Biology subjects

Wagner, A.

Publications and source records attributed to Wagner, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Rapid femtosecond laser trenching accelerates cryo-electron tomography lamella workflows

Transmission electron microscopy requires electron-transparent specimens, typically below 200 nm thick. For cellular and tissue samples this is typically achieved by focused ion-beam (FIB) thinning to form lamellae. Milling times scale with specimen thickness, and high-pressure-frozen specimens more than 10 m thick can take up to 2 hours per lamella. We present a pre-processing approach in which bulk material is removed by ultrashort-pulse laser ablation prior to milling, rapidly generating large trenches in vitrified grids under cryogenic conditions and leaving material to be finished by conventional milling to yield electron-transparent lamellae. Laser ablation completes in seconds (4 seconds per pattern), compared with minutes to tens of minutes for FIB trench milling, and introduces negligible damage to the final lamellae. By replacing the most time-intensive, contamination-sensitive bulk-removal steps with seconds-long laser trenching, this open-source approach reduces time-to-lamellae, increasing throughput and preserves compatibility with standard cryo-FIB/SEM polishing and cryo-ET data-collection pipelines.

biophysics↗

bioRxiv2026

Tumbly: An Open-Source Home-Cage Device for Time-Restricted Feeding in Mice

Time-restricted feeding (TRF) is a common tool for studying metabolism, circadian rhythms, aging, and neurodegeneration. Here, we describe a new device called Tumbly, an automated device for performing TRF studies in mouse home-cages. Tumbly controls access to rodent diets at user-set times via a programmable servo-driven food hopper. Tumbly is also battery powered and compatible with multiple types of vivarium home-cages, due to a removable faceplate that can be customized to fit different cage dimensions without modification. Finally, Tumbly is open-source and affordable, with all design files available online, allowing researchers to build and customize it. As a validation test, we deployed Tumbly in two labs at separate institutions, one with singly housed mice (n = 6, within-subject) and one with group-housed mice (n = 22, between-group). We measured daily body weight and food intake. In both labs, mice on an 8-hour TRF schedule maintained their body weight and ate similar amounts of food compared to ad libitum feeding. Our quantified error rates were also low, with no device failures over >1000 hours of operation. We conclude that Tumbly provides a reliable, low-cost option for running TRF studies in mice.

neuroscience↗

bioRxiv2026

Human cerebral assembloids as a model to study the interaction of glioblastoma with resident glial cells

Glioblastoma (GB) is a highly invasive brain cancer characterized by extensive peritumoral glial reactivity. Although GB infiltration and recurrence remain major therapeutic challenges, the contribution of GB-induced gliosis to tumor progression is poorly understood. To investigate glia-GB interactions, we developed human iPSC-derived cerebral forebrain assembloids containing innate microglia and macroglia and transplanted them with GB cells. The model recapitulates key tumor features observed in patients, including distinct tumor regions, reactive gliosis, and altered tissue stiffness. Single-cell RNA sequencing revealed an unexpected enrichment of extracellular vesicles (EVs) in peritumoral reactive glia, which we validated in patient samples. EV inhibition impaired formation of the peritumoral niche, whereas EVs from GB-containing assembloids induced glial reactivity in GB-free assembloids. Furthermore, the modularity of our system enables incorporation of additional niche components, such as monocytes, allowing investigation of the monocyte-glia-tumor axis and providing a powerful platform to dissect mechanisms regulating the GB microenvironment.

neuroscience↗

bioRxiv2026

Profiling of intestinal T cells reveals antigen-specific dependency on early-life programming for the generation of microbiota-reactive Tregs

The generation of appropriate responses to the intestinal microbiota is an essential aspect of immune homeostasis. Studies of microbes that elicit stereotyped, antigen-specific CD4 T cell responses support the view that immune recognition of the microbiota is specific, relatively limited, and largely skewed toward Tregs, but recent profiling efforts have suggested that recognition of the microbiota may be broader and less regulatory. Such studies have mostly tracked responses to single antigens or used mice that acquire their microbiota after weaning, which has made it difficult to generalize the rules that govern microbiota-mediated T cell responses. Here we comprehensively profile, for the first time, the T cell response to the microbiota at homeostasis, and develop a panel of tools to track multiple, endogenous microbiota-reactive CD4 T cell populations in vivo. Our profiling reveals a broad microbiota-reactive T cell response that is Treg-biased but that also contains non-Treg responses to specific microbes. We find that certain microbiota-reactive T cells require priming during early-life to efficiently establish their Treg identity, while others do not. Our findings illustrate an unappreciated antigen-specific complexity in programming of microbiota-reactive T cells and underscore the importance of studying responses to the microbiota under homeostatic conditions.

immunology↗

bioRxiv2026

Extracting interpretable single-cell metabolic states with graph-guided representation learning

Metabolism shapes cellular function and state, yet measuring single-cell metabolic states at scale remains a challenge. We present Metabolic Representation Net (MeRN), a graph-guided variational autoencoder that leverages prior metabolic knowledge as a topology graph to learn latent representations of metabolic state and reaction activity from single-cell transcriptomes. MeRN's scalable estimation of reaction activity enables the definition of data-driven pathways (DDPs): context-specific metabolic modules supported by transcriptomic evidence and agnostic of standard pathway definitions. Using DDPs, we introduce the weakest link analysis to identify metabolic network rewiring. MeRN recovers metabolic zonation in the mouse intestine, links a folate deficiency-induced break in de novo purine synthesis to embryonic neural tube defects, shows cytokines with similar non-metabolic effects can elicit divergent T cell metabolism, and identifies metabolic drivers of T cell exhaustion and therapy response in human cancers. Our results establish MeRN as a unified method for metabolic analysis of single-cell transcriptomes.

bioinformatics↗

bioRxiv2026

Folate deficiency disrupts key metabolic transitions within the developing neural ectoderm

Despite long-standing epidemiological associations, the mechanism linking folate availability to gestational neural tube defects remains unclear, partly because measuring and interpreting metabolic activity in dynamic biological systems remains challenging. Here, we apply a deep-learning-based graph-guided variational autoencoder (MeRN; Metabolic Representation Network) to infer single-cell metabolic activity and states from scRNA-seq data of mouse embryogenesis. By analyzing folate-deficient embryogenesis from E7.0 to E9.0, we identify a transient state within the nascent neural lineage that is acutely sensitive to folate availability, leading to an interconnected disruption between key bioenergetic pathways and de novo purine biosynthesis. Moreover, metabolically induced growth defects lead to permanent morphological disruptions along the dorsal-ventral axis, which we confirm by generating whole-embryo fate maps using a prime-editing-based lineage recorder (PEtracer). Collectively, our results establish a highly scalable framework for interpreting dynamic changes in embryonic metabolism and elucidating the mechanistic bases underlying environmentally linked congenital disorders.

genomics↗

bioRxiv2026

Bacterial secreted products selectively inhibit non-symbiotic fungi in bees

Microbial interactions play an important role in shaping microbiome assembly, such as by limiting invasion by harmful organisms that can directly affect the host or disrupt microbiome-associated benefits. Such interactions have been observed across systems, including in the microbiomes of key pollinators such as stingless bees. In Scaptotrigona depilis, bacteria associated with the larval diet inhibit potentially pathogenic filamentous fungi while allowing beneficial yeast symbionts to persist. The mechanisms underlying these effects, however, remain unclear. Here, we combined conditioned media (cell-free supernatant) assays with genomic and metabolomic analyses to investigate whether bacterial secreted products mediate these effects in the bee microbiome. Our results show that bacterial secreted products, particularly from prevalent bacterial taxa such as Apilactobacillus kunkeei, strongly affect fungal growth. Filamentous fungi, including the pathogen Aspergillus, were consistently inhibited, partly through substrate acidification driven by organic acids, but also through additional acidity-independent factors. In contrast, yeast responses were more variable: a non-symbiotic Zygosaccharomyces was inhibited by bacterial metabolites under near-neutral pH, whereas the symbiotic Zygosaccharomyces required for larval development was maintained or promoted under acid-conditioned media. Genomic analyses revealed limited canonical antifungal biosynthetic clusters in the most prevalent bacteria in the larval diet, while metabolomics identified extracellular peptide-like compounds across strains, suggesting a role for non-canonical secreted products. Together, these results show that bacterial secreted products play a key role in selectively shaping fungal communities in the stingless bee larval diet, providing first hints on a mechanistic basis for how microbial interactions structure this ecosystem.

microbiology↗

bioRxiv2026

Long-term stability of cellular-resolution brain-computer interface recordings after stroke

Implantable brain-computer interfaces (iBCIs) with single-neuron resolution are showing great promise for restoring mobility and communication in individuals with spinal cord injury or motor neuron disease. Stroke is the most common cause of acquired brain injury and a major contributor to long-term disability, making chronic stroke a highly relevant indication for iBCIs. However, whether stable intracortical recordings can be obtained from the structurally lesioned human brain is unknown. We report recordings from four 64-channel microelectrode arrays implanted in a participant with chronic aphasia after a large left-hemispheric stroke. The arrays targeted right-hemispheric frontoparietal regions homotopic to the damaged left-hemispheric language network. Across 111 sessions spanning 1,240 days, unit yield and signal quality remained stable. Waveform-based tracking reliably identified individual units across sessions, including across extended recording gaps. Short- and long-term unit stability was comparable to previous reports from iBCI participants without structural brain lesions, and tracked units showed consistent spiking properties across sessions. Our findings provide the first evidence that single-neuron recordings can remain stable over the long term in the stroke-lesioned human brain. They establish the feasibility of chronic, cellular-resolution iBCIs after stroke and support the development of neurorestorative applications for deficits caused by structural brain injury.

neuroscience↗

bioRxiv2026

Regulatory scope shapes the adaptive landscapes of bacterial transcription factor binding sites

Transcription factors (TFs) span a regulatory hierarchy from local regulators that control one or few genes to global regulators that regulate hundreds. Local TFs typically operate through few, highly specific binding sites; global TFs through many of varying affinity. Whether this difference in TF biology systematically affects TFBS evolution is unclear. Here, we address this question by studying experimentally mapped adaptive landscapes of transcription factor binding sites (TFBSs) for five bacterial TFs that differ in their regulatory scope -- TetR (local), LasR (semi-global), and CRP, Fis, and IHF (global). All landscapes are rugged and epistatic, but three topographic properties vary systematically with regulatory scope. First, mean mutational robustness increases from local to global TFs. Second, high-regulation-strength peaks are clustered in the local landscape but dispersed in the global ones. Third, adaptive evolution reaches high adaptive peaks more readily in local landscapes. All five landscapes also harbor many evolvability-enhancing (EE) mutations, which increase the likelihood that subsequent mutations are beneficial. The fraction of these mutations is approximately an order of magnitude higher than previously reported for a protein landscape. Populations experiencing EE mutations consistently reach higher regulation strength, an effect that is strongest in the local TetR landscape. Together, these results identify regulatory scope as an organising axis of TFBS adaptive landscapes.

evolutionary biology↗

bioRxiv2026

Negative Autoregulation Promotes the Evolution of Strong Environmental Switching

Living systems rely on gene regulatory circuits to respond to environmental change. Such circuits often act as molecular switches: OFF in the absence of a cue, and ON in its presence. We do not know how a circuits regulatory architecture affect its ability to evolve such responsiveness. In nature, a very frequent and simple regulatory architecture involves a transcriptional regulator that negatively regulates its own expression. To study how such autoregulation affects adaptive evolution, we engineered E. coli circuits in which a target gene is regulated by the repressor TetR with (A-) or without (A0) negative autoregulation of TetR. We evolved TetR in both architectures toward responsiveness to a novel environment, embodied by a novel inducer of TetR. Early during their evolution, TetR circuits with negative autoregulation evolved stronger environmental switching. A combination of high throughput DNA sequencing, protein engineering, and biophysical modeling showed why. Only A-circuits favored TetR alleles that interact strongly with both the inducer and DNA. Such alleles combine strong repression caused by strong DNA binding with strong derepression caused by strong inducer binding, the defining property of a strong environmental response. Our biophysical model shows that negative autoregulation helps to create this regulatory regime. As a result, only A- circuits favor alleles that create strong molecular switches. Altogether, our work shows that even the simplest form of gene regulation can change the topography of a fitness landscape, and enable new modes of evolutionary change.

evolutionary biology↗

bioRxiv2026

Preserved Barrier Integrity and Altered Immune Profiles in Chronic Cannabis Users: Potential Roles of Δ9-Tetrahydrocannabinol

Although cannabinoids such as delta-9-tetrahydrocannabinol (THC) are generally immunosuppressive in preclinical models, chronic cannabis use in humans is paradoxically associated with increased infection risk and systemic inflammation. In this study, we demonstrate that THC directly strengthens intestinal epithelial barrier function in vitro by increasing trans-epithelial electrical resistance in a concentration-dependent manner in Caco-2 monolayers. In a cross-sectional study of chronic cannabis users via smoking or snorting compared with non-using controls, plasma lipopolysaccharide (LPS), and microbial translocation-driven inflammatory cytokines (IL-23, MCP-1, IL-8) were significantly reduced, while some cytokines (IL-6, IL-1{beta}, TNF-, IL-10) remained unchanged. Concurrently, users exhibited elevated macrophage-derived chemokine (MDC) and homeostatic cytokines IL-15 and IL-21, markedly suppressed IL-7 and IL-4. Plasma IL-15 and MDC levels correlated with consumption intensity, and IL-23, IL-7, and IP-10 correlated with age of first use or during heaviest use. These findings suggest that habitual cannabis use may protect gut barrier integrity and reduce microbial translocation and associated inflammation, while simultaneously disrupting systemic immune homeostasis through selective cytokine dysregulation. This dual, dose-dependent immunomodulatory profile highlights the complex balance between potential benefits and risks in both recreational and therapeutic cannabis use.

immunology↗

bioRxiv2026

Oral exposure to Perfluorooctanoic acid disrupts the microbiota-gut-liver axis and enhances the severity of chemically induced colitis in mice.

Inflammatory bowel diseases (IBD) affect millions of patients worldwide and impair quality of life. Although genetic and environmental factors are known to disrupt the gastrointestinal (GI) epithelial barrier and increase susceptibility to IBD, the precise contribution of specific environmental exposures remains unclear. Per- and polyfluoroalkyl substances (PFAS), or "forever chemicals," are widely used in consumer products and contaminate food and water sources, resulting in chronic oral exposure worldwide. Perfluorooctanoic acid (PFOA), a common PFAS, has been epidemiologically associated with the development of IBD, particularly in older adults. Here, we assessed the effects of oral PFOA exposure on the GI tract, liver, and susceptibility to colitis. C57BL/6 mice were exposed to PFOA (0.1 mg/kg or 1.0 mg/kg) beginning at weaning (post-natal day [P]21) for a time course of 4 or 8 weeks. GI physiology/pathology (Ussing chambers; histology), expression of pro-inflammatory cytokines (qPCR), microbiota composition (16S sequencing), bile acids production (qPCR; LC/MS), and liver pathology (histology) were assessed. Colitis susceptibility was evaluated in genetically predisposed (IL10 knockout) mice, and in induced (dextran sodium sulfate [DSS]) mouse models following PFOA exposure (8 weeks at 1.0 mg/kg). Oral PFOA exposure increased intestinal permeability, mildly increased cytokine expression, altered gut microbiota composition, disrupted liver and serum bile acids, and caused hepatic hypertrophy at higher doses and longer exposure. Although PFOA did not increase disease susceptibility in genetically predisposed Il10 KO mice, it significantly worsened DSS-induced colitis, but only in male mice. Together, these findings demonstrate that early-life PFOA exposure disrupts the gut-liver axis and may contribute to colitis development in a sex dependent manner.

pharmacology and toxicology↗

bioRxiv2026

Vagus nerve stimulation limits colonic inflammation through distinct neuroimmune circuitry shaped by inflammatory history

Bidirectional communication between the nervous and immune systems has been demonstrated to limit or enhance immune cell function across organ systems and conditions. Although these neuroimmune circuits can become activated as an anti-inflammatory reflex, vagus nerve stimulation (VNS) reduces inflammation in models of endotoxemia, rheumatoid arthritis, and intestinal inflammation. In the spleen during endotoxemia, VNS activates a "cholinergic anti-inflammatory pathway" (CAIP), whereby choline acetyltransferase (ChAT)-expressing T cells release acetylcholine to reduce macrophage activation. VNS can also drive CAIP-independent pathways to reduce inflammation in the spleen and intestinal tract, although the circuitry modulating colonic inflammation remains underexplored. Here, we demonstrate that left cervical VNS reduces acute LPS-induced inflammation, evidenced by reduced Tnfa expression in colon and spleen and decreased circulating TNF. In the colon, these protective effects required efferent but not afferent VNS and were independent of ChAT+ T cells, IL10, {beta}-adrenergic signaling, and colonic sympathetic innervation. Critically, the ability of VNS to modulate colonic inflammation depended on prior tissue-specific inflammation. Mice recovering from DSS colitis, despite near-complete histological recovery, were refractory to the protective effects of VNS in the colon. This lack of efficacy in the colon was not reflected in measures of inflammation in the spleen or serum, highlighting the need for target-organ-specific monitoring. This loss of efficacy after colonic inflammation was transient, with restoration occurring upon complete recovery. These findings demonstrate that VNS efficacy in colonic inflammation depends on circuitry distinct from canonical systemic anti-inflammatory pathways, and that tissue responsiveness is shaped by anatomical site and inflammatory history. Key PointsO_LIElectrical stimulation of the left cervical vagus nerve reduces LPS-induced inflammation in the mouse colon. C_LIO_LIThis colonic anti-inflammatory effect requires vagal efferents but not afferent signaling. C_LIO_LIUnlike canonical splenic anti-inflammatory pathways, the colonic response does not require ChAT+ T cells, IL-10, {beta}-adrenergic signaling, or local sympathetic innervation. C_LIO_LIRecent DSS colitis abolishes colonic responsiveness to VNS despite preserved splenic and systemic anti-inflammatory effects. C_LIO_LIRecovery of VNS anti-inflammatory efficacy after colitis shows that neuroimmune responsiveness in the colon is dynamically shaped by inflammatory history. C_LI

immunology↗

bioRxiv2026

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

biophysics↗

bioRxiv2026

A right-hemispheric language network at single-neuron resolution

Human language depends on highly specialized left-hemispheric brain networks. Damage to these networks causes severe language impairments (aphasia), one of the most common, debilitating and costly consequences of left-hemispheric brain injury, especially stroke. The limited recovery of aphasia despite intensive rehabilitation efforts emphasizes the need to understand the basis of residual language abilities at the single-neuron level, which has remained unexplored so far. Here, we report large-scale microelectrode recordings with single-unit resolution over a period of ten months from the right-hemispheric prefrontal and parietal association cortex of an individual with stroke-induced chronic non-fluent aphasia. Single neurons exhibited regionally specific responses during comprehension, retrieval and articulation of words, the core operations of language. Distinct subpopulations encoded linguistic information in a task-specific manner, despite correlated firing patterns across tasks. Both single-neuron activity and temporally coordinated population dynamics were predicted by semantic and phonological embeddings derived from large language models (LLMs), revealing a regional dissociation in which semantic features preferentially accounted for prefrontal activity and phonological features for parietal activity. Our findings suggest that right-hemispheric circuits, homotopic to the left language network, can support language processing through structured, functionally organised activity at the level of single neurons. This study opens an avenue for developing mechanistically specific neurorehabilitation and neurorestorative strategies for aphasia, such as brain-computer interfaces (BCIs), that leverage right-hemispheric language resources.

neuroscience↗

bioRxiv2026

High Resolution Multi-Pass Astral Analyzer Quantification Enables Highly Multiplexed 35-Plex Tandem Mass Tag Proteomics

Tandem mass tags (TMT) allow highly multiplexed and thus high-throughput, precisely quantitative proteomic analysis. Incorporation of additional deuterated reporter channels has near-doubled the multiplexation achieved with Thermo Scientific TMTpro reagents from 18 to 35-plex but requires extremely high [~]100k analyzer resolving power at m/z 128 to differentiate and quantify reporter ion channels, far beyond any single reflection time-of-flight analyzer, and exceeding the multi-reflection Thermo Scientific Astral analyzer in its standard operation. A multi-pass mode of Astral operation has been developed for the Thermo Scientific Orbitrap Astral Zoom mass spectrometer that triples the ion path to 90 m, more than doubling resolving power for a narrow m/z range. This "TMT HR mode" has been integrated into a new method of TMT proteomic analysis that splits regular MS2 analysis of labeled peptides into paired measurements comprising wide mass range scans for peptide identification, and TMT HR mode scans for reporter ion quantification. The method has been shown to accurately quantify 32-plex labeled HeLa protein lysate and provide far greater depth of analysis as state-of-the-art Orbitrap-only methods, while analysis of 11-plex labeled yeast showed no analytical depth sacrificed vs regular Orbitrap Astral TMT analysis. Further comparative measurements of a 2-cell line 35-plex sample demonstrated greater analytical depth, and similar quantitative precision, to "gold standard" Orbitrap MS3 methods.

biochemistry↗

bioRxiv2025

Metabolic zonation and characterization of tissue slices with spatial transcriptomics

The exchanges of small molecules between cells and their environments are essential for the formation of functioning tissues. To study them at scale, we developed Harreman (Basque for "receive and give"), an algorithm for identifying metabolic crosstalk from spatially resolved transcriptomics of intact tissues. Unlike previous methods, which primarily focus on the secretion or reception of protein signals, Harreman reconstructs molecular metabolic crosstalk based on the co-localized expression of metabolite transporters. By utilizing a series of increasingly detailed models for testing spatial correlation, Harreman provides insight at multiple levels: a) coarse partition of the tissue into regions sharing metabolic characteristics; b) identification of metabolic exchanges within each region; and c) inference of the cell subsets involved in those exchanges. Harreman identified a sodium/calcium exchange at the tumor boundary in human lung metastases of human renal cancers, and associated it with nearby pro-inflammatory macrophages. In the mouse model of DSS-induced colitis, Harreman identified vitamin A and lysophosphatidylcholine transport at the interface of the epithelial monolayer as major signals associated with regeneration. Harreman is available at https://github.com/YosefLab/Harreman.

bioinformatics↗

bioRxiv2025

Strong promoters are mutationally robust

Mutational robustness is the persistence of a phenotype upon mutation. It facilitates molecular evolution and has been characterized in a variety of biological systems, but studies of prokaryotic promoters are limited. Prokaryotic promoters are non-coding DNA sequences that regulate transcription. The main housekeeping promoters ({sigma}70) share two sequence motifs called the -35 and the -10 box that are spaced 17{+/-}1 base pairs (bps) apart. The sequence of these boxes, their distance, and the existence of multiple nearby boxes can determine promoter strength, the ability of a promoter to drive high levels of transcription. Here we used computational modelling to show that the mutational robustness of {sigma}70 promoters - the persistence of promoter strength upon mutation - correlates with promoter strength itself. Mutational robustness is also influenced by potential overlaps between -10 and -35 boxes, and by specific positions within the boxes. It is higher when the boxes are exactly 17 bps apart. These findings can be partially explained by the flexibility of -35 boxes, which enables adjacent bases to create overlapping and new -35 boxes. Our work can help to engineer synthetic promoters for strength and robustness, and to understand the dynamics of promoter evolution.

evolutionary biology↗