技术资料

Variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes can confound the diagnosis and treatment of suspected Lynch syndrome (LS) patients. To aid the reclassification of VUS,we developed the in cellulo analysis of MMR variants (inCAMA) and used known control variants to calibrate this assay such that results can be readily applied as functional evidence by expert classification panels. We used CRISPR gene engineering to introduce known pathogenic and benign variants into the MSH6 or MSH2 loci in human embryonic stem cells and assessed their effects on cellular MMR repair and damage response functions. Our functional assay successfully discerned known pathogenic and benign variants. Using these results and performing a linear regression analysis with available odds of pathogenicity scores for the known calibration variants,we created equations that can generate a functional odds of pathogenicity score for any future MSH6 or MSH2 variant tested. In summary,inCAMA represents a new,calibrated assay for testing the function of virtually any MSH6 or MSH2 variant. The conversion of assay results directly into odds of pathogenicity scores makes it possible to use any PS3 or BS3 evidence strength level toward the reclassification of VUS. View Publication

ABSTRACTExpression of SRY-box transcription factor 17 (Sox17) in the endodermal region caudal to the hepatic diverticulum during late gastrulation is necessary for hepato-pancreato-biliary system formation. Analysis of an allelic series of promoter-proximal mutations near the transcription start site (TSS) 2 of Sox17 in mouse has revealed that gallbladder (GB) and extrahepatic bile duct (EHBD) development is exquisitely sensitive to Sox17 expression levels. Deletion of a SOX17-binding cis-regulatory element in the TSS2 promoter impairs GB and EHBD development by reducing outgrowth of the nascent biliary bud. These findings reveal the existence of a SOX17-dependent autoregulatory loop that drives Sox17 expression above a critical threshold concentration necessary for GB and EHBD development to occur,and that minor impairments in Sox17 gene expression are sufficient to impair the expression of SOX17-regulated genes in the nascent GB and EHBD system,impairing or preventing development. Summary: A SOX17-binding cis-regulatory element in transcription start site 2 of Sox17 forms a positive autoregulatory feedback loop necessary for gallbladder and extra-hepatic bile duct formation. View Publication

Eicosanoids are key players in inflammatory diseases and cancer. Targeting their production by inhibiting Group IVA cytosolic phospholipase A2 (cPLA2α) offers a promising approach for cancer therapy. In this study,we synthesize a second generation of thiazolyl ketone inhibitors of cPLA2α starting with compound GK470 (AVX235) and test their in vitro and cellular activities. We identify a more potent and selective lead molecule,GK420 (AVX420),which we test in parallel with AVX235 and a structurally unrelated compound,AVX002 for inhibition of cell viability across a panel of cancer cell lines. From this,we show that activity of polycomb group repressive complex 2 is a key molecular determinant of sensitivity to cPLA2α inhibition,while resistance depends on antioxidant response pathways. Consistent with these results,we show that elevated intracellular reactive oxygen species and activating transcription factor 4 target gene expression precede cell death in AVX420-sensitive T-cell acute lymphoblastic leukemia cells. Our findings imply cPLA2α may support cancer by mitigating oxidative stress and inhibiting tumor suppressor expression and suggest that AVX420 has potential for treating acute leukemias and other cancers that are susceptible to oxidative cell death. Inhibition of group IVA cytosolic phospholipase A2 (cPLA2α) is a potential approach for cancer treatment. Here,the authors report thiazolyl ketone inhibitors of cPLA2α starting from compound GK470 (AVX235),and the discovery of a more potent and selective lead,GK420 (AVX420),with significant chemotherapeutic properties. View Publication

Background:Neural stem cells (NSCs) disrupt with aging,contributing to neurodegeneration. Ginsenoside Rg1 (Rg1),a compound found in Ginseng,is known for its anti-aging effects; however,its role in the progression of aging NSCs remains unclear. Therefore,this investigation explored the impact of Rg1 on the growth and maturation of aging NSC and elucidated its underlying molecular mechanisms.Methods:Initially,mouse models of brain aging were successfully established using D-galactose (D-gal) injection. Mice received Rg1 treatment along with D-gal administration. Brain tissues and NSCs were isolated and analyzed for pathological changes,gene expression,and cellular function. In vitro,experiments used aging NSCs treated with Rg1 to assess cell viability,proliferation,differentiation,and senescence markers. Results:D-gal triggered aging-related pathological alterations in mouse brains,elevated acetylcholinesterase levels,upregulated senescence genes,and inhibited NSC proliferation (p < 0.05). However,Rg1 treatment mitigated D-gal-induced effects,delayed brain aging,and improved NSC function. In vitro,Rg1 significantly increased cell viability,promoted NSC proliferation and differentiation,reduced senescent neurons,and downregulated p53 and p21 genes (p < 0.05). Conclusions:Rg1 demonstrates anti-aging properties in D-gal-induced mouse brain aging,promoting the proliferation and differentiation of NSCs,and downregulating the p53-p21 signaling pathway. View Publication

AbstractNeuroinflammation impacts on the progression of amyotrophic lateral sclerosis (ALS),a fatal neurodegenerative disorder. Specialized pro-resolving mediators trigger the resolution of inflammation. We investigate the specialized pro-resolving mediator blood profile and their receptors’ expression in peripheral blood mononuclear cells in relation to survival in ALS. People living with ALS (pwALS) were stratified based on bulbar versus limb onset and on key progression metrics using a latent class model,to separate faster progressing from slower progressing ALS. Specialized pro-resolving mediator blood concentrations were measured at baseline and in one additional visit in 20 pwALS and 10 non-neurological controls (Cohort 1). Flow cytometry was used to study the GPR32 and GPR18 resolvin receptors’ expression in peripheral blood mononuclear cells from 40 pwALS and 20 non-neurological controls (Cohort 2) at baseline and in two additional visits in 17 pwALS. Survival analysis was performed using Cox proportional hazards models,including known clinical predictors and GPR32 and GPR18 mononuclear cell expression. Differential expression and linear discriminant analyses showed that plasma resolvins were able to distinguish phenotypic variants of ALS from non-neurological controls. RvE3 was elevated in blood from pwALS,whilst RvD1,RvE3,RvT4 and RvD1n-3 DPA were upregulated in A-S and RvD2 in A-F. Compared to non-neurological controls,GPR32 was upregulated in monocytes expressing the active inflammation-suppressing CD11b+ integrin from fast-progressing pwALS,including those with bulbar onset disease (P < 0.0024),whilst GPR32 and GPR18 were downregulated in most B and T cell subtypes. Only GPR18 was upregulated in naïve double positive Tregs,memory cytotoxic Tregs,senescent late memory B cells and late senescent CD8+ T cells from pwALS compared to non-neurological controls (P < 0.0431). Higher GPR32 and GPR18 median expression in blood mononuclear cells was associated with longer survival,with GPR32 expression in classical monocytes (hazard ratio: 0.11,P = 0.003) and unswitched memory B cells (hazard ratio: 0.44,P = 0.008) showing the most significant association,along with known clinical predictors. Low levels of resolvins and downregulation of their membrane receptors in blood mononuclear cells are linked to a faster progression of ALS. Higher mononuclear cell expression of resolvin receptors is a predictor of longer survival. These findings suggest a lipid-mediated neuroprotective response that could be harnessed to develop novel therapeutic strategies and biomarkers for ALS. Yildiz et al. describe the blood profile of specialized lipid mediators involved in inflammation resolution and of GPR32/GPR18 membrane receptors in blood mononuclear cells from patients with amyotrophic lateral sclerosis with a fast and slow rate of disease progression. Plasma resolvins were able to distinguish phenotypic variants of amyotrophic lateral sclerosis,and higher GPR32/GPR18 median expression was associated with longer survival. Graphical Abstract Graphical Abstract View Publication

Genomic imprinting is required for sexual reproduction and embryonic development of mammals,in which,differentially methylated regions (DMRs) regulate the parent-specific monoallelic expression of imprinted genes. Numerous studies on imprinted genes have highlighted their critical roles in development. However,what imprinting network is essential for development is still unclear. Here,we establish a stepwise system to reconstruct a development-related imprinting network,in which diploid embryonic stem cells (ESCs) are derived by fusing between parthenogenetic (PG)- and androgenetic (AG)-haploid embryonic stem cells (haESCs) with different DMR deletions (termed Ha-Ha-fusion system),followed by tetraploid complementation to produce all-haESC fetuses. Diploid ESCs fused between PG-haESCs carrying 8 maternally-derived DMR deletions and AG-haESCs with 2 paternally-derived DMR deletions give rise to live pups efficiently,among which,one lives to weaning. Strikingly,diploid ESCs derived from the fusion of PG-haESCs with 7 maternal DMR deletions and AG-haESCs with 2 paternal DMR deletions and maternal Snrpn-DMR deletion also support full-term embryonic development. Moreover,embryos reconstructed by injection of AG-haESCs with hypomethylated H19-DMR into oocytes with H19-DMR deletion develop into live mice sustaining inverted allelic gene expression. Together,our findings indicate that restoration of monoallelic expression of 10 imprinted regions is adequate for the full-term development of all-haESC pups,and it works irrespective of their parental origins. Meanwhile,Ha-Ha-fusion system provides a useful tool for deciphering imprinting regulation networks during embryonic development. View Publication

The impact of chirality on immune response has attracted great interest in cancer vaccine research recently. However,the study of chiral synthetic polypeptide hydrogels as cancer vaccines as well as of the impact of biomaterials themselves for antitumor immunotherapy has rarely been reported. Here,we show the key role of residue chirality of polypeptide hydrogels in antitumor immunity and local immune microenvironment regulation. Compared to poly(γ-ethyl-L-glutamate)-based hydrogels (L-Gel),poly(γ-ethyl-D-glutamate)-based hydrogels (D-Gel) induces enhanced level of immune cell infiltration. However,D-Gel causes higher levels of suppressive markers on antigen-presenting cells and even induces stronger T cell exhaustion than L-Gel. Finally,D-Gel establishes a local chronic inflammatory and immunosuppressive microenvironment and shows insufficient anti-tumor effects. Conversely,the milder host immune responses induced by L-Gel leads to more effective tumor inhibition. This study provides insights on the role of residue chirality in the regulation of local immune microenvironment and affecting antitumor immune response. The impact of chemical chirality on immune response attracts attention in cancer vaccine design recently. Here this group reports the chirality of poly(γ-ethyl-D-glutamate)-based hydrogel exhibiting higher levels of suppression on antigen-presenting cells and inducing stronger T cell exhaustion than L-Gel eventually leading to insufficient anti-tumor efficacy. View Publication

Stem cells are special cells with the distinctive capability to self-renew,forming a new pool of undifferentiated stem cells. They are also able to differentiate into lineage-specific cell types that are specialized and matured. Thus,stem cells are considered as the building blocks of tissues and organs in which they reside. Among the many types of stem cells,hematopoietic stem cells (HSCs) are the most studied adult stem cells and are considered as a promising source of cells for applications in the clinical and basic sciences. Historically,research on HSCs was initiated in the 1940s,where in a groundbreaking experiment,intravenously injected bone marrow (BM) cells prevented the death of irradiated mice by restoring blood cell production. Since then,HSCs have been studied and utilized in medical therapies and research for over several decades. Over time,more sophisticated tools have been developed to evaluate the behaviour of specifically purified subsets of hematopoietic cells that have the capacity to produce blood cells. One of the established tools is the colony-forming units (CFUs) assay. This assay facilitates the identification,enumeration,and analysis of colonies formed by differentiated hematopoietic stem and progenitor cells (HSPCs) from myeloid,erythroid and lymphoid lineages. Hence,the CFUs assay is a fundamental in vitro platform that allows functional studies on the lineage potential of an individual HSPCs. The outcomes of such studies are crucial in providing critical insights into hematopoiesis. In this review,we explore the fundamental discoveries concerning the CFUs assay by covering the following aspects: (i) the historical overview of the CFUs assay for the study of clonal hematopoiesis involving multilineage potential of HSPCs,(ii) its use in various experimental models comprising humans,mice/rodents,zebrafish and induced pluripotent stem cells (iPSCs) and (iii) research gaps and future direction concerning the role of CFUs assay in clinical and basic sciences. Overall,the CFUs assay confers a transformative platform for a better understanding of HSPCs biology in governing hematopoiesis. View Publication

Cystic diseases,especially autosomal dominant polycystic kidney disease (ADPKD; incidence approx. 1/1000),are a leading cause of renal failure,caused by appearance and growth of renal cysts that can lead to renal failure in middle age. Most ADPKD cases are caused by mutations in PKD1 or PKD2,encoding polycystin-1 (PC1) and polycystin-2 (PC2). PC1 is a mechanosensor that controls PC2,a Ca2+-permeable cation channel that,by regulating cytoplasmic Ca2+,prevents adenylyl cyclase producing cyst-promoting concentrations of cAMP. In other systems,there is evidence that PC2 interacts with TRPM3. We therefore examined the effect of pharmacological activators and inhibitors of TRPM3 on cyst formation in cultured mouse kidney rudiments exposed to a range of concentrations of forskolin,a cAMP-elevating drug commonly used experimentally to induce cysts in cultured kidneys. We found that TRPM3 inhibitors (isosakuranetin,primidone,diclofenac) increased cyst formation,while TRPM3 activators (CIM0216 and nifedipine) greatly reduced cyst formation and reduced the sensitivity of kidneys to forskolin. These preclinical,in-vitro data suggest that TRPM3 may be a promising target in ADPKD management. View Publication

Hyalocytes are macrophage-like cells residing in the eye vitreous cortex. Even though hyalocytes have been firstly described in the mid-Nineteenth century,they have been poorly explored. Recent researches highlighted hyalocyte involvement in both physiological and pathological processes of the vitreoretinal interface. Nonetheless,the majority of works involving hyalocyte cultures were carried out in animals,while fewer studies were performed on humans because their isolation requires vitrectomy. The aim of this study was to differentiate human induced pluripotent stem cells (iPSCs) into hyalocytes as a non-invasive method to continuously obtain cells. iPSCs were first differentiated into hematopoietic stem/progenitor cells (HSPCs) and then into macrophages. Macrophages were either left untreated (NT) or treated with ascorbic acid (AA) alone or combined with bFGF and/or TGF-β1. Additionally,macrophages were cultured in the presence of a pool of vitreous bodies from vitrectomies. Cells were analyzed for morphology and then for gene and protein expression through qRT-PCR,immunofluorescence,Western Blot,and flow cytometry. Similar to cells treated with the vitreous body,macrophages treated with AA alone or in combination with bFGF exhibited a more elongated shape compared to NT or cells treated with TGF-β1. Additionally,these treatments resulted in gene expression downregulation for S100A4,S100A10,S100B,and CX3CR1,while upregulating COL6A1,HLA-DRA,and CD74. At the protein level,S100B,CD14,and CD49d were downregulated with all treatments,while collagen VI and HLA-DR were upregulated. This work demonstrates that hyalocytes can be differentiated by treatment of iPSC-derived macrophages with ascorbic acid for a period of 21 days.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13577-025-01182-2. View Publication

BackgroundCytokine induced memory-like natural killer (CIML NK) cells combined with an IL-15 super-agonist (N-803) are a novel modality to treat relapsed/refractory head and neck cancer.MethodsWe report data from a phase I trial of haploidentical CIML NK cells combined with N-803 with or without ipilimumab (IPI) in relapsed/refractory head and neck cancer patients after a median of 6 prior lines of therapy. The trial adhered to a 3 + 3 dose de-escalation design,with primary endpoint being safety. High-resolution immunophenotypic and transcriptional profiling characterized the NK cells and their interacting partners in vivo.ResultsThe primary safety endpoint was established,with dose-limiting toxicity in 1/10 patients. A transient disease control rate correlated with donor NK cell expansion,the latter occurring irrespective of IPI. The combination of CIML NK cells with N-803 and IPI was associated with increased early NK cell proliferation,contraction of Treg: Tcon,rapid recovery of recipient CD8+ T cells,and subsequent accelerated rejection of donor NK cells.ConclusionsCIML NK cells combined with N-803 and ipilimumab to treat head and neck cancer is safe,and associated with a more proliferative NK cell phenotype. However,the combination leads to reduced HLA mismatched NK cell persistence,resulting in an important limitation affecting NK cell combination therapies in clinical trials. These results inform evaluation of CIML NK therapy for advanced malignancies,with considerations for combination with IPI.Trial RegistrationNCT04290546.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13045-025-01669-3. View Publication

BackgroundCartilage injuries pose significant challenges in horses and often lead to post-traumatic osteoarthritis (PTOA). Despite the advances in surgical and regenerative techniques,the result in most cases is the formation of a fibrocartilage repair tissue. Cell-based cartilage therapies are mainly focused on equine bone marrow-derived mesenchymal stem cells (eBMSCs) as they are easily accessible,and multipotent. Nonetheless,alternative allogeneic sources,for example equine umbilical cord matrix mesenchymal stromal cells (eUCMSCs),hold promise given their non-invasive and readily accessible nature. Considerable research has been dedicated to exploring chondroinductive factors (e.g.,peptides and small compounds),aiming to replace growth factors for inducing chondrogenesis. However,these factors have not yet translated to the equine community. Therefore,in the current study,we selected from the literature two promising peptides,CM10 and CK2.1,and two promising compounds,kartogenin and SM04690,and assessed their chondroinductive potential with both eBMSCs and eUCMSCs. In addition,the chondroinductive potential of eBMSCs was evaluated in monolayer and spheroid culture in both hypoxia and normoxia in response to dexamethasone and/or transforming growth factor beta 3 (TGF-β3).ResultsFollowing 21 days of culture,none of the evaluated chondrogenic factors resulted in a higher gene expression of chondrogenic markers compared to the positive or negative controls with eBMSCs or eUCMSCs. Interestingly,spheroid culture in hypoxia with dexamethasone treatment (without TGF-β or any compound or peptide) was sufficient to induce the chondrogenic differentiation of eBMSCs.ConclusionBased on cell response to the positive control,in the conditions employed in the current study,eBMSCs may be preferred over eUCMSCs for chondrogenesis. The current study supports the use of spheroid culture,and the use of dexamethasone over TGF-β or any of the compounds or peptides tested here from the prior literature to drive chondrogenesis with eBMSCs. View Publication