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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2021

Quantifying Cellular Pluripotency and Pathway Robustness through Forman-Ricci Curvature

In stem cell biology, cellular pluripotency describes the capacity of a given cell to differentiate into multiple cell types. From a statistical physics perspective, entropy provides a statistical measure of randomness and has been demonstrated as a way to quantitate pluripotency when considering biological gene networks. Furthermore, recent theoretical work has established a relationship between Ricci curvature (a geometric measure of "flatness") and entropy (also related to robustness), which one can exploit to link the geometric quantity of curvature to the statistical quantity of entropy. Therefore, this study seeks to explore Ricci curvature in biological gene networks as a descriptor of pluripotency and robustness among gene pathways. Here, we investigate Forman-Ricci curvature, a combinatorial discretization of Ricci curvature, along with network entropy, to explore the relationship of the two quantities as they occur in gene networks. First, we demonstrate our approach on an experiment of stem cell gene expression data. As expected, we find Ricci curvature directly correlates with network entropy, suggesting Ricci curvature could serve as an indicator for cellular pluripotency much like entropy. Second, we measure Forman-Ricci curvature in a dataset of cancer and non-cancer cells from melanoma patients. We again find Ricci curvature is increased in the cancer state, reflecting increased pluripotency or "stemness". Further, we locally examine curvature on the gene level to identify several genes and gene pathways with known relevance to melanoma. In turn, we conclude Forman-Ricci curvature provides valuable biological information related to pluripotency and pathway functionality. In particular, the advantages of this geometric approach are promising for extension to higher-order topological structures in order to represent more complex features of biological systems.

systems biology↗

bioRxiv2021

Computational simulations reveal that Abl activity controls cohesiveness of actin networks in growth cones

Extensive studies of growing axons have revealed many individual components and protein interactions that guide neuronal morphogenesis. Despite this, however, we lack any clear picture of the emergent mechanism by which this nanometer-scale biochemistry generates the multi-micron scale morphology and cell biology of axon growth and guidance in vivo. To address this, we studied the downstream effects of the Abl signaling pathway using a computer simulation software (MEDYAN) that accounts for mechanochemical dynamics of active polymers. Previous studies implicate two Abl effectors, Arp2/3 and Enabled, in Abl-dependent axon guidance decisions. We now find that Abl alters actin architecture primarily by activating Arp2/3, while Enabled plays a more limited role. Our simulations show that simulations mimicking modest levels of Abl activity bear striking similarity to actin profiles obtained experimentally from live-imaging of actin in wild type axons in vivo. Using a graph-theoretical filament-filament contact analysis, moreover, we find that networks mimicking hyperactivity of Abl (enhanced Arp2/3) are fragmented into smaller domains of actin that interact weakly with each other, consistent with the pattern of actin fragmentation observed upon Abl overexpression in vivo. Two perturbative simulations further confirm that high Arp2/3 actin networks are mechanically disconnected and fail to mount a cohesive response to perturbation. Taken together, these data provide a molecular-level picture of how the large-scale organization of the axonal cytoskeleton arises from the biophysics of actin networks. Highlight summaryHow do single-molecule dynamics produce multi-micron scale changes in actin organization in an extending axon? Comparison of computational simulations to in vivo data suggests that Abl kinase and Arp2/3 expand actomyosin networks by fragmenting into multiple domains, thus toggling the axon between states of local vs global internal connectivity.

cell biology↗

bioRxiv2023

A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.

cell biology↗

bioRxiv2025

Identification of a VPS29 isoform with restricted association to Retriever and Retromer accessory proteins through auto-inhibition.

The endosomal-lysosomal network is a hub of organelles that orchestrate the dynamic sorting of hundreds of integral membrane proteins to maintain cellular homeostasis. VPS29 is a central conductor of this network through its assembly into Retromer, Retriever and Commander endosomal sorting complexes, and its role in regulating RAB GTPase activity. Two VPS29 isoforms have been described, VPS29A and VPS29B, that differ solely in their amino-terminal sequences. Here we identify a third VPS29 isoform, which we term VPS29C, that harbours an extended amino-terminal sequence compared to VPS29A and VPS29B. Through a combination of AlphaFold predictive modelling, in vitro complex reconstitution, mass spectrometry and molecular cell biology, we find that the amino-terminal VPS29C extension constitutes an autoinhibitory sequence that limits access to a hydrophobic groove necessary for effector protein recruitment to Retromer, and association with Retriever and Commander. VPS29C is therefore unique in its ability to uncouple Retromer-dependent cargo sorting from the broader roles of VPS29A and VPS29B in regulating the endosomal-lysosomal network through accessory protein recruitment. Our identification and characterisation of VPS29C points to additional complexity in the differential subunit assembly of Retromer, an important consideration given the increasing interest in Retromer as a potential therapeutic target in neurodegenerative diseases. SIGNIFICANCE STATEMENTThe endosomal-lysosomal network is essential for normal cellular function with network defects being associated with numerous neurodegenerative diseases. Two heterotrimeric complexes, Retromer and Retriever, control transmembrane protein recycling through the network. Of these, reduced Retromer expression is observed in Alzheimers disease and Retromer mutations lead to familial Parkinsons disease. Here, we identify and characterise a new isoform of VPS29, a subunit shared between Retromer and Retriever. We reveal how this isoform, VPS29C, adopts an auto-inhibitory conformation to limit its association into Retriever and restrict the binding of VPS29C-containing Retromer to accessory proteins vital for regulating network function. By revealing added complexity in Retromer assembly and function, we provide new insight into Retromers potential as a therapeutic target in neurodegenerative diseases.

cell biology↗

bioRxiv2020

Adaptability and evolution of the cell polarization machinery in budding yeast

How can a self-organized cellular function evolve, adapt to perturbations, and acquire new sub-functions? To make progress in answering these basic questions of evolutionary cell biology, we analyze, as a concrete example, the cell polarity machinery of Saccharomyces cerevisiae. This cellular module exhibits an intriguing resilience: it remains operational under genetic perturbations and recovers quickly and reproducibly from the deletion of one of its key components. Using a combination of modeling, conceptual theory, and experiments, we show that multiple, redundant self-organization mechanisms coexist within the protein network underlying cell polarization and are responsible for the modules resilience and adaptability. Based on our mechanistic understanding of polarity establishment, we hypothesize how scaffold proteins, by introducing new connections in the existing network, can increase the redundancy of mechanisms and thus increase the evolvability of other network components. Moreover, our work suggests how a complex, redundant cellular module could have evolved from a more rudimental ancestral form.

evolutionary biology↗

bioRxiv2024

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets.

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and a huge variety of lipid species, Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of the different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. Harnessing a new form of SMA linked to fluorescein, which we call FSMA, we demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially the result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

bioengineering↗

bioRxiv2023

Downregulation of lysosomal trafficking in ARPE19 cells leads to decreased transfection efficiency at high passage

PURPOSEARPE19 cells are a commonly used cell culture model for the study of retinal pigment epithelial cell biology and pathologies. However, numerous studies have demonstrated that ARPE19 undergo morphologic, transcriptomic and genomic alterations over time and with increasing passage number. Herein, we explore the mechanisms underlying increased resistance to the delivery of exogenous genetic material via transfection in ARPE19 cells using mass spectrometry. METHODSARPE19 cells (N=5 wells/reagent) were seeded in 6-well plates at passages 24 through 30. At 70% confluency an mCherry reporter construct was delivered via transfection using Lipofectamine 3000, Lipofectamine LTX, Lipofectamine Stem, or PEI (polyethylenimine) reagents. After 72 hours, transfection efficiency was quantified by fluorescence microscopy and flow cytometry. Mass spectrometry and immunofluorescence of ARPE19 cells were performed at passages 24 and 30 to evaluate altered protein synthesis and localization between passage numbers. RESULTSARPE19 transfection showed a maximum transfection efficiency of 32.4% at P26 using Lipofectamine 3000 reagent. All lipofectamine based reagents demonstrated statistically significant decreases in transfection efficiency between passages 24 and 30. Mass spectrometry analysis revealed 18 differentially expressed proteins, including down-regulation of clathrin light chain B (CLTB) and legumain (LGMN) that was confirmed via immunofluorescence imaging, which indicated altered intracellular localization. CONCLUSIONSARPE19 cells demonstrate passage number dependent changes in lipofectamine-based transfection efficiency. Mass spectrometry and immunofluorescence indicates the observed decrease in transfection efficiency involves the dysregulation of endocytosis and intracellular endolysosomal trafficking at later passages. TRANSLATIONAL RELEVANCEThis study contributes to mounting evidence for changes in ARPE19 cell physiology with increasing passage number. This information is of value for the continued use of ARPE19 cells as a model system for RPE biology and the development of therapeutics.

cell biology↗

bioRxiv2019

Loss of function variants in PCYT1A causing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation.

AbstractSpondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD) is a rare autosomal recessive disorder of the skeleton and the retina caused by biallelic variants in PCYT1A, encoding the nuclear enzyme CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes the rate-limiting step in phosphatidylcholine (PC) biosynthesis by the Kennedy pathway. As a first step in understanding the consequences of PCYT1A variants on SMD-CRD pathophysiology, we generated and characterized a series of cellular models for SMD-CRD, including CRISPR-edited PCYT1A-null HEK293 and ATDC5 cell lines. Immunoblot and PC synthesis assays of cultured skin fibroblasts from SMD-CRD patient cell lines revealed patient genotype-specific reductions in CCT steady state levels (10-75% of wild-type) and choline incorporation into PC (22-54% of wild-type). While PCYT1A-null HEK293 cells exhibited fewer and larger lipid droplets in response to oleate loading than their wild-type counterparts, SMD-CRD patient fibroblasts (p.Ser323Argfs*38 homozygotes) failed to show significant differences in lipid droplet numbers or sizes as compared to controls. Lipid droplet phenotypes in PCYT1A-null HEK293 cells were rescued by transfection with wild-type, p.Ala99Val, and p.Tyr240His human PCYT1A cDNAs. While both edited cellular models had normal morphology and proliferation rates compared to unedited controls, Pcyt1a-null ATDC5 cells demonstrated accelerated rates of chondrocyte differentiation as compared to their wild-type counterparts. Lipidomics revealed changes in 75-200 lipid levels in PCYT1A-null HEK293 and ATDC5 cells or in SMD-CRD patient fibroblasts as compared to wild-type controls. The specific lipids altered and extent of change varied by cell type. Importantly, both PCYT1A-null HEK293 cells and SMD-CRD patient fibroblast cell lines had decreased phosphatidylcholine:phosphatidylethanolamine (PC:PE) ratios and decreased levels of several lysophosphatidylcholine (LPC) species as compared to wild-type controls, suggesting compensatory PC production through increased LPC remodeling by LPCAT or decreased conversion of PC to LPC by phospholipase A2. Our results show that all tested PCYT1A alleles associated with SMD-CRD are hypomorphic and suggest involvement of PCYT1A in chondrocyte differentiation, PC:PE ratio maintenance and LPC metabolism, and lipid droplet formation. Author SummaryRare genetic disorders can reveal the function of genes on an organismal scale. When normal gene activity is lost, patients can experience a range of symptoms, often dependent on the residual activity of the encoded protein. Rare variants in the gene PCYT1A can cause multiple inherited disorders, including a disorder of the skeleton and the retina characterized by short stature, bone abnormalities, and blindness. PCYT1A is required for normal cellular function, particularly lipid metabolism, but the role of this gene in human disease is still poorly understood. To determine consequences of genetic variants in patients with this disorder, we made and studied a series of cellular models, including cells cultured from patients and CRISPR-edited cell lines lacking normal copies of PCYT1A. Here we show that patient variants lead to reduced PCYT1A expression and/or function and have adverse consequences on cell biology and lipid metabolism that are often cell-type specific. This work advances understanding of the role of lipid metabolism in skeletal and eye development.

genetics↗

bioRxiv2020

Stem cell lineage survival as a noisy competition for niche access

Understanding to what extent stem cell potential is a cell-intrinsic property, or an emergent behavior coming from global tissue dynamics and geometry, is a key outstanding question of stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially-localized niche, giving rise to a "stochastic conveyor-belt" model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favourably repositioned. Importantly, even when assuming that all cells in a tissue molecularly equivalent, the model predicts a common ("universal") functional dependence of the long-term clonal survival probability on the position within the niche, as well as the emergence of a well-defined number of "functional" stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets on pubertal mammary gland tips, embryonic kidney tips as well homeostatic intestinal crypt, and find good quantitative agreement for the number of functional stem cells in each organ, as well as the predicted functional dependence of the competition.

biophysics↗

bioRxiv2020

ExBoX: a simple Boolean exclusion strategy to drive expression in neurons

The advent of modern single-cell biology has revealed the striking molecular diversity of cell populations once thought to be more homogeneous. This newly appreciated complexity has made intersectional genetic approaches essential to understanding and probing cellular heterogeneity at the functional level. Here we build on previous knowledge to develop a simple AAV-based approach to define specific subpopulations of cells by Boolean exclusion logic (AND NOT). This Expression by Boolean Exclusion (ExBoX) system encodes for a gene of interest which is turned On by a particular recombinase (Cre or FlpO) and turned Off by another. ExBoX allows for the specific transcription of a gene of interest in cells expressing only the activating recombinase, but not in cells expressing both. We show the ability of the ExBoX system to tightly regulate expression of fluorescent reporters in vitro and in vivo, and further demonstrate the adaptability of the system by achieving expression of a variety of virally-delivered coding sequences in the mouse brain. This simple strategy will expand the molecular toolkit available for cell- and time-specific gene expression in a variety of systems. Summary statementUbina et al. describe the generation of a novel AAV-based intersectional approach to define and target specific subpopulations of cells in time and space via Expression by Boolean Exclusion (ExBoX).

neuroscience↗

bioRxiv2021

A novel transposable element based authentication protocol for Drosophila cell lines

Drosophila cell lines are used by researchers to investigate various cell biological phenomena. It is crucial to exercise good cell culture practice. Poor handling can lead to both inter- and intraspecies cross-contamination. Prolonged culturing can lead to introduction of large- and small-scale genomic changes. These factors, therefore, make it imperative that methods to authenticate Drosophila cell lines are developed to ensure reproducibility. Mammalian cell line authentication is reliant on short tandem repeat (STR) profiling, however the relatively low STR mutation rate in D. melanogaster at the individual level is likely to preclude the value of this technique. In contrast, transposable elements (TE) are highly polymorphic among individual flies and abundant in Drosophila cell lines. Therefore, we investigated the utility of TE insertions as markers to discriminate Drosophila cell lines derived from the same or different donor genotypes, divergent sub-lines of the same cell line, and from other insect cell lines. We developed a PCR-based next-generation sequencing protocol to cluster cell lines based on the genome-wide distribution of a limited number of diagnostic TE families. We determined the distribution of five TE families in S2R+, S2-DRSC, S2-DGRC, Kc167, ML-DmBG3-c2, mbn2, CME W1 Cl.8+, and OSS Drosophila cell lines. Two independent downstream analyses of the NGS data yielded similar clustering of these cell lines. Double-blind testing of the protocol reliably identified various Drosophila cell lines. In addition, our data indicate minimal changes with respect to the genome-wide distribution of these five TE families when cells are passaged for at least 50 times. The protocol developed can accurately identify and distinguish the numerous Drosophila cell lines available to the research community, thereby aiding reproducible Drosophila cell culture research.

genetics↗

bioRxiv2023

Assembly of tight junction belts by surface condensation and actin elongation

Formation of biomolecular condensates via phase separation enables compartmentation of many cellular processes. However, how cells can control condensation at specific locations to create complex cellular structures remains poorly understood. Here, we investigated the mechanism of tight junction formation, which involves condensation of scaffold proteins at cell-cell contacts and elongation of the condensates into a belt around the cellular perimeter. Using cell biology, reconstitution, and thermodynamic theory, we discovered that cells use surface phase transitions to control local condensation at the membrane far below bulk saturation. Surface condensation of junctional ZO-scaffold proteins is mediated by receptor binding and regulated by the receptors oligomerization state. Functionally, ZO surface condensation is directly coupled to actin polymerization and bundling, which drives elongation of receptor-ZO-actin condensates similar to tight junction belt formation in cells. We conclude that surface phase transitions provide a robust mechanism to control the position and shape of protein condensates. One-Sentence SummaryLocal surface binding of cytosolic scaffold proteins provides spatial control of protein condensation to assemble adhesion junctions.

biophysics↗

bioRxiv2023

Peds1 deficiency in zebrafish results in myeloid cell apoptosis and exacerbated inflammation

Plasmalogens are glycerophospholipids with a vinyl ether bond that confers unique properties. Recent identification of the gene encoding PEDS1, the desaturase generating the vinyl ether bond, enables evaluation of the role of plasmalogens in health and disease. Here, we report that Peds1-deficient zebrafish larvae display delayed development, increased basal inflammation, normal hematopoietic stem and progenitor cell emergence, and cell-autonomous myeloid cell apoptosis. In a sterile acute inflammation model, Peds1-deficient larvae exhibited impaired inflammation resolution and tissue regeneration, increased interleukin-1{beta} and NF-{kappa}B activities, and elevated ROS levels at the wound site. Abnormal immune cell recruitment, neutrophil persistence, and fewer but predominantly pro-inflammatory macrophages was observed. Chronic skin inflammation worsened in Peds1-deficient larvae but was mitigated by exogenous plasmalogen, which also alleviated hyper-susceptibility to bacterial infection, as did pharmacological inhibition of caspase-3 and colony-stimulating factor 3-induced myelopoiesis. Overall, our results highlight an important role for plasmalogens in myeloid cell biology and inflammation. KeypointsO_LIPlasmalogens are crucial for cell autonomous survival, recruitment and activation of neutrophils and macrophages. C_LIO_LIPlasmalogen production aids inflammation resolution, while supplementation reduces inflammation and boosts bacterial clearance. C_LI

immunology↗

bioRxiv2024

RNA regulates cohesiveness and porosity of a biological condensate

Biological condensates have emerged as key elements of a biological cell function, concentrating disparate biomolecules to accomplish specific biological tasks. RNA was identified as a key ingredient of such condensates, however, its effect on the physical properties of the condensate was found to depend on the condensates composition while its effect on the microstructure has remained elusive. Here, we characterize the physical properties and the microstructure of a protein-RNA condensate by means of large-scale coarse-grained (CG) molecular dynamics simulations. By developing a custom CG model of RNA compatible with a popular CG model of proteins, we systematically investigate the structural, thermodynamic, and kinetic properties of condensate droplets containing thousands of individual protein and RNA molecules over a range of temperatures. While we find RNA to increase the condensates cohesiveness, its effect on the condensates fluidity is more nuanced with longer molecules compacting the condensate and making it less fluid. We show that a biological condensate has a sponge-like morphology of interconnected channels of size that increases with temperature and decreases in the presence of RNA. Our results suggest that longer RNA form a dynamic scaffold within a condensate, regulating not only its fluidity but also permeability to intruder molecules.

biophysics↗

bioRxiv2024

Generic rules of lumen nucleation and fusion in epithelial organoids

Many internal organs in the body harbor a fluid-filled lumen. The mechanisms of lumens initiation and fusion have been reported as dependent on organ-type during organogenesis. In contrast, the physics of lumen suggests that force balance between luminal pressure and cell mechanics could lead to conserved rules which may unify their self-organisation. However, this hypothesis lacks experimental evidence. Here we compare lumen dynamics for three different systems (MDCK cysts, pancreatic spheres, and epiblast cysts) by using quantitative cell biology, microfabrication and theory. We report that initial cell number determines the maximum number of lumens but does not impact the steady state which is a final single lumen. In addition, lumen numbers exhibit two phases over time, a nucleation phase followed by a fusion phase. In the nucleation phase, lumens form between two cells in pancreatic and MDCK cysts whereas they form at the rosette stage between ten cells in epiblasts. In the second phase, lumens fuse by an increase in lumen volume for pancreatic spheres and MDCK cysts, whereas cell convergent directional motion leads to lumens fusion in epiblasts. We show that these phenomena are associated to the luminal hydrostatic pressure. We support these results with theoretical arguments and numerical simulations. We finally use MDCK cysts to manipulate cell adhesion and lumen volume and we successfully reproduce the fusion dynamics of pancreatic spheres and epiblasts. Our results reveal self-organisation rules of lumens across systems with relevance for morphogenesis during development and for the design of synthetic organs.

biophysics↗

bioRxiv2025

High-resolution digital dissociation of brain tumors with deep multimodal autoencoder

In single cell biology, the complexity of tissues may hinder lineage cell mapping or tumor microenvi-ronment decomposition, requiring digital dissociation of bulk tissues. Many deconvolution methods focus on transcriptomic assay, not easily applicable to other omics due to ambiguous cell markers and reference-to-target difference. Here, we present MODE, a multimodal autoencoder pipeline linking multi-dimensional features to jointly predict personalized multi-omic profiles and cellular compositions, using pseudo-bulk data constructed by internal non-transcriptomic reference and external scRNA-seq data. MODE was evaluated through rigorous simulation experiments and real multiomic data from multiple tissue types, outperforming nine deconvolution pipelines with superior generalizability and fidelity.

bioinformatics↗

bioRxiv2025

Defining effective strategies to integrate multi-sample single-nucleus ATAC-seq datasets via a multimodal-guided approach

BackgroundChromatin accessibility, measured via single-nucleus Assay for Transposase-Accessible Chromatin with sequencing (snATAC-seq), can reveal the underpinnings of transcriptional regulation across heterogeneous cell states. As the number and scale of snATAC-seq datasets increases, we need robust computational pipelines to integrate samples within a dataset and datasets across studies. These integration pipelines should correct cell-state-obfuscating technical effects while conserving underlying biological cell states, as has been shown for single-cell RNA-seq (scRNA-seq) pipelines. However, scRNA-seq integration methods have performed inconsistently on snATAC-seq datasets, potentially due to sparsity and genomic feature differences. ResultsUsing single-nucleus multimodal datasets profiling ATAC and RNA simultaneously, we can measure snATAC-seq integration method performance by comparison to independently integrated snRNA-seq gold standard embeddings and annotations. Here, we benchmark 58 pipelines, incorporating 7 integration methods plus 1 embedding correction method with 5 feature sets. Using our command-line tool, we assessed 5 multimodal datasets at 3 different resolutions using 2 novel metrics to determine the best practices for multi-sample snATAC-seq integration. ATAC features outperformed Gene Activity Score (GAS) features, and embedding correction with Harmony was generally useful. SnapATAC2, PeakVI, and ArchRs iterative Latent Semantic Indexing (LSI) performed well. ConclusionsWe recommend SnapATAC2 + Harmony with pre-defined ENCODE candidate cis-regulatory element (cCRE) features as a first-pass pipeline given its metric performance, generalizability of features, and method resource-efficiency. This and other high-performing pipelines will guide future comprehensive gene regulation maps.

genomics↗

bioRxiv2025

Simultaneous detection and quantification of adenine nucleotides in mammalian cultured cells by HPLC

Adenine nucleotides, including ATP, ADP, ADP-ribose, AMP, NAD+ and NADH, play central roles in cellular homeostasis and involved in multiple metabolic and signaling pathways. Owing to their broad functional relevance in cell biology, the accurate quantification of these metabolites is essential for diverse research areas such as bioenergetics, cell signaling and cancer biology. Several analytical methods have been described for the measurement of adenine nucleotides, ranging from enzymatic assays to mass spectrometry-based approaches. In this study, we developed a reverse-phase high-performance liquid chromatography (RP-HPLC) method with UV-Vis detection that enables the simultaneous quantification of ATP, ADP, ADP-ribose, AMP, NAD+ and NADH. This method is simple, sensitive within the physiological concentration range of all analytes, and capable of detecting biologically relevant changes in ATP, AMP and NAD+ levels induced by pharmacological treatments. Therefore, it presents an accessible and reliable alternative for quantification of these nucleotides in biological samples.

biochemistry↗