OBJECTIVE: To explore the effect and mechanism of DNA methyltransferase 1 (DNMT1) knockout on the inhibition of acute myeloid leukemia (AML) by natural killer (NK) cells. METHODS: The peripheral blood NK cells of AML patients and controls were collected, and the mRNA and protein level of DNMT1 were measured by PCR and Western blot, respectively. The DNMT1 knockout mice were constructed to obtain NK^DNMT1-/- cells. The NK cells were stimulated with interleukin (IL)-12, IL-15, and IL-18 to construct memory NK cells, and then the interferon-γ (IFN-γ) levels were measured by ELISA. After co-culturing with memory NK cells and HL60 cells, the killing effect of NK^DNMT1-/- cells on HL60 cells was detected by LDH assay. Then, the HL60 cell apoptosis and NK cell NKG2D level were measured by flow cytometry. The perforin and granzyme B protein levels of NK cells were measured by Western blot. The AML model mice were constructed by injecting HL60 cells into the tail vein, meanwhile, memory NK cells were also injected, and then the mouse weights, CD33 positive rates, and survival time were detected. RESULTS: The mRNA and protein levels of DNMT1 in NK cells of AML patients were significantly higher than those in the control group (both P < 0.01), while the IFN-γ level induced by interleukin was significantly lower than that in the control group (P < 0.05). Compared with NK^DNMT1+/+ cells, the ability of NK^DNMT1-/- cells to secrete IFN-γ after interleukin stimulation was significantly increased (P < 0.05). The killing and apoptosis-inducing effects of NK^DNMT1-/- cells on HL60 cells were significantly stronger than those of NK^DNMT1+/+ cells (both P < 0.05). The NKG2D level and expression of perforin and granzyme B of NK^DNMT1-/- cells were significantly increased compared with NK^DNMT1+/+ cells (all P < 0.05). Compared with AML mice injected with NK^DNMT1+/+ cells, AML mice injected with NK^DNMT1-/- cells showed significantly increased body weight, decreased CD33 positive rate, and prolonged survival time (all P < 0.05). CONCLUSION Knocking out DNMT1 can enhance the inhibitory effect of NK cells on AML, which may be related to enhancing NK cell memory function.
Killer Cells, Natural/metabolism* ; Animals ; Leukemia, Myeloid, Acute ; Humans ; DNA (Cytosine-5-)-Methyltransferase 1 ; Mice ; Mice, Knockout ; HL-60 Cells ; Apoptosis ; Interferon-gamma/metabolism* ; Granzymes/metabolism* ; Perforin/metabolism* ; NK Cell Lectin-Like Receptor Subfamily K/metabolism*

OBJECTIVE: To explore the effect of bear bile powder (BBP) on acute lung injury (ALI) and the underlying mechanism. METHODS: The chemical constituents of BBP were analyzed by ultra-high-pressure liquid chromatography-mass spectrometry (UPLC-MS). After 7 days of adaptive feeding, 50 mice were randomly divided into 5 groups by a random number table (n=10): normal control (NC), lipopolysaccharide (LPS), dexamethasone (Dex), low-, and high-dose BBP groups. The dosing cycle was 9 days. On the 12th and 14th days, 20 µL of Staphylococcus aureus solution (bacterial concentration of 1 × 10^-7 CFU/mL) was given by nasal drip after 1 h of intragastric administration, and the mice in the NC group was given the same dose of phosphated buffered saline (PBS) solution. On the 16th day, after 1 h intragastric administration, 100 µL of LPS solution (1 mg/mL) was given by tracheal intubation, and the same dose of PBS solution was given to the NC group. Lung tissue was obtained to measure the myeloperoxidase (MPO) activity, the lung wet/dry weight ratio and expressions of CD14 and other related proteins. The lower lobe of the right lung was obtained for pathological examination. The concentrations of inflammatory cytokines including interleukin (IL)-6, tumour necrosis factor α (TNF-α ) and IL-1β in the bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay, and the number of neutrophils was counted. The colonic contents of the mice were analyzed by 16 sRNA technique and the contents of short-chain fatty acids (SCFAs) were measured by gas chromatograph-mass spectrometer (GC-MS). RESULTS: UPLC-MS revealed that the chemical components of BBP samples were mainly tauroursodeoxycholic acid and taurochenodeoxycholic acid sodium salt. BBP reduced the activity of MPO, concentrations of inflammatory cytokines, and inhibited the expression of CD14 protein, thus suppressing the activation of NF-κB pathway (P<0.05). The lung histopathological results indicated that BBP significantly reduced the degree of neutrophil infiltration, cell shedding, necrosis, and alveolar cavity depression. Moreover, BBP effectively regulated the composition of the intestinal microflora and increased the production of SCFAs, which contributed to its treatment effect (P<0.05). CONCLUSIONS BBP alleviates lung injury in ALI mouse through inhibiting activation of NF-κB pathway and decreasing expression of CD14 protein. BBP may promote recovery of ALI by improving the structure of intestinal flora and enhancing metabolic function of intestinal flora.
Animals ; Acute Lung Injury/pathology* ; Lipopolysaccharides ; Ursidae ; Gastrointestinal Microbiome/drug effects* ; Bile/chemistry* ; Lipopolysaccharide Receptors/metabolism* ; Powders ; Male ; Lung/drug effects* ; Mice ; Peroxidase/metabolism* ; Signal Transduction/drug effects* ; Cytokines/metabolism*

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin regions, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at the single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences on TF binding. Notably, cFOOT-seq, combined with FootTrack analysis, enables de novo prediction of TF binding sites and tracking of TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.
Transcription Factors/genetics* ; Humans ; Chromatin/genetics* ; Animals ; Binding Sites ; Mice ; DNA Footprinting/methods*

Objective To explore the otherness of orthotopic injection of cell suspensions and transplantation of tumor tissue blocks to establish orthotopic implantation models of hepatocellular carcinoma in mice,and to provide a technical reference for the establishment of an orthotopic implantation model.Methods Healthy KM mice were divided into four groups:group A,direct injection of H22 cells;group B,direct injection of H22 ascitic cells;group C,transplantation of tissues;and group D,direct injection of saline.Activity and weight changes were observed regularly in each group and survival times were recorded.Liver tumor formation,tumor size,abdominal organ adhesion degree,and metastasis were observed in all groups.B-ultrasound imaging was performed,concentrations of alpha fetoprotein(AFP)and abnormal prothrombin(DCP)were detected,and liver histopathological changes were detected by hematoxylin and eosin staining.Results Mice molding operation time in groups A,B,and C were(3.36±0.44)min,(3.30±0.41)min,and(5.68±0.65)min,respectively.After modeling for 25 days,the rates of model formation in groups A,B,and C were all 100.0%.Severe abdominal adhesions occurred in 40.0%of mice in group A and 60.0%in group B,but in no mice in group C or D.Ascites occurred in 40.0%,100.0%,and 0.0%and abdominal wall tumors in 30.0%,60.0%,and 0.0%of mice in groups A,B,and C,respectively,while 40.0%of mice in group B also had liver metastasis.B-ultrasound imaging,detection of serum AFP and DCP levels,and histopathological result showed smooth liver margins,uneven echo and slightly lower echo mass,maintained high AFP and DCP secretion,and large numbers of inflammatory cells and tumor cells in mice in groups A,B,and C.Conclusions At day 25,all three methods can thus be used to establish orthotopic transplantation models of HCC.Among these,inj ection of cell suspensions demonstrated the advantage of simplicity in operation and the presence of multiple metastatic nodules within the liver,compared to transplantation of tumor tissue.Conversely,transplantation of tumor tissue showed the advantage of causing less impact on the abdomen and other organs when compared to inj ection of cell suspensions.

Objective:To compare the efficacy of amide proton transfer-weighted (APTw) imaging with time-dependent diffusion MRI (td-dMRI) in the diagnosis of malignant breast lesions.Methods:This study was a cross-sectional study. The clinical, pathological and imaging data of patients with breast lesions admitted to the First Affiliated Hospital of Zhengzhou University from March to August 2023 were prospectively analyzed. All patients firstly underwent T 2WI, diffusion-weighted imaging, followed by dynamic contrast-enhanced MRI (DCE-MRI), and finally APTw imaging and td-dMRI were performed for breast lesions using DCE-MRI as reference. Reconstructed images from APTw imaging measured lesions with a frequency shift of 3.5 ppm asymmetric magnetic susceptibility MTR asym(+3.5 ppm). The apparent diffusion coefficient (ADC) values at different oscillating frequency gradients (ADC PGSE, ADC 17 Hz, ADC 33 Hz values) were measured using reconstructed td-dMRI images. Independent sample t-test was used to compare APTw imaging, td-dMRI parameter differences between benign and malignant breast tumors, breast malignant tumors with different molecular types [estrogen receptor (ER) negative and positive, progesterone receptor (PR) negative and positive, human epidermal growth factor receptor (HER-2) negative and positive, proliferation index (Ki-67) low and high expression] and different histological grades (grade Ⅱ and Ⅲ). Receiver operating characteristic curve and area under the curve (AUC) were used to evaluate the efficacy of APTw imaging and td-dMRI parameters in differentiating benign and malignant breast tumors, molecular classification and histological grading of malignant breast lesions. Results:There were 171 lesions in 171 patients, including 103 malignant lesions and 68 benign lesions. Histological grades were grade Ⅱ in 51 cases and grade Ⅲ in 38 cases of 89 cases of invasive carcinoma. Totally 98 cases of malignant lesions were included in molecular typing analysis, 36 cases were ER negative and 62 cases were ER positive. PR was negative in 51 cases and positive in 47 cases. There were 33 negative HER-2 patients, 65 positive HER-2 patients. There were 50 cases of low Ki-67 expression and 48 cases of high Ki-67 expression. The MTR asym(+3.5 ppm) value of malignant breast lesions was higher than that of benign lesions ( t=5.76, P<0.001), and the ADC PGSE, ADC 17 Hz and ADC 33 Hz values were lower than those of benign breast lesions ( t was 4.84, 4.62, 4.01, respectively, all P<0.001). MTR asym(+3.5 ppm) had the highest AUC value (0.83) and the highest specificity (90.38%), and ADC PGSE had the highest sensitivity (85.86%). There were no significant differences in MTR asym(+3.5 ppm), ADC PGSE, ADC 17 Hz and ADC 33 Hz between grade Ⅱ and grade Ⅲ histological grades of malignant breast lesions (all P>0.05). The ADC PGSE value of ER negative was higher than that of ER positive ( t=2.34, P=0.018), and the AUC for distinguishing ER positive from negative was 0.64. The ADC PGSE and ADC 17 Hz values of PR negative were higher than those of PR positive ( t=2.87, 2.81, P=0.004, 0.006, respectively), and their AUCs for identifying PR positive versus negative breast malignant lesions were 0.68 and 0.67, respectively. The ADC 33 Hz value of negative HER-2 was lower than that of positive HER-2 ( t=3.00, P=0.003), and the AUC for distinguishing positive and negative HER-2 was 0.67. There were no significant differences in other parameters among different subtypes of breast malignant lesions (all P>0.05). Conclusion:Compared with td-dMRI, APTw imaging is more effective in differentiating benign and malignant lesions of breast tumors, and ADC values at different gradient oscillation frequencies obtained by td-dMRI show better diagnostic efficacy in differentiating different molecular types of breast malignant lesions.

Objective To analyze the effect of Meleis transition theory in the intervention of family caregivers of advanced lung cancer patients.Methods From January 2022 to June 2023,94 patients with advanced lung cancer and their family caregivers treated in 4 hospitals of a tertiary A hospital in Zhejiang Province were selected by convenience sampling method,and they were divided into a control group(n=47)and an experimental group(n=47)according to random number table method.The family caregivers of the experimental group received a four-week intervention based on the Meleis transition theory on the basis of routine nursing education,including role recognition,disease knowledge,life care,psychological support,and social resource connection.Family caregivers in the control group received routine nursing education,caregiving education and guidance.The differences in the readiness,caring ability,psychological burden and patients'quality of life were compared between the 2 groups.Results There was no lost follow-up cases in the control group and experimental group.After intervention,the preparation,ability,and quality of life of family caregivers in the experimental group were better than those in the control group,and the psychological burden of caregivers was significantly lower than that in the control group(P＜0.05).Conclusion The intervention of caregiving ability of family caregivers based on Meleis transition theory can effectively improve the caregiving ability of caregivers,reduce the psychological burden of caregivers,improve the quality of care,and improve the quality of life of patients.

Traditional acute kidney injury (AKI) classifications, which are centered around semi-anatomical lines, can no longer capture the complexity of AKI. By employing strategies to identify predictive and prognostic enrichment targets, experts could gain a deeper comprehension of AKI’s pathophysiology, allowing for the development of treatment-specific targets and enhancing individualized care. Subphenotyping, which is enriched with AKI biomarkers, holds insights into distinct risk profiles and tailored treatment strategies that redefine AKI and contribute to improved clinical management. The utilization of biomarkers such as N-acetyl-β-D-glucosaminidase, tissue inhibitor of metalloprotease-2·insulin-like growth factor-binding protein 7, kidney injury molecule-1, and liver fatty acid-binding protein garnered significant attention as a means to predict subclinical AKI. Novel biomarkers offer promise in predicting persistent AKI, with urinary motif chemokine ligand 14 displaying significant sensitivity and specificity. Furthermore, they serve as predictive markers for weaning patients from acute dialysis and offer valuable insights into distinct AKI subgroups. The proposed management of AKI, which is encapsulated in a structured flowchart, bridges the gap between research and clinical practice. It streamlines the utilization of biomarkers and subphenotyping, promising a future in which AKI is swiftly identified and managed with unprecedented precision. Incorporating kidney biomarkers into strategies for early AKI detection and the initiation of AKI care bundles has proven to be more effective than using care bundles without these novel biomarkers. This comprehensive approach represents a significant stride toward precision medicine, enabling the identification of high-risk subphenotypes in patients with AKI.

Hepatocellular carcinoma(HCC)is one of the most common tumor types and remains a major clinical challenge.Increasing evidence has revealed that mitophagy inhibitors can enhance the effect of chemotherapy on HCC.However,few mitophagy inhibitors have been approved for clinical use in humans.Pyrimethamine(Pyr)is used to treat infections caused by protozoan parasites.Recent studies have reported that Pyr may be beneficial in the treatment of various tumors.However,its mechanism of action is still not clearly defined.Here,we found that blocking mitophagy sensitized cells to Pyr-induced apoptosis.Mechanistically,Pyr potently induced the accumulation of autophagosomes by inhibiting autophagosome-lysosome fusion in human HCC cells.In vitro and in vivo studies revealed that Pyr blocked autophagosome-lysosome fusion by upregulating BNIP3 to inhibit synaptosomal-associated protein 29(SNAP29)-vesicle-associated membrane protein 8(VAMP8)interaction.Moreover,Pyr acted synergistically with sorafenib(Sora)to induce apoptosis and inhibit HCC proliferation in vitro and in vivo.Pyr enhances the sensitivity of HCC cells to Sora,a common chemotherapeutic,by inhibiting mitophagy.Thus,these results provide new insights into the mechanism of action of Pyr and imply that Pyr could potentially be further developed as a novel mitophagy inhibitor.Notably,Pyr and Sora combination therapy could be a promising treatment for malignant HCC.

OBJECTIVE: To explore the mechanisms of large-conductance calcium-activated potassium channel (BKCa) involved in inflammatory response in sepsis. METHODS: The serum levels of BKCa were measured by enzyme-linked immunosorbent assay (ELISA) in patients with sepsis (28 cases), patients with common infection (25 cases) and healthy people (25 cases). The relationship between levels of BKCa and acute physiology and chronic health evaluation II (APACHE II) were analyzed. Cultured RAW 264.7 cells were stimulated by lipopolysaccharide (LPS). In some experiments, a cell model of sepsis was constructed using Nigericin as the second stimulus signal. The mRNA and protein expressions of BKCa in RAW 264.7 cells stimulated with LPS (0, 50, 100, 1 000 μg/L) were measured by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. RAW 264.7 cells were transfected with small interfering RNA of BKCa (siRNA-BKCa), and the levels of caspase-1 precursor (pro-caspase-1), interleukin-1β precursor (pro-IL-1β) in cell, and the levels of caspase-1 p20, IL-1β p17 of cell culture medium, and NOD-like receptor protein 3 (NLRP3), nuclear factor-κB (NF-κB) were measured by Western blotting. The apoptosis were detected by staining with propidium iodide (PI), the release rate of lactate dehydrogenase (LDH) were measured, and the expression of apoptotic protein Gasdermin D (GSDMD) was measured by Western blotting to evaluate the effect of silencing BKCa on cell pyrosis. RESULTS: The level of serum BKCa in patients with sepsis was significantly higher than that in patients with common infection and health peoples (ng/L: 165.2±25.9 vs. 102.5±25.9, 98.8±20.0, both P < 0.05). In addition, the level of serum BKCa in patients with sepsis was significantly positively correlated with APACHE II score (r = 0.453, P = 0.013). LPS could construct a sepsis cell model by which LPS could promote BKCa expression in mRNA and protein with a concentration-dependent manner. The mRNA and protein expressions of BKCa in the cells stimulated by 1 000 μg/L LPS were significantly higher than that in the blank group (0 μg/L) [BKCa mRNA (2^-ΔΔCt): 3.00±0.36 vs. 1.00±0.16, BKCa/β-actin: 1.30±0.16 vs. 0.37±0.09, both P < 0.05]. Compared with the control group, the ratios of caspase-1 p20/pro-caspase-1 and IL-1β p17/pro-IL-1β in the model group were significantly increased (caspase-1 p20/pro-caspase-1: 0.83±0.12 vs. 0.27±0.05, IL-1β p17/pro-IL-1β: 0.77±0.12 vs. 0.23±0.12, both P < 0.05), however, transfection of siRNA-BKCa induced the decrease both of them (caspase-1 p20/pro-capase-1: 0.23±0.12 vs. 0.83±0.12, IL-1β p17/pro-IL-1β: 0.13±0.05 vs. 0.77±0.12, both P < 0.05). Compared with the control group, the number of apoptotic cells, LDH release rate and GSDMD expression in the model group were significantly increased [LDH release rate: (30.60±8.40)% vs. (15.20±7.10)%, GSDMD-N/GSDMD-FL: 2.10±0.16 vs. 1.00±0.16, both P < 0.05], however, transfection of siRNA-BKCa induced the decrease both of them [LDH release rate: (15.60±7.30)% vs. (30.60±8.40)%, GSDMD-N/GSDMD-FL: 1.13±0.17 vs. 2.10±0.16, both P < 0.05]. The mRNA and protein expressions of NLRP3 in sepsis cells were significantly higher than those in the control group [NLRP3 mRNA (2^-ΔΔCt): 2.06±0.17 vs. 1.00±0.24, NLRP3/GAPDH: 0.46±0.05 vs. 0.15±0.04, both P < 0.05]. However, the expression of NLRP3 after siRNA-BKCa transfection was significantly lower than that in model group [NLRP3 mRNA (2^-ΔΔCt): 1.57±0.09 vs. 2.06±0.17, NLRP3/GAPDH: 0.19±0.02 vs. 0.46±0.05, both P < 0.05]. Compared with the control group, the NF-κB p65 nuclear transfer of sepsis cell were significantly increased (NF-κB p65/Histone: 0.73±0.12 vs. 0.23±0.09, P < 0.05). However, the NF-κB p65 expression in the nucleus were decreased after siRNA-BKCa transfection (NF-κB p65/Histone: 0.20±0.03 vs. 0.73±0.12, P < 0.05). CONCLUSIONS BKCa is involved in the pathogenesis of sepsis, and its possible mechanism is to activate NF-κB/NLRP3/caspase-1 signaling pathway to induce inflammatory factor production and cell death.
Humans ; Histones ; Caspase 1 ; Large-Conductance Calcium-Activated Potassium Channels ; Lipopolysaccharides ; NF-kappa B ; NLR Family, Pyrin Domain-Containing 3 Protein ; L-Lactate Dehydrogenase ; Sepsis ; RNA, Small Interfering ; Caspases

As a recognized rare and highly fatal disease, hereditary angioedema (HAE) is difficult to diagnose and characterized by recurrent edema involving the head, limbs, genitals and larynx, etc. Diagnosis of HAE is not difficult. However, low incidence and lack of clinical characteristics lead to difficulty of doctors on timely diagnosis and correct intervention for HAE patients. Therefore, it is crucial to improve the awareness of this disease and prevent its recurrence. for HAE patients. In view of absent cognition of doctors and the general public on HAE, patients often suffer from sudden death or become disabled due to laryngeal edema which cannot be treated in time. Thus, based on the Internet mobile terminal platform, the team set up an all-day rapid emergency response system which is provided for HAE patients by setting up "one-click help". The aim is to offer optimization on overall management of HAE and designed the intelligent follow-up management to provide timely assistance and specialized suggestion for patients with acute attacks.
Humans ; Angioedemas, Hereditary/drug therapy*