Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

Objective: To assess the correlation between HMGB1 expression and the patient prognosis in a multicancer context. Methods: The potential oncogenic role of HMGB1 was explored in forty tumors through the TCGA, GEO, and Oncomine datasets. We analyzed the clinical prognostic value and antitumor immunotherapy of HMGB1 in a multicancer context using GEO (GSE111636). Results: High expression of HMGB1 is present in multicancer cases, and its low expression is closely associated with the prognostic survival of patients, in terms of both overall and disease-free survival in ACC and LUAD. Further investigation revealed that the high expression of gastric and lung cancer is closely associated with low risk and better prognosis of patients based on COX and Kaplan–Meier analysis of OS, FP and PPS. HMGB1 expression was found to be significantly correlated with cancer-associated fibroblast and CD8⁺ T cell infiltration in the TME. The analysis of GO functional annotation/KEGG pathways indicates that HMGB1 may regulate tumor immunity-related pathways, such as the tumor immunotherapy response in colorectal cancer. The function of four genes as hubs are confirmed by in vitro HMGB1 knockdown which led to inhibition of cell proliferation and metastasis in SW620 and SW480 cells. Conclusion: HMGB1 is a potential novel biomarker for improving clinical prognosis and antitumor immunotherapy efficacy. CDK1, HMGB2, SSRP1, and H2AFV may serve as key nodes for HMGB1 in colorectal cancer.

The aim of this study was to evaluate the association between Ferredoxin 1 (FDX1) expression and the prognostic survival of tumor patients and predict the efficacy of immunotherapy response to antitumor drug sensitivity. FDX1 plays an oncogenic role in thirty-three types of tumors, based on TCGA and GEO databases, and further experimental validation in vitro was provided through multiple cell lines. FDX1 was expressed highly in multiple types of cancer and differently linked to the survival prognosis of tumorous patients. A high phosphorylation level was correlated with the FDX1 site of S177 in lung cancer. FDX1 exhibited a significant association with infiltrated cancer-associated fibroblasts and CD8⁺ T cells. Moreover, FDX1 demonstrated correlations with immune and molecular subtypes, as well as functional enrichments in GO/KEGG pathways. Additionally, FDX1 displayed relationships with the tumor mutational burden (TMB), microsatellite instability (MSI), DNA methylation, and RNA and DNA synthesis (RNAss/DNAss) within the tumor microenvironment. Notably, FDX1 exhibited a strong connection with immune checkpoint genes in the co-expression network. The validity of these findings was further confirmed through Western blotting, RT-qPCR, and flow cytometry experiments conducted on WM115 and A375 tumor cells. Elevated FDX1 expression has been linked to the enhanced effectiveness of PD-L1 blockade immunotherapy in melanoma, as observed in the GSE22155 and GSE172320 cohorts. Autodocking simulations have suggested that FDX1 may influence drug resistance by affecting the binding sites of antitumor drugs. Collectively, these findings propose that FDX1 could serve as a novel and valuable biomarker and represent an immunotherapeutic target for augmenting immune responses in various human cancers when used in combination with immune checkpoint inhibitors.

Aims Interventions using prebiotic inulin‐type fructans (ITFs) are widely prescribed to modulate the gut microbiota composition and activity to promote health. However, the impacts of ITFs on post‐antibiotic reconstitution of the gut microbiome remain incompletely understood. The aim of the present study was to investigate the effects of ITFs supplementation on intestinal inflammation, the composition of the intestinal microbiota and the colonic transcriptome after antibiotic treatment. Methods and Results Male BALB/c mice were subjected to an antibiotic cocktail (ABx) treatment for 7 days, and their microbiomes were then reconstituted either spontaneously or with ITFs supplementation (5%) for 14 days. Our data showed that ITFs supplementation delayed the recovery of antibiotic‐induced colitis compared with the spontaneous recovery. Neither ITFs supplementation nor spontaneous recovery could restore the microbial community composition at the genus level back to its initial composition. ITFs supplementation increased the relative abundance of some beneficial bacteria and butyrate levels, but resulted in selective blooms of some opportunistic pathogens and elevated the pathways associated with diseases linked to gut microbiota function. Both ITFs supplementation and spontaneous recovery could restore the colonic transcriptome nearly to the initial profile to a certain extent; however, ITFs supplementation delayed the restoration of the immunoglobulin genes compared to spontaneous recovery. Conclusion These data showed that post‐antibiotic ITFs consumption did not always lead to beneficial effects but might lead to potential adverse effects in the context of dysbiosis. Significance and Impact of the Study These findings highlighted that caution is required when supplementing ITFs to restore intestinal homeostasis in the context of dysbiosis resulting from broad‐spectrum antibiotics.

Background: Adoptive cell therapy (ACT), including CAR-T and TCR-T therapies, shows promise for cancer treatment, depending on infused T cell expansion, persistence and activity. We previously characterized four T-cell subsets (T_N, T_SCM, T_CM and T_EM) and their miRNA profiles. Objectives: This study investigates miR-143-3p’s role in T cell differentiation. Methods: Using qPCR, we analyzed miR-143-3p expression. Target genes were validated by dual-luciferase assays. Functional assays assessed differentiation markers, proliferation, apoptosis and cytokine secretion. Results: miR-143-3p was upregulated in early-differentiated T_SCM but downregulated during progression. We confirmed ABL2 and PAG1 as direct targets suppressed by miR-143-3p. Overexpression increased early markers (LEF1, CCR7 and CD62L) while decreasing late markers (EOMES, KLRG1 and CD45RO). It also enhanced proliferation, reduced apoptosis and suppressed cytokine secretion. Conclusions: miR-143-3p promotes T_SCM differentiation and inhibits progressive differentiation by targeting ABL2/PAG1, suggesting new ACT optimization strategies.