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Multicolor fate mapping of microglia reveals polyclonal proliferation, heterogeneity, and cell-cell interactions after ischemic stroke in mice
Get PDFMicroglial proliferation is a principal element of the inflammatory response to brain ischemia. However, the precise proliferation dynamics, phenotype acquisition, and functional consequences of newly emerging microglia are not yet understood. Using multicolor fate mapping and computational methods, we here demonstrate that microglia exhibit polyclonal proliferation in the ischemic lesion of female mice. The peak number of clones occurs at 14 days, while the largest clones are observed at 4 weeks post-stroke. Whole-cell patch-clamp recordings of microglia reveal a homogeneous acute response to ischemia with a pattern of outward and inward currents that evolves over time. In the resolution phase, 8 weeks post-stroke, microglial cells within one clone share similar membrane properties, while neighboring microglia from different clones display more heterogeneous electrophysiological profiles. Super-resolution microscopy and live-cell imaging unmask various forms of cell-cell interactions between microglial cells from different clones. Overall, this study demonstrates the polyclonal proliferation of microglia after cerebral ischemia and suggests that clonality contributes to their functional heterogeneity. Thus, targeting clones with specific functional phenotypes may have potential for future therapeutic modulation of microglia after stroke
Open-source cardiac MR fingerprinting
No full textMagnetic Resonance (MR) raw data acquired with an open-source cardiac MR Fingerprinting (cMRF) sequence of a phantom at four different MR scanners. More details can be found here: https://github.com/PTB-MR/cMRF. The colormaps are taken from https://zenodo.org/records/11185704 because zenodo_get failed on trying to download this record in a jupyter notebook. Additionally cMRF data was acquired in three volunteers who were scanned at two different scanners. Cartesian and golden radial cine data was acquired to verify the anatomical features seen in the quantitative maps
Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation [TESLA-seq]
No full textORGANISM: Homo sapiens.
EXPERIMENT TYPE: Other.
SUMMARY: Transcriptional enhancers are non-coding DNA elements that regulate gene transcription in a temporal and tissue-specific manner. Despite advances in computational and experimental methods, identifying enhancers and their target genes essential for specific biological processes remains challenging. Determining target genes for enhancers is also complex and often relies on indirect, low-resolution, and/or assumptive methodologies. To identify and functionally perturb enhancers at their endogenous sites without altering their sequence, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma development. This screen allowed the identification of CRISPRa- responsive elements (CaREs) that alter cellular growth within the 2 Mb genomic region. To determine CaRE target genes, we developed TESLA-seq (TargEted- SingLe- cell- Activation), which combines CRISPRa screening with targeted single-cell RNA-sequencing, and identified functional CaRE-target gene pairs. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements already active in the system, we found many CaREs and target connections normally active only in other tissue types or with no previous evidence and induced out of context by CRISPRa. This highlights the power of TESLA-seq to reveal gene regulatory networks active outside of a given experimental system.
OVERALL DESIGN: TESLA-seq was performed as described in details in the manuscript. We selected 222 top-scoring significant CaREs from the bulk phenotypic screen. For each of them, we selected gRNAs with the highest fold change. These gRNAs, together with 52 control gRNAs (total of 1098 in Table S5), were ordered as oligos from Twist Bioscience, cloned, and sequenced as described for the phenotypic screen. The lentiviral production was done as in the viability screen. SHSY-5Y-VPR line was infected at 85% in both experiments as determined by the BD Rhapsody scanner after staining with Calcein AM (Thermo Fisher Scientific #C1430) and Draq7 (Thermo Fisher Scientific #564904) according to the manufacturer's protocol. Single-cell capture and cDNA synthesis were performed using the BD RhapsodyTM Single-Cell Analysis System according to the manufacturer's instructions. Capture probes for 146 transcripts corresponding to 78 genes in the 6MB genomic space (+/- 3MB from the PHOX2B TSS) on chr4 were designed by BD. The targets were enriched and the library was prepared according to the BD mRNA Targeted Library Preparation protocol. Paired-end sequencing (2x75nt) was performed on a NextSeq 500/550 using a HighOutput v2 Kit for 150 cycles with a 20% PhiX spike-in. Two replicates of this experiment are submited: TESLA-seq replicate_1 and TESLA-seq_replicate_2
Capturing global pet dog gut microbial diversity and hundreds of near-finished bacterial genomes by using long-read metagenomics in a Shanghai cohort
Get PDFPet dogs are considered part of the family, and understanding their gut microbiomes can provide insights into both animal and household health. Most comprehensive studies, however, relied on short-read sequencing, resulting in fragmented MAGs that miss mobile elements, antimicrobial-resistance genes, and ribosomal genes. Here, we applied deep long-read metagenomics (polished with short-reads) to fecal samples from 51 urban pet dogs in Shanghai, generating 2,676 MAGs—representing 320 bacterial species—, of which ∼72% achieved near-finished quality, often improving on the corresponding reference public genome. Comparisons with external datasets showed that our Shanghai-based MAG catalog is representative of pet dogs worldwide (median read mapping of >90%). Moreover, we recovered circular extrachromosomal elements, including those linked to antimicrobial resistance, which were also detected in external dog gut datasets. In conclusion, we provide a high-quality reference resource and demonstrate the power of deep long-read metagenomics to resolve microbial diversity in complex host-associated microbiomes
The 2025 ESC myocarditis and pericarditis guidelines - what clinicians needs to know
No full textThe 2025 European Society of Cardiology (ESC) Guidelines on Myocarditis and Pericarditis represent the first unified framework addressing both conditions under a single guideline. For the first time, myocarditis and pericarditis are integrated into the concept of inflammatory myopericardial syndrome (IMPS), acknowledging their overlapping aetiologies and forms of these contiguous cardiac structures.(1) This document provides a critical summary of the main novelties, focusing on implications for clinical practice
Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution
Get PDFGene amplification in the form of extrachromosomal DNA (ecDNA) is a frequent driver in multiple cancer types. As ecDNA lack centromeres, their mitotic segregation does not follow traditional inheritance principles. However, the mechanisms that govern ecDNA fate following mitosis remain unclear. We found that ecDNA undergo numerical and structural optimization under increased selective pressure, with mitotic chromosomal tethering, or detachment, dictating ecDNA fate. When tethered, ecDNA aggregates promote uneven distribution into the newly formed daughter cells, thereby driving inter-cellular numerical heterogeneity and rapid increase of amplification under selective pressure. Mitotically detached ecDNA frequently encapsulate within micronuclei of variable size and content that appear to be highly fragile. Strikingly, ecDNA enclosed in very small micronuclei, which we term nanonuclei, are being actively degraded through autophagy. Together with ongoing structural rearrangements, nanonuclear ecDNA degradation promotes their structural evolution, which facilitates cancer cell adaptation. Our work highlights ecDNA aggregation, micronucleation, and degradation, as pivotal events in directing cancer genome evolution trajectories
The two faces of MyoD: repressor and activator of gene expression during myogenesis
Get PDFMyoD is well known for its ability to reprogram a broad range of cell types into myogenic cells and for its pioneer function in activating the myogenic program during muscle development and regeneration. The basic helix–loop–helix (bHLH) protein achieves this by directly binding to E-boxes in DNA and recruiting proteins like histone acetyltransferases and the SWI/SNF chromatin remodeling complex. Interestingly, Nicoletti and colleagues (doi:10.1101/gad.352708.125) report in this issue of Genes & Development an unexpected finding; namely, that MyoD can also act as a repressor. This repressive activity is E-box-independent, meaning that MyoD can be indirectly recruited to distinct sites in chromatin. Transcription factor motifs enriched at these sites correspond to E2F, NF-Y, and Jun/Fos motifs. The genes that are repressed by this noncanonical MyoD function control nonmyogenic fates and participate in cell cycle regulation as well as proliferation. At such sites, MyoD binding is associated with chromatin compaction and repression of transcription
Brolucizumab and platelet activation and reactivity
Get PDFPURPOSE: This study explores the potential interaction of brolucizumab with platelets and its effects on platelet activation and reactivity, crucial in retinal vasculitis and retinal vascular occlusion. Safety concerns remain of interest, although brolucizumab showed superior retinal efficacy and reduced injection frequency compared to other licensed anti-VEGF agents. METHODS: Resting and activated platelets of healthy volunteers were pretreated with brolucizumab at the following concentrations 0.6 µg/mL, 3 µg/mL, 6 µg/mL, 300 µg/mL, and 3000 µ/mL or its solvent or PBS. The surface expression of platelet activation markers GPIIb/IIIa and P-selectin was determined by multispectral imaging flow cytometry, which combines flow cytometry and fluorescence microscopy. Two different methods were used to examine the interaction of brolucizumab with platelets: 1) A cross-pretreatment experiment was performed with FITC-labeled brolucizumab and bevacizumab; 2) Resting and activated platelets were pretreated with brolucizumab or its solvent or PBS, followed by anti-brolucizumab antibody generated by rabbit immunization. RESULTS: Brolucizumab did not significantly affect platelet activation compared to solvent or PBS, across a range of concentrations. No significant upregulation of CD62P and no activation of the fibrinogen receptor (GPIIb/IIa) were observed in resting and TRAP-activated platelets. After pretreatment with PBS, the level of brolucizumab-FITC was significantly lower in comparison to bevacizumab-FITC (normalized MFI = 3.32, CI = 3.16-3.48 vs. normalized MFI = 7.19, CI = 7.04-7.35; < 0.001). Both brolucizumab- and bevacizumab-FITC were downregulated after pretreatment with brolucizumab or bevacizumab compared to pretreatment with PBS. Antibodies against brolucizumab did not show any significant difference between pretreatment with brolucizumab and its solvent in resting and TRAP-activated platelets. CONCLUSION: Brolucizumab does not appear to directly affect platelet activation or reactivity to thrombin receptor agonists. No platelet interaction was observed after increasing brolucizumab concentrations or anti-brolucizumab antibodies in resting and activated platelets. However, brolucizumab might be taken up in platelets
Rifaximin-induced changes in the gut microbiome associated to improvement of neurotransmission alterations and learning in rats with chronic liver disease
Get PDFRifaximin, a gut-targeted antibiotic, improves cognitive function and reduces the risk of hepatic encephalopathy (HE), yet its effects on the gut-brain axis remain unknown. This study explores how rifaximin influences gut microbiota functions and its association with cognitive function and molecular alterations in rats with liver injury. Liver injury was induced by chronic administration of carbon tetrachloride (CCl4), and rifaximin was administered daily. Fecal samples were collected after eight weeks of CCl4 administration, and taxonomic and functional changes in the gut microbiome were analyzed. Rifaximin altered microbiota diversity and composition, increasing α diversity in liver-injured rats but reducing diversity in healthy rats. It influenced microbiota interactions with neurotransmission alterations, where Dorea, Lachnospiraceae A2, and possibly Erysipelotricaceae might be important contributors. Functionally, butyric acid levels negatively correlated with gene orthologues associated with GABA, tryptophan, and glutamate degradation pathways. In healthy rats, fecal short-chain fatty acid (SCFA) levels were positively correlated with each other, a pattern absent in other groups. Rifaximin significantly influenced gut microbiota and promoted bacterial groups linked to improved cognition and neurotransmission in liver disease. Our findings underscored the direct relationship between a healthy microbiome and the maintenance of balanced SCFA concentrations
Automated classification of cellular expression in multiplexed imaging data with Nimbus
No full textMultiplexed imaging offers a powerful approach to characterize the spatial topography of tissues in both health and disease. To analyze such data, the specific combination of markers that are present in each cell must be enumerated to enable accurate phenotyping, a process that often relies on unsupervised clustering. We constructed the Pan-Multiplex (Pan-M) dataset containing 197 million distinct annotations of marker expression across 15 different cell types. We used Pan-M to create Nimbus, a deep learning model to predict marker positivity from multiplexed image data. Nimbus is a pretrained model that uses the underlying images to classify marker expression of individual cells as positive or negative across distinct cell types, from different tissues, acquired using different microscope platforms, without requiring any retraining. We demonstrate that Nimbus predictions capture the underlying staining patterns of the full diversity of markers present in Pan-M, and that Nimbus matches or exceeds the accuracy of previous approaches that must be retrained on each dataset. We then show how Nimbus predictions can be integrated with downstream clustering algorithms to robustly identify cell subtypes in image data. We have open-sourced Nimbus and Pan-M to enable community use at https://github.com/angelolab/Nimbus-Inference