Biology subjects

Wagner, A.

Publications and source records attributed to Wagner, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Conserved 'late' effector genes from Leptosphaeria maculans inducing gene-for-gene quantitative resistance in Brassica napus semi-winter genotypes

Leptosphaeria maculans is a phytopathogenic fungus responsible for stem canker on Brassica napus. Its infectious cycle goes through an early phase of leaf infection and a late phase of colonization and infection of the stem. The disease is mainly controlled by plant genetic resistances targeting a limited set of early fungal effector genes overexpressed during leaf infection and located in dynamic repeat-rich genomic regions. Thus, these resistances can be rapidly overcome by the pathogen. To find new sources of resistance, we focused on late effector genes, expressed during stem infection and located in gene-rich regions. A previous study revealed a quantitative resistance in the stem partly relying on a gene-for-gene interaction with a late effector gene. In this study, we deciphered whether all late effector genes shared the same genomic and evolutionary characteristics and if they could be more stable than early effector genes, rendering the resistances they trigger more durable. In addition, as previous studies highlighted new criteria for selecting late effectors and suggested B. napus semi-winter genotypes as an interesting genetic pool for uncovering resistance sources, we selected six new late effector gene candidates and screened an enlarged panel of semi-winter genotypes. We revealed that early and late effector genes diverged for most of their genomic characteristics supplying for the hypothesis of late effector genes being more conserved. Moreover, we revealed new resistance sources to late effector genes, almost all belonging to the semi-winter genetic pool, validating their importance to uncover new resistance sources.

plant biology↗

bioRxiv2025

De-novo promoters emerge more readily from random DNA than from genomic DNA

Promoters are DNA sequences that help to initiate transcription. Point mutations can create de-novo promoters, which can consequently transcribe inactive genes or create novel transcripts. We know little about how de-novo promoters emerge in genomic DNA, especially compared to random DNA that has never been subjected to selection. Here, we assayed the promoter activity of 17,129 random, synthetic DNA sequences and 91,866 E. coli genomic DNA sequences. Genomic DNA encodes ~1.3 times more promoters than random DNA. We then studied 584,573 point mutations in 225 random and 60 genomic sequences, and asked how they cause the emergence of de-novo promoters. We find that de-novo promoters emerge ~3 times more readily from random DNA than from genomic DNA. The reason is that the genome contains fewer proto-binding sites for transcriptional activators than random DNA. Our work shows that the evolutionary history of a DNA sequence introduces substantial biases in its evolutionary potential, especially in the likelihood that mutations create new and potentially adaptive transcripts.

evolutionary biology↗

bioRxiv2025

Inhibitor fluorination pattern tunes chemically induced protein dimerization

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite Trypanosoma brucei through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.

biophysics↗

bioRxiv2025

The fitness landscape of the E.coli lac operator is highly rugged in two different environments

We know little about the fitness landscapes of bacterial operators, regulatory DNA elements that are crucial to regulate metabolic genes like those of the lac operon for lactose utilization. For example, we do not know whether adaptive evolution could easily create strong operators from weak ones or from non-regulatory DNA. To find out, we used CRISPR-Cas-assisted genome editing, bulk competition, and high-throughput sequencing to map the fitness landscape of more than 140,000 lac operator variants in two chemical environments that harbor lactose or glycerol as sole carbon sources. Both landscapes are highly rugged and contain thousands of fitness peaks, which allow only 2 percent of evolving populations to reach a high fitness peak. The landscapes share only 15 percent of fitness peaks. Our work illustrates that landscape ruggedness caused by epistasis can represent an important obstacle to adaptive evolution of regulatory sequences. It also shows that a simple environmental change can substantially affect fitness landscape topography.

evolutionary biology↗

bioRxiv2025

Decoding Lymphangioleiomyomatosis (LAM) Niche Environment via Integrative Analysis of Single Cell Multiomics and Spatial Transcriptomics

Lymphangioleiomyomatosis (LAM) is a rare, progressive lung disease characterized by cystic destruction and metastatic growth of smooth muscle-like cells. Despite advances in understanding its genetic basis, the cellular heterogeneity, regulatory mechanisms, and microenvironmental interactions driving LAM progression remain poorly defined. In this study, we employed an integrative multi-omics approach combining single-cell RNA sequencing (scRNA-seq), single-nucleus ATAC sequencing (snATAC-seq), and high-resolution spatial transcriptomics (Visium, Visium HD, and Xenium) to decode the LAM niche in its native environment. We identified two spatially and functionally distinct LAM subtypes: LAMCORE1 and LAMCORE2. LAMCORE1 cells exhibited a uterine smooth muscle-like phenotype, expressing associated markers (ACTA2, MYH11) and were enriched in MTORC1 signaling and myogenic pathways, supporting a uterine origin. In contrast, LAMCORE2 cells displayed fibroblast-like features, with upregulated extracellular matrix (ECM) remodeling genes (COL1A1, MMP11) and epithelial-to-mesenchymal transition (EMT) pathways, suggesting a role in niche formation. Pseudotime and regulon analyses revealed dynamic transitions between these subtypes, driven by distinct transcriptional networks (e.g., HOX/PBX in LAMCORE1, TWIST/ZEB in LAMCORE2). The presence of the two distinct LAM subtypes was further validated by RNAscope and immunofluorescence microscopy. We identified LAM-associated fibroblasts (LAFs) as activated stromal cells expressing canonical markers (FAP, S100A4, VIM, IGFBP7, SPARC) and localized within LAM lesions. Subpopulations of LAFs, LAF-seed (proximal to LAMCORE1) and LAF-niche (surrounding LAM niches), exhibited unique functional profiles, including ECM deposition, TGF-{beta} signaling, and myofibroblast activation. Regulatory network analysis pinpointed EGR1 as a central hub governing LAF phenotype. Our comprehensive spatial profiling revealed niche structures dominated by LAMCORE1 cells and surrounded by lymphatic endothelial cells (LECs), LAFs, scattered LAMCORE2 cells, macrophages, and reprogrammed alveolar epithelial cells (AT2). ECM remodeling and aberrant organization of cable-like structures (-smooth muscle actin+) of the LAM niches were further validated by second harmonic generation microscopy. These findings provide a high-resolution blueprint of LAM pathogenesis, highlighting the interplay between uterine-derived LAMCORE cells, activated fibroblasts, and the remodeled lung microenvironment. They significantly enhance our understanding of the LAM niche microenvironment and offer insights into potential therapeutic targets and strategies for managing this complex disease.

genomics↗

bioRxiv2025

Intra-Tumoral Treg Ablation Unleashes NK Cell-Mediated Control of CD8 T Cell-Resistant Tumors

Cancer cells frequently lose MHC I to evade CD8+ T cell recognition. While Natural Killer (NK) cells are poised to target MHC I-deficient cancer cells, MHC I loss alone is often insufficient to unleash fully effective NK cell responses. Here we show that selective intra-tumoral (IT) ablation of regulatory T (Treg) cells elicited potent antitumor NK cell responses that controlled MHC I-deficient and even MHC I+ cancers. Tregs controlled the activation, maturation, and anti-tumor cytotoxic activity of NK cells within the tumor microenvironment. Mechanistically, IT Tregs prevented the cDC2-dependent induction of IL-2 production by CD4+ Tconv cells that was necessary for NK cell activation. Systemically administered antibodies that selectively depleted IT Tregs similarly empowered NK- dependent tumor control. These findings expand the breadth of Treg-mediated cancer immunosuppression to encompass antitumor NK cells and suggest that targeting Tregs in tumors can control CD8+ T cell-resistant cancers. One Sentence SummaryIT Treg ablation drives NK cell tumor control via CD4+ Tconv-derived IL-2, eliminating MHC I+/MHC I- cancers without systemic toxicity.

immunology↗

bioRxiv2025

In vitro model of human subcutaneous adipocyte spheroids for studying mitochondrial dysfunction and mitochondria activating compounds

Mitochondrial abnormalities drive subcutaneous white adipose tissue dysfunction in obesity, leading to metabolic complications. A key challenge in obesity research is the lack of in vitro models to study human adipocyte mitochondria. Here, we establish a human subcutaneous adipocyte spheroid model to characterize mitochondrial metabolism under obesity-relevant conditions and drug exposure. Human preadipocyte spheroids were differentiated in ultra-low attachment plates for 3 weeks in media free of thiazolidinediones. The differentiated adipocyte spheroids showed increased lipid accumulation, adipogenic gene expression, mitochondrial respiration and adiponectin secretion, and a proper response to hormonal stimulation. Lipid mixture administration during differentiation induced metabolic disturbances including mitochondrial respiration failure associated with increased mitochondrial biogenesis. Post-differentiation treatment with rosiglitazone, a peroxisome proliferator-activated receptor {gamma} agonist, improved mitochondrial bioenergetics and adiponectin secretion in lipid mixture-administered adipocyte spheroids. Our model enables precise measurement of adipocyte mitochondria under various conditions, providing a platform for mitochondria-related research and drug discovery in obesity.

molecular biology↗

bioRxiv2025

Poxvirus attack of anti-viral defense pathways unleashes an effector-triggered NF-κB response

Effector-triggered immunity (ETI) is a form of pathogen sensing that involves detection of pathogen-encoded virulence factors or "effectors". To discover novel ETI pathways in mammals, we developed a screening approach in which individual virulence factors are expressed in human monocytes and transcriptional responses are assessed by RNA-seq. Using this approach, we identify a poxvirus effector, myxoma virus M3.1, which elicits an anti-viral NF-{kappa}B response. We find that NF-{kappa}B is unleashed by an ETI pathway that senses M3.1 attack of two anti-viral complexes: ZAP and TBK1. NF-{kappa}B activation occurs because the proteins inhibited by M3.1-- N4BP1, ZC3H12A, and TBK1--are negative regulators of NF-{kappa}B. Our results illustrate how negative regulators can function as pathogen sensors and establish a systematic approach for the discovery of ETI pathways.

immunology↗

bioRxiv2025

Distinct roles for thymic stromal lymphopoietin (TSLP) and IL-33 in experimental eosinophilic esophagitis

RationaleThymic stromal lymphopoietin (TSLP) and IL-33 are alarmins implicated in EoE pathogenesis by activating multiple cells including mast cells (MCs). Whether TSLP or IL-33 have a role in EoE and whether their activities are distinct requires further investigation. MethodsExperimental EoE was induced in wild type (WT) Il33-/-and Crlf2-/- mice. TSLP or IL-5 were neutralized using antibodies. Esophageal histopathology was determined by H&E, anti-Ki67, anti-CD31 and anti-MBP staining. Esophageal RNA was subjected to RNA sequencing. Bone marrow-derived MCs were activated with TSLP and IL-13 was determined (ELISA) ResultsTSLP and IL-33 were overexpressed in human and experimental EoE. Human and mouse esophageal MCs displayed the highest level of Crlf2 (TSLPR) compared to other immune cells. Crlf2-/- mice were nearly-completely protected from EoE, and TSLP neutralization resulted in decreased basal cell proliferation, eosinophilia, lamina propria thickening and vascularization. Induction of experimental EoE in Il33-/- mice resulted in reduced eosinophilia but no alterations in tissue remodeling were observed compared to WT mice. RNA sequencing revealed that TSLP regulates the expression of key genes associated with human EoE (e.g. eotaxins, Il19, Klk5, Flg, Il36rn, Il1r2) and suggest a role for TSLP in regulating IL-1 signaling, barrier integrity and epithelial cell differentiation. Experimental EoE was characterized by a MC-associated gene signature and elevated MCs. Activation of MCs with TSLP resulted in secretion of IL-13. ConclusionTSLP and IL-33 have non-redundant functions in experimental EoE. This study highlights TSLP as an upstream regulator of IL-13 and a potential therapeutic target for EoE.

immunology↗

bioRxiv2025

Tracing social mechanisms and interregional connections in Early Bronze Age Societies in Lower Austria

In this study, we present the results of archaeogenetic investigations of Early Bronze Age individuals from Lower Austria, specifically associated with the Un[e]tice and Unterwolbling cultural groups. Through analysing newly generated genome-wide data of 138 individuals, we explore the social structure and genetic relationships within and between these communities. Our results reveal a predominantly patrilocal society with non-strict female exogamic practices. Additionally, Identity-by-Descent (IBD) analysis detects long-distance genetic connections, emphasizing the complex network of interactions in Central Europe during this period. Despite shared social dynamics, notable genetic distinctions emerge between the Un[e]tice and Unterwolbling groups. These insights contribute to our understanding of Bronze Age population interconnections and call for a nuanced interpretation of social dynamics in this historical context.

genetics↗

bioRxiv2025

Morphology and distribution of a new scale mechanoreceptor type in olive-headed sea snakes (Hydrophis major)

Snakes are known for their superb sensory specialisations but are not widely appreciated for their sense of touch despite emerging evidence of tactile specialisation among sea snakes. This is partly due to the challenges in quantifying such small and numerous scale mechanoreceptors or scale sensilla across individuals. By using a novel application of gel-based 3D profilometry (GelSight scanner) in combination with scanning electron-and light microscopy, we comprehensively quantified the morphology and distribution of scale mechanoreceptors in a sea snake, Hydrophis major (Hydrophiinae), for the first time. We discovered a new type of scale mechanoreceptor distinguished by its larger size (43.65 {+/-} 22.57 m height) and asymmetrical peak shape, created by a thickening in the cornified outer layers of the epidermis. Asymmetrical peaks contain a dermal papilla with central cells, indicative of meissner-like corpuscles that underlie smooth dome-shaped mechanoreceptors (31.88 {+/-} 19.03 m height) typically found in both sea snakes, but is positioned slightly off centre from the tallest point of the asymmetrical peak. Smooth domes are concentrated anterior-posteriorly on the head with the highest densities on the rostrum and nasal scales (9.83 {+/-} 1.88 and 5.24 {+/-} 2.85 per mm{superscript 2}, respectively). Asymmetrical peaks are rarer; detected only on the dorsal and lateral sides of the head and most common on the postocular scale (mean density 1.26 {+/-} 0.72 per mm{superscript 2}). We suggest functional differences in mechanosensory capabilities: asymmetrical peaks may serve primarily proprioceptive purposes whereas smooth domes are used for hydrodynamic reception.

ecology↗

bioRxiv2025

CD38 expression by neonatal human naïve CD4+ T cells shapes their distinct metabolic state and high regulatory T cell potential

Neonatal life is marked by rapid antigen exposure, necessitating establishment of peripheral immune tolerance via conversion of naive CD4+ T cells into regulatory T cells (Tregs). Here, we demonstrate heighted capacity for FOXP3 expression and tolerogenic function among cord blood versus adult blood naive CD4+ T cells and that this is linked to their unique metabolic profile and elevated expression of the NADase, CD38. Early life naive CD4+ T cells demonstrate a metabolic preference for glycolysis, which directly facilitates their differentiation trajectory. We reveal an age-dependent gradient in CD38 levels on naive CD4+ T cells and show that high CD38 expression contributes to both the glycolytic state and tolerogenic potential of neonatal CD4+ T cells, effects that are mediated at least in part via the NAD-dependent deacetylase SIRT1. Thus, the early life window for peripheral tolerance in humans is critically enabled by the immunometabolic state of the naive CD4+ compartment.

immunology↗

bioRxiv2024

STAT1 Promotes PD-L1 Activation and Tumor Growth in Lymphangioleiomyomatosis

Lymphangioleiomyomatosis (LAM) is a cystic lung disease that primarily affects women. LAM is caused by the invasion of metastatic smooth muscle-like cells into the lung parenchyma, leading to abnormal cell proliferation, lung remodeling and progressive respiratory failure. LAM cells have TSC gene mutations, which occur sporadically or in people with Tuberous Sclerosis Complex. Although it is known that hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) due to TSC2 gene mutations contributes to aberrant cell growth in LAM lung, tumor origin and invasive mechanism remain unclear. To determine molecular drivers responsible for aberrant LAM cell growth, we performed integrative single-cell transcriptomic analysis and predicted that STAT1 interacts with Pre-B cell leukemia transcription factor (PBX1) to regulate LAM cell survival. Here, we show activation of STAT1 and STAT3 proteins in TSC2- deficient LAM models. Fludarabine, a potent STAT1 inhibitor, induced the death of TSC2- deficient cells, increased caspase-3 cleavage, and phosphorylation of necroptosis marker RIP1. Fludarabine treatment impeded lung colonization of TSC2-deficient cells and uterine tumor progression, associated with reduced percentage of PCNA-positive cells in vivo. Interestingly, IFN-{gamma} treatment increased STAT1 phosphorylation and PD-L1 expression, indicating that STAT1 aids TSC2-deficient tumor cells in evading immune surveillance in LAM. Our findings indicate that STAT1 signaling is critical for LAM cell survival and could be targeted to treat LAM and other mTORC1 hyperactive tumors.

cancer biology↗

bioRxiv2024

The adaptive landscapes of three global Escherichia coli transcriptional regulators

The evolution of gene regulation is a major source of evolutionary adaptation and innovation, particularly when organisms encounter new or changing environments. Central to this process is the emergence of new transcription factor binding sites (TFBSs). Adaptive landscapes provide a powerful framework to study such emergence by linking regulatory DNA sequences to their transcriptional outputs. Although several landscapes have been characterized for DNA, RNA, and proteins, large-scale in vivo adaptive landscapes for bacterial TFBSs remain scarce. Here, we address this gap by experimentally mapping the first comprehensive in vivo regulatory landscapes for three global transcription factors in Escherichia coli: CRP, Fis, and IHF. Using a massively parallel reporter assay, we quantify the regulation strength of more than 30,000 TFBS variants for each factor and reconstruct their adaptive landscapes. All three landscapes are highly rugged and exhibit pervasive epistasis, with thousands of local peaks distributed broadly across sequence space. This ruggedness contrasts sharply with the much smoother TFBS landscapes of eukaryotes. It suggests greater constraints on the evolution of prokaryotic gene regulation. Nonetheless, evolutionary simulations show that [~]10% of evolving populations can reach a peak of strong regulation, a proportion that is significantly greater than in comparable random landscapes. Adaptive evolution starting from the same DNA sequence can attain different high peaks, and some peaks are reached more frequently than others. Together, our results show that de novo adaptive evolution of new gene regulation in bacteria is feasible, but subject to a blend of chance, historical contingency, and evolutionary biases.

evolutionary biology↗

bioRxiv2024

Entangled adaptive landscapes facilitate the evolution of gene regulation by exaptation

Exaptation, the co-option of existing traits for new functions, is a central process in Darwinian evolution. However, the molecular changes leading to exaptations remain unclear. We investigated the potential of bacterial transcription factor binding sites (TFBSs) to evolve exaptively for the three global E. coli transcription factors (TFs) CRP, Fis, and IHF. Using a massively parallel reporter assay, we mapped three combinatorially complete adaptive landscapes, encompassing all intermediate sequences between three pairs of strong TFBSs for each TF. Our results revealed that these landscapes are smooth and navigable, with a monotonic relationship between mutations and their impact on gene regulation. Starting from a strong TFBS for one of our TFs, Darwinian evolution can create a strong TFBS for another TF through a small number of individually adaptive mutations. Notably, most intermediate genotypes are prone to transcriptional crosstalk - gene regulation mediated by both TFs. Because our landscapes are smooth, Darwinian evolution can also easily create TFBSs that show such crosstalk whenever it is adaptive. We also present evidence of exaptive evolution and crosstalk from an analysis of bacterial genomes. Our study presents the first in vivo evidence that new TFBSs can evolve exaptively through multiple small and adaptive mutational steps. It also highlights the importance of regulatory crosstalk for the diversification of gene regulation.

evolutionary biology↗

bioRxiv2024

AI-readiness for Biomedical Data: Bridge2AI Recommendations

Biomedical research is rapidly adopting artificial intelligence (AI). Yet the inherent complexity of biomedical data preparation requires implementing actionable, robust criteria for ethical and explainable AI (XAI) at the "pre-model" stage, encompassing data acquisition, detailed transformations, and ethical governance. Simple conformance to FAIR (Findable, Accessible, Interoperable, Reusable) Principles is insufficient. Here, we define criteria and practices for reliable AI-readiness of biomedical data, developed by the NIH Bridge to Artificial Intelligence (Bridge2AI) Standards Working Group across seven core dimensions of dataset AI-readiness: FAIRness, Provenance, Characterization, Ethics, Pre-model Explainability, Sustainability, and Computability. Conformance to these criteria provides a basis for pre-model scientific rigor and ethical integrity, mitigating downstream risks of bias and error prior to AI modeling. We apply and evaluate these standards across all four Bridge2AI flagship datasets, spanning functional genomics to clinical medicine, and encode them in machine-actionable metadata bound to the datasets. This framework sets a benchmark for preparing ethical, reusable datasets in biomedical AI and provides standardized methods for reliable pre-model data evaluation.

bioinformatics↗

bioRxiv2024

New components of the community based DNA-repair mechanism in Sulfolobales

After exposure to UV light, Sulfolobus acidocaldarius cells aggregate in a species-specific manner to exchange DNA and repair double-strand breaks via homologous recombination. The formation of cell-cell interactions is mediated by Ups pili. DNA exchange subsequently occurs through the Ced system, which imports DNA. To identify novel players in these processes, we investigated several genes upregulated after UV exposure by creating in-frame deletion mutants and performing cell aggregation and DNA exchange assays. This led to the identification of two novel components involved in the Ups and Ced systems: UpsC, a minor pilin of the Ups pili, and CedD, a VirD4-like ATPase essential for DNA import. Altogether, these findings provide new insights into the fascinating DNA damage response of Sulfolobales.

microbiology↗

bioRxiv2024

The glycolytic reaction PGAM unexpectedly restrains Th17 pathogenicity and Th17-dependent autoimmunity

Glucose metabolism is a critical regulator of T cell function, largely thought to support their activation and effector differentiation. Here, we investigate the relevance of individual glycolytic reactions in determining the pathogenicity of T helper 17 (Th17) cells using single-cell RNA-seq and Compass, an algorithm we previously developed for estimating metabolic flux from single-cell transcriptomes. Surprisingly, Compass predicted that the metabolic shunt between 3-phosphoglycerate (3PG) and 2-phosphoglycerate (2PG) is inversely correlated with pathogenicity in these cells, whereas both its upstream and downstream reactions were positively correlated. Perturbation of phosphoglycerate mutase (PGAM), an enzyme required for 3PG to 2PG conversion, resulted in an increase in protein expression of IL2, IL17, and TNFa, as well as induction of a pathogenic gene expression program. Consistent with PGAM playing a pro-regulatory role, inhibiting PGAM in Th17 cells resulted in exacerbated autoimmune responses in the adoptive transfer model of experimental autoimmune encephalomyelitis (EAE). Finally, we further investigated the effects of modulating glucose concentration on Th17 cells in culture. Th17 cells differentiated under high- and low-glucose conditions substantially differed in their metabolic and effector transcriptomic programs, both central to Th17 function. Importantly, the PGAM-dependent gene module marks the least pathogenic state of Th17 cells irrespective of glucose concentration. Overall, our study identifies PGAM, contrary to other glycolytic enzymes, as a negative regulator of Th17 pathogenicity.

immunology↗