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Adipocyte autophagy-mediated signals in the control of macrophage phenotype during diet-induced obesity
No full textA hallmark of obesity is a pathological expansion of white adipose tissue that is accompanied by an increase in local inflammation and fibrosis. Autophagy is increased during obesity in adipose tissue, however, its role remains incompletely understood. Here, we report that autophagy is a critical regulator of pathological white adipose tissue remodelling and inflammation in diet-induced obese mice. The absence of adipocyte autophagy substantially exacerbates pericellular fibrosis specifically in gonadal white adipose tissue, ameliorating metabolic syndrome. Notably, changes in tissue architecture correlate with increased infiltration of macrophages and autophagy-dependent rewiring of adipocyte metabolism
Self-organized vascularized human liver spheroids: Serum-free culture conditions and use as tissue building blocks
No full textEngineering vascularized human liver tissue for in vitro models and in vivo applications remains a major challenge. Here, we describe a scalable approach to generate human liver spheroids with self-organized, lumen-containing vascular networks and demonstrate their use as building blocks for fabricating vascularized tissue layers. Spheroids were formed from HepaRG liver cells, human umbilical vein endothelial cells (HUVECs), and adipose tissue-derived mesenchymal stem cells (MSCs). The inclusion of MSCs prevented spatial segregation of hepatic and endothelial compartments and enabled endothelial network formation. We present two media that are suitable for culturing these spheroids: a serum-reduced medium and a defined serum-free medium containing GibcoTM KnockOut serum replacement. These media supported the long-term maintenance of hepatocytes in a metabolically active, relatively mature state, as well as the persistence of endothelial networks. Endothelial cell identity and organization were confirmed by VE-cadherin and ICAM-2 immunostaining and by transmission electron microscopy, which revealed adherens junctions and luminal morphologies consistent with a capillary-like organization. Spheroid-derived HUVECs established anastomoses with external endothelial channels in microfluidic devices. Moreover, endothelial sprouts emerging from the spheroids formed inter-spheroid connections within permissive hydrogels (fibrin or collagen-methylcellulose), a process that depended on the inter-spheroid distance. Finally, we demonstrate the fabrication of planar tissue layers with vascularly interconnected spheroids. Together, we identify key conditions, including cellular ratios, medium formulations, biomaterials, and spatial design criteria that enable the generation and assembly of vascularized liver spheroids as scalable tissue building blocks for tissue engineering applications
Evolutionarily conserved transcriptional regulators control monoaminergic neuron development
Get PDFTo what extent conserved developmental programs specify homologous cell types is a central question in biology. Here, we address this by focusing on reconstructing monoaminergic neuron development in Drosophila melanogaster embryo using time-resolved single-cell genomics, spatial transcript mapping with hybridisation chain reaction, and targeted metabolomics. We uncover a regulatory landscape in which specific transcription factors are activated before biosynthetic enzymes, establishing a prospective temporal architecture for monoaminergic fate specification. Comparative analyses of developmental single-cell atlases from zebrafish and sea urchin indicate that components of this machinery are conserved across ~550 million years of bilaterian evolution with orthologous transcription factors showing similar temporal dynamics. Together, these findings point to a putatively conserved regulatory core that interfaces with other context-dependent transcription factors; this interplay accommodates monoaminergic multifunction and subtype diversity across distinct neuroanatomies
Quantitative analysis of gadolinium deposits in liver tissue of patients after single or multiple gadolinium-based contrast agent application
Get PDFOBJECTIVES: Gadolinium-based contrast agents (GBCAs) are widely used in magnetic resonance imaging. Concerns exist regarding gadolinium deposition and its potential histopathologic tissue alterations, especially after repeated administrations of linear, less stable GBCAs. This study aimed to quantify gadolinium mass fractions in liver specimens of subjects exposed to GBCAs in correlation with histopathologic features.
MATERIALS AND METHODS: In this Institutional Review Board-approved study, mass fractions of gadolinium in human liver specimens ω(Gd) from 25 subjects who underwent liver tumor resection surgery and had received GBCA (1 to 9 times over 4 y), were quantitatively analyzed using inductively coupled plasma-mass spectrometry (ICP-MS). Histomorphology was assessed based on the nonalcoholic fatty liver disease activity score (NAS). Linear regression analyses were performed with ω(Gd), time and dosage metrics, and histopathologic parameters.
RESULTS: The median interval between last GBCA administration and surgery (T) was 14 days (range: 1 to 69 d). Gadolinium was detected in all liver samples (ω(Gd), median: 0.348 µg/g; range: 0.120 to 0.874 µg/g). No significant correlation was found between ω(Gd) and histologic scores, including inflammation and fibrosis. A strong negative correlation was found between ω(Gd) and ln(T) (P < 0.001). A positive correlation existed between ω(Gd) and the number (P = 0.010) but not the cumulative dose of previous GBCA administrations (P = 0.205).
CONCLUSIONS: Our results suggest that after intravenous administration of GBCA, a small fraction of gadolinium is retained in the liver over a time period of at least several weeks. A relationship was observed between Gadolinium retention and the number of GBCA administrations, but not with the cumulative dose and the degree of fatty liver disease
Phosphoprofile reorganization of the actin binding protein Drebrin during long term depression
No full textDrebrin (DBN), an actin-binding protein critical for the structural integrity and function of dendritic spines, is highly phosphorylated at steady state in neurons. Here, we investigate the phosphorylation dynamics of DBN in the context of chemically induced long-term depression (cLTD), a synaptic plasticity model mimicking activity-dependent weakening of synapses. Using biochemical analyses and mass spectrometry analyses, we show that DBN undergoes rapid and robust changes in phosphorylation following cLTD induction. Notably, cLTD triggers a marked decrease in many DBN phosphorylation sites, accompanied by proteolytic cleavage of the protein, suggesting a tightly regulated mechanism linking post-translational modification to structural remodelling of the synapse. Our findings highlight the dynamic regulation of DBN by phosphorylation during synaptic depression and support its potential role as a modulator of activity-dependent synaptic plasticity
IMMProveCF_public
No full textThis repository contains all code and data used for the analysis reported in the manuscript: "CFTR modulator therapy drives microbiome restructuring through improved host physiology in cystic fibrosis: the IMMProveCF phase IV trial". Repository Content: .R files – Contain functions used throughout the analysis.; .Rmd files – Ordered according to the analysis presented in the manuscript. These include code for statistical tests and figure generation to fully reproduce the findings.; .rds files – Contain annotated but raw count 16S sequencing data combined with clinical metadata as a phyloseq object.; renv.lock – Captures exact package versions; renv/settings.json – Stores renv settings; .Rproj – Helps with project organization
Live-cell quantitative monitoring reveals distinct, high-affinity Gβγ regulations of GIRK2 and GIRK1/2 channels
Get PDFG(i/o) protein-coupled receptors (GPCRs) inhibit cardiac and neuronal excitability via G protein-activated K+ channels (GIRK), assembled by combinations of GIRK1 - GIRK4 subunits. GIRKs are activated by direct binding of the Gβγ dimer of inhibitory G(i/o) proteins. However, key aspects of this textbook signaling pathway remain debated. Recent studies suggested no G(i/o)-GIRK pre-coupling and low (>250 µM) Gβγ-GIRK interaction affinity, contradicting earlier sub-µM estimates and implying low signaling efficiency. We show that Gγ prenylation, which mediates Gβγ membrane attachment required for GIRK activation, also contributes to the Gβγ-GIRK interaction, explaining the poor affinity obtained with non-prenylated Gβγ. Using quantitative protein titration and electrophysiology in live Xenopus oocytes, Gβγ affinity for homotetrameric GIRK2 ranges from 4-30 µM. Heterotetrameric GIRK1/2 shows a higher Gβγ apparent affinity due to the Gβγ-docking site (anchor) in GIRK1, which enriches Gβγ at the channel. Biochemical approaches and molecular dynamic simulations reveal that the Gβγ anchor is formed by interacting N-terminal and distal C-terminal domains of the GIRK1 subunits, distinct from the Gβγ-binding “activation” site(s) underlying channel opening. Thus, the affinity of Gβγ-GIRK interaction is within the expected physiological range, while dynamic pre-coupling of Gβγ to GIRK1-containing channels through high-affinity interactions further enhances the GPCR-G(i/o)-GIRK signaling efficiency
AP1 is a pioneer transcription factor that programmes cell fate through MADS-domain protein tetramerisation
Get PDFBACKGROUND: In animals, pioneer transcription factors (TFs) have long been known to be crucial molecular players in programming cell fate. However, in plants much less is known about this functional class of TFs and how they mechanistically alter local chromatin architecture in order to reprogramme gene regulation to orchestrate cell fate changes. RESULTS: Here, we provide evidence that APETALA1 (AP1) functions as a pioneer TF in Arabidopsis thaliana, facilitated by tetramerisation. Using an integrated combination of multi-omics and high-resolution imaging approaches on both wild-type and AP1 mutant transgenic plants, we show that tetramerisation assists AP1 in providing access to, and enhancing the binding of AP1 to, closed chromatin in vivo. This, in turn, allows for a switching of the chromatin state from closed to open to ensure access to target DNA sequences needed for organ specification. CONCLUSIONS: These novel insights provide a mechanistic basis for how AP1 functions as a pioneer factor to reprogramme stem cells towards a “floral ground state”, increasing our understanding of how MADS-domain TFs function as combinatorial units during early Arabidopsis thaliana reproductive development
Barriers to care for people with unclear visual loss - data from a tertiary-level-of-care neuroinflammation center
Get PDFBACKGROUND: Visual symptoms are common in people with multiple sclerosis. The revised 2024 McDonald criteria include the optic nerve as a fifth anatomical region, underscoring the need for specific diagnostics. Although optical coherence tomography (OCT) and visual evoked potentials (VEP) are available, the extent of their routine pre-referral use is insufficiently documented. We evaluated pre-referral utilization and hypothesized that specific diagnostics are used less often than non-specific diagnostics and that differences are not explained by demographics alone. METHODS: Retrospective cross-sectional study of 305 patients referred for visual symptoms to a tertiary neuroimmunology clinic in Germany. Analyses focused on people with multiple sclerosis (n = 112) and disease controls with neuromyelitis optica spectrum disorders or myelin oligodendrocyte glycoprotein-associated disease (pwNM; n = 36). RESULTS: In people with multiple sclerosis, only 6.2% received OCT and 33% VEP for their visual complaints, compared to unspecific diagnostics such as cranial magnetic resonance imaging (58%) and lumbar puncture (42%) – independent of demographic factors. CONCLUSION: The pre-referral use of specific neurovisual tests in people with multiple sclerosis with visual symptoms was low relative to non-specific procedures. This suggests heterogeneous integration of neurovisual testing across care levels. In light of the revised McDonald Criteria 2024, prospective multicenter studies should examine implementation and clinical impact
Dynamic nanoscale architecture of synaptic vesicle fusion in mouse hippocampal neurons
Get PDFSynaptic vesicle (SV) fusion is not only tightly coordinated but also happens at a millisecond timescale. Competing models for fusion initiation and propagation suggest tight docking and hemifusion of SVs or localized lipid rearrangements leading to tip-like membrane contacts. Yet, a direct nanoscale examination of the full SV fusion sequence has been lacking. Here, we establish a workflow for timed in situ cryo-electron tomography of optogenetically stimulated mouse neurons to capture the complete SV fusion sequence - from SV recruitment to fusion pore formation, opening and collapse - with near-native structural preservation. Notably, tethered SVs directly undergo fusion initiation via stalk formation, without preceding tight docking or SV flattening. The plasma membrane forms a minimal dimple during fusion initiation, contradicting preceding models that invoke strong membrane bending prior to fusion. In addition, we observe filaments linking fusing SVs to adjacent SVs, indicating a physical link between fusion and SV resupply