METTL3 mediates bone marrow mesenchymal stem cell adipogenesis to promote chemoresistance in acute myeloid leukaemia.

Adipogenesis of bone marrow mesenchymal stem cells (MSCs) promotes chemoresistance of acute myeloid leukaemia (AML) cells. MSCs from AML patients (AML-MSCs) display enhanced adipogenesis compared with bone marrow MSCs from healthy donors. However, the precise molecular mechanism by which adipogenesis of MSCs from AML marrow differs from normal counterparts remains obscure. We found that METTL3 significantly inhibits MSC adipogenesis. Here, we aimed to identify the molecular mechanism linking METTL3 and MSC adipogenesis. Analysis of m6 A epigenetic changes in MSCs determined via RIP-qPCR and MeRIP-qPCR indicated that METTL3 affects AKT protein expression in MSCs by mediating m6 A modification of AKT1-mRNA. Downregulated METTL3 expression in AML-MSCs induced an increase in AKT protein, resulting in enhanced MSC adipogenesis, thereby contributing to chemoresistance in AML cells. Therefore, targeting AKT regulation by mRNA modification in MSC adipogenesis might provide a novel therapeutic strategy to overcome AML chemoresistance.

SUVmax of 18FDG PET/CT Predicts Histological Grade of Lung Adenocarcinoma.

OBJECTIVES: The relationship between the 18FDG PET-CT maximum standard uptake value (SUVmax) and the type of lung adenocarcinoma is still not established. The aim of this study was to investigate the relationship between SUVmax value and histological grade and pathological subtype of lung adenocarcinoma, and to determine the optimum SUVmax cutoffs for distinguishing different histological grades. MATERIALS AND METHODS: The data of 618 lung adenocarcinoma patients were retrospectively analyzed. The relationship between SUVmax measured on preoperative 18FDG-PET-CT and the histological grade and pathological subtype was examined. The Kruskal-Wallis test was used to compare differences among groups, and the Bonferroni-Dunn test for pairwise comparison among groups. ROC analysis was applied to determine the optimal cut-off values for distinguishing different groups. In addition, the cut-off value was verified in an independent cohort of 85 consecutive lung adenocarcinoma cases. RESULTS: The SUVmax was significantly different between the low, intermediate, and high-grade groups(p < .001). SUVmax value increased with increase in the degree of malignancy. The optimal cut-off value for identifying low-grade tumors was 2.01 (sensitivity 90.4%, specificity 86.9%, area under the curve [AUC] = 0.928, 95% confidence interval: 0.91-0.95; p < .001). The optimal cutoff SUVmax value for identifying high-grade tumors was 7.41 (sensitivity 79.8%, specificity 73.5%, AUC = 0.830, 95% confidence interval: 0.79-0.87; p < .001). The validation experiment showed that the coincidence rate was 88.89% in the low-level group, 64.15% in the middle-level group, and 78.57% in the high-level group. CONCLUSION: SUVmax can be used to predict pathological subtype and histological grade of lung adenocarcinoma. Thus, 18FDG PET-CT can serve as a noninvasive tool for precise diagnosis and help in the preoperative formulation of patient-specific treatment strategies.

Hepatitis B virus X protein promotes liver cell pyroptosis under oxidative stress through NLRP3 inflammasome activation.

OBJECTIVE: Hepatitis B virus X protein (HBx) is a pivotal factor for HBV-induced hepatitis. Herein, we sought to investigate HBx-mediated NLR pyrin domain containing 3 (NLRP3) inflammasome activation and pyroptosis under oxidative stress. METHODS: The effect of HBx on the NLRP3 inflammasome was analyzed by enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence in hepatic HL7702 cells. Pyroptosis was evaluated by western blotting, lactate dehydrogenase release, propidium iodide staining, and transmission electron microscopy. NLRP3 expression in the inflammasome from liver tissues was assessed by immunohistochemistry. RESULTS: In hydrogen peroxide (H2O2)-stimulated HL7702 cells, HBx triggered the release of pro-inflammatory mediators apoptosis-associated speck-like protein containing a CARD (ASC), interleukin (IL)-1ß, IL-18, and high-mobility group box 1 (HMGB1); activated NLRP3; and initiated pro-inflammatory cell death (pyroptosis). HBx localized to the mitochondria, where it induced mitochondrial damage and production of mitochondrial reactive oxygen species (mitoROS). Treatment of HL7702 cells with a mitoROS scavenger attenuated HBx-induced NLRP3 activation and pyroptosis. Expression levels of NLRP3, ASC, and IL-1ß in liver tissues from patients were positively correlated with HBV DNA concentration. CONCLUSIONS: The NLRP3 inflammasome was activated by elevated mitoROS levels and mediated HBx-induced liver inflammation and hepatocellular pyroptosis under H2O2-stress conditions.

Effect of HBx on inflammation and mitochondrial oxidative stress in mouse hepatocytes.

Hepatitis B virus × protein (HBx) serves an important role in the pathogenesis of the hepatitis B virus infection. Previous studies have reported that the interaction between HBx and hepatocyte mitochondria is an important factor leading to liver cell injury and apoptosis, ultimately inducing the formation of liver cancer. In the present study, a mouse model expressing HBx was constructed using hydrodynamic in vivo transfection based on the interaction between HBx and cytochrome c oxidase (COX) subunit III. The specific mechanism of HBx-induced oxidative stress in mouse hepatocytes and the subsequent effect on mitochondrial function and inflammatory injury was assessed. The results demonstrated that HBx reduced the activity of COX and the expression of superoxide dismutase and upregulated the expression of malondialdehyde, NF-κB and phospho-AKT, thus increasing oxidative stress. In addition, HBx induced an increase in interleukin (IL)-6, IL-1ß and IL-18 expression levels, which created an inflammatory microenvironment in the liver, further promoting hepatocyte inflammatory injury. Therefore, it was proposed that HBx may affect hepatocyte mitochondrial respiration by reducing the activity of cytochrome c oxidase, leading to mitochondrial dysfunction and inducing hepatocyte inflammation and injury.

12-Lipoxygenase promotes epithelial-mesenchymal transition via the Wnt/ß-catenin signaling pathway in gastric cancer cells.

BACKGROUND: 12-Lipoxygenase (12-LOX) plays a major role in the progression and metastasis of various types of cancer. In gastric cancer (GC), the expression level of 12-LOX is significantly up-regulated; however, its function, and underlying mechanism of action remain unclear. METHODS: The mRNA and protein expression levels of 12-LOX were assessed using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analyses, respectively, in GC cell lines. 12-LOX expression was stably up-regulated using lentiviral vector in BGC823 and MGC803 cells, and cell-counting kit-8 (CCK8), colony formation, and invasion assays were performed to verify the function of 12-LOX in proliferation and metastasis. In addition, the expression levels of epithelial-mesenchymal transition (EMT) differentiation markers and downstream targets of the Wnt/ß-catenin signaling pathway were examined by Western blotting. A nude mouse model of tumor growth and metastasis was established to investigate the role of 12-LOX in vivo. RESULTS: Our findings demonstrate that 12-LOX mRNA and protein were highly expressed in GC cell lines. 12-LOX overexpression promoted GC cell proliferation, migration, and invasion both in vitro and in vivo. In addition, up-regulation of 12-LOX promoted the EMT in GC cells, as reflected by a decrease in E-cadherin expression and an increase in N-cadherin and Snail expression. 12-LOX overexpression in GC cells also increased the expression of multiple downstream targets of the Wnt/ß-catenin signaling pathway. CONCLUSION: These findings revealed that 12-LOX functions as an oncogene in promoting GC cell proliferation and metastasis in vitro and in vivo. In addition, 12-LOX might regulate the EMT via the Wnt/ß-catenin signaling pathway, indicating a potential role for 12-LOX as a target in GC treatment.

Biodistribution characteristics and SPECT imaging of (99m)Tc-RET and (99m)Tc-REG in human lung cancer xenografts.

OBJECTIVE: To investigate the biodistribution and single-photon emission computed tomography (SPECT) imaging of (99m)Tc-labeled arginine-glutamic acid-threonine (RET) and arginine-glutamic acid-glycine (REG) in nude mice bearing human lung cancer xenografts. MATERIALS AND METHODS: RET and REG were labeled directly with (99m)Tc and their binding efficiency to tumor cells was measured in human nonsmall cell lung cancer H1299 cells. After intravenously injecting (99m)Tc-RET and (99m)Tc-REG into normal mice and nude mice bearing human lung cancer xenografts, their biodistribution was measured at different postinjection times, and percentages of injected dose per gram tissue (% ID/g) of organs of interest were calculated. The mice bearing H1299 lung cancer xenografts were scanned by SPECT at different times following the (99m)Tc-RET or (99m)Tc-REG injection. RESULTS: The radiochemical purity of (99m)Tc-RET and (99m)Tc-REG was 93.15%±2.02% and 92.90%±2.86%, respectively. The binding rate of (99m)Tc-RET and (99m)Tc-REG to H1299 cells was 3.56%±0.37% and 2.32%±0.31%, respectively. The uptake of (99m)Tc-RET and (99m)Tc-REG in tumor was 4.96±1.05% ID/g at 4 hours postinjection and 1.95±0.73% ID/g at 2 hours postinjection, respectively. Tumors in nude mice could be best imaged at 4.5-6 hours postinjection of (99m)Tc-RET. CONCLUSION: (99m)Tc-RET has a higher binding rate to H1299 cells than (99m)Tc-REG and might be used as a potential lung cancer imaging agent.

Novel esophageal squamous cell carcinoma bone metastatic clone isolated by scintigraphy, X ray and micro PET/CT.

AIM: To establish a Chinese esophageal squamous cell carcinoma (ESCC) cell line with high bone metastasis potency using (99m)Tc-methylene diphosphonate ((99m)Tc-MDP) micro-pinhole scintigraphy, X ray and micro-positron emission tomography/computed tomography (PET/CT) for exploring the mechanism of occurrence and development in esophageal cancer. METHODS: The cells came from a BALB/c nu/nu immunodeficient mouse, and oncogenic tumor tissue was from a surgical specimen from a 61-year-old male patient with ESCC. The cell growth curve was mapped and analysis of chromosome karyotype was performed. Approximately 1 × 106 oncogenic cells were injected into the left cardiac ventricle of immunodeficient mice. The bone metastatic lesions of tumor-bearing mice were detected by (99m)Tc-MDP scintigraphy, micro-PET/CT and X-ray, and were resected from the mice under deep anesthesia. The bone metastatic cells in the lesions were used for culture and for repeated intracardiac inoculation. This in vivo/in vitro experimental metastasis study was repeated for four cycles. All of the suspicious bone sites were confirmed by pathology. Real-time polymerase chain reaction was used to compare the gene expression in the parental cells and in the bone metastatic clone. RESULTS: The surgical specimen was implanted subcutaneously in immunodeficient mice and the tumorigenesis rate was 100%. First-passage oncogenic cells were named CEK-Sq-1. The chromosome karyotype analysis of the cell line was hypotriploid. The bone metastasis rate went from 20% with the first-passage oncogenic cells via intracardiac inoculation to 90% after four cycles. The established bone metastasis clone named CEK-Sq-1BM had a high potential to metastasize in bone, including mandible, humerus, thoracic and lumbar vertebrae, scapula and femur. The bone metastasis lesions were successfully detected by micro-pinhole bone scintigraphy, micro-PET/CT, and X-ray. The sensitivity, specificity and accuracy of the micro-pinhole scintigraphy, X-ray, and micro-PET/CT imaging examinations were: 89.66%/32%/80%, 88.2%/100%/89.2%, and 88.75%/77.5%/87.5%, respectively. Some gene expression difference was found between parental and bone metastasis cells. CONCLUSION: This newly established Chinese ESCC cell line and animal model may provide a useful tool for the study of the pathogenesis and development of esophageal carcinoma.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation.

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia. .

Tissue-targeting lead generation and optimization from random and directed screening of technetium-99m labeled tripeptide complex libraries in vivo.

BACKGROUND: Screening libraries against a molecular target in vitro are idealized models that cannot reflect the real state in vivo where biomolecules coexist and interact. C-terminal amide tripeptides labelled with Technetium-99m can provide a unique noninvasive approach to trace a large number of compounds in vivo. METHODS: The C-terminal amide tripeptide libraries were synthesized on Rink Amide-MBHA resin using iterative and pooling protocol. Technetium (V) oxo core [TcO(3+)] was bound to each tripeptide via 4 deprotonated nitrogen atoms to form a library of 8000 (99m)Tc tripeptoid complexes. The radiocombinatorial screening (RCS) in vivo was carried out on SD rats and A549 tumour bearing mice. RESULTS: Signals of tissue distribution and metabolism of libraries were recorded by counting or imaging and tissue targeting leads identified by both random and directed RCS. Among them, (99m)Tc RPA, (99m)Tc VIG and (99m)Tc RES had specific tissue targeting in kidney, liver and tumour respectively. The percent injected dose per gram tissue of (99m)Tc labelled leads in their target tissue was highly structure dependent. Because the nontarget tissue binding and the metabolism of (99m)Tc tripeptoid sublibraries were simultaneously monitored successfully by RCS, the interference of background activity was limited to the lowest level. Optimization of renal function agent from the labelled libraries was carried out by directed screening. (99m)Tc DSG was finally identified the most promising agent for renal function studies. CONCLUSIONS: RCS in vivo is a powerful tool for the discovery of tissue targeting drugs. The potential screening bias is probably the major limitation of labelled libraries.

Half-metallic ferromagnetism and structural stability of zincblende phases of the transition-metal chalcogenides.

An accurate density-functional method is used to study systematically half-metallic ferromagnetism and stability of zincblende phases of 3d-transition-metal chalcogenides. The zincblende CrTe, CrSe, and VTe phases are found to be excellent half-metallic ferromagnets with large half-metallic gaps (up to 0.88 eV). They are mechanically stable and approximately 0.31-0.53 eV per formula unit higher in total energy than the corresponding nickel-arsenide ground-state phases, and therefore would be grown epitaxially in the form of films and layers thick enough for spintronic applications.