Biology subjects

Nordenfelt, P.

Publications and source records attributed to Nordenfelt, P..

17 recordsLinked to original sources

bioRxiv2026

NucleiSky enables cross-scale multimodal registration of microscopy data using nuclei constellations

Integrating tissue-level organisation with sub-cellular resolution and molecular information often requires combining multiple microscopy modalities and scales. However, aligning images acquired with different modalities, settings, or instruments remains challenging. Here, we introduce NucleiSky, a microscopy image registration framework that utilises the spatial arrangement of nuclei or other landmarks as an intrinsic biological fingerprint. NucleiSky represents images as constellations of centroids and aligns them using geometric algorithms and spatial consensus scoring. In benchmark datasets, NucleiSky could localise query regions within larger reference images using as few as five nuclei. We show that NucleiSky can locate high-magnification fields of view within low-magnification overview scans, map these alignments to additional channels, support live brightfield-to-fixed registration using synthetic nuclear labels, and guide microscope retargeting. We further show that the same constellation-matching principle can be extended to 3D localisation and to non-nuclear landmarks. These findings establish local landmark geometry as an intrinsic spatial fingerprint that enables localisation and registration across imaging scales, modalities and microscopy platforms. NucleiSky is available as an open-source Python package and as notebook-based applications.

Cell Biology↗

bioRxiv2026

Mechanical memory of confinement pressure governs expansion size in epithelial monolayers

Epithelial tissues undergo rapid expansion during development, repair, and morphogenesis, yet how tissue-scale growth is coordinated to re-establish homeostasis remains unclear. Here, we show that large epithelial monolayers confined at a wide range of initial densities and mechanochemical states robustly converge to the same final size and density upon release, despite differences in initial cell size, YAP activity, and cell number dynamics. To investigate the underlying mechanism, we combined quantitative experiments with a mechanochemical agent-based model in which mechanical pressure arising from confinement acts as a tissue-scale signal that modulates intracellular cell-cycle activity over time. Using this framework, we show that transient mechanical relaxation during confinement selectively elevates cell-cycle activity in higher-density tissues at the time of release, accelerating early expansion without disrupting final homeostatic outcomes. Together, these results reveal how epithelial tissues coordinate collective growth and robustly restore homeostasis during expansion. SignificanceMechanisms that re-establish homeostasis and coordinate cell proliferation in expanding epithelial tissues such as during development and tissue regeneration remain poorly understood. Here, we used a large 2D epithelial expansion model and agent-based modelling to demonstrate history-dependent regulation of colony growth, mediated by pressure sensing. We find that, typically, equilibration of pressure during confinement just before the onset of expansion results in all expanding tissues of a given starting size reaching a target density and final size independent of starting cell numbers. By transiently reducing actomyosin contractility during confinement, we selectively accelerate the expansion of high-density tissues. Our findings provide key insights into physical mechanisms that govern organ development and tissue repair.

cell biology↗

bioRxiv2025

ECM-stiffness mediated persistent fibroblast activation requires integrin and formin dependent chromatin remodeling

Transient activation of fibroblasts into contractile myofibroblasts is essential for extracellular matrix (ECM) production and remodeling during wound healing and tissue regeneration. While ECM-dependent mechanisms mediating transient activation is well studied, how fibroblasts switch from transient to a persistently activated state and drive fibrosis and aberrant tissue repair in diseases such as cancer is less understood. Here, we show that human cancer-associated fibroblasts (CAFs) switch from transient to persistently activated states upon prolonged exposure to stiff ECMs and stiffness-dependent secreted factors. This switch is accompanied by activation of ECM-stiffness-dependent mechanotransduction pathways and changes in the nuclear architecture and its association with chromatin. Mechanistically, we identify two pathways required for this switch-ECM ligand binding and complete activation of {beta}1 integrins smoothens the nuclear lamina during prolonged exposure, increases nuclear YAP, and reduces lamin-chromatin contacts while in parallel, exposure to the stiff ECM activates the formin mDia2 and independent of alterations in the nuclear architecture alters lamin-chromatin coupling, likely through its role in assembling nuclear actin. Importantly, we find that blocking either pathway prevents persistent myofibroblast activation, which is rescued by inhibition of histone deacetylases, indicating that dynamic chromatin modifications act downstream of these ECM-dependent pathways to maintain the persistently activated state. These findings link integrin-based ECM sensing to chromatin remodeling and fibroblast memory, with implications for stromal plasticity in the tumor microenvironment.

cell biology↗

bioRxiv2025

Smart Microscopy: Current Implementations and a Roadmap for Interoperability

Smart microscopy is transforming life sciences by automating experimental imaging workflows and enabling real-time adaptation based on feedback from images and other data streams. This shift increases throughput, improves reproducibility, and expands the functional capabilities of microscopes. However, the current landscape is highly fragmented. Academic researchers often develop custom solutions for specific scientific needs, while industry offerings are typically proprietary and tied to specific hardware. This diversity, while fostering innovation, also creates major challenges in interoperability, reproducibility, and standardization, which slows progress and adaption. This article presents a collaborative effort between academic and industry leaders to survey the current state of smart microscopy, highlight representative implementations, and identify common technical and organizational barriers. We propose a framework for greater interoperability based on shared standards, modular software design, and community-driven development. Our goal is to support collaboration across the field and lay the groundwork for a more connected, reusable, and accessible smart microscopy ecosystem. We conclude with a call to action for researchers, hardware developers, and institutions to join in building an open, interoperable foundation that will unlock the full potential of smart microscopy in life science research.

cell biology↗

bioRxiv2024

Cardiomyocyte antisense transcription regulates exon usage in the elastic spring region of Titin to modulate sarcomere function

BackgroundThe spring-like sarcomere protein Titin (TTN) is a key determinant of cardiac passive stiffness and diastolic function. Alternative splicing of TTN I-band exons produce protein isoforms with variable size and elasticity, but the mechanisms regulating TTN exon skipping and isoform composition in the human heart are not well studied. Non-coding RNA transcripts from the antisense strand of protein-coding genes have been shown to regulate alternative splicing of the sense gene. The TTN gene locus harbours >80 antisense transcripts with unknown function in the human heart. The aim of this study was to determine if TTN antisense transcripts play a role in alternative splicing of TTN. MethodsRNA-sequencing and RNA in situ hybridization (ISH) of cardiac tissue from unused organ donor hearts (n=7) and human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) were used to determine the expression and localization of TTN antisense transcripts. The effect of siRNA-mediated knock down of TTN antisense transcripts on TTN exon usage in iPS-CMs was determined using RNA-sequencing. Live cell imaging with sarcomere tracking was used to analyze the effect of antisense transcript knock down on sarcomere length, organization and contraction dynamics. RNA ISH, immunofluorescence and high content microscopy was performed in iPS-CMs to study the interaction between antisense transcripts, TTN mRNA and splice factor protein RBM20. ResultsIn mapping TTN antisense transcription, we found that TTN-AS1-276 was the predominant transcript in the human heart and that it was mainly localized in cardiomyocyte nuclear chromatin. Knock down of TTN-AS1-276 in human iPS-CMs resulted in decreased interaction between the splicing factor RBM20 and TTN pre-mRNA, decreased TTN I-band exon skipping, and markedly lower expression of the less elastic TTN isoform N2B. The effect on TTN exon usage was independent of sense-antisense exon overlap and polymerase II elongation rate. Furthermore, knockdown resulted in longer sarcomeres with preserved alignment, improved fractional shortening and relaxation times. ConclusionsWe demonstrate a role for the cardiac TTN antisense transcript TTN-AS1-276 in facilitating alternative splicing of TTN and regulating sarcomere properties. This transcript could constitute a target for improving cardiac passive stiffness and diastolic function in conditions such as heart failure with preserved ejection fraction.

molecular biology↗

bioRxiv2024

Cellular and biophysical barriers to lipid nanoparticle mediated delivery of RNA to the cytosol

Lipid nanoparticle (LNP) formulation was the first approved delivery strategy for liver-targeted siRNA delivery and currently represent the most advanced platform for delivery of therapeutic mRNA in clinical use, after the approval of mRNA-based vaccines against the SARS-CoV-2 coronavirus. Still, poor efficiency of LNPs to promote cytosolic delivery of both mRNA and siRNA after internalization by target cells limits their potency and thus clinical application as RNA delivery vehicles - especially in extrahepatic tissues and tumors. It is still unclear how endosomal escape of LNP-delivered RNA cargo occurs, and why only a minute proportion of all therapeutic molecules reach the cytoplasm. Here, we explored the intracellular sorting, integrity and endosomal escape of both mRNA and siRNA loaded LNPs to characterize the nature of RNA release from endosomal compartments and pinpoint current bottlenecks. Using live-cell imaging and super-resolution microscopy of LNPs with fluorescently labeled RNA payload, we identify multiple distinct steps of substantial inefficiency in the cytosolic delivery of nucleic acid cargoes. We demonstrate that membrane damages marked by recruitment of galectins are conducive to cytosolic RNA delivery, while membrane perturbations recruiting the ESCRT machinery do not permit endosomal escape. By quantitative single-vesicle analysis, we show that only a small fraction of the nucleic acid cargo contained in the endosome is released to the cytosol upon LNP-triggered membrane damage detected by galectins. Unexpectedly, we also observe that only a subset of damaged endosomes contain RNA payload. Through FRET and super-resolution microscopy, using LNPs formulated with both fluorescently labeled ionizable lipid (MC3-BODIPY) and RNA, we find that RNA cargo and ionizable lipid segregate during endosomal sorting - both within single endosomes and to different endosomal compartments. Finally, we visualize localized MC3-BODIPY enrichment in endosomal membranes and membrane damage in direct proximity to siRNA-LNPs tethered to the luminal vesicle membrane. Taken together, this work has identified multiple mechanistically distinct barriers limiting intracellular RNA delivery by LNPs.

bioengineering↗

bioRxiv2023

Dissecting the properties of circulating IgG against Group A Streptococcus through a combined systems antigenomics-serology workflow

Most individuals maintain circulating antibodies against various pathogenic bacteria as a consequence of previous exposures. However, it remains unclear to what extent these antibodies contribute to host protection. This knowledge gap is linked to the need for better methods to characterize antimicrobial polyclonal antibodies, including their antigen and epitope repertoires, subclass distribution, glycosylation status, and effector functions. Here, we showcase a generic mass spectrometry-based strategy that couples systems antigenomics and systems serology to characterize human antibodies directly in clinical samples. The method is based on automated affinity purification workflows coupled to an integrated suite of high-resolution MS-based quantitative, structural- and glyco-proteomics readouts. We focused on Streptococcus pyogenes (Group A Streptococcus; GAS), a major human pathogen still awaiting an approved vaccine. Our methodology reveals that both healthy and GAS infected individuals have circulating Immunoglobulin G (IgG) against a subset of genomically conserved streptococcal proteins, including numerous toxins and virulence factors. The antigen repertoire targeted by these antibodies was relatively constant across healthy individuals, but considerably changed in GAS bacteremia. Detailed analysis of the antigen-specific IgG indicates inter-individual variation regarding titers, subclass distributions, and Fc-signaling capacity, but not in epitope and Fc-glycosylation patterns. Importantly, we show that the IgG subclass has a major impact on the ability of GAS-antibodies to trigger immune signaling, in an antigen- and Fc receptor-specific fashion. Overall, these results uncover exceeding complexity in the properties of GAS-specific IgG, and showcase our methodology as high-throughput and flexible workflow to understand adaptive immune responses to bacterial pathogens. Significance statementMost people develop polyclonal antibodies against bacterial pathogens during infections but their structural and functional properties are poorly understood. Here, we showcase a combined systems antigenomics and systems serology strategy to quantify key antibody properties directly in clinical samples. We applied this method to characterize polyclonal antibody responses against Streptococcus pyogenes, a major human pathogen. We mapped the antigen and epitope landscape of anti-streptococcal antibodies circulating in healthy adult plasma, and their changes during blood infections. We further demonstrate the analytical power of our approach to resolve individual variations in the structure and effector functions of antigen-specific antibodies, including a dependency between immunoglobulin subclass and Fc-signaling capacity.

immunology↗

bioRxiv2023

The increased hinge flexibility of an IgG1-IgG3 hybrid monoclonal enhances Fc-mediated protection against group A streptococci

Antibodies are central to the immune response against microbes. We have previously generated a protective IgG1 monoclonal antibody targeting the M protein, a critical virulence factor of Streptococcus pyogenes. Here, we generated this antibody in all human IgG subclasses and evaluated their function. Despite significantly reduced binding, the IgG3 subclass antibody demonstrated remarkably enhanced opsonic function. We hypothesized that increased Fc flexibility could explain this improved efficacy. We engineered a hybrid IgG subclass antibody, IgGh, containing the backbone of IgG1 with the hinge of IgG3, leaving the Fabs unchanged. The IgGh maintained a similar binding ability as IgG1 while gaining the strong opsonic function seen with IgG3. Molecular dynamics simulations of the different antibodies showed altered IgG Fab-antigen interactions, reflecting the differences observed in affinity. More importantly, when the antibodies were bound to the antigen, the simulations showed that the Fc of both IgGh and IgG3 exhibited extensive movement in 3D space relative to the M protein. The increased flexibility of IgGh directly translated to enhanced opsonic function and significantly increased the protection against infection with Streptococcus pyogenes in mice. Our findings demonstrate how altering Fc flexibility can improve Fc-mediated opsonic function and how modifications in the constant domain can regulate Fab-antigen interactions. In addition, the enhanced in vivo function of a more flexible IgG provides new therapeutic opportunities for monoclonal antibodies. One sentence summaryAntibody Fc flexibility in 3D space correlates with efficient Fc-mediated phagocytosis of streptococci

immunology↗

bioRxiv2023

Protective non-neutralizing mAbs targets conserved opsonic epitopes on SARS-CoV-2 variants

Antibodies play a central role in the immune defense against SARS-CoV-2. There is substantial evidence supporting that Fc-mediated effector functions of anti-spike antibodies contribute to anti-SARS-Cov-2 immunity. We have previously shown that two non-neutralizing but opsonic mAbs, Ab81 and Ab94, are protective against lethal Wuhan SARS-CoV-2 infection in mice. The protective effect was comparable to a potent neutralizing antibody, Ab59. Here, we hypothesized that, unlike the neutralizing antibodies, non-neutralizing opsonic antibodies would have a higher likelihood of retaining their function to the mutated variants, potentially functioning as broadly protective mAbs. Most of the mutations on the SARS-CoV-2 variants cluster on neutralizing epitopes, leaving other epitopes unaltered. We observed that neutralizing antibodies lost binding to Omicron. In contrast, seven non-neutralizing opsonic antibodies retained nanomolar affinity towards Omicron, BA.2, BA.4, and BA.5. Focusing on the two protective non-neutralizing antibodies Ab81 and Ab94, we showed that they maintain their strong reactivity even to XBB, XBB1.5, and BQ1.1. In the case of Ab94, interestingly, it even has increased affinity towards all variants except for XBB, which is comparable to WT. Finally, we show that Ab94 and Ab81 have potent Fc-mediated functions in vitro against the XBB and BQ1.1 and that combining the mAbs in a cocktail further enhances the effect. These results show that protective non-neutralizing mAbs such as Ab94 and Ab81 can be a viable strategy for anti-SARS-CoV-2 mAb therapies against current and possibly future SARS-CoV-2 variants and that opsonic epitopes could have implications for vaccine design.

immunology↗

bioRxiv2022

Invasive streptococcal infection can lead to the generation of cross-strain opsonic antibodies

IntroductionThe human pathogen Streptococcus pyogenes causes substantial morbidity and mortality. It is unclear if antibodies developed after infections with this pathogen are opsonic and if they are strain-specific or more broadly protective. Here, we quantified the opsonic antibody response following invasive S. pyogenes infection. Materials and MethodsFour patients with S. pyogenes bacteremia between 2018-2020 at Sk[a]ne University Hospital in Lund, Sweden, were prospectively enrolled. Acute and convalescent sera were obtained, and the S. pyogenes isolates were genome-sequenced (emm118, emm85, and two emm1). Quantitative antibody binding and phagocytosis assays were used to evaluate isolate-dependent opsonic antibody function in response to infection. ResultsAntibody binding increased modestly against the infecting isolate and across emm types in convalescent compared to acute sera for all patients. For two patients, phagocytosis increased in convalescent serum for both the infecting isolate and across types. The increase was only across types for one patient, and one had no improvement. No correlation to the clinical outcomes was observed. ConclusionInvasive S. pyogenes infections result in a modestly increased antibody binding with differential opsonic capacity, both non-functional binding and broadly opsonic binding across types. These findings question the dogma that an invasive infection should lead to a strong type-specific antibody increase rather than a more modest but broadly reactive response, as seen in these patients. Furthermore, our results indicate that an increase in antibody titers might not be indicative of an opsonic response and highlight the importance of evaluating antibody function in S. pyogenes infections.

immunology↗

bioRxiv2022

Data-driven microscopy allows for automated targeted acquisition of relevant data withhigher fidelity

Light microscopy is a powerful single-cell technique that allows for quantitative spatial information at subcellular resolution. However, unlike flow cytometry and single-cell sequencing techniques, microscopy has issues achieving high-quality population-wide sample characterization while maintaining high resolution. Here, we present a general framework, data-driven microscopy (DDM), that uses population-wide cell characterization to enable data-driven high-fidelity imaging of relevant phenotypes. DDM combines data-independent and data-dependent steps to synergistically enhance data acquired using different imaging modalities. As proof-of-concept, we apply DDM with plugins for improved high-content screening and live adaptive microscopy. DDM also allows for easy correlative imaging in other systems with a plugin that uses the spatial relationship of the sample population for automated registration. We believe DDM will be a valuable approach for reducing human bias, increasing reproducibility, and placing singlecell characteristics in the context of the sample population when interpreting microscopy data, leading to an overall increase in data fidelity.

systems biology↗

bioRxiv2022

Group A streptococci induce high-affinity M protein-fibronectin interaction when specific human antibodies are bound

Group A streptococcus (GAS) is a highly adapted, humanspecific pathogen that is known to manipulate the immune system through various mechanisms. GAS M protein constitutes a primary target of the immune system due to its spatial configuration and dominance on the bacterial surface. Antibody responses targeting the M protein have been shown to favor the conserved C region. Such antibodies circumvent antigenic escape and efficiently bind to various M types. The ability of GAS to bind to fibronectin (Fn), a high molecular weight glycoprotein of the extracellular matrix, has long been known to be essential for the pathogens evolutionary success and fitness. However, some strains lack the ability to efficiently bind Fn. Instead, they have been found to inefficiently bind Fn via the M protein A-B domains. Here, we show that human antibodies can induce a high-affinity Fn-binding state in M proteins, likely by enhancing the weak A-B domain binding. The antibodies bind to a conserved region of M proteins, and the high-affinity binding only occurs on the individual M proteins with bound specific antibodies. By allowing the binding of antibodies to a certain region in M, and thereby enhancing Fn-binding, GAS exploits the human humoral immune response to efficiently bind Fn without needing to waste energy on the production of additional proteins - potentially giving such strains an evolutionary advantage.

immunology↗

bioRxiv2021

Opsonization by non-neutralizing antibodies can confer protection to SARS-CoV-2 despite Spike-dependent modulation of phagocytosis

Spike-specific antibodies are central to effective COVID19 immunity. Research efforts have focused on antibodies that neutralize the ACE2-Spike interaction but not on non-neutralizing antibodies. Antibody-dependent phagocytosis is an immune mechanism enhanced by opsonization, where typically, more bound antibodies trigger a stronger phagocyte response. Here, we show that Spike-specific antibodies, dependent on concentration, can either enhance or reduce Spike-bead phagocytosis by monocytes independently of the antibody neutralization potential. Surprisingly, we find that both convalescent patient plasma and patient-derived monoclonal antibodies lead to maximum opsonization already at low levels of bound antibodies and is reduced as antibody binding to Spike protein increases. Moreover, we show that this Spike-dependent modulation of opsonization seems to affect the outcome in an experimental SARS-CoV-2 infection model. These results suggest that the levels of anti-Spike antibodies could influence monocyte-mediated immune functions and propose that non-neutralizing antibodies could confer protection to SARS-CoV-2 infection by mediating phago-cytosis.

immunology↗

bioRxiv2021

Bacterial clearance caused by a single anti-M protein antibody simultaneously binding two separate epitopes

Group A streptococci have evolved multiple strategies to evade human antibodies, making it challenging to create effective vaccines or antibody treatments. Here, we have generated antibodies derived from the memory B cells of an individual who had successfully cleared a group A streptococcal infection. The antibodies bind with high affinity in the central region of the surface-bound M protein. Such antibodies are typically non-opsonic. However, one antibody could effectively promote vital immune functions, including phagocytosis and in vivo protection. Remarkably, this antibody primarily interacts through a bivalent dual-Fab cis mode, where the Fabs bind to two distinct epitopes in the M protein. The dual-Fab cis binding phenomenon is conserved across different groups of M types. In contrast, other antibodies binding with normal single-Fab mode to the same region can not bypass the M proteins virulent effects. A broadly binding, protective monoclonal antibody could be a candidate for anti-streptococcal therapy. Our findings highlight the concept of dual-Fab cis binding as a means to access conserved, and normally non-opsonic regions, for protective antibody targeting.

immunology↗

bioRxiv2020

Binding site localization on non-homogeneous cell surfaces using topological image averaging

Antibody binding to cell surface proteins plays a crucial role in immunity and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines. In the present work, we have developed a method for imaging-based localization of binding sites on cellular surface proteins. The cellular morphology typically varies, and antibodies often bind in a non-homogenous manner, making traditional particle-averaging strategies challenging for accurate native antibody localization. Nanometer-scale resolution is achieved through localization in one dimension, namely the distance from a bound ligand to a reference surface, by using topological image averaging. Our results show that this method is well suited for antibody binding site measurements on native cell surface morphology.

immunology↗

bioRxiv2020

A predictive model of antibody binding in the presence of IgG-interacting bacterial surface proteins

Many bacteria can interfere with how antibodies bind to their surfaces. This bacterial antibody targeting makes it challenging to predict the immunological function of bacteria-associated antibodies. The M and M-like proteins of group A streptococci exhibit IgGFc-binding regions, which they use to reverse IgG binding orientation depending on the host environment. Unraveling the mechanism behind these binding characteristics may identify conditions under which bound IgG can drive an efficient immune response. Here, we have developed a biophysical model for describing these complex protein-antibody interactions. We show how the model can be used as a tool for studying various IgG samples behaviour by performing in silico simulations and correlating this data with experimental measurements. Besides its use for mechanistic understanding, this model could potentially be used as a tool to predict the effect, as well as aid in the development of antibody treatments. We illustrate this by simulating how IgG binding in serum is altered as specified amounts of monoclonal or pooled IgG is added. Phagocytosis experiments link this altered antibody binding to a physiological function and demonstrate that it is possible to predict the effect of an IgG treatment with our model. Our study gives a mechanistic understanding of bacterial antibody targeting and provides a tool for predicting the effect of antibody treatments.

microbiology↗

bioRxiv2019

High-sensitivity assessment of phagocytosis by persistent association-based normalization

Phagocytosis is measured as a functional outcome in many research fields but can be challenging to quantify accurately, with no robust method available for cross-laboratory reproducibility. Here, we identified a simple, measurable parameter - persistent prey-phagocyte association - to use for normalization and dose-response analysis. We apply this in a straightforward analytical method, persistent association-based normalization (PAN), where first the multiplicity of prey (MOP) ratio needed to elicit half of the phagocytes to associate persistently (MOP50) is determined. MOP50 is then applied to normalize for experimental factors, with adhesion and internalization analyzed separately. We use THP-1 cells and different prey and opsonization conditions to compare the PAN method to standard ways of assessing phagocytosis, and find it better in all cases, with increased robustness, sensitivity, and reproducibility. The approach is easily incorporated into most existing phagocytosis assays and allows for reproducibly comparing results across experiments and laboratories with high sensitivity.

cell biology↗