Biology subjects

Held, M.

Publications and source records attributed to Held, M..

16 recordsLinked to original sources

bioRxiv2026

BeatRatePy - Automated Hight-Throughput Python-Based Beat Rate Quantification and Generation of Speed and Contraction Traces

Analysing cardiac motion from label free imaging can be difficult, especially when recordings vary in quality or show complex movement. We developed a Python-based pipeline that automatically measures beat rate, contraction, speed from transmitted light timelapse data. The workflow runs in a Jupyter notebook and uses straightforward steps that can be followed easily, making the analysis accessible including to users with limited coding experience. Using human iPSC-derived cardiomyocytes imaged on a Zeiss Cell Discoverer 7, the pipeline produced beat rates, contraction and contraction speed measurements that matched expected behaviour. To test wider use, we applied the same workflow to killifish larvae heart recordings and public datasets, changing only basic input parameters. The pipeline performed consistently across these different systems and did not require changes to the underlying code. By supporting clear parameter control and use across laboratories, microscopy platforms and repositories, this approach strengthens reproducible analysis of cardiac motion from label free imaging.

cell biology↗

bioRxiv2026

Chimerophore antibiotics: engineered multimodal host defense peptides

Ribosomally synthesized host defense peptides (HDPs) are promising candidates for novel antibiotics. However, non-lytic HDPs, which target intracellular machinery, remain underexploited due to limitations including low potency in serum and narrow activity spectra. To enhance their therapeutic profile, we fused non-lytic HDPs generating "chimerophores" with multimodal mechanisms of action (MOAs). Using a high-throughput self-screening platform (Mex), we synthesized and evaluated a combinatorial library of 99,235 variants, identifying over 30,300 active chimerophores, vastly expanding the functional space of chimeric HDPs. Functional screening of 18 chimerophores revealed candidates with potent, broad-spectrum activity displaying serum-tolerance, low cytotoxicity, orthogonal uptake pathways and multimodality, such as simultaneously targeting of ribosomes and DNA. Integrating these distinct mechanisms into a single molecule allowed lead candidate cp9 to suppress the emergence of resistance in Pseudomonas aeruginosa, establishing a scalable platform for the systematic engineering of next-generation multimodal antibiotics.

microbiology↗

bioRxiv2026

Bloodstream-associated Salmonella Typhimurium and Enteritidis iNTS pathovariants hyper-replicate in human macrophages

Invasive non-typhoidal Salmonella (iNTS) are a major cause of bloodstream infections in sub-Saharan Africa, yet the host-pathogen interaction mechanisms remain poorly understood. Here, we developed and optimised a human macrophage infection model based on PMA-differentiated THP-1 cells to investigate infection dynamics of clinically relevant Salmonella Typhimurium and Salmonella Enteritidis strains. We compared intracellular survival and replication of gastroenteritis-associated and bloodstream-associated pathovariants, including S. Typhimurium ST313 Lineage 2, the novel S. Typhimurium ST313 Lineage 3 and the understudied S. Enteritidis Central/Eastern African (CEAC) clades. Our results reveal that the CEAC S. Enteritidis and ST313 S. Typhimurium iNTS pathovariants hyper-replicate within host cells, compared to global epidemic isolates. The cellular model achieved robust pro-inflammatory polarisation of human macrophages, while revealing limitations in modelling macrophage plasticity. Overall, this work defines clear differences in intracellular behaviour of Salmonella pathovariants and establishes a robust experimental framework for future studies on invasive disease pathogenesis and therapeutic interventions that target iNTS bacteria.

microbiology↗

bioRxiv2025

Single-cell phenotypic analysis and multiplet detection through incorporation of microscopy data into cellenONE-based single-cell proteomic data analysis

The reliability of single-cell proteomics (SCP) is intrinsically linked to the fidelity of cell isolation; however, the identification of co-isolated cells (doublets or multiplets) remains a persistent challenge for single-cell (sc-) omics. While single-cell transcriptomics has established probabilistic frameworks for doublet detection, these methods are ill-suited for the sparser throughput of SCP datasets. This work presents scpImaging, a novel computational pipeline that repurposes the latent microscopy data routinely generated during cellenONE-based sample preparation to provide deterministic quality control (QC) and high-content phenotypic profiling. We demonstrate that standard proteomic quality metrics for SCP (e.g., peptide count, signal intensity) paradoxically favour retaining doublets. In contrast, scpImaging applies machine-learning-based cell segmentation (Cellpose-SAM) to identify and exclude these artefacts with high precision. Furthermore, the framework integrates morphological metrics generated using the open-source CellProfiler, with proteomic abundance data, enabling joint analysis that links cell shape and texture to molecular cell state. Provided as an open-source R package, scpImaging offers a scalable, automated solution for enhancing data integrity and adding a phenotypic dimension to SCP experiments without increasing experimental cost or complexity.

systems biology↗

bioRxiv2025

The SI compartment model describes embolism spreading in networks of vessels and bordered pits in angiosperm xylem

Plant xylem consists of a network of interconnected vessels, through which water is transported under negative pressure. Filling of vessels with air, or embolism, disturbs this transport process and, in extreme cases, leads to tree mortality. Despite this significance, embolism propagation dynamics are still poorly understood, primarily because xylem is opaque to direct observation. Furthermore, existing models of embolism spreading build excessively on physiological and anatomical parameters, and many misrepresent the inter-vessel pit membrane as a 2D surface. Here, we first extend these physiological models by implementing the pit membrane as a 3D object. Then, we introduce a susceptible-infected (SI) model, a simple stochastic model for tracking spreading through a population, for embolism propagation. After correctly fitting the spreading probability, our SI model reproduces vulnerability curves produced by both the physiological model and empirical data, highlighting that the SI model can address embolism spreading dynamics in plant species, for which detailed physiological data are not available. Furthermore, relating the SI model to the physiological one allows interpreting embolism spreading as a directed percolation process. Elucidating the exact mapping between directed percolation and embolism spreading will likely yield new fundamental insights into the relationships between xylem network architecture and embolism dynamics.

plant biology↗

bioRxiv2025

Topical Formulation of Repurposed FDA-Approved Compounds Inhibits Pseudomonas aeruginosa ExoU and Improves Corneal Infection Outcomes

Microbial keratitis, infection of the cornea, cause by ExotoxinU (ExoU) strains of Pseudomonas aeruginosa have a poor clinical outcome and response to antimicrobials. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. Using a high-throughput microscopy platform, we screened 53 P. aeruginosa keratitis isolates, confirming broad efficacy of these inhibitors in exoU strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an anti-virulence strategy for the treatment of P. aeruginosa infections in humans and veterinary medicine.

microbiology↗

bioRxiv2025

Digital PCR detection of Mycobacterium tuberculosis and HIV-1 co-localization in spinal tuberculosis biopsies

BackgroundMycobacterium tuberculosis (Mtb) and HIV-1 co-infection in tissues is suggested to favour reciprocal replication, infection and reservoir expansion. Yet, confirmation of this detrimental synergism in diseased tissues is limited. MethodsIn this prospective study of 25 adults investigated for spinal tuberculosis (STB) in South Africa (13 (52%) people living with HIV-1 (PLWH), on antiretroviral treatment) 25 open surgery or CT-guided biopsies were collected and portioned into 93 segments. Extracted DNA was analysed by droplet digital PCR (ddPCR) to detect and quantify Mtb complex (MTBC) (rpoB, IS6110), HIV-1 (pol, gag) and Human (RPP30) gene copies. ddPCR sensitivity for Mtb was validated against Xpert-Ultra and culture. Total biopsy and intra-biopsy variation in pathogen DNA abundance, co-detection, and relationships to human cellularity, HIV status, and peripheral viral load (VL) evaluated. ResultsddPCR detected MTBC DNA in biopsies from 10/10 (100%) culture-confirmed STB, 5/6 (83%) Xpert Ultra-confirmed STB and 4/9 (44%) diagnosed Not STB (all 4 with previous pulmonary TB). Detected MTBC ranged from 8-59,144 rpoB copies/biopsy. RpoB copies/million human cells were higher in biopsies from PLWH (p=0.0096) and positively correlated with matched-segment HIV-1 pol copies/million cells (r=0.40; p=0.0003), but not VL. HIV-1 DNA was detected in all PLWH biopsies, four with undetectable VL. HIV-1 pol copies/million cells were higher in segments with MTBC DNA co-detected (p=0.011) and also correlated with VL (r=0.91; p=0.0003). ConclusionsReciprocal relationships exists between Mtb and HIV-1 abundance in spinal tissue. Findings support investigating TB-HIV co-infected tissue segments to characterise how the immune microenvironment impacts HIV-1/Mtb reservoir persistence and/or expansion.

molecular biology↗

bioRxiv2025

Prediction of metabolic dynamics through deep learning and high-throughput multiomics data

Synthetic biologys remarkable potential to tackle important societal problems is held back by our inability to predictably engineer biological systems. Here, we collected one of the largest public multiomics synthetic biology datasets generated to date, and used it to train a novel deep learning algorithm able to predict product and metabolic dynamics with great accuracy, starting to approach the predictive capabilities found in physics and chemistry. We were able to predict production time series with 90-99% accuracy, and final production with 96% accuracy. Further, we were able to produce good predictions for a majority of extracellular metabolites, and twenty different intracellular metabolites. These predictions were provided for a target of industrial relevance: a non-model yeast (Pichia kudriavzevii) engineered to produce large amounts of malonic acid, a desirable biomanufacturing target. This approach is generally applicable to any host, pathway, and product because all required knowledge is inferred from experimental data.

bioengineering↗

bioRxiv2024

Development of antisense tools to study Bodo saltans and its intracellular symbiont

Obligate symbioses are common in nature and present a particular challenge for functional genetic analysis. In many cases, the host is a non-model species with poor tools for genetic manipulation and the symbiont cannot be cultured or its gene expression manipulated to investigate function. Here we investigated the potential for using antisense inhibition to analyse host and symbiont gene function within an obligate aquatic symbiosis. We focused on the kinetoplastid host Bodo saltans and its bacterial symbiont, Candidatus Bodocaedibacter vickermanii, a member of Rickettsiales. We conclude that antisense inhibition is not feasible in the B. saltans and its symbiont, as the holobiont feeds on the antisense molecules - and increases in numbers - upon treatment with the antisense construct. Although our approach has proven unsuccessful, we have developed an array of protocols which can be used to study the biology of this microeukaryote and its microbial associates.

molecular biology↗

bioRxiv2023

Aminergic and peptidergic modulation of Insulin-Producing Cells in Drosophila

Insulin plays a critical role in maintaining metabolic homeostasis. Since metabolic demands are highly dynamic, insulin release needs to be constantly adjusted. These adjustments are mediated by different pathways, most prominently the blood glucose level, but also by feedforward signals from motor circuits and different neuromodulatory systems. Here, we analyze how neuromodulatory inputs control the activity of the main source of insulin in Drosophila - a population of Insulin-Producing Cells (IPCs) located in the brain. IPCs are functionally analogous to mammalian pancreatic beta cells, but their location makes them accessible for in vivo recordings in intact animals. We characterized functional inputs to IPCs using single-nucleus RNA sequencing analysis, anatomical receptor expression mapping, connectomics, and an optogenetics-based intrinsic pharmacology approach. Our results show that the IPC population expresses a variety of receptors for neuromodulators and classical neurotransmitters. Interestingly, IPCs exhibit heterogeneous receptor profiles, suggesting that the IPC population can be modulated differentially. This is supported by electrophysiological recordings from IPCs, which we performed while activating different populations of modulatory neurons. Our analysis revealed that some modulatory inputs have heterogeneous effects on the IPC activity, such that they inhibit one subset of IPCs, while exciting another. Monitoring calcium activity across the IPC population uncovered that these heterogeneous responses occur simultaneously. Certain neuromodulatory populations shifted the IPC population activity towards an excited state, while others shifted it towards inhibition. Taken together, we provide a comprehensive, multi-level analysis of neuromodulation in the insulinergic system of Drosophila.

neuroscience↗

bioRxiv2022

Signal peptide efficiency: from high-throughput data to prediction and explanation

The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ~12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the in silico evaluation of signal peptides.

synthetic biology↗

bioRxiv2022

SATINN: An automated neural network-based classification of testicular sections allows for high-throughput histopathology of mouse mutants

MotivationThe mammalian testis is a complex organ with a hierarchical organization that changes smoothly and stereotypically over time in normal adults. While testis histology is already an invaluable tool for identifying and describing developmental differences in evolution and disease, methods for standardized, digital image analysis of testis are needed to expand the utility of this approach. ResultsWe developed SATINN (Software for Analysis of Testis Images with Neural Networks), a multi-level framework for automated analysis of multiplexed immunofluorescence images from mouse testis. This approach uses a convolutional neural network (CNN) to classify nuclei from seminiferous tubules into 7 distinct cell types with an accuracy of 94.2%. These cell classifications are then used in a second-level tubule CNN, which places seminiferous tubules into one of 7 distinct tubule stages with 90.4% accuracy. We further describe numerous cell- and tubule-level statistics that can be derived from wildtype testis. Finally, we demonstrate how the classifiers and derived statistics can be used to rapidly and precisely describe pathology by applying our methods to image data from two mutant mouse lines. Our results demonstrate the feasibility and potential of using computer-assisted analysis for testis histology, an area poised to evolve rapidly on the back of emerging, spatially-resolved genomic and proteomic technologies. Availability and implementationScripts to apply the methods described here are available from http://github.com/conradlab/SATINN.

bioinformatics↗

bioRxiv2021

Real-time respiration changes as a viability indicator for rapid antibiotic susceptibility testing in a microfluidic chamber array

Rapid identification of a pathogen and the measurement of its antibiotic susceptibility are key elements in the diagnostic process of bacterial infections. Microfluidic technologies offer great control over handling and manipulation of low sample volumes with the possibility to study microbial cultures on the single-cell level. Downscaling the dimensions of cultivation systems directly results in a lower number of bacteria required for antibiotic susceptibility testing (AST) and thus in a reduction of the time to result. The developed platform presented in this work allows the reading of pathogen resistance profiles within 2-3 hours based on the changes of the dissolved oxygen levels during bacterial cultivation. The platform contains hundreds of individual growth chambers prefilled with a hydrogel containing oxygen-sensing nanoprobes and different concentrations of antibiotic compounds. The performance of the microfluidic platform is tested using quality control Escherichia coli strains (ATCC 25922 and ATCC 35218) in response to different clinically relevant antibiotics. The achieved results are in agreement with values given in clinical reference guides and independent measurements using a clinical AST protocol. Finally, the platform is successfully used for AST of an E. coli clinical isolate obtained from a patient blood culture.

microbiology↗

bioRxiv2018

Intracellular mechanisms of fungal space searching in microenvironments

The underlying intracellular mechanisms involved in the fungal growth received considerable attention, but the experimental and theoretical work did not take into account the modulation of these processes by constraining microenvironments similar to many natural fungal habitats. To fill this gap in the scientific knowledge, we used time-lapse live-cell imaging of Neurospora crassa growth in custom-built confining microfluidics environments. We show that the position and dynamics of the Spitzenkorper-microtubules system in constraining environments differs markedly from that associated with unconstrained growth. First, when hyphae encounter an obstacle at shallow angles, the Spitzenkorper moves from its central position in the apical dome off-axis towards a contact with the obstacle, thus functioning as a compass preserving the directional memory of the initial growth. The trajectory of Spitzenkorper is also followed by microtubules, resulting in a cutting corners pattern of the cytoskeleton in constrained geometries. Second, when an obstacle blocks a hypha at nearnormal incidence, the Spitzenkorper-microtubule system temporarily disintegrates, followed by the formation of two equivalent systems in the proto-hyphae - the basis of obstacle-induced branching. Third, a hypha, passing a lateral opening along a wall, continues to grow largely unperturbed while a lateral proto-hypha gradually branches into the opening, which starts forming its own Spitzenkorper-microtubule system. These observations suggest that the Spitzenkorper-microtubules system conserves the directional memory of the hyphae when they navigate around obstacles, but in the absence of the Spitzenkorper-microtubule system during constrainment-induced apical splitting and lateral branching, the probable driving force of obstacle-induced branching is the isotropic turgor pressure.

cell biology↗

bioRxiv2017

Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy

Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.

developmental biology↗

bioRxiv2017

Pollen tube growth and guidance: Occam’s razor sharpened on a molecular AGP Rosetta Stone

Occams Razor suggests a new model of pollen tube tip growth based on a novel Hechtian oscillator that integrates: (1) a periplasmic AGP-Ca2+ calcium capacitor with tip-localised arabinogalactan glycoproteins (AGPs); (2) tip-focussed cytosolic Ca2+oscillations; (3) Hechtian strands evidence of adhesion between the plasma membrane and the cell wall of the growing tip. Thus Hechtian adhesion, as a piconewton force transducer, couples the internal stress of a rapidly growing wall to the plasma membrane. Such Hechtian transduction via stretch-activated Ca2+ channels and H+-ATPase proton efflux dissociating periplasmic AGP-Ca2+, creates a Ca2+ influx that activates exocytosis of wall precursors. In effect a highly simplified primary cell wall regulates its own synthesis and a Hechtian growth oscillator regulates overall tip growth. By analogy with the Rosetta Stone that translates trilingual inscriptions as a single identical proclamation, the Hechtian Hypothesis translates classical AGPs and their roles as a Ca2+ capacitor, pollen tube guide and wall plasticiser into a simple but widely applicable model of tip growth. Even wider ramifications of the Hechtian oscillator may implicate AGPs in osmosensing or gravisensing and other tropisms, leading us yet further towards the Holy Grail of plant growth.

biochemistry↗