Biology subjects

Gustafsson, N.

Publications and source records attributed to Gustafsson, N..

7 recordsLinked to original sources

bioRxiv2026

Multi-Site Reproducibility Study of 3D High-Content Analysis with Dual-View Oblique Plane Microscopy

High content imaging is being applied to achieve quantitative fluorescence readouts in increasingly complex 3-dimensional (3D) cell culture models such as spheroids and organoids. Compared to conventional 2D assays, 3D assays better represent biological heterogeneity but require more complex sample preparation, 3D imaging and 3D image analysis that can affect the accuracy and precision of such assays. We used spheroids formed from the NRAS-activated melanoma cell line 19161 modified to express an ERK kinase translocation reporter (KTR) as an exemplar 3D phenotypic assay carried out in 96-well plates. The spheroids were treated with the ERK activator TPA and a range of concentrations of the MEK inhibitor Binimetinib. 3D live-cell imaging with sub-cellular spatial resolution was performed using a dual-view oblique plane microscope (dOPM) - a form of single-objective light-sheet microscope - and the experiment was performed separately at 4 different institutes. The results were analysed using an identical 3D analysis pipeline and parameters. We assessed the variation in assay readout using a linear mixed effects model. Random variance at the well level was negligible (SD = 0.0048 relative to range of KTR biosensor readout at reference site of 0.17), indicating low technical noise. Treatment effects were dose-dependent and highly statistically significant compared to DMSO control across all sites (Dunnett-corrected p < 0.001). The range in KTR readout between the minimum (3.5 M Binimetinib) and maximum (100 nM TPA) treatments varied between 59 to 96% relative to the reference site. Measured bias in KTR readout between sites was between 6 and 12% of the range of the reference site. This study quantifies the reproducibility of a 3D live spheroid-based assay employing a fluorescence biosensor requiring readout out at the per-cell level using the dOPM platform and discusses areas where experimental protocol could be improved in the future to further improve reproducibility.

bioengineering↗

bioRxiv2025

Tartrate-resistant acid phosphatase (TRAP/ACP5) sex-specifically regulates bone maintenance in old mice, but the anabolic effects of mechanical loading is regulated in a sex-independent way

Tartrate-resistant acid phosphatase (TRAP/ACP5), primarily known as an osteoclast marker, has emerged as a critical regulator of skeletal integrity, regulating sex-specific bone growth, and bones response to mechanical load in young adult male mice. In this study, we investigated the sex-specific roles of TRAP in bone structure and response to mechanical stimuli in old (19-month-old) wild-type (WT) and TRAP-deficient (TRAP-/-) mice using micro-computed tomography, serum bone turnover markers, in vivo axial mechanical loading, and in vitro mechanotransduction assays. Our findings revealed that TRAP-/- mice of both sexes maintained shorter tibiae than WT mice independent of sex. Notably, male, but not female, TRAP-/- mice have increased trabecular bone volume fraction and cortical bone area compared to WT, indicative of disrupted bone remodelling processes in male mice. Interestingly, TRAP-deficiency substantially impaired the anabolic bone response to mechanical loading, affecting both trabecular and cortical compartments in both sexes, indicating that when challenged, TRAP is important for bone formation also in female mice. Mechanical stimulation in vitro of hematopoietic progenitor cells from WT and TRAP-/- mice revealed that the increased ATP-release in response to mechanical stimulation was only disrupted in male mice, while mechanically induced increase in osteoclast formation was inhibited in TRAP-/- mice of both sexes. These results highlight the importance of TRAP in maintaining trabecular architecture and cortical bone in male mice and underscore its critical function in mediating adaptive responses to mechanical loading of both sexes, during aging. Future investigations should focus on elucidation of TRAP-dependent pathways as potential therapeutic targets to counteract age-related deficits in bone adaptation and remodelling.

developmental biology↗

bioRxiv2025

Multimodal profiling unveils a reversible basal-like breast cancer cell state resistant to AKT inhibition

The PI3K/AKT/mTOR pathway is central to cell metabolism and growth. However, pharmacological inhibition of the pathway is not uniformly effective across cancer types, or even within a single cancer model. In this study, we leverage oblique plane microscopy of triple negative breast cancer organoids, as well as lineage tracing to uncover a source of heterogeneity. Non-genetic resistance to AKT inhibition is associated with basal cell features of normal breast epithelium and the master transcription factor of basal cell state, {Delta}Np63, is sufficient to confer resistance. Cells can transition between states within four weeks and therefore, AKT inhibition only delays tumour growth, with tumours rich in KRT14+ cells resulting. Thus, under selection, triple negative breast cancer exploits a repertoire of cell states inherent to the breast.

cancer biology↗

bioRxiv2025

Tumbleweed: an artificial motor protein that walks along a DNA track

Summary ParagraphMolecular motors are fundamental to life1-6 because of their ability to convert chemical energy into mechanical work, an ability that is conferred by the chemical and structural complexity of their constituent proteins. Scientists have long sought to create artificial protein motors that may reveal insights into how biological motors function. While artificial molecular motors based on small molecules7 and DNA8,9 have been developed, creating an artificial motor protein has remained an elusive goal in synthetic biology10. Here we demonstrate the realization of an artificial protein motor called Tumbleweed (TW) that walks directionally along a DNA track under external control. TW consists of three legs, each with a ligand-gated DNA-binding domain that enables selective interaction with specific sites along a DNA track11. Using single-molecule fluorescence assays and a programmable microfluidic device, we show that TW steps directionally along a designed DNA track in response to a defined sequence of ligand inputs. We built our TW molecular walker using a modular approach, combining existing proteins with known properties to achieve emergent motor function, similar to how Nature evolves new proteins. Our design strategy thus offers a a platform for engineering advanced and dynamic protein functionality. Our demonstration of TW walking represents a step toward developing fully autonomous protein motors and opens new avenues for uncovering and leveraging the principles by which biological motors transduce chemical energy into motion.

biophysics↗

bioRxiv2020

Super-Beacons: open-source probes with spontaneous tunable blinking compatible with live-cell super-resolution microscopy

Localization based super-resolution microscopy relies on the detection of individual molecules cycling between fluorescent and non-fluorescent states. These transitions are commonly regulated by high-intensity illumination, imposing constrains to imaging hardware and producing sample photodamage. Here, we propose single-molecule self-quenching as a mechanism to generate spontaneous photoswitching independent of illumination. To demonstrate this principle, we developed a new class of DNA-based open-source Super-Resolution probes named Super-Beacons, with photoswitching kinetics that can be tuned structurally, thermally and chemically. The potential of these probes for live-cell friendly Super-Resolution Microscopy without high-illumination or toxic imaging buffers is revealed by imaging Interferon Inducible Transmembrane proteins (IFITMs) at sub-100nm resolutions.

cell biology↗

bioRxiv2018

NanoJ: a high-performance open-source super-resolution microscopy toolbox

Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was de-veloped for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image re-construction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

bioinformatics↗

bioRxiv2017

A hidden Markov model approach to characterizing the photo-switching behavior of fluorophores

Fluorescing molecules (fluorophores) that stochastically switch between photon-emitting and dark states underpin some of the most celebrated advancements in super-resolution microscopy. While this stochastic behavior has been heavily exploited, full characterization of the underlying models can potentially drive forward further imaging methodologies. Under the assumption that fluorophores move between fluorescing and dark states as continuous time Markov processes, the goal is to use a sequence of images to select a model and estimate the transition rates. We use a hidden Markov model to relate the observed discrete time signal to the hidden continuous time process. With imaging involving several repeat exposures of the fluorophore, we show the observed signal depends on both the current and past states of the hidden process, producing emission probabilities that depend on the transition rate parameters to be estimated. To tackle this unusual coupling of the transition and emission probabilities, we conceive transmission (transition-emission) matrices that capture all dependencies of the model. We provide a scheme of computing these matrices and adapt the forward-backward algorithm to compute a likelihood which is readily optimized to provide rate estimates. When confronted with several model proposals, combining this procedure with the Bayesian Information Criterion provides accurate model selection.

biophysics↗