cells, where virus inclusion bodies are formed by an RNA-binding phosphoprotein (NS2) of Bluetongue virus to serve as sites for subviral particle assembly and virus maturation. We show that NS2 undergoes LLPS that is dependent on protein phosphorylation and RNA-binding and that LLPS occurrence is accompanied by a change in protein secondary structure. Site-directed mutagenesis identified two critical arginine residues in NS2 responsible for specific RNA binding and thus for NS2-RNA complex driven LLPS. Reverse genetics identified the same residues as essential for VIB assembly in infected cells and virus viability. Our findings suggest that a specific arginine-RNA interaction in the context of a phosphorylated state drives LLPS in this, and possibly other, virus infections.

All-ukrainian conference on molecular and cell biology with international participation, dedicated to the heroic struggle of the ukrainian people against the russian invaders

The All-Ukrainian Conference on Molecular and Cell Biology with international participation was held as an online event on the Zoom platform, from the 15th to the 17th of June 2022. The purpose of this event was to encourage Ukrainian scientists, to give the opportunity to colleagues from abroad to demonstrate their support to Ukraine, and to keep the scientific process ongoing even in the background of the war. Scientists of every career stage could take part in any way convenient to them. Seven sections of bioscience were available at the Conference, namely: Microbiology and Biotechnology, Genetics and Epigenetics, Molecular biology and Biorganic chemistry, Molecular oncology, Molecular physiology and Biophysics, Cell biology and System biology, and Bioinformatics. 123 scientists from Ukraine and abroad have registered for participation in the conference. 38 oral presentations, 14 poster presentations, 10 Keynote Lectures, and 1 Special Lecture were included in the Conference program. Scientists from all over Ukraine, several European countries Poland, Chech Republic, Germany, Italy, Sweden, Finland), and USA participated in the Conference. On the third day the Panel Discussion on the topic “Viruses, evolution, and the struggle for survival. Human progress in diagnosis, therapy, and prevention”, was performed. The abstract book, based on the Conference materials is published online.

An Insight into Pyrazole-containing Compounds: Synthesis and Pharmacological Activities

Background: Heterocyclic scaffolds have gained exceptional devotion in the recent years due to their marked utility in bio-organic field. Among these, pyrazole remains a privileged scaffold as broad array of medicinally active agents encompasses this heterocycle as a core nucleus. Pyrazole is a five-membered, aromatic ring containing two nitrogen atoms at adjacent positions are readily able to show interactions with numerous receptor (s), enzymes located on the target cells in biological system. Pyrazole-containing compounds are acknowledged with anticyclooxygenases (anti-inflammatory), carbonic anhydrase inhibitor, α-glycosidase inhibitor, and cholinesterase enzymes inhibitor and anti-DNA gyrases activities. Noticeably, rimonabant, phenylbutazone, fipronil, difenamizole, celecoxib, antipyrine, fezolamide and betazole are few representatives of pyrazole containing drugs. Objectives: The manuscript aims to review the detailed synthetic approaches applied for the synthesis of pyrazole derivatives. In particular, we examine recent scientific finding on antimicrobial, anti-tubercular, antiviral, anticancer and anti-inflammatory perspectives of pyrazole containing analogues. Methods: Pyrazole analogues have been widely explored by scientific community as large number of papers has been published in this regard. Numerous pyrazole-containing analogues have been designed, synthesized, and screened for their in vitro and in vivo bio-efficacy and many of them endowed with commendable pharmacological activities. Pyrazole analogues with superior applications as antiviral, anticancer, and anti-inflammatory efficacy have also been well documented in patents granted to this heterocyclic nucleus. Results: This review outlines the recent advances in medicinal chemistry of pyrazole analogues with a special emphasize of structure-activity relationships to afford ideas for the rational drug-design and discovery and their impact on desired pharmacological applications. Conclusion: The information provided in this manuscript may help the medicinal chemists to generate robust pyrazole analogues with high efficacy.

Saccharomyces cerevisiae as a Model System for Eukaryotic Cell Biology, from Cell Cycle Control to DNA Damage Response.

The yeast Saccharomyces cerevisiae has been used for bread making and beer brewing for thousands of years. In addition, its ease of manipulation, well-annotated genome, expansive molecular toolbox, and its strong conservation of basic eukaryotic biology also make it a prime model for eukaryotic cell biology and genetics. In this review, we discuss the characteristics that made yeast such an extensively used model organism and specifically focus on the DNA damage response pathway as a prime example of how research in S. cerevisiae helped elucidate a highly conserved biological process. In addition, we also highlight differences in the DNA damage response of S. cerevisiae and humans and discuss the challenges of using S. cerevisiae as a model system.

Diffusion dynamics in branched spherical structure

Diffusion on a spherical surface with trapping is a common phenomenon in cell biology and porous systems. In this paper, we study the diffusion dynamics in a branched spherical structure and explore the influence of the geometry of the structure on the diffusion process. The process is a spherical movement that occurs only for a fixed radius and is interspersed with a radial motion inward and outward the sphere. Two scenarios govern the transport process in the spherical cavity: free diffusion and diffusion under external velocity. The diffusion dynamics is described by using the concepts of probability density function (PDF) and mean square displacement (MSD) by Fokker–Planck equation in a spherical coordinate system. The effects of dead ends, sphere curvature, and velocity on PDF and MSD are analyzed numerically in detail. We find a transient non-Gaussian distribution and sub-diffusion regime governing the angular dynamics. The results show that the diffusion dynamics strengthens as the curvature of the spherical surface increases and an external force is exerted in the same direction of the motion.

Computational investigation for deformation of lipid membrane by BAR proteins due to electrostatic interaction

Deformation of lipid membrane by peripheral BAR (Bin/Amphiphysin/RVs) proteins due to electrostatic interaction between them has an important implication in field of cell-biological systems. We study the deformation behavior of a lipid membrane sheet subjected to electrostatic traction with the help of a very simple finite element elastic model. The lipid membrane is considered as an elastic sheet subjected to the electrostatic traction derived from the literature for the interaction between the lipid membrane and a single BAR protein. Both the small and large deformation behavior of the lipid membrane is considered. To predict small or infinitesimal deformation of the membrane, we use Hooke’s law and large deformation is predicted by Neo-Hookean strain energy density function. The small deformation behavior obtained from the present finite element model does not support the scaffolding mechanism of a single BAR protein binding with the lipid membrane. However, the large deformation behavior of the membrane predicts very well the scaffolding mechanism for the wide range of BAR proteins involved in cell-biological systems. The present model can be used for understanding deformation of any kind of elastic sheet driven by electrostatic forces.