The authors have withdrawn this preprint because the co-authors were not in agreement about posting the work in its current form. The authors request that this work not be cited. Please contact the corresponding author (Dr. Xunlei Kang, kangxu@health.missouri.edu ) with questions.

Chimeric antigen receptor T cell therapy (CAR-T) has revolutionized the treatment of hematologic malignancies, but its success in solid tumors, particularly those of the digestive system, remains limited. Tumors of the gastrointestinal system, including gastric, colorectal, esophageal, hepatic, and pancreatic malignancies, represent a significant global health burden with high morbidity and mortality. Recent advances in antigen selection, chimeric antigen receptor design, delivery techniques, and combinatorial approaches have sparked renewed interest in CAR-T immunotherapy for these cancers. This article discusses recent progress in CAR-T development across the major digestive system tumors, outlines tumor-specific targets and clinical trials, highlights prevailing challenges and potential solutions, and proposes strategic directions for the next generation of CAR-T therapies in solid tumors.

Leukaemic stem cells (LSCs) reside in protective bone marrow (BM) niches that promote therapeutic resistance and relapse. Here we characterized longitudinal BM niches supporting LSC survival, distinguishing the metaphysis from the central marrow. Quiescent LSCs preferentially localized to the metaphysis and exhibited reduced stemness and aggressiveness upon mobilization to the central marrow. Targeting DPP4 in acute myeloid leukaemia (AML) cells altered CXCL12 gradients at three spatial scales. Systemically, reversal of the BM-peripheral blood CXCL12 gradient confined AML cells within the BM. At the BM level, disruption of the metaphysis-central marrow gradient displaced LSCs from their protective niche. At the microscale, loss of the CXCL12 gradient between N-cadherin+ stromal cells and the surrounding matrix impaired LSC recruitment. These effects arise from the CXCL12-DPP4-GPC3 axis, in which DPP4 truncates and inactivates CXCL12, whereas stromal GPC3 restrains DPP4 activity. Modulating this axis disrupts niche protection and enhances therapeutic vulnerability in AML.

Pituitary Neuroendocrine Tumors (PitNETs) are the most frequently diagnosed intracranial neoplasms in adults. The World Health Organization’s 2022 fifth edition classification of pituitary neuroendocrine tumours (PitNETs) maintains an immunohistochemistry based taxonomy that places molecular biology at the centre of diagnosis. This framework provides a solid basis for subtype classification and therapy development. Methylation, defined as the transfer of a methyl group (CH₃) to DNA bases, histone side chains, or RNA nucleotides, is an epigenetic modification that has emerged as a key mechanism in neoplastic transformation. In this review, we synthesise current knowledge on histone, DNA, and RNA methylation in pituitary tumourigenesis, describing their distinct roles and mutual molecular crosstalk. By examining how these epigenetic modifications promote tumour initiation and progression, we assess their potential as drug targets and their translational applicability. Our objective is to propose new research directions and precision treatment strategies that exploit methylation related vulnerabilities, with the goal of improving clinical outcomes for patients with PitNETs.

Background: Acute myeloid leukemia (AML) is sustained by a rare population of leukemic stem cells (LSCs) that reside within protective bone marrow (BM) niches. These LSCs are responsible for disease propagation, therapy resistance, and relapse. While the endosteal and sinusoidal niches have been well characterized, the longitudinal spatial organization of the BM—particularly the distinction between metaphysis and central marrow—has not been systematically investigated in the context of AML. A better understanding of how spatial cues influence LSC behavior is critical for developing effective niche-targeted therapies. Methods: We utilized murine AML models (MLL-AF9 and AML1-ETO9a) with genetic manipulation of Dpp4, Cxcl12, and Gpc3, specifically in leukemic cells or niche-resident N-cadherin⁺ mesenchymal stromal cells (MSCs). Using flow cytometry, immunofluorescence microscopy, cytokine profiling, and functional transplantation assays, we evaluated the distribution, proliferation, apoptosis, and stemness of LSCs across different BM regions—proximal/distal metaphysis and central marrow. Single-cell RNA sequencing (scRNA-seq) was performed on non-hematopoietic stromal compartments from the metaphysis to identify Cxcl12-producing cells and niche-specific regulatory factors. Results: We found that LSCs preferentially localize to the proximal and distal metaphysis (PM/DM), where they exhibit quiescence and high leukemogenic potential. In contrast, LSCs in the central marrow (CM) display increased cell cycle activity and reduced self-renewal. Genetic deletion of Dpp4 in AML cells disrupted a previously unrecognized CXCL12 gradient across the BM, causing LSCs to redistribute from the metaphysis to the CM. This redistribution triggered increased proliferation and apoptosis, indicative of functional exhaustion. Notably, Dpp4 deletion also reversed the CXCL12 gradient between BM and peripheral blood, confining AML cells within the marrow and dramatically reducing peripheral dissemination. scRNA-seq analysis identified N-cadherin⁺ MSCs as the primary Cxcl12-expressing niche population within the metaphysis. These cells also express high levels of Glypican-3 (GPC3), a known DPP4 inhibitor. Mechanistically, GPC3 preserved CXCL12 activity locally by inhibiting DPP4 enzymatic degradation, establishing a localized CXCL12-rich environment that supports LSC maintenance. Conditional deletion of either Cxcl12 or Gpc3 in N-cadherin⁺ MSCs phenocopied the effects of Dpp4 loss, leading to LSC displacement, exhaustion, and impaired leukemogenesis. Transcriptomic profiling confirmed convergence of Dpp4-deficient and Cxcl12/Gpc3-depleted models, with shared downregulation of stemness-related pathways and upregulation of metabolic stress and cell death signatures. Conclusions: This study defines a novel spatial regulatory mechanism in AML, where the CXCL12–DPP4–GPC3 axis governs LSC localization between the metaphysis and central marrow. By disrupting this axis, LSCs are mobilized out of protective niches and driven toward exhaustion. Furthermore, reversing the CXCL12 gradient restricts AML cells to the marrow, reducing dissemination and prolonging survival. These findings not only uncover a previously underappreciated dimension of BM niche organization but also highlight DPP4 inhibition as a rational therapeutic approach to target the AML stem cell–niche interaction. Given that DPP4 inhibitors are already FDA-approved for other indications, these insights hold strong translational potential for rapid clinical application in AML.

Acute myeloid leukemia (AML) often enters remission after chemotherapy but frequently relapses due to chemotherapy-resistant leukemic stem cells (LSCs). Relapsed AML remains largely unresponsive to current therapies and carries a poor prognosis. We developed a large-language model (LLM) agent that incorporates multi-modal data to nominate druggable therapeutic targets for AML. We identified that higher expression of AGTR2 (encoding AT2R) is associated with better chemotherapy response and longer survival. In functional studies of 68 primary human AML samples, we found that LSCs consistently lacked AT2R expression. Across both CD34-expressing and -non-expressing AML samples, AT2R expression positively correlated with CD34 and CD117 expression. In patient-derived xenograft (PDX) models using 21 primary AML samples, AT2R⁻ cells initiated leukemia, whereas AT2R⁺ cells failed to do so. AT2R⁻ cells gave rise to both AT2R⁺ and AT2R⁻ progeny, suggesting hierarchical differentiation. Following chemotherapy in AML PDX mice, bone marrow analysis showed a marked enrichment of AT2R⁻ cells and depletion of AT2R⁺ cells, indicating that AT2R⁻ cells drive minimal residual disease and relapse. These results support the role of AT2R absence as a marker of LSCs. We observed reduced AT2R expression in AML cells compared to healthy peripheral blood and bone marrow mononuclear cells, suggesting a tumor suppressor role. Whole-genome sequencing of AML patients revealed no functional mutations in AGTR2 . However, 3D chromatin and epigenetic analyses uncovered frequent chromatin rearrangements involving AGTR2 promoter–silencer interactions, indicating epigenetic silencing as a likely mechanism for AT2R downregulation in AML. To validate the tumor suppressor role of AT2R, we developed murine AML models driven by MLL-AF9 or AML1-ETO9a fusions with either Agtr2 knockdown or enforced expression. Agtr2 knockdown accelerated leukemogenesis, while enforced Agtr2 expression delayed AML progression. In these models, enforced Agtr2 expression reduced LSC frequency, impaired cell cycle progression, and decreased AML stemness, as confirmed by limiting dilution assays and analysis of LSC-enriched populations. Mechanistically, enforced Agtr2 expression suppressed fatty acid metabolism – a key driver of AML stemness and growth – and inhibited downstream signaling pathways, including GSK3, PI3K/AKT, and Wnt/β-catenin. This led to reduced SREBF1 activity, confirmed by protein level changes and CUT&Tag assays. We tested buloxibutid (C21), a small-molecule AT2R agonist currently in phase II trials for idiopathic pulmonary fibrosis, in AML PDX models derived from 20 de novo and 6 relapsed AML samples. C21 significantly inhibited AML progression and enhanced the efficacy of chemotherapy, particularly in relapsed AML models.

Acute myeloid leukemia (AML) often achieves remission after therapy; however, relapse – driven by chemotherapy-resistant leukemic stem cells (LSCs) – remains a major cause of mortality. LSCs closely resemble hematopoietic stem cells (HSCs) in immunophenotype, making selective targeting difficult and highlighting an unmet need in AML therapy. To address this, we compared gene and protein expression profiles of LSCs and HSCs using public datasets and primary human AML samples across diverse subtypes. MILR1 (encoding Allergin-1) was consistently upregulated in LSC-enriched populations compared to non-LSC populations and HSPCs. In patient-derived xenograft (PDX) models, Allergin-1⁺ AML cells initiated leukemia, whereas Allergin-1⁻ cells did not. Knockdown of Allergin-1 significantly impaired leukemogenesis in PDX mice, establishing Allergin-1 as a functional LSC marker. Using murine models of MLL-AF9-driven AML, Milr1 knockout (KO) delayed leukemia progression and prolonged survival without affecting normal hematopoiesis. Transcriptomic analysis revealed two key effects of Milr1 loss: (1) reduced SHP-1/MAPK signaling, impairing AML stemness, and (2) enhanced natural killer (NK) cell-mediated immune activation. NK cells from Milr1-KO AML mice exhibited increased activation markers and cytotoxicity in co-culture assays, whereas T cell responses remained unchanged. This likely reflects NK cells' reliance on germline-encoded ligands, unlike T cells' dependence on neoantigen presentation. Surface proteomic analysis showed Milr1-KO AML cells exhibited decreased Lgals9 and Cdh2 and increased expression of MHC genes, Stamf7, and Fcgr1, indicating altered immunogenicity likely driven by SHP-1 suppression-induced cellular stress. To therapeutically target Allergin-1, we generated a panel of rabbit-derived monoclonal antibodies (mAbs). From 50 candidates, we identified a high-affinity mAb (Allergin-1 hAb) that antagonizes Allergin-1 activation. Molecular docking predicted that Allergin-1 hAb engages critical residues within the D1 domain of Allergin-1, blocking ligand interaction. In humanized PDX models, Allergin-1 hAb treatment significantly reduced AML burden compared to control IgG, supporting its therapeutic potential. Conclusion: Allergin-1 is a leukemia-specific functional marker that promotes stemness and immune evasion in AML. Targeting Allergin-1 with monoclonal antibodies represents a promising therapeutic strategy for AML treatment.

Chromatin remodeler chromodomain helicase DNA-binding protein 8 (CHD8) defines a subtype of autism that is associated with immune disorders. It remains unknown whether CHD8 plays a cell-intrinsic role in immune cells such as regulatory T cells (Tregs) that maintain immune tolerance through suppressing CD4+ and CD8+ effector T cells. Treg-specific conditional CHD8-deficient mice were generated by crossing Chd8Flox/Flox mice with Foxp3YFP−cre transgenic mice. Effects of CHD8 deficiency were investigated using hematoxylin and eosin (H E) staining, flow cytometry, and multi-omics, including RNA-sequencing (RNA-seq), assay for transposase-accessible chromatin sequencing (ATAC-seq), and chromatin immunoprecipitation sequencing (CHIP-seq). We found that Treg-specific CHD8 deletion led to early, fatal inflammation owing to increased CD4+ and CD8+ effector T cells. CHD8 deletion did not alter Treg homeostasis but increased their functional plasticity with elevated expression of effector T cell cytokines. CHIP-seq of Tregs uncovered that CHD8 binding genes were enriched in phosphatidylinositol-3 kinase (PI3K)–protein kinase B (Akt)–mammalian target of rapamycin (mTOR) signaling and several other pathways. RNA-seq and ATAC-seq revealed that CHD8 deletion upregulated a number of pathways, notably mammalian target of rapamycin complex 1 (mTORC1) signaling and its mediated glycolysis that have been reported to promote Treg plasticity. Integrating RNA-seq data with CHIP-seq and ATAC-seq data identified a number of CHD8 target genes whose expression depends on CHD8 direct binding-mediated chromatin remodeling. Our findings suggest that CHD8 plays an important role in maintaining Treg fitness through genetic and epigenetic mechanisms to control autoimmunity, which may have important implications in immune changes in autism.

Clonal hematopoiesis (CH), arising from hematopoietic progenitor cells with acquired mutations, resulting in clonal expansion, and displaying functionally altered hematopoietic cells, is traditionally associated with adverse health outcomes of numerous conditions. However, CH may not always be pathological - our study found that CH associated with DNMT3A and TP53 promotes tissue regeneration after acute injury, indicating the positive effects of CH. Genetics analysis of 125,966 patients revealed that individuals with TET2 and JAK2 CH show worse prognosis in acute cardiovascular diseases, while no significant association was observed among individuals with DNMT3A and TP53 mutations. In mouse models of myocardial infarction, hindlimb ischemia, and muscle injury, we utilized competitive bone marrow transplantation to mimic CH with Dnmt3a and Trp53 mutations. Mice with CH demonstrated enhanced tissue repair compared to controls. Single-cell RNA sequencing of recovering tissues and residing hematopoietic cells indicated that clonal hematopoiesis was associated with the presence of diverse hematopoietic cell populations exhibiting pro-regenerative functions. Notably, macrophages from CH mice showed increased expression of VEGF, contributing to improved tissue repair. Signaling analysis showed that suppressed E2F family transcription factors drive higher expression of Vegfa . These results suggest that CH may play a role in promoting recovery following ischemic injury and highlight its potential as a therapeutic strategy for improving outcomes in ischemic disease.

Clonal hematopoiesis (CH), arising from hematopoietic progenitor cells with acquired mutations, resulting in clonal expansion, and displaying functionally altered hematopoietic cells, is traditionally associated with adverse health outcomes of numerous conditions. However, CH may not always be pathological - our study found that CH promotes post-injury tissue regeneration, indicating the positive effects of CH. In mouse models of myocardial infarction, hindlimb ischemia, and muscle injury, we utilized competitive bone marrow transplantation to mimic CH with Trp53 mutation. Mice with CH demonstrated enhanced tissue repair compared to controls. Single-cell RNA sequencing of recovering tissues and residing hematopoietic cells indicated that mutant macrophages were associated with enhanced pro-angiogenic and phagocytic capability. Macrophages from CH mice showed increased expression of VEGF, contributing to improved tissue repair. Signaling analysis showed that suppressed E2F drives higher expression of Vegfa . These results suggest that CH may play a role in promoting recovery following ischemic injury and highlight its potential as a therapeutic strategy for improving outcomes in ischemic disease. ### Competing Interest Statement The authors have declared no competing interest.

Clinical treatment of acute myeloid leukemia (AML) largely relies on intensive chemotherapy. However, the application of chemotherapy is often hindered by cardiotoxicity. Patient sequence data revealed that angiotensin II receptor type 1 ( AGTR1 ) is a shared target between AML and cardiovascular disease (CVD). We found that inhibiting AGTR1 sensitized AML to chemotherapy and protected the heart against chemotherapy-induced cardiotoxicity in a human AML cell–transplanted mouse model. These effects were regulated by the AGTR1-Notch1 axis in AML cells and cardiomyocytes from mice. In mouse cardiomyocytes, AGTR1 was hyperactivated by AML and chemotherapy. AML leukemogenesis increased the expression of the angiotensin-converting enzyme and led to increased production of angiotensin II, the ligand of AGTR1, in an MLL-AF9–driven AML mouse model. In this model, the AGTR1-Notch1 axis regulated a variety of genes involved with cell stemness and chemotherapy resistance. AML cell stemness was reduced after Agtr1a deletion in the mouse AML cell transplant model. Mechanistically, Agtr1a deletion decreased γ-secretase formation, which is required for transmembrane Notch1 cleavage and release of the Notch1 intracellular domain into the nucleus. Using multiomics, we identified AGTR1-Notch1 signaling downstream genes and found decreased binding between these gene sequences with Notch1 and chromatin enhancers, as well as increased binding with silencers. These findings describe an AML/CVD association that may be used to improve AML treatment.

The processes that govern leukemia progression and remission are poorly understood. Our research reveals that the CXCL12 gradient, traditionally associated with LSC quiescence and survival, critically determines LSC localization and associated behavior. Specifically, CXCL12 guides LSCs to either the quiescent niche in the metaphysis, characterized by N-cadherin-expressing mesenchymal stromal cells (N-cad+ MSCs), or the proliferative niche in the central marrow (CM), marked by sinusoidal endothelial cells and associated stromal cells. We identified that the CXCL12 gradient is finely regulated by the interplay between dipeptidyl peptidase 4 (DPP4) on LSCs and glypican-3 (GPC3) on N-cad+ MSCs. DPP4 deactivates CXCL12, while GPC3 inhibits DPP4, resulting in a higher CXCL12 concentration in the metaphysis and a lower concentration in the CM. This differential gradient facilitates leukemia progression by promoting LSC quiescence and survival in the metaphysis versus relative proliferation and apoptosis in the CM. Depletion of Dpp4 from LSCs or Cxcl12 from N-cad+ MSCs disrupts this gradient, mobilizing LSCs from the metaphysis to the CM and significantly hindering leukemia development. Our findings redefine the role of CXCL12 in LSC behavior and provide a clearer understanding of leukemia progression. This novel insight highlights the potential for targeted therapeutic strategies that disrupt the CXCL12 gradient to treat minimal residual LSCs, offering a promising path toward a lasting cure for acute myeloid leukemia (AML). ### Competing Interest Statement The authors have declared no competing interest.

Introduction: Patients with acute myeloid leukemia (AML) frequently develop significant cardiac toxicity when receiving chemotherapy with poor prognosis, but the mechanisms remain largely unknown. Our in silico analysis revealed that angiotensin II receptor type 1 (AT1R) is abundantly expressed in AML and significantly increased in the blood of AML patients. It is known that AT1R is associated with the pathogenesis of cardiomyopathy. Hypothesis: We hypothesize that inhibiting AT1R can simultaneously suppress AML development and reduce chemotherapy-induced cardiotoxicity. Methods and Results: Inhibition of AT1R inhibition using specific AT1R inhibitor or AT1R knockout (KO) substantially reduced AML stemness and boosted chemotherapy efficacy for AML. Mechanistically, AT1R regulates γ-secretase activity, which is responsible for Notch1 transcription factor activation. AT1R KO effectively reduced the expressions of Notch1 downstream genes responsible for AML stemness, cell cycle,and proliferation. When Notch1 was re-introduced into AT1R KO AML cells, the expressions of these Notch1 downstream genes were rescued. Additionally, chromatin structure omics (HiC) and epigenetic omics (CUT&RUN)analysis revealed that 3D-chromatin structural and epigenetic alteration of multiple genes were associated with chemotherapy resistance. In fact, human AML patient derived xenograft model showedAT1R inhibition simultaneously boosted chemotherapy for AML while reducingchemotherapy-induced cardiotoxicity. Further mechanistic study demonstrated that the expression of angiotensin-converting enzyme was significantly increased in the cellsurface of AML with increased blood Ang II level in a mouse model, thus, may contributing to chemotherapy-induced cardiac toxicity. Conclusions: Together, these data demonstrated that AT1R-mediated signaling plays a key role in AML development and chemotherapy-induced cardiotoxicity. These findings indicate that the high incidence of cardiomyopathy in AML was increased AT1R-mediated signaling and targeting AT1R signaling could be a novel strategy for simultaneously suppressingleukemia, boosting chemotherapy efficacy, and preventing chemotherapy-inducedcardiotoxicity.

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings 1-5 . Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad) Hi Pdgfrα + mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cad Lo Albumin + hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs' reduction in the adult bone marrow (BM) 6,7 , depletion of Cxcl12 via Cdh2 CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

The increasing demand for immune cell applications, both in clinical settings and in research laboratories, has highlighted the critical need for cryopreservation (banking) methods for T cells. While conventional techniques such as freezing with liquid nitrogen remain prevalent, they pose significant challenges including high equipment costs, safety considerations, and logistical hurdles in transportation. Our cryopreservation medium, C80EZ®, represents a novel approach, leveraging biocompatible polysaccharides as cryoprotectants to enable safe storage at −80°C. This paper presents a comprehensive series of tests assessing the effectiveness of C80EZ® in shielding T cells from the detrimental effects of cryopreservation. Importantly, our findings demonstrate that C80EZ® not only ensures the survival of T cells, with a particular emphasis on preserving the CD8+ subsets, but also maintains their critical function in targeting and eliminating cancer cells.

As a transcription factor in the RUNT domain core-binding factor family, RUNX1 is crucial in multiple stages of hematopoiesis, and its mutation can cause familial platelet disorder with a predisposition to acute myeloid leukemia. Previous work has established that RUNX1 is involved in the maturation of megakaryocytes (MKs) and the production of platelets. Recent studies have shown that there exists a subpopulation of hematopoietic stem cells (HSCs) with relatively high expression of von Willebrand factor and CD41 at the apex of the HSC hierarchy, termed MK-HSCs, which can give rise to MKs without going through the traditional differentiation trajectory from HSC via MPP (multipotent progenitors) and MEP (megakaryocyte-erythroid progenitor). Here, by using Runx1F/FMx1-Cre mouse model, we discovered that the MK-HSC to MK direct differentiation can occur within 1 cell division, and RUNX1 is an important regulator in the process. Runx1 knockout results in a drastic decrease in platelet counts and a severe defect in the differentiation from MK-HSCs to MKs. Single cell RNA sequencing (RNAseq) analysis shows that MK-HSCs have a distinct gene expression signature compared with non-MK-HSCs, and Runx1 deletion alters the platelet and MK-related gene expression in MK-HSCs. Furthermore, bulk RNAseq and Cut&Run analyses show that RUNX1 binds to multiple essential MK or platelet developmental genes, such as Spi1, Selp, and Itga2b and regulates their expressions in MK-HSCs. Thus, by modulating the expression of MK-related genes, RUNX1 governs the direct differentiation from MK-HSCs to MKs and platelets.

Leukemic-stem-cell-specific targeting may improve the survival of patients with acute myeloid leukemia (AML) by avoiding the ablative effects of standard regimens on normal hematopoiesis. Herein, we perform an unbiased screening of compounds targeting cell surface proteins and identify clinically used DPP4 inhibitors as strong suppressors of AML development in both murine AML models and primary human AML cells xenograft model. We find in retrovirus-induced AML mouse models that DPP4-deficient AML cell-transplanted mice exhibit delay and reversal of AML development, whereas deletion of DPP4 has no significant effect on normal hematopoiesis. DPP4 activates and sustains survival of AML stem cells that are critical for AML development in both human and animal models via binding with Src kinase and activation of nuclear factor κB (NF-κB) signaling. Thus, inhibition of DPP4 is a potential therapeutic strategy against AML development through suppression of survival and stemness of AML cells.

CRISPR/Cas9 and associated base editing systems are used to precisely modify target genes in diverse cell types and organisms. However, CRISPR/Cas-induced mutants often contain small insertions and deletions (indels) or single nucleotide variations (SNVs) at edited sites with different genotypes, and gene editing frequency (GEF) typically varies between editing sites and editing systems. Therefore, methods are needed to identify mutants and their genotypes, and evaluate GEF. Herein, we describe a novel SMART approach for simultaneous analysis of CRISPR/Cas-induced mutants, genotypes and GEF, using DNA-guided Thermus thermophilus Argonaute (TtAgo) in vitro cleavage-mediated quantitative real-time PCR (qPCR) or digital PCR. SMART proved versatile for induced mutant identification, genotyping, and GEF evaluation using CRISPR/Cas9 gene-editing offspring lines harbouring the rice RNA-directed DNA methylation 3-like (RdDM3l) gene. SMART is higher specific, sensitive, and accurate than previously methods. SMART detected all types of mutations, including small indels, large indels and nucleotide substitutions, with good sensitivity regardless of mutation type. TtAgo has few strict requirements or restrictions for target sequences, such as restriction sites, GC content or protospacer-adjacent motifs (PAMs). SMART facilitates versatile CRISPR/Cas-induced mutant screening, genotyping, and GEF quantification, and it could be developed for clinical detection of rare mutations involving SNVs and indels.

Background:Gliomas are the most common malignant tumors of the central nervous system. However, the inherited genetic variation in gliomas is presently unclear. Therefore, this study investigated the association of the rs2071559 and rs2239702 gene polymorphisms with glioma susceptibility in Chinese patients.Methods:In this study, a case-control approach was used to compare and analyze whether two genes, rs2071559 and rs2239702, were associated with the risk of glioma formation.Results:The cases and controls were matched for sex, smoking status, and family history of cancer using single nucleotide polymorphisms. Specific rs2071559 and rs2239702 alleles were found much more frequently in the glioma group than in the control group (P < 0.001 and P = 0.014, respectively).Conclusions:These findings suggest that specific rs2071559 and rs2239702 polymorphisms are associated with a higher risk of glioma development; the risk allele is C in rs2071559 or A in rs2239702. Moreover, the kinase-insert-domain-containing receptor may act as a suppressor of tumor progression.

BACKGROUND:Hematopoietic stem cell (HSC) therapy has shown promise for tissue regeneration after ischemia. Therefore, there is a need to understand mechanisms underlying endogenous HSCs activation in response to ischemic stress and coordination of angiogenesis and repair. SHP-1 plays important roles in HSC quiescence and differentiation by regulation of TGF-β1 signaling. TGF-β1 promotes angiogenesis by stimulating stem cells to secrete growth factors to initiate the formation of blood vessels and later aid in their maturation. We propose that SHP-1 responds to ischemia stress in HSC and progenitor cells (HSPC) via regulation of TGF-β1.METHODS:A mouse hind limb ischemia model was used. Local blood perfusion in the limbs was determined using laser doppler perfusion imaging. The number of positive blood vessels per square millimeter, as well as blood vessel diameter (μm) and area (μm2), were calculated. Hematopoietic cells were analyzed using flow cytometry. The bone marrow transplantation assay was performed to measure HSC reconstitution.RESULTS:After femoral artery ligation, TGF-β1 was initially decreased in the bone marrow by day 3 of ischemia, followed by an increase on day 7. This pattern was opposite to that in the peripheral blood, which is concordant with the response of HSC to ischemic stress. In contrast, SHP-1 deficiency in HSC is associated with irreversible activation of HSPCs in the bone marrow and increased circulating HSPCs in peripheral blood following limb ischemia. In addition, there was augmented auto-induction of TGF-β1 and sustained inactivation of SHP-1-Smad2 signaling, which impacted TGF-β1 expression in HSPCs in circulation. Importantly, restoration of normal T GF-β1 oscillations helped in the recovery of limb repair and function.CONCLUSIONS:HSPC-SHP-1-mediated regulation of TGF-β1 in both bone marrow and peripheral blood is required for a normal response to ischemic stress.