技术资料

Mucus stasis is a pathologic hallmark of muco-obstructive diseases,including cystic fibrosis (CF). Mucins,the principal component of mucus,are extensively modified with hydroxyl (O)-linked glycans,which are largely terminated by sialic acid. Sialic acid is a negatively charged monosaccharide and contributes to the biochemical/biophysical properties of mucins. Reports suggest that mucin sialylation may be altered in CF; however,the consequences of reduced sialylation on mucus clearance have not been fully determined. Here,we investigated the consequences of reduced sialylation on the charge state and conformation of the most prominent airway mucin,MUC5B,and defined the functional consequences of reduced sialylation on mucociliary transport (MCT). Reduced sialylation contributed to a lower charged MUC5B form and decreased polymer expansion. The inhibition of total mucin sialylation de novo impaired MCT in primary human bronchial epithelial cells and rat airways,and specific α-2,3 sialylation blockade was sufficient to recapitulate these findings. Finally,we show that ST3 beta-galactoside alpha-2,3-sialyltransferase (ST3Gal1) expression is downregulated in CF and partially restored by correcting CFTR via Elexacaftor/Tezacaftor/Ivacaftor treatment. Overall,this study demonstrates the importance of mucin sialylation in mucus clearance and identifies decreased sialylation by ST3Gal1 as a possible therapeutic target in CF and potentially other muco-obstructive diseases. Subject terms: Biophysical chemistry,Glycobiology,Respiration View Publication

Human iPSC-derived cardiomyocytes (hiPSC-CMs) have proven invaluable for cardiac disease modeling and regeneration. Challenges with quality,inter-batch consistency,cryopreservation and scale remain,reducing experimental reproducibility and clinical translation. Here,we report a robust stirred suspension cardiac differentiation protocol,and we perform extensive morphological and functional characterization of the resulting bioreactor-differentiated iPSC-CMs (bCMs). Across multiple different iPSC lines,the protocol produces 1.2E6/mL bCMs with ~94% purity. bCMs have high viability after cryo-recovery (>90%) and predominantly ventricular identity. Compared to standard monolayer-differentiated CMs,bCMs are more reproducible across batches and have more mature functional properties. The protocol also works with magnetically stirred spinner flasks,which are more economical and scalable than bioreactors. Minor protocol modifications generate cardiac organoids fully in suspension culture. These reproducible,scalable,and resource-efficient approaches to generate iPSC-CMs and organoids will expand their applications,and our benchmark data will enable comparison to cells produced by other cardiac differentiation protocols. Subject terms: Cardiovascular biology,Induced pluripotent stem cells,Cardiovascular models View Publication

Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features,we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT),a specialized DNA polymerase intrinsic to VDJ recombination,broadly expressed within CD34 + progenitors prior to B/T cell emergence. While these TdT + cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype,their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84 lo GMPs demonstrates robust lymphoid potentials ex vivo,while still retaining significant myeloid differentiation capacity,akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors,further defining the lympho-myeloid axis in human hematopoiesis. Subject terms: Lymphopoiesis,Systems analysis,Proteomic analysis,Myelopoiesis View Publication

Acute myeloid leukaemia (AML) is a disease of the haematopoietic stem cells(HSCs) that is characterised by the uncontrolled proliferation and impaired differentiation of normal haematopoietic stem/progenitor cells. Several pathways that control the proliferation and differentiation of HSCs are impaired in AML. Activation of the Wnt/beta-catenin signalling pathway has been shown in AML and beta-catenin,which is thought to be the key element of this pathway,has been frequently highlighted. The present study was designed to determine beta-catenin expression levels and beta-catenin-related genes in AML. In this study,beta-catenin gene expression levels were determined in 19 AML patients and 3 controls by qRT-PCR. Transcriptome analysis was performed on AML grouped according to beta-catenin expression levels. Differentially expressed genes(DEGs) were investigated in detail using the Database for Annotation Visualisation and Integrated Discovery(DAVID),Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG),STRING online tools. The transcriptome profiles of our AML samples showed different molecular signature profiles according to their beta-catenin levels(high-low). A total of 20 genes have been identified as hub genes. Among these,TTK,HJURP,KIF14,BTF3,RPL17 and RSL1D1 were found to be associated with beta-catenin and poor survival in AML. Furthermore,for the first time in our study,the ELOV6 gene,which is the most highly up-regulated gene in human AML samples,was correlated with a poor prognosis via high beta-catenin levels. It is suggested that the identification of beta-catenin-related gene profiles in AML may help to select new therapeutic targets for the treatment of AML. View Publication

Autoimmune thyroid disease (AITD) is a common autoimmune disease. In a GWAS meta-analysis of 110,945 cases and 1,084,290 controls,290 sequence variants at 225 loci are associated with AITD. Of these variants,115 are previously unreported. Multiomics analysis yields 235 candidate genes outside the MHC-region and the findings highlight the importance of genes involved in T-cell regulation. A rare 5’-UTR variant (rs781745126-T,MAF = 0.13% in Iceland) in LAG3 has the largest effect (OR = 3.42,P = 2.2 × 10 −16 ) and generates a novel start codon for an open reading frame upstream of the canonical protein translation initiation site. rs781745126-T reduces mRNA and surface expression of the inhibitory immune checkpoint LAG-3 co-receptor on activated lymphocyte subsets and halves LAG-3 levels in plasma among heterozygotes. All three homozygous carriers of rs781745126-T have AITD,of whom one also has two other T-cell mediated diseases,that is vitiligo and type 1 diabetes. rs781745126-T associates nominally with vitiligo (OR = 5.1,P = 6.5 × 10 −3 ) but not with type 1 diabetes. Thus,the effect of rs781745126-T is akin to drugs that inhibit LAG-3,which unleash immune responses and can have thyroid dysfunction and vitiligo as adverse events. This illustrates how a multiomics approach can reveal potential drug targets and safety concerns. Subject terms: Genetics research,Disease genetics,Thyroid diseases,Genome-wide association studies,Gene expression View Publication

DNA methylation plays an important role in various biological processes,including cell differentiation,ageing,and cancer development. The most important methylation in mammals is 5-methylcytosine mostly occurring in the context of CpG dinucleotides. Sequencing methods such as whole-genome bisulfite sequencing successfully detect 5-methylcytosine DNA modifications. However,they suffer from the serious drawbacks of short read lengths and might introduce an amplification bias. Here we present Rockfish,a deep learning algorithm that significantly improves read-level 5-methylcytosine detection by using Nanopore sequencing. Rockfish is compared with other methods based on Nanopore sequencing on R9.4.1 and R10.4.1 datasets. There is an increase in the single-base accuracy and the F1 measure of up to 5 percentage points on R.9.4.1 datasets,and up to 0.82 percentage points on R10.4.1 datasets. Moreover,Rockfish shows a high correlation with whole-genome bisulfite sequencing,requires lower read depth,and achieves higher confidence in biologically important regions such as CpG-rich promoters while being computationally efficient. Its superior performance in human and mouse samples highlights its versatility for studying 5-methylcytosine methylation across varied organisms and diseases. Finally,its adaptable architecture ensures compatibility with new versions of pores and chemistry as well as modification types. Subject terms: Genome informatics,Epigenomics,Computational models,DNA sequencing,DNA methylation View Publication

p47 phox -deficient chronic granulomatous disease (p47-CGD) is a primary immunodeficiency caused by mutations in the neutrophil cytosolic factor 1 ( NCF1 ) gene,resulting in defective NADPH oxidase function in phagocytes. Due to its complex genomic context,the NCF1 locus is not suited for safe gene editing with current genome editing technologies. Therefore,we developed a targeted NCF1 coding sequence knock-in by CRISPR-Cas9 ribonucleoprotein and viral vector template delivery,to restore p47 phox expression under the control of the endogenous NCF2 locus. NCF2 encodes for p67 phox,an NADPH oxidase subunit that closely interacts with p47 phox and is predominantly expressed in myeloid cells. This approach restored p47 phox expression and NADPH oxidase function in p47-CGD patient hematopoietic stem and progenitor cells (HSPCs) and in p47 phox -deficient mouse HSPCs,with the transgene expression following a myeloid differentiation pattern. Adeno-associated viral vectors performed favorably over integration-deficient lentiviral vectors for template delivery,with fewer off-target integrations and higher correction efficacy in HSPCs. Such myeloid-directed gene editing is promising for clinical CGD gene therapy,as it leads to the co-expression of p47 phox and p67 phox,ensuring spatiotemporal and near-physiological transgene expression in myeloid cells. View Publication

RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA-chromatin interactions remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast,small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note,snoRNA-chromatin interaction was associated with chromatin modifications and occurred independently of the classical snoRNA-RNP complex. Two C/D box snoRNAs,namely SNORD118 and SNORD3A,displayed high frequency of trans -association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells,but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and primary patient samples. Notably,this effect was leukemia specific with less impact on healthy CD34+ hematopoietic stem and progenitor cells. These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation. Subject terms: Acute myeloid leukaemia,Cancer epigenetics View Publication

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise,with a negligible affinity for mismatched substrates,but its low cellular targeting efficiency limits therapeutic use. Here,we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency,knock-in rates,and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally,we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor,highlighting its therapeutic utility. Subject terms: CRISPR-Cas9 genome editing,Molecular medicine,Genetic engineering,CRISPR-Cas9 genome editing View Publication

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy,contingent upon the species is crucial to circumvent transgene silencing,necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly,transgene silencing occurred while using the CAG promoter,contrary to conventional understanding,whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations,confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally,GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats,thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2,mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations,with implications for future research in gene-engineered rat models. The online version contains supplementary material available at 10.1186/s12917-024-04123-7. View Publication