Biology subjects

Wrona, A.

Publications and source records attributed to Wrona, A..

6 recordsLinked to original sources

bioRxiv2026

Aerosol delivery-based serial femtosecond crystallography

Serial femtosecond crystallography (SFX) has revolutionised structural biology by enabling direct visualization of conformational dynamics of biomacromolecules at near-physiological temperatures. However, conventional SFX sample delivery at X-ray free-electron laser (XFEL) facilities introduces a significant X-ray scattering background, which may limit the data quality and resolution. Here, we introduce an aerosol delivery-based method that drastically reduces the back-ground scattering by minimising the liquid environment surrounding the nanocrystals. We validate our method by solving the structure of Cydia pomonella granulovirus nanocrystals at 1.9 [A] resolution, achieving orders-of-magnitude lower background scattering compared to a liquid jet-based method. Structural comparison with the liquid jet-based model revealed similar overall structure, suggesting that the native structure is largely preserved despite dehydration during aerosolisation. Our method enables efficient SFX studies, particularly pump-probe time-resolved SFX on protein nanocrystals with enhanced signal-to-noise ratios, as well as high-throughput small molecule SFX (smSFX) applications for pharmaceuticals and functional materials.

biophysics↗

bioRxiv2025

Therapeutic Potential of Neural Rosette Stem Cell-Derived Small Extracellular Vesicles in the Management of Brain Edema

Small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) have gained significant interest for their potential roles in neuroprotection, brain edema management, and the preservation of blood-brain barrier (BBB) integrity. These nanoscale particles facilitate intercellular communication and carry a diverse array of bioactive molecules, including proteins and RNAs, which influence various neurobiological processes. The composition of their cargo depends on the parental cell type, affecting their interactions with recipient cells. In this study, we characterized the cargo of sEVs derived from neural rosette stem cells (NRSCs) at both transcriptomic and proteomic levels and compared it to sEVs derived from cardiomyocyte progenitor cells (CMPCs) and human pluripotent stem cells (hPSCs). Furthermore, we assessed the therapeutic effects of NRSC-derived sEVs in a rat model of transient middle cerebral artery occlusion (tMCAO) to investigate neuronal injury and the progression of brain edema. Our proteomic and miRNA analyses revealed that the sEVs from NRSCs, CMPCs, and hPSCs possess distinct cargo compositions. In an in vitro neuronal ischemia model, NRSC-derived sEVs significantly enhanced cell viability, while in an endothelial model, they expedited the restoration of cell barrier integrity. Furthermore, the in vivo studies utilizing the rat model of tMCAO showed that intravenous administration of NRSC-derived sEVs at 3, 24 and 48 hours after reperfusion resulted in a significantly faster resolution of brain edema. These findings underscore the influence of the parental cell type on sEV composition and highlight the therapeutic potential of NRSC-derived sEVs in treating brain edema, paving the way for novel therapeutic strategies.

neuroscience↗

bioRxiv2025

Efficient Generation of Expandable Dorsal Forebrain Neural Rosette Stem Cell Lines

Neural stem cells (NSCs) represent an interesting option for developing in vitro disease models and drug screening assays due to their differentiation capacity into neurons and glial cells. Additionally, NSCs are under investigation in on-going clinical trials for treatment of various human neurological disorders. NSCs can be isolated from the central nervous system or derived in vitro from human pluripotent stem cells (hPSCs). However, the current methods for generating NSCs typically include a phase of neural rosette formation and subsequent manual isolation of these tiny structures. As this is a laborious process characterized by operator-dependent variability and scalability challenges, there is a pressing need to develop optimized and scalable protocols to obtain pure NSC populations. In this study, we present a new method for generating highly pure and expandable dorsal forebrain FOXG1+OTX2+TLE4+SOX5+ neural rosette stem cell (NRSC) lines without the necessity for manual isolation of rosette structures. Our findings demonstrate the reproducibility of this protocol through the characterization of different NRSC lines over multiple passages, highlighting the robustness of the process. These NRSCs can be expanded for at least 12 passages without compromising their rosette-formation capacity or their initial dorsal forebrain identity. Furthermore, we show the differentiation capacity of these NRSCs to generate pure populations of TUBB3+ neurons, and under specific conditions, their ability to differentiate into early glial progenitor cells including GFAP+ astrocytes and O4+ oligodendrocytes. Collectively, these results show the capabilities of our protocol to generate an expandable NRSC population suitable for in vitro disease modeling and drug screening, while also suggesting a viable strategy for scalable NRSC production for clinical application.

neuroscience↗

bioRxiv2024

Crystal structure of a bacterial photoactivated adenylate cyclase determined at room temperature by serial femtosecond crystallography

OaPAC is a recently discovered blue-light using flavin adenosine dinucleotide (BLUF) photoactivated adenylate cyclase from the cyanobacterium Oscillatoria acuminata that uses adenosine triphosphate and translates the light signal into the production of cyclic adenosine monophosphate. Here, we report the crystal structures of the enzyme in the absence of its natural substrate determined from room temperature serial crystallography data collected at both an X-ray free electron laser and a synchrotron and we compare them with the cryo macromolecular crystallography structures obtained at a synchrotron by us and others. These results reveal slight differences in the structure of the enzyme due to data collection at different temperatures and X-ray sources. We further investigate the effect of the Y6 mutation in the blue-light using flavin adenosine dinucleotide domain, a mutation which results in a rearrangement of the hydrogen-bond network around the flavin and a notable rotation of the side-chain of the critical Q48 residue. These studies pave the way for ps - ms time-resolved serial crystallography experiments at X-ray free electron lasers and synchrotrons in order to determine the early structural intermediates and correlate them with the well-studied ps - ms spectroscopic intermediates. SynopsisStructures of the dark-adapted state of a photoactivated adenylate cyclase are determined from serial crystallography (SX) data collected at room temperature at an X-ray free electron laser (XFEL) and a synchrotron and are compared with cryo macromolecular crystallography (MX) synchrotron structures obtained by us and others. These structures of the wild-type enzyme in combination with the cryo MX synchrotron structure of a light-sensor domain mutant provide insight into the hydrogen bond network rearrangement upon blue-light illumination and pave the way for the determination of structural intermediates of the enzyme by time-resolved SX.

biophysics↗

bioRxiv2023

Light-induced Trpin/Metout switching during BLUF domain activation in ATP-bound photoactivatable adenylate cyclase OaPAC

The understanding of signal transduction mechanisms in photoreceptor proteins is essential for elucidating how living organisms respond to light as environmental stimuli. In this study, we investigated the ATP binding, photoactivation and signal transduction process in the photoactivatable adenylate cyclase from Oscillatoria acuminata (OaPAC) upon blue light excitation. Structural models with ATP bound in the active site of native OaPAC at cryogenic as well as room temperature are presented. ATP is found in one conformation at cryogenic- and in two conformations at ambient-temperature, and is bound in a non-productive conformation. However, FTIR spectroscopic experiments confirm that the non-productive conformation is the native binding mode in dark state OaPAC and that transition to a productive conformation for ATP turnover only occurs after light activation. A combination of time-resolved crystallography experiments at synchrotron and X-ray Free Electron Lasers sheds light on the initial events around the Flavin Adenine Dinucleotide (FAD) chromophore in the light-sensitive BLUF domain of OaPAC. Initial changes involve the highly conserved amino acids Tyr6, Gln48 and Met92. Crucially, the Gln48 side chain performs a 180{degrees} rotation during activation, leading to the stabilization of the FAD chromophore. Cryo-trapping experiments allowed us to investigate a late light-activated state of the reaction and revealed significant conformational changes in the BLUF domain around the FAD chromophore. In particular, a Trpin/Metout transition upon illumination is observed for the first time in the BLUF domain and its role in signal transmission via -helix 3 and 4 in the linker region between sensor and effector domain is discussed.

biochemistry↗

bioRxiv2022

Mix-and-extrude using 3D printed nozzles for time-resolved membrane protein crystallography

Time-resolved crystallography enabled the visualization of protein molecular motion during reaction. While light is commonly used to initiate reactions in time-resolved crystallography, only a small number of proteins can in fact be activated by light. However, many biological reactions can be triggered by the interaction of proteins with ligands. The sample delivery method presented here uses a mix-and-extrude approach based on 3D printed microchannels in conjunction with a micronozzle to study the dynamics of samples in viscous media that can be triggered by diffusive mixing. The device design allows for mixing of ligands and protein crystals in a time window of 2 to 20 seconds. The device characterization using a model system (fluorescence quenching of iq-mEmerald proteins by copper ions) demonstrated that ligand and protein crystals, each within the lipidic cubic phase, can be mixed efficiently. The potential use of this approach for time-resolved membrane protein crystallography to support in the development of new drugs is also discussed. Synopsis3D printed mixing-HVE devices address time-resolved membrane protein crystallography challenges via compact dual-flow LCP injection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/517685v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12f6f68org.highwire.dtl.DTLVardef@13b198eorg.highwire.dtl.DTLVardef@10abe5eorg.highwire.dtl.DTLVardef@5810ee_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗