[Characterization and identification of chemical components from Euodiae Fructus based on UHPLC-Q-TOF-MS].

A sensitive and efficient ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry(UHPLC-Q-TOF-MS) approach was established. Based on the self-developed information library, the chemical components from Euodiae Fructus were systematically characterized and identified. The chromatographic separation conditions(e. g., stationary phase,mobile phase, column temperature, and elution gradient) and MS detection conditions(nozzle voltage, capillary voltage, fragmentor,and collision energy) were optimized. Ultimately, an HSS T3 column(2. 1 mm×100 mm, 1. 8 µm) maintained at 35 ℃ was used,and 0. 1% formic acid water-acetonitrile at the flow rate of 0. 4 m L·min~(-1) was used as the mobile phase. Electrospray ionization was adopted to collect the positive and negative ion mass spectrometry data in Auto MS/MS mode. According to the reference compound comparison, fragment ion information interpretation, literature, and retrieval in the self-developed information library, 92 compounds were characterized or derived from the decoction of Euodiae Fructus, including 33 alkaloids, 23 flavonoids, 12 terpenoids, 12phenylpropanoids, and 12 others. Among them, 17 compounds were identified by comparison with the reference compounds, and 11compounds were unreported from Euodiae Fructus. This study realizes the rapid characterization and identification of multi-class chemical components in the decoction of Euodiae Fructus and provides a reference for the studies regarding its effective substances and quality control.

Evaluation and comparison of liquid chromatography/high-resolution mass spectrometry platforms for the separation and characterization of ginsenosides from the leaves of Panax ginseng.

The measurement of data repeatability in small-molecule metabolites acquired within and among different liquid chromatography-mass spectrometry (LC-MS) platforms is crucial for data sharing or data transfer in natural products research. This work was designed to investigate and evaluate the separation and detection performance of three commercial high-resolution LC-MS platforms (e.g., Agilent 6550 QTOF, Waters Vion IM-QTOF, and Thermo Scientific Orbitrap Exploris 120) using 68 ginsenoside references and the extract of Panax ginseng leaf. The retention time (tR), measured on these three platforms (under the same chromatography condition), showed good stability in different concentration tests, and within/among different instruments for both intra-day and inter-day precision examinations. Correlation in tR of ginsenosides was also highly determined on these three platforms. In spite of the different mass analyzers involved, these three platforms gave the accurate mass determination ability, especially enhanced resolution gained because of the ion mobility (IM) separation facilitated by IM-quadrupole time-of-flight. The current study has systematically evaluated the separation and MS detection performance enabled by three high-resolution LC-MS platforms taking ginsenosides as the template, and the reported findings can benefit the researchers for the selection of analytical platforms and the purpose of data sharing or data transfer.

Marchaetoglobins A-D: Four Cytochalasans with Proangiogenic Activity from the Marine-Sponge-Associated Fungus Chaetomium globosum 162105.

Four new cytochalasans, marchaetoglobins A-D (1-4), along with five known compounds (5-9), were isolated from the marine-sponge-associated fungus Chaetomium globosum 162105. Compounds 1-4 represent examples of 19,20-seco-chaetoglobosins, of which compound 1 is the first furan-containing cytochalasan. Their structures and absolute configurations were elucidated by extensive spectroscopic analyses and electronic circular dichroism calculations. Compounds 5, 8, and 9 displayed weak to moderate antibacterial activities against Bacillus thuringiensis, Edwardsiella piscicida, Vibrio alginolyticus, and Pseudomonas syringae pv. actinidiae with minimum inhibitory concentration values ranging from 5 to 25 µg/mL. In addition, compounds 2, 3, and 5 showed potent in vivo proangiogenic activity in transgenic zebrafish, comparable to the positive control.

Marcytoglobosins A and B, Cytochalasans From a Marine Sponge Associated Chaetomium globosum 162105 Fungus.

Two new cytochalasans, marcytoglobosins A (1) and B (2) were isolated from the marine sponge associated fungus Chaetomium globosum 162105, along with six known compounds (3-8). The complete structures of two new compounds were determined based on 1D/2D NMR and HR-MS spectroscopic analyses coupled with ECD calculations. All eight isolates were evaluated for their antibacterial activity. Among them, compounds 3-8 displayed antibacterial effects against Staphylococcus epidermidis, Bacillus thuringiensis, Pseudomonas syringae pv. Actinidiae, Vibrio alginolyticus, and Edwardsiella piscicida with minimum inhibitory concentration (MIC) values ranging from 10 to 25 µg/mL.

Cardiovascular adverse events associated with immune checkpoint inhibitors: A retrospective multicenter cohort study.

BACKGROUND: Over the past decade, immune checkpoint inhibitors (ICIs) have significantly transformed cancer treatment. However, ICIs inevitably may cause a spectrum of immune-related adverse events, among which cardiovascular toxicity, particularly myocarditis, while infrequent, has garnered increasing attention due to its high fatality rate. METHODS: We conducted a multicenter retrospective study to characterize ICI-associated cardiovascular adverse events. Logistic regression was performed to explore the risk factors for the development of myocarditis and severe myocarditis. Receiver operating characteristic curves were conducted to assess the diagnostic abilities of cardiac biomarkers to distinguish different cardiovascular toxicities, and the performance and calibration were evaluated using Hosmer-Lemeshow test. RESULTS: Forty-four patients were identified, including thirty-five myocarditis, five heart failure, three arrhythmias, and one myocardial infarction. Compared with other patients, myocarditis patients had higher cardiac troponin-I (cTnI) levels (p < 0.001), higher creatine kinase levels (p = 0.003), higher creatine kinase isoenzyme-MB (CK-MB) levels (p = 0.013), and shorter time to the incidence of adverse cardiovascular events (p = 0.022) after ICI treatment. Twenty-one patients (60%) were classified as severe myocarditis, and they presented higher cardiac troponin I (cTnI) levels (p = 0.013), higher N-terminal pro-B-type natriuretic peptide levels (p = 0.031), higher creatine kinase levels (p = 0.018), higher CK-MB levels (p = 0.026), and higher neutrophil to lymphocyte ratio (NLR) levels (p = 0.016) compared to non-severe myocarditis patients after ICI treatment. Multivariate logistic regression showed that CK-MB (adjusted odds ratio [OR]: 1.775, 95% confidence interval [CI]: 1.055-2.984, p = 0.031) was the independent risk factor of the development of ICI-associated myocarditis, and cTnI (adjusted OR: 1.021, 95% CI: 1.002-1.039, p = 0.03) and NLR (adjusted OR: 1.890, 95% CI: 1.026-3.483, p = 0.041) were the independent risk factors of ICI-associated severe myocarditis. The receiver operating characteristic curve showed an area under curve of 0.785 (95% CI: 0.642 to 0.928, p = 0.013) for CK-MB, 0.765 (95% CI: 0.601 to 0.929, p = 0.013) for cTnI, and 0.773 for NLR (95% CI: 0.597 to 0.948, p = 0.016). CONCLUSIONS: Elevated CK-MB after ICI treatment is the independent risk factor for the incidence of ICI-associated myocarditis, and elevated cTnI and NLR after ICI treatment are the independent risk factors for the development of ICI-associated severe myocarditis. CK-MB, cTnI, and NLR demonstrated a promising predictive utility for the identification of ICI-associated myocarditis and severe myocarditis.

Meroterpenoids from Marine Sponge Hyrtios sp. and Their Anticancer Activity against Human Colorectal Cancer Cells.

Two new meroterpenoids, hyrtamide A (1) and hyrfarnediol A (2), along with two known ones, 3-farnesyl-4-hydroxybenzoic acid methyl ester (3) and dictyoceratin C (4), were isolated from a South China Sea sponge Hyrtios sp. Their structures were elucidated by NMR and MS data. Compounds 2-4 exhibited weak cytotoxicity against human colorectal cancer cells (HCT-116), showing IC50 values of 41.6, 45.0, and 37.3 µM, respectively. Furthermore, compounds 3 and 4 significantly suppressed the invasion of HCT-116 cells while also downregulating the expression of vascular endothelial growth factor receptor 1 (VEGFR-1) and vimentin proteins, which are key markers associated with angiogenesis and epithelial-mesenchymal transition (EMT). Our findings suggest that compounds 3 and 4 may exert their anti-invasive effects on tumor cells by inhibiting the expression of VEGFR-1 and impeding the process of EMT.

Mutagenetic analysis of the biosynthetic pathway of tetramate bripiodionen bearing 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione skeleton.

BACKGROUND: Natural tetramates are a family of hybrid polyketides bearing tetramic acid (pyrrolidine-2,4-dione) moiety exhibiting a broad range of bioactivities. Biosynthesis of tetramates in microorganisms is normally directed by hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) machineries, which form the tetramic acid ring by recruiting trans- or cis-acting thioesterase-like Dieckmann cyclase in bacteria. There are a group of tetramates with unique skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, which remain to be investigated for their biosynthetic logics. RESULTS: Herein, the tetramate type compounds bripiodionen (BPD) and its new analog, featuring the rare skeleton of 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione, were discovered from the sponge symbiotic bacterial Streptomyces reniochalinae LHW50302. Gene deletion and mutant complementation revealed the production of BPDs being correlated with a PKS-NRPS biosynthetic gene cluster (BGC), in which a Dieckmann cyclase gene bpdE was identified by sit-directed mutations. According to bioinformatic analysis, the tetramic acid moiety of BPDs should be formed on an atypical NRPS module constituted by two discrete proteins, including the C (condensation)-A (adenylation)-T (thiolation) domains of BpdC and the A-T domains of BpdD. Further site-directed mutagenetic analysis confirmed the natural silence of the A domain in BpdC and the functional necessities of the two T domains, therefore suggesting that an unusual aminoacyl transthiolation should occur between the T domains of two NRPS subunits. Additionally, characterization of a LuxR type regulator gene led to seven- to eight-fold increasement of BPDs production. The study presents the first biosynthesis case of the natural molecule with 3-(2H-pyran-2-ylidene)pyrrolidine-2,4-dione skeleton. Genomic mining using BpdD as probe reveals that the aminoacyl transthiolation between separate NRPS subunits should occur in a certain population of NRPSs in nature.

Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia.

BACKGROUND: Ferroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown. METHODS: Lipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo. RESULTS: We first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models. CONCLUSIONS: Our study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.

Isolation and absolute configuration of alkylpyridine alkaloids from the marine sponge Hippospongia lachne.

Marine sponges are well known as prolific producers of structurally diverse molecules with valuable pharmacological potential. As part of our ongoing program to discover bioactive compounds from marine sponges collected from the Xisha Islands in the South China Sea, a chemical study on the specimens of Hippospongia lachne was conducted. As a result, eight undescribed compounds, including four zwitterionic alkylpyridinium salts, hippospondines A-D (1-4), and four 3-alkylpyridine alkaloids, hippospondines E (5), F (6), and (±)-hippospondine G (7), were isolated from the marine sponge H. lachne, together with one known 3-alkylpyridine alkaloid (8). The undescribed structures were elucidated by HRESIMS, NMR, DP4+ and CP3 probability analysis, and the Snatzke's method. Hippospondines A-D (1-4) represent the rare example of inner salt type alkylpyridinium alkaloid with a farnesyl moiety. Compounds 1-3 and 8 were subjected to cytotoxic and lymphocyte proliferation assays. Compound 3 exhibited a weak promotion effect on the ConA-induced T lymphocyte proliferation.

The novel HLA-B*40:555 allele identified by Sanger dideoxy nucleotide sequencing in a Chinese individual.

HLA-B*40:555 differs from HLA-B*40:01:02:01 by one nucleotide in exon 3.

m5 C regulator-mediated methylation modification patterns and tumor microenvironment infiltration characteristics in acute myeloid leukemia.

BACKGROUND: Recently, many studies have been conducted to examine immune response modification at epigenetic level, but the candidate effect of RNA 5-methylcytosine (m5 C) modification on tumor microenvironment (TME) of acute myeloid leukemia (AML) is still unknown at present. METHODS: We assessed the patterns of m5 C modification among 417 AML cases by using nine m5 C regulators. Thereafter, we associated those identified modification patterns with TME cell infiltration features. Additionally, stepwise regression and LASSO Cox regression analyses were conducted for quantifying patterns of m5 C modification among AML cases to establish the m5 C-score. Meanwhile, we validated the expression of genes in the m5C-score model by qRT-PCR. Finally, the present work analyzed the association between m5 C-score and AML clinical characteristics and prognostic outcomes. RESULTS: In total, three different patterns of m5 C modification (m5 C-clusters) were identified, and highly differentiated TME cell infiltration features were also identified. On this basis, evaluating patterns of m5 C modification in single cancer samples was important for evaluating the immune/stromal activities in TME and for predicting prognosis. In addition, the m5 C-score was established, which showed a close relation with the overall survival (OS) of test and training set samples. Moreover, multivariate Cox analysis suggested that our constructed m5 C-score served as the independent predicting factor for the prognosis of AML (hazard ratio = 1.57, 95% confidence interval = 1.38-1.79, p < 1e-5 ). CONCLUSIONS: This study shows that m5 C modification may be one of the key roles in the formation of diversity and complexity of TME. Meanwhile, assessing the patterns of m5 C modification among individual cancer samples is of great importance, which provides insights into cell infiltration features within TME, thereby helping to develop relevant immunotherapy and predict patient prognostic outcomes.

Exploring 3-O-glycosylations of 20(R)-dammarane ginsenosides and the catalytic mechanism underlying the stereoselectivity with the combined assistance of AlphaFold 2 and molecular docking.

Glycosylation at C3-OH is the favorable modification for pharmaceutical activities and diversity expansion of 20(R)-dammarane ginsenosides. The 3-O-glycosylation, exclusively occurring in 20(R)-PPD ginsenosides, has never been achieved in 20(R)-PPT ginsenosides. Herein, 3-O-glycosylation of 20(R)-PPT enabled by a glycosyltransferase (GT) OsSGT2 was achieved with the combined assistance of AlphaFold 2 and molecular docking. Firstly, we combined AlphaFold2 algorithm and molecular docking to predict interactions between 20(R)-PPT and candidate GTs. A catalytically favorable binding geometry was thus identified in the OsSGT2-20(R)-PPT complex, suggesting OsSGT2 might act on 20(R)-PPT. The enzymatic assays demonstrated that OsSGT2 reacted with varied sugar donors to form 20(R)-PPT 3-O-glycosides, exhibiting donor promiscuity. Additionally, OsSGT2 displayed acceptor promiscuity, catalyzing 3-O-glucosylation of 20(R/S)-PPT, 20(R/S)-PPD and 20(R/S)-Rh1, respectively. Protein engineering on OsSGT2 was thus performed to probe its catalytic mechanism underlying its stereoselectivity. The W207A mutant preferred 20(S)-dammarane aglycons, while F395Q/A396G(QG) displayed a conversion enhancement towards both 20(R/S)-dammarane aglycons. The QG mutant was then used to synthesize 20(R)-PPT 3-O-glucoside, which displayed a moderate angiotensin-converting enzyme inhibitory effect with an IC50 of 27.5 ± 4.7 µM, superior to that of its 20(S)-epimer, with the combined assistance of target fishing and reverse docking. The water solubility of 20(R)-PPT 3-O-glucoside increased as well.

Spatial Distribution and Characterization of the Small-Molecule Metabolites and In Situ Hydrolyzed Oligosaccharides in the Rhizome of Glycyrrhiza uralensis by Desorption Electrospray Ionization-Mass Spectrometry Imaging and High-Resolution Liquid Chromatography-Mass Spectrometry.

Characterization and spatial distribution studies of the metabolome in plants are crucial for revealing the physiology of plants and developing functional foods. Using the rhizome of Glycyrrhiza uralensis as a case, we integrated desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) and high-resolution liquid chromatography/mass spectrometry approaches aimed at characterizing and locating both the small molecules and the macromolecular polysaccharides. Under the optimal conditions, 21 flavonoids and 12 triterpenoids were detected and characterized in different tissues of the rhizome and another 19 components were characterized exclusively by DESI-MSI. Combined with hydrophilic interaction chromatography/ion mobility-quadrupole time-of-flight mass spectrometry, eight different degrees of polymerization of oligosaccharides (after in situ acid hydrolysis) were characterized from the rhizome of G. uralensis. Majority of these metabolites are located in the cortex, phloem, and medulla, which lays the foundation for understanding the physiology of G. uralensis. The useful information can benefit the sustainable utilization and further development of Glycyrrhiza resource.

LC-MS-Guided Isolation of Cyanogripeptides A-C, Cyclolipopeptides with ß-Methyl-Leucine Residues, from an Actinoalloteichus cyanogriseus LHW52806.

Cyanogripeptides A-C (1-3), three new cyclolipopeptides with unusual ß-methyl-leucine residues, were identified from an Actinoalloteichus cyanogriseus LHW52806 using an LC-MS-guided strategy. The structures of compounds 1-3 were elucidated by 1D/2D NMR, HR-MS/MS, and the advanced Marfey's method. The absolute configuration of the ß-methyl-leucine residue was determined by a combination of stereoselective biosynthesis of (2S,3R)-ß-methyl-leucine, racemization to its epimer (2R,3R)-ß-methyl-leucine, and the advanced Marfey's method. The biosynthetic pathway of cyanogripeptides was deduced by analyzing the genome of A. cyanogriseus LHW52806. Compound 3 exhibited antibacterial activity against Helicobacter pylori G27, Helicobacter pylori 26695, and Mycolicibacterium smegmatis ATCC607 with MIC values of 32 µg/mL.

Comparative Characterization of the Ginsenosides from Six Panax Herbal Extracts and Their In Vitro Rat Gut Microbial Metabolites by Advanced Liquid Chromatography-Mass Spectrometry Approaches.

Ginseng extracts are extensively used as raw materials for food supplements and herbal medicines. This study aimed to characterize ginsenosides obtained from six Panax plant extracts (Panax ginseng, red ginseng, Panax quinquefolius, Panax notoginseng, Panax japonicus, and Panax japonicus var. major) and compared them with their in vitro metabolic profiles mediated by rat intestinal microbiota. Ultrahigh-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) with scheduled multiple reaction monitoring (sMRM) quantitation methods were developed to characterize and compare the ginsenoside composition of the different extracts. After in vitro incubation, 248 ginsenosides/metabolites were identified by UHPLC/IM-QTOF-MS in six biotransformed samples. Deglycosylation was determined to be the main metabolic pathway of ginsenosides, and protopanaxadiol-type and oleanolic acid-type saponins were easier to be easily metabolized. Compared with the ginsenosides in plant extracts, those remaining in six biotransformed samples were considerably fewer after biotransformation for 8 h. However, the compositional differences in four subtypes of the ginsenosides among the six Panax plants became more distinct.

Hipposponols A and B, two new 9, 11-secosterols from the marine sponge Hippospongia lachne de Laubenfels.

Two new 9,11-secosterols, hipposponols A (1) and B (2), together with five known analogues, aplidiasterol B (3), (3ß,5α,6ß)-3,5,6-triol-cholest-7-ene (4), (3ß,5α,6ß,22E)-3,5,6-triol-ergosta-7,22-diene (5), and one pair of inseparable C-24 epimers of (3ß,5α,6ß,22E)-3,5,6-triol-stigmasta-7,22-diene (6/7), were isolated from the marine sponge Hippospongia lachne de Laubenfels. The structures of isolated compounds were extensively elucidated based on HRESIMS and NMR data. Compounds 2 - 5 showed cytotoxicity against PC9 cells with IC50 values ranging from 34.1 ± 0.9 to 38.9 ± 1.0 µM and compound 4 displayed cytotoxicity against MCF-7 cells with IC50 value of 39.0 ± 0.4 µM.

Natural sesquiterpene quinone/quinols: chemistry, biological activity, and synthesis.

Covering: 2010 to 2021Sesquiterpene quinone/quinols (SQs) are characterized by a C15-sesquiterpenoid unit incorporating a C6-benzoquinone/quinol moiety. Numerous unprecedented carbon skeletons have been constructed with various connection patterns between the two parts. The potent anti-cancer, anti-inflammatory, anti-microbial, anti-viral, and fibrinolytic activities of SQs are associated with their diverse structures. The representative avarol has even entered the stage of clinical phase II research as an anti-HIV agent, and was developed as paramedic medicine against psoriasis. This review provides an overall summary of 558 new natural SQs discovered between 2010 and 2021, including seven groups and sixteen structure-type subgroups, which comprehensively recapitulates their chemical structures, spectral characteristics, source organisms, biological activities, synthesis, and biosynthesis, aiming to expand the application scope of this unique natural product resource.

DNA hypermethylation-induced miR-182 silence targets BCL2 and HOXA9 to facilitate the self-renewal of leukemia stem cell, accelerate acute myeloid leukemia progression, and determine the sensitivity of BCL2 inhibitor venetoclax.

Rationale: microRNAs (miRNAs) are frequently deregulated and play important roles in the pathogenesis and progression of acute myeloid leukemia (AML). miR-182 functions as an onco-miRNA or tumor suppressor miRNA in the context of different cancers. However, whether miR-182 affects the self-renewal of leukemia stem cells (LSCs) and normal hematopoietic stem progenitor cells (HSPCs) is unknown. Methods: Bisulfite sequencing was used to analyze the methylation status at pri-miR-182 promoter. Lineage-negative HSPCs were isolated from miR-182 knockout (182KO) and wild-type (182WT) mice to construct MLL-AF9-transformed AML model. The effects of miR-182 depletion on the overall survival and function of LSC were analyzed in this mouse model in vivo. Results: miR-182-5p (miR-182) expression was lower in AML blasts than normal controls (NCs) with hypermethylation observed at putative pri-miR-182 promoter in AML blasts but unmethylation in NCs. Overexpression of miR-182 inhibited proliferation, reduced colony formation, and induced apoptosis in leukemic cells. In addition, depletion of miR-182 accelerated the development and shortened the overall survival (OS) in MLL-AF9-transformed murine AML through increasing LSC frequency and self-renewal ability. Consistently, overexpression of miR-182 attenuated AML development and extended the OS in the murine AML model. Most importantly, miR-182 was likely dispensable for normal hematopoiesis. Mechanistically, we identified BCL2 and HOXA9 as two key targets of miR-182 in this context. Most importantly, AML patients with miR-182 unmethylation had high expression of miR-182 followed by low protein expression of BCL2 and resistance to BCL2 inhibitor venetoclax (Ven) in vitro. Conclusions: Our results suggest that miR-182 is a potential therapeutic target for AML patients through attenuating the self-renewal of LSC but not HSPC. miR-182 promoter methylation could determine the sensitivity of Ven treatment and provide a potential biomarker for it.

Circular RNA PVT1 Regulates Cell Proliferation, Migration, and Apoptosis by Stabilizing c-Myc and Downstream Target CXCR4 Expression in Acute Myeloid Leukemia

Objective: This study aimed to investigate the role and mechanism of circular RNA PVT1 (circPVT1) in patients with acute myeloid leukemia (AML). Materials and Methods: The expression of circPVT1 in 23 patients with de novo AML (not acute promyelocytic leukemia, not APL) and cell lines were detected by RT-qPCR. Loss of function assays were carried out to explore the influence of silenced circPVT1 on the proliferation, migration, and apoptosis in the THP-1 cell line. CCK-8 assays, trans-well assays, and annexin V/PI staining assays were performed to assess proliferation, migration, and apoptosis, respectively. Results: CircPVT1 was highly expressed in AML patients and myeloid cell lines compared to healthy controls. Higher expression of circPVT1 was related to shorter overall survival (OS) and relapse-free survival (RFS) in AML patients. Cell viability and migration were inhibited and apoptosis was increased when circPVT1 was knocked down in THP-1 cells. Knockdown of circPVT1 resulted in marked suppression of c-Myc protein with no significant change in mRNA levels. We also found that circPVT1 knockdown markedly increased the phosphorylation of c-Myc Thr-58, which was responsible for c-Myc degradation. Silencing of c-Myc caused a significant decrease in CXCR4 mRNA and protein expression, whereas the overexpression of c-Myc caused the opposite result, suggesting that CXCR4 is a target molecule of c-Myc. Finally, we found that overexpression of c-Myc could partially reverse circPVT1 knockdown-induced anti-tumor effects on THP-1 cells in vitro. Conclusion: Our findings showed that circPVT1 was highly expressed in AML patients and was related to shorter OS and RFS. CircPVT1 may exert an oncogenic effect in THP-1 cells by stabilizing c-Myc protein expression and downstream target CXCR4 expression. These data indicate that circPVT1 may be a promising therapeutic target for AML.

Tentative exploration of pharmacodynamic substances: Pharmacological effects, chemical compositions, and multi-components pharmacokinetic characteristics of ESZWD in CHF-HKYd rats.

The chemical components of Xin'an famous prescription Ershen Zhenwu Decoction (ESZWD) are still unclear. The results showed that ESZWD could significantly reduce left ventricular end diastolic diameter, decrease N-terminal pro-brain natriuretic peptide (NT-proBNP), angiotensinII, aldosterone, reactive oxygen species, and malondialdehyde, increase serum superoxide dismutase, while had no significant effect on inflammatory factors. Ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis detected 30 prototype components in model rats' serum, mainly including alkaloids, saponins, terpenoids, tanshinones, phenols. UPLC-MS/MS successfully detected the pharmacokinetic parameters of four components, and correlation analysis shows that there are negative correlations between four compounds and serum NT-proBNP. Thirty components of ESZWD may play a therapeutic role in chronic heart failure with heart-kidney Yang deficiency (CHF-HKYd) by improving myocardial injury, reducing oxidative stress levels, and inhibiting activation of the RAAS system in rats. Salsolinol, aconitine, paeoniflorin, and miltrione are equipped with potential characteristics as pharmacodynamic substances for ESZWD in treating CHF-HKYd. Additionally, the constituents of ESZWD in CHF-HKYd rats are different from normal rats, which provided a reference for the selection of subjects for further study.