Search Results (49)

Conventional methods for generating knock-out or knock-in mammalian cell models using CRISPR-Cas9 genome editing often require tedious single-cell clone selection and expansion. In this study, we develop and optimise rapid and robust strategies to engineer homozygous fluorescent reporter knock-in cell pools with precise genome editing, circumventing clonal variability inherent to traditional approaches. To reduce false-positive cells associated with random integration, we optimise the design of donor DNA by removing the start codon of the fluorescent reporter and incorporating a self-cleaving T2A peptide system. Using fluorescence-assisted cell sorting (FACS), we efficiently identify and isolate the desired homozygous fluorescent knock-in clones, establishing stable cell pools that preserve parental cell line heterogeneity and faithfully reflect endogenous transcriptional regulation of the target gene. We evaluate the knock-in efficiency and rate of undesired random integration in the electroporation method with either a dual-plasmid system (sgRNA and donor DNA in two separate vectors) or a single-plasmid system (sgRNA and donor DNA combined in one vector). We further demonstrate that coupling our single-plasmid construct with an integrase-deficient lentivirus vector (IDLV) packaging system efficiently generates fluorescent knock-in reporter cell pools, offering flexibility between electroporation and lentivirus transduction methods. Notably, compared to the electroporation methods, the IDLV system significantly minimises random integration. Moreover, the resulting reporter cell lines are compatible with most of the available genome-wide sgRNA libraries, enabling unbiased CRISPR screens to identify key transcriptional regulators of a gene of interest. Overall, our methodologies provide a powerful genetic tool for rapid and robust generation of fluorescent reporter knock-in cell pools with precise genome editing by CRISPR-Cas9 for various research purposes. Full article

Atherosclerosis, a chronic inflammatory disease characterized by lipid accumulation and immune cell infiltration, is linked to plaque formation and cardiovascular events. While traditionally associated with lipid metabolism and endothelial dysfunction, recent research highlights the roles of autophagy and clonal hematopoiesis (CH) in its pathogenesis. Autophagy, a cellular process crucial for degrading damaged components, regulates macrophage homeostasis and inflammation, both of which are pivotal in atherosclerosis. In macrophages, autophagy influences lipid metabolism, cytokine regulation, and oxidative stress, helping to prevent plaque instability. Defective autophagy exacerbates inflammation, impairs cholesterol efflux, and accelerates disease progression. Additionally, autophagic processes in endothelial cells and smooth muscle cells further contribute to atherosclerotic pathology. Recent studies also emphasize the interplay between autophagy and CH, wherein somatic mutations in genes like TET2, JAK2, and DNMT3A drive immune cell expansion and enhance inflammatory responses in atherosclerotic plaques. These mutations modify macrophage function, intensifying the inflammatory environment and accelerating atherosclerosis. Chaperone-mediated autophagy (CMA), a selective form of autophagy, also plays a critical role in regulating macrophage inflammation by degrading pro-inflammatory cytokines and oxidized low-density lipoprotein (ox-LDL). Impaired CMA activity leads to the accumulation of these substrates, activating the NLRP3 inflammasome and worsening inflammation. Preclinical studies suggest that pharmacologically activating CMA may mitigate atherosclerosis progression. In animal models, reduced CMA activity accelerates plaque instability and increases inflammation. This review highlights the importance of autophagic regulation in macrophages, focusing on its role in inflammation, plaque formation, and the contributions of CH. Building upon current advances, we propose a hypothesis in which autophagy, programmed cell death, and clonal hematopoiesis form a critical intrinsic axis that modulates the fundamental functions of macrophages, playing a complex role in the development of atherosclerosis. Understanding these mechanisms offers potential therapeutic strategies targeting autophagy and inflammation to reduce the burden of atherosclerotic cardiovascular disease. Full article

Patients with chronic, indolent myeloproliferative neoplasms (MPNs) are at risk for transformation to highly lethal leukemia, although targetable mechanisms driving progression remain elusive. We discovered that the High Mobility Group A1 (HMGA1) gene is up-regulated with MPN progression in patients and required for evolution into myelofibrosis (MF) or acute myeloid leukemia (AML) in preclinical models. HMGA1 encodes the HMGA1 epigenetic regulators that modulate the chromatin state during embryogenesis and tissue regeneration. While HMGA1 is silenced in most differentiated cells, it becomes aberrantly re-expressed in JAK2 mutant (JAK2-V617F) MPN, with the highest levels after transformation to secondary MF or AML. Here, we review recent work highlighting HMGA1 function in MPN progression. Though underlying mechanisms continue to emerge, increasing evidence suggests that HMGA1 functions as a “chromatin key” required to “unlock” regions of the genome involved in clonal expansion and progression in MPN. Together, these findings illuminate HMGA1 as a driver of MPN progression and a promising therapeutic target. Full article

Clonal hematopoiesis (CH) is associated with an increased risk of developing myeloid neoplasms (MNs) such as myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML). In general, CH comprises clonal hematopoiesis of indeterminate potential (CHIP) and clonal cytopenia of undetermined significance (CCUS). It is an age-related phenomenon characterized by the presence of somatic mutations in hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) that acquire a fitness advantage under selection pressure. Individuals with CHIP have an absolute risk of 0.5–1.0% per year for progressing to MDS or AML. Inflammation, smoking, cytotoxic therapy, and radiation can promote the process of clonal expansion and leukemic transformation. Of note, exposure to chemotherapy or radiation for patients with solid tumors or lymphomas can increase the risk of therapy-related MN. Beyond hematological malignancies, CH also serves as an independent risk factor for heart disease, stroke, chronic obstructive pulmonary disease, and chronic kidney disease. Prognostic models such as the CH risk score and MN-prediction models can provide a framework for risk stratification and clinical management of CHIP/CCUS and identify high-risk individuals who may benefit from close surveillance. For CH or related disorders, therapeutic strategies targeting specific CH-associated mutations and specific selection pressure may have a potential role in the future. Full article

Treatment-resistant asthma remains an unresolved clinical problem and a challenge for current medical science. Consequently, there is a growing and urgent need to develop novel or alternative therapeutic options for the treatment of asthma. The research problem raised in this study was to assess and compare mycophenolate mofetil (MMF), an inhibitor of inosine monophosphate dehydrogenase, and tofacitinib (TFB), a Janus kinase inhibitor, for anti-asthmatic properties, and consequently to determine whether these agents may have potential as alternative options for treatment of allergic asthma. For this purpose, we assessed the effect of administration of MMF and TFB on the development of a mouse model of allergic airway inflammation (AAI) and accompanying CD4⁺ (cluster of differentiation 4) T-cell immune response in the lung-draining mediastinal lymph nodes (MLNs) and lungs, i.e., in the inductive and effector sites, respectively, of the immune response underlying the development of allergic asthma. The results from a histopathological scoring system demonstrated that the administration of MMF and TFB did not prevent or abolish ovalbumin-induced AAI, but strongly attenuated its severity. The pulmonary function tests revealed that the treatment with MMF and TFB significantly reduced methacholine-induced bronchoconstriction. These results indicate that the treatment with TFB and MMF attenuated the development of ovalbumin-induced AAI. The magnitude of the anti-asthmatic effect was comparable between both agents. The study revealed that the impairment of the clonal expansion of effector CD4⁺ T cells in the MLNs is a critical event in the mechanism underlying the anti-asthmatic effect of MMF and TFB. Apart from this, the findings of the study strongly suggest that the suppression of the interleukin-33/suppression of tumorigenicity-2 signaling pathway may constitute an additional mechanism responsible for producing this effect. In turn, the results indicate that the anti-asthmatic action induced by the studied agents is not mediated by the generation of forkhead box protein 3-expressing CD4⁺ regulatory T cells. Clinical implication of the results: the results suggest that MMF and TFB may exert anti-asthmatic action, and thus they may be considered therapeutic options for the treatment of allergic asthma cases resistant to conventional/existing treatment. Full article

The ramet system is a typical structural type in the life history of clonal plants. This massive structure is formed by many similar ramets connected by underground rhizomes, which are independent and mutually influential. Therefore, the ramet system is unique to bamboo forests, and its role in the construction, maintenance, and productivity of bamboo populations is irreplaceable. Mulch management is a high-level cultivation model for bamboo forests that is used to cultivate bamboo shoots. However, the basic conditions of bamboo ramet systems in this managed model are poorly understood. This study analyzed the underground rhizome morphology, bud bank, and branching of bamboo ramets in a Phyllostachys praecox C.D. Chu et C.S. Chao ‘Prevernalis’ forest to explore the growth patterns of bamboo ramets in high-level management fields. In mulched bamboo forests, the bamboo rhizomes, distributed in intermediate positions of the bamboo ramet system, were long with many lateral buds and branches, and those at the initial and distal ends were short with few lateral buds and branches. The initial end of the ramet system reduced the ramet system, the intermediate part expanded the ramet system, and the distal end promoted ramet system regeneration. Owing to the continuous reduction, expansion, and renewal of ramet systems, the bamboo rhizome system demonstrates mobility and adaptability. This study found that a higher level of bamboo forest management increased the possibility of artificial fragmentation of the ramet system and that improving the efficiency of the ramet system was beneficial for maintaining its high vitality. Thus, this study provides a crucial reference for guiding the precise regulation of bamboo ramet systems in artificial bamboo forests. Full article

The tree fern Culcita macrocarpa, a threatened Iberian–Macaronesian endemism, represents the sole European species of the order Cyatheales. Considered a Tertiary relict of European Palaeotropical flora, its evolutionary history and genetic diversity, potentially influenced by presumed high clonal propagation, remain largely unknown. This study elucidates the phylogeographic history of C. macrocarpa, assessing the impact of vegetative reproduction on population dynamics and genetic variability. We provide genetic data from eight newly identified nuclear microsatellite loci and one plastid DNA region for 17 populations spanning the species’ range, together with species distribution modeling data. Microsatellites reveal pervasive clonality in C. macrocarpa, which has varied among populations. We assess the impact of clonality on genetic diversity and evaluate how estimates of intra-population genetic diversity indices and genetic structuring are affected by the chosen definition of “individual” (focusing exclusively on genetically distinct individuals, genets, as opposed to considering all independent clonal replicates, ramets). We identify two main population groups, one in the northern Iberian Peninsula and the other in the Macaronesian archipelagos and southern Iberian Peninsula. Within each group, we found relict populations (in the Azores and the Cantabrian Cornice) as well as recent originated populations. This population structure suggests colonization dynamics in which recent populations originated from one or a few genets of relict populations and became established through intra-gametophytic self-fertilization and vegetative expansion. DAPC analysis facilitated the identification of alleles that most significantly contributed to the observed population structure. The current Andalusian populations appear to have originated from colonization events from the Azores and the Cantabrian Cornice. Our findings suggest that C. macrocarpa persisted through the Last Glacial Maximum in two refugia: the Azores and the Cantabrian Cornice. Colonization into new areas occurred presumably from these refuges, generating two large population groups with structured genetic diversity. This study underscores the significance of clonality in establishing new populations and shaping genetic structure. Full article

Despite the notable achievements of programmed death 1 (PD-1) antibodies in treating various cancers, the overall efficacy remains limited in the majority of colorectal cancer (CRC) cases. Metabolism reprogramming of tumors inhibits the tricarboxylic acid (TCA) cycle, leading to down-regulation of fumarate hydratase (FH), which is related to poor prognosis in CRC patients. By establishing a tumor-bearing mouse model of CRC with Fh1 expression deficiency, we confirmed that the therapeutic effect of PD-1 antibodies alone was suboptimal in mice with low Fh1 expression, which was improved by combination with a protein invertase subtilisin/kexin 9 (PCSK9) inhibitor. Mechanistically, FH binds to Ras-related nucleoprotein (RAN), which inhibits the nuclear import of the PCSK9 transcription factor SREBF1/2, thus reducing the expression of PCSK9. This leads to increased clonal expansion of CD8+ T cells while the number of Tregs remains unchanged, and the expression of PD-L1 does not change significantly, thus enhancing the immunotherapy response. On the contrary, the expression of PCSK9 increased in CRC cells with low FH expression, which antagonized the effects of immunotherapy. Overall, CRC patients with low FH expression may benefit from combinatorial therapy with PD-1 antibodies and PCSK9 inhibitors to enhance the curative effect. Full article

Background: Suppression of HBV DNA, inhibition of HBV surface (HBsAg) production and therapeutic vaccination to reverse HBV-specific T-cell exhaustion in chronic HBV patients are likely required to achieve a functional cure. In the AAV-HBV mouse model, therapeutic vaccination can be effective in clearing HBV when HBsAg levels are low. Using a single-cell approach, we investigated the liver immune environment with different levels of HBsAg and sustained HBsAg loss through treatment with a GalNAc-HBV-siRNA followed by therapeutic vaccination. Methods: AAV-HBV-transduced C57BL/6 mice were treated with GalNAc-HBV-siRNA to lower HBsAg levels and then vaccinated using a DNA vaccine. We used single-cell RNA and V(D)J sequencing to understand liver immune microenvironment changes. Results: GalNAc-HBV-siRNA, followed by therapeutic vaccination, achieved sustained HBsAg loss in all mice. This was accompanied by CD4 follicular helper T-cell induction, polyclonal activation of CD8 T cells and clonal expansion of plasma cells that were responsible for antibody production. Conclusions: This study provides novel insights into liver immune changes at the single-cell level, highlighting the correlation between induced reduction of HBsAg levels and clonal expansion of CD4, CD8 T cells and plasma cells in the liver upon HBV siRNA and subsequent therapeutic vaccination. Full article

Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells. Full article

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction. Full article

Murine hematopoietic stem and progenitor cells (HSPCs) are commonly used as model systems during gene therapeutic retroviral vector development and preclinical biosafety assessment. Here, we developed cell culture conditions to maintain stemness and prevent differentiation during HSPC culture. We used the small compounds A83-01, pomalidomide, and UM171 (APU). Highly purified LSK SLAM cells expanded in medium containing SCF, IL-3, FLT3-L, and IL-11 but rapidly differentiated to myeloid progenitors and mast cells. The supplementation of APU attenuated the differentiation and preserved the stemness of HSPCs. The TGF$\beta$ inhibitor A83-01 was identified as the major effector. It significantly inhibited the mast-cell-associated expression of Fc$\epsilon$R1$\alpha$ and the transcription of genes regulating the formation of granules and promoted a 3800-fold expansion of LSK cells. As a functional readout, we used expanded HSPCs in state-of-the-art genotoxicity assays. Like fresh cells, APU-expanded HSPCs transduced with a mutagenic retroviral vector developed a myeloid differentiation block with clonal restriction and dysregulated oncogenic transcriptomic signatures due to vector integration near the high-risk locus Mecom. Thus, expanded HSPCs might serve as a novel cell source for retroviral vector testing and genotoxicity studies. Full article

Multiple Myeloma (MM) and its preexisting stage, termed Monoclonal Gammopathy of Undetermined Significance (MGUS), have long been considered mainly as genomic diseases. However, the bone changes observed in both conditions have led to a reassessment of the role of the bone microenvironment, mainly the endosteal niche in their genesis. Here, we consider the disruption of the endosteal niche in the bone marrow, that is, the shift of the endosteal niche from an osteoblastic to an osteoclastic profile produced by bone senescence and inflammaging, as the key element. Thus, this disrupted endosteal niche is proposed to represent the permissive microenvironment necessary not only for the emergence of MM from MGUS but also for the emergence and maintenance of MGUS. Moreover, the excess of osteoclasts would favor the presentation of antigens (Ag) into the endosteal niche because osteoclasts are Ag-presenting cells. As such, they could significantly stimulate the presentation of some specific Ag and the clonal expansion of the stimulated cells as well as favor the expansion of such selected clones because osteoclasts are immunosuppressive. We also discuss this scenario in the Gaucher disease, in which the high incidence of MGUS and MM makes it a good model both at the bone level and the immunological level. Finally, we envisage that this endosteal niche disruption would increase the stochasticity (epigenetic and genetic instability) in the selected clones, according to our Tissue Disruption-induced cell Stochasticity (TiDiS) theory. Full article

Photodynamic therapy (PDT) is a two-stage treatment relying on cytotoxicity induced by photoexcitation of a nontoxic dye, called photosensitizer (PS). Using 5-aminolevulinic acid (5-ALA), the pro-drug of PS protoporphyrin IX, we investigated the impact of PDT on hepatocellular carcinoma (HCC). Optimal 5-ALA PDT dose was determined on three HCC cell lines by analyzing cell death after treatment with varying doses. HCC-patient-derived tumor hepatocytes and healthy donor liver myofibroblasts were treated with optimal 5-ALA PDT doses. The proliferation of cancer cells and healthy donor immune cells cultured with 5-ALA-PDT-treated conditioned media was analyzed. Finally, therapy efficacy on humanized SCID mice model of HCC was investigated. 5-ALA PDT induced a dose-dependent decrease in viability, with an up-to-four-fold reduction in viability of patient tumor hepatocytes. The 5-ALA PDT treated conditioned media induced immune cell clonal expansion. 5-ALA PDT has no impact on myofibroblasts in terms of viability, while their activation decreased cancer cell proliferation and reduced the tumor growth rate of the in vivo model. For the first time, 5-ALA PDT has been validated on primary patient tumor hepatocytes and donor healthy liver myofibroblasts. 5-ALA PDT may be an effective anti-HCC therapy, which might induce an anti-tumor immune response. Full article

The TP53 gene is a major player in cancer formation, and it is considered the most important tumor suppressor gene. The p53 protein acts as a transcription factor, and it is involved in DNA repair, senescence, cell-cycle control, autophagy, and apoptosis. Beyond cancer, there is evidence that TP53 is associated with fertility, aging, and longevity. Additionally, more evidence exists that genetic variants in TP53 are associated with environmental adaptation. Special TP53 amino-acid residues or pathogenic TP53 mutations seem to be adaptive for animals living in hypoxic and cold environments or having been exposed to starvation, respectively. At the somatic level, it has recently been proven that multiple cancer genes, including TP53, are under positive selection in healthy human tissues. It is not clear why these driver mutations do not transform these tissues into cancerous ones. Other studies have shown that elephants have multiple TP53 copies, probably this being the reason for the very low cancer incidence in these large animals. This may explain the famous Peto’s paradox. This review discusses in detail the multilevel role of TP53 in adaptation, according to the published evidence. This role is complicated, and it extends from cells to individuals and to populations. Full article