Hyperforin is the compound responsible for the effectiveness of St. John\'s wort (Hypericum perforatum) as an antidepressant, but its complete biosynthesis remains unknown. Gene discovery based on co-expression analysis of bulk RNA-sequencing data or genome mining failed to discover the missing steps in hyperforin biosynthesis. Here we sequenced the 1.54 Gb tetraploid H. perforatum genome assembled into 32 chromosomes with scaffold N50 value of 42.44 Mb. By single-cell RNA-seq, we identified a type of cells, Hyper cells, wherein hyperforin biosynthesis de novo takes place in both leaves and flowers. Through pathway reconstitution in yeast and tobacco, we identify and characterize four transmembrane prenyltransferases (HpPT1-4) to resolve hyperforin biosynthesis, which localize to the plastid envelope. The hyperforin polycyclic scaffold is created by a reaction cascade involving an irregular isoprenoid coupling and a tandem cyclization. Our findings reveal how and where hyperforin is biosynthesized that enables synthetic-biology reconstitution of the complete pathway. These results deepen our comprehension of specialized metabolism at the cellular level, and we anticipate acceleration of pathway elucidation in plant metabolism.

OBJECTIVE: While meibomian gland dysfunction (MGD) is widely recognized as a major cause of evaporative dry eye disease, little is known about normal gland differentiation and lipid synthesis or the mechanism underlying gland atrophy and abnormal lipid secretion. The purpose of this study was to use single-cell and spatial transcriptomics to probe changes in cell composition, differentiation, and gene expression associated with two murine models of MGD: age-related gland atrophy in wild-type mice and altered meibum quality in acyl-CoA wax alcohol acyltransferase 2 (Awat2) knockout (KO) mice.
METHODS: Young (6 month) and old (22 month) wild type, C57Bl/6 mice and young (3 month) and old (13 month) Awat2 KO mice were used in these studies. For single-cell analysis, the tarsal plate was dissected from the upper and lower eyelids, and single cells isolated and submitted to the UCI Genomic Core, while for the spatial analysis frozen tissue sections were shipped to Resolve Biosciences on dry ice and sections probed in duplicate using a meibomian gland specific, 100 gene Molecular Chartography panel.
RESULTS: Analysis of gene expression patterns identified the stratified expression of lipogenic genes during meibocyte differentiation, which may control the progressive synthesis of meibum lipids; an age-related decrease in meibocytes; and increased immune cell infiltration. Additionally, we detected unique immune cell populations in the Awat2 KO mouse suggesting activation of psoriasis-like, inflammatory pathways perhaps caused by ductal dilation and hyperplasia.
CONCLUSIONS: Together these findings support novel mechanism controlling gland function and dysfunction.

Neuropathic pain is characterised by periodic or continuous hyperalgesia, numbness, or allodynia, and results from insults to the somatosensory nervous system. Peripheral nerve injury induces transcriptional reprogramming in peripheral sensory neurons, contributing to increased spinal nociceptive input and the development of neuropathic pain. Effective treatment for neuropathic pain remains an unmet medical need as current therapeutics offer limited effectiveness and have undesirable effects. Understanding transcriptional changes in peripheral nerve injury-induced neuropathy might offer a path for novel analgesics. Our literature search identified 65 papers exploring transcriptomic changes post-peripheral nerve injury, many of which were conducted in animal models. We scrutinize their transcriptional changes data and conduct gene ontology enrichment analysis to reveal their common functional profile. Focusing on genes involved in \'sensory perception of pain\' (GO:0019233), we identified transcriptional changes for different ion channels, receptors, and neurotransmitters, shedding light on its role in nociception. Examining peripheral sensory neurons subtype-specific transcriptional reprograming and regeneration-associated genes, we delved into downstream regulation of hypersensitivity. Identifying the temporal program of transcription regulatory mechanisms might help develop better therapeutics to target them effectively and selectively, thus preventing the development of neuropathic pain without affecting other physiological functions.

Idiopathic subglottic stenosis (ISGS) is a rare fibrotic disease of the upper trachea with an unknown pathomechanism. It typically affects adult Caucasian female patients, leading to severe airway constrictions caused by progressive scar formation and inflammation with clinical symptoms of dyspnoea, stridor and potential changes to the voice. Endoscopic treatment frequently leads to recurrence, whereas surgical resection and reconstruction provides excellent long-term functional outcome. This study aimed to identify so far unrecognized pathologic aspects of ISGS using single cell RNA sequencing. Our scRNAseq analysis uncovered the cellular composition of the subglottic scar tissue, including the presence of a pathologic, profibrotic fibroblast subtype and the presence of Schwann cells in a profibrotic state. In addition, a pathology-associated increase of plasma cells was identified. Using extended bioinformatics analyses, we decoded pathology-associated changes of factors of the extracellular matrix. Our data identified ongoing fibrotic processes in ISGS and provide novel insights on the contribution of fibroblasts, Schwann cells and plasma cells to the pathogenesis of ISGS. This knowledge could impact the development of novel approaches for diagnosis and therapy of ISGS.

Background: Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. Muscle degeneration involves a complex interplay between multiple cell lineages spatially located within areas of damage, termed the degenerative niche, including inflammatory cells, satellite cells (SCs) and fibro-adipogenic precursor cells (FAPs). FAPs are mesenchymal stem cell which have a pivotal role in muscle homeostasis as they can either promote muscle regeneration or contribute to muscle degeneration by expanding fibrotic and fatty tissue. Although it has been described that FAPs could have a different behavior in DMD patients than in healthy controls, the molecular pathways regulating their function as well as their gene expression profile are unknown. Methods: We used single-cell RNA sequencing (scRNAseq) with 10X Genomics and Illumina technology to elucidate the differences in the transcriptional profile of isolated FAPs from healthy and DMD patients. Results: Gene signatures in FAPs from both groups revealed transcriptional differences. Seurat analysis categorized cell clusters as proliferative FAPs, regulatory FAPs, inflammatory FAPs, and myofibroblasts. Differentially expressed genes (DEGs) between healthy and DMD FAPs included upregulated genes CHI3L1, EFEMP1, MFAP5, and TGFBR2 in DMD. Functional analysis highlighted distinctions in system development, wound healing, and cytoskeletal organization in control FAPs, while extracellular organization, degradation, and collagen degradation were upregulated in DMD FAPs. Validation of DEGs in additional samples (n = 9) using qPCR reinforced the specific impact of pathological settings on FAP heterogeneity, reflecting their distinct contribution to fibro or fatty degeneration in vivo. Conclusion: Using the single-cell RNA seq from human samples provide new opportunities to study cellular coordination to further understand the regulation of muscle homeostasis and degeneration that occurs in muscular dystrophies.

Cuproptosis is a novel type to regulate cell death with copper-dependent manner, and has been reported to involve in the occurrence and development of various malignant tumors. However, the association between cuproptosis and the tumor microenvironment (TME) of clear cell renal cell carcinoma (ccRCC) remained unclear. To address this question, we integrated the single cell RNA sequencing (scRNA-seq) datasets of ccRCC across different stages, systematically examined the distinctive expression patterns of cuproptosis-related genes (CRGs) within the TME of ccRCC, and explored the crucial signatures using the spatial transcriptome sequencing (ST-seq) dataset. The cuproptosis activities reduced in cancer tissues along with the ccRCC development, and recovered after therapy. We identified HILPDA+ ccRCC1 subtype, characterized with hypoxia, as cuproptosis susceptible cells associated with a better prognosis. The main co-expression modules of HILPDA+ ccRCC1 subtype highlighted the role in anion transport, response to oxygen species and PD-L1-PD-1 pathway. Furthermore, the immunosuppressive cells might interact with HILPDA+ ccRCC1 subtype via HAVCR2-LGALS9, C3-C3AR1, HLA-A-CD8B and HLA-C-CD8A axises to shape the cuproptosis-related TME landscape. In summary, we anticipate that this study will offer valuable insights and potential strategies of cuproptosis for therapy of ccRCC.

BACKGROUND: Tracheal adenoid cystic carcinoma (ACC) is a slow growing yet aggressive malignancy with high rates of local recurrence as well as distant metastasis. Tracheal ACC exhibit a low mutation burden along with high mutational diversity, and generally do not respond well to chemotherapeutics.
METHODS: We present a rare case of primary tracheal ACC initially presenting with nonspecific cervicalgia and globus sensation that was ultimately treated with tracheal resection followed by chemoradiation. Immune profiling of intratumoral T-cell receptor (TCR) repertoire was subsequently performed using single cell RNA sequencing (scRNAseq).
RESULTS: We describe a rare case of primary tracheal adenoid cystic carcinoma highlighting several management principles as well as providing new insights into intratumor T cell populations.
CONCLUSIONS: Primary tracheal ACC is most commonly treated with surgical resection followed by adjuvant therapy. Further characterization of the tumor immune microenvironment is necessary to better understand ACC disease biology and to identify potential therapeutic targets.

BACKGROUND: Metabolite-associated cell communications play critical roles in maintaining human biological function. However, most existing tools and resources focus only on ligand-receptor interaction pairs where both partners are proteinaceous, neglecting other non-protein molecules. To address this gap, we introduce the MRCLinkdb database and algorithm, which aggregates and organizes data related to non-protein L-R interactions in cell-cell communication, providing a valuable resource for predicting intercellular communication based on metabolite-related ligand-receptor interactions.
RESULTS: Here, we manually curated the metabolite-ligand-receptor (ML-R) interactions from the literature and known databases, ultimately collecting over 790 human and 670 mouse ML-R interactions. Additionally, we compiled information on over 1900 enzymes and 260 transporter entries associated with these metabolites. We developed Metabolite-Receptor based Cell Link Database (MRCLinkdb) to store these ML-R interactions data. Meanwhile, the platform also offers extensive information for presenting ML-R interactions, including fundamental metabolite information and the overall expression landscape of metabolite-associated gene sets (such as receptor, enzymes, and transporter proteins) based on single-cell transcriptomics sequencing (covering 35 human and 26 mouse tissues, 52 human and 44 mouse cell types) and bulk RNA-seq/microarray data (encompassing 62 human and 39 mouse tissues). Furthermore, MRCLinkdb introduces a web server dedicated to the analysis of intercellular communication based on ML-R interactions. MRCLinkdb is freely available at https://www.cellknowledge.com.cn/mrclinkdb/ .
CONCLUSIONS: In addition to supplementing ligand-receptor databases, MRCLinkdb may provide new perspectives for decoding the intercellular communication and advancing related prediction tools based on ML-R interactions.

Abdominal aortic aneurysm (AAA) is a highly lethal cardiovascular disease that currently lacks effective pharmacological treatment given the complex pathophysiology of the disease. Here, single-cell RNA-sequencing data from patients with AAA and a mouse model are analyzed, which reveals pivotal pathological changes, including the M1-like polarization of macrophages and the loss of contractile function in smooth muscle cells (SMCs). Both cell types express the integrin αvβ3, allowing for their dual targeting with a single rationally designed molecule. To this end, a biocompatible nanodrug, which is termed EVMS@R-HNC, that consists of the multifunctional drug everolimus (EVMS) encapsulated by the hepatitis B virus core protein modifies to contain the RGD sequence to specifically bind to integrin αvβ3 is designed. Both in vitro and in vivo results show that EVMS@R-HNC can target macrophages as well as SMCs. Upon binding of the nanodrug, the EVMS is released intracellularly where it exhibits multiple functions, including inhibiting M1 macrophage polarization, thereby suppressing the self-propagating inflammatory cascade and immune microenvironment imbalance, while preserving the normal contractile function of SMCs. Collectively, these results suggest that EVMS@R-HNC presents a highly promising therapeutic approach for the management of AAA.

BACKGROUND: Neutrophils play an essential role in Alzheimer\'s disease (AD) pathology. However, the extent of their heterogeneity remains poorly explored, particularly in the context of developing novel therapies targeting these cells.
RESULTS: We investigate the population structure of neutrophils purified from peripheral blood samples of AD mice. Utilizing single cell RNA sequencing, we comprehensively map neutrophil populations into six distinct clusters and find that the Neu-5 subset is specially enriched in AD mice. This subset exhibits fewer specific granules and a lower mature score. Gene ontology (GO) analysis reveals that genes involved in cytokine-mediated signaling are downregulated in the Neu-5 cluster. Furthermore, we identify the Ccrl2 gene is specifically upregulated in this subgroup, which is confirmed by flow cytometry in AD mice. Finally, immunohistochemical staining indicates that CCRL2 protein is increased in the brains of AD mice.
CONCLUSIONS: We identify a unique CCRL2 positive neutrophil cluster, that is specifically enriched in the peripheral blood of AD mice.