Mutation of the ALS-/FTD-Associated RNA-Binding Protein FUS Affects Axonal Development.

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.

Fast, multicolour optical sectioning over extended fields of view with patterned illumination and machine learning.

Structured illumination can reject out-of-focus signal from a sample, enabling high-speed and high-contrast imaging over large areas with widefield detection optics. However, this optical sectioning technique is currently limited by image reconstruction artefacts and poor performance at low signal-to-noise ratios. We combine multicolour interferometric pattern generation with machine learning to achieve high-contrast, real-time reconstruction of image data that is robust to background noise and sample motion. We validate the method in silico and demonstrate imaging of diverse specimens, from fixed and live biological samples to synthetic biosystems, reconstructing data live at 11 Hz across a 44 × 44µm2 field of view, and demonstrate image acquisition speeds exceeding 154 Hz.

Molecular Mechanisms of Cationic Fusogenic Liposome Interactions with Bacterial Envelopes.

Although fusogenic liposomes offer a promising approach for the delivery of antibiotic payloads across the cell envelope of Gram-negative bacteria, there is still a limited understanding of the individual nanocarrier interactions with the bacterial target. Using super-resolution microscopy, we characterize the interaction dynamics of positively charged fusogenic liposomes with Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. The liposomes merge with the outer membrane (OM) of Gram-negative bacteria, while attachment or lipid internalization is observed in Gram-positive cells. Employing total internal reflection fluorescence microscopy, we demonstrated liposome fusion with model supported lipid bilayers. For whole E. coli cells, however, we observed heterogeneous membrane integrations, primarily involving liposome attachment and hemifusion events. With increasing lipopolysaccharide length, the likelihood of full-fusion events was reduced. The integration of artificial lipids into the OM of Gram-negative cells led to membrane destabilization, resulting in decreased bacterial vitality, membrane detachment, and improved codelivery of vancomycin─an effective antibiotic against Gram-positive cells. These findings provide significant insights into the interactions of individual nanocarriers with bacterial envelopes at the single-cell level, uncovering effects that would be missed in bulk measurements. This highlights the importance of conducting single-particle and single-cell investigations to assess the performance of next-generation drug delivery platforms.

Attachment discs of the diving bell spider Argyroneta aquatica.

To adhere their silk threads for the construction of webs and to fix the dragline, spiders produce attachment discs of piriform silk. Uniquely, the aquatic spider Argyroneta aquatica spends its entire life cycle underwater. Therefore, it has to glue its attachment discs to substrates underwater. Here we show that Argyroneta aquatica applies its thread anchors within an air layer around the spinnerets maintained by superhydrophobic setae. During spinning, symmetric movements of the spinnerets ensure retaining air in the contact area. The flat structure of the attachment discs is thought to facilitate fast curing of the piriform adhesive cement and improves the resistance against drag forces. Pull-off tests on draglines connected with attachment discs on different hydrophilic substrates point to dragline rupture as the failure mode. The Young´s modulus of the dragline (8.3 GPa) is within the range as in terrestrial spiders. The shown structural and behavioral adaptations can be the model for new artificial underwater gluing devices.

Diversity of dynamic voltage patterns in neuronal dendrites revealed by nanopipette electrophysiology.

Dendrites and dendritic spines are the essential cellular compartments in neuronal communication, conveying information through transient voltage signals. Our understanding of these compartmentalized voltage dynamics in fine, distal neuronal dendrites remains poor due to the difficulties inherent to accessing and stably recording from such small, nanoscale cellular compartments for a sustained time. To overcome these challenges, we use nanopipettes that permit long and stable recordings directly from fine neuronal dendrites. We reveal a diversity of voltage dynamics present locally in dendrites, such as spontaneous voltage transients, bursting events and oscillating periods of silence and firing activity, all of which we characterized using segmentation analysis. Remarkably, we find that neuronal dendrites can display spontaneous hyperpolarisation events, and sustain transient hyperpolarised states. The voltage patterns were activity-dependent, with a stronger dependency on synaptic activity than on action potentials. Long-time recordings of fine dendritic protrusions show complex voltage dynamics that may represent a previously unexplored contribution to dendritic computations.

Multiparametric Sensing of Outer Membrane Vesicle-Derived Supported Lipid Bilayers Demonstrates the Specificity of Bacteriophage Interactions.

The use of bacteriophages, viruses that specifically infect bacteria, as antibiotics has become an area of great interest in recent years as the effectiveness of conventional antibiotics recedes. The detection of phage interactions with specific bacteria in a rapid and quantitative way is key for identifying phages of interest for novel antimicrobials. Outer membrane vesicles (OMVs) derived from Gram-negative bacteria can be used to make supported lipid bilayers (SLBs) and therefore in vitro membrane models that contain naturally occurring components of the bacterial outer membrane. In this study, we employed Escherichia coli OMV derived SLBs and use both fluorescent imaging and mechanical sensing techniques to show their interactions with T4 phage. We also integrate these bilayers with microelectrode arrays (MEAs) functionalized with the conducting polymer PEDOT:PSS and show that the pore forming interactions of the phages with the SLBs can be monitored using electrical impedance spectroscopy. To highlight our ability to detect specific phage interactions, we also generate SLBs using OMVs derived from Citrobacter rodentium, which is resistant to T4 phage infection, and identify their lack of interaction with the phage. The work presented here shows how interactions occurring between the phages and these complex SLB systems can be monitored using a range of experimental techniques. We believe this approach can be used to identify phages that work against bacterial strains of interest, as well as more generally to monitor any pore forming structure (such as defensins) interacting with bacterial outer membranes, and thus aid in the development of next generation antimicrobials.

α-Synuclein fibril and synaptic vesicle interactions lead to vesicle destruction and increased lipid-associated fibril uptake into iPSC-derived neurons.

Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson's disease patient's brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.

Open-source software package for on-the-fly deskewing and live viewing of volumetric lightsheet microscopy data.

Oblique plane microscopy, OPM, is a form of lightsheet microscopy that permits volumetric imaging of biological samples at high temporal and spatial resolution. However, the imaging geometry of OPM, and related variants of light sheet microscopy, distorts the coordinate frame of the presented image sections with respect to the real space coordinate frame in which the sample is moved. This makes live viewing and practical operation of such microscopes difficult. We present an open-source software package that utilises GPU acceleration and multiprocessing to transform the display of OPM imaging data in real time to produce a live extended depth of field projection. Image stacks can be acquired, processed and plotted at rates of several Hz, making live operation of OPMs, and similar microscopes, more user friendly and intuitive.

ERnet: a tool for the semantic segmentation and quantitative analysis of endoplasmic reticulum topology.

The ability to quantify structural changes of the endoplasmic reticulum (ER) is crucial for understanding the structure and function of this organelle. However, the rapid movement and complex topology of ER networks make this challenging. Here, we construct a state-of-the-art semantic segmentation method that we call ERnet for the automatic classification of sheet and tubular ER domains inside individual cells. Data are skeletonized and represented by connectivity graphs, enabling precise and efficient quantification of network connectivity. ERnet generates metrics on topology and integrity of ER structures and quantifies structural change in response to genetic or metabolic manipulation. We validate ERnet using data obtained by various ER-imaging methods from different cell types as well as ground truth images of synthetic ER structures. ERnet can be deployed in an automatic high-throughput and unbiased fashion and identifies subtle changes in ER phenotypes that may inform on disease progression and response to therapy.

Feasibility of Coacervate-Like Nanostructure for Instant Drug Nanoformulation.

Despite the enormous advancements in nanomedicine research, a limited number of nanoformulations are available on the market, and few have been translated to clinics. An easily scalable, sustainable, and cost-effective manufacturing strategy and long-term stability for storage are crucial for successful translation. Here, we report a system and method to instantly formulate NF achieved with a nanoscale polyelectrolyte coacervate-like system, consisting of anionic pseudopeptide poly(l-lysine isophthalamide) derivatives, polyethylenimine, and doxorubicin (Dox) via simple "mix-and-go" addition of precursor solutions in seconds. The coacervate-like nanosystem shows enhanced intracellular delivery of Dox to patient-derived multidrug-resistant (MDR) cells in 3D tumor spheroids. The results demonstrate the feasibility of an instant drug formulation using a coacervate-like nanosystem. We envisage that this technique can be widely utilized in the nanomedicine field to bypass the special requirement of large-scale production and elongated shelf life of nanomaterials.

Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research.

As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

Single-Molecule Sizing through Nanocavity Confinement.

An approach relying on nanocavity confinement is developed in this paper for the sizing of nanoscale particles and single biomolecules in solution. The approach, termed nanocavity diffusional sizing (NDS), measures particle residence times within nanofluidic cavities to determine their hydrodynamic radii. Using theoretical modeling and simulations, we show that the residence time of particles within nanocavities above a critical time scale depends on the diffusion coefficient of the particle, which allows the estimation of the particle's size. We demonstrate this approach experimentally through the measurement of particle residence times within nanofluidic cavities using single-molecule confocal microscopy. Our data show that the residence times scale linearly with the sizes of nanoscale colloids, protein aggregates, and single DNA oligonucleotides. NDS thus constitutes a new single molecule optofluidic approach that allows rapid and quantitative sizing of nanoscale particles for potential applications in nanobiotechnology, biophysics, and clinical diagnostics.

Machine learning assisted interferometric structured illumination microscopy for dynamic biological imaging.

Structured Illumination Microscopy, SIM, is one of the most powerful optical imaging methods available to visualize biological environments at subcellular resolution. Its limitations stem from a difficulty of imaging in multiple color channels at once, which reduces imaging speed. Furthermore, there is substantial experimental complexity in setting up SIM systems, preventing a widespread adoption. Here, we present Machine-learning Assisted, Interferometric Structured Illumination Microscopy, MAI-SIM, as an easy-to-implement method for live cell super-resolution imaging at high speed and in multiple colors. The instrument is based on an interferometer design in which illumination patterns are generated, rotated, and stepped in phase through movement of a single galvanometric mirror element. The design is robust, flexible, and works for all wavelengths. We complement the unique properties of the microscope with an open source machine-learning toolbox that permits real-time reconstructions to be performed, providing instant visualization of super-resolved images from live biological samples.

Lessons from 10 years' experience running the Fiom KID-DNA database, a voluntary DNA-linking register for donor-conceived people and donors in The Netherlands.

Worldwide, there is increasing acknowledgment of the importance of getting access to ancestry information. More and more countries facilitate access to this information through law changes and voluntary contact-services. In the Netherlands, the state-funded Fiom KID-DNA database was established in 2010 to facilitate information and/or contact exchange between those people who are genetically related as a result of donor-assisted conception. By the end of 2021, 846 donors and 2355 donor-conceived people are registered in the database. For 25% of the donors a link was found with one or more donor-conceived people, and 39% of the donor-conceived people were linked to a donor-profile. Fiom offers support by professionally qualified staff throughout the entire process from registration to contact to donor-conceived people, donors and their relatives. During the period of more than 10 years several challenges emerged; how does a state-funded DNA database function in the area of commercial DNA databases?; what can be learned from the continuous growing donor-conceived half-siblings networks?; how to deal with malpractices from the past and how to cope with ageing donors?

Hydrotalcite-Derived Copper-Based Oxygen Carrier Materials for Efficient Chemical-Looping Combustion of Solid Fuels with CO2 Capture.

Chemical-looping combustion (CLC) is a promising technology that utilizes metal oxides as oxygen carriers for the combustion of fossil fuels to CO2 and H2O, with CO2 readily sequestrated after the condensation of steam. Thermally stable and reactive metal oxides are desirable as oxygen carrier materials for the CLC processes. Here, we report the performance of Cu-based mixed oxides derived from hydrotalcite (also known as layered double hydroxides) precursors as oxygen carriers for the combustion of solid fuels. Two types of CLC processes were demonstrated, including chemical looping oxygen uncoupling (CLOU) and in situ gasification (iG-CLC) in the presence of steam. The Cu-based oxygen carriers showed high performance for the combustion of two solid fuels (a lignite and a bituminous coal), maintaining high thermal stability, fast reaction kinetics, and reversible oxygen release and storage over multiple redox cycles. Slight deactivation and sintering of the oxygen carrier occurred after redox cycles at an very high operation temperature of 985 °C. We expect that our material design strategy will inspire the development of better oxygen carrier materials for a variety of chemical looping processes for the clean conversion of fossil fuels with efficient CO2 capture.

Precursor engineering of hydrotalcite-derived redox sorbents for reversible and stable thermochemical oxygen storage.

Chemical looping processes based on multiple-step reduction and oxidation of metal oxides hold great promise for a variety of energy applications, such as CO2 capture and conversion, gas separation, energy storage, and redox catalytic processes. Copper-based mixed oxides are one of the most promising candidate materials with a high oxygen storage capacity. However, the structural deterioration and sintering at high temperatures is one key scientific challenge. Herein, we report a precursor engineering approach to prepare durable copper-based redox sorbents for use in thermochemical looping processes for combustion and gas purification. Calcination of the CuMgAl hydrotalcite precursors formed mixed metal oxides consisting of CuO nanoparticles dispersed in the Mg-Al oxide support which inhibited the formation of copper aluminates during redox cycling. The copper-based redox sorbents demonstrated enhanced reaction rates, stable O2 storage capacity over 500 redox cycles at 900 °C, and efficient gas purification over a broad temperature range. We expect that our materials design strategy has broad implications on synthesis and engineering of mixed metal oxides for a range of thermochemical processes and redox catalytic applications.

Protein Condensation, Cellular Organization, and Spatiotemporal Regulation of Cytoplasmic Properties.

The cytoplasm is an aqueous, highly crowded solution of active macromolecules. Its properties influence the behavior of proteins, including their folding, motion, and interactions. In particular, proteins in the cytoplasm can interact to form phase-separated assemblies, so-called biomolecular condensates. The interplay between cytoplasmic properties and protein condensation is critical in a number of functional contexts and is the subject of this review. The authors first describe how cytoplasmic properties can affect protein behavior, in particular condensate formation, and then describe the functional implications of this interplay in three cellular contexts, which exemplify how protein self-organization can be adapted to support certain physiological phenotypes. The authors then describe the formation of RNA-protein condensates in highly polarized cells such as neurons, where condensates play a critical role in the regulation of local protein synthesis, and describe how different stressors trigger extensive reorganization of the cytoplasm, both through signaling pathways and through direct stress-induced changes in cytoplasmic properties. Finally, the authors describe changes in protein behavior and cytoplasmic properties that may occur in extremophiles, in particular organisms that have adapted to inhabit environments of extreme temperature, and discuss the implications and functional importance of these changes.

Near-native state imaging by cryo-soft-X-ray tomography reveals remodelling of multiple cellular organelles during HSV-1 infection.

Herpes simplex virus-1 (HSV-1) is a large, enveloped DNA virus and its assembly in the cell is a complex multi-step process during which viral particles interact with numerous cellular compartments such as the nucleus and organelles of the secretory pathway. Transmission electron microscopy and fluorescence microscopy are commonly used to study HSV-1 infection. However, 2D imaging limits our understanding of the 3D geometric changes to cellular compartments that accompany infection and sample processing can introduce morphological artefacts that complicate interpretation. In this study, we used soft X-ray tomography to observe differences in whole-cell architecture between HSV-1 infected and uninfected cells. To protect the near-native structure of cellular compartments we used a non-disruptive sample preparation technique involving rapid cryopreservation, and a fluorescent reporter virus was used to facilitate correlation of structural changes with the stage of infection in individual cells. We observed viral capsids and assembly intermediates interacting with nuclear and cytoplasmic membranes. Additionally, we observed differences in the morphology of specific organelles between uninfected and infected cells. The local concentration of cytoplasmic vesicles at the juxtanuclear compartment increased and their mean width decreased as infection proceeded, and lipid droplets transiently increased in size. Furthermore, mitochondria in infected cells were elongated and highly branched, suggesting that HSV-1 infection alters the dynamics of mitochondrial fission/fusion. Our results demonstrate that high-resolution 3D images of cellular compartments can be captured in a near-native state using soft X-ray tomography and have revealed that infection causes striking changes to the morphology of intracellular organelles.

A method for the fast and photon-efficient analysis of time-domain fluorescence lifetime image data over large dynamic ranges.

Fluorescence lifetime imaging (FLIM) allows the quantification of sub-cellular processes in situ, in living cells. A number of approaches have been developed to extract the lifetime from time-domain FLIM data, but they are often limited in terms of speed, photon efficiency, precision or the dynamic range of lifetimes they can measure. Here, we focus on one of the best performing methods in the field, the centre-of-mass method (CMM), that conveys advantages in terms of speed and photon efficiency over others. In this paper, however, we identify a loss of photon efficiency of CMM for short lifetimes when background noise is present. We subsequently present a new development and generalization of CMM that provides for the rapid and accurate extraction of fluorescence lifetime over a large lifetime dynamic range. We provide software tools to simulate, validate and analyse FLIM data sets and compare the performance of our approach against the standard CMM and the commonly employed least-square minimization (LSM) methods. Our method features a better photon efficiency than standard CMM and LSM and is robust in the presence of background noise. The algorithm is applicable to any time-domain FLIM data set.

Nanoscale Features of Tunable Bacterial Outer Membrane Models Revealed by Correlative Microscopy.

The rise of antibiotic resistance is a growing worldwide human health issue, with major socioeconomic implications. An understanding of the interactions occurring at the bacterial membrane is crucial for the generation of new antibiotics. Supported lipid bilayers (SLBs) made from reconstituted lipid vesicles have been used to mimic these membranes, but their utility has been restricted by the simplistic nature of these systems. A breakthrough in the field has come with the use of outer membrane vesicles derived from Gram-negative bacteria to form SLBs, thus providing a more physiologically relevant system. These complex bilayer systems hold promise but have not yet been fully characterized in terms of their composition, ratio of natural to synthetic components, and membrane protein content. Here, we use correlative atomic force microscopy (AFM) with structured illumination microscopy (SIM) for the accurate mapping of complex lipid bilayers that consist of a synthetic fraction and a fraction of lipids derived from Escherichia coli outer membrane vesicles (OMVs). We exploit the high resolution and molecular specificity that SIM can offer to identify areas of interest in these bilayers and the enhanced resolution that AFM provides to create detailed topography maps of the bilayers. We are thus able to understand the way in which the two different lipid fractions (natural and synthetic) mix within the bilayers, and we can quantify the amount of bacterial membrane incorporated into the bilayer. We prove the system's tunability by generating bilayers made using OMVs engineered to contain a green fluorescent protein (GFP) binding nanobody fused with the porin OmpA. We are able to directly visualize protein-protein interactions between GFP and the nanobody complex. Our work sets the foundation for accurately understanding the composition and properties of OMV-derived SLBs to generate a high-resolution platform for investigating bacterial membrane interactions for the development of next-generation antibiotics.