[Correlation of miR-155 Expression with Drug Sensitivity of FLT3-ITD+ Acute Myeloid Leukemia Cell Line and Its Mechanism].

OBJECTIVE: To investigate the correlation of miR-155 expression with drug sensitivity of FLT3-ITD+ acute myeloid leukemia (AML) cell line and its potential regulatory mechanism. METHODS: By knocking out miR-155 gene in FLT3-ITD+ AML cell line MV411 through CRISPR/Cas9 gene-editing technology, monoclonal cells were screened. The genotype of these monoclonal cells was validated by PCR and Sanger sequencing. The expression of mature miRNA was measured by RT-qPCR. The treatment response of doxorubicin, quizartinib and midostaurin were measured by MTT assay and IC50 of these drugs were calculated to identify the sensitivity. Transcriptome sequencing was used to analyze change of mRNA level in MV411 cells after miR-155 knockout, gene set enrichment analysis to analyze change of signaling pathway, and Western blot to verify expressions of key molecules in signaling pathway. RESULTS: Four heterozygotes with gene knockout and one heterozygote with gene insertion were obtained through PCR screening and Sanger sequencing. RT-qPCR results showed that the expression of mature miR-155 in the monoclonal cells was significantly lower than wild-type clones. MTT results showed that the sensitivity of MV411 cells to various anti FLT3-ITD+ AML drugs increased significantly after miR-155 knockout compared with wild-type clones. RNA sequencing showed that the mTOR signaling pathway and Wnt signaling pathway were inhibited after miR-155 knockout. Western blot showed that the expressions of key molecules p-mTOR, Wnt5α and ß-catenin in signaling pathway were down-regulated. CONCLUSION: Drug sensitivity of MV411 cells to doxorubicin, quizartinib and midostaurin can be enhanced significantly after miR-155 knockout, which is related to the inhibition of multiple signaling pathways including mTOR and Wnt signaling pathways.

Oleanane and 30-noroleanane triterpenoids from the roots of Paeonia lactiflora.

An investigation of EtOAc extract from the roots of Paeonia lactiflora yielded three new 30-noroleanane triterpenoids paeonenoides L-N (1-3) and one new oleanane triterpenoid paeonenoide O (4) together with 7 known compounds (5-11). Extensive spectrographic experiments were applied to identify the structures of 1-4, and their absolute configurations were unambiguously determined by theoretical calculations of ECD spectra, as well as the single-crystal X-ray diffraction analysis. Compounds 8, 9 and 10 were isolated from the Paeonia genus for the first time. Moreover, compounds 8, 9 and 11 showed inhibitory activities against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages with the IC50 values of 72. 17 ± 4.74, 30.02 ± 2.03 and 28.34 ± 1.85 µM, respectively.

Arabidopsis Leaf Chloroplasts Have a Specific Sphingolipidome.

Sphingolipids are ubiquitous in eukaryotes and certain prokaryotes, where they serve as vital components of biological membranes and bioactive molecules. Chloroplasts have complex membrane structures that play crucial roles in photosynthesis, but their specific sphingolipidome remains unreported. In this study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to analyze the sphingolipidome of purified Arabidopsis thaliana chloroplasts. We detected 92 chloroplast sphingolipids. The chloroplast sphingolipidome differed from total leaf (TL) samples, with a higher content of free long-chain bases and hydroxyceramides and a greater proportion of complex sphingolipids with 16C fatty acid (FA) forms. Notably, chloroplast glucosylceramides were predominantly the d18:1 h16:0 and t18:1 h16:0 forms rather than the 24C FA form found in TL and other cellular structures. Comparing the sphingolipidomes of different cellular structures underscores the inhomogeneity of the intracellular distribution of sphingolipids. This provides a robust reference for further elucidating the function of sphingolipids in plant cells.

Aberrant accumulation of ceramides in mitochondria triggers cell death by inducing autophagy in Arabidopsis.

Sphingolipids are membrane lipids and play critical roles in signal transduction. Ceramides are central components of sphingolipid metabolism that are involved in cell death. However, the mechanism of ceramides regulating cell death in plants remains unclear. Here, we found that ceramides accumulated in mitochondria of accelerated cell death 5 mutant (acd5), and expression of mitochondrion-localized ceramide kinase (ACD5) suppressed mitochondrial ceramide accumulation and the acd5 cell death phenotype. Using immuno-electron microscopy, we observed hyperaccumulation of ceramides in acer acd5 double mutants, which are characterized by mutations in both ACER (alkaline ceramidase) and ACD5 genes. The results confirmed that plants with specific ceramide accumulation exhibited localization of ceramides to mitochondria, resulting in an increase in mitochondrial reactive oxygen species production. Interestingly, when compared with the wild type, autophagy-deficient mutants showed stronger resistance to ceramide-induced cell death. Lipid profiling analysis demonstrated that plants with ceramide accumulation exhibited a significant increase in phosphatidylethanolamine levels. Furthermore, exogenous ceramide treatment or endogenous ceramide accumulation induces autophagy. When exposed to exogenous ceramides, an increase in the level of the autophagy-specific ubiquitin-like protein, ATG8e, associated with mitochondria, where it directly bound to ceramides. Taken together, we propose that the accumulation of ceramides in mitochondria can induce cell death by regulating autophagy.

Orosomucoid proteins limit endoplasmic reticulum stress in plants.

Sphingolipids are cell membrane components and signaling molecules that induce endoplasmic reticulum (ER) stress responses, but the underlying mechanism is unknown. Orosomucoid proteins (ORMs) negatively regulate serine palmitoyltransferase activity, thus helping maintain proper sphingolipid levels in humans, yeast, and plants. In this report, we explored the roles of ORMs in regulating ER stress in Arabidopsis thaliana. Loss of ORM1 and ORM2 function caused constitutive activation of the unfolded protein response (UPR), as did treatment with the ceramide synthase inhibitor Fumonisin B1 (FB1) or ceramides. FB1 treatment induced the transcription factor bZIP28 to relocate from the ER membrane to the nucleus. The transcription factor WRKY75 positively regulates the UPR and physically interacted with bZIP28. We also found that the orm mutants showed impaired ER-associated degradation (ERAD), blocking the degradation of misfolded MILDEW RESISTANCE LOCUS-O 12 (MLO-12). ORM1 and ORM2 bind to EMS-MUTAGENIZED BRI1 SUPPRESSOR 7 (EBS7), a plant-specific component of the Arabidopsis ERAD complex, and regulate its stability. These data strongly suggest that ORMs in the ER membrane play vital roles in the UPR and ERAD pathways to prevent ER stress in Arabidopsis. Our results reveal that ORMs coordinate sphingolipid homeostasis with ER quality control and play a role in stress responses.

Is it safe for the spinal metastasis patients with preoperative deep vein thrombosis to use low-molecular-weight heparin before surgery? A prospective study.

Spine surgeons should weigh the risks of anticoagulants against their benefits in preventing deep venous thrombosis (DVT), as they may increase the risk of bleeding. Spinal metastasis patients undergoing decompression with fixation are at a high risk for DVT, which may occur preoperatively. Therefore, anticoagulants should be administered preoperatively. This study aimed to evaluate the safety of the administration of anticoagulants in treating spinal metastasis patients with preoperative DVT. Therefore, we prospectively investigated the prevalence of DVT in these patients. Patients who were diagnosed with preoperative DVT were included in an anticoagulant group. Subcutaneous low-molecular-weight heparin (LMWH) was administered. Patients without DVT were included in a non-anticoagulant group. Data on patient information, clinical parameters, blood test results, and bleeding complications were also collected. Moreover, the safety of anticoagulants was analyzed. The prevalence of preoperative DVT was 8.0%. None of the patients developed pulmonary thromboembolism. Furthermore, no significant differences in blood loss, drainage volume, hemoglobin levels, number of transfusions, or preoperative trans-catheter arterial embolization were observed between the two groups. None of the patients developed major bleeding. However, two patients experienced wound hematoma and one experienced incisional bleeding in the non-anticoagulant group. Therefore, LMWH is safe for spinal metastasis patients. Future randomized controlled trials should be conducted to evaluate the validity of perioperative prophylactic anticoagulation therapy in these patients.

[Advances in animal models of chronic heart failure and its applications in traditional Chinese medicine].

Chronic heart failure(CHF) is a series of clinical syndromes in which various heart diseases progress to their end stage. Its morbidity and mortality are increasing year by year, which seriously threatens people's life and health. The diseases causing CHF are complex and varied, such as coronary heart disease, hypertension, diabetes, cardiomyopathy and so on. It is of great significance to establish animal models of CHF according to different etiologies to explore the pathogenesis of CHF and develop drugs to prevent and treat CHF induced by different diseases. Therefore, based on the classification of the etiology of CHF, this paper summarizes the animal models of CHF widely used in recent 10 years, and the application of these animal models in traditional Chinese medicine(TCM) research, in order to provide ideas and strategies for studying the pathogenesis and treatment of CHF, and provide ideas for TCM modernization research.

(±)-Yanhusuomide A, a pair of ornithine-fused benzylisoquinoline enantiomers from Corydalis yanhusuo.

(±)-Yanhusuomide A (1), a novel enantiomeric pair of ornithine-fused benzylisoquinoline, were characterized from the dried tubers of Corydalis yanhusuo, along with a biogenetically related intermediate oblongine (2). Yanhusuomide A features an unprecedented skeleton based on a benzylisoquinoline coupled with an ornithine derivative to form a rare 5,6-dihydro-4H-pyrido[3,4,5-de]quinazoline motif. Plausible biosynthetic pathway of 1 was proposed, and (±)-yanhusuomide A (1) presented potential inhibitory bioactivity against acetylcholinesterase (AChE) with IC50 = 14.07 ± 2.38 µM. The simulation of molecular docking displayed that 1 generated strong interaction with Asp-74 and Trp-86 residues of AChE through attractive charge of the quaternary nitrogen.

[Progress of target determination and mechanism of bioactive components of traditional Chinese medicine].

The pharmacodynamic substances of traditional Chinese medicine(TCM) are the basis for the research of TCM and the development of innovative drugs. However, the lack of clarity of targets and molecular mechanisms is the bottleneck problem that restricts the research of pharmacodynamic substances of TCM. Bioactive components are the material basis of the efficacy of TCM, which exert activity by regulating the corresponding targets. Therefore, it is very important to identify the targets of the bioactive components to elucidate the pharmacological mechanism of TCM. Proteins are the most important drug targets, and study of the interaction between the proteins and bioactive components of TCM plays a key role in the development of pharmacological mechanism of TCM. In recent years, the main techniques for detecting the interaction between the bioactive components and proteins include surface plasmon resonance, fluorescence resonance energy transfer, bio-layer interference, molecular docking, proteome chip, target fishing, target mutant, and protein crystallization techniques, etc. This review summarized the biological target detection techniques and their applications in locating the targets of the bioactive components in TCM in the last decade, and this paper will provide useful strategies to elucidate the pharmacological mechanisms of TCM.

Penicipurate A, a new polyketide derivative from the endophytic fungus Penicillium purpurogenum.

A new polyketide derivative containing a 3-hydroxydecanoic acid ester moiety, penicipurate A (1), was purified from the solid cultures of the fungus Penicillium purpurogenum, a fungal strain endophytic in the leaves of Edgeworthia chrysantha. The structure of 1 was established by spectroscopic methods, including UV, IR, HRESIMS, 1D, and 2D NMR and 13C NMR chemical shifts calculations coupled with DP4+ analysis, as well as the chemical degradation method. Compound 1 showed moderate inhibitory activity against pancreatic lipase (PL) with an IC50 value of 9.61 ± 1.42 µM.

[Trace therapeutic substances of traditional Chinese medicine: great resources of innovative drugs derived from traditional Chinese medicine].

The traditional Chinese medicine(TCM) contains very complex constituents. Besides the major constituents, there are a large number of unclear trace constituents with novel skeletons and potent bioactivities, which have been regarded as one of the important therapeutic substances and the great resources of innovative drugs derived from TCM. The present review highlighted that the development of the trace therapeutic substances of TCM is closely depends on the advanced technologies for their identification, isolation, structure elucidation, and bioactivity evaluation. Additionally, this paper reviewed the novel trace compounds derived from Chinese herbal medicines which have been published in Organic Letters during 2001-2021, and summarized the important licensed drugs originated from the trace therapeutic substances and the discovery and development of trace therapeutic substances of 8 kinds of Chinese herbal medicines. This review provides references for the research and development of TCM therapeutic substances and innovative drugs.

Two New Nor-seco-Isodhilarane-Type Meroterpenoids from the Endophytic Fungus Penicillium purpurogenum.

Two new nor-seco isodhilarane meroterpenoids (NSIMs), purpurogenolides F (1) and G (2), along with three known meroterpenoid analogs (3-5), were isolated from the cultures of an endophytic fungus, Penicillium purpurogenum. Structures and absolute configurations of the new NSIMs were determined based on extensive spectroscopic data analyses, including HR-ESI-MS, UV, IR, NMR chemical shift calculations together with DP4+ probability analysis, as well as ECD calculations. All the isolated meroterpenoids were assessed for their anti-inflammatory activities, and compound 4 exhibited moderate inhibitory activity against the nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 cells with an IC50 value of 20.85±2.31 µM.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Factors associated with adherence to colonoscopy among individuals who were positive in the preliminary screening for colorectal neoplasms.

OBJECTIVES: This study aimed to investigate the potential factors associated with adherence to colonoscopy among participants who were preliminarily screened positive in a community-based colorectal cancer screening program in China. METHODS: This study analyzed data from 1219 out of 6971 community residents who were identified as positive cases by the well-validated high-risk factor questionnaire (HRFQ) or fecal immunochemical test (FIT) in the preliminary screening stage for colorectal neoplasms. Patients showing adherence to colonoscopy were defined as those who received positive results in a preliminary screening for colorectal neoplasms and later received a colonoscopy examination as required. The associations of social-demographic factors, lifestyle behaviors, history of diabetes, body mass index (BMI), and risk factors in the HRFQ with adherence to colonoscopy were evaluated using logistic regression models. RESULTS: Among 1219 participants who preliminarily screened positive, the top five risk factors reported by the participants were chronic constipation (25.9%), hematochezia (23.5%), family history of CRC in first-degree relatives (22.1%), chronic diarrhea (21.8%), and history of polyps (16.6%). Around 14.2% of participants who preliminarily screened positive reported three or more risk factors, and the proportion was 26.2% among participants who were positive according to both HRFQ and FIT. Among all participants who were preliminarily screened positive, the multivariable results showed that those who were married (OR = 1.58, 95% CI: 1.12, 2.25, p = 0.01), had chronic diarrhea (OR = 1.34, 95% CI: 1.00, 1.78, p = 0.047), and had a positive FIT (OR = 1.60, 95% CI: 1.21, 2.10, p < 0.001 for patients who were negative according to HRFQ but positive according to FIT; OR = 2.12, 95% CI: 1.33, 2.78, p = 0.002 for patients who were positive for both HRFQ and FIT) were more likely to adhere to colonoscopy, while participants with a history of cancer (OR: 0.50, 95% CI: 0.31, 0.79, p = 0.003) were less likely to adhere to colonoscopy. The results among participants who were tested positive according to only HRFQ were similar to those among all participants who were tested positive according to HRFQ or FIT. However, among participants who were tested positive according to only FIT, we only found that those who were married (OR = 2.52, 95% CI: 1.08, 5.90, p = 0.033) had a higher odds of adhering to colonoscopy, while those with a history of diabetes (OR = 0.35, 95% CI: 0.13, 0.96, p = 0.042) were less likely to adhere to colonoscopy. CONCLUSION: Our findings provide evidence supporting the development of tailored interventional strategies that aim to improve adherence to colonoscopy for individuals with a high risk of colorectal neoplasms. Both barriers and facilitators associated with adherence to colonoscopy should be considered in supportive systems and health policies. However, further well-designed prospective studies are warranted to confirm our findings.

Bioactive neolignans and lignans from the roots of Paeonia lactiflora.

Two new neolignans and one new lignan (1-3) were obtained from the roots of Paeonia lactiflora. Their structures were unambiguously elucidated based on extensive spectroscopic analysis, single-crystal X-ray crystallography, and the calculated and experimental electronic circular dichroism (ECD) spectra. Compound 1 was a racemic mixture and successfully resolved into the anticipated enantiomers via chiral-phase HPLC. Compound 3 demonstrated moderate inhibitory activity against human carboxylesterase 2A1 (hCES2A1) with an IC50 value of 7.28 ± 0.94 µmol·-1.

[Effect of MiR-155 Knockout Mediated by Dual sgRNAs on Drug Sensitivity of FLT3-ITD+AML].

OBJECTIVE: Two sgRNAs transfected FLT3-ITD+AML cell line MV411 with different binding sites were introduced into CRISPR/cas9 to obtain MV411 cells with miR-155 gene knockout. To compare the efficiency of miR-155 gene knockout by single and double sgRNA transfection and their effects on cell phenotypes. METHODS: The lentiviral vectors were generated containing either single sgRNA or dual sgRNAs and packaged into lentivirus particles. PCR was conducted to measure gene editing efficiency, and miR-155 expression was evaluated by qPCR. CCK-8 assay was used to evaluate the cell proliferation, and calculate drug sensitivity of cells to adriamycin and quizartinib. Annexin V-APC/7-AAD staining was used to label cell apoptosis induced by adriamycin and quizartinib. RESULTS: In the dual sgRNAs transfected cells, a cleavage band could be observed, meaning the success of gene editing. Compared with the single sgRNA transfected MV411 cells, the expression level of mature miR-155-5p was lower in the dual sgRNA transfected cells. And, dual sgRNA transfected MV411 were more sensitive to adriamycin and quizartinib with lower IC50 and higher apoptosis rate. CONCLUSION: The inhibition rate of miR-155 gene expression transfected by dual sgRNA is higher than that by single sgRNA. Dual sgRNA transfection can inhibit cell proliferation, reverse drug resistance, and induce apoptosis more significantly. Compared with single sgRNA transfection, dual sgRNA transfection is a highly efficient gene editing scheme.

Seco and Nor-seco Isodhilarane-Type Meroterpenoids from Penicillium purpurogenum and the Configuration Revisions of Related Compounds.

Seco and nor-seco isodhilarane-type meroterpenoids (SIMs and NSIMs) are mainly found in Penicillium fungi and have been characterized by highly congested polycyclic skeletons and a broad range of bioactivities. However, the literature reports inconsistent configuration assignments for some SIMs and NSIMs, due to their complex polycyclic systems and multichiral centers. Herein, we described eight SIMs and NSIMs isolated from the EtOAc extract of Penicillium purpurogenum, which led to the configuration revisions of purpurogenolide C (1a), berkeleyacetal B (2a), chrysogenolide F (3a), and berkeleyacetal C (4a) as compounds 1-4, respectively. Furthermore, extensive re-evaluation of the experimental and computational 13C NMR chemical shifts of the reported 39 SIMs and NSIMs provided an empirical approach for determining the C-9 relative configuration, according to the 13C NMR chemical shifts of C-9, which contributed to the configuration revisions of another three SIMs (5a and 6a) and NSIMs (7a), denoted as compounds 5-7, respectively. Biological assays indicated that compound 3 exhibited cytotoxic activity against HepG2 and A549 cell lines with IC50 values of 5.58 and 6.80 µM, respectively. Compounds 2-4, 8, 9, and 32 showed moderate hepatoprotective activity at 10 µM in the APAP-induced HepG2 cell injury model.

Ceramides regulate defense response by binding to RbohD in Arabidopsis.

Sphingolipids, a class of bioactive lipids, play a critical role in signal transduction. Ceramides, which are central components of sphingolipid metabolism, are involved in plant development and defense. However, the mechanistic link between ceramides and downstream signaling remains unclear. Here, the mutation of alkaline ceramidase in a ceramide kinase mutant acd5 resulted in spontaneous programmed cell death early in development and was accompanied by ceramide accumulation, while other types of sphingolipids, such as long chain base, glucosylceramide, and glycosyl inositol phosphorylceramide, remained at the same level as the wild-type plants. Analysis of the transcriptome indicated that genes related to the salicylic acid (SA) pathway and oxidative stress pathway were induced dramatically in acer acd5 plants. Comparison of the level of reactive oxygen species (ROS), SA, and ceramides in the wild-type and acer acd5 plants at different developmental stages indicated that the acer acd5 mutant exhibited constitutive activation of SA and ROS signaling, which occurred simultaneously with the alteration of ceramides. Overexpressing NahG in the acer acd5 mutant could completely suppress its cell death and ceramide accumulation, while benzo-(1,2,3)-thiadiazole-7-carbothioc acid S-methyl ester treatment restored its phenotype again. Moreover, we found that the plasma membrane of acer acd5 mutant was the main site of ROS production. Ceramides accumulated in the plasma membrane of acer acd5, directly binding and activating the NADPH oxidase RbohD and promoting hydrogen peroxide generation and SA- or defense-related gene activation. Our data illustrated that ceramides play an essential role in plant defense.

13,13a-seco-protoberberines from the tubers of Corydalis yanhusuo and their anti-inflammatory activity.

Six undescribed protoberberine derivatives including two pairs of enantiomers, named yanhusanines G-L, along with fifteen reported protoberberine alkaloids, were isolated from the tubers of Corydalis yanhusuo. Among them, yanhusanines H-L feature a unique 13,13a-seco skeleton which is rare in nature. Their structural elucidations were achieved by extensive spectroscopic analysis and quantum chemistry calculations. A biogenetic route for yanhusanines H-L was proposed. Bioassay results showed that yanhusanine J exhibited potent inhibitory effect against the nitric oxide (NO) production in lipopolysaccharide (LPS) induced RAW 264.7 cells (IC50 = 2.25 ± 1.32 µM). Western blot analysis demonstrated that yanhusanine J exerted its anti-inflammatory effect via suppressing the nuclear factor kappa B (NF-κB) pathway, together with the decrease of the inflammatory factors TNF-α, IL-6 and IL-1ß. Furthermore, molecular simulation docking indicated that yanhusanine J had strong interaction with the active site of the inducible nitric oxide synthase (iNOS) protein.

Bioactive sesquineolignans from the twigs of Litsea cubeba.

In a continuing search for biological natural products with structure diversity from traditional Chinese herbs, five new sesquineolignans (1-5) were isolated from an ethyl acetate extract of the twigs of Litsea cubeba. Their structures were elucidated based on MS, 1D and 2D NMR spectroscopic data, as well as experimental electronic circular dichroism (ECD) spectra. Compounds 1-5 showed moderate inhibitory effects against LPS-induced NO production in RAW264.7 macrophages, with IC50 values of 16.2, 20.2, 22.1, 15.1, and 16.6 µmol·L-1, respectively.