Biology subjects

Bauer, R.

Publications and source records attributed to Bauer, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Survival of Streptococcus anginosus in blood is linked to hydrogen peroxide production

Streptococcus anginosus is an opportunistic pathogen causing bacteremia, respiratory tract infections, abscesses, odontogenic infections and has recently been associated with gastric cancer. While numerous genes encoding putative virulence factors have been detected in whole genome sequences, a detailed molecular characterization is missing for most of them. To address putative virulence determinants, we investigated hydrogen peroxide production by S. anginosus and assessed its functional significance for survival in human blood. In Streptococcus pneumoniae the pyruvate oxidase, encoded by the spxB gene, is responsible for H2O2 production and represents a major virulence factor. Interestingly, S. anginosus harbors two genetic variants of the spxB gene, one with high homology to the S. pneumoniae spxB (sequence identity: approx. 94 %) gene, while the other appears to be specific for S. anginosus. Strains carrying the S. pneumoniae variant demonstrate higher H2O2 production (10-20 M) compared to strains harboring the anginosus variant (<1 M). Incubation of S. anginosus strains in human blood showed that bacterial survival was correlated to the amount of hydrogen peroxide production. Assessing H2O2 production in an S. anginosus strain carrying a deletion of the CcpA regulator revealed the control of spxB and H2O2 production by carbon catabolite repression. In conclusion, we found evidence for the presence of a major S. pneumoniae virulence factor in S. anginosus, and we were able to demonstrate that it increases the ability of S. anginosus to survive in human blood.

microbiology↗

bioRxiv2026

Sex-Dependent Proteomic Remodelling During ex vivo Degeneration of Young and Aged Murine Peripheral Nerves

IntroductionPeripheral nerve ageing leads to profound proteomic remodelling, with shifts in metabolic and inflammatory signalling pathways resembling changes that occur during nerve degeneration and regeneration following injury. Moreover, aged nerves exhibit impaired degeneration and regeneration, contributing to age-related peripheral neuropathies that show sex-specific differences in prevalence. However, it remains unclear whether these alterations arise from intrinsic nerve changes or an altered systemic environment. Therefore, we investigated the impact of sex on age-related proteome changes and nerve-intrinsic proteomic responses in young and aged male and female nerves using an ex vivo degeneration model. MethodsMass spectrometry-based proteomics were performed on young and old nerves from male and female animals, as well as on contralateral nerves after seven days of ex vivo nerve degeneration. A comparative bioinformatic analysis was then used to identify changes during ageing and ex vivo nerve degeneration that were independent of sex, as well as changes that were sex-specific. ResultsEx vivo nerve degeneration induced extensive proteome remodelling in mouse sciatic nerves that was largely independent of age and sex. Principal component and clustering analyses clearly separated intact from degenerated nerves, while revealing only subtle age- and sex-related effects, with more pronounced ageing-associated changes in males. Approximately 20% of age-regulated proteins and 7-10% of degeneration-regulated proteins exhibited sex-specific expression patterns. Degeneration was characterised by increased abundance of lysosomal and repair-associated proteins alongside reduced myelin and axonal proteins, consistent with active tissue remodelling. In aged nerves, impaired protein clearance and partial pre-activation of degeneration-associated pathways suggested altered injury responses. Comparative analyses demonstrated positive correlations of protein abundance changes between ex vivo and in vivo degeneration datasets, although the temporal dynamics were altered in aged nerves. Pathway enrichment analyses identified coordinated regulation of metabolic, RNA-processing and vesicular transport pathways, while ageing was associated with enhanced immune signalling and reduced lipid metabolism. Sex-specific analyses revealed stronger inflammatory signatures in males, whereas females exhibited enrichment of metabolic pathways, including folate biosynthesis. ConclusionThese findings reveal distinct sex-specific molecular features of peripheral nerve ageing, characterised by enhanced inflammatory signalling in males and metabolic adaptations that may confer resilience in females. Our datasets provide a comprehensive molecular resource of sex-dependent changes in peripheral nerve ageing and nerve-intrinsic injury responses, offering a foundation for identifying therapeutic strategies to promote healthy peripheral nerve ageing. Plain English summaryAge-related peripheral neuropathies are common disorders that can cause pain, numbness, weakness and reduced mobility, affecting millions of people worldwide. They become more common from around the age of 50 and affect men and women differently. These conditions are thought to result from age-related changes in the structure and function of peripheral nerves, which reduce their ability to repair themselves after injury. In this study, we used advanced protein analysis (proteomics) to investigate how ageing affects peripheral nerves in male and female mice. We also used an ex vivo model, in which nerves are studied outside the body, to examine how age and sex influence the molecular changes that occur during nerve degeneration. We found that degeneration caused widespread changes in the proteins present in the sciatic nerve in both young and old mice. Most of these changes were similar in males and females, but some important differences emerged. Male nerves showed stronger signs of inflammation, whereas female nerves showed increased activity of metabolic pathways, including those involved in folate metabolism. Ageing nerves also appeared less able to remove damaged material and showed signs of activating degeneration-related processes even before injury. Overall, the ex vivo model reproduced many of the molecular changes seen after nerve injury in living animals, although it did not fully capture the inflammatory response, suggesting that signals from the rest of the body, including factors carried in the blood, also contribute to nerve degeneration. HighlightsO_LIEx vivo nerve degeneration caused major protein changes in young and old mouse sciatic nerves. C_LIO_LIMost degeneration-related protein changes were shared between males and females. C_LIO_LIAgeing altered the nerve proteome, with stronger ageing-related shifts in males. C_LIO_LIMale nerves showed stronger inflammatory and immune-related signatures. C_LIO_LIFemale nerves showed enrichment of metabolic pathways, including folate biosynthesis, and ex vivo degeneration did not fully reproduce the inflammatory response seen after injury in vivo. C_LI

neuroscience↗

bioRxiv2026

A Multicenter Confirmatory Randomized-Controlled Study of rhNRGβ1 Protein Replacement Therapy in a Murine Model of NF2-related Schwannomatosis

BackgroundPrevious exploratory studies identified recombinant human Neuregulin-1 {beta} (rhNRG{beta}1) as a promising therapeutic strategy for inhibiting the growth of Nf2-deficient schwannomas by promoting cellular differentiation. Because robust confirmation across independent laboratories is essential for advancing promising preclinical findings toward clinical translation, we conducted a multicenter, randomized, controlled confirmatory study under stringent preclinical standards. MethodsIn a pre-registered trial (DOI: 10.17590/asr.0000304), 216 mice (Nf2-flox;P0-Cre;Nefh- Cre) were randomized at three independent research sites. Following a standardized sciatic nerve crush, mice received systemic rhNRG{beta}1 (10 {micro}g/kg) or vehicle for 13 weeks. Rigorous quality measures included double-blinding, standardized surgery, centralized data management, and an automated Fiji macro for objective nerve thickness quantification (Primary Outcome). Secondary molecular outcomes included Western blot and in-depth, quantitative proteomics and phosphoproteomics. All methods were SOP-based for reproducible and comparable results across the three study centers ResultsThe primary confirmatory analysis revealed no reduction in nerve thickness in the rhNRG{beta}1 group (pbest case imputation = 0.076 and pworst case imputation = 0.533). Secondary analyses via quantitative Western blotting and DIA proteomics demonstrated that core biochemical markers of Schwann cell differentiation (MBP, ERBB2) remained unchanged across all centers. Based on the absence of macroscopic or primary biochemical effects, further histological analysis was omitted to avoid scientific redundancy. High-depth profiling of a predefined 60-protein functional marker panel confirmed a remarkably stable tumor proteome across all replication sites and both sexes, with no evidence of coordinated changes in key downstream oncogenic signaling pathways (Hippo/YAP, mTORC1, and RTK-Ras-MAPK) or metabolic signaling cascades. These findings indicate an absence of measurable target engagement under our tested dosing regimen, potentially reflecting pharmacokinetic or tissue-delivery limitations rather than an invalidation of the underlying biological pathway. ConclusionDespite high statistical power and rigorous methodology, this study could not confirm rhNRG{beta}1 as a robust therapeutic candidate for schwannoma growth arrest or shrinkage. These findings suggest that previously reported therapeutic effects were either highly context- dependent or could not be reproduced under adequately powered, rigorously controlled experimental conditions. As underpowered preclinical studies are more susceptible to random biological variation, our results highlight the importance of sufficient sample sizes alongside robust experimental design. Our study underscores the value of trial-like methodological standards in preclinical therapeutic evaluation to identify ineffective interventions (dead ends) early and strengthen translational decision-making. Although we could not confirm the previously reported efficacy of rhNRG{beta}1, the multicenter framework established here provides a methodological benchmark for robust preclinical testing in translational oncology, with the potential to improve reproducibility and the success of therapies progressing to early-phase clinical trials. From a translational perspective, these findings provide a robust foundation for optimizing future rhNRG{beta}1-based therapeutic approaches through improved dosing, delivery routes, and treatment schedules.

cancer biology↗

bioRxiv2026

Genetic interaction approaches reveal emerging roles of innexins in development : Insights from a novel pannier–innexin-2 interaction during Drosophila embryogenesis

Effective communication between cells is essential for the typical development and behaviour of an organism. In this context, gap junctions represent the most universally preserved components at cellular membranes of multicellular organisms, facilitating metabolic and electrical connections between cells. Disruptions in these junctions have been linked to various developmental abnormalities and pathological conditions in humans. The invertebrate gap junction proteins, referred to as innexins, exhibit conserved cellular and molecular mechanisms of functioning with their vertebrate counterparts, known as connexins. Consequently, they provide valuable means for studying and understanding the functions of gap junctions in development. In the Drosophila embryo, innexin-2 is expressed in the amnioserosa and ectoderm, where it is required for epithelial morphogenesis. Genetic depletion of innexin-2 results in cuticular defects and embryonic lethality. Pannier, a GATA family transcription factor, is a key regulator of dorsal tissue development in Drosophila and is expressed in the amnioserosa, dorsal ectoderm and the dorsal vessel during embryogenesis. Pannier mutants exhibit defects in dorsal closure, cuticle formation, and cardiac specification. Although substantial evidence from vertebrate systems indicate that connexin expression is regulated by transcription factors such as GATA4, Nkx2.5, Tbx2, Tbx3, and Tbx5, whether a similar regulatory relationship exists between these transcription factors and gap junction proteins in Drosophila remains unknown. In this study, we investigate how innexin mediated intercellular communication impacts pannier dependent morphogenetic processes during Drosophila embryogenesis.

Developmental Biology↗

bioRxiv2026

A rapid, low-cost approach to solid immersion lens fabrication for enhanced resolution in optical microscopy

Solid immersion lenses (SILs) enhance the spatial resolution of an optical microscope by increasing the effective numerical aperture (NA) without physical modification of the objective lens. However, SIL application remains limited by cost, fragility, and accessibility. We present a rapid, single-step fabrication process to create optical quality hemispherical SILs using consumer-grade UV-curable transparent resin which reduces material costs by over five orders of magnitude relative to commercial glass counterparts. Our method produced resin SILs within seconds which can be easily implemented into conventional microscopy setups for increasing the effective NA. Quantitative imaging of USAF resolution targets and histology muscle preparations demonstrated a resolution enhancement approaching theoretical limits and comparable performance to N-BK7 glass SILs. This enabled visualisation of features usually below the diffraction limit of low NA dry objectives at a fraction of the cost of otherwise required high-powered objective lenses. To demonstrate accessibility and translational potential, our workflow was taught in a practical tutorial of an international microscopy course, where non-expert participants successfully fabricated, characterised, and applied SILs within a single session, reporting high confidence in independent implementation. We established ultra-low-cost resin SILs as a practical, scalable option to enhance the spatial resolution of routine optical microscopes and as an accessible and cost-effective platform for optics education.

biophysics↗

bioRxiv2026

Membrane lipid composition and endocytosis modulate Wingless release from secreting cells

Wnts are secreted signalling molecules that regulate development and adult homeostasis. Most Wnts carry a lipid moiety that must be shielded from the aqueous environment. In the secretory pathway, this is achieved by a hydrophobic tunnel in Wntless, a multipass transmembrane protein. However, the Wnt lipid moiety must be released from Wntless before Wnts can engage with Frizzled receptors on receiving cells. Here we address the cell biological basis of Wnt-Wntless dissociation, using as a model the secretion of Drosophila Wingless in wing primordia. Super-resolution microscopy shows that Wingless first reaches the apical surface before being re-internalized to reach, without Wntless, specialized Rab7/Rab4-positive endosomes. From there Wingless traffics to the basolateral membrane where it can engage with glypicans to form a basolateral gradient. Acute inhibition of endocytosis, either with a temperature-sensitive dynamin mutant or a novel optogenetic means of inhibiting clathrin, leads to apical Wingless release in abnormal punctae devoid of Wntless, suggesting that Wingless-Wntless dissociation commences at the apical surface, perhaps because of a distinct lipid composition there. Indeed, similar looking punctae are produced upon genetic abrogation of the ceramide synthase Schlank, specifically in Wingless-producing cells. These punctae resemble insoluble aggregates that form in vitro upon detergent removal. Accordingly, punctae formation can be prevented by shielding the Wingless lipid, in vivo with excess Dally-like protein (Dlp) or in vitro with liposomes. Our results show that membrane lipid composition modulates the orderly transfer of Wingless lipid from Wntless to the inner endosomal surface thus preventing aggregation and ensuring seamless secretion in the basolateral space.

cell biology↗

bioRxiv2026

Convergent molecular signatures of ageing and injury in the peripheral nervous system

Peripheral nervous system (PNS) ageing is marked by structural and functional decline, yet it remains unclear whether ageing constitutes a distinct biological programme or reflects a chronic injury-like state. To address this, we performed an unbiased, comparative molecular analysis of PNS ageing, neuroprotective dietary restriction (DR), and nerve injury. We conducted transcriptomic and proteomic profiling of peripheral nerves from young, old and geriatric mice fed ad libitum or subjected to long-term DR, and proteomics of nerves collected at multiple time points following injury. Age-associated molecular changes followed both linear and non-linear trajectories, and DR partially attenuated these ageing-related alterations. Notably, ageing-and injury-induced proteomic signatures showed considerable similarities, supporting the concept that an aged nerve resembles an injured nerve. Together, our study provides the most comprehensive molecular resource of PNS changes during ageing, DR, and injury, enabling the definition of key molecular signatures underlying PNS physiology. All datasets are integrated into the "PNS-omics Viewer", a Shiny web application designed to facilitate data mining of the herein presented datasets (tba).

neuroscience↗

bioRxiv2025

Distinct Effects of Aging and Klotho Deletion on the Choroid Plexus

Klotho (Kl) is an anti-aging protein primarily produced in the kidney and the choroid plexus (CP, where it regulates cerebrospinal fluid composition and exerts neuroprotective effects. Here, we investigated the age-dependent consequences of a CP-specific Kl deletion on CP structure and function using mice lacking KL exclusively in CP epithelial cells (Kl{Delta}CP). In control mice, aging markedly disrupted CP architecture and cilia organization both in the lateral (LV-CP) and fourth ventricle (FV-CP). While CP-specific Kl deletion alone caused no major structural changes it induced region- and age-dependent calcification: FV-CP calcification increased in both aged and young Kl{Delta}CP mice, whereas LV-CP calcification emerged only in older Kl{Delta}CP mice. Proteomic analysis of the CP and hippocampus revealed mild molecular alterations, suggesting compensatory mechanisms that preserve structural and functional stability despite calcium dysregulation. Consistently, Kl{Delta}CP mice exhibited no significant behavioral or cognitive deficits. Overall, Kl deficiency sensitizes the CP to age-related calcification prior to overt structural decline, revealing a region-specific and functional link between Klotho, calcium imbalance, and brain aging.

neuroscience↗

bioRxiv2025

Low-Cost 3D Printed Optics for Super-Resolution Multifocal Structured Illumination Microscopy

We present a low-cost 3D printing method of fabricating optical quality lenslet arrays for integration in a multifocal structured illumination microscope (mSIM), achieving fluorescence imaging below the optical diffraction limit. We detail the design and manufacturing processes to produce high-quality 3D printed optics, showing their comparable surface roughness of 30 {+/-} 2.5 nm for the 3D printed elements compared to 37 {+/-} 1.4 nm for commercial glass optics. A 3D printed lenslet array with a honeycomb geometry and 1.2 mm lenslet diameter was compared to a high-end glass commercial lenslet array with 250 {micro}m lenslet diameter and a lower cost commercial lenslet array with a 1.2 mm by 1.6 mm lenslet footprint. The imaging performance of the different optics was benchmarked using a custom mSIM setup by quantifying the beam profile homogeneity and the experimental lateral resolution. The mSIM setup incorporating the different microlens arrays was tested using a commercial bovine pulmonary artery endothelial cell specimen, highlighting an achievable resolution enhancement from 229 nm {+/-} 11 nm with widefield illumination to 137 {+/-} 11 nm using the high-end commercial microlens array and 134 nm {+/-} 9 nm using the 3D printed honeycomb lenslet array. Advantages of improved background rejection through the custom lenslet geometry are discussed, highlighting the super-resolution microscope performance achievable through custom low-cost 3D printed optics.

biophysics↗

bioRxiv2025

Spatial Expression Pattern and Cellular Organisation of Gap Junctions in Third Instar Wing Imaginal Discs of Drosophila melanogaster

The Drosophila wing imaginal disc serves as a powerful model to study intercellular communication during development. In our study, we report and discuss the expression pattern and cellular distribution of innexin-1, innexin-2 and innexin-3 in the cells of the wing imaginal discs. Our immunohistochemical data show that all three innexins are broadly expressed across the membranes of both the disc proper and peripodial epithelial cells of the wing disc. The stainings further reveal that, within the disc proper epithelium, junctional proteins are arranged in a clear apico-basal hierarchy: cadherins at the apical surface, followed by septate junction proteins, with innexins localised sub-apically beneath these components. All three innexins are enriched within this sub-apical domain, and are additionally detected at mid-and baso-lateral sites in varying levels. Notably, innexin-2 exhibits partial colocalization with coracle, a septate junction-associated protein, suggesting a functional association. In the peripodial epithelium, innexins are detected in distinct punctate patterns across cell membranes, implying heterogeneity in their molecular characteristics. To validate these expression patterns, we carried out tissue-specific RNAi-mediated knockdowns using the pannier-Gal4 driver targeting the notum, a structurally and functionally important but underexplored region in innexin research. Knockdown of innexin-2 and innexin-3 led to complete loss of their expression within this region. Notably, silencing of innexin-2 also affected the expression of septate junction associated proteins and innexin-3 knockdown was accompanied by a significant reduction in disc size and altered morphology. These findings depict and confirm the presence of innexins in the notum region and also indicate that individual innexins may have distinct or shared functional roles within the same tissue domain of expression. Their localization to specific membrane domains is likely to underlie their differential modes of action. Although previous studies have demonstrated the functional involvement of gap junctions in various aspects of normal wing development in Drosophila, a description of the arrangement of innexins on the third instar wing discs is required for better understanding of their roles. Our study addresses this gap by providing a comprehensive analysis of the cellular localisation and organisation of gap junctions, specifically innexin-1,-2 and-3, within the third instar wing discs, thereby supporting and extending existing knowledge.

cell biology↗

bioRxiv2025

Targeting the homeodomain of ceramide-synthase can ameliorate insulin resistance

Multiple studies have linked ceramide accumulation with insulin resistance and diabetes. Ceramide Synthases (CerS) are at the center of ceramide de novo formation. Impaired CerS activity leads to lower ceramide and resolves insulin resistance. Drosophila has only one CerS, named Schlank, which contains a catalytic lag1p motif and, like many CerS, a homeodomain regulating lipid homeostasis. How CerS homeodomains are associated with diabetes has been little studied. Here we demonstrate that, depending on the respective mutation in the CerS homeodomain high sugar diet (HSD)-induced insulin resistance is exacerbated or ameliorated. HSD shifts the profile of sphingolipids towards polyunsaturated longer sphingoid bases, systemic insulin signaling is reduced, as indicated by nuclear accumulation of FoxO, and secretion of insulin-like peptide 2 (DILP2) is impaired. Expression of a CerS variant with a mutation in the nuclear localization signal 2 within its homeodomain in the fat body improves systemic insulin signaling and DILP2 release. Thus, the CerS homeodomain may be a potential target to attenuate insulin resistance.

genetics↗

bioRxiv2025

Open Benchmarking for Cell-Based Multiscale Models: Lessons from a Community Initiative

The emergence of virtual human twins (VHT) in biomedical research has sparked interest in multiscale in silico modelling frameworks, particularly in their application bridging cellular to tissue levels. Among the diverse array of multiscale modelling tools, off-lattice center-based agent-based models (CBM) offer a promising approach due to their depiction of cells in 3D space, closely resembling biological reality. Despite the proliferation of CBM tools addressing various biomedical challenges, a comprehensive and systematic comparison among them has been elusive. This paper presents a community-driven benchmark initiative aimed at evaluating and comparing CBM for biomedical applications, akin to successful efforts in other scientific domains such as the Critical Assessment of Protein Structure Prediction (CASP). Enlisting developers from leading tools like BioDynaMo, Chaste, PhysiCell,TiSim, and CompuTiX, we devised a benchmark scope, defined metrics, and established reference datasets to ensure a meaningful and equitable evaluation. Unit tests targeting different solvers within these tools were designed, ranging from diffusion and mechanics to cell cycle simulations and growth scenarios. Results from these tests demonstrate varying tool implementations in handling diffusion, mechanics, and cell cycle equations, emphasising the need for standardised benchmarks and interoperability. Discussions among the community underscore the necessity for defining gold standards, fostering interoperability, and drawing lessons from analogous benchmarking experiences. The outcomes, disseminated through a public platform in collaboration with OpenEBench, aim to catalyse advancements in computational biology, offering a comprehensive resource for tool evaluation and guiding future developments in cell-level simulations. This initiative endeavours to strengthen and expand the computational biology simulation community through continued dissemination and performance-oriented benchmarking efforts to enable the use of VHT in biomedicine.

systems biology↗

bioRxiv2025

A large 3D-printed integrated lens-biprism element enhances contrast in transmission stereomicroscopy

Stereomicroscopes are routinely used across disciplines for material and surface characterisation due to their simplicity of use and minimal specimen preparation requirements. However, the stereomicroscope transillumination design is suboptimal, as a single incident beam at the specimen plane is shared and transmitted via two laterally offset detection axes. This flaw limits life science applications due to the transparent nature of samples which results in poor contrast images. We use a single additional element in the illumination path to correct the illumination uniformity across the field of view and, by doing so, enhance image contrast and facilitate detection of refractive structures in transparent biological specimens. We designed and fabricated an integrated lens-biprism element using low-cost, consumer-grade 3D printing methods and consumables. This 3D printed lens-biprism distributed diverging rays from a single incandescent light source into two parallel beams that converged at the specimen plane and transmitted through the respective left and right detection axes of a stereomicroscope. This improved transillumination setup increased the image contrast by up to 67.62% compared to the conventional stereomicroscope setup. We demonstrated the benefit of the lens-biprism element by visualising dynamic cellular events in live tissue and discerning refractive structures more easily in transparent specimens.

biophysics↗

bioRxiv2025

Coupled equilibria of dimerization and lipid binding modulate SARS Cov 2 Orf9b interactions and interferon response

Open Reading Frame 9b (Orf9b), an accessory protein of SARS-CoV and -2, is involved in innate immune suppression through its binding to the mitochondrial receptor Translocase of Outer Membrane 70 (Tom70). Previous structural studies of Orf9b in isolation revealed a {beta}-sheet-rich homodimer, however, structures of Orf9b in complex with Tom70 revealed a monomeric helical fold. Here, we developed a biophysical model that quantifies how Orf9b switches between these conformations and binds to Tom70, a requirement for suppressing the type 1 interferon response. We used this model to characterize the effect of lipid binding and mutations in variants of concern to the Orf9b:Tom70 equilibrium. We found that the binding of a lipid to the Orf9b homodimer biases the Orf9b monomer:dimer equilibrium towards the dimer by reducing the dimer dissociation rate [~]100-fold. We also found that mutations in variants of concern can alter different microscopic rate constants without significantly affecting binding to Tom70. Together our results highlight how perturbations to different steps in these coupled equilibria can affect the apparent affinity of Orf9b to Tom70, with potential downstream implications for interferon signaling in coronavirus infection.

biochemistry↗

bioRxiv2025

SEVI Fibrils are Induced by Bacterial Surface Molecules and Exert Antimicrobial Activity Against ESKAPE Pathogens

The semen-derived enhancer of viral infection (SEVI) is an amyloid fibril formed by the self-assembly of the PAP248-286 peptide, a cleavage product of prostatic acid phosphatase, which is naturally present in human seminal plasma. Beyond its previously described role in HIV transmission, findings highlight a physiological function for SEVI in innate immunity. As many amyloids display antimicrobial effects, we tested SEVI fibrils for antibacterial activity against microbial ESKAPE pathogens and urogenital bacteria, including Pseudomonas aeruginosa, Klebsiella quasipneumoniae, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes. SEVI exhibited direct dose-dependent antibacterial effects in radial diffusion and survival assays, with activity observed at physiologically relevant concentrations. Bacterial surface molecules such as lipopolysaccharides and lipoteichoic acid induced the formation of SEVI fibrils, as confirmed by kinetic assays. Preincubation with epigallocatechin gallate, a fibril disruptor, abolished SEVIs antibacterial activity, pointing to the importance of its fibrillar structure. Mechanistic studies and electron microscopy revealed limited bacterial membrane disruption and the intracellular accumulation of polyphosphate granules in P. aeruginosa, indicating a stress response. In conclusion, SEVI exerts a potent antibacterial activity against pathogens found in the urogenital tract, indicating a potential physiological role in vaginal mucosal immunity.

microbiology↗

bioRxiv2024

A fully 3D-printed optical microscope for low-cost histological imaging

We present the manufacture and characterisation of a fully 3D printed, low-cost optical microscope using both a 3D printed chassis and 3D printed illumination and imaging optics. The required commercial components, consisting of a basic camera for image acquisition and light emitting diode controlled by a Raspberry Pi for illumination, are integrated into the 3D printed microscope with the full design shown for ease of replication. Our 3D printed microscope uses a single 3D printed objective lens with a 2.9x magnification and a numerical aperture of 0.07. To benchmark the imaging performance of the system, we used standard test targets and histological specimens, namely, a Giemsa-stained blood smear sample and a thin section of mouse kidney stained with Haemotoxylin and Eosin. We demonstrated that sub-cellular resolution was obtained, and we corroborated this by imaging individual red blood cells and intricate anatomical details of the stained mouse kidney section. All of this was achieved using entirely 3D printed hardware and optics, at a fraction of the cost of a commercial brightfield microscope, while presenting remarkable potential for customisation and increased accessibility for diagnostic imaging applications.

biophysics↗

bioRxiv2024

Lipid Disbalance Affects Neuronal Dendrite Growth and Maintenance in a Human Ceramide Synthase Disease Model

The brain is susceptible to disturbances in lipid metabolism. Among the rare, genetically-linked epilepsies Progressive Myoclonic Epilepsy Type 8 (PME8), associated with the loss of Ceramide Synthase (CerS) activity, causes epileptic symptoms accompanied by neurodegenerative traits. We show that expression of a disease-causing cerS allele in Drosophila sensory neurons yielded developmental and degenerative dendrite loss. In cerS mutants, C18-C24 ceramides and membrane-forming complex sphingolipids, into which ceramides are converted, were reduced. At the same time bioactive signaling lipids including (dh)Sphingosine-1-P, deriving from the CerS substrate, were increased. To clarifying the etiology of PME8, we thus performed in vivo experiments to cell-autonomously rescue the individual metabolic alterations. We report that restoring specific long-chain ceramides while in parallel decreasing (dh)Sphingosine-1-P fully rescues the cerS mutant phenotype. Thus, despite the complex metabolic alterations, our data provide essential information about the metabolic origin of PME8 and delineate a potential therapy.

neuroscience↗

bioRxiv2024

G-CSF and IL-6 drive myeloid dysregulation during severe viral infection

Dysregulated myeloid states are associated with disease severity in both sepsis and COVID-19. However, their relevance in non-COVID-19 viral infection, the factors driving their induction, and their role in tissue injury remain poorly understood. We performed a meta-analysis of 1,622,180 myeloid cells from 890 COVID-19 or sepsis patients and controls across 19 published blood scRNA-seq datasets, which revealed severity-associated gene programs in both neutrophils and monocytes pointing to emergency myelopoiesis (EM). Using published bulk transcriptional data from 562 individuals with non-COVID-19 viral disease, we show that these signatures are similarly upregulated during severe influenza and RSV infection. Analysis of transcriptional and proteomic responses in tocilizumab-treated COVID-19 patients show that IL-6 signaling blockade results in a partial reduction of EM signatures and a compensatory increase in the growth factor G-CSF. Using a cellular model of human myelopoiesis, we show that both IL-6 and G-CSF stimulate the production of myeloid cells that express EM signatures in vitro. Using a mouse model of severe influenza infection, we demonstrate the effect of IL-6 and G-CSF signaling blockade on EM-associated myeloid cells, and highlight the opposing effects of EM-induced neutrophils and monocytes on tissue injury. Our study demonstrates the link between systemic cytokines and myeloid dysregulation during severe infection in humans, and highlights the cooperative role of IL-6 and G-CSF signaling in driving infection-induced myelopoiesis.

immunology↗