Effect and mechanism of C-terminal cysteine on the properties of HEV p222 protein.

Preliminary investigations have demonstrated that the cysteines located at the C-terminus of HEV ORF2 protein exhibits disulfide bonding capability during virus-like particles (VLPs) assembly. However, the effect and mechanism underlying the pairing of disulfide bonds formed by C627, C630, and C638 remains unclear. The p222 protein encompasses C-terminus and serves as a representative of HEV ORF2 to investigate the specific impacts of C627, C630, and C638. The three cysteines were subjected to site-directed mutagenesis and expressed in prokaryotes; Both the mutated proteins and p222 underwent polymerization except for p222A; Surprisingly, only p222 was observed as abundant spherical particles under transmission electron microscope (TEM); Stability and immunogenicity of the p222 exhibited higher than other mutated proteins; LC/MS/MS analysis identified four disulfide bonds in the p222. The novel findings suggest that the three cysteines contribute to structural and functional properties of ORF2 protein, highlighting the indispensability of each cysteine.

Erratum: Overexpression of ZEB1 and YAP1 is related to poor prognosis in patients with gliomas with different IDH1 status.

[This corrects the article on p. 138 in vol. 16, PMID: 37559682.].

Overexpression of ZEB1 and YAP1 is related to poor prognosis in patients with gliomas with different IDH1 status.

OBJECTIVE: Whether there is a correlation between zinc-finger E-box-binding homolog 1 (ZEB1) and Yes-associated protein 1 (YAP1) with clinical outcome in gliomas remains unclear. Hence, this study aimed to investigate the effects of ZEB1 and YAP1 on the prognosis of human gliomas and its relationship with the isocitrate dehydrogenase 1 (IDH1) gene state. METHODS: Immunohistochemical staining was used to record the expression levels of ZEB1, YAP1, and p-YAP1 in 122 cases of low-grade glioma (LGGs) and 69 cases of glioblastoma (GBMs). The correlations of ZEB1 and YAP1 with pathological data were determined by Pearson's Chi-square test. Spearman correlation analysis was then used for analyzing the relationship among YAP1, ZEB1, and IDH1 gene status. The effects of ZEB1 and YAP1 on prognosis were investigated through survival analysis. RESULTS: We detected high ZEB1 expression levels in 29 LGGs (23.8%) and 39 GBMs (56.5%), and high YAP1 expression levels in 22 LGGs (18.0%) and 44 of GBM (63.8%). These results revealed that the protein expression levels of ZEB1 and YAP1 were higher in GBM (P < 0.001). There was a significantly positive correlation between ZEB1 and YAP1 (P < 0.001; r = 0.533). High ZEB1 expression was related to tumor grade (P < 0.001) and Ki-67 (P = 0.0037). YAP1 overexpression was correlated with Ki-67 (P < 0.001), P53 (P = 0.009), tumor grade (P < 0.001), and tumor location (P = 0.018). Patients with ZEB1 and YAP1 high expression had worse overall survival (OS) (P < 0.001). The multivariate analysis showed that YAP1 was an independent prognostic factor for OS. In the LGG group, worse OS were observed in glioma patients with elevated YAP1 expression level. Spearman correlation analysis revealed no association between ZEB1 expression and IDH1 state (P = 0.360; r = -0.084), and YAP1 expression had a negative correlation with IDH1 mutation (P < 0.001, r = -0.364). CONCLUSIONS: Our study showed that ZEB1 and YAP1 were significantly activated in GBM, and patients with high ZEB1 and YAP1 expression had worse OS. ZEB1 expression was significantly correlated with YAP1 in glioma. ZEB1 and YAP1 coexpression may serve as a useful prognostic biomarker for glioma, and aberrant YAP1 expression may be associated with IDH1 gene state.

Widely Targeted Metabolomics Reveals the Effects of Soil on the Metabolites in Dioscorea opposita Thunb.

Chinese yam (Dioscorea opposita Thunb. cv. Tiegun), a type of homologous medicinal plant, mainly grows in sandy soil (SCY) and loessial soil (LCY). However, the effects of the soil on the metabolites in SCY and LCY remain unclear. Herein, this study aims to comprehensively elucidate the metabolites in SCY and LCY. A UPLC-MS/MS-based, widely targeted metabolomics approach was adapted to compare the chemical composition of SCY and LCY. A total of 988 metabolites were detected, including 443 primary metabolites, 510 secondary metabolites, and 35 other compounds. Notably, 177 differential metabolites (classified into 12 categories) were identified between SCY and LCY; among them, 85.9% (152 differential metabolites) were upregulated in LCY. LCY significantly increased the contents of primary metabolites such as 38 lipids and 6 nucleotides and derivatives, as well as some secondary metabolites such as 36 flavonoids, 28 phenolic acids, 13 alkaloids, and 6 tannins. The results indicate that loessial soil can improve the nutritional and medicinal value of D. opposita.

Aniline-induced production of aniline-containing polyketides and related bicyclic polyketides by the Yellow River wetland-derived fungus Talaromyces funiculosus.

Introduction and Methods: Silencing gene activation can effectively enrich the diversity of fungal secondary metabolites. Results and Discussion: Cultivation of the Yellow River wetland-derived fungus Talaromyces funiculosus HPU-Y01 with aniline led to the isolation of one new aniline-containing polyketide tanicutone A (1), two new bicyclic polyketides tanicutones B-C (2-3), a new related trienoic acid 8-methyldeca-2,4,6-trienoic acid (5), and a known compound 4. The planar structures and configurations of 1-5 were determined by NMR, MS, and ECD calculations. Compound 2 featured a key aldehyde group and showed promising inhibitory activity against Vibrio parahaemolyticus with a minimum inhibitory concentration (MIC) value of 0.17 µg/mL. This is a rare report of aniline-induced fungal production of tetrahydronaphthone polyketides.

DLK1-directed chimeric antigen receptor T-cell therapy for hepatocellular carcinoma.

BACKGROUND: Delta-like homologue 1 (DLK1), a transmembrane protein, is highly expressed in hepatocellular carcinoma (HCC). We explored whether DLK1-directed chimeric antigen receptor (CAR) T cells can specifically eliminate DLK1-positive HCC cells and serve as a therapeutic strategy for HCC immunotherapy. METHODS: We first characterized a homemade anti-human DLK1 monoclonal antibody, sequenced the single-chain Fragment variable (scFv) and integrated it into the second-generation CAR lentiviral vector, and then developed the DLK1-directed CAR-T cells. The cytotoxic activities of DLK1-directed CAR-T cells against different HCC cells were evaluated in vitro and in vivo. RESULTS: The genetically modified human T cells with the DLK1-directed CARs produced cytotoxic activity against DLK1-positive HCC cells. Additionally, the DLK1-directed CARs enhanced T cell proliferation and activation in a DLK1-dependent manner. Interestingly, the DLK1-targeted CAR-T cells significantly inhibited both subcutaneous and peritoneal xenograft tumours derived from human liver cancer cell lines HepG2 or Huh-7. CONCLUSION: DLK1-directed CAR-T cells specifically suppresses DLK1-positive HCC cells in vitro and in vivo. This study provides a novel transmembrane antigen DLK1 as a potential therapeutic target appropriate for CAR-T cell therapy, which may be further developed as a clinical therapeutic strategy for HCC immunotherapy.

Analysis of the expression and prognostic value of MT1-MMP, ß1-integrin and YAP1 in glioma.

Increased expression of membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14) is associated with the development of many cancers. MT1-MMP may promote the entry of yes-associated protein1 (YAP1) into the nucleus by regulating the regulation of ß1-integrin. The purpose of this study was to investigate the effects of MT1-MMP, ß1-integrin and YAP1 on the prognosis of gliomas. The expression of proteins was detected by bioinformatics and immunohistochemistry. The relationship between three proteins and clinicopathological parameters was analyzed by the χ 2 test. Survival analysis was used to investigate the effects of three proteins on prognosis. The results showed that high expressions of MT1-MMP, ß1-integrin and YAP1 were found in glioblastoma (GBM) compared with lower-grade glioma (LGG). There was a significantly positive correlation between MT1-MMP and ß1-integrin (r = 0.387), MT1-MMP and YAP1 (r = 0.443), ß1-integrin and YAP1 (r = 0.348). Survival analysis showed that patients with overexpression of MT1-MMP, ß1-integrin and YAP1 had a worse prognosis. YAP1 expression was the independent prognostic factor for progression-free survival (PFS). There was a statistical correlation between the expression of MT1-MMP and YAP1 and isocitrate dehydrogenase 1 (IDHl) mutation. Thus, this study suggested that MT1-MMP, ß1-integrin and YAP1, as tumor suppressors, are expected to be promising prognostic biomarkers and therapeutic targets for glioma patients.

The Expression and Prognostic Value of ILK and YAP1 in Glioma.

Integrin-linked kinase (ILK) is a widely expressed serine/threonine-protein kinase that has been implicated in cancer development, progression, and metastasis. Yes-associated protein (YAP), as the most important effector of Hippo signaling pathway, which is considered to be a tumor suppressor pathway, acts as an oncogene in a variety of human cancers. The present study aimed to explore the expression of ILK and YAP1, the relationship between them, and the effect of ILK, YAP1 on prognosis in gliomas. Immunohistochemistry was used to examine the expression of ILK and YAP1. The χ2 test analyzes the relationship between ILK, YAP1, and pathologic parameters. The Spearman correlation analyzes the relationship between ILK and YAP1. Survival analysis was used to investigate the effect of ILK and YAP1 on prognosis. High expression of ILK was associated with the age above 50 (P=0.003), higher World Health Organization (WHO) grade (P<0.001), recurrence (P<0.001), and Ki-67 expression≥10% (P<0.001). High expression of YAP1 was associated with higher WHO grade (P<0.001), recurrence (P=0.043), and Ki-67 expression ≥10% (P=0.037). In lower grade gliomas, the high expression rate of ILK in isocitrate dehydrogenase 1 wild-type was higher than that in isocitrate dehydrogenase 1 mutant (P=0.048). The high expression rate of YAP1 in 1p19q non-codeletion was higher than that in 1p19q codeletion (P=0.022). There was a positive correlation between ILK and YAP1 (r=0.344). The patients with high expression of ILK and YAP1 had worse OS and PFS. As an upstream factor of the Hippo signaling pathway, ILK may affect the development and prognosis of gliomas by regulating YAP1.

Precursor-Directed Biosynthesis of Talaroenamine Derivatives Using a Yellow River Wetland-Derived Penicillium malacosphaerulum.

Precursor-directed biosynthesis was used to introduce selected aniline derivatives into the talaroenamine pathway, which had recently been defined from a Yellow River wetland-derived Penicillim malacosphaerulum HPU-J01. The known talaroenamine B (1) and six previously undescribed talaroenamine derivatives, talaroenamines F-K (2-7), were generated and structurally characterized. The aniline derivatives are introduced via nonenzymatic addition to the reactive intermediate cyclohexanedione. Compound 2 was active against Bacillus cereus with an MIC value of 0.85 µg/mL.

Expression of YAP1 and pSTAT3-S727 and their prognostic value in glioma.

AIMS: A growing research demonstrated that YAP1 played important roles in gliomagenesis. We explored the expression of YAP1 and STAT3, the relationship between them and the effect of YAP1, STAT3 on prognosis in glioma. METHODS: Expression of YAP1, p-YAP1, STAT3, pSTAT3-S727 and pSTAT3-Y705 in 141 cases of low-grade gliomas (LGG) and 74 cases of high-grade gliomas (HGG) of surgical specimens were measured by immunohistochemistry. Pearson's X2 test was used to determine the correlation between immunohistochemical expressions and clinicopathological parameters. Pearson's or Spearman correlation test was used to determine the association between these proteins expression. Survival analysis was used to investigate the effect of these proteins on prognosis. RESULTS: High expressions of YAP1, STAT3, pSTAT3-S727 and pSTAT3-Y705 were found in HGG compared with LGG (p=0.000). High expressions of YAP1, STAT3, pSTAT3-S727 and pSTAT3-Y705 were found in 63.5%, 59.5%, 66.2% and 31.1% cases of HGG, respectively. YAP1 expression was associated to tumour location, Ki-67 and P53, STAT3 expression was related with Ki-67 and P53, and the expression of pSTAT3-S727 was associated with Ki-67. There was a significantly positive correlation between YAP1 and pSTAT3-S727 (p<0.0001; r=0.5663). Survival analysis revealed that patients with YAP1 and pSTAT3-S727 coexpression had worse overall survival (OS) and progression-free survival (PFS) (p<0.0001). Tumour grade, age, Ki-67 and YAP1 expression were independent prognostic factors for OS. In LGG group, both YAP1 and pSTAT3-S727 expressions were negative correlation with IDH1 mutation, YAP1 and pSTAT3-S727 coexpression showed worse OS and PFS of glioma patients. CONCLUSION: Our research showed that YAP1 and STAT3 were significantly activated in HGG compared with LGG. YAP1 significantly correlated with pSTAT3-S727 in glioma, YAP1 and pSTAT3-S727 coexpression may serve as a reliable prognostic biomarker and therapeutic target for glioma.

Rapid identification of chemical compositions in callicarpa kwangtungensis Chun by ultra-high-performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry.

Callicarpa kwangtungensis Chun is a traditional Chinese medicine that has various therapeutic effects. Despite its wide use in Chinese medicine, the study is still quite limited, especially its chemical compositions. In this research, an ultra-high-pressure liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry tandem mass spectrometry method was utilized to analyze its chemical compositions for the first time. As a result, a total of 124 compounds, including 20 phenylethanoid glycosides, 31 flavonoids, 36 organic acids, 26 terpenoids and 11 phenols, were identified or tentatively characterized in 30 min. Among them, 49 compounds, including 5 phenylethanoid glycosides, 12 flavonoids, 16 organic acids, 12 terpenoids, and 4 phenols, were identified in Callicarpa kwangtungensis Chun for the first time. Besides, the fragmentation pathways were also discussed. This research established a rapid and reliable method to analyze the chemical compositions of complicated herb without the process of isolation, and provide abundant information on the chemical material basis for further bioactivity and quality control studies.

Three new labdane-type diterpenoids from Callicarpa macrophylla Vahl.

Three new labdane-type diterpenoids, callicapene M3-M5 (1-3) were isolated from the Callicarpa macrophylla Vahl. Their structures were identified by spectroscopic method. The isolated compounds were evaluated for inhibitory activity on NO production in LPS-activated RAW 264.7 macrophage cells by using MTT assays. Compounds 1-3 showed potent inhibitory activity, with IC50 value of 48.15, 46.31 and 38.72 µM respectively.

4-Iminooxazolidin-2-one as a Bioisostere of the Cyanohydrin Moiety: Inhibitors of Enterovirus 71 3C Protease.

A recently reported potent inhibitor of enterovirus 71 3C protease, ( R)-1, was found to have stability and potential toxicity issues due to the presence of a cyanohydrin moiety. Modifying the labile cyanohydrin moiety, by serendipity, led to the discovery of 4-iminooxazolidin-2-one-based inhibitors 4e and 4g with potent inhibitory activity and significantly improved stability. In vivo pharmacokinetic studies of 4e also demonstrated high plasma exposure and moderate half-life. These compounds have shown potential of becoming anti-EV71 drug candidates.

SCFFBW7-mediated degradation of Brg1 suppresses gastric cancer metastasis.

Brg1/SMARCA4 serves as the ATPase and the helicase catalytic subunit for the multi-component SWI/SNF chromatin remodeling complex, which plays a pivotal role in governing chromatin structure and gene transcription. However, the upstream signaling pathways regulating Brg1 protein stability and its physiological contribution to carcinogenesis remain largely elusive. Here we report that Brg1 is a bona fide ubiquitin substrate of SCFFBW7. We reveal that CK1δ phosphorylates Brg1 at Ser31/Ser35 residues to facilitate the binding of Brg1 to FBW7, leading to ubiquitination-mediated degradation. In keeping with a tumor suppressive role of FBW7 in human gastric cancer, we find an inverse correlation between FBW7 and Brg1 expression in human gastric cancer clinical samples. Mechanistically, we find that stabilization of Brg1 in gastric cancer cells suppresses E-cadherin expression, subsequently promoting gastric cancer metastasis. Hence, this previously unknown FBW7/Brg1 signaling axis provides the molecular basis and the rationale to target Brg1 in FBW7-compromised human gastric cancers.

Structure of the Enterovirus 71 3C Protease in Complex with NK-1.8k and Indications for the Development of Antienterovirus Protease Inhibitor.

Hand-foot-and-mouth disease (HFMD), caused by enterovirus, is a threat to public health worldwide. To date, enterovirus 71 (EV71) has been one of the major causative agents of HFMD in the Pacific-Asia region, and outbreaks with EV71 cause millions of infections. However, no drug is currently available for clinical therapeutics. In our previous works, we developed a set of protease inhibitors (PIs) targeting the EV71 3C protease (3Cpro). Among these are NK-1.8k and NK-1.9k, which have various active groups and high potencies and selectivities. In the study described here, we determined the structures of the PI NK-1.8k in complex with wild-type (WT) and drug-resistant EV71 3Cpro Comparison of these structures with the structure of unliganded EV71 3Cpro and its complex with AG7088 indicated that the mutation of N69 to a serine residue destabilized the S2 pocket. Thus, the mutation influenced the cleavage activity of EV71 3Cpro and the inhibitory activity of NK-1.8k in an in vitro protease assay and highlighted that site 69 is an additional key site for PI design. More information for the optimization of the P1' to P4 groups of PIs was also obtained from these structures. Together with the results of our previous works, these in-depth results elucidate the inhibitory mechanism of PIs and shed light to develop PIs for the clinical treatment of infections caused by EV71 and other enteroviruses.

Inhibition of enterovirus 71 replication by an α-hydroxy-nitrile derivative NK-1.9k.

Enterovirus 71 (EV71) is one of the major etiological agents of human hand-foot-and-mouth disease (HFMD) worldwide. EV71 infection in young children and people with immunodeficiency causes severe symptoms with a high fatality rates. However, there is still no approved drugs to treat such infections. Based on our previous report of a peptide-aldehyde anti-EV71 protease, we present here a highly specific α-hydroxy-nitrile derivative NK-1.9k, which inhibited the proliferation of multiple EV71 strains and coxsackievirus A16 (CVA16) in various cells with EC50 of 37.0 nM with low cytotoxicity (CC50 > 200 µM). The hydroxy-nitrile covalent warhead conferred NK-1.9k high potency and selectivity to interact with the cysteine residue of the active site of the viral protease. We also documented the resistance to NK-1.9k with a N69S mutation in EV71 3Cpro. The combination of NK-1.9k and EV71 polymerase or entry inhibitors produced strong synergistic antiviral effects. Collectively, our findings suggest our compounds can potentially be developed as drugs for the treatment of HFMD.

Structure-activity relationship study of peptidomimetic aldehydes as enterovirus 71 3C protease inhibitors.

A series of peptidomimetic aldehydes were designed, synthesized, and evaluated for their biochemical activity against 3C protease (3Cpro) and anti-enterovirus 71 (EV71) activity in vitro. Molecular docking revealed that 5s (IC50 = 0.22 ± 0.07 µM, EC50 = 0.18 ± 0.05 µM) could bind well to the active site of EV71 3Cpro, which was consistent with the biological data compared to reference 5a (IC50 = 0.54 ± 0.02 µM, EC50 = 0.26 ± 0.07 µM). Structure and relationship study led to the discovery of aldehyde 5x (IC50 = 0.10 ± 0.02 µM, EC50 = 0.11 ± 0.07 µM), which exhibited the most potent 3Cpro inhibitory and antiviral activity.

Synthesis and biological properties of polyamine modified flavonoids as hepatocellular carcinoma inhibitors.

A series of polyamine conjugates of flavonoids with a naphthalene motif were synthesized and evaluated for their anti-hepatocellular carcinoma properties using in vitro and in vivo assays. Compound 8a displayed favorable selectivity between hepatocellular carcinoma cells and normal hepatocyte cells, and the combination of 8a with aspirin resulted in additive inhibition of in vitro tumor cell growth and migration. The 8a-aspirin combination also inhibited H22 liver tumor growth and pulmonary metastasis and improved body weight index in animal models. Preliminary mechanistic studies indicated that 8a increased the expression of apoptosis-related proteins such as p-p38, p-JNK, p53 and Bcl-2, an effect that was further amplified by aspirin. Therefore, a cocktail therapy of flavonoid-polyamine conjugates with aspirin has potential use as an antitumor therapy.

Cyanohydrin as an Anchoring Group for Potent and Selective Inhibitors of Enterovirus 71 3C Protease.

Cyanohydrin derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. Compared with the reported inhibitors, cyanohydrins (1S,2S,2'S,5S)-16 and (1R,2S,2'S,5S)-16 exhibited significantly improved activity and attractive selectivity profiles against other proteases, which were a result of the specific interactions between the cyanohydrin moiety and the catalytic site of 3C(pro). Cyanohydrin as an anchoring group with high selectivity and excellent inhibitory activity represents a useful choice for cysteine protease inhibitors.

Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C.

Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 µM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy.