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Enteric nervous system-derived VIP restrains differentiation of LGR(5)(+) stem cells toward the secretory lineage impeding type 2 immune programs
No full textBarrier homeostasis relies on a finely tuned interplay between the immune system, epithelial cells and commensal microbiota. Beyond these regulators, the enteric nervous system has recently emerged as a central hub coordinating intestinal immune responses, although its role in epithelial differentiation has remained largely unexplored. Here, we identify a neuroepithelial circuit in which vasoactive intestinal peptide (VIP)-positive enteric neurons act on VIPR1(+) epithelial stem cells to restrain both their proliferation and secretory lineage differentiation. Disruption of this pathway leads to an expansion of tuft cells, enhanced interleukin (IL)-25 production, activation of group 2 innate lymphoid cells (ILC2s) and induction of a type 2 immune response resembling worm expulsion. This phenotype occurs independently of the microbiota but is modulated by the IL-25R-ILC2-IL-13 axis and dietary solid food intake. Our findings expose the enteric nervous system as a critical regulator of epithelial fate decisions and immune balance, complementing established mechanisms that safeguard barrier integrity and mucosal homeostasis
The histone modifier KANSL2 is an actionable biomarker in multiple myeloma
No full textEpigenetic aberrations are key drivers of multiple myeloma (MM), yet targeted therapies exploiting epigenetic alterations have not been established. By integrating clinical and molecular MM patient data sets with an unbiased genetic in vivo screen, we identified KAT8 regulatory NSL complex subunit 2 (KANSL2) as a histone posttranslational modification (PTM)-associated candidate oncogene. High expression of KANSL2 was associated with adverse prognosis in MM patients. Genetic gain and loss of function models identified a protective role of KANSL2 towards genotoxic stress. By transcriptomics, proteomics and quantitative acetylome profiling, we identified a KANSL2-dependent specific molecular program targetable by acetylation-related modifiers. High KANSL2 levels increased sensitivity to the histone deacetylase (HDAC) inhibitor panobinostat and bromodomain and extra-terminal motif (BET) inhibitor OTX-015 and their combination. Ex vivo drug response profiling in relapsed/refractory MM patient samples confirmed that high KANSL2 expression is associated with selective MM cell killing by HDAC and BET inhibitors. Collectively, these findings position KANSL2 as a mediator of chemotherapy resistance and actionable biomarker for response to drugs targeting its epigenetic program
Butyrate rescues cardiac metabolic dysfunction in hypertensive heart failure with preserved ejection fraction
No full textDiastolic dysfunction in heart failure with preserved ejection fraction (HFpEF) is characterized by metabolic inflexibility. Unlike systolic heart failure, where ketone bodies support energy homeostasis, the failing heart in HFpEF lacks well-characterized alternative fuels to meet its high ATP demand. Here, we show that butyrate, a microbiota-derived short-chain fatty acid, serves as an ancillary energy source and improves diastolic function. Although cardiac power was preserved in rats with HFpEF, both experimental and human HFpEF hearts exhibited an impaired expression of proteins in mitochondrial electron transport chain and oxidative phosphorylation. Additionally, accumulation of 3-hydroxy-butyrate (BOH) in rat and also human HFpEF indicated that ketones do not rescue the cardiac energetic deficit. In HFpEF patients from the UK Biobank, higher BOH levels were associated with increased mortality, particularly those with hypertension. Applying 13C-butyrate to isolated perfused hearts with and without HFpEF resulted in isotope incorporation in butyryl-CoA and downstream TCA intermediates and thus proving its active metabolization. Butyrate was efficiently oxidized by cardiomyocytes and was overtaking BOH and amino acids in supporting respiration. Finaly, chronic butyrate supplementation improved survival, enhanced diastolic function, and reduced fibrosis and inflammation in HFpEF rats despite persistent hypertension. These findings identify butyrate as a compensatory fuel and a promising therapeutic candidate in energetically compromised HFpEF
Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors
Get PDFThe immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34⁺ HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours
Time-resolved mapping of myocardial stiffness using 2D multifrequency spiral MR elastography with and without external vibration
Get PDFPURPOSE: There is a clinical need for stiffness mapping of the heart; however, current cardiac magnetic resonance elastography (cMRE) has limited spatiotemporal resolution. Therefore, we developed 2D spiral multifrequency MRE of the human heart and conducted a study to analyze the consistency and reproducibility of motion-encoding and stiffness mapping with and without external vibration. METHODS: Eleven healthy volunteers were studied using single-slice gradient-echo spiral cMRE with cardiac triggering and encoding of harmonic shear wave fields at 70, 80, and 90 Hz frequency generated by either external drivers or endogenous heart motion. Tissue displacement was monitored synchronized to the cardiac cycle, and frequency-resolved shear wave velocity (SWS) maps were reconstructed as a proxy for left ventricular (LV) stiffness variations. After several days, all subjects underwent repeat scanning for reproducibility analysis based on intraclass correlation coefficients (ICCs). RESULTS: cMRE with external vibration showed LV SWS to be highest in end-systole (ES) (2.17 ± 0.23 m/s), followed by diastole (DIA) (1.94 ± 0.15 m/s) and isovolumetric contraction (IVC) (1.78 ± 0.18 m/s). ICCs decreased with distance from the R-wave from excellent (0.93) in IVC to moderate (0.68) in ES. Without external vibration, sufficient LV harmonic displacement amplitudes permitted SWS reconstruction, resulting in similar SWS, but lower ICC values than with external vibration in IVC and ES. CONCLUSIONS: Multifrequency cMRE offers high spatiotemporal resolution and reproducibility with external vibration. In addition, the technique allows the encoding of endogenous shear waves during cardiac phases with pronounced wall motion
Segmenting whole-body MRI and CT for multiorgan anatomic structure delineation
Get PDFPURPOSE: To develop and validate MRSegmentator, a retrospective cross-modality deep learning model for multiorgan segmentation of MRI scans. MATERIALS AND METHODS: This retrospective study trained MRSegmentator on 1,200 manually annotated UK Biobank Dixon MRI sequences (50 participants), 221 in-house abdominal MRI sequences (177 patients), and 1228 CT scans from the TotalSegmentator-CT dataset. A human-in-the-loop annotation workflow leveraged cross-modality transfer learning from an existing CT segmentation model to segment 40 anatomic structures. The model's performance was evaluated on 900 MRI sequences from 50 participants in the German National Cohort (NAKO), 60 MRI sequences from AMOS22 dataset, and 29 MRI sequences from TotalSegmentator-MRI. Reference standard manual annotations were used for comparison. Metrics to assess segmentation quality included Dice Similarity Coefficient (DSC). Statistical analyses included organ-and sequence-specific mean ± SD reporting and two-sided t tests for demographic effects. RESULTS: 139 participants were evaluated; demographic information was available for 70 (mean age 52.7 years ± 14.0 [SD], 36 female). Across all test datasets, MRSegmentator demonstrated high class wise DSC for well-defined organs (lungs: 0.81-0.96, heart: 0.81-0.94) and organs with anatomic variability (liver: 0.82-0.96, kidneys: 0.77-0.95). Smaller structures showed lower DSC (portal/splenic veins: 0.64-0.78, adrenal glands: 0.56-0.69). The average DSC on the external testing using NAKO data, ranged from 0.85 ± 0.08 for T2-HASTE to 0.91 ± 0.05 for in-phase sequences. The model generalized well to CT, achieving mean DSC of 0.84 ± 0.12 on AMOS CT data. CONCLUSION: MRSegmentator accurately segmented 40 anatomic structures on MRI and generalized to CT; outperforming existing open-source tools
Neurobehavioral mechanisms of fear and anxiety in multiple sclerosis
Get PDFBACKGROUND: Anxiety is a common yet often underdiagnosed and undertreated comorbidity in multiple sclerosis (MS). While altered fear processing is a hallmark of anxiety in other populations, its neurobehavioral mechanisms in MS remain poorly understood. This study investigates the extent to which neurobehavioral mechanisms of fear generalization contribute to anxiety in MS. METHODS: We recruited 18 persons with MS (PwMS) and anxiety, 36 PwMS without anxiety, and 23 healthy persons (HPs). Participants completed a functional MRI (fMRI) fear generalization task to assess fear processing and diffusion-weighted MRI for graph-based structural connectome analyses. RESULTS: Consistent with findings in non-MS anxiety populations, PwMS with anxiety exhibit fear overgeneralization, perceiving non-threating stimuli as threatening. A machine learning model trained on HPs in a multivariate pattern analysis (MVPA) cross-decoding approach accurately predicts behavioral fear generalization in both MS groups using whole-brain fMRI fear response patterns. Regional fMRI prediction and graph-based structural connectivity analyses reveal that fear response activity and structural network integrity of partially overlapping areas, such as hippocampus (for fear stimulus comparison) and anterior insula (for fear excitation), are crucial for MS fear generalization. Reduced network integrity in such regions is a direct indicator of MS anxiety. CONCLUSIONS: Our findings demonstrate that MS anxiety is substantially characterized by fear overgeneralization. The fact that a machine learning model trained to associate fMRI fear response patterns with fear ratings in HPs predicts fear ratings from fMRI data across MS groups using an MVPA cross-decoding approach suggests that generic fear processing mechanisms substantially contribute to anxiety in MS
Extrachromosomal DNA-driven oncogene dosage heterogeneity promotes rapid adaptation to therapy in MYCN-amplified cancers
No full textExtrachromosomal DNA (ecDNA) amplification enhances intercellular oncogene dosage variability and accelerates tumor evolution by violating foundational principles of genetic inheritance through its asymmetric mitotic segregation. Spotlighting highrisk neuroblastoma, we demonstrate how ecDNA amplification undermines the clinical efficacy of current therapies in cancers with extrachromosomal MYCN amplification. Integrating theoretical models of oncogene copy number–dependent fitness with single-cell ecDNA quantification and phenotype analyses, we reveal that ecDNA copy-number heterogeneity drives phenotypic diversity and determines treatment sensitivity through mechanisms unattainable by chromosomal oncogene amplification. We demonstrate that ecDNA copy number directly influences cell fate decisions in cancer cell lines, patient-derived xenografts, and primary neuroblastomas, illustrating how extrachromosomal oncogene dosage–driven phenotypic diversity offers a strong evolutionary advantage under therapeutic pressure. Furthermore, we identify senescent cells with reduced ecDNA copy numbers as a source of treatment resistance in neuroblastomas and outline a strategy for their targeted elimination to improve the treatment of MYCN-amplified cancers. SIGNIFICANCE: ecDNA-driven tumor genome evolution provides a major challenge to curative cancer therapies. We demonstrate that ecDNA copy-number dynamics drives treatment resistance by promoting oncogene dosage–dependent phenotypic heterogeneity in MYCN-amplified cancers. Exploiting phenotype-specific vulnerabilities of ecDNA cells, therefore, presents a powerful strategy to overcome treatment resistance
Cryosectioning-enhanced super-resolution microscopy for single-protein imaging across cells and tissues
Get PDFDNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) enables nanoscale imaging with virtually unlimited multiplexing and molecular counting. Here, we address challenges, such as variable imaging performance and target accessibility, that can limit its broader applicability. Specifically, we enhance its capacity for robust single-protein imaging and molecular counting by optimizing the integration of total internal reflection f luorescence microscopy with physical sectioning, in particular, Tokuyasu cryosectioning. Our method, tomographic and kinetically enhanced DNA-PAINT (tkPAINT), achieves 3 nm localization precision across diverse samples, enhanced imager binding, and improved cellular integrity. tkPAINT can facilitate molecular counting with DNA-PAINT inside the nucleus, as demonstrated through its quantification of the in situ abundance of RNA Polymerase II in both HeLa cells as well as mouse tissues. Anticipating that tkPAINT could become a versatile tool for the exploration of biomolecular organization and interactions across cells and tissues, we also demonstrate its capacity to support multiplexing, multimodal targeting of proteins and nucleic acids, and three-dimensional (3D) imaging
Dysfunctional mitochondria in ageing T cells: a perspective on mitochondrial quality control mechanisms
Get PDFDysfunctional mitochondria are a hallmark of T cell ageing and contribute to organismal ageing. This arises from the accumulation of reactive oxygen species (ROS), impaired mitochondrial dynamics, and inefficient removal of dysfunctional mitochondria. Both cell-intrinsic and cell-extrinsic mechanisms for removing mitochondria and their byproducts have been identified in T cells. In this review, we explore how T cells manage mitochondrial damage through changes in mitochondrial metabolism, mitophagy, asymmetric mitochondrial inheritance, and mitochondrial transfer, highlighting the impact of these mechanisms on T cell ageing and overall organismal ageing. We also discuss current therapeutic strategies aimed at removing dysfunctional mitochondria and their byproducts and propose potential new therapeutic targets that may reverse immune ageing or organismal ageing