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Biology subjects

Brunner, M.

Publications and source records attributed to Brunner, M..

At least 19 recordsLinked to original sources

Multiscale mapping of venous remodelling in idiopathic pulmonary fibrosis

The integrity of the pulmonary vasculature is a key determinant of lung health, yet challenges in visualisation and quantification have hindered interrogation of its pathophysiological role in chronic lung disease. Here, we align recent advances in microscale image acquisition with novel computer vision-based vessel segmentation models to demonstrate expansion of the bronchial and pulmonary veins across varying severities of tissue remodelling in idiopathic pulmonary fibrosis (IPF). Relating these imaging findings to molecular data in control, mild, and severe fibrosis, we show that bronchial venous endothelial cells expand beyond their physiological peribronchial niche even in mild disease, acquiring persistent angiogenic, inflammatory, and matrix-remodelling programmes that define a specialised fibrovascular-immune interface. Finally, we relate these microscale and molecular observations to clinical CT imaging, demonstrating that intrapulmonary vein enlargement independently associates with worsened survival across three IPF cohorts. Collectively, our multimodal, multiscale approach connects previously unresolved three-dimensional venous architecture to its molecular endothelial correlate, establishing venous enlargement as a prognostically significant and clinically relevant feature of IPF. Our analytical approach also demonstrates how biological discoveries made in intact ex vivo human organs can translate into measurable phenotypes in living patients, providing a template for other organs and diseases.

pathology↗

Cell cycle-coupled CK1δ turnover, autoinhibition, and activity

Casein kinase 1{delta} (CK1{delta}) is a ubiquitously expressed kinase involved in diverse cellular processes, including cell cycle regulation. CK1{delta} activity is attenuated by (auto)phosphorylation. However, inhibitory phosphorylation is efficiently opposed by cellular phosphatases as CK1{delta} accumulates in its hypophosphorylated, active state. CK1{delta} is a target of the nuclear ubiquitin ligase APC/C-CDH1, yet the kinase is apparently stable. Thus, the physiological relevance of CK1{delta} (auto)phosphorylation, autoinhibition, and regulated turnover has remained unclear. Here we show that CK1{delta} activity and abundance are coordinated in a cell cycle-dependent manner. During G1, assembled CK1{delta} kinase is stable while free active kinase is degraded. In S phase, unassembled CK1{delta} is no longer degraded, likely to support functions in DNA damage signaling. Upon mitotic entry, the downregulation of phosphatases promotes CK1{delta} (auto)phosphorylation and consequent autoinhibition, thereby preserving a pool of kinase to rapidly reestablish the post-mitotic steady state.

biochemistry↗

Single cell decomposition of multicellular aging programs associated with impaired lung regeneration

Aging impairs the regenerative capacity of mammalian organs and is a major risk factor for organ fibrosis. Mechanisms underlying persistent fibrosis after lung injury in old individuals remain unclear. We used longitudinal single-cell RNA-seq after lung injury and dissected aging effects computationally and experimentally at baseline and during repair. In old mice, sustained fibroblast activation in the resolution phase of fibrosis was associated with prolonged epithelial senescence and persistent epithelial-mesenchymal crosstalk. Single-cell interpretable tensor decomposition analysis revealed that aging most strongly affected T/B-lymphocytes and macrophages. Notably, we identified a Granzyme K-high CD8+ T cell state that was unique to aged mice, co-localized with epithelial progenitors, and its co-culture or Gzmk treatments in lung organoids impaired progenitor function by inducing stem cell senescence. In summary, our study highlights the effects of immune aging on epithelial progenitor function and provides a time-resolved high resolution map of lung regeneration in the context of aging.

cell biology↗

CK1a-Mediated Two-Step Subunit Remodeling of the Circadian FRQ-FRH Complex

The circadian clock of Neurospora operates through a negative feedback loop in which FRQ, along with FRH and CK1a, inhibits its transcriptional activator, WCC, via phosphorylation. CK1a, anchored to FRQ, hyperphosphorylates FRQ at its IDRs in a slow, temperature-independent manner, forming a module suited for molecular timekeeping. However, the molecular processes triggered by FRQs hyperphosphorylation have remained unclear. We show that FRH, the folded binding partner of disordered FRQ, decodes FRQs time-dependent phosphorylation state by triggering a two-step remodeling of the FRQ-FRH complex: initially, two FRH molecules bind a FRQ dimer, keeping it inactive by blocking its interaction with WCC. Gradual phosphorylation induces with a delay the dissociation of one FRH, exposing a binding site for WCC and activating the complex. Following a time delay, attributable to the slow and stochastic nature of phosphorylation, the release of the second FRH promotes nuclear export and subsequent degradation of FRQ. This stepwise remodeling ensures precise activation and inactivation of FRQ and positions FRH as a hub for decoding temporal phosphorylation information.

cell biology↗

Unraveling new characteristics of γδT cells using scRNA-seq in TCR KO chicken

The characterization of T cells in chickens has proven to be challenging, primarily due to the lack of specific markers to differentiate classical Th1, Th2, and Th17 subsets. Furthermore, chickens possess a notably high proportion of {gamma}{delta} T cells, making them a unique model for investigating the poorly understood role of these cells not only in chickens but also in mammals. To gain deeper insights into the functions and characteristics of the {beta} and {gamma}{delta} T cell subsets in chickens, whole transcriptome analysis (WTA) on CD3+ single cells isolated from wild type (WT), TCR C{beta} knockout (KO) and TCR C{gamma} KO chickens at embryonic day (ED) 18, day (d) 3, and d14 post-hatch was conducted. The results indicate that {gamma}{delta} T cells exhibit cytolytic activity in both TCR C{beta} KO and WT chickens. A distinct cluster of {gamma}{delta} T cells expressing genes associated with the interferon pathway was revealed in TCR C{beta} KO chickens, which may contribute to the severe phenotype in these chickens, which is characterized by severe inflammation of spleen, gut and stomach. Additionally, novel gene markers were identified that precisely define the {beta} and {gamma}{delta} T cell subsets. These findings provide new insights into chicken T cell biology and contribute to a better understanding of the mechanisms underlying the severe phenotype observed in TCR C{beta} KO chickens.

immunology↗

Nuclear CK1δ as a Critical Determinant of PER:CRY Complex Dynamics and Circadian Period

The mammalian circadian clock is governed by a feedback loop in which the transcription activator CLOCK:BMAL1 induces expression of its inhibitors, PERs and CRYs, which form a complex with CK1{delta}, the main circadian kinase. However, the spatiotemporal dynamics of this feedback loop and the precise role of CK1{delta} remain incompletely understood. Using an inducible overexpression system, we show that nuclear availability of CK1{delta} is limited by both rapid nuclear degradation and active export of unassembled kinase, while cytoplasmic kinase is readily available for association with PERs. We demonstrate that CK1{delta}-mediated phosphorylation may disrupt PER2-CRY1 interaction thereby resulting in cytoplasmic PER2 dimers containing substoichiometric amounts of CRY1. Analysis of endogenous PER2 localization in the context of an intact circadian clock reveals that PER2 accumulates in the cytoplasm late in the circadian cycle. Based on these findings, we propose that cytoplasmic accumulation of PER:CRY:CK1{delta} complexes contributes to the clearance of nuclear PER2, while the CK1{delta}-dependent release of CRY1 into the nucleus may sustain CLOCK:BMAL1 repression on DNA supporting the transition from the early to the late repressive phase.

biochemistry↗

Type I and type III interferon receptor knockout chickens: Novel models for unraveling interferon dynamics in influenza infection

The rapid cross-species transmission of highly pathogenic avian influenza presents a significant zoonotic threat. Elucidating the avian interferon (IFN) system, the primary antiviral defense in chickens, is critical for controlling the virus at its source and preventing its spillover into humans and other species. We engineered type I (IFN-/{beta}) and type III (IFN-{lambda}) IFN receptor knockout chickens to dissect the role of IFNs in viral infections. Results revealed that type I IFN predominantly modulates innate immune cell populations, T cell subsets, and their contribution to antibody production following immunization under physiological conditions. In ovo and in vivo challenge experiments utilizing diverse influenza A virus strains demonstrated strain-specific roles of both IFN-/{beta} and IFN-{lambda} in orchestrating viral pathogenesis, immunological responses, and tissue-tropism effects. Notably, type I IFN was particularly crucial in the initial defense mechanisms against H3N1 avian influenza A virus infection. These novel models offer unprecedented insights into avian IFN biology within the context of avian influenza, which is essential for developing more effective strategies to prevent and control this public health challenge.

immunology↗

Regulation of B cell migration to the Bursa of Fabricius by CCR7 and cell adhesion molecules in chicken embryonic development

The development of functional B lymphocytes during chicken embryogenesis relies on a series of tightly regulated processes. Precursor B cells migrate from the spleen via the blood to the bursa of Fabricius, where they colonize the bursal follicles to undergo further maturation and differentiation. To better understand the molecular mechanisms underlying early B cell migration in the chicken embryo, transcriptome analysis of B cells isolated from the spleen, blood, and bursa at embryonic days (ED) 12, ED14, and ED16 was performed. These findings suggest that sphingosine-1-phosphate (S1P) and its receptors regulate B cell presence in the bloodstream, while CCR7 and CXCR4 guide B cells to the bursa. Additionally, integrins and cell adhesion molecules, such as PECAM1, appear to facilitate transendothelial migration into the bursal mesenchyme. This study highlights a coordinated interplay between chemokines, integrins and cell adhesion molecules involved in B cell recruitment and colonization of the bursa microenvironment. These findings enhance our understanding of early B cell migration and shed light on the mechanisms governing B cell trafficking during chicken embryonic development.

immunology↗

Radioproteomics stratifies molecular response to antifibrotic treatment in pulmonary fibrosis

Antifibrotic therapy with nintedanib is the clinical mainstay in the treatment of progressive fibrosing interstitial lung disease (ILD). High-dimensional medical image analysis, known as radiomics, provides quantitative insights into organ-scale pathophysiology, generating digital disease fingerprints. Here, we used an integrative analysis of radiomic and proteomic profiles (radioproteomics) to assess whether changes in radiomic signatures can stratify the degree of antifibrotic response to nintedanib in (experimental) fibrosing ILD. Unsupervised clustering of delta radiomic profiles revealed two distinct imaging phenotypes in mice treated with nintedanib, contrary to conventional densitometry readouts, which showed a more uniform response. Integrative analysis of delta radiomics and proteomics demonstrated that these phenotypes reflected different treatment response states, as further evidenced on transcriptional and cellular levels. Importantly, radioproteomics signatures paralleled disease- and drug related biological pathway activity with high specificity, including extracellular matrix (ECM) remodeling, cell cycle activity, wound healing, and metabolic activity. Evaluation of the preclinical molecular response-defining features, particularly those linked to ECM remodeling, in a cohort of nintedanib-treated fibrosing ILD patients, accurately stratified patients based on their extent of lung function decline. In conclusion, delta radiomics has great potential to serve as a non-invasive and readily accessible surrogate of molecular response phenotypes in fibrosing ILD. This could pave the way for personalized treatment strategies and improved patient outcomes.

molecular biology↗

Pseudospatial transcriptional gradient analysis of hypothalamic ependymal cells: towards a new tanycyte classification

The ependyma lining the third ventricle (3V) in the mediobasal hypothalamus is recognized as a critical player in controlling energy balance and glucose homeostasis. Its molecularly distinct cell types, including diverse tanycyte subpopulations and typical ependymal cells, confer a high functional heterogeneity. The study of gene expression profiles and dynamics of ependymal cells has the potential to uncover fundamental mechanisms and pathways involved in metabolic regulation. Here, we cataloged 5481 hypothalamic ependymocytes using FACS-assisted single-cell RNA sequencing from fed, 12h-fasted, and 24h-fasted adult male mice. First, standard clustering analysis revealed the limitation of the current characterization regarding the different ependymal cell subpopulations along the 3V. Indeed, while typical ependymal cells and {beta}2-tanycytes are sharply defined at the molecular level, other subpopulations (i.e., {beta}1-, 2-, and 1 tanycytes) display fuzzy boundaries and very few specific markers. Moreover, we observed that 12h- and 24h-fasting dynamically modulate gene expression, increasing tanycyte subgroup heterogeneity. Secondly, pseudospatial trajectory analysis based on peculiar UMAP neuroanatomical distribution improved the identification of tanycyte markers, distinguishing specific versus overlapping features and better segregating tanycyte specific versus standard functions. Intriguingly, we discovered numerous functions related to tanycyte-neuron and tanycyte-synapse interactions with modulation by energy balance. Finally, combining pseudospatial analysis and gene regulatory network inference, we observed that fasting dynamically shifts patterns in gene expression and transcription activity along the 3V, creating a metabolic and functional switch for some subpopulations. Altogether, this data provides a mechanism through which energy status leads to distinct cell type-specific responses along the 3V and gives new insights into molecular diversity underlying tanycyte classification.

neuroscience↗

CK1δ homeostasis by activity-dependent shuttling and degradation of orphan kinase

Casein kinase 1{delta} (CK1{delta}) is a simple monomeric enzyme involved in the regulation of a variety of functions, including signal transduction, the circadian clock, and the cell cycle. Although CK1{delta} is targeted by the ubiquitin ligase APC/CCdh1 is not understood how CK1{delta} expression is regulated to support its multiple functions. Here, we show that kinase activity controls CK1{delta} homeostasis by coordinating two competing processes: export from the nucleus to ensure distribution of CK1{delta} between its assembly partners, and proteasomal degradation of unassembled CK1{delta} in the nucleus to keep the amount of active, potentially deleterious orphan kinase low. During mitosis, CK1{delta} is released from centrosomes and stabilized by (auto)phosphorylation to preserve it for the subsequent G1 phase. TeaserCompetitive nuclear export and nuclear degradation of active CK1{delta} ensure efficient partner interaction and keep unassembled kinase levels low.

biochemistry↗

Transcription activator WCC recruits deacetylase HDA3 to control transcription dynamics and bursting in Neurospora

RNA polymerase II initiates transcription either randomly or in bursts. We examined the light-dependent transcriptional activator White Collar Complex (WCC) of Neurospora to characterize the transcriptional dynamics of the strong vivid (vvd) promoter and the weaker frequency (frq) promoter. We show that WCC is not only an activator but also represses transcription by recruiting histone deacetylase 3 (HDA3). Our data suggest that bursts of frq transcription are governed by a long-lived refractory state established and maintained by WCC and HDA3 at the core promoter, whereas transcription of vvd is determined by WCC binding dynamics at an upstream activating sequence. Thus, in addition to stochastic binding of transcription factors, transcription factor-mediated repression may also influence transcriptional bursting. TEASERBalanced interaction of transcription factor with coactivator and corepressors determines transcription dynamics and bursting.

molecular biology↗

Antisense transcription of the Neurospora frequency gene is rhythmically regulated by CSP-1 repressor but dispensable for clock function

The circadian clock of Neurospora crassa is based on a negative transcriptional/translational feedback loops. The frequency (frq) gene controls the morning-specific rhythmic transcription of a sense RNA encoding FRQ, the negative element of the core circadian feedback loop. In addition, a long noncoding antisense RNA, qrf, is rhythmically transcribed in an evening-specific manner. It has been reported that the qrf rhythm relies on transcriptional interference with frq transcription and that complete suppression of qrf transcription impairs the circadian clock. We show here that qrf transcription is dispensable for circadian clock function. Rather, the eveningspecific transcriptional rhythm of qrf is mediated by the morning-specific repressor CSP-1. Since CSP-1 expression is induced by light and glucose, this suggests a rhythmic coordination of qrf transcription with metabolism. However, a possible physiological significance for the circadian clock remains unclear, as suitable assays are not available.

molecular biology↗

Adaptation to starvation requires a flexible circadian clockwork in Neurospora crassa

The circadian clock governs rhythmic cellular functions by driving expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain a rhythmic physiology and preserve fitness even under long-term nutritional stress.

cell biology↗

Data-driven modelling captures dynamics of the circadian clock ofNeurospora crassa

Eukaryotic circadian clocks are based on self-sustaining, cell-autonomous oscillatory feedback loops that can synchronize with the environment via recurrent stimuli (zeitgebers) such as light. The components of biological clocks and their network interactions are becoming increasingly known, calling for a quantitative understanding of their role for clock function. However, the development of data-driven mathematical clock models has remained limited by the lack of sufficiently accurate data. Here we present a comprehensive model of the circadian clock of Neurospora crassa that describe free-running oscillations in constant darkness and entrainment in light-dark cycles. To parameterize the model, we measured high-resolution time courses of luciferase reporters of morning and evening specific clock genes in WT and a mutant strain. Fitting the model to such comprehensive data allowed estimating parameters governing circadian phase, period length and amplitude, and the response of genes to light cues. Our model suggests that functional maturation of the core clock protein Frequency (FRQ) causes a delay in negative feedback that is critical for generating circadian rhythms.

systems biology↗

APRANK: computational prioritization of antigenic proteins and peptides from complete pathogen proteomes

Availability of highly parallelized immunoassays has renewed interest in the discovery of serology biomarkers for infectious diseases. Protein and peptide microarrays now provide a rapid, high-throughput platform for immunological testing and validation of potential antigens and B-cell epitopes. However, there is still a need for tools to prioritize and select relevant probes when designing these arrays. In this work we describe a computational method called APRANK (Antigenic Protein and Peptide Ranker) which integrates multiple molecular features to prioritize antigenic targets in a given pathogen proteome. These features include subcellular localization, presence of repetitive motifs, natively disordered regions, secondary structure, transmembrane spans and predicted interaction with the immune system. We applied this method to prioritize potentially antigenic proteins and peptides in a number of bacteria and protozoa causing human diseases: Borrelia burgdorferi (Lyme disease), Brucella melitensis (Brucellosis), Coxiella burnetii (Q fever), Escherichia coli (Gastroenteritis), Francisella tularensis (Tularemia), Leishmania braziliensis (Leishmaniasis), Leptospira interrogans (Leptospirosis), Mycobacterium leprae (Leprae), Mycobacterium tuberculosis (Tuberculosis), Plasmodium falciparum (Malaria), Porphyromonas gingivalis (Periodontal disease), Staphylococcus aureus (Bacteremia), Streptococcus pyogenes (Group A Streptococcal infections), Toxoplasma gondii (Toxoplasmosis) and Trypanosoma cruzi (Chagas Disease). We have tested this integrative method using non-parametric ROC-curves and made an unbiased validation using an independent data set. We found that APRANK is successful in predicting antigenicity for all pathogen species tested, facilitating the production of antigen-enriched protein subsets. We make APRANK available to facilitate the identification of novel diagnostic antigens in infectious diseases.

immunology↗

Interleukin-3 is a predictive marker for severity and outcome during SARS-CoV-2 infections

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a worldwide health threat. Here, we report that low plasma interleukin-3 (IL-3) levels were associated with increased severity and mortality during SARS-CoV-2 infections. IL-3 promoted the recruitment of antiviral circulating plasmacytoid dendritic cells (pDCs) into the airways by stimulating CXCL12 secretion from pulmonary CD123+ epithelial cells. This study identifies IL-3 as a predictive disease marker and potential therapeutic target for SARS-CoV-2 infections.Competing Interest StatementThe authors have declared no competing interest.View Full Text

immunology↗

Multiple random phosphorylations provide long delays and switches in circadian clocks

Theory predicts that self-sustained oscillations require robust delays and nonlinearities (ultrasensitivity). Delayed negative feedback loops with switch-like inhibition of transcription constitute the core of eukaryotic circadian clock. The kinetics of core clock proteins such as PER2 in mammals and FRQ in Neurospora crassa is governed by multiple phosphorylations. We investigate how multiple, slow and random phosphorylations control delay and molecular switches. We model phosphorylations of intrinsically disordered clock proteins (IDPs) using conceptual models of sequential and distributive phosphorylations. Our models help to understand the underlying mechanisms leading to delays and ultrasensitivity. The model shows temporal and steady state switches for the free kinase and the phosphoprotein. We show that random phosphorylations and sequestration mechanisms allow high Hill coefficients required for self-sustained oscillations.

systems biology↗