Biology subjects

Mueller, S.

Publications and source records attributed to Mueller, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Temporal dynamics of neuroplasticity and neurodegeneration in the central auditory system following noise-induced hearing loss: A multimodal imaging and histological study

Noise-induced hearing loss (NIHL) is a sensorineural disorder that triggers profound neuroplastic and neurodegenerative consequences in the central auditory nervous system (CNS). This study examined how neuronal density, axonal integrity and glutamatergic and GABAergic neurotransmission are affected after acute overstimulation in the central inferior colliculus (CIC) and the ventral medial geniculate body of the thalamus (MGV). To achieve this, a correlative multimodal approach combining audiometric, magnetic resonance imaging (MRI) and histological biomarkers was performed. Adult mice were noise-exposed to broadband white noise (5-20 kHz) for 3 hours at either high (115 dB SPL) or moderate (90 dB SPL) intensity, while unexposed mice were used as controls. Separate cohorts of mice were investigated 1-, 7-, 56- and 84-days post-exposure using in vivo magnetic resonance imaging (MRI) techniques: Voxel-based morphometry (VBM) of gray matter density (GMD), diffusion MRI (dMRI) of microstructure and connectivity, and single-voxel proton magnetic resonance spectroscopy (1H-MRS) for glutamate and GABA quantification. Frequency-specific auditory brainstem responses (ABR) were recorded at 4, 8, 16 and 32 kHz before and after exposure to examine hearing threshold (HT) shifts. Following imaging, brains were processed for fluorescence immunohistochemistry (FIHC) targeting Neuronal Nuclear Protein (NeuN), 4,6-diamidino-2-fenilindo (DAPI), Neurofilament (SMI312), vesicular GABA transporter (VGAT), and vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2). HTs were significantly elevated 7d, 56d and 84d after 115 dB noise exposure, suggesting a NIHL phenotype. Neurofilament density significantly increased in the CIC and MGV 1d after 115 dB noise exposure, and returned to baseline at later time points. dMRI transient microstructural alterations were observed 7d after 90 dB noise exposure. Glutamate and GABA decreases 84d after 90 dB exposure were also detected. Moreover, correlation analysis between audiometric, histological and MRI datasets revealed scarce relationships between the investigated parameters. These findings advance our understanding of the CNS adaptations of NIHL and emphasizes the need for more sensitive and integrative approaches to identify robust biomarkers of central auditory dysfunction.

neuroscience↗

bioRxiv2025

Overcoming Ligand Discovery Challenges: Developing Peptide-Based Tracers for SPSB2

Developing new E3 ligase ligands for the design of heterobivalent molecules, such as PROteolysis TArgeting Chimeras (PROTACs), requires careful evaluation of target engagement (TE). Characterizing protein-protein interactions (PPIs) is therefore essential in drug discovery, as it enables the assessment of ligand binding to sites that are often difficult to target. Degrons, peptide motifs recognized by E3 ligases, may serve as valuable starting points for designing E3 ligands. However, many degrons are highly polar and lack intrinsic membrane permeability, requiring alternative strategies for efficient cellular delivery. In this study, we used the SPRY domain-containing SOCS box protein 2 (SPSB2) E3 ligase as a model system to develop TE strategies for in vitro and in cellulo using polar degron-based peptides. By conjugating various polycationic cell-penetrating peptides (CPPs) to the degron sequence, we present a study demonstrating efficient cellular delivery. We obtained a high-resolution crystal structure and used various biophysical techniques to assess the influence of each modification, while confocal microscopy and BRET-based assays confirmed successful cellular delivery as well as potent target engagement. O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/673904v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@191848borg.highwire.dtl.DTLVardef@94fae6org.highwire.dtl.DTLVardef@e43016org.highwire.dtl.DTLVardef@110f9f0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Click. Screen. Degrade. A Miniaturized D2B Workflow for rapid PROTAC Discovery

Targeted protein degradation is one of the fastest developing fields in medicinal chemistry and chemical biology. Despite significant development in assay technologies and inhibitor discovery, the development of PROTACs remains a challenging endeavor since rational design approaches remain widely elusive. Our workflow eliminates the rate-limiting step of classic synthesis, namely compound purification, and pairs it with high-throughput, semi-automated plate-based synthesis, and direct cellular assay evaluation. We applied this direct-to-biology approach to four diverse targets demonstrating the general applicability of this technology. PROTAC synthesis was realized by using the highly efficient copper-catalyzed azide-alkyne cycloaddition reaction. This simplified reaction setup allowed synthesis in the nanomole scale with reaction volumes as low as 5 L. This high throughput approach enables the synthesis and testing of hundreds of PROTACs within a few days, allowing for a comprehensive assessment of target degradability and the identification of the most suitable E3 ligase for degrader development.

biochemistry↗

bioRxiv2025

Un-LOK-ing a new approach for conformational selective targeting of STK10 (LOK)

STK10 (serine/threonine kinase 10, LOK), is an important regulator of diverse cellular processes, such as cell cycle progression or lymphocyte migration. STK10 has emerged as a potential therapeutic target for diseases associated with impaired cell migration and cell division. Here we present a late-stage optimization of a macrocyclic pyrazolo[1,5-a]pyrimidine scaffold that led to a urea-based lead series targeting the back-pocket of STK10. Co-crystal structure analysis of 23 revealed that the optimized macrocycles adopted a unique binding mode that protrudes deep into the back pocket of STK10. Compound 23 exhibited potent on-target activity in biophysical and activity assays and displayed nanomolar activity for STK10 in cells. In addition, 23 shows good selectivity against the kinome and remarkably also against the closely related kinase SLK (STE20-like kinase). Therefore, we propose that targeting the unique and largely extended pocket in STK10 represents an opportunity to develop highly selective STK10 inhibitors. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/666149v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@36f039org.highwire.dtl.DTLVardef@d55025org.highwire.dtl.DTLVardef@80c007org.highwire.dtl.DTLVardef@bf2471_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Image-Based Meta- and Mega-Analysis (IBMMA): A Unified Framework for Large-Scale, Multi-Site, Neuroimaging Data Analysis

The increasing scale and complexity of neuroimaging datasets aggregated from multiple study sites present substantial analytic challenges, as existing statistical analysis tools struggle to handle missing voxel-data, suffer from limited computational speed and inefficient memory allocation, and are restricted in the types of statistical designs they are able to model. We introduce Image-Based Meta- & Mega-Analysis (IBMMA), a novel software package implemented in R and Python that provides a unified framework for analyzing diverse neuroimaging features, efficiently handles large-scale datasets through parallel processing, offers flexible statistical modeling options, and properly manages missing voxel-data commonly encountered in multi-site studies. IBMMA produced stronger effect sizes and revealed findings in brain regions that traditional software overlooked due to missing voxel-data resulting in gaps in brain coverage. IBMMA has the potential to accelerate discoveries in neuroscience and enhance the clinical utility of neuroimaging findings.

neuroscience↗

bioRxiv2025

MINDNet: Proximity interactome of the MICOS complex revealing a multifaceted network orchestrating mitochondrial biogenesis

The Mitochondrial contact site and cristae organizing system (MICOS) complex is a multisubunit complex regulating mitochondrial inner membrane (IM) architecture, which is enriched at crista junctions (CJs) and required for cristae membrane dynamics. It modulates various mitochondrial processes including protein and lipid transport and is causally linked to a variety of human diseases. To gain a broad overview of the various pathways modulated by the MICOS complex, we examined its molecular neighbourhood. For this, we employed proximity biotinylation assays using APEX2 fused to four MICOS subunits (MIC10, MIC13, MIC26 and MIC27) in the respective mammalian knockout cells. These four MICOS-APEX2 fusion proteins integrated into the native MICOS complex and properly localised as revealed by electron microscopy combined with DAB staining and STED super-resolution nanoscopy. Here, we identify 119 common and 50 unique proteins, termed MICOS NanoDomain Network (MINDNet) encompassing the versatile proximity proteome of the MIC10/MIC13/MIC26/MIC27 subcomplex playing multifaceted mitochondrial functions. The MINDNet revealed a large number of OXPHOS proteins, protein translocases of the IM and OM, mitochondrial ribosomal proteins and solute carrier family transporters. Using the cues obtained from the proximity interaction studies, we investigated the role of all the MICOS proteins in modulating the function of the OXPHOS complexes. Among all the MICOS proteins, MIC10 and MIC60 consistently regulated the assembly and activity of the OXPHOS complexes. Overall, we propose that the MICOS complex integrates numerous spatial and temporal cues to regulate the dynamic microenvironment, along with IM architecture, which are involved in multiple pathways controlling mitochondrial biogenesis.

cell biology↗

bioRxiv2025

Harmonized Protocol for Subfield Segmentation in the Hippocampal Body on High-Resolution in vivo MRI from the Hippocampal Subfields Group (HSG)

Hippocampal subfields differentially develop and age, and they vary in vulnerability to neurodegenerative diseases. Innovation in high-resolution imaging has accelerated clinical research on human hippocampal subfields, but substantial differences in segmentation protocols impede comparisons of results across laboratories. The Hippocampal Subfields Group (HSG) is an international organization seeking to address this issue by developing a histologically-valid, reliable, and freely available segmentation protocol for high-resolution T2-weighted 3 tesla MRI (http://www.hippocampalsubfields.com). Here, we report the first portion of the protocol focused on subfields in the hippocampal body; protocols for the head and tail are in development. The body protocol includes definitions of the internal boundaries between subiculum, Cornu Ammonis (CA) 1-3 subfields, and dentate gyrus, in addition to the external boundaries of the hippocampus apart from surrounding white matter and cerebrospinal fluid. The segmentation protocol is based on a novel histological reference data set labeled by multiple expert neuroanatomists. With broad participation of the research community, we voted on the segmentation protocol via online survey, which included detailed protocol information, feasibility testing, demonstration videos, example segmentations, and labeled histology. All boundary definitions were rated as having high clarity and reached consensus agreement by Delphi procedure. The harmonized body protocol yielded high inter- and intra-rater reliability. In the present paper we report the procedures to develop and test the protocol, as well as the detailed procedures for manual segmentation using the harmonized protocol. The harmonized protocol will significantly facilitate cross-study comparisons and provide increased insight into the structure and function of hippocampal subfields across the lifespan and in neurodegenerative diseases.

neuroscience↗

bioRxiv2025

Directing stem cell differentiation by chromatin state approximation

A prime goal of regenerative medicine is to replace dysfunctional cells in the body. To design protocols for producing target cells in the laboratory, one may need to consider exponentially large combinations of culture components. Here, we investigated the potential of iteratively approximating the target phenotype by quantifying the distance between chromatin profiles (ATAC-seq) of differentiating cells in vitro and their in-vivo counterparts. We tested this approach on the well-studied generation of erythroblasts from haematopoietic stem cells, evaluating a fixed number of components over two sequential differentiation rounds (8x8 protocols). We found that the most erythroblast-like cells upon the first round yielded the most erythroblast-like cells at the second round, suggesting that greedy selection by chromatin approximation can be a viable optimisation strategy. Furthermore, by analysing regulatory sequences in incompletely reprogrammed chromatin regions, we uncovered transcriptional regulators linked to roadblocks in differentiation and made a data-driven selection of ligands that further improved erythropoiesis. In future, our methodology can help craft notoriously difficult cells in vitro, such as B cells.

genomics↗

bioRxiv2025

Covalent targeting leads to the development of LIMK1 isoform-selective inhibitors

Selectivity for closely related isoforms of protein kinases is a major challenge in the design of drugs and chemical probes. Covalent targeting of unique cysteines is a potential strategy to achieve selectivity for highly conserved binding sites. Here, we used a pan-LIMK inhibitor to selectively probe LIMK1 over LIMK2 by targeting the LIMK1-specific cysteine C349 located in the glycine-rich loop region. Binding kinetics of both non-covalent and covalent LIMK inhibitors were investigated, and the fast on-rate and small size of type-I inhibitors were used in the design of a covalent LIMK1 inhibitor. The developed cell-active, isoform-selective LIMK1 inhibitor showed excellent proteome-wide selectivity in pull-down assays, enabling studies of LIMK1 isoform-selective functions in cellular model systems and providing a versatile chemical tool for studies of the LIMK signalling pathway.

biochemistry↗

bioRxiv2025

Development of pyrazolopyrimidine based macrocyclic kinase inhibitors targeting AAK1

Since the outbreak of SARS-CoV-2 in recent years, our society has become more aware that zoonotic diseases pose a real threat. Therefore, the demand for small molecules that target host proteins, essential for viral entry and replication, has increased as an interesting strategy for the development of antiviral agents, as these agents may be effective against several different pathogens. NAK kinases is one such potential target family because they are involved in a variety of cellular functions, hijacked by viruses to invade host cells, such as clathrin-mediated endocytosis. A large number of different inhibitors have already been reported targeting NAK kinases, but there are still no compounds that selectively target AAK1 over other NAK family members, in particular the closely related family member BIKE. Here, we developed a series of pyrazolo[1,5-a]pyrimidine-based macrocyclic NAK inhibitors, starting from the acyclic AAK1 inhibitor LP-935509. Through a structure-guided activity relationship study within the NAK family, we identified potent AAK1 inhibitors 16, 18 and 27, which show promising selectivity within the NAK family. The inhibitors showed a potent inhibition of the phosphorylation of the AP-2 complex and the antiviral activity of the compounds was evaluated against various RNA viruses.

biochemistry↗

bioRxiv2025

biscot: an Optimal Transport framework for multimodal bacterial single-cell data analysis

Computational optimal transport-based approaches have emerged as promising tools for the integration and interpretation of complex single-cell data. In this study, we introduce an integrative Optimal Transport (OT) framework for spatiotemporal and multi-omics bacterial single-cell analysis using Gaussian Mixture Model (GMM) OT, termed biscot (bacterial integrative single-cell optimal transport). We show that biscot, equipped with a novel global-to-local GMM initialization, outperforms classical OT and entropically-regularized OT methods both in terms of speed and accuracy for disentangling complex bacterial communities mixtures from single-cell flow cytometry data. When applied to time-series flow cytometry data from Bacillus subtilis, our framework delivers robust and biologically meaningful results, effectively capturing subtle phenotypic shifts in spore populations transitioning from inactive to active growth states. biscot also allows multi-omics integration of flow cytometry and unpaired bacterial single-cell RNA sequencing (scRNA-seq) data, enabling the alignment of individual gene expression profiles to the cytometric data. For an unpaired flow cytometry/scRNA-seq dataset of Bacillus subtilis cells, we validate the biological plausibility of inferred gene expression patterns with relevant marker genes, including spoVID and nin, closely aligning with observed cellular states. Overall, our framework thus provides not only dynamic tracking of phenotypic cell states but aligns cell states with detailed transcriptomic information from scRNA-seq, demonstrating its potential to advance microbial single-cell research. biscot will be made publicly available on GitHub.

bioinformatics↗

bioRxiv2025

Mapping Tumor Microenvironment and Treatment Response of Diffuse Midline Glioma Using Multiplexed Immunofluorescence and AI Models

BackgroundDespite its clinical promise in non-solid tumor, immunotherapy is yet to show significant clinical efficacy for brain tumors including pediatric diffuse midline glioma (DMG). This indicated the need to fully explore DMG immune tumor microenvironment (TME). MethodWhole brains (49 DMGs, 20 non-DMG, 10 non-malignant) from 79 pediatric patients were used to establish a tissue microarray (918 cores) representing primary, metastatic, and adjacent healthy sites. CellDIVE MxIF multiplex assay was used to probe for 33 immune and cell type markers. RNA sequencing (n=62 patients) defined additional immune signatures. Findings were validated using patient plasma and DMG PDX models. Our annotated single-cell atlas was used to train a spatial AI model to predict antigens from H&E staining. FindingsWe found enrichment of M1-activated microglia in primary versus adjacent healthy tissue. PD1 positive cells were significantly (p<0.01) higher in tumor compared to adjacent controls. This was validated by mRNA profiling, further indicating two distinct groups with top 35 significant (p<0.05) genes revealing synaptic signature in the metastatic cohort. We stratified the patient cohort by treatment. Imipridone cohort (n=5) showed decreased progenitor (Nestin+, Vimentin+, and SOX2+) and increased macrophages/microglia infiltration. Increased T and B cells was validated in patient plasma following imipridone therapy. Combination therapy of imipridone and immunotherapy (n=7) resulted in increased myeloid (Iba1, CD68, CD163) and lymphoid (CD3, CD8) cells. Enhanced immune engagement was validated in DMG PDX models. Machine learning resulted in a spatial AI model capable of predicting 22 antigens using H&E slides. InterpretationsDMG tumors maintain a cold immune microenvironment, which is nevertheless dynamic and responsive to therapy, indicating the need to explore combination therapies. AI-assisted antigen detection is suitable for rapid interpretation of clinical biospecimens. FundingThis work was supported by Rising Tide, SNF, LilaBean Foundation, Swifty Foundation, Swiss to Cure DIPG and Yuvaan Tiwari Foundation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=45 SRC="FIGDIR/small/644698v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@198b382org.highwire.dtl.DTLVardef@312e0forg.highwire.dtl.DTLVardef@c71b82org.highwire.dtl.DTLVardef@1df1c35_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

cancer biology↗

bioRxiv2025

A double-staining automated flow cytometry method for real-time monitoring of bacteria in continuous bioreactors

In industrial biotechnology, cell density is a critical parameter that influences key process control variables such as feed rate, harvest timing, and product recovery. Traditional biomass measurements are indirect, incapable of quantifying population heterogeneity, and often prone to inaccuracies due to manual handling and interference from culture media compounds. This study introduces an automated flow cytometry approach to enable continuous, real-time monitoring of bacterial cultures in a continuous bioreactor. Our method employs a double-staining protocol that combines DAPI for assessing total DNA content with Alexa Fluor 488 via Click-iT technology to detect the percentage of cells with active DNA replication through EdU incorporation. This streamlined protocol, which integrates cell fixation, permeabilization, staining, and measurement, was applied to three Gram-negative strains: Bradyrhizobium sp., Escherichia coli, and Stenotrophomonas rhizophila. The approach successfully captured both growth dynamics and cell cycle progression, providing rapid and quantitative insights into culture composition. The enhanced temporal resolution achieved by this method facilitates timely adjustments of process parameters, ultimately improving bioprocess efficiency and product quality. These results underscore the potential of automated flow cytometry as a powerful tool for real-time monitoring and control in industrial biotechnology.

microbiology↗

bioRxiv2025

Cannibalism Shapes Biofilm Structure and Composition in Bacillus subtilis

In Bacillus subtilis colony biofilms, phenotypic diversification confers tissue-like properties and enhanced competitive fitness within a structural framework that allows both colony expansion and long-term survival via endospore formation. Cannibalism is thought to delay sporulation by enabling one subpopulation to produce the sporulation delay protein SDP, the sporulation killing factor SKF and the epipeptide EPE. These toxins are thought to lyse susceptible nonproducers, thereby releasing nutrients to prevent premature sporulation. However, the molecular mechanisms orchestrating this bacterial programmed cell death during biofilm development are poorly understood. Here, we comprehensively characterized mutants defective in either toxin production or the corresponding autoimmunity by a multiscale approach, combining luminescence reporters, colony biopsy, multi-parameter flow cytometry and MALDI-mass spectrometry imaging to resolve cannibalism function and distribution. The toxins are produced in distinct, only partially overlapping areas of the colony and interdepend in their spatial distribution. Both EPE and SDP, but not SKF, are crucial for delaying sporulation. Loss of EPE or SDP autoimmunity resulted in severe morphological changes and stress-induced occurrence of suppressor mutants. The absence of all three toxins led to small, hyper-sporulating colonies with excessive wrinkle formation, indicating that cannibalism is essential for maintaining biofilm structure and lateral expansion. Our results provide the first evidence for the complex interactions between the cannibalism toxins that shape biofilm architecture through bacterial programmed cell death. Localized toxin production and their spatial distribution affect the spatiotemporal organization, morphology and subpopulation dynamics within B. subtilis biofilms. ImportanceProgrammed cell death (PCD) is a ubiquitous and crucial mechanism to structure eukaryotic multicellular tissues. PCD-like processes have also been described in bacteria, but their contribution to the multicellular development is poorly understood. Cannibalism in Bacillus subtilis has been described as a sporulation delay strategy, in which one subpopulation produces antimicrobial peptides that kill susceptible nonproducing siblings. Their lysis is thought to release nutrients that delay the sporulation in the producing subpopulation. This study comprehensively analyses the role of the three cannibalism toxins in shaping colony biofilms. By combining MALDI-mass spectrometry imaging, colony biopsy, flow cytometry, and luminescence reporters, we demonstrate that cannibalism toxins are crucial for biofilm structure. They show a discrete and interdependent localization within the biofilm. While cannibalism inhibits sporulation and causes severe envelope stress within colonies, our data challenges the established role of cannibalism-dependent killing as the mechanism behind this sporulation delay.

microbiology↗

bioRxiv2025

Mobile species' responses to surrounding land use generate trade-offs among nature's contributions to people

Agricultural landscapes provide material, non-material and regulating contributions that affect human wellbeing. The responses of these natures contributions to people (NCP) to land-use patterns depend on supporting biota with different habitat requirements, generating trade-offs and synergies. Predictions of NCP trade-offs could inform land-use decisions, but these do not typically account for the effects of land-use patterns on the movement of NCP-providing species, nor for interactions among NCP providers. To explore spatial trade-offs and synergies in eight indicators of NCP, we used Bayesian models that allow for interactions among land uses and among NCP using data from 150 grassland sites across rural Germany. We found that spatial arrangements of forest and open habitat influenced many NCP: acoustic diversity, birdwatching potential, natural enemy abundance and pollination. In particular, the amount and proximity of land uses in the surrounding landscape, especially forest and open habitat, drove the supply of most NCP. However, NCP provided by smaller-bodied taxa (herbivory and pathogen infection) responded weakly to landscape factors. Multiple NCP provided by a given taxon responded differently to their surrounding landscape (e.g. bird-provided caterpillar predation and seed predation), leading to trade-offs among NCP over short distances ([≤]60 m). These were caused by different rates and directions of response to amount and location of land uses. Resulting spatial predictions revealed that grassland-dominated or mixed forest-grassland areas better maximize beneficial NCP and minimize detrimental NCP across landscapes than purely forest-dominated areas. This suggests promoting semi-natural vegetation in agricultural landscapes to provide greater-than-additive benefits to net NCP supply. Significance StatementLand use affects natures contributions to people (NCP), including those provided by mobile species, in complex ways. Variation in the responses of species to the amount and location of land uses results in NCP trade-offs across landscapes, but decision-makers lack the capacity to spatially predict these trade-offs. We predict how the supply of both beneficial and detrimental NCP and associated trade-offs vary across diverse rural landscapes and find that grassland-dominated or mixed forest-grassland areas deliver higher net NCP supply than forest-dominated areas in our study system. Our findings support policies for promoting semi-natural vegetation cover in agricultural landscapes, as these may provide non-additive benefits for NCP.

ecology↗

bioRxiv2025

Type VI secretion phenotypic heterogeneity ensures trade-off between antibacterial activity and resistance in Enteroaggregative E. coli

The type VI secretion system (T6SS) is a nanoweapon deployed by bacteria to deliver effectors into target cells, allowing niche colonization or host invasion. In entero-aggregative Escherichia coli (EAEC), the expression of the T6SS gene cluster is responsive to iron starvation by the Fur regulator, and to Dam-dependent methylation. However, fluorescence microscopy recordings showed that the T6SS displays heterogeneous expression in a clonal population. The population is composed of three types of cells in a stable equilibrium: non producers (OFF), producers, and assemblers (ON). Single-cell analyses further showed that the activation state is transmitted to the progeny, but the overall distribution respects the equilibrium suggesting a genetic control of heterogeneity. Mutagenesis of Fur-binding boxes and GATC sites revealed that specific promoter elements control this heterogeneity. Phenotypic characterization of the ON and OFF subpopulations demonstrated that ON cells eliminate susceptible target cells, whereas OFF cells survive attacks from T6SS-defensive bacteria. The heterogeneity of EAEC T6SS is therefore an attenuation strategy conferring a trade-off between antibacterial efficiency and survival in polymicrobial environments.

microbiology↗

bioRxiv2025

Repurposing of the RIPK1 selective benzooxazepin-4-one scaffold for the development of a type-III LIMK1/2 inhibitor

Benzoxazepinones have been extensively studied as exclusively selective RIP kinase 1 inhibitors. This scaffold binds as a type-III inhibitor targeting the C-out/DFG-out conformation. This inactive conformation results in a large expansion of the kinase back pocket, a conformation that has also been reported for LIM kinases. Scaffold hopping is common in the design of orthosteric kinase inhibitors, but has not been explored in the design of allosteric inhibitors, mainly due to the typically exclusive selectivity of type III inhibitors. Here, we hypothesized that the shared structural properties of LIMKs and RIPKs could lead to novel type III LIMK inhibitors using the benzoxazepinone scaffold. We report the discovery of a novel LIMK1/2 inhibitor that relies on this scaffold-based approach. The discovered compound 10 showed low nanomolar potency on LIMK1/2 and exceptional selectivity, as confirmed by a comprehensive selectivity panel with residual RIPK activity as the only off-target. The study provides one of the few examples for scaffold hopping for type-III inhibitors which are usually associated with exclusive target selectivity. Table of contents (TOC) graphics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/636296v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@19bad48org.highwire.dtl.DTLVardef@18d8929org.highwire.dtl.DTLVardef@1280043org.highwire.dtl.DTLVardef@1296ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Esophageal adenocarcinoma relapse after chemoradiation is dominated by a basal-like subtype

Neoadjuvant chemoradiation therapy (RCT) is a frequently used treatment regimen for esophageal adenocarcinoma (EAC); however, the response varies dramatically, and resistance is a clinical challenge. We aimed to identify the molecular mechanisms underlying RCT resistance. We established a mouse xenograft RCT model with human EAC cell lines representing different response groups, and tested enhanced genomic instability as a potential evolutionary modulator by reducing BRCA2 function. Xenografts that relapsed after RCT displayed upregulation of stress response keratins, including KRT6 and KRT16 connected with a basal-like transcriptomic/ proteomic phenotype. We screened our cohort of 728 patients with EAC and found significantly shorter overall survival for patients with KRT6-high tumors, driven by patients receiving neoadjuvant treatment. Overall, we identified a basal-like cell state in EAC that reflects RCT relapse. The basal-like subtype is a marker of treatment failure, providing a new avenue for translational research to overcome RCT resistance.

cancer biology↗