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Müller, P.

Publications and source records attributed to Müller, P..

At least 19 recordsLinked to original sources
bioRxiv2026

Temperature scaling of developmental tempo reveals reaction-dominated morphogen patterning

A central question in developmental biology is how embryonic patterning remains robust when developmental tempo changes. In ectotherms, temperature strongly alters developmental speed and biochemical reaction rates, whereas diffusion is expected to change only weakly. How morphogen gradients required for patterning remain correctly scaled under these conditions is unclear. We addressed this problem in medaka embryos by quantifying developmental speed, morphogen clearance, diffusion, and endogenous Nodal dynamics across a broad temperature range. Developmental tempo, morphogen production, clearance, and tissue movement increased approximately two- to threefold per 10{degrees}C, whereas effective morphogen diffusion remained largely unchanged. Despite this mismatch, germ-layer proportions and morphogen patterning remained robust. Our mathematical simulations suggest that this robustness arises because the system operates in a reaction-dominated regime, in which changes in reaction and growth kinetics - rather than diffusion - are sufficient to rescale morphogen gradients. Together, our experiments and simulations provide new insights into how morphogen gradients adjust to maintain accurate tissue patterning across changes in developmental time and tempo.

developmental biology↗
bioRxiv2026

Multimodal 3D imaging reveals a central role for lysosomes indissolution of cholesterol crystals by macrophages

Formation of cholesterol crystals (CCs) is a key event during the development of atherosclerosis, but the molecular mechanisms of their degradation within cells are poorly understood. By incorporating the fluorescent cholesterol analogue TopFluor-Cholesterol (TF-Chol) into CCs, we were able to visualize the uptake of CCs in macrophages using correlative fluorescence and soft X-ray microscopy. Using quantitative 3D live-cell imaging, we show that CCs are processed in late endosomes and lysosomes (LE/Lys), resulting in formation of TF-Chol containing lipid droplets (LDs) over time. Inhibition of lysosomal sterol export with U18666A caused accumulation of TF-Chol in LE/Lys, and inhibition of lysosomal acidification with bafilomycin A1 led to reduced dissolution of the CCs. Using a novel assay combined with 3D image processing, we show that large CCs in contact with macrophages are processed via lysosomal exocytosis followed by extracellular and intracellular degradation of CCs. Treating macrophages with a fluorescent version of cyclodextrin (CD) promoted the dissolution of CCs and enhanced the formation of LDs enriched with TF-Chol. The majority of fluorescent CD co-localized with a marker for LE/Lys during this process, suggesting that intracellular delivery to LE/Lys may contribute to the dissolution of CCs. Dehydroergosterol (DHE) is an intrinsically fluorescent sterol closely mimicking the properties and behavior of cholesterol. DHE is known to self-associate into aggregates and crystals, and by using fluorescence spectroscopy and specialized ultraviolet (UV) microscopy, we found that CD enhances the dissolution of DHE crystals in vitro and in cells. Together, our findings highlight the lysosomal pathway as responsible for dissolution of CCs, cholesterol trafficking, and efflux in macrophages.

biochemistry↗
bioRxiv2026

Antibody-mediated inhibition of ADAMTS-7 reduces experimental atherosclerosis

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

physiology↗
bioRxiv2026

Single-cell profiling reveals tumor grade-dependent immune remodeling in BRAFV600E-driven glioneuronal tumors

BRAFV600E is the key driver variant in epilepsy-associated glioneuronal tumors (GNTs). These tumors often share MAPK/PI3K hyperactivation, a generally benign biological course and rare occurrence of malignant variants. We aimed to characterize the poorly defined immune cell milieu of GNTs with distinct biological behavior. We mapped cellular heterogeneity of the tumor microenvironment (TME) using single-cell transcriptomics on murine models of low-grade (LG-GNT; BRAFV600E/AKTA) and high-grade (HG-GNT; BRAFV600E/AKTA/Trp53KO) tumors, generated via intraventricular in utero electroporation (IUE). Furthermore, ex vivo functional assays with CSF1R-mediated myeloid depletion were utilized to assess the role of identified signaling molecules on tumor viability. We observed a striking, grade-dependent immunological dichotomy: LG-GNT exhibited a permissive niche with prominent surveillance by T cells and pro-inflammatory microglia. In contrast, HG-GNT TME was characterized by a restricted T cell infiltration, massively dominated by myeloid cell infiltrates. Differential gene expression analysis identified Spp1 (osteopontin) as a key mediator of this immunosuppressive HG-GNT TME, exclusively expressed in microglia and border-associated macrophages (BAMs). Crucially, ex vivo functional assays demonstrated that recombinant SPP1 enhances tumor viability through a paracrine mechanism. These findings suggest fundamentally distinct immune activation (a) stimulated by aberrant MAPK/PI3K signaling in LG-GNT, versus (b) malignant tumor feature-driven, e.g. through necrosis in HG-GNT. In the latter, SPP1 signaling creates the immunosuppressive niche. Consequently, while modulating the pro-inflammatory niche may mitigate tumor-related epileptogenicity in LG-GNTs, targeting the SPP1-myeloid axis may restore anti-tumor immunity in HG-GNTs.

neuroscience↗
bioRxiv2025

An RNA ligase shapes transcriptional profiles, neural function, and behaviour in the developing larval zebrafish

RNA ligases are essential for the repair, splicing, and editing of RNA across various biological systems. Recently, a new enzyme that catalyses 5-3 RNA ligation - RNA ligase 1 (Rlig1) - was identified in vitro. However, the in vivo biological functions of Rlig1 have remained elusive. Here, we reveal the role of Rlig1 during vertebrate development using embryonic and larval zebrafish as a model system. We found that rlig1 mRNA is maternally deposited and present ubiquitously during early embryogenesis, whereas at larval stages it localises to the brain and eyes. Interestingly, CRISPR/Cas9-generated rlig1 knockout zebrafish exhibited no overt morphological abnormalities, but showed reduced behavioural responsiveness to visual stimuli along with massively perturbed transcriptomes and widespread dysregulation of core metabolic and translational pathways. Brain-wide calcium imaging in rlig1 knockout larvae revealed decreased neuronal activity in key regions for visual processing, consistent with the observed behavioural defects. Together, our findings identify a role for Rlig1 in maintaining the integrity and function of the nervous system and uncover a new link between neuronal RNA processing, development, and sensory-motor computation.

developmental biology↗
bioRxiv2025

Genomic Evidence for a Novel Introduction and Intra-host Diversity of DENV-2 in Dar es Salaam, Tanzania

Dengue virus (DENV) poses a growing risk in Tanzania, yet its diversity in mosquitoes is poorly understood. Using Nanopore sequencing, we recovered full coding sequences from six DENV-2 positive mosquito pools collected in Dar es Salaam outside outbreak periods. Phylogenetic analysis placed the sequences in a distinct monophyletic clade within genotype II, separate from strains linked to Tanzanias 2014 outbreak. Instead, they clustered with Asian lineages, showing closest relatedness to DENV-2 from Kenya (2013) and India (2014), with divergence estimated around 2010. Variant profiling identified 212 low-frequency intra-host variants, largely nonsynonymous changes in NS3 and NS5. These results suggest an unrecognized introduction of genotype II now circulating silently in local mosquito populations. Our findings highlight the value of genomic surveillance in vectors for early detection of arboviral threats, even when human cases are absent.

genomics↗
bioRxiv2025

Mosquito diversity and arbovirus circulation at the Taï National Park, western Cote d'Ivoire

Understanding the ecological and virological dynamics of mosquito populations across different landscapes is essential for predicting and mitigating the risk of arbovirus spillover. From December 2021 to September 2022, we conducted an entomological and molecular survey across sylvatic, transition, and urban zones in the Tai region of western Cote dIvoire. We collected 4,414 mosquitoes, representing 29 taxa, and grouped them into 1,553 pools of the same taxa for virus detection and identification. While Eretmapoditis sp. were dominant across all zones, their species composition varied between the zones, with Er. fraseri being most abundant in the sylvatic and urban zones. In the transition zone Er. quinquevittatus was the dominant species. Abundance of other species also varied across zones. Aedes aegypti and Anopheles sp. were most frequent in the urban zone with An. gambiae s.l. and An. paludis being the most common ones. Molecular screening revealed flavivirus RNA being present in multiple pools, with the highest detection rates in the transition zone. We were able to assemble three flavivirus contigs, including Cimo flaviviruses II and VIII, known as insect-specific flaviviruses that can influence vector competence. Notably, Anopheles sp. had the highest estimated infection rates in the sylvatic and transition zones, suggesting a potential role in arbovirus ecology. Our findings suggest that Eretmapodites sp., particularly Er. fraseri, may serve as key bridge vectors due to their widespread presence, mammalian feeding behaviour and potential virus competence. The transition zone emerged as a hotspot for arbovirus diversity and vector-host interactions. This study provides the first comprehensive ecological and molecular characterization of mosquito communities in the Tai region since more than twenty years and underscores the importance of longitudinal and habitat-specific surveillance for anticipating spillover risks. Author summaryMosquitoes are important to study because they can carry viruses that spread to humans and animals. These viruses, known as arboviruses, often come from wildlife and can spill over into people, especially when natural and human environments overlap. From late 2021 to 2022, we collected and studied mosquitoes in western Cote dIvoire, across forest, village-forest edges, and towns. We wanted to understand which mosquito species were present, how their populations varied across different landscapes, and whether they carried any viruses. We found that one group of mosquitoes, called Eretmapodites, was common in all areas, with different species dominating in different zones. We also found that some mosquito groups, including Anopheles, carried viruses that could potentially affect humans or influence how diseases spread. The area between forest and town, the transition zone, showed the greatest variety of viruses and mosquitoes. Our work helps show how changes in land use and mosquito ecology can affect the risk of disease outbreaks. By understanding where and when mosquitoes are most likely to carry viruses, we can improve surveillance and better prepare for future disease emergence. This is especially important in regions where people and wildlife live close together.

ecology↗
bioRxiv2025

High-Throughput Mechanomic Screening Reveals Novel Regulators of Single-Cell Mechanics

The mechanical properties of cells are dynamic, allowing them to adjust to different needs in different biological contexts. In recent years, advanced biophysical techniques have enabled the rapid, high-throughput assessment of single-cell mechanics, providing new insights into the regulation of the mechanical cell phenotype. However, the molecular mechanisms by which cells maintain and regulate their mechanical properties remain poorly understood. Here, we present a genome-scale RNA interference (RNAi) screen investigating the roles of kinase and phosphatase genes in regulating single-cell mechanics using Real-Time Fluorescence and Deformability Cytometry (RT-FDC). Our screen identified 80 known and novel mechanical regulators across diverse cellular functions from 214 targeted genes, leveraging RT-FDCs unique capabilities for comprehensive, high-throughput mechanical phenotyping with single-cell and cell cycle resolution. These findings refine our understanding of how signaling pathways coordinate structural determinants of cell mechanical phenotypes and provide a starting point for uncovering new molecular targets involved in biomechanical regulation across diverse biological systems. SIGNIFICANCECell mechanical properties are tightly regulated and play pivotal roles in processes ranging from tissue morphogenesis to disease progression. Despite their importance, the genetic regulation of single-cell mechanics remains largely unexplored. This study represents one of only a few large-scale mechanomic investigations conducted to date. It is the first study to leverage RT-FDCs unique capability for high-throughput mechanical phenotyping with single-cell and cell cycle resolution to detect gene impacts that may be overlooked in lower-throughput or population-level studies. The mechanical genes identified here provide valuable data points for understanding how cells control their mechanical state and serve as a foundation for future studies exploring the molecular basis of biomechanical regulation.

biophysics↗
bioRxiv2024

Spatial and temporal characteristics of laboratory-induced Anopheles coluzzii swarms: shape, structure and flight kinematics

Anopheles mosquitoes mate at sunset in aerial swarms. The development of mating-based methods for effective malaria vector control requires a good knowledge of the flight behaviour of Anopheles species in mating swarms. However, the process of how swarms are formed and maintained remains poorly understood. Here, we characterized the three-dimensional spatial and temporal flight kinematics of Anopheles coluzzii males swarming above a ground marker. We observed that the location, shape and volume of swarms were highly stereotypic, consistent over the swarming duration, regardless of the number of individuals in the swarm. The swarm had an elliptical cone shape, and we observed a differential spatial distribution of flight kinematics parameters within the swarm volume. Among these parameters, only swarm density varied with swarm size. Using a sensory system-informed model, we show that swarm location and shape can accurately be modelled based on visual perception of the marker. To control swarm height, swarming individuals maintain an optical angle of the marker ranging from 24{degrees} to 55{degrees}. Limiting the viewing angle deviation to 4.5% of the maximum value results in the observed elliptical cone swarm shape. We discuss the implications of these finding in mating success, speciation and for vector control.

animal behavior and cognition↗
bioRxiv2024

Transformation of the drug ibuprofen by Priestia megaterium: Reversible glycosylation and generation of hydroxylated metabolites

As one of the most-consumed drugs worldwide, ibuprofen (IBU) reaches the environment in considerable amounts as environmental pollutant, necessitating studies of its further biotransformation as potential removal mechanism. Therefore, we screened bacteria with known capabilities to degrade aromatic environmental pollutants, belonging to the genera Bacillus, Priestia (formerly also Bacillus) Paenibacillus, Mycobacterium, and Cupriavidus, for their ability to transform ibuprofen. We identified five transformation products, namely 2-hydroxyibuprofen, carboxyibuprofen, ibuprofen pyranoside, 2-hydroxyibuprofen pyranoside, and 4-carboxy--methylbenzene-acetic acid. Based on our screening results, we focused on ibuprofen biotransformation by Priestia megaterium SBUG 518 with regard to structure of transformation products and bacterial physiology. Biotransformation reactions by P. megaterium involved (A) the hydroxylation of the isobutyl side chain at two positions, and (B) conjugate formation via esterification with a sugar molecule of the carboxylic group of ibuprofen and an ibuprofen hydroxylation product. Glycosylation seems to be a detoxification process, since the ibuprofen conjugate (ibuprofen pyranoside) was considerably less toxic than the parent compound to P. megaterium SBUG 518. Based on proteome profile changes and inhibition assays, cytochrome P450 systems are likely crucial for ibuprofen transformation in P. megaterium SBUG 518. The toxic effect of ibuprofen appears to be caused by interference of the drug with different physiological pathways, including especially sporulation, as well as amino acid and fatty acid metabolism. ImportanceIbuprofen is a highly consumed drug, and, as it reaches the environment in high quantities, also an environmental pollutant. It is therefore of great interest how microorganisms transform this drug and react to it. Here, we screened several bacteria for their ability to transform ibuprofen. Priestia megaterium SBUG 518 emerged as highly capable and was therefore studied in greater detail. We show that P. megaterium transforms ibuprofen via two main pathways, hydrolyzation and reversible conjugation. These pathways bear resemblance to those in humans. Ibuprofen likely impacts the physiology of P. megaterium on several levels, including spore formation. Taken together, P. megaterium SBUG 518 is well suited as a model organism to study bacterial ibuprofen metabolism.

microbiology↗
bioRxiv2024

Beyond comparison: Brillouin microscopy and AFM-based indentation reveal divergent insights into the mechanical profile of the murine retina

Mechanical tissue properties increasingly serve as pivotal phenotypic characteristics that are subject to change during development or pathological progression. The quantification of such material properties often relies on physical contact between a load-applying probe and an exposed sample surface. For most tissues, these requirements necessitate animal sacrifice, tissue dissection and sectioning. These invasive procedures bear the risk of yielding mechanical properties that do not portray the physiological mechanical state of a tissue within a functioning organism. Brillouin microscopy has emerged as a non-invasive, optical technique that allows to assess mechanical cell and tissue properties with high spatio-temporal resolution. In optically transparent specimens, this technique does not require animal sacrifice, tissue dissection or sectioning. However, the extent to which results obtained from Brillouin microscopy allow to infer conclusions about potential results obtained with a contact-based technique, and vice versa, is unclear. Potential sources for discrepancies include the varying characteristic temporal and spatial scales, the directionality of measurement, environmental factors, and mechanical moduli probed. In this work, we addressed those aspects by quantifying the mechanical properties of acutely dissected murine retinal tissues using Brillouin microscopy and atomic force microscopy (AFM)-based indentation measurements. Our results show a distinct mechanical profile of the retinal layers with respect to the Brillouin frequency shift, the Brillouin linewidth and the apparent Youngs modulus. Contrary to previous reports, our findings do not support a simple correlative relationship between Brillouin frequency shift and apparent Youngs modulus. Additionally, the divergent sensitivity of Brillouin microscopy and AFM-indentation measurements to cross-linking or changes post mortem underscores the dangers of assuming both methods can be generally used interchangeably. In conclusion, our study advocates for viewing Brillouin microscopy and AFM-based indentation measurements as complementary tools, discouraging direct comparisons a priori and suggesting their combined use for a more comprehensive understanding of tissue mechanical properties.

biophysics↗
bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗
bioRxiv2023

Tuning of granulopoietic signaling by de novo designed agonists

Enhancing cytokine-based therapies by systematically tuning how an agonist associates its receptor is emerging as a powerful new concept in drug discovery. Here, we report the design and characterization of agonists that tune the granulocyte-colony stimulating factor receptor (G-CSFR) activity, which is central for the proliferation and granulocytic differentiation of hematopoietic stem cells. Using design agonists, we study the impact of varying the receptor-binding affinity and dimerization geometry on receptor association, downstream signaling, and cellular response. Hence, we achieved agonists with altered signaling specificities that are hyper-thermostable, can outcompete the native ligand (G-CSF), and bias granulopoietic differentiation over triggering proliferation. Furthermore, the design agonists differentially modulate the kinetics and amplitudes of signal transduction pathways, and gene expression patterns. Unlike G-CSF, they achieve selective activation of gene sets with hematopoietic functions with minimal unwanted effects on immunomodulatory signaling. These findings demonstrate the potential of dissecting the complex G-CSFR signaling, and open up ways for new therapeutic applications for designed cytokines. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/568662v3_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@af56e3org.highwire.dtl.DTLVardef@171920forg.highwire.dtl.DTLVardef@12c2c0aorg.highwire.dtl.DTLVardef@ff7556_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗
bioRxiv2023

Synthesis and characterization of novel intrinsically fluorescent analogs of cholesterol with improved photophysical properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physico-chemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present novel intrinsically fluorescent sterols containing four conjugated double bonds in the ring system and either a hydroxy or a keto group in the C3 position. Synthesis of these probes involves dehydrogenation of 7-dehydrocholesterol using mercury(II) acetate, Swern oxidation/dehydrogenation, and stereoselective Luche reduction. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that the analog with a 3-hydroxy-group like cholesterol can condense fatty acyl chains and form hydrogen bonds to water molecules at the bilayer interface. The emission of both probes is red-shifted by 80-120 nm compared to the widely used sterol analogs dehydroergosterol or cholestatrienol. This allows for their imaging on conventional microscopes, as we here show in giant unilamellar vesicles. These experiments reveal a preferred partitioning of both sterol probes into the biologically relevant liquid-ordered phase. In conclusion, we present a synthesis strategy leading to novel intrinsically fluorescent sterol probes with close resemblance of cholesterol. Their improved photophysical properties will allow for live-cell imaging of sterol transport in the future.

biophysics↗
bioRxiv2023

Axonopathy and altered synaptic development in early hippocampal epileptogenesis of Dravet syndrome

Dravet syndrome caused by SCN1A variants is a severe developmental epileptic encephalopathy (DEE) characterized by pharmaco-resistant epileptic seizures and progressive neurodevelopmental decline with cognitive impairment and autism-spectrum-traits. Numerous preceding studies indicate that the initial pathophysiology due to impaired NaV1.1 function mainly derives from reduced interneuron firing leading to a network hyperexcitability (Bender et al. 2012). However, little is known how epileptogenesis and generally disease pathogenesis progress from the inborn molecular defect to infantile seizure onset. We address this question in a Dravet mouse model by comprehensive single-cell RNA sequencing and selected downstream analysis via single-cell electrophysiology, histology, live cell imaging and electron microscopy. Our data reveal a continuum of early primary (preseizure) and secondary (post-seizure onset) transcriptomic changes in various cell populations in the hippocampus. Focusing on cornu ammonis, we find a number of transcriptional pathways that are dysregulated including synaptic transmembrane adhesion molecules of the neurexin superfamily and voltage-gated ion channels. Further investigations support an ultrastructural and functional axonopathy and synaptopathy of parvalbumin interneurons. These processes precede somatic firing impairment and seizures suggesting they underlie fundamental early-phase disease mechanisms. Taken together we provide a cellularly resolved transcriptomic resource of early disease phases of Dravet syndrome and demonstrate epileptogenesis beyond NaV1.1 loss-of-function during an early developmental time window of CNS maturation. Altogether these data establish proof-of principle that the concept of epileptogenesis, originally devised for acquired forms of epilepsy, similarly applies to genetic epilepsies and DEEs.

neuroscience↗
bioRxiv2023

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes

Pyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor(R) G2 (IG2), a first-in-class dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insects mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2s public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose. Therefore, we tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with established mixed-function oxidases and Vgsc-L995F knockdown resistance alleles were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 hours overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding. Both prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI: 0.23-0.46) and 0.43 (95% CI: 0.25-0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI: 0.22-0.41). We demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes. Author summaryMalaria remains a serious problem in many tropical and sub-tropical areas, affecting the welfare and health of many individuals. Since 2016, malaria has increased and the emergence of mosquitoes that are resistant to different classes of insecticides used in vector control tools may have contributed to some of this increase. Therefore, insecticides with a different mode of action are required to manage vector resistance to insecticides used for public health vector control. One of the main resistance mechanisms is metabolic resistance where mosquitoes upregulate detoxification enzymes to break down insecticides. Chlorfenapyr is a pyrrole-pro-insecticide that is metabolised by these detoxification enzymes from chlorfenapyr to tralopyril that disrupts mitochondrial function in mosquitoes. We therefore hypothesized that the metabolites of chlorfenapyr may also have an effect on Plasmodia since they, too possess mitochondria and this could reduce the development of Plasmodium in mosquitoes that survived a sub-lethal dose of chlorfenapyr. In this study we established and evaluated a modified WHO tunnel assay to investigate the effect of chlorfenapyr in Plasmodium-infected Anopheles mosquitoes. In this bioassay, we found that chlorfenapyr substantially reduces the proportion of Plasmodium-infected mosquitoes at doses sub-lethal to mosquitoes. Our findings demonstrate that chlorfenapyr provides additional benefits beyond mosquito killing although the mechanism of action requires further elucidation.

cell biology↗
bioRxiv2023

The novel coronary artery disease risk factor ADAMTS-7 modulates atherosclerotic plaque formation by degradation of TIMP-1

BackgroundThe ADAMTS7 locus was genome-wide significantly associated with coronary artery disease (CAD). Lack of the extracellular matrix (ECM) protease ADAMTS-7 was shown to reduce atherosclerotic plaque formation. ObjectiveTo identify molecular mechanisms and downstream targets of ADAMTS-7 mediating risk of atherosclerosis. MethodsTargets of ADAMTS-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques from Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. Putative targets were validated using immunofluorescence, in vitro degradation assays, co-immunoprecipitation, and Forster resonance energy transfer (FRET)-based protein-protein interaction assays. ADAMTS7 expression was measured in fibrous caps of human carotid artery plaques. ResultsIn humans, ADAMTS7 expression was higher in caps of unstable as compared to stable carotid plaques. Compared to Apoe-/- mice, atherosclerotic aortas of Apoe-/- mice lacking Adamts-7 (Apoe-/-Adamts7-/-) contained higher protein levels of tissue inhibitor of metalloproteases 1 (Timp-1). In co-immunoprecipitation experiments, the catalytic domain of ADAMTS-7 bound to TIMP-1, which was degraded in the presence of ADAMTS-7 in vitro. ADAMTS-7 reduced the inhibitory capacity of TIMP-1 at its canonical target matrix metalloprotease 9 (MMP-9) As a downstream mechanism, we investigated collagen content in plaques of Apoe-/- and Apoe-/-Adamts7-/- mice after Western diet. Picrosirius red staining of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. In order to facilitate high-throughput screening for ADAMTS-7 inhibitors with the aim to decrease TIMP-1 degradation, we designed a FRET-based assay targeting the ADAMTS-7 catalytic site. ConclusionADAMTS-7, which is induced in unstable atherosclerotic plaques, decreases TIMP-1 stability reducing its inhibitory effect on MMP-9, which is known to promote collagen degradation and is likewise genome-wide significantly associated with CAD. Disrupting the interaction of ADAMTS-7 and TIMP-1 might be a strategy to increase collagen content and plaque stability for reduction of atherosclerosis-related events.

molecular biology↗
bioRxiv2022

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a causal factor in regeneration failure. We demonstrate that the SLRPs Chondroadherin, Fibromodulin, Lumican, and Prolargin are enriched in human, but not zebrafish, CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer structural and mechanical properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as previously unknown inhibitory ECM factors in the human CNS that impair axon regeneration by modifying tissue mechanics and structure. ONE SENTENCE SUMMARYComposition, structural organization, and mechanical properties of the injury ECM direct central nervous system regeneration.

neuroscience↗