BACKGROUND: Ischemic acute kidney injury (AKI) is a common syndrome associated with considerable mortality and healthcare costs. Up to now, the underlying pathogenesis of ischemic AKI remains incompletely understood, and specific strategies for early diagnosis and treatment of ischemic AKI are still lacking. Here, this study aimed to define the transcriptomic landscape of AKI patients through single-cell RNA sequencing (scRNA-seq) analysis in kidneys. METHODS: In this study, scRNA-seq technology was applied to kidneys from two ischemic AKI patients, and three human public scRNA-seq datasets were collected as controls. Differentially expressed genes (DEGs) and cell clusters of kidneys were determined. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, as well as the ligand-receptor interaction between cells, were performed. We also validated several DEGs expression in kidneys from human ischemic AKI and ischemia/reperfusion (I/R) injury induced AKI mice through immunohistochemistry staining. RESULTS: 15 distinct cell clusters were determined in kidney from subjects of ischemic AKI and control. The injured proximal tubules (PT) displayed a proapoptotic and proinflammatory phenotype. PT cells of ischemic AKI had up-regulation of novel pro-apoptotic genes including USP47 , RASSF4 , EBAG9 , IER3 , SASH1 , SEPTIN7 , and NUB1 , which have not been reported in ischemic AKI previously. Several hub genes were validated in kidneys from human AKI and renal I/R injury mice, respectively. Furthermore, PT highly expressed DEGs enriched in endoplasmic reticulum stress, autophagy, and retinoic acid-inducible gene I (RIG-I) signaling. DEGs overexpressed in other tubular cells were primarily enriched in nucleotide-binding and oligomerization domain (NOD)-like receptor signaling, estrogen signaling, interleukin (IL)-12 signaling, and IL-17 signaling. Overexpressed genes in kidney-resident immune cells including macrophages, natural killer T (NKT) cells, monocytes, and dendritic cells were associated with leukocyte activation, chemotaxis, cell adhesion, and complement activation. In addition, the ligand-receptor interactions analysis revealed prominent communications between macrophages and monocytes with other cells in the process of ischemic AKI. CONCLUSION Together, this study reveals distinct cell-specific transcriptomic atlas of kidney in ischemic AKI patients, altered signaling pathways, and potential cell-cell crosstalk in the development of AKI. These data reveal new insights into the pathogenesis and potential therapeutic strategies in ischemic AKI.
Humans ; Mice ; Animals ; Transcriptome/genetics* ; Ligands ; Kidney/metabolism* ; Acute Kidney Injury/metabolism* ; Ischemia/metabolism* ; Reperfusion Injury/metabolism* ; Sequence Analysis, RNA ; Adaptor Proteins, Signal Transducing/metabolism* ; Tumor Suppressor Proteins/metabolism*

BACKGROUND: Previous research demonstrated that a homozygous mutation of g.136372044G>A (S12N) in caspase recruitment domain family member 9 ( CARD9 ) is critical for producing Aspergillus fumigatus -induced ( Af -induced) T helper 2 (T H 2)-mediated responses in allergic bronchopulmonary aspergillosis (ABPA). However, it remains unclear whether the CARD9S12N mutation, especially the heterozygous occurrence, predisposes the host to ABPA. METHODS: A total of 61 ABPA patients and 264 controls (including 156 healthy controls and 108 asthma patients) were recruited for sequencing the CARD9 locus to clarify whether patients with this heterozygous single-nucleotide polymorphisms are predisposed to the development of ABPA. A series of in vivo and in vitro experiments, such as quantitative real-time polymerase chain reaction, flow cytometry, and RNA isolation and quantification, were used to illuminate the involved mechanism of the disease. RESULTS: The presence of the p.S12N mutation was associated with a significant risk of ABPA in ABPA patients when compared with healthy controls and asthma patients, regardless of Aspergillus sensitivity. Relative to healthy controls without relevant allergies, the mutation of p.S12N was associated with a significant risk of ABPA (OR: 2.69 and 4.17 for GA and AA genotypes, P = 0.003 and 0.029, respectively). Compared with patients with asthma, ABPA patients had a significantly higher heterozygous mutation (GA genotype), indicating that p.S12N might be a significant ABPA-susceptibility locus ( aspergillus sensitized asthma: OR: 3.02, P = 0.009; aspergillus unsensitized asthma: OR: 2.94, P = 0.005). The mutant allele was preferentially expressed in ABPA patients with heterozygous CARD9S12N , which contributes to its functional alterations to facilitate Af -induced T H 2-mediated ABPA development. In terms of mechanism, Card9 wild-type ( Card9WT ) expression levels decreased significantly due to Af -induced decay of its messenger RNA compared to the heterozygous Card9S12N . In addition, ABPA patients with heterozygous CARD9S12N had increased Af -induced interleukin-5 production. CONCLUSION Our study provides the genetic evidence showing that the heterozygous mutation of CARD9S12N , followed by allele expression imbalance of CARD9S12N , facilitates the development of ABPA.
Humans ; Aspergillosis, Allergic Bronchopulmonary/complications* ; Aspergillus fumigatus/genetics* ; Asthma/genetics* ; Aspergillus ; Mutation/genetics* ; CARD Signaling Adaptor Proteins/genetics*

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adaptor protein that plays an important role in the clearance of proteins to be degraded as well as in the maintenance of cellular proteostasis. p62 protein has multiple functional domains, which interact with several downstream proteins to precisely regulate multiple signaling pathways, thereby linking p62 to oxidative defense systems, inflammatory responses and nutrient sensing. Studies have shown that mutation or abnormal expression of p62 is closely related to the occurrence and development of various diseases, including neurodegenerative diseases, tumors, infectious diseases, genetic diseases and chronic diseases. This review summarizes the structural features and molecular functions of p62. Moreover, we systematically introduce its multiple functions in protein homeostasis and regulation of signaling pathways. Furthermore, the complexity and versatility of p62 in the occurrence and development of diseases are summarized, with the aim to provide a reference for understanding the function of p62 protein and facilitating related disease research.
Humans ; Autophagy/genetics* ; Sequestosome-1 Protein/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; Signal Transduction ; Neoplasms/genetics*

OBJECTIVES: To study the efficacy and safety of repeated application of rituximab (RTX) at a low dose (200 mg/m²) versus the recommended dose (375 mg/m²) for remission maintenance in frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS). METHODS: A randomized controlled trial was conducted for 29 children with FRNS/SDNS who received systemic treatment in the Department of Nephrology, Anhui Provincial Children's Hospital, from September 2020 to December 2021. These children were divided into a recommended dose group (n=14) and a low dose group (n=15) using a random number table. The two groups were compared in terms of general characteristics, changes in CD19 expression after RTX treatment, number of relapses, glucocorticoid dose, adverse reactions of RTX, and hospital costs. RESULTS: After RTX treatment, both the low dose group and the recommended dose group achieved B-lymphocyte depletion and had significant reductions in the number of relapses and glucocorticoid dose (P<0.05). The low dose group had a comparable clinical effect to the recommended dose group after RTX treatment (P>0.05), and the low dose group had a significant reduction in hospital costs for the second, third, and fourth times of hospitalization (P<0.05). There were no serious adverse reactions in either group during RTX treatment and late follow-up, and there was no significant difference in adverse reactions between the two groups (P>0.05). CONCLUSIONS Repeated RTX treatment at a low dose has comparable clinical efficacy and safety to that at the recommended dose and can significantly reduce the number of FRNS/SDNS relapses and the amount of glucocorticoids used, with little adverse effect throughout the treatment cycle. Therefore, it holds promise for clinical application.
Humans ; Child ; Nephrotic Syndrome/drug therapy* ; Rituximab/adverse effects* ; Glucocorticoids/adverse effects* ; Prospective Studies ; Adaptor Proteins, Signal Transducing

Humans ; Adenoma, Pleomorphic/genetics* ; Adenoma ; Salivary Gland Neoplasms/genetics* ; Adaptor Proteins, Signal Transducing

OBJECTIVE: Bo investigate the regulatory relationship between NKD1 and YWHAE and the mechanism of NKD1 for promoting tumor cell proliferation. METHODS: HCT116 cells transfected with pcDNA3.0-NKD1 plasmid, SW620 cells transfected with NKD1 siRNA, HCT116 cells with stable NKD1 overexpression (HCT116-NKD1 cells), SW620 cells with nkd1knockout (SW620-nkd1^-/- cells), and SW620-nkd1^-/- cells transfected with pcDNA3.0-YWHAE plasmid were examined for changes in mRNA and protein expression levels of YWHAE using qRT-PCR and Western blotting. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NKD1 to the promoter region of YWHAE gene. The regulatory effect of NKD1 on YWHAE gene promoter activity was analyzed by dual-luciferase reporter gene assay, and the interaction between NKD1 and YWHAE was analyzed with immunofluorescence assay. The regulatory effect of NKD1 on glucose uptake was examined in the tumor cells. RESULTS: In HCT116 cells, overexpression of NKD1 significantly enhanced the expression of YWHAE at both the mRNA and protein levels, while NKD1 knockout decreased its expression in SW620 cells (P < 0.001). ChIP assay showed that NKD1 protein was capable of binding to the YWHAE promoter sequence; dual luciferase reporter gene assay showed that NKD1 overexpression (or knockdown) in the colon cancer cells significantly enhanced (or reduced) the transcriptional activity of YWHAE promoter (P < 0.05). Immunofluorescence assay demonstrated the binding of NKD1 and YWHAE proteins in colon cancer cells. NKD1 knockout significantly reduced glucose uptake in colon cancer cells (P < 0.01), while YWHAE overexpression restored the glucose uptake in NKD1-knockout cells (P < 0.05). CONCLUSION NKD1 protein activates the transcriptional activity of YWHAE gene to promote glucose uptake in colon cancer cells.
Humans ; Colonic Neoplasms ; HCT116 Cells ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; RNA, Messenger ; Glucose ; Calcium-Binding Proteins/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; 14-3-3 Proteins/metabolism*

BACKGROUND: Small cell lung cancer (SCLC) with high c-Myc expression is prone to relapse and metastasis, leading to extremely low survival rate. Cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor Abemaciclib plays a key role in the treatment of tumors, but the effects and mechanisms on SCLC remain unclear. This study was to analyze the effect and molecular mechanism of Abemaciclib in inhibiting proliferation, migration and invasion of SCLC with high c-Myc expression, with a view to expanding a new direction for reducing the recurrence and metastasis. METHODS: Proteins interacting with CDK4/6 were predicted using the STRING database. The expressions of CDK4/6 and c-Myc in 31 cases of SCLC cancer tissues and paired adjacent normal tissues were analyzed by immunohistochemistry. The effects of Abemaciclib on the proliferation, invasion and migration of SCLC were detected by CCK-8, colony formation assay, Transwell and migration assay. Western blot was used to detect the expressions of CDK4/6 and related transcription factors. Flow cytometry was used to analyze the effects of Abemaciclib on the cell cycle and checkpoint of SCLC. RESULTS: The expression of CDK4/6 was associated with c-Myc by STRING protein interaction network. c-Myc can directly modalize achaete-scute complex homolog 1 (ASCL1), neuronal differentiation 1 (NEUROD1) and Yes-associated protein 1 (YAP1). Moreover, CDK4 and c-Myc regulate the expression of programmed cell death ligand 1 (PD-L1). Immunohistochemistry showed that the expressions of CDK4/6 and c-Myc in cancer tissues were higher than those in adjacent tissues(P<0.0001). CCK-8, colony formation assay, Transwell and migration assay verified that Abemaciclib could effectively inhibit the proliferation, invasion and migration of SBC-2 and H446OE(P<0.0001). Western blot analysis further showed that Abemaciclib not only inhibited CDK4 (P<0.05) and CDK6 (P<0.05), but also affected c-Myc (P<0.05), ASCL1 (P<0.05), NEUROD1 (P<0.05) and YAP1 (P<0.05), which are related to SCLC invasion and metastasis. Flow cytometry showed that Abemaciclib not only inhibited the cell cycle progression of SCLC cells (P<0.0001), but also significantly increased PD-L1 expression on SBC-2 (P<0.01) and H446OE (P<0.001). CONCLUSIONS Abemaciclib significantly inhibits the proliferation, invasion, migration and cell cycle progression of SCLC by inhibiting the expressions of CDK4/6, c-Myc, ASCL1, YAP1 and NEUROD1. Abemaciclib can also increase the expression of PD-L1 in SCLC.
Humans ; Small Cell Lung Carcinoma ; B7-H1 Antigen ; Sincalide ; Lung Neoplasms ; Neoplasm Recurrence, Local ; Transcription Factors ; Adaptor Proteins, Signal Transducing ; Cell Proliferation

Tumor-associated macrophages (TAMs) play crucial roles in tumor progression and immune responses. However, mechanisms of driving TAMs to antitumor function remain unknown. Here, transcriptome profiling analysis of human oral cancer tissues indicated that regulator of G protein signaling 12 (RGS12) regulates pathologic processes and immune-related pathways. Mice with RGS12 knockout in macrophages displayed decreased M1 TAMs in oral cancer tissues, and extensive proliferation and invasion of oral cancer cells. RGS12 increased the M1 macrophages with features of increased ciliated cell number and cilia length. Mechanistically, RGS12 associates with and activates MYC binding protein 2 (MYCBP2) to degrade the cilia protein kinesin family member 2A (KIF2A) in TAMs. Our results demonstrate that RGS12 is an essential oral cancer biomarker and regulator for immunosuppressive TAMs activation.
Mice ; Humans ; Animals ; Tumor-Associated Macrophages/metabolism* ; Carcinoma, Squamous Cell ; Squamous Cell Carcinoma of Head and Neck ; Mouth Neoplasms ; GTP-Binding Proteins/metabolism* ; Head and Neck Neoplasms ; Ubiquitin-Protein Ligases/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; RGS Proteins/metabolism* ; Kinesins/metabolism* ; Repressor Proteins/metabolism*

As an important part of tumor microenvironment, neutrophils are poorly understood due to their spatiotemporal heterogeneity in tumorigenesis. Here we defined, at single-cell resolution, CD44-CXCR2- neutrophils as tumor-specific neutrophils (tsNeus) in both mouse and human gastric cancer (GC). We uncovered a Hippo regulon in neutrophils with unique YAP signature genes (e.g., ICAM1, CD14, EGR1) distinct from those identified in epithelial and/or cancer cells. Importantly, knockout of YAP/TAZ in neutrophils impaired their differentiation into CD54+ tsNeus and reduced their antitumor activity, leading to accelerated GC progression. Moreover, the relative amounts of CD54+ tsNeus were found to be negatively associated with GC progression and positively associated with patient survival. Interestingly, GC patients receiving neoadjuvant chemotherapy had increased numbers of CD54+ tsNeus. Furthermore, pharmacologically enhancing YAP activity selectively activated neutrophils to suppress refractory GC, with no significant inflammation-related side effects. Thus, our work characterized tumor-specific neutrophils in GC and revealed an essential role of YAP/TAZ-CD54 axis in tsNeus, opening a new possibility to develop neutrophil-based antitumor therapeutics.
Humans ; Animals ; Mice ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Stomach Neoplasms/pathology* ; Neutrophils/pathology* ; Signal Transduction/genetics* ; YAP-Signaling Proteins ; Tumor Microenvironment ; Hyaluronan Receptors/genetics*

Although the mTOR-4E-BP1 signaling pathway is implicated in aging and aging-related disorders, the role of 4E-BP1 in regulating human stem cell homeostasis remains largely unknown. Here, we report that the expression of 4E-BP1 decreases along with the senescence of human mesenchymal stem cells (hMSCs). Genetic inactivation of 4E-BP1 in hMSCs compromises mitochondrial respiration, increases mitochondrial reactive oxygen species (ROS) production, and accelerates cellular senescence. Mechanistically, the absence of 4E-BP1 destabilizes proteins in mitochondrial respiration complexes, especially several key subunits of complex III including UQCRC2. Ectopic expression of 4E-BP1 attenuates mitochondrial abnormalities and alleviates cellular senescence in 4E-BP1-deficient hMSCs as well as in physiologically aged hMSCs. These f indings together demonstrate that 4E-BP1 functions as a geroprotector to mitigate human stem cell senescence and maintain mitochondrial homeostasis, particularly for the mitochondrial respiration complex III, thus providing a new potential target to counteract human stem cell senescence.
Mesenchymal Stem Cells/physiology* ; Cellular Senescence ; Homeostasis ; Cell Cycle Proteins/metabolism* ; Adaptor Proteins, Signal Transducing/metabolism* ; Mitochondria/metabolism* ; Electron Transport Complex III/metabolism* ; Humans ; Cells, Cultured