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A dataset of spatially resolved transcriptomics of post-natal cardiac development in mice.
The post-natal development and maturation of the mammalian heart involve highly intricate processes that remain incompletely understood, particularly concerning the molecular signature and roles of the diverse cell types involved. In this study, we present a comprehensive dataset generated from murine hearts at three key post-natal developmental stages using Spatio-Temporal Enhanced Resolution Omics-Sequencing (Stereo-seq), an advanced spatially resolved transcriptomic technology. This dataset encompasses spatial transcriptomes of approximately 0.186 million individual cells within intact sections of murine hearts at post-natal developmental stages. Our dataset serves as a valuable resource for investigating the mechanisms underlying mammalian heart development and maturation. Through initial analyses, we identified distinct cell types and their spatial distributions, including 93,826 cardiomyocytes within a single heart section. This extensive dataset provides researchers with opportunities for data mining and facilitates diverse analyses, including studies on transcriptional regulation, cell-to-cell communication, and the functional activities of genes and signalling molecules during critical phases of heart development.
FORMIC ACID
This dataset contains raw LC–MS data generated during the evaluation of formic acid (FA) pre-treatment for serum and plasma sample preparation in untargeted metabolomics. Samples were extracted using acetonitrile (MeCN) with and without 1% (v/v) FA pre-treatment, and a control set was prepared by post-spiking FA into extracts after MeCN precipitation. Analyses were performed using reversed-phase LC–MS (RPLC–MS) in both positive and negative electrospray ionisation modes, as well as anion-exchange chromatography MS (AEC–MS) in negative mode. The dataset includes all raw files from these test extractions, enabling comparison of metabolite feature detection and signal profiles across the three extraction conditions.
Covalently constrained 'Di-Gembodies' enable parallel structure solutions by cryo-EM.
Whilst cryo-electron microscopy(cryo-EM) has become a routine methodology in structural biology, obtaining high-resolution cryo-EM structures of small proteins (<100 kDa) and increasing overall throughput remain challenging. One approach to augment protein size and improve particle alignment involves the use of binding proteins or protein-based scaffolds. However, a given imaging scaffold or linking module may prove inadequate for structure solution and availability of such scaffolds remains limited. Here, we describe a strategy that exploits covalent dimerization of nanobodies to trap an engineered, predisposed nanobody-to-nanobody interface, giving Di-Gembodies as modular constructs created in homomeric and heteromeric forms. By exploiting side-chain-to-side-chain assembly, they can simultaneously display two copies of the same or two distinct proteins through a subunit interface that provides sufficient constraint required for cryo-EM structure determination. We validate this method with multiple soluble and membrane structural targets, down to 14 kDa, demonstrating a flexible and scalable platform for expanded protein structure determination.
Lysosomal free sialic acid storage disorder iPSC-derived neural cells display altered glycosphingolipid metabolism.
Lysosomal free sialic acid storage disorder (FSASD) is a rare neurodegenerative disease caused by biallelic mutations in SLC17A5, encoding the lysosomal sialic acid exporter, SLC17A5 (sialin). While the involvement of oligodendroglia in FSASD pathogenesis is established, the roles of other neural cell types remain elusive. In this study, we utilized radial glial cells (iRGCs), immature and mature astrocytes (iIAs and iMAs, respectively), and cortical neurons (iCNs) differentiated from induced pluripotent stem cells (iPSCs) derived from two individuals with FSASD, alongside two independent healthy donors for comparison. We employed a multifaceted profiling approach, including the assessment of cellular glycosphingolipids (GSLs), transcriptomics focused on GSL metabolism genes, and 4-methylumbelliferone-based lysosomal enzyme activity measurements. Our findings revealed significant elevations in free sialic acid levels across all FSASD cell types, indicating that iPSCs and derived iRGCs, iIAs, iMAs and iCNs may be used to model FSASD in vitro. We observed significant alterations in the abundance of specific GSL species, predominantly in mature astrocytes, with fewer changes in other cell types. Transcriptomic analyses uncovered differential expression of genes involved in GSL catabolism, including those encoding glycohydrolases. Enzyme assays corroborated the transcriptomic findings, showing heightened glycohydrolase activities, particularly in mature astrocytes. Collectively, these data may help refine our understanding of neural cell phenotypes and potential contributors to selective vulnerability in FSASD.
Compartmentalization proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation.
Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.
A Data-Driven Perspective on Bioisostere Evaluation: Mapping the Benzene Bioisostere Landscape with BioSTAR.
The bioisostere landscape is continually expanding, with new scaffolds emerging as alternatives in drug design. Increasingly, medicinal chemists face the challenge of selecting and prioritising these bioisosteres, often relying on personal experience and anecdotal evidence. In this Perspective, we lay out a data-driven approach to analyze the bioisostere landscape, using benzene bioisosteres as a representative example, and quantitatively compare replacements based on their impact on bioactivity, solubility, and metabolic stability. To support the findings of the analysis, we highlight recent and particularly elegant examples of benzene bioisostere applications while identifying areas where further development could significantly benefit the community. By providing this Perspective and associated data-mining workflow (BioSTAR), we aim to support more informed decision-making in bioisosteric replacement selection in drug design and inspire future innovations in bioisostere design.
Steric control of signaling bias in the immunometabolic receptor GPR84.
Biased signaling in G protein-coupled receptors offers therapeutic promise, yet rational design of biased ligands remains challenging due to limited mechanistic understanding. Here, we report a molecular framework for controlling signaling bias at the immunometabolic receptor GPR84. We identified three structurally-matched ligands (OX04529, OX04954, and OX04539) with varying steric profiles that exhibit comparable Gi protein activation but dramatically different β-arrestin recruitment capacities. A high-resolution cryo-EM structure of GPR84-Gi in complex with OX04529, complemented by molecular dynamics simulations and targeted mutagenesis, revealed that steric interactions between ligand substituents and Leu3366.52 and Phe1875.47 indirectly disrupt a critical polar network involving Tyr3326.48, Asn1043.36 and Asn3627.45 essential for β-arrestin recruitment. Based on these insights, we developed a steric-dependent model that enabled rational design of G protein-biased agonists with predictable β-arrestin recruitment profiles. This mechanistic framework provides a blueprint for designing biased agonists with customized signaling profiles at GPR84 and potentially other class A GPCRs.
Lysosomal proteases and their role in signaling pathways
Lysosomes contain more than 50 acid hydrolases and are the major location for degradation of both intracellular and extracellular macromolecules. However, lysosomes also play a key role in cell signaling processes, for example, trafficking via the endosomal/lysosomal pathway, regulation of autophagy, growth factor degradation, and through their involvement in antigen presentation. In this chapter, we summarize the classification of lysosomal proteases and their roles in cell signaling, with particular focus on the cysteine, serine, and aspartic cathepsins. In addition, we summarize how disruption of these processes may be linked to specific diseases, including neurodegenerative disease, cardiovascular disease, cancer, and inflammation.
Seedless: on-the-fly pulse calculation for NMR experiments.
NMR experiments require sequences of radio frequency (RF) pulses to manipulate nuclear spins. Signal is lost due to non-uniform excitation of nuclear spins resonating at different energies (chemical shifts) and inhomogeneity in the RF unavoidably generated by hardware over the sample volume. To overcome this, we present Seedless, a tool to calculate NMR pulses that compensate for these effects to enhance control of magnetisation and boost signal. As calculations take only a few seconds using an optimised GRadient Ascent Pulse Engineering (GRAPE) implementation, this now allows pulses to be generated in a few seconds, allowing them to be optimised for individual samples and spectrometers ("on-the-fly"). Each calculated pulse requires bands of chemical shift to be identified, over which one of 4 transforms will be performed, selected from a set that covers all commonly used applications. Using imaging experiments, we demonstrate our pulses effectively both increase the size of the coil volume and signal-to-noise in all experiments. We illustrate the approach by showing sensitivity gains in 1, 2 and 3D applications suitable for chemical and biological NMR. Seedless provides a means to enhance sensitivity in all pulse sequences in a manner that can be tailored to different samples and hardware being used.
Neuro-Cells Mitigate Amyloid Plaque Formation and Behavioral Deficits in the APPswe/PS1dE9 Model of Alzheimer Disease While Also Reducing IL-6 Production in Human Monocytes
Neuroinflammation is a key feature of Alzheimer’s disease (AD), and stem cell therapies have emerged as promising candidates due to their immunomodulatory properties. Neuro-Cells (NC), a combination of unmodified mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), have demonstrated therapeutic potential in models of central nervous system (CNS) injury and neurodegeneration. Here, we studied the effects of NC in APPswe/PS1dE9 mice, an AD mouse model. Twelve-month-old APPswe/PS1dE9 mice or their wild-type littermates were injected with NC or vehicle into the cisterna magna. Five to six weeks post-injection, cognitive, locomotor, and emotional behaviors were assessed. The brain was stained for amyloid plaque density using Congo red, and for astrogliosis using DAPI and GFAP staining. Gene expression of immune activation markers (Il-1β, Il-6, Cd45, Tnf) and plasticity markers (Tubβ3, Bace1, Trem2, Stat3) was examined in the prefrontal cortex. IL-6 secretion was measured in cultured human monocytes following endotoxin challenge and NC treatment. Untreated APPswe/PS1dE9 mice displayed impaired learning in the conditioned taste aversion test, reduced object exploration, and anxiety-like behavior, which were improved in the NC-treated mutants. NC treatment normalized the expression of several immune and plasticity markers and reduced the density of GFAP-positive cells in the hippocampus and thalamus. NC treatment decreased amyloid plaque density in the hippocampus and thalamus, targeting plaques of <100 μm2. Additionally, NC treatment suppressed IL-6 secretion by human monocytes. Thus, NC treatment alleviated behavioral deficits and reduced amyloid plaque formation in APPswe/PS1dE9 mice, likely via anti-inflammatory mechanisms. The reduction in IL-6 production in human monocytes further supports the potential of NC therapy for the treatment of AD.