SEARCH · bioRxiv Science

Results for “cell biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,459 records · Page 81Linked to original sources

bioRxiv2024

Connecting Transcriptomics with Computational Modeling to Reveal Developmental Adaptations in the Human Pediatric Myocardium

BackgroundNearly 1% or 1.3 million babies are born with congenital heart disease (CHD) globally each year - many of whom will require palliative or corrective heart surgery within the first few years of life. A detailed understanding of cardiac maturation can help to expand our knowledge on cardiac diseases that develop during gestation, identify age-appropriate cardiovascular drug therapies, and inform clinical care decisions related to surgical repair, myocardial preservation, or postoperative management. Yet, to date, our knowledge of the temporal changes that cardiomyocytes undergo during postnatal development is largely limited to animal models. MethodsRight atrial tissue samples were collected from n=117 neonatal, infant, and pediatric patients undergoing correct surgery due to (acyanotic) CHD. Patients were stratified into five age groups: neonate (0-30 days), infant (31-364 days), toddler to preschool (1-5 years), school age (6-11 years), and adolescent to young adults (12-32 years). We measured age-dependent adaptations in cardiac gene expression, and used computational modeling to simulate action potential and calcium transients. ResultsEnrichment of differentially expressed genes (DEG) was explored, revealing age-dependent changes in several key biological processes (cell cycle, cell division, mitosis), cardiac ion channels, and calcium handling genes. Gene-associated changes in ionic currents exhibited both linear trends and sudden shifts across developmental stages, with changes in calcium handling (INCX) and repolarization (IK1) most strongly associated with an age-dependent decrease in the action potential plateau potential and increase in triangulation, respectively. We also note a shift in repolarization reserve, with lower IKr expression in younger patients, a finding likely tied to the increased amplitude of IKs triggered by elevated sympathetic activation in pediatric patients. ConclusionThis study provides valuable insights into age-dependent changes in human cardiac gene expression and electrophysiology among patients with CHD, shedding light on molecular mechanisms underlying cardiac development and function across different developmental stages.

cell biology↗

bioRxiv2024

Plant Accessible Tissue Clearing Solvent System (PATCSOS) for 3-D Imaging of Whole Plants

Tissue clearing is a technique to make the inner structure of opaque tissue visible to achieve 3-dimensional (3-D) tissue imaging by unifying the refractive indexes of most of the cell components. Tissue clearing is widely used in animal tissue imaging, where whole body 3-D imaging has been realized. However, it has not been widely used in plant research. Most plant tissue clearing protocols have their disadvantages, including low efficiency, not being fluorescence-friendly and poor transparency on tissues with a high degree of lignification. In this work, we developed a new plant tissue clearing method for whole plant imaging, named Plant Accessible Tissue Clearing Solvent System (PATCSOS), which was based on the Polyethylene Glycol-associated Solvent System (PEGASOS). The PATCSOS method realized extensive transparency of plant tissues, including the flower, leaf, stem, root, and seed of Arabidopsis thaliana, with high efficiency. The PATCSOS method consists of four main steps: fixation, decolorization/delipidation, dehydration, and clearing. Subsequently a rapid and efficient clearing of mature plant tissue can be achieved. With PATCSOS, we can image Arabidopsis seedling in their entirety in 3-D using endogenous cellulose autofluorescence. Whats more, the PATCSOS method is compatible with fluorescence protein imaging and GUS staining, which greatly expands the applicability of this method. We also imaged intact Nicotiana benthamiana leaf and Zea mays embryos. Our results showed that the PATCSOS clearing method is an excellent tool to study plant development and cell biology.

plant biology↗

bioRxiv2024

Direct single-molecule detection and super-resolution imaging with a low-cost portable smartphone-based microscope

We present a novel, low-cost, portable smartphone-based fluorescence microscope capable of directly detecting single molecules without signal amplification. The setup leverages the image sensors and data handling capacity of mass-produced smartphones, making it adaptable to any smartphone and capable of detecting single molecules across the visible spectral range. We showcase this capability through single-molecule measurements on DNA origami models and super-resolution microscopy of biological cells by single-molecule localization microscopy. This development paves the way for biotechnology innovations making use of massively distributed or personalized assays with single-molecule sensitivity with the potential to revolutionize digital bioassays, point-of-care diagnostics, field expeditions, STEM outreach, and life science education.

biophysics↗

bioRxiv2024

Environment by environment interactions (ExE) differ across genetic backgrounds (ExExG)

While the terms "gene-by-gene interaction" (GxG) and "gene-by-environment interaction" (GxE) are widely recognized in the fields of quantitative and evolutionary genetics, "environment-byenvironment interaction" (ExE) is a term used less often. In this study, we find that environmentby-environment interactions are a meaningful driver of phenotypes, and moreover, that they differ across different genotypes (suggestive of ExExG). To support this conclusion, we analyzed a large dataset of roughly 1,000 mutant yeast strains with varying degrees of resistance to different antifungal drugs. Our findings reveal that the effectiveness of a drug combination, relative to single drugs, often differs across drug resistant mutants. Remarkably, even mutants that differ by only a single nucleotide change can have dramatically different drug x drug (ExE) interactions. We also introduce a new framework that more accurately predicts the direction and magnitude of ExE interactions for some mutants. Understanding how ExE interactions change across genotypes (ExExG) is crucial not only for modeling the evolution of pathogenic microbes, but also for enhancing our knowledge of the underlying cell biology and the sources of phenotypic variance within populations. While the significance of ExExG interactions has been overlooked in evolutionary and population genetics, these fields and others stand to benefit from understanding how these interactions shape the complex behavior of living systems.

evolutionary biology↗

bioRxiv2024

Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

physiology↗

bioRxiv2024

A pluripotent stem cell platform for in vitro systems genetics studies of mouse development

The directed differentiation of pluripotent stem cells (PSCs) from panels of genetically diverse individuals is emerging as a powerful experimental system for characterizing the impact of natural genetic variation on developing cell types and tissues. Here, we establish new PSC lines and experimental approaches for modeling embryonic development in a genetically diverse, outbred mouse stock (Diversity Outbred mice). We show that a range of inbred and outbred PSC lines can be stably maintained in the primed pluripotent state (epiblast stem cells -- EpiSCs) and establish the contribution of genetic variation to phenotypic differences in gene regulation and directed differentiation. Using pooled in vitro fertilization, we generate and characterize a genetic reference panel of Diversity Outbred PSCs (n = 230). Finally, we demonstrate the feasibility of pooled culture of Diversity Outbred EpiSCs as "cell villages", which can facilitate the differentiation of large numbers of EpiSC lines for forward genetic screens. These data can complement and inform similar efforts within the stem cell biology and human genetics communities to model the impact of natural genetic variation on phenotypic variation and disease-risk.

developmental biology↗

bioRxiv2024

A DNA-based molecular clamp for probing protein interactions and structure under force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

bioengineering↗

bioRxiv2024

Giant pore formation in vesicles under msPEF-induced electroporation: role of charging time and waveform

Giant unilamellar vesicle is the closest possible prototypical model for investigating membrane electrodeformation and electroporation in biological cells. In this paper, the effect of membrane charging time on vesicle electroporation, an unresolved issue, is exclusively investigated under milli-second pulsed-electric-field (msPEF) of different waveforms, using numerical simulations. The existing analytical models uncover several fundamental features of cell or vesicle electroporation, but remain far from being realistic as electrode-formation effects were neglected. Our numerical approach, which implements the effect of electric stretching on membrane tension and precise calculation of pore energy, successfully predicts the formation of giant pores of O({micro}m) size as observed in past experiments. The poration zone is found to extend up to certain angles from the poles, termed critical angles. Increase in charging time delays pore formation, decreases the pore density as well as trim downs the poration zone. Counterintuitively, this effect promotes significant pore growth. Thus, more pores evolve into giant pores resulting in higher fractional pore area. Moreover, there exists a cut-off charging time above which pore formation is completely inhibited. This phenomenon is particularly pronounced with bipolar pulses. Comparisons with the past experimental results reveal that electrodeformation-poration-induced membrane surface area variation and that induced by electroporation evolves in a similar fashion. Therefore, although the agreements are qualitative, the present electroporation model can be used as the simplest tool to predict the transient electrodeformation of a porated vesicle in the laboratory experiments.

biophysics↗

bioRxiv2024

DNA walk of specific fused oncogenes exhibit distinct fractal geometric characteristics in nucleotide patterns

Background/ObjectivesThe complex system of cancer has led to an emphasis on understanding the more general causal relationship within the disease. In this context, concepts of symmetry and symmetry-breaking in distinct biological cell features or components have been examined as an approach to cancer investigation. However, there can be possible limitations in directly interpreting the symmetry-based approach from a physical viewpoint due to the lack of understanding of physical laws governing symmetry in complex systems like cancer. MethodsFractal geometry and DNA walk representation were employed to investigate the geometric features i.e., self-similarity and heterogeneity in DNA nucleotide coding sequences of wild-type and mutated oncogenes, tumour-suppressor, and other unclassified genes. The mutation-facilitated self-similar and heterogenous features were quantified by the fractal dimension and lacunarity coefficient measures, respectively. Additionally, the geometrical orderedness and disorderedness in the analyzed sequences were interpreted from the combination of the fractal measures. ResultsThe findings showed distinct fractal geometric features in the case of fusion mutations. It also highlights the possible interpretation of the observed fractal features as geometric analogues concerning explicit observations corresponding to specific cancer types. In addition, the two-dimensional multi-fractal analysis highlighted the presence of a single exponent in the scaling of mutation-mediated gene sequence self-similarity/complexity and heterogeneity. ConclusionsThe approach identified mutation-induced geometric features in gene sequences, demonstrating the potential of DNA walks and fractal analysis in translational research regarding cancer. The findings suggest that investigating fractal parameters can capture unique geometric features in nucleotide sequences, contributing to the understanding of cancers molecular complexity.

bioinformatics↗

bioRxiv2024

Cryo-ET reveals the in situ architecture of the polar tube invasion apparatus from microsporidian parasites

Microsporidia are divergent fungal pathogens that employ a harpoon-like apparatus called the polar tube (PT) to invade host cells. The PT architecture and its association with neighboring organelles remain poorly understood. Here, we use cryo-electron tomography to investigate the structural cell biology of the PT in dormant spores from the human-infecting microsporidian species, Encephalitozoon intestinalis. Segmentation and subtomogram averaging of the PT reveal at least four layers: two protein-based layers surrounded by a membrane, and filled with a dense core. Regularly spaced protein filaments form the structural skeleton of the PT. Combining cryo-electron tomography with cellular modeling, we propose a model for the 3-dimensional organization of the polaroplast, an organelle that is continuous with the membrane layer that envelops the PT. Our results reveal the ultrastructure of the microsporidian invasion apparatus in situ, laying the foundation for understanding infection mechanisms.

microbiology↗

bioRxiv2024

Single influenza A viruses induce nanoscale cellular reprogramming at the virus-cell interface

Viruses, as nanoscale entities with limited proteomes, must efficiently infect target cells using their available resources. During infection, individual virions induce specific cellular signaling within the virus-cell interface, a nanoscale patch of the plasma membrane in contact with the virus. However, virus-induced receptor recruitment and cellular activation are transient processes that occur within minutes and at the nanoscale level. Hence, the temporal and spatial kinetics of such early events often remain poorly understood due to technical limitations. To address this challenge, we developed a novel protocol to covalently immobilize unmodified influenza A viruses on glass surfaces before exposing them to live epithelial cells. This approach extends the observation time for virus-plasma membrane interaction while preserving the viruses native state for uncompromised cell interaction. Using single-molecule super-resolution microscopy, we investigated virus-receptor interaction showing that viral receptors are not immobilized by the virus but rather slowed down, which leads to a specific local receptor accumulation and turnover. We further followed the dynamics of clathrin-mediated endocytosis at the single-virus level and demonstrate the recruitment of adaptor protein 2 (AP-2), previously thought to be uninvolved in influenza A virus infection. Finally, we examined the nanoscale organization of the actin cytoskeleton at the virus-binding site, showing a local and dynamic response of the cellular actin cortex to the infecting virus. Our findings provide novel insights into the fundamental process of virus-cell interaction and demonstrate the versatility and potential impact of our approach on virus-cell biology.

microbiology↗

bioRxiv2024

Integrative spatiotemporal modeling of biomolecular processes: application to the assembly of the Nuclear Pore Complex

Dynamic processes involving biomolecules are essential for the function of the cell. Here, we introduce an integrative method for computing models of these processes based on multiple heterogeneous sources of information, including time-resolved experimental data and physical models of dynamic processes. We first compute integrative structure models at fixed time points and then optimally select and connect these snapshots into a series of trajectories that optimize the likelihood of both the snapshots and transitions between them. The method is demonstrated by application to the assembly process of the human Nuclear Pore Complex in the context of the reforming nuclear envelope during mitotic cell division, based on live-cell correlated electron tomography, bulk fluorescence correlation spectroscopy-calibrated quantitative live imaging, and a structural model of the fully-assembled Nuclear Pore Complex. Modeling of the assembly process improves the model precision over static integrative structure modeling alone. The method is applicable to a wide range of time-dependent systems in cell biology, and is available to the broader scientific community through an implementation in the open source Integrative Modeling Platform software.

biophysics↗

bioRxiv2024

Overcoming fixation and permeabilization challenges in flow cytometry by optical barcoding and multi-pass acquisition

The fixation and permeabilization of cells are essential for labeling intracellular biomarkers in flow cytometry. However, these chemical treatments often alter fragile targets, such as cell surface and fluorescent proteins, and can destroy chemically-sensitive fluorescent labels. This reduces measurement accuracy and introduces compromises into sample workflows, leading to losses in data quality. Here, we demonstrate a novel multi-pass flow cytometry approach to address this long-standing problem. Our technique utilizes individual cell barcoding with laser particles, enabling sequential analysis of the same cells with single-cell resolution maintained. Chemically-fragile protein markers and their fluorochrome conjugates are measured prior to destructive sample processing and adjoined to subsequent measurements of intracellular markers after fixation and permeabilization. We demonstrate the effectiveness of our technique in accurately measuring intracellular fluorescent proteins and methanol-sensitive antigens and fluorophores, along with various surface and intracellular markers. This approach significantly enhances assay flexibility, enabling accurate and comprehensive cell analysis without the constraints of conventional one-time measurement flow cytometry. This innovation paves new avenues in flow cytometry for a wide range of applications in immuno-oncology, stem cell research, and cell biology.

bioengineering↗

bioRxiv2024

The GenPPI tool enhanced Protein Interaction Network Generation with Machine Learning-Based Protein Similarity Inference

AbstractO_ST_ABSBackgroundC_ST_ABSComputational prediction of protein-protein interactions (PPIs) is crucial for understanding cell biology and drug development, offering an alternative to costly experimental methods. The original GenPPi software advanced ab initio PPI network prediction from bacterial genomes but was limited by its reliance on high sequence similarity. This work introduces GenPPi 1.5 to enhance these predictive capabilities. ResultsGenPPi 1.5 incorporates a Random Forest (RF) algorithm, trained on 60 biophysical features from amino acid propensity indices, to classify protein similarity even in low sequence identity scenarios (targeting >65% identity). To manage computational complexity from the increased interactions generated by the RF model, especially in extensive conserved phylogenetic profiles, we developed and integrated the Reduced Interaction Sampling (RIS) algorithm. RIS stochastically samples interactions within these profiles, optimizing performance for complete genome analysis. Extensive simulations across various configurations validated the methodology. RF integration significantly broadened GenPPis predictive power; application to Buchnera aphidicola showed up to 62% overlap with STRING database interactions. Analysis of RIS demonstrated that while introducing some randomness, critical node identification remains robust, particularly for Top N values[≥] 100, indicating minimal compromise to network integrity. ConclusionThe combination of Machine Learning (RF) and the RIS algorithm in GenPPi 1.5 represents a significant advancement. It overcomes the highsimilarity dependency of the previous version while efficiently handling complex genomes. GenPPi 1.5 provides a robust and scalable alignment-free PPI prediction solution, enabling users to train custom models tailored to specific genomic contexts. GenPPi is freely available on our website https://genppi.facom.ufu.br/, its source code is hosted on GitHub https://github.com/santosardr/genppi, and it can be easily installed via the Python Package Index using the command pip install genppipy.

bioinformatics↗

bioRxiv2024

Cell Geometry and Membrane Protein Crowding Constrain Growth Rate, Overflow Metabolism, Respiration, and Maintenance Energy.

The rules of prokaryotic cell design remain elusive. Here, a theory is presented for interpreting growth rate, overflow metabolism, respiration efficiency, and maintenance energy flux based on cell dimensions, membrane protein crowding, and metabolism. The theory employs biophysical properties and systems analysis to successfully interpret phenotypes of Escherichia coli K-12 strains MG1655 and NCM3722. These strains are genetically similar but differ in surface area-to-volume (SA:V) ratios ([~]30%), growth rate on glucose ([~]40%), and overflow-inducing growth rates ([~]80%). Six predictions were tested and validated using experimental phenomics, proteomics, and mutant data. Analyses did not require assumptions regarding cytosolic macromolecular crowding highlighting the distinct properties of the theory. Cell geometry and membrane protein crowding are significant biophysical constraints of cell biology.

systems biology↗

bioRxiv2024

SCRATCH: A programmable, open-hardware, benchtop robot that automatically scratches cultured tissues to investigate cell migration, healing, and tissue sculpting.

Despite the widespread popularity of the scratch assay, where a pipette is dragged through cultured tissue to create an injury gap to study cell migration and healing, the manual nature of the assay carries significant drawbacks. So much of the process depends on individual manual technique, which can complicate quantification, reduce throughput, and limit the versatility and reproducibility of the approach. Here, we present a truly open-source, low-cost, accessible, and robotic scratching platform that addresses all of the core issues. Compatible with nearly all standard cell culture dishes and usable directly in a sterile culture hood, our robot makes highly reproducible scratches in a variety of complex cultured tissues with high throughput. Moreover, we demonstrate how scratching can be programmed to precisely remove areas of tissue to sculpt arbitrary tissue and wound shapes, as well as enable truly complex co-culture experiments. This system significantly improves the usefulness of the conventional scratch assay, and opens up new possibilities in complex tissue engineering and cell biological assays for realistic wound healing and migration research.

bioengineering↗

bioRxiv2024

LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.

biophysics↗

bioRxiv2024

DiatOmicBase, a gene-centered platform to mine functional omics data across diatom genomes

Diatoms are prominent microalgae found in all aquatic environments. Over the last 20 years, thanks to the availability of genomic and genetic resources, diatom species such as Phaeodactylum tricornutum have emerged as valuable experimental model systems for exploring topics ranging from evolution to cell biology, (eco)physiology and biotechnology. Since the first genome sequencing in 2008, numerous genome-enabled datasets have been generated, based on RNA-Seq and proteomics, epigenomes, and ecotype variant analysis. Unfortunately, these resources, generated by various laboratories, are often in disparate formats and challenging to access and analyze. Here we present DiatOmicBase, a genome portal gathering comprehensive omics resources from P. tricornutum and two other diatoms to facilitate the exploration of dispersed public datasets and the design of new experiments based on the prior-art. DiatOmicBase provides gene annotations, transcriptomic profiles and a genome browser with ecotype variants, histone and methylation marks, transposable elements, non-coding RNAs, and read densities from RNA-Seq experiments. We developed a semi-automatically updated transcriptomic module to explore both publicly available RNA-Seq experiments and users private datasets. Using gene-level expression data, users can perform exploratory data analysis, differential expression, pathway analysis, biclustering, and co-expression network analysis. Users can create heatmaps to visualize precomputed comparisons for selected gene subsets. Automatic access to other bioinformatic resources and tools for diatom comparative and functional genomics is also provided. Focusing on the resources currently centralized for P. tricornutum, we showcase several examples of how DiatOmicBase strengthens molecular research on diatoms, making these organisms accessible to a broad research community. Significance statementIn recent years, diatoms have become the subject of increasing interest because of their ecological importance and their biotechnological potential for natural products such as pigments and polyunsaturated fatty acids. Here, we present an interactive web-based server that integrates public diatom omics data (genomics, transcriptomics, epigenomics, proteomics, sequence variants) to connect individual diatom genes to broader-scale functional processes.

genomics↗