Biology subjects

Schilling, M.

Publications and source records attributed to Schilling, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Mind the gap between functional groups and surface of magnetic nanoparticles for highly specific magnetic-based protein assays in biological medium

Magnetic readout-based assays are compatible with unprocessed biological samples as unbound background molecules do not interfere with magnetic signal. Yet, a true challenge is their poor specificity and susceptibility of magnetic nanoparticles (MNPs) to clusters in complex biological media, hampering their true advancement. Here, we demonstrate that the spatial organization of functional groups at the external periphery of custom magnetic nanoparticles by harnessing ultra-dense double-stranded DNA results in an efficient antibody conjugation with good accessibility toward antigen. By labeling our MNPs with anti-S protein neutralizing IgG antibody, we showcase the detection of S1 subunit of SARS-CoV-2 Spike protein in a wash-free fashion in less than five minutes in nM regime using magnetic particle spectrometer. By mixing our IgG-labelled MNPs with DMEM cell culture (10-20% FBS serum), we sense the S1 proteins in a one-pot fashion with high specificity. Our results show that by having the ultra-dense dsDNA shell on MNPs, the entropic cost of an irreversible protein binding to particle surface is high, thus allowing the formation of dynamic protein corona on the DNA shell that can be replaced with S1 protein with high affinity. When the azide moieties are placed at the close proximity of MNPs by using non-functional dsDNA, antibody conjugation becomes inefficient, to a level not sufficient for S1 protein detection. Our study highlights the importance of spatial organization of functional moieties on the nanoscale on magnetic nanoparticles for highly specific assays in biologically complex media.

biochemistry↗

bioRxiv2026

Integration of proteomic data from cell lines and tumors

Cancer cell lines are widely used in preclinical research, yet the clinical translation of findings from cell lines remains limited. Identifying cell lines that best resemble patient tumors requires integration of molecular profiles across biologically distinct sample types. Advances in transcriptomic integration have demonstrated the potential of deep learning for aligning data across different sample types. However, comparable approaches for proteomic data integration remain lacking, potentially because of the prevalence of missing values in proteomic datasets. Here, we introduce ProtInt, a deep learning-based framework that integrates proteomic data by combining principles from proteomic imputation and transcriptomic integration methods. We applied ProtInt to integrate label-free proteomic profiles from 771 cancer cell lines and 550 treatment-naive tumors, and showed that ProtInt outperformed batch correction and transcriptomic integration methods. Comparison of the cell line proteomes before and after integration revealed recurrent increase of proteins associated with immune reaction and reduction of proteins involved in mitochondrial gene expression as proteomes of cell lines were adapted to resemble tumors. These results establish ProtInt as a framework for joint analysis of proteomic datasets across distinct sample types and may facilitate the identification of cell lines best suited for clinically relevant studies.

bioinformatics↗

bioRxiv2026

In vivo analyses reveal rapid and permissive lipid transport between the ER and mitochondria

Interorganelle lipid transport is essential for mitochondrial membrane biogenesis and function, yet its kinetics and substrate selectivity remain poorly understood in vivo. Here, we developed two complementary approaches to quantify lipid trafficking from the endoplasmic reticulum (ER) to mitochondria in yeast. Metabolic labeling combined with organelle fractionation revealed that newly synthesized phospholipids rapidly accumulate in mitochondria, with 20-35% of newly synthesized molecules detected in mitochondrial fractions within minutes of synthesis. To directly quantify lipid flux, we established a synthetic transport assay based on the production of heterologous galactolipids absent from yeast. This approach revealed an ER-to-mitochondria transport flux of approximately 2.6 x 105 lipid molecules per cell per minute. Remarkably, galactolipids were transported with high efficiency despite their absence from fungal membranes, indicating limited substrate selectivity of ER-mitochondria lipid transport pathways. Together, these complementary assays provide quantitative tools to investigate intracellular lipid transport and reveal the rapid and permissive nature of lipid exchange between the ER and mitochondria. SummaryUsing complementary metabolic labeling and synthetic lipid reporter assays, we quantitatively measured ER-mitochondria lipid transport in yeast. Our results reveal rapid lipid exchange, high transport fluxes and limited substrate selectivity, indicating that mitochondrial lipid trafficking efficiently accommodates structurally diverse membrane lipids.

Cell Biology↗

bioRxiv2026

Loss of the Y chromosome drives epigenetic and transcriptomic plasticity in lung adenocarcinoma

Loss of the Y chromosome (LOY) is associated with poor survival across multiple solid tumors, yet the underlying molecular mechanisms remain poorly understood. Here, we identify LOY as a central driver of lineage plasticity and epigenetic heterogeneity in lung adenocarcinoma. Integrating multi-omic profiling of primary samples with isogenic cellular models, we show that LOY triggers epithelial-to-mesenchymal transition (EMT). Mechanistically, LOY causes haploinsufficiency of dosage-sensitive regulators, leading to widespread DNA hypomethylation at EMT gene promoters, including THY1 and LOX. Single-cell multi-omic analyses demonstrate that LOY induces epigenetic heterogeneity, destabilizes the chromatin landscape, and increases lineage plasticity, enabling rapid cellular adaptation to metabolic and genotoxic stress. Moreover, LOY-induced plasticity facilitates tumor engraftment and metastatic dissemination in vivo. These findings establish Y-linked gene dosage as a critical guardian of epigenetic stability, providing a mechanistic rationale for how its loss amplifies phenotypic diversity and lineage plasticity, ultimately driving adverse clinical outcomes in LOY patients.

cancer biology↗

bioRxiv2026

A novel genetically distinct Amdoparvovirus in Sorex araneus in the United Kingdom highlights an unexplored ancestral link

Amdoparvoviruses have historically been documented almost exclusively in carnivores, with recent detections in bats. However, endogenous viral elements in rodent genomes suggest a more ancient and taxonomically broader evolutionary history. Despite this, small mammals have never been systematically surveyed for extant amdoparvovirus infections. In this study, we used whole genome sequencing to screen four different shrew species and wild American mink in the UK, which may act as a reservoir host for amdoparvoviruses. We identified a highly divergent amdoparvovirus in native common shrews (Sorex araneus) from northern England, tentatively named Shrew parvovirus 1(SP 1). Classical amdoparvovirus sequences were also detected in wild American mink (Neogale vison), confirming the presence of known amdoparvovirus strains in UK mustelids. Phylogenetic analysis revealed that the shrew virus, SP 1, forms a distinct clade, suggesting ancient divergence or long-term cryptic circulation in small mammal reservoirs. These findings fundamentally challenge the hypothesis that amdoparvoviruses are carnivore-restricted pathogens and underscore the importance of systematic wildlife surveillance for understanding viral host range evolution and assessing spillover risks.

evolutionary biology↗

bioRxiv2025

CochleaNet: deep learning-based image analysis for cochlear connectomics and gene therapy

With the emergence of gene and optogenetic therapies targeting deafness, the comprehensive analysis of the molecular anatomy and physiology of the cochlea has become ever more important. Here, we introduce CochleaNet, a deep learning-based framework to analyze volumetric imaging data obtained by light-sheet microscopy of decalcified, cleared and fluorescently labeled cochleae. CochleaNet covers the workflow from reconstruction of the cochlea to segmentation of inner hair cells, spiral ganglion neurons and their afferent synapses, to analyzing the expression of gene therapy products. We validated CochleaNet by comparison to manual image analysis. Trained on high isotropic resolution mouse data, CochleaNet was also applicable to the cochlea of the gerbil, another relevant animal model, and lower-resolution mouse data from a commercially available microscope. We conclude that the combination of light-sheet microscopy and image analysis with CochleaNet paves the way for rapid and reliable quantification of cochlear molecular anatomy and preclinical gene therapy outcomes.

neuroscience↗

bioRxiv2025

msBayesImpute: A Versatile Framework for Addressing Missing Values in Biomedical Mass Spectrometry Proteomics Data

Advancements in mass spectrometry (MS) technologies have significantly improved the ability to quantify proteins and analyse their modifications. However, MS-based proteomics datasets frequently encounter missing values due to a complex interplay of missing at random (MAR) and missing not at random (MNAR) mechanisms. If unaddressed, such missing data can result in information loss and biased outcomes in data pre-processing, such as normalisation, as well as subsequent analyses and interpretations. Few approaches effectively address both MAR and MNAR, and those that do often necessitate manual tuning of mixture percentages between them or rely on two-group experimental designs. To enhance the handling of missing values, we developed msBayesImpute, an innovative computational method that integrates Bayesian factorization with probabilistic dropout models. We evaluated msBayesImpute against several popular imputation methods using both simulated missing values and those generated through a dilution series experiment on samples from lung cancer patients. Our comprehensive benchmark demonstrated superior performance in reconstructing missing values, estimating normalization factors, and identifying differentially expressed proteins across varying levels of missingness. Notably, msBayesImpute does not require predefined experimental designs and is scalable to large-scale studies. This versatility positions msBayesImpute as an effective and robust tool for enhancing the utility of MS datasets in biological research.

bioinformatics↗

bioRxiv2025

Decoupling of plastid and endomembrane homeoviscous response to low temperature and darkness in Phaeodactylum tricornutum

Understanding how marine diatoms adapt to fluctuating environmental conditions is critical for deciphering their ecological success. Here, we investigated the lipidomic responses of the model diatom Phaeodactylum tricornutum to low temperatures (10 {degrees}C), darkness, and combined stress. Using a detailed lipidomics approach, we revealed significant remodeling of plastidial and extraplastidial lipid species. Plastidial lipids showed increased unsaturation levels, particularly the 16:3-to-16:4 fatty acid switch, only under light conditions. This light-dependent adaptation suggests a tightly regulated mechanism that aligns lipid remodeling with photosynthetic activity. Phospholipids in the endomembrane system exhibited less dependency on light, with notable changes even under extended dark periods. These findings highlight distinct strategies employed by diatoms to maintain membrane fluidity and protect cellular processes during stress. Unlike plants, P. tricornutum lacks polygalactosyldiacylglycerols, underscoring a divergent adaptation pathway. Our results demonstrate that P. tricornutum adapts to cold and dark environments through lipid composition changes, supporting survival in variable marine habitats. This study provides critical insights into diatom resilience, paving the way for understanding their role in global biogeochemical cycles. HighlightLow temperature and darkness trigger distinct lipidomic remodeling in Phaeodactylum tricornutum, revealing species-specific adaptive mechanisms that maintain membrane integrity and enhance resilience under extreme environmental conditions.

physiology↗

bioRxiv2025

Mismatch-Assisted Toehold Exchange Cascades for Magnetic Nanoparticle-Based Nucleic Acid Diagnostics

Sensitive, simple, and rapid detection of nucleic acid sequences at point-of-care (POC) settings is still an unmet quest. Magnetic readout assays combined with toehold-mediated strand displacement (TMSD)-based circuits are amplification- and wash-free, essential features for contributing to this demand. Nevertheless, nonenzymatic TMSD circuits are slow with low sensitivity for early disease diagnostics. Here, we propose novel mismatch-assisted toehold exchange (MATE) magnetic cascades, wherein magnetic susceptibility increases by dissociation of magnetic nanoparticles (MNPs) from engineered magnetic clusters upon detecting nucleic acid target in solution. The MATE relies on the generation of an allosteric toehold (TH) by spontaneous dissociation to efficiently recycle the target, amplify magnetic signal output, and enhance the assays kinetics. We show that introducing a mismatch in the allosteric TH domain enhances the overall declustering kinetics 7-fold, as also confirmed with oxDNA simulations, with the largest effect gained for the mismatch closest to where the branch migration by the target ends. By integrating MATE into magnetic diagnostics cascades, we demonstrate 3.6-fold better limit-of-detection (LoD) and 12-fold shorter assay time compared to our previous circuit design. Our work makes a major leap towards bringing MNP-based diagnostics much closer to the clinical POC settings by offering a simple, rapid, isothermal, and nonenzymatic assay workflow.

biochemistry↗

bioRxiv2024

DGTS overproduced in seed plants is excluded from plastid membranes and promotes endomembrane expansion

Plants and algae must adapt to environmental changes, facing various stresses that negatively impact their growth and development. One common stress is phosphate (Pi) deficiency, which is often in limiting quantity in the environment. In response to Pi deficiency, these organisms increase Pi uptake and remobilize intracellular Pi. Phospholipids are degraded to provide Pi and replaced by non-phosphorous lipids, such as glycolipids or betaine lipids. During the evolution, seed plants lost their capacity to synthesize betaine lipid. By expressing BTA1 genes, involved in the synthesis of diacylglyceryl-N,N,N-trimethyl-homoserine (DGTS), from different species, our work shows that DGTS can be produced in seed plants. In Arabidopsis, expressing BTA1 under a phosphate starvation-inducible promoter resulted in limited DGTS production without having any impact on plant growth or lipid remodeling. In transient expression systems in Nicotiana benthamiana, leaves were able to accumulate DGTS up to 20 % of their glycerolipid content at a slight expense of galactolipid and phospholipid production. At the subcellular level, we showed that DGTS is absent from plastid and seems to be enriched in endomembrane, driving an ER membrane proliferation. Finally, DGTS synthesis pathway seems to compete with PC synthesis via the Kennedy pathway but does not seem to be derived from PC diacylglycerol backbone and therefore does not interfere with the eukaryotic pathway involved in galactolipid synthesis.

plant biology↗

bioRxiv2024

Transovarial transmission in field caught mosquitoes identifies a mechanism for the establishment of Usutu virus in a temperate country

Usutu virus (USUV) is an emerging zoonotic flavivirus in Europe, and the first zoonotic mosquito-borne virus to be confirmed in animal hosts in the United Kingdom (UK). Phylogenetic analysis of USUV in the three years following its initial detection in 2020 indicated that the virus is overwintering in the UK. In 2023, USUV was identified outside Greater London for the first time. Therefore, USUV should now be considered endemic within southeast England. Surveillance of avian hosts and mosquito vectors has been insufficient to elucidate the mechanism by which USUV has persisted through temperate winters. It is likely that mosquitoes play a significant role in facilitating the establishment of USUV in temperate areas, as is the case for related mosquito-borne viruses. Here we undertake enhanced targeted vector surveillance at the index site to investigate the role of mosquitoes in facilitating USUV establishment in the UK. Between 2021 and 2024 inclusive, we detected USUV in host-seeking adult female Culex pipiens s.l. (n = 8/554 pools), a key vector of the virus in Europe. During 2023, enhanced surveillance detected transovarial transmission of USUV in wild Cx. pipiens s.l. (n = 1/202 pools), by screening adults following captive rearing of mosquito larvae collected from the field. This is, to our knowledge, the first description of vertical transmission of USUV in an arthropod vector. Consequently, transovarial transmission should be considered a viable mechanism for the persistence of USUV in temperate areas. Our results highlight the importance of undertaking detailed vector surveillance, across life stages, to inform the epidemiology of vector-borne viruses.

microbiology↗

bioRxiv2024

Quantification of transcript isoforms at the single-cell level using SCALPEL

Single-cell RNA sequencing (scRNA-seq) has facilitated the study of gene expression and the development of new tools to quantify transcript in individual cells. Yet, most of these methods have low sensitivity and accuracy. Here we present SCALPEL, a Nextflow-based tool to quantify and characterize transcript isoforms at the single-cell level using standard 3 based scRNA-seq data. SCALPEL predictions have higher sensitivity than other tools and can be validated experimentally. We have used SCALPEL to study the changes in isoform usage during mouse spermatogenesis and in the differentiation of induced pluripotent stem cells (iPSCs) to neural progenitors. These analyses allow the identification of novel cell populations that cannot be defined using conventional gene expression profiles, confirm known changes in 3 UTR length during cell differentiation, and identify cell-type specific miRNA signatures controlling isoform expression in individual cells. Together, our work highlights how SCALPEL expands the current scRNA-seq toolset to explore post-transcriptional gene regulation in individual cells from different species, tissues, and technologies to investigate the variability and the specificity of gene regulatory mechanisms at the single-cell level.

bioinformatics↗

bioRxiv2024

AtVPS13M1 is involved in lipid remodeling in low phosphate and is located at the mitochondria surface in plants

VPS13 are conserved lipid transporters with multiple subcellular localizations playing key roles in many fundamental cellular processes. While the localization and function of VPS13 have been extensively investigated in yeast and animals, little is known about their counterparts in plants, particularly regarding their role in stress response. In this study, we characterized AtVPS13M1, one of the four VPS13 paralogs of the flowering plant Arabidopsis thaliana. We show that AtVPS13M1 binds and transports glycerolipids with a low specificity in vitro. AtVPS13M1 interferes with phospholipids degradation in response to phosphate starvation, a nutrient stress that triggers a massive remodeling of membrane lipids. AtVPS13M1 is mainly expressed in young dividing and vascular tissues. Finally, we show that AtVPS13M1 is mainly located at the surface of mitochondria in leaves. Overall, our work highlights the conserved role in lipid transport of VPS13 in plants, reveals their importance in nutrient stress response and opens important perspectives for the understanding of lipid remodeling mechanisms and for the characterization of this protein family in plants.

cell biology↗

bioRxiv2024

Lysis buffer selection guidance for mass spectrometry-based global proteomics including studies on the intersection of signal transduction and metabolism

Prerequisite for a successful proteomics experiment is a high-quality lysis of the sample of interest, resulting in a large number of identified proteins as well as a high coverage of protein sequences. Therefore, the choice of suitable lysis conditions is crucial. Many buffers were previously employed in proteomics studies, yet a comprehensive comparison of lysate preparation conditions was so far missing. In this study, we compared the efficiency of four commonly used lysis buffers, containing the agents NP40, SDS, urea or GdnHCl, in four different types of biological samples (suspension and adherent cell lines, primary mouse cells and mouse liver tissue). After liquid chromatography-mass spectrometry (LC-MS) measurement and MaxQuant analysis, we compared chromatograms, intensities, number of identified proteins and the localization of the identified proteins. Overall, SDS emerged as the most reliable reagent, ensuring stable performance and reproducibility across diverse samples. Furthermore, our data advocated for a dual-sample lysis approach, including that the resulting pellet is lysed again after the initial lysis with a urea lysis buffer and subsequently both lysates are combined for a single LC-MS run to maximize the proteome coverage. However, none of the investigated lysis buffers proved to be superior in every category, indicating that the lysis buffer of choice depends on the proteins of interest and on the biological question. Further, we demonstrated with our systematic studies the establishment of conditions that allows to perform global proteomics and affinity purification-based interactome characterization from the same lysate. In sum our results provide guidance for the best-suited lysis buffer for mass spectrometry-based proteomics depending on the question of interest.

systems biology↗

bioRxiv2024

Phototropin connects blue light perception to starch metabolism in green algae

In photosynthetic organisms light acts as an environmental signal to control their development and physiology, and as energy source to drive the conversion of CO2 into carbohydrates used for growth or storage. The main storage carbohydrate in green algae is starch, which accumulates during the day and is broken down at night to meet cellular energy demands. The signalling role of light quality in the regulation of starch accumulation remains unexplored. Here, we identify PHOTOTROPIN-MEDIATED SIGNALLING KINASE 1 (PMSK1) as a key regulator of starch metabolism in Chlamydomonas reinhardtii. In its phosphorylated form (PMSK1-P), it activates GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE (GAP1), promoting starch biosynthesis. We show that blue light, perceived by PHOTOTROPIN, induces PMSK1 dephosphorylation that in turn represses GAP1 mRNA levels and reduces starch accumulation. These findings reveal a novel blue light-mediated signaling pathway that advances our understanding of photoreceptor-controlled carbon metabolism in microalgae. One-Sentence SummaryBlue light perception by PHOTOTROPIN triggers kinase-mediated signaling to inhibit starch accumulation in the green alga Chlamydomonas.

plant biology↗

bioRxiv2023

Basal MET Phosphorylation is an Indicator of Hepatocyte Dysregulation in Liver Disease

Chronic liver diseases are worldwide on the rise. Due to the rapidly increasing incidence, in particular in Western countries, Non-alcoholic fatty liver disease (NAFLD) is gaining importance as the disease can develop into hepatocellular carcinoma. Lipid accumulation in hepatocytes has been identified as the characteristic structural change in NAFLD development, but molecular mechanisms responsible for disease progression remained unresolved. Here, we uncover in primary hepatocytes from a preclinical model fed with a Western diet (WD) a strong downregulation of the PI3K-AKT pathway and an upregulation of the MAPK pathway. Dynamic pathway modeling of hepatocyte growth factor (HGF) signal transduction combined with global proteomics identifies that an elevated basal MET phosphorylation rate is the main driver of altered signaling leading to increased proliferation of WD-hepatocytes. Model-adaptation to patient-derived hepatocytes reveal patient-specific variability in basal MET phosphorylation, which correlates with patient outcome after liver surgery. Thus, dysregulated basal MET phosphorylation could be an indicator for the health status of the liver and thereby inform on the risk of a patient to suffer from liver failure after surgery.

systems biology↗

bioRxiv2023

Cooperative dynamics of DNA grafted magnetic nanoparticles optimize magnetic biosensing and coupling to DNA origami

Magnetic nanoparticles (MNPs) enable unique capabilities for biosensing and actuation via coupling to DNA origami, yet how DNA grafting density affects their dynamics and accessibility remains poorly understood. Here, we demonstrate functionalization of MNPs with single-stranded DNA (ssDNA) via click chemistry conjugation with tunable grafting density. Several complementary methods show that particle translational and rotational dynamics exhibit a sigmoidal dependence on ssDNA grafting density. At low densities ssDNA strands are coiled and cause small changes to particle dynamics, while at high densities they form polymer brushes that cooperatively change particle dynamics. Intermediate ssDNA densities show the highest magnetic biosensing sensitivity for detection of target nucleic acids. Finally, we demonstrate that MNPs with high grafting densities are required to efficiently couple them to DNA origami. These results together establish ssDNA grafting density as a critical parameter for functionalization of MNPs for use in a broad range of applications.

biochemistry↗

bioRxiv2023

Hypoxia dampens innate immune signalling at early time points and increases Zika virus replication in iPSC-derived macrophages

Type I interferons (IFNs) are the major host defence against viral infection and are induced following activation of cell surface or intracellular pattern recognition receptors, including retinoic-acid-inducible gene I (RIGI)-like receptors (RLRs). All cellular processes are shaped by the microenvironment and one important factor is the local oxygen tension. The majority of published studies on IFN signalling are conducted under atmospheric (18%) oxygen conditions, that do not reflect the physiological oxygen levels in most organs (1-5% O2). We studied the effect of low oxygen on IFN induction and signalling in induced Pluripotent Stem Cell (iPSC)-derived macrophages as a model for tissue-resident macrophages and assessed the consequence for Zika virus (ZIKV) replication. Hypoxic conditions dampened the expression of interferon-stimulated genes (ISGs) following RLR stimulation or IFN treatment at early time points. RNA-sequencing and bio-informatic analysis uncovered several pathways including changes in transcription factor availability, the presence of HIF binding sites in promoter regions, and CpG content that may contribute to the reduced ISG expression. Importantly, hypoxic conditions increased ZIKV replication at early time points, emphasizing the importance of understanding how low oxygen conditions in the local microenvironment affect pathogen sensing and host defence.

microbiology↗