技术资料

The scarcity of reliable devices for diagnosis of Animal African trypanosomiasis (AAT) presents a limitation to control of the disease. Existing high-sensitivity technologies such as PCR are costly,laborious,time-consuming,complex,and require skilled personnel. Hence,utilisation of most diagnostics for AAT is impracticable in rural areas,where the disease occurs. A more accessible point-of-care test (POCT) capable of detecting cryptic active infection,without relying on expensive equipment,would facilitate AAT detection. In turn,early management,would reduce disease incidence and severity. Today,several ongoing research projects aim at modifying complex immunoassays into POCTs. In this context,we report the development of an antigen (Ag) detection sandwich ELISA prototype for diagnosis of T . congolense infections,which is comprised of nanobody (Nb) and monoclonal antibody (mAb) reagents. The Nb474H used here,originated from a past study. Briefly,the Nb was engineered starting from mRNA of peripheral blood lymphocytes of an alpaca immunized with soluble lysate of Trypanosoma congolense (TC13). T . congolense glycosomal fructose-1,6-bisphosphate aldolase ( Tco ALD) was discovered as the cognate Ag of Nb474H. In this study,splenocytes were harvested from a mouse immunized with recombinant Tco ALD and fused with NS01 cells to generate a hybridoma library. Random screening of the library on Tco ALD retrieved a lone binder,designated IgM8A2. Using Nb474H as Ag-capture reagent in combination with the IgM8A2 monoclonal antibody Ag-detection reagent resulted in a tool that effectively detects native Tco ALD released during infection by T . congolense parasites. Hitherto,development of POCT for detection of active trypanosome infection is elusive. The Nanobody/Monoclonal Antibody (Nb/mAb) “hybrid” sandwich technology offers prospects for exploration,using the unique specificity of Nb as a key determinant in Ag capturing,while using the versatility of monoclonal Ab to adapt to various detection conditions. 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

Current protocols generate highly pure human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet,a risk of arrhythmias exists when hiPSC‐CMs are injected into large animal models. Thus,understanding hiPSC‐CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC‐CM maturation. Here,we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC‐CM maturation in 3‐dimensional suspension culture. Three‐dimensional cultures of hiPSC‐CMs were treated with Nutlin‐3a (p53 activator,10 μM),LOM612 (FOXO relocator,5 μM),AS1842856 (FOXO inhibitor,1 μM),or RCM‐1 (FOXM1 inhibitor,1 μM),starting 2 days after onset of beating,with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC‐CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation,in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac‐specific markers such as TNNT2. In contrast,FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility,oxygen consumption,and voltage peak maximum upstroke velocity,in n=3 to 6 per group for all assays. Further,by single‐cell RNA sequencing of n=2 LOM612‐treated cells compared with dimethyl sulfoxide,LOM612‐mediated FOXO activation promoted expression of cardiac maturational pathways. We show that p53 activation promotes FOXO and suppresses FOXM1 during 3‐dimensional hiPSC‐CM maturation. These results expand our understanding of hiPSC‐CM maturational mechanisms in a clinically‐relevant 3‐dimensional culture system. 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

The development of haematopoiesis involves the coordinated action of numerous genes,some of which are implicated in haematological malignancies. However,the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here,we report a previously uncharacterised gene in haematopoiesis,identified by screening mutant embryonic stem cells. The gene,‘ attenuated haematopoietic development ( Ahed )’,encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward,leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed -deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model,we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways,and published databases present cancer patients with somatic mutations in Ahed . Collectively,our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis,implicating its association with malignancies. Subject terms: Lymphopoiesis,Development,Haematopoietic stem cells,Differentiation View Publication

Hypoxia-activated prodrug (HAP) is a promising candidate for highly tumor-specific chemotherapy. However,the oxygenation heterogeneity and dense extracellular matrix (ECM) of tumor,as well as the potential resistance to chemotherapy,have severely impeded the resulting overall efficacy of HAP. A HAP potentiating strategy is proposed based on ultrasound responsive nanodroplets (PTP@PLGA),which is composed of protoporphyrin (PpIX),perfluoropropane (PFP) and a typical HAP,tirapazamine (TPZ). The intense vaporization of PFP upon ultrasound irradiation can magnify the sonomechanical effect,which loosens the ECM to promote the penetration of TPZ into the deep hypoxic region. Meanwhile,the PpIX enabled sonodynamic effect can further reduce the oxygen level,thus activating the TPZ in the relatively normoxic region as well. Surprisingly,abovementioned ultrasound effect also results in the downregulation of the stemness of cancer cells,which is highly associated with drug-refractoriness. This work manifests an ideal example of ultrasound-based nanotechnology for potentiating HAP and also reveals the potential acoustic effect of intervening cancer stem-like cells. The online version contains supplementary material available at 10.1186/s12951-024-02623-0. View Publication

The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However,the systems-level effects of AS-NMD in this context are poorly understood. We developed an R package,factR2,which offers a comprehensive suite of AS-NMD analysis functions. Using this tool,we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably,this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes. The online version contains supplementary material available at 10.1186/s13059-024-03305-8. View Publication

Mononuclear phagocytes (MNP),including macrophages and dendritic cells form an essential component of primary responses to environmental hazards and toxic exposures. This is particularly important in disease conditions such as asthma and allergic airway disease,where many different cell types are present. In this study,we differentiated CD34+ haematopoietic stem cells towards different populations of MNP in an effort to understand how different cell subtypes present in inflammatory disease microenvironments respond to the common allergen house dust mite (HDM). Using single cell mRNA sequencing,we demonstrate that macrophage subtypes MC SPP1+ and MLC MARCO+ display different patterns of gene expression after HDM challenge,noted especially for the chemokines CXCL5,CXCL8,CCL5 and CCL15. MLC CD206Hi alternatively activated macrophages displayed the greatest changes in expression,while neutrophil and monocyte populations did not respond. Further work investigated how pollutant diesel exhaust particles could modify these transcriptional responses and revealed that CXC but not CC type chemokines were further upregulated. Through the use of diesel particles with adsorbed material removed,we suggest that soluble pollutants on these particles are the active constituents responsible for the modifying effects on HDM. This study highlights that environmental exposures may influence tissue responses dependent on which MNP cell type is present,and that these should be considerations when modelling such events in vitro. Understanding the nuanced responsiveness of different immune cell types to allergen and pollutant exposure also contributes to a better understanding of how these exposures influence the development and exacerbation of human disease. View Publication

Successful cell and gene therapy clinical trials have resulted in the US Food and Drug Administration and European Medicines Agency approving their use for treatment of patients with certain types of cancers and monogenetic diseases. These novel therapies,which rely heavily on lentiviral vectors to deliver therapeutic transgenes to patient cells,have driven additional investigations,increasing demand for both pre-clinical and current Good Manufacturing Practices-grade viral vectors. To better support novel studies by improving current production methods,we report the development of a genetically modified HEK293T-based cell line that is null for expression of both Protein Kinase R and Beta-2 microglobulin and grows in suspension using serum-free media,SJ293TS-DPB. Absence of Protein Kinase R increased anti-sense lentiviral vector titers by more than 7-fold,while absence of Beta-2 microglobulin,a key component of major histocompatibility complex class I molecules,has been reported to reduce the immunogenicity of lentiviral particles. Furthermore,we describe an improved methodology for culturing SJ293TS-DPB that facilitates expansion,reduces handling,and increases titers by 2-fold compared with previous methods. SJ293TS-DPB stably produced lentiviral vectors for over 4 months and generated lentiviral vectors that efficiently transduce healthy human donor T cells and CD34 + hematopoietic stem cells. View Publication

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group,respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1,7,14,28,35,42,49,56,and 63 posttransplantation. BLI signals were detected in all recipients,including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However,the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore,our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts,as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy. View Publication

Chronic obstructive pulmonary disease (COPD) is characterised by persistent airway inflammation even after cigarette smoking cessation. Neutrophil extracellular traps (NETs) have been implicated in COPD severity and acute airway inflammation induced by short-term cigarette smoke (CS). However,whether and how NETs contribute to sustained airway inflammation in COPD remain unclear. This study aimed to elucidate the immunoregulatory mechanism of NETs in COPD,employing human neutrophils,airway epithelial cells (AECs),dendritic cells (DCs),and a long-term CS-induced COPD mouse model,alongside cyclic guanosine monophosphate-adenosine monophosphate synthase and toll-like receptor 9 knockout mice ( cGAS -−/−,TLR9 −/− ); Additionally,bronchoalveolar lavage fluid (BALF) of COPD patients was examined. Neutrophils from COPD patients released greater cigarette smoke extract (CSE)-induced NETs (CSE-NETs) due to mitochondrial respiratory chain dysfunction. These CSE-NETs,containing oxidatively-damaged DNA (NETs-DNA),promoted AECs proliferation,nuclear factor kappa B (NF-κB) activation,NF-κB-dependent cytokines and type-I interferons production,and DC maturation,which were ameliorated/reversed by silencing/inhibition of cGAS/TLR9. In the COPD mouse model,blocking NETs-DNA-sensing via cGAS − /− and TLR9 − /− mice,inhibiting NETosis using mitoTEMPO,and degrading NETs-DNA with DNase-I,respectively,reduced NETs infiltrations,airway inflammation,NF-κB activation and NF-κB-dependent cytokines,but not type-I interferons due to IFN-α/β receptor degradation. Elevated NETs components (myeloperoxidase and neutrophil elastase activity) in BALF of COPD smokers correlated with disease severity and NF-κB-dependent cytokine levels,but not type-I interferon levels. In conclusion,NETs-DNA promotes NF-κB-dependent autoimmunity via cGAS/TLR9 in long-term CS exposure-induced COPD. Therefore,targeting NETs-DNA and cGAS/TLR9 emerges as a potential strategy to alleviate persistent airway inflammation in COPD. Subject terms: Inflammation,Respiratory tract diseases View Publication