Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

Osteoarthritis (OA), a joint disease, burdens global healthcare due to aging and obesity. Recent studies show that extracellular vesicles (EVs) from bone marrow-derived mesenchymal stem cells (BMSCs) contribute to joint homeostasis and OA management. However, the impact of donor age on BMSC-derived EV efficacy remains underexplored. In this study, we investigated EV efficacy from young BMSCs (2-month-old) in mitigating OA, contrasting them with EVs from aged BMSCs (27-month-old). The study used destabilisation of the medial meniscus (DMM) surgery on mouse knee joints to induce accelerated OA. Cartilage degeneration markers and senescence markers' expression levels were investigated in response to EV treatment. The therapeutic impact of EVs on chondrocytes under inflammatory responses was also evaluated. Despite having similar morphologies, EVs from young BMSCs markedly decreased senescence and improved chondroprotection by activating the PTEN pathway while simultaneously suppressing the upregulation of the PI3K/AKT pathways, proving to be more effective than those from older BMSCs in vitro. Furthermore, intraperitoneal injections of EVs from young donors significantly mitigated OA progression by preserving cartilage and reducing synovitis in a surgical OA model using DMM in mice. These findings highlight that donor age as a critical determinant in the therapeutic potential of BMSC-derived EVs for clinical use in OA treatment.

Periodontal ligament stem cells (PDLSCs) are key cells that suppress periodontal damage during both the progression and recovery stages of periodontitis. Although substantial evidence has demonstrated that incubation under an inflammatory condition may accelerate senescence of PDLSCs, whether cellular senescence in response to inflammatory incubation contributes to cell dysfunction remain unexplored. In this study, we first observed inflammation-caused PDLSC senescence in periodontitis based on comparisons of matched patients, and this cellular senescence was demonstrated in healthy cells that were subjected to inflammatory conditions. We subsequently designed further experiments to investigate the possible mechanism underlying inflammation-induced PDLSC senescence with a particular focus on the role of long noncoding RNAs (lncRNAs). LncRNA microarray analysis and functional gain/loss studies revealed SLC30A4-AS1 as a regulator of inflammation-mediated PDLSC senescence. By full-length transcriptome sequencing, we found that SLC30A4-AS1 interacted with SRSF3 to affect the alternative splicing (AS) of TP53BP1 and alter the expression of TP53BP1-204. Further functional studies showed that decreased expression of TP53BP1-204 reversed PDLSC senescence, and SLC30A4-AS1 overexpression-induced PDLSC senescence was abolished by TP53BP1-204 knockdown. Our data suggest for the first time that SLC30A4-AS1 plays a key role in regulating PDLSC senescence in inflammatory environments by modulating the AS of TP53BP1.

Ageing of the endometrium is a critical factor that affects reproductive health, yet its intricate mechanisms remain poorly explored. In this study, we performed transcriptome profiling and experimental verification of endometrium and endometrial organoids from young and advanced age females, to elucidate the underlying mechanisms and to explore novel treatment strategies for endometrial ageing. First, we found that age-associated decline in endometrial functions including fibrosis and diminished receptivity, already exists in reproductive age. Subsequently, based on RNA-seq analysis, we identified several changes in molecular processes affected by age, including fibrosis, imbalanced inflammatory status including Th1 bias in secretory phase, cellular senescence and abnormal signalling transduction in key pathways, with all processes been further validated by molecular experiments. Finally, we uncovered for the first time that PI3K-AKT-FOXO1 signalling pathway is overactivated in ageing endometrium and is closely correlated with fibrosis and impaired receptivity characteristics of ageing endometrium. Blocking or activation of PI3K by LY294002 or 740Y-P could attenuate the effect of ageing or accelerate dysfunction of endometrial organoids. This discovery is expected to bring new breakthroughs for understanding the pathophysiological processes associated with endometrial ageing, as well as treatment strategies to improve reproductive outcomes in women of advanced reproductive age.

CTCF plays a vital role in shaping chromatin structure and regulating gene expression. Clinical studies have associated CTCF mutations with congenital developmental abnormalities, including congenital cardiomyopathy. In this study, we investigated the impact of the homozygous CTCF-R567W (Ctcf^R567W/R567W) mutation on cardiac tissue morphogenesis during mouse embryonic development. Our results reveal significant impairments in heart development, characterised by ventricular muscle trabecular hyperplasia and reduced ventricular cavity sizes. We also observe a marked downregulation of genes involved in sarcomere assembly, calcium ion transport, and mitochondrial function in heart tissues from homozygous mice. Furthermore, the Ctcf^R567W/R567W mutation disrupts CTCF's interaction with chromatin, resulting in alterations to topologically associating domain (TAD) structure within specific genomic regions and diminishing crucial promoter-enhancer interactions necessary for cardiac development. Additionally, we find that the heterozygous CTCF-R567W (Ctcf^+/R567W) mutation significantly compromises cardiac contractility in 8-week-old mice. This study elucidates the mechanism by which the CTCF-R567W mutation hampers cardiac development, underscoring the essential role of CTCF-R567 in embryonic heart development and maturation.

The aim is to explore the mechanisms underlying pain development in chronic prostatitis and identify therapeutic targets for pain management in patients with chronic prostatitis. RNA sequence of the spinal cord dorsal horns and proteomic analysis of spinal macrophages of experimental autoimmune prostatitis (EAP) mice were conducted to identify pain-related genes, proteins and signalling pathways. The clodronate liposome, CXCR3 and P-STAT3 inhibitors, NGF antibody and cromolyn sodium were used to investigate the roles of the CXCL10/CXCR3, JAK/STAT3 and NGF/TrKA pathways in spinal macrophage recruitment and pain response. Finally, prostate tissues from benign prostate hyperplasia (BPH) patients were collected to validate the aforementioned results. Neuron and astrocyte-derived CXCL10 was associated with spinal macrophage recruitment, and CXCL10/CXCR3 axis could regulate the chemotaxis of macrophage to the spinal cord in EAP mice. Results of proteomic analysis found that CXCL10 could regulate the JAK/STAT3 pathway to mediate neuroinflammation in EAP, which was validated in vivo and in vitro experiments. The number of mast cells and expressions of NGF, TrKA and PGP9.5 increased in the prostates of EAP mice and BPH patients, and targeting NGF could reduce spinal macrophage recruitment and pain response. NGF was the triggering factor to induce chemotaxis of spinal macrophages and neuroinflammation, and the CXCL10/CXCR3 axis and JAK/STAT3 pathway was involved in spinal macrophage recruitment and infiltration, which provided therapeutic targets for pain management.

T-cell acute lymphoblastic leukaemia (T-ALL) is a heterogeneous malignant disease with high relapse and mortality rates. To characterise the multiomics features of T-ALL, we conducted integrative analyses using single-cell RNA, TCR and chromatin accessibility sequencing on pre- and post-treatment peripheral blood and bone marrow samples of the same patients. We found that there is transcriptional rewiring of gene regulatory networks in T-ALL cells. Some transcription factors, such as TCF3 and KLF3, showed differences in activity and expression levels between T-ALL and normal T cells and were associated with the prognosis of T-ALL patients. Furthermore, we identified multiple malignant TCR clonotypes among the T-ALL cells, where the clonotypes consisted of distinct combinations of the same TCR α and β chain per patient. The T-ALL cells displayed clonotype-specific immature thymocyte cellular characteristics and response to chemotherapy. Remarkably, T-ALL cells with an orphan TCRβ chain displayed the strongest stemness and resistance to chemotherapy. Our study provided transcriptome and epigenome characterisation of T-ALL cells categorised by TCR clonotypes, which may be helpful for the development of novel predictive markers to evaluate treatment effectiveness for T-ALL.

Sensorineural hearing loss is mainly caused by damage to hair cells (HC), which cannot be regenerated spontaneously in adult mammals once damaged. Cochlear Lgr5⁺ progenitors are characterised by HC regeneration capacity in neonatal mice, and we previously screened several new genes that might induce HC regeneration from Lgr5⁺ progenitors. Net1, a guanine nucleotide exchange factor, is one of the screened new genes and is particularly active in cancer cells and is involved in cell proliferation and differentiation. Here, to explore in vivo roles of Net1 in HC regeneration, Net1^loxp/loxp mice were constructed and crossed with Lgr5^CreER/+ mice to conditionally overexpress (cOE) Net1 in cochlear Lgr5⁺ progenitors. We observed a large number of ectopic HCs in Lgr5^CreER/+Net1^loxp/loxp mouse cochlea, which showed a dose-dependent effect. Moreover, the EdU assay was unable to detect any EdU⁺/Sox2⁺ supporting cells, while lineage tracing showed significantly more regenerated tdTomato⁺ HCs in Lgr5^CreER/+Net1^loxp/loxptdTomato mice, which indicated that Net1 cOE enhanced HC regeneration by inducing the direct trans-differentiation of Lgr5⁺ progenitors rather than mitotic HC regeneration. Additionally, qPCR results showed that the transcription factors related to HC regeneration, including Atoh1, Gfi1 and Pou4f3, were significantly upregulated and are probably the mechanism behind the HC regeneration induced by Net1. In conclusion, our study provides new evidence for the role of Net1 in enhancing HC regeneration in the neonatal mouse cochlea.

Currently, there is no specific treatment for diabetes-induced osteoporosis (DOP). Our study identified diabetes-induced cellular senescence, marked by elevated activity of senescence-associated β-galactosidase. Targeting senescent cells holds promise for osteoporosis treatment. We demonstrated that scutellarin (SCU) effectively mitigated bone loss in DOP mice, and co-treatment with SCU significantly reduced diabetes-induced senescence in LepR+MSCs. Furthermore, our research highlighted the role of Nrf2 in SCU's anti-senescence effects on bone. The deletion of Nrf2 impaired SCU's ability to alleviate DOP. Mechanistically, SCU enhances Ezh2 expression and increases H3K27me3 activity at the Keap1 promoter region, leading to Keap1 repression and enhanced Nrf2-ARE signalling. Additionally, SCU notably inhibited cellular senescence and diabetes-related osteoporosis, these effects were significantly reduced in Ezh2^LepRcre conditional knockout models. These findings suggest that the Ezh2-Nrf2 signalling axis is crucial for mediating SCU's beneficial effects in this context. Overall, our discoveries provide insights into the mechanisms underlying DOP and propose a potential preventive strategy for this condition.

The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)–transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats–associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.

Ovarian endometrioma (OEM), a particularly severe form of endometriosis, is an oestrogen-dependent condition often associated with pain and infertility. The mechanisms by which OEM impairs fertility, particularly through its direct impact on oocyte-cumulus cell (CC) communication and related pathways, remain poorly understood. This study investigates the impact of OEM on oocyte-CC communication and explores melatonin's therapeutic potential. We used a mouse model of OEM and employed ovarian transcriptome and gene set enrichment analyses to identify disrupted gene pathways, alongside phalloidin staining for cytoskeletal analysis, gap junction coupling analysis for intercellular communication, and mitochondrial function assessments for cellular metabolism. Our results showed that OEM significantly impairs steroidogenesis and cumulus cell function, leading to increased apoptosis, disrupted transzonal projections (TZPs), and impaired antioxidant transfer to oocytes. This culminates in oxidative stress, mitochondrial dysfunction, and compromised ATP production. OEM oocytes also exhibited severe abnormalities, including DNA damage, maturation defects, spindle assembly disruptions, and increased aneuploidy. This study identifies disrupted TZPs as a key pathological feature in OEM and highlights melatonin's potential to restore intercellular communication, mitigate oxidative damage, and improve reproductive outcomes.

Genetic diseases have long posed significant challenges, with limited breakthroughs in treatment. Recent advances in gene editing technologies offer new possibilities in gene therapy for the treatment of inherited disorders. However, traditional gene editing methods have limitations that hinder their potential for clinical use, such as limited editing capabilities and the production of unintended byproducts. To overcome these limitations, prime editing (PE) has been developed as a powerful tool for precise and efficient genome modification. In this review, we provide an overview of the latest advancements in PE and its potential applications in the treatment of inherited disorders. Furthermore, we examine the current delivery vehicles employed for delivering PE systems in vitro and in vivo, and analyze their respective benefits and limitations. Ultimately, we discuss the challenges that need to be addressed to fully unlock the potential of PE for the remission or cure of genetic diseases.