A robust high-throughput functional screening assay for plant pathogen effectors using the TMV-GFP vector.

Uncovering the function of phytopathogen effectors is crucial for understanding mechanisms of pathogen pathogenicity and for improving our ability to protect plants from diseases. An increasing number of effectors have been predicted in various plant pathogens. Functional characterization of these effectors has become a major focus in the study of plant-pathogen interactions. In this study, we designed a novel screening system that combines the TMV (tobacco mosaic virus)-GFP vector and Agrobacterium-mediated transient expression in the model plant Nicotiana benthamiana. This system enables the rapid identification of effectors that interfere with plant immunity. The biological function of these effectors can be easily evaluated by observing the GFP fluorescence signal using a UV lamp within just a few days. To evaluate the TMV-GFP system, we initially tested it with well-described virulence and avirulence type III effectors from the bacterial pathogen Ralstonia solanacearum. After proving the accuracy and efficiency of the TMV-GFP system, we successfully screened a novel virulence effector, RipS1, using this approach. Furthermore, using the TMV-GFP system, we reproduced consistent results with previously known cytoplasmic effectors from a diverse array of pathogens. Additionally, we demonstrated the effectiveness of the TMV-GFP system in identifying apoplastic effectors. The easy operation, time-saving nature, broad effectiveness, and low technical requirements of the TMV-GFP system make it a promising approach for high-throughput screening of effectors with immune interference activity from various pathogens.

Targeting RORγ inhibits the growth and metastasis of hepatocellular carcinoma.

Approximately 80%-90% of hepatocellular carcinomas (HCC) occur in a premalignant environment of fibrosis and abnormal extracellular matrix (ECM), highlighting an essential role of ECM in the tumorigenesis and progress of HCC. However, the determinants of ECM in HCC are poorly defined. Here, we show that nuclear receptor RORγ is highly expressed and amplified in HCC tumors. RORγ functions as an essential activator of the matrisome program via directly driving the expression of major ECM genes in HCC cells. Elevated RORγ increases fibronectin-1 deposition, cell-matrix adhesion, and collagen production, creating a favorable microenvironment to boost liver cancer metastasis. Moreover, RORγ antagonists effectively inhibit tumor growth and metastasis in multiple HCC xenografts and immune-intact models, and they effectively sensitize HCC tumors to sorafenib therapy in mice. Notably, elevated RORγ expression is associated with ECM remodeling and metastasis in patients with HCC. Taken together, we identify RORγ as a key player of ECM remodeling in HCC and as an attractive therapeutic target for advanced HCC.

Cytosolic DNA initiates a vicious circle of aging-related endothelial inflammation and mitochondrial dysfunction via STING: the inhibitory effect of Cilostazol.

Endothelial senescence, aging-related inflammation, and mitochondrial dysfunction are prominent features of vascular aging and contribute to the development of aging-associated vascular disease. Accumulating evidence indicates that DNA damage occurs in aging vascular cells, especially in endothelial cells (ECs). However, the mechanism of EC senescence has not been completely elucidated, and so far, there is no specific drug in the clinic to treat EC senescence and vascular aging. Here we show that various aging stimuli induce nuclear DNA and mitochondrial damage in ECs, thus facilitating the release of cytoplasmic free DNA (cfDNA), which activates the DNA-sensing adapter protein STING. STING activation led to a senescence-associated secretory phenotype (SASP), thereby releasing pro-aging cytokines and cfDNA to further exacerbate mitochondrial damage and EC senescence, thus forming a vicious circle, all of which can be suppressed by STING knockdown or inhibition. Using next-generation RNA sequencing, we demonstrate that STING activation stimulates, whereas STING inhibition disrupts pathways associated with cell senescence and SASP. In vivo studies unravel that endothelial-specific Sting deficiency alleviates aging-related endothelial inflammation and mitochondrial dysfunction and prevents the development of atherosclerosis in mice. By screening FDA-approved vasoprotective drugs, we identified Cilostazol as a new STING inhibitor that attenuates aging-related endothelial inflammation both in vitro and in vivo. We demonstrated that Cilostazol significantly inhibited STING translocation from the ER to the Golgi apparatus during STING activation by targeting S162 and S243 residues of STING. These results disclose the deleterious effects of a cfDNA-STING-SASP-cfDNA vicious circle on EC senescence and atherogenesis and suggest that the STING pathway is a promising therapeutic target for vascular aging-related diseases. A proposed model illustrates the central role of STING in mediating a vicious circle of cfDNA-STING-SASP-cfDNA to aggravate age-related endothelial inflammation and mitochondrial damage.

RAD54B inhibits vascular endothelial senescence via suppression of CHK1/p53/p21 pathway.

RAD54B belongs to the SNF2/SWI2 superfamily, participating in homologous recombination repair. DNA damage is the central driver of aging, but there is no direct evidence of an association between RAD54B and vascular aging. The present study sought to investigate the role and mechanisms of RAD54B in endothelial senescence. In senescent animal models, including spontaneously hypertensive rats, normal aging mice, and D-gal-induced senescent mice, and senescent cell models induced by H2O2, D-gal, and culture, RAD54B was remarkably downregulated. Knockdown of RAD54B increased the expression of p53 and p21, increased the ratio of SA-ß-gal-positive cells, and decreased the proportion of EdU-positive cells. Conversely, overexpression of RAD54B reversed the senescent phenotypes stimulated by H2O2 and delayed replicative endothelial senescence. Mechanistically, silencing RAD54B compensatorily increased the expression of RAD51/XRCC4, which remained unchanged in H2O2-induced senescence. RAD54B lacking the SNF2 domain could still reverse the increasing expression of p53/p21 induced by H2O2. RAD54B reduced γH2A.X expression and inhibited the expression and phosphorylation of CHK1. In conclusion, RAD54B exerts a direct protective effect against DNA damage through enhancing homologous recombination repair in endothelial senescence, resulting in inhibition of the downstream CHK1/p53/p21 pathway, suggesting that RAD54B may be a potential therapeutic target for vascular aging-associated diseases.

Application of transabdominal ultrasound- and laparoscopy-guided percutaneous microwave ablation for treating uterine fibroids: 24-month follow-up outcomes.

PURPOSE: To determine the ablation efficacy of transabdominal ultrasound- and laparoscopy-guided percutaneous microwave ablation (PMWA), to investigate whether the risk of damage to adjacent organs and endometrium due to this technique can be reduced or even avoided. We also evaluated the clinical efficacy of this technique in the treatment of uterine fibroids of different sizes and at different locations over a 24-month follow-up period. METHODS: This study included 50 patients with uterine fibroids who underwent transabdominal ultrasound- and laparoscopy-guided PMWA from August 2018 to July 2020. Lesions were confirmed by pathology. The technical efficacy and complications of PMWA were assessed. The lesion diameter, lesion volume, lesion location, and contrast-enhanced ultrasound (CEUS) features before PMWA and within 24 h after PMWA were recorded. Magnetic resonance imaging (MRI) was used for follow-up at 3 and 6 months after PMWA. Transvaginal ultrasound was used for follow-up at 24 months after PMWA. RESULTS: A total of 50 patients with uterine fibroids received treatment. The median ablation rate of uterine fibroids was 97.21%. The mean lesion volume reduction rates were 32.63%, 57.26%, and 92.64% at 3, 6, and 24 months after treatment, respectively. The size and location of uterine fibroids did not significantly affect the ablation rate and the rate of lesion volume reduction. No major complication was found during and after the procedure. CONCLUSION: Transabdominal ultrasound- and laparoscopy-guided PMWA can be utilized to safely enhance the ablation rate while minimizing ablation time and avoiding harm to adjacent organs and the endometrium. This technique is applicable for treating uterine fibroids of different sizes and at varying locations. TRIAL REGISTRATION NUMBER: ChiCTR-IPR-17011910, and date of trial registration: 08/07/2017.

Genetic Diversity of Ralstonia solanacearum Causing Tobacco Bacterial Wilt in Fujian Province and Identification of Biocontrol Streptomyces sp.

Tobacco bacterial wilt is a highly destructive soilborne disease caused by the Ralstonia solanacearum species complex, exhibiting a significant risk to global flue-cured tobacco cultivation and resulting in substantial economic loss. In this study, 77 isolates were collected from three prominent flue-cured tobacco cultivation areas in Fujian, China (Nanping, Sanming, and Longyan), in 2021 and 2022. The isolated strains were classified through phylotype-specific multiplex polymerase chain reaction (Pmx-PCR) and physiological tests. The analysis showed that all the strains were associated with phylotype I, race 1, and biovar III. Subsequent phylogenetic analysis using partial egl gene sequences classified the 77 isolates into 5 distinct sequevars: 13, 15, 16, 17, and 34. Notably, a remarkable predominance of sequevar 15 was observed in Fujian Province, while sequevar 16 was first reported on tobacco in China, which was identified in other plants, expanding the understanding of its host range and distribution in the country. In addition, a Streptomyces strain extracted from the rhizosphere soil of tobacco was found to inhibit the growth of multiple sequevars of tobacco R. solanacearum, indicating its broad-spectrum antagonistic properties. Furthermore, pot experiments showed that the strain St35 effectively controlled tobacco bacterial wilt. The isolate St35 was conclusively identified as Streptomyces gancidicus according to the morphological and genetic features. In summary, the present study demonstrated the genetic diversity and distribution of tobacco R. solanacearum strains in the Fujian province of China, as well as the identification of a candidate biological control agent for the management of tobacco bacterial wilt.

Endothelial Dysfunction in Atherosclerotic Cardiovascular Diseases and Beyond: From Mechanism to Pharmacotherapies.

The endothelium, a cellular monolayer lining the blood vessel wall, plays a critical role in maintaining multiorgan health and homeostasis. Endothelial functions in health include dynamic maintenance of vascular tone, angiogenesis, hemostasis, and the provision of an antioxidant, anti-inflammatory, and antithrombotic interface. Dysfunction of the vascular endothelium presents with impaired endothelium-dependent vasodilation, heightened oxidative stress, chronic inflammation, leukocyte adhesion and hyperpermeability, and endothelial cell senescence. Recent studies have implicated altered endothelial cell metabolism and endothelial-to-mesenchymal transition as new features of endothelial dysfunction. Endothelial dysfunction is regarded as a hallmark of many diverse human panvascular diseases, including atherosclerosis, hypertension, and diabetes. Endothelial dysfunction has also been implicated in severe coronavirus disease 2019. Many clinically used pharmacotherapies, ranging from traditional lipid-lowering drugs, antihypertensive drugs, and antidiabetic drugs to proprotein convertase subtilisin/kexin type 9 inhibitors and interleukin 1ß monoclonal antibodies, counter endothelial dysfunction as part of their clinical benefits. The regulation of endothelial dysfunction by noncoding RNAs has provided novel insights into these newly described regulators of endothelial dysfunction, thus yielding potential new therapeutic approaches. Altogether, a better understanding of the versatile (dys)functions of endothelial cells will not only deepen our comprehension of human diseases but also accelerate effective therapeutic drug discovery. In this review, we provide a timely overview of the multiple layers of endothelial function, describe the consequences and mechanisms of endothelial dysfunction, and identify pathways to effective targeted therapies. SIGNIFICANCE STATEMENT: The endothelium was initially considered to be a semipermeable biomechanical barrier and gatekeeper of vascular health. In recent decades, a deepened understanding of the biological functions of the endothelium has led to its recognition as a ubiquitous tissue regulating vascular tone, cell behavior, innate immunity, cell-cell interactions, and cell metabolism in the vessel wall. Endothelial dysfunction is the hallmark of cardiovascular, metabolic, and emerging infectious diseases. Pharmacotherapies targeting endothelial dysfunction have potential for treatment of cardiovascular and many other diseases.

Inhibition of miR-214-3p attenuates ferroptosis in myocardial infarction via regulating ME2.

Myocardial infarction (MI) contributes to an increased risk of incident heart failure and sudden death, but there is still a lack of effective treatment in clinic. Recently, growing evidence has indicated that abnormal expression of microRNAs (miRNAs) plays a crucial role in cardiovascular diseases. In this research, the involvement of miRNA-214-3p in MI was explored. A mouse model of MI was established by ligation of the left anterior descending coronary artery, and primary cultures of neonatal rat cardiomyocytes (NRCMs) were submitted to hypoxic treatment to stimulate cellular injury in vitro. Our results showed that miR-214-3p level was significantly upregulated in the infarcted region of mouse hearts and in NRCMs exposed to hypoxia, accompanying with an obvious elevation of ferroptosis. Inhibition of miR-214-3p by antagomir injection improved cardiac function, decreased infarct size, and attenuated iron accumulation and oxidant stress in myocardial tissues. MiR-214-3p could also promote ferroptosis and cellular impairments in NRCMs, while miR-214-3p inhibitor effectively protected cells from hypoxia. Furthermore, dual luciferase reporter gene assay revealed that malic enzyme 2 (ME2) is a direct target of miR-214-3p. In cardiomyocytes, overexpression of ME2 ameliorated the detrimental effects and excessive ferroptosis induced by miR-214-3p mimic, whereas ME2 depletion compromised the protective role of miR-214-3p inhibitor against hypoxic injury and ferroptosis. These findings suggest that miR-214-3p contributes to enhanced ferroptosis during MI at least partially via suppressing ME2. Inhibition of miR-214-3p may be a new approach for tackling MI.

Retraction notice to "Berberine ameliorates renal injury in diabetic C57BL/6 mice: Involvement of suppression of SphK-S1P signaling pathway" [Arch. Biochem. Biophys. 502(2010) 112-120].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. This article shows significant data duplication and overlap with Liu, Weihua et al., Effects of berberine on matrix accumulation and NF-kappa B signal pathway in alloxan-induced diabetic mice with renal injury. European Journal of Pharmacology. 2010 Jul 25; 638(1-3):150-5 (https://doi.org/10.1016/j.ejphar.2010.04.033) without adequate referencing. Although there is a slight difference in the methodology section regarding alloxan-induced diabetes models in the two articles, there is a clear overlap between Table 2 of Lan, Tian et al. (2010); and Tables 1 and 2 of Liu, Weihua et al. (2010). The two manuscripts were submitted from the same laboratory in the same year.

Regulation of toll-like receptor (TLR) signaling pathways in atherosclerosis: from mechanisms to targeted therapeutics.

Atherosclerosis, one of the life-threatening cardiovascular diseases (CVDs), has been demonstrated to be a chronic inflammatory disease, and inflammatory and immune processes are involved in the origin and development of the disease. Toll-like receptors (TLRs), a class of pattern recognition receptors that trigger innate immune responses by identifying pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), regulate numerous acute and chronic inflammatory diseases. Recent studies reveal that TLRs have a vital role in the occurrence and development of atherosclerosis, including the initiation of endothelial dysfunction, interaction of various immune cells, and activation of a number of other inflammatory pathways. We herein summarize some other inflammatory signaling pathways, protein molecules, and cellular responses associated with TLRs, such as NLRP3, Nrf2, PCSK9, autophagy, pyroptosis and necroptosis, which are also involved in the development of AS. Targeting TLRs and their regulated inflammatory events could be a promising new strategy for the treatment of atherosclerotic CVDs. Novel drugs that exert therapeutic effects on AS through TLRs and their related pathways are increasingly being developed. In this article, we comprehensively review the current knowledge of TLR signaling pathways in atherosclerosis and actively seek potential therapeutic strategies using TLRs as a breakthrough point in the prevention and therapy of atherosclerosis.

SZC-6, a small-molecule activator of SIRT3, attenuates cardiac hypertrophy in mice.

Sirtuin3 (SIRT3), a class III histone deacetylase, is implicated in various cardiovascular diseases as a novel therapeutic target. SIRT3 has been proven to be cardioprotective in a model of Ang II-induced cardiac hypertrophy. However, a few small-molecule compounds targeting deacetylases could activate SIRT3. In this study, we generated a novel SIRT3 activator, 3-(2-bromo-4-hydroxyphenyl)-7-hydroxy-2H-chromen-2-one (SZC-6), through structural optimization of the first SIRT3 agonist C12. We demonstrated that SZC-6 directly bound to SIRT3 with Kd value of 15 µM, and increased SIRT3 deacetylation activity with EC50 value of 23.2 ± 3.3 µM. In neonatal rat cardiomyocytes (NRCMs), pretreatment with SZC-6 (10, 20, 40 µM) dose-dependently attenuated isoproterenol (ISO)-induced hypertrophic responses. Administration of SZC-6 (20, 40 and 60 mg·kg-1·d-1, s.c.) for 2 weeks starting from one week prior ISO treatment dose-dependently reversed ISO-induced impairment of diastolic and systolic cardiac function in wild-type mice, but not in SIRT3 knockdown mice. We showed that SZC-6 (10, 20, 40 µM) dose-dependently inhibited cardiac fibroblast proliferation and differentiation into myofibroblasts, which was abolished in SIRT3-knockdown mice. We further revealed that activation of SIRT3 by SZC-6 increased ATP production and rate of mitochondrial oxygen consumption, and reduced ROS, improving mitochondrial function in ISO-treated NRCMs. We also found that SZC-6 dose-dependently enhanced LKB1 phosphorylation, thereby promoting AMPK activation to inhibit Drp1-dependent mitochondrial fragmentation. Taken together, these results demonstrate that SZC-6 is a novel SIRT3 agonist with potential value in the treatment of cardiac hypertrophy partly through activation of the LKB1-AMPK pathway.

JMJD6 protects against isoproterenol-induced cardiac hypertrophy via inhibition of NF-κB activation by demethylating R149 of the p65 subunit.

Histone modification plays an important role in pathological cardiac hypertrophy and heart failure. In this study we investigated the role of a histone arginine demethylase, Jumonji C domain-containing protein 6 (JMJD6) in pathological cardiac hypertrophy. Cardiac hypertrophy was induced in rats by subcutaneous injection of isoproterenol (ISO, 1.2 mg·kg-1·d-1) for a week. At the end of the experiment, the rats underwent echocardiography, followed by euthanasia and heart collection. We found that JMJD6 levels were compensatorily increased in ISO-induced hypertrophic cardiac tissues, but reduced in patients with heart failure with reduced ejection fraction (HFrEF). Furthermore, we demonstrated that JMJD6 overexpression significantly attenuated ISO-induced hypertrophy in neonatal rat cardiomyocytes (NRCMs) evidenced by the decreased cardiomyocyte surface area and hypertrophic genes expression. Cardiac-specific JMJD6 overexpression in rats protected the hearts against ISO-induced cardiac hypertrophy and fibrosis, and rescued cardiac function. Conversely, depletion of JMJD6 by single-guide RNA (sgRNA) exacerbated ISO-induced hypertrophic responses in NRCMs. We revealed that JMJD6 interacted with NF-κB p65 in cytoplasm and reduced nuclear levels of p65 under hypertrophic stimulation in vivo and in vitro. Mechanistically, JMJD6 bound to p65 and demethylated p65 at the R149 residue to inhibit the nuclear translocation of p65, thus inactivating NF-κB signaling and protecting against pathological cardiac hypertrophy. In addition, we found that JMJD6 demethylated histone H3R8, which might be a new histone substrate of JMJD6. These results suggest that JMJD6 may be a potential target for therapeutic interventions in cardiac hypertrophy and heart failure.

Cigarettes smoking and e-cigarettes using among university students: a cross-section survey in Guangzhou, China, 2021.

BACKGROUND: There is an increase in the use of cigarettes and e-cigarettes worldwide, and the similar trends may be observed in young adults. Since 2014, e-cigarettes have become the most commonly used nicotine products among young adults (Sun et al., JAMA Netw Open 4:e2118788, 2021). With the increase in e-cigarette use and the decrease in use of cigarettes and other tobacco products, however, there is limited information about Chinese smokers, e-cigarettes users and trends in cigarettes and e-cigarettes use among university students. Therefore, our objective was to investigate the using status of cigarettes, e-cigarettes and smoking behavior among the students from 7 universities in Guangzhou, China. METHODS: Students at 7 different universities in Guangzhou were investigated online in 2021 through a cross-sectional survey. A total of 10,008 students were recruited and after screening, 9361 participants were adopted in our statistics. Descriptive analysis, Chi-square analysis, and multiple logistic regression analysis were used to explore the smoking status and influencing factors. RESULTS: The average age of the 9361 university students was 22.4 years (SD = 3.6). 58.3% of participants were male. 29.8% of the participants smoked or used e-cigarettes. Among the smokers and users of e-cigarettes, 16.7% were e-cigarettes only users, 35.0% were cigarettes only users, and 48.3% were dual users. Males were more likely to smoke or use e-cigarettes. Medical students, students from prestigious Chinese universities, and students with higher levels of education were less likely. Students with unhealthy lifestyles (e.g., drinking alcohol frequently, playing video games excessively, staying up late frequently) were more likely to smoke or use e-cigarettes. Emotion can have significant impacts on both cigarettes and e-cigarettes dual users when choosing cigarettes or e-cigarettes to use. More than half of dual users said they would choose cigarettes when they were depressed and e-cigarettes when they were happy. CONCLUSION: We identified factors influencing the use of cigarettes and e-cigarettes among university students in Guangzhou, China. Gender, education level background, specialization, lifestyle habits and emotion all influenced the use of cigarettes and e-cigarettes among university students in Guangzhou, China. Male, low education level, from non-prestigious Chinese universities or vocational schools, non-medical specialization, and presence of unhealthy lifestyles were influencing factors for the use of cigarettes and e-cigarettes among university students in Guangzhou and students with these factors were more likely to smoke or use e-cigarettes. Besides, emotions can influence dual users' choice of products. This study provides more information to better understand young people's preferences for cigarettes and e-cigarettes by elucidating the characteristics of cigarettes and e-cigarettes use, as well as related influencing factors, among university students in Guangzhou. Further research involving more variables connected to the use of cigarettes and e-cigarettes will be required in our future study.

Signal Suppression in LC-ESI-MS/MS from Concomitant Medications and Its Impact on Quantitative Studies: An Example Using Metformin and Glyburide.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been widely used in the quantitative analysis of drugs. The ubiquitous concomitant drug scenario in the clinic has spawned a large number of co-analyses based on this technique. However, signal suppression caused by concomitant drugs during electrospray ionization may affect the quantification accuracy of analytes, which has not received enough attention. In this study, metformin (MET) and glyburide (GLY) were co-eluted by the conventional optimization of chromatographic conditions to illustrate the effect of signal suppression caused by the combined drugs on the quantitative analysis. The response of MET was not affected by GLY over the investigated concentration range. However, the GLY signal could be suppressed by about 30% in the presence of MET, affecting its pharmacokinetic analysis in simulated samples. As an attempt to solve the suppression of GLY by co-eluting MET, dilution can alleviate the suppression. However, this method still has limitations due to the sacrifice of sensitivity. The stable isotope-labeled internal standard could play a role in correction and improve the quantitative accuracy of GLY, which was further confirmed in the pharmacokinetic study of simulated samples. This study provided an example model to illustrate the possible effect of clinical drug combination on LC-MS/MS drug quantitative analysis and investigated the effective methods to solve this problem.

A conserved type III effector RipB is recognized in tobacco and contributes to Ralstonia solanacearum virulence in susceptible host plants.

Ralstonia solanacearum, the causal agent of bacterial wilt, causes devastating diseases in a wide range of plants including potato, tomato, pepper and tobacco. The pathogen delivers approximately 70 type III effectors (T3Es) into plant cells during infection. In this study, we confirmed that a T3E RipB is recognized in tobacco. We further demonstrated that RipB is conserved among R. solanacearum isolates and five different ripB alleles are all recognized in tobacco. The ripB from GMI1000 was transformed into susceptible host Arabidopsis, and a defect in root development was observed in ripB-transgenic plants. Pathogen inoculation assays showed that ripB expression promoted plant susceptibility to R. solanacearum infection, indicating that RipB contributes to pathogen virulence in Arabidopsis. Expression of ripB in roq1 mutant partially suppressed reactive oxygen species production, confirming that RipB interferes with plant basal defense. Interestingly, ripB expression promoted cytokinin-related gene expression in Arabidopsis, suggesting a role of cytokinin signaling pathway in plant-R. solanacearum interactions. Finally, RipB harbors potential 14-3-3 binding motifs, but the associations between RipB and 14-3-3 proteins were undetectable in yeast two-hybrid assay. Together, our results demonstrate that multiple ripB alleles are recognized in Nicotiana, and RipB suppresses basal defense in susceptible host to promote R. solanacearum infection.

Loss-of-Function Genetic Screening Identifies Aldolase A as an Essential Driver for Liver Cancer Cell Growth Under Hypoxia.

BACKGROUND AND AIMS: Hypoxia is a common feature of the tumor microenvironment (TME), which promotes tumor progression, metastasis, and therapeutic drug resistance through a myriad of cell activities in tumor and stroma cells. While targeting hypoxic TME is emerging as a promising strategy for treating solid tumors, preclinical development of this approach is lacking in the study of HCC. APPROACH AND RESULTS: From a genome-wide CRISPR/CRISPR-associated 9 gene knockout screening, we identified aldolase A (ALDOA), a key enzyme in glycolysis and gluconeogenesis, as an essential driver for HCC cell growth under hypoxia. Knockdown of ALDOA in HCC cells leads to lactate depletion and consequently inhibits tumor growth. Supplementation with lactate partly rescues the inhibitory effects mediated by ALDOA knockdown. Upon hypoxia, ALDOA is induced by hypoxia-inducible factor-1α and fat mass and obesity-associated protein-mediated N6 -methyladenosine modification through transcriptional and posttranscriptional regulation, respectively. Analysis of The Cancer Genome Atlas shows that elevated levels of ALDOA are significantly correlated with poor prognosis of patients with HCC. In a screen of Food and Drug Administration-approved drugs based on structured hierarchical virtual platforms, we identified the sulfamonomethoxine derivative compound 5 (cpd-5) as a potential inhibitor to target ALDOA, evidenced by the antitumor activity of cpd-5 in preclinical patient-derived xenograft models of HCC. CONCLUSIONS: Our work identifies ALDOA as an essential driver for HCC cell growth under hypoxia, and we demonstrate that inhibition of ALDOA in the hypoxic TME is a promising therapeutic strategy for treating HCC.

Therapeutic targeting RORγ with natural product N-hydroxyapiosporamide for small cell lung cancer by reprogramming neuroendocrine fate.

Small cell lung cancer (SCLC) is an aggressive and exceptionally fatal disease. Unlike non- small cell lung cancer (NSCLC), no targetable genetic driver events have been identified in SCLC to date. Here, we investigate the function of RAR-related orphan receptor gamma (RORγ) and identified the anti-cancer activity of its natural inhibitor against SCLC and illustrate the underlying mechanism. We show that RORγ depletion affected cell growth both in 2-D cell proliferation and 3-D organoids formation. Natural marine product N-hydroxyapiosporamide (N-hydap) directly bound to RORγ and inhibited its transcriptional activity, leading to the blocking of transmission process of RORγ signaling. Gene expression profiling analysis revealed that N-hydap reprograms neuroendocrine fate via inhibiting RORγ activity in SCLC. Chromatin immunoprecipitation analysis showed that N-hydap strongly reduced RORγ occupancy and transcriptional activation-linked histone marks H3K27ac on the promoter and/or enhancer sites of neurogenesis markers gene including aurora kinase a (AURKA), delta like canonical Notch ligand 3 (DLL3) and tubulin beta 3 class III (TUBB3). Therapeutically, N-hydap exhibited a strong inhibitory effect on tumor growth and did not show significant toxicity in SCLC mice xenograft models. Taken together, RORγ could be an attractive target for SCLC and thus N-hydap can be a promising therapeutic drug candidate for SCLC by inhibiting the RORγ activation.

MiRNA-339-5p promotes isoproterenol-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling.

MicroRNAs (miRNAs) regulate multiple biological processes and participate in various cardiovascular diseases. This study aims to investigate the role of miR-339-5p in cardiomyocyte hypertrophy and the involved mechanism. Neonatal rat cardiomyocytes (NRCMs) were cultured and stimulated with isoproterenol (ISO). The hypertrophic responses were monitored by measuring the cell surface area and expression of hypertrophic markers including ß-myosin heavy chain (ß-MHC) and atrial natriuretic factor (ANF). Bioinformatic prediction tools and dual-luciferase reporter assay were performed to identify the target gene of miR-339-5p. Quantitative real-time polymerase chain reaction and western blot analysis were used to determine the levels of miR-339-5p and its downstream effectors. Our data showed that miR-339-5p was upregulated during cardiomyocyte hypertrophy triggered by ISO. MiR-339-5p overexpression resulted in enlargement of cell size and increased the levels of hypertrophic markers. In contrast, inhibition of miR-339-5p significantly attenuated ISO-induced hypertrophic responses of NRCMs. Valosin-containing protein (VCP), a suppressor of cardiac hypertrophy via inhibiting mechanistic target of rapamycin (mTOR) signaling, was validated as a target of miR-339-5p. MiR-339-5p suppressed VCP protein expression, leading to elevated phosphorylation of mTOR and ribosomal protein S6 kinase (S6K). VCP depletion activated the mTOR/S6K cascade and could compromise the anti-hypertrophic effects of miR-339-5p inhibitor. Additionally, the hypertrophic responses caused by miR-339-5p was alleviated in the presence of mTOR inhibitor rapamycin. In conclusion, our research revealed that miR-339-5p promoted ISO-induced cardiomyocyte hypertrophy by targeting VCP to activate the mTOR signaling, suggesting a promising therapeutic intervention by interfering miR-339-5p.

The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG.

N6-methyladenosine (m6A) is the most abundant posttranscriptional methylation modification that occurs in mRNA and modulates the fine-tuning of various biological processes in mammalian development and human diseases. In this study we investigated the role of m6A modification in the osteogenesis of mesenchymal stem cells (MSCs), and the possible mechanisms by which m6A modification regulated the processes of osteoporosis and bone necrosis. We performed systematic analysis of the differential gene signatures in patients with osteoporosis and bone necrosis and conducted m6A-RNA immunoprecipitation (m6A-RIP) sequencing to identify the potential regulatory genes involved in osteogenesis. We showed that fat mass and obesity (FTO), a primary m6A demethylase, was significantly downregulated in patients with osteoporosis and osteonecrosis. During the differentiation of human MSCs into osteoblasts, FTO was markedly upregulated. Both depletion of FTO and application of the FTO inhibitor FB23 or FB23-2 impaired osteogenic differentiation of human MSCs. Knockout of FTO in mice resulted in decreased bone mineral density and impaired bone formation. PPARG, a biomarker for osteoporosis, was identified as a critical downstream target of FTO. We further revealed that FTO mediated m6A demethylation in the 3'UTR of PPARG mRNA, and reduced PPARG mRNA stability in an YTHDF1-dependent manner. Overexpression of PPARG alleviated FTO-mediated osteogenic differentiation of MSCs, whereas knockdown of PPARG promoted FTO-induced expression of the osteoblast biomarkers ALPL and OPN during osteogenic differentiation. Taken together, this study demonstrates the functional significance of the FTO-PPARG axis in promoting the osteogenesis of human MSCs and sheds light on the role of m6A modification in mediating osteoporosis and osteonecrosis.

The novel STAT3 inhibitor WZ-2-033 causes regression of human triple-negative breast cancer and gastric cancer xenografts.

Hyperactive signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in human triple-negative breast cancer (TNBC) and gastric cancer, leading to uncontrolled tumor growth, resistance to chemotherapy, and poor prognosis. Thus, inhibition of STAT3 signaling is a promising therapeutic approach for both TNBC and gastric cancer, which have high incidences and mortality and limited effective therapeutic approaches. Here, we report a small molecule, WZ-2-033, capable of inhibiting STAT3 activation and dimerization and STAT3-related malignant transformation. We present in vitro evidence from surface plasmon resonance analysis that WZ-2-033 interacts with the STAT3 protein and from confocal imaging that WZ-2-033 disrupts HA-STAT3 and Flag-STAT3 dimerization in intact cells. WZ-2-033 suppresses STAT3-DNA-binding activity but has no effect on STAT5-DNA binding. WZ-2-033 inhibits the phosphorylation and nuclear accumulation of pY705-STAT3 and consequently suppresses STAT3-dependent transcriptional activity and the expression of STAT3 downstream genes. Moreover, WZ-2-033 significantly inhibited the proliferation, colony survival, migration, and invasion of TNBC cells and gastric cancer cells with aberrant STAT3 activation. Furthermore, administration of WZ-2-033 in vivo induced a significant antitumor response in mouse models of TNBC and gastric cancer that correlated with the inhibition of constitutively active STAT3 and the suppression of known STAT3 downstream genes. Thus, our study provides a novel STAT3 inhibitor with significant antitumor activity in human TNBC and gastric cancer harboring persistently active STAT3.