Oncolytic viruses (OVs) are widely studied for their ability to lyse cancer cells and prime immune responses; however, the immune consequences triggered by OVs remain incompletely understood. Here, we discover that oncolytic VSVΔ51 treatment suppresses the T cell receptor signaling of tumor-infiltrating T cells. Mechanistically, VSVΔ51-infected cancer cells upregulate PCSK9 secretion, which triggers lysosomal degradation of major histocompatibility complex (MHC)-I in bystander cells. PCSK9 inhibition synergizes with VSVΔ51 treatment to suppress tumor growth in multiple colorectal cancer models and induce complete regression in a microsatellite-stable (MSS) tumor model. This combination fosters stem-like CD8+ T cells and establishes anti-tumor memory. Engineered VSVΔ51 expressing anti-PCSK9 single-chain variable fragments improves intra-tumor viral replication, sustains anti-tumor CD8+ T cell memory, and enhances anti-PD-1 therapy efficacy. Our results identify the role of PCSK9 in the immunosuppressive feedback following viral infection and propose a strategy for engineered oncolytic virotherapy.

Neurological injuries and neurodegenerative disorders, including spinal cord injury, traumatic brain injury, stroke, and Parkinson's disease remain largely incurable. In the central nervous system (CNS), a self-reinforcing cascade of neuroinflammation, oxidative stress, blood-brain barrier breakdown, and glial fibrotic scarring restricts long-distance axonal regrowth and graft survival. The peripheral nervous system (PNS) exhibits greater intrinsic regenerative potential, yet critical-length defects remain challenging and have driven the development of clinically relevant conduit designs. This review provides an overview of the microenvironment following CNS injury and summarizes the key design requirements for engineered repair matrices, while highlighting lessons from advanced peripheral nerve guidance conduits. Injectable extracellular matrix (ECM)-mimetic and smart hydrogels can conformally fill CNS cavities, modulate immune and redox cascades, restore vascular function, and provide permissive niches for neural stem/progenitor and endothelial cells. CNS-compatible bioinks and 3D bioprinting enable the fabrication of neurovascular architectures and multicellular constructs with controlled mechanics, topology, and circuit geometry. Advances in nerve guidance conduits inform translation of PNS principles to the brain and spinal cord. Organoid-based strategies, including vascularized organoids, biomaterial-supported grafts, and organoid-neuroelectronic interfaces, suggest routes toward modular biohybrid constructs. Integrating pathology-informed biomaterials, biofabrication, and organoid engineering offers a roadmap for neural circuit reconstruction.

Despite extensive global efforts to eradicate cervical cancer through improved screening and widespread HPV vaccination, the disease continues to claim over 350 000 lives annually. Persistent infection with high-risk HPV genotypes, primarily HPV-16 and HPV-18, is the principal etiological driver, with viral DNA integration into the host genome representing a critical event in malignant progression. In this study, HPV integration was investigated using 25 RNA-seq datasets from HPV-positive cervical cancer cell lines and 2 datasets from the HPV-negative C-33A cell line. Analysis revealed a recurrent HPV-16 integration hotspot on Chromosome 6 within the CLIC5 gene. This integration was validated by qRT-PCR and Sanger sequencing. Subsequent gene expression analysis demonstrated significant CLIC5 upregulation in HPV-16-positive cell lines, with a 21-fold increase in CaSki and a 4-fold increase in SiHa compared to HPV-negative and HPV-18-positive controls. These findings delineate a distinct HPV-16 integration pattern and its associated transcriptional impact. The identification and validation of this strain-specific integration site nominates HPV-16 integration at the CLIC5 locus as a potential biomarker for HPV-driven cervical cancer.

To develop novel hydropyrimidine linked amide-pyridine hybrids as anticancer agents.

Orofacial and limb muscles differ in embryonic origin and regenerative capacity. Neuromuscular junction (NMJ) regeneration is critical for muscle restoration both histologically and functionally. The relative potential of orofacial and limb muscles to form postsynaptic apparatuses remains elusive. While the role of fibro-adipogenic progenitors (FAPs) in NMJ regeneration has been discussed in limb muscles, it remains unexplored in orofacial muscles.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by loss of fragile X messenger ribonucleoprotein (FMRP). In most cases, this results from a CGG expansion exceeding 200 repeats in the 5' untranslated region of the fragile X messenger ribonucleoprotein 1 (FMR1) gene, known as the "full mutation". While the trinucleotide expansion has long been thought to induce epigenetic silencing of this locus, studies have shown that many males with a full mutation still express FMR1 mRNA. However, these individuals produce little to no FMRP protein, due to mechanisms that remain unclear. Mis-splicing of FMR1 transcripts with an expanded CGG tract has recently been proposed as a potential mechanism underlying the absence of FMRP in FXS tissues despite the presence of gene transcripts.

CRISPR-based gene editing holds promise for treating genetic diseases, yet its application to lung disorders has been hindered by the challenges of pulmonary delivery. Inspired by the modularity and biocompatibility of amino acid-derived chemistries, we report the combinatorial synthesis of 960 ionizable lipids incorporating chemically diverse backbones from both proteinogenic and non-proteinogenic α-amino acids. Through high-throughput screening and structure-function analysis, we identify CHCha-10, a cyclohexyl amino acid-derived lipid that forms biodegradable nanoparticles capable of efficiently delivering mRNA-based gene editors to lung epithelial cells. Following intratracheal administration, CHCha-10 nanoparticles exhibit enhanced mucus penetration and epithelial-specific transfection in both mice and ferrets. Here, as a functional application, we demonstrate in vivo base editing in the lung via inhalation. Delivery of adenine base editor mRNA and guide RNA targeting the CFTR G542X mutation restores CFTR expression and chloride channel function in G542X human airway epithelial cells, mouse-derived intestinal organoids and the lungs of cystic fibrosis mice. This work establishes a chemically modular design framework for ionizable lipids and a translatable platform for RNA-based pulmonary gene correction.

Cellular diversity is governed not only by the transcriptome but also by multiple layers of epigenomic regulation, including nucleosome occupancy, chromatin states and genome architecture1-3. Here, to comprehensively understand how these regulatory modalities converge to shape cellular identity, we developed a single-cell four-omics sequencing method that enables parallel profiling of genome conformation, histone modifications, chromatin accessibility and gene expression within the same cell (CHARM). Applying CHARM to mouse embryonic stem cells and cortical tissues, we reconstructed integrated epigenome profiles, uncovering distinct cell-cycle dynamics of chromatin accessibility and histone modification, and spatial clustering of regulatory elements in three-dimensional nuclear space. Leveraging an interpretable machine learning model, we further identified thousands of enhancer-promoter linkages with high accuracy that modulate gene expression in a cell-type- and subtype-specific manner. Together, CHARM enables integrative dissection of the three-dimensional epigenome at single-cell resolution, providing a versatile platform for decoding the regulatory landscape across diverse cells in complex tissues.

Haploidentical hematopoietic stem cell transplant (Haplo-HSCT) has emerged as an alternative for patients lacking matched human leukocyte antigen (HLA) donors. The scarcity of stem cell donor registries and cord banks across the Eastern Mediterranean (EM) region has led to increased need of Haplo-HSCT as a mean to overcome this limitation. In this study, we aimed to assess trends in Haplo-HSCT utilization within the Eastern Mediterranean Blood and Marrow Transplantation (EMBMT) registry from 2012 to 2024, and describe key clinical and transplant-related factors associated with patient outcomes. We conducted a retrospective, multicenter registry-based analysis of patients who had their first un-manipulated Haplo-HSCT in ten EMBMT centers across six countries, who responded and accepted to participate in the study. Due to the variability in time-to-event reporting across different centers, survival and relapse-related outcomes were analyzed descriptively. Continuous variables were summarized using medians and ranges, and categorical variables were described by counts and percentages. The association between individual baseline variables and patient survival, was conducted using a univariate analysis utilizing the chi-square test. Categorical variables such as patient and transplant characteristics were compared against survival status at last follow-up. All variables were further evaluated in the multivariate analysis. Overall survival (OS) and progression-free survival (PFS) were estimated using Kaplan-Meier survival analysis. Between 2012 and 2024, a total of 673 patients received haplo-HSCT across ten centers, in six EMBMT-registered countries. Median age was 25 years, and the predominant indication for haplo-HSCT was leukemia (64.3%). Transplant was done using stem cells collected from peripheral blood in majority of cases (65.7%), and Total Body Irradiation (TBI) was utilized in 37.6% of all patient. 76.5% had malignant hematological disorders, while 23.5% had non-malignant disorders. Relapse was reported in 232 patients (34.5%) and death in 220 patients (32.7%). Of these, 88 deaths (12.8%) were attributed to disease progression. Haplo-HSCT activity increased steadily over the study period, with a marked increase in the last few years, reflecting broader regional adoption. PFS was 66% and 60% at 1- and 2- years after transplantation. Also, the 1-year and 2-year post-transplant OS was 71% and 65%, respectively. Multivariate analysis identified disease type and chronic GVHD as factors associated with improved outcomes. Despite variability across centers, outcomes were consistent with international benchmarks, underscoring the growing feasibility and therapeutic value of haplo-HSCT in the EMRO region. Haplo-HSCT is increasingly adopted across the EMRO region as the primary alternative donor HSCT modality. Our findings confirm its clinical efficacy, with outcomes comparable to international benchmarks. These results underscore the importance of continued regional collaboration and the development of personalized strategies to address challenges such as graft-versus-host disease (GVHD), disease relapse, and disparities in access to transplantation services.

The Jiangquan black pigs, a new breed of swine obtained by introducing traits from Duroc pigs into Yimeng black pigs, exhibits fast growth rates and high meat quality. To understand how daily weight gain influences muscle development in this breed, we analyzed longissimus dorsi muscle cell subpopulations from Jiangquan black pigs using snRNA and bulk RNA sequencing. Thirteen distinct cell types (e.g., muscle stem cells, satellite cells, fibroblasts) were identified, and marker genes (PAX7, MYOD, MYOG) were found to exhibit stage-specific expression during differentiation. Pseudotime analysis revealed the differentiation trajectories of these cell populations, while cell cycle analysis uncovered the higher mitotic activity in satellite cells of the fast-growth versus slow-growth groups. Furthermore, cell communication analysis highlighted the interactions between muscle cells and other cell types. Finally, intergroup analysis revealed that 2,466 and 2,597 genes were differentially expressed in muscle stem cells and muscle satellite cells, respectively. These genes were enriched in disease-related pathways. This study provides a single-cell resolution atlas of porcine muscle development, offering insights into the genetic regulation of growth and potential targets for breeding optimization.

Mechanical stimulation plays a crucial role in odontoblast differentiation. However, the underlying molecular mechanisms remain unclear. We have previously shown that hydrostatic pressure (HP) applied to stem cells from human exfoliated deciduous teeth (SHED) promotes odontoblast differentiation by translocating RUNX2 and increasing WNT16 expression through PIEZO1 signaling. In this study, we further explored the downstream signaling cascade linking PIEZO1 activation and odontoblast differentiation. HP stimulation increased the expression of odontoblast differentiation markers PANX3 and DSPP, as shown by qPCR, and enhanced Alizarin Red staining-results significantly suppressed by siRNA targeting either PIEZO1 or WNT16. RT-PCR analysis revealed that, among the two known human WNT16 isoforms, only WNT16b was expressed in SHED. qPCR demonstrated that HP-induced WNT16 expression was reduced by siPIEZO1 and further decreased by siRUNX2. Promoter analysis identified four RUNX2-binding sites within the upstream region of WNT16. A luciferase reporter assay using plasmids containing the WNT16 promoter showed that RUNX2 overexpression in HEK293 cells significantly increased luciferase activity. Moreover, HaloChIP assays with a HaloTag-RUNX2 expression vector confirmed RUNX2's binding to the WNT16 promoter. These findings suggest that PIEZO1-mediated mechanical stress promotes odontoblast differentiation through the RUNX2-dependent transcriptional activation of WNT16.

Inflammatory bowel disease (IBD) is secondary to an abnormal immune response to the microbiota. To study this, models of host-microbe interactions that represent mucosal bacterial communities and inter-patient diversity are required. Human intestinal organoids (HIOs) are an established model to investigate epithelial responses. Here, we describe a technique of culturing bacteria directly from the sites of inflammation in IBD, while simultaneously sampling host tissue. We generated HIOs from a cohort of newly diagnosed paediatric IBD patients, without confounding treatments or comorbidities, and explored their response to site-specific bacteria. A unique biobank of matched HIOs and cultured mucosa-attached bacteria was established from 27 paediatric patients. Transcriptional profiling revealed differential gene expression between control and IBD-derived organoids. We used microinjection to introduce bacteria to the apical surface of the epithelium, to determine the effect of bacteria on host epithelial cells. We measured survival and growth of bacteria within the HIOs and tested several related bacterial isolates for their impact on the epithelium. An isolate from a control patient stimulated inflammatory signalling pathways but this was not observed in response to a closely related isolate originating from an IBD patient. This study demonstrates the feasibility of isolating bacteria and generating organoids from the same biopsy tissue, to explore personalised host-microbe interactions. The microinjections, while labour-intensive, demonstrate that closely related bacteria can induce very different epithelial responses, with downstream implications for immune response. This highlights the importance of understanding host-microbe interactions in a strain- and site-specific manner and developing techniques for personalised microbiome-based therapeutics.

Keratinocytes are increasingly recognized as central regulators of cutaneous immune responses and key contributors to maintaining immune homeostasis. However, whether and how epidermal stem and progenitor cells (EPSCs) actively suppress proinflammatory signaling pathways to prevent excessive inflammation and maintain epidermal immune quiescence remains unclear. Here, we generated a conditional knockout mouse model (K14-CreERT; Supt6fl/fl) to investigate the role of SPT6, a transcription elongation factor, in epidermal and immune homeostasis. Loss of SPT6 in basal keratinocytes led to spontaneous, psoriasis-like skin inflammation, characterized by epidermal hyperplasia, immune cell infiltration, parakeratosis, and hyperkeratosis. SPT6-deficient mice also exhibited significantly delayed wound healing accompanied by impaired Wnt signaling. Moreover, single-cell RNA sequencing revealed distinct keratinocyte subpopulations with inflammatory signatures, elevated NF-κB signaling, and suppressed Wnt signaling. Mechanistically, SPT6 suppresses NF-κB signaling by binding to an enhancer of the RELA gene and preventing its positive transcriptional feedback loop. These findings support a new paradigm in which the default state of the skin may be primed for inflammation, and active suppression by factors such as SPT6 is required to maintain epidermal homeostasis. Taken together, the results of our study reveal a previously unrecognized role for SPT6 as a key regulator of epidermal immune quiescence and tissue integrity.

Selective elimination of suboptimal cells is critical for the developmental integrity of early mammalian embryogenesis. Cell competition is a non-autonomous quality control in which 'winner' cells outcompete viable but suboptimal 'loser' cells based on fitness differences. Here we investigate cell competition dynamics using mosaic mouse gastruloids, a 3D embryonic stem cell-based model of gastrulation. Introducing just two Trp53-deficient supercompetitor cells suffices to impair growth in neighbouring wild-type cells through mitochondrial apoptosis. Competition is tightly restricted to a developmental transition stage between primed pluripotency and early gastrulation and involves gene regulatory networks of pluripotency exit. Heterochronic gastruloids from developmental stage-shifted cells, EpiGastruloids, and dynamic p53-degrons reveal that both winners and losers must reside within this permissive stage, during which acute relative p53 protein levels determine competitive outcomes. These findings advance our understanding of cell fitness evaluation and establish gastruloids as a powerful 3D model for investigating developmental stage-specific cell competition in mammalian embryogenesis.

Bladder cancer is a common urogenital malignancy that remains difficult to treat, particularly due to therapeutic resistance, such as resistance to cisplatin, in which cancer stem cells (CSCs) play a central role. This study investigates the combination of 5-aminolevulinic acid-mediated photodynamic therapy (5-ALA-PDT) and berbamine as a potential multimodal treatment strategy using the bladder cancer cell lines RT112 and J82, their cisplatin-resistant variants, and generated CSC-like cells. Berbamine is a natural plant compound and was confirmed in this study to have anticancer properties by inhibiting cell migration and invasion, and by inducing apoptosis. This study also showed that berbamine enhances the accumulation of protoporphyrin IX (PpIX), the photosensitizer induced by 5-ALA. 5-ALA-PDT destroys cancer cells by stimulating PpIX via 635 nm red laser light to produce reactive oxygen species (ROS). This was found to happen in all tested cell lines, whereas berbamine could modulate the cell destruction in a concentration-dependent manner and was influenced by the specific biological characteristics of the tested cell variants. CSCs showed the strongest response to the combination therapy approach, suggesting that they may represent more vulnerable cell variants to the tested treatment. Cisplatin-resistant cell lines could also be treated successfully with 5-ALA-PDT, whereas berbamine could enhance its efficacy in the cisplatin-resistant J82 LTT. These findings suggest that the combination treatment of 5-ALA-PDT and berbamine may serve as a promising approach to overcome therapeutic resistance in bladder cancer, particularly in cisplatin-resistant and CSC-enriched tumour types.