Xl019, a novel JAK inhibitor, suppressed osteoclasts differentiation induced by RANKL through MAPK signaling pathway.

Inbonemetabolism,osteoclastsare the only cellscapableofresorbingbone. Hyperactivity of osteoclasts may lead to osteolytic disease like osteoporosis and arthritis. Although there are several drugs for the treatment of osteolytic diseases, they have limitations and a variety of side effects. An inhibitor of Janus kinase (JAK), XL019, has shown promising results in the treatment of myelofibrosis and other cancers. But whether it can functionally impact osteoclast activity has not been proven. In this study, the effects of XL019 on osteoclastogenesis and the mechanism pathway were investigated in vitro. It was found that XL019 could impair osteoclasts formation, interfere with bone resorption ability and downregulate the osteoclast-specific genes and proteins expression. Furthermore, Western blot and molecular docking studies demonstrated that XL019 inhibited RANKL-induced osteoclastogenesis by suppressing MAPK signaling. A molecular docking analysis explained how XL019 binds to MAPK pathway factors. In addition, titanium particles induced calvarial osteolysis in mice further confirming its beneficial effect on bone homeostasis in vivo. In conclusion, this study demonstrates that Osteoclastactivity canbeeffectivelyinhibitedby XL019viaMAPK signalingpathway,making it a promising alternative pharmacologicaltreatmentfor bone metabolicdisorders.

Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation.

Glycosyltransferases are nature's versatile tools to tailor the functionalities of proteins, carbohydrates, lipids, and small molecules by transferring sugars. Prominent substrates are hydroxycoumarins such as scopoletin, which serve as natural plant protection agents. Similarly, C13-apocarotenoids, which are oxidative degradation products of carotenoids/xanthophylls, protect plants by repelling pests and attracting pest predators. We show that C13-apocarotenoids interact with the plant glycosyltransferase NbUGT72AY1 and induce conformational changes in the enzyme catalytic center ultimately reducing its inherent UDP-α-d-glucose glucohydrolase activity and increasing its catalytic activity for productive hydroxycoumarin substrates. By contrast, C13-apocarotenoids show no effect on the catalytic activity toward monolignol lignin precursors, which are competitive substrates. In vivo studies in tobacco plants (Nicotiana benthamiana) confirmed increased glycosylation activity upon apocarotenoid supplementation. Thus, hydroxycoumarins and apocarotenoids represent specialized damage-associated molecular patterns, as they each provide precise information about the plant compartments damaged by pathogen attack. The molecular basis for the C13-apocarotenoid-mediated interplay of two plant protective mechanisms and their function as allosteric enhancers opens up potential applications of the natural products in agriculture and pharmaceutical industry.

Segnet Network Algorithm-Based Ultrasound Images in the Diagnosis of Gallbladder Stones Complicated with Gallbladder Carcinoma and the Relationship between P16 Expression with Gallbladder Carcinoma.

The study focused on how to improve the diagnostic coincidence rate of patients with gallbladder stones and gallbladder cancer based on an optimized Segnet network algorithm and the relationship of gallbladder cancer with multiple tumor suppressor 1 (P16). 300 patients diagnosed with gallbladder cancer in the hospital were selected as the research subjects. The pyramid pooling operation was incorporated into the original Segnet network algorithm, and its performance was evaluated, factoring into the intersection of union (IoU), algorithm precision (Pre), and recall rate (Recall). After 8 hours of fasting, conventional ultrasound and contrast-enhanced ultrasound examinations were performed, and the images were evaluated by three experienced ultrasound diagnosticians. The positive signal of P16 immunohistochemical staining was brownish yellow, which was generally concentrated in the nucleus, and a small part was located in the cytoplasm. In each slice, ten visual fields were selected. Then, they were observed under a high-power mirror, and the number was counted. It was found that the optimized Segnet network algorithm increased the IoU by 7.3%, the precision by 8.2%, and the recall rate by 11.1%. The diagnostic coincidence rates of conventional ultrasound and contrast-enhanced ultrasound examinations for gallbladder cancer were 78.13% (25/32) and 87.5% (25/32), respectively. The positive expression rate of P16 in gallbladder adenocarcinoma (47.06%) was significantly lower than that of acute cholecystitis with gallbladder stones (84.38%) and gallbladder polyps (67.16%) (P < 0.05). The positive expression rate of P16 in patients with stage III and stage IV (33.33% and 40%) was significantly lower than that in patients with stages I and II (87.5% and 80%) (P < 0.05). The positive expression rate of P16 in high differentiation (86.67%) was significantly higher than that of moderate differentiation (40%) and poor differentiation (28.57%) (P < 0.05). In short, contrast-enhanced ultrasound can effectively improve the diagnostic coincidence rate of gallbladder cancer, and the expression of P16 in gallbladder cancer is closely related to tumor staging and differentiation.

Six Uridine-Diphosphate Glycosyltransferases Catalyze the Glycosylation of Bioactive C13-Apocarotenols.

C13-apocarotenoids (norisoprenoids) are carotenoid-derived oxidation products that perform important physiological functions in plants. Although their biosynthetic pathways have been extensively studied, their metabolism including glycosylation remains poorly understood. Candidate uridine-diphosphate glycosyltransferase genes (UGTs) were selected based on their high transcript abundance in comparison with other UGTs in vegetative tissues of Nicotiana benthamiana and peppermint (Mentha × piperita), as these tissues are rich sources of apocarotenoid glucosides. Hydroxylated C13-apocarotenol substrates were produced by P450-catalyzed biotransformation and microbial/plant enzyme systems were established for the synthesis of glycosides. Natural substrates were identified by physiological aglycone libraries prepared from isolated plant glycosides. In total, we identified six UGTs that catalyze the glucosylation of C13-apocarotenols, where Glc is bound either to the cyclohexene ring or the butane side chain. MpUGT86C10 is a superior novel enzyme that catalyzes the glucosylation of allelopathic 3-hydroxy-α-damascone, 3-oxo-α-ionol, 3-oxo-7,8-dihydro-α-ionol (Blumenol C), and 3-hydroxy-7,8-dihydro-ß-ionol, whereas a germination test demonstrated the higher phytotoxic potential of a norisoprenoid glucoside in comparison to its aglycone. Glycosylation of C13-apocarotenoids has several functions in plants, including increased allelopathic activity of the aglycone, facilitating exudation by roots and allowing symbiosis with arbuscular mycorrhizal fungi. The results enable in-depth analysis of the roles of glycosylated norisoprenoid allelochemicals, the physiological functions of apocarotenoids during arbuscular mycorrhizal colonization, and the associated maintenance of carotenoid homeostasis.

Glucosylation of the phytoalexin N-feruloyl tyramine modulates the levels of pathogen-responsive metabolites in Nicotiana benthamiana.

Enzyme promiscuity, a common property of many uridine diphosphate sugar-dependent glycosyltransferases (UGTs) that convert small molecules, significantly hinders the identification of natural substrates and therefore the characterization of the physiological role of enzymes. In this paper we present a simple but effective strategy to identify endogenous substrates of plant UGTs using LC-MS-guided targeted glycoside analysis of transgenic plants. We successfully identified natural substrates of two promiscuous Nicotiana benthamiana UGTs (NbUGT73A24 and NbUGT73A25), orthologues of pathogen-induced tobacco UGT (TOGT) from Nicotiana tabacum, which is involved in the hypersensitive reaction. While in N. tabacum, TOGT glucosylated scopoletin after treatment with salicylate, fungal elicitors and the tobacco mosaic virus, NbUGT73A24 and NbUGT73A25 produced glucosides of phytoalexin N-feruloyl tyramine, which may strengthen cell walls to prevent the intrusion of pathogens, and flavonols after agroinfiltration of the corresponding genes in N. benthamiana. Enzymatic glucosylation of fractions of a physiological aglycone library confirmed the biological substrates of UGTs. In addition, overexpression of both genes in N. benthamiana produced clear lesions on the leaves and led to a significantly reduced content of pathogen-induced plant metabolites such as phenylalanine and tryptophan. Our results revealed some additional biological functions of TOGT enzymes and indicated a multifunctional role of UGTs in plant resistance.

MicroRNA-325-3p inhibits cell proliferation and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma by down-regulation of aquaporin 5.

BACKGROUND: Hepatitis B virus (HBV) infection is acknowledged as the main cause of hepatocellular carcinoma (HCC). Moreover, previous studies have revealed that microRNAs (miRNAs) widely participate in regulation of various cellular processes, such as viral replication. Hence, the purpose of this study was to investigate the roles of aquaporin 5 (AQP5) and miR-325-3p in the proliferation and apoptosis of HBV-related HCC cells. METHODS: AQP5 and miR-325-3p expression in both normal and HBV-HCC tissues or cells (both Huh7-1.3 and HepG2.2.15) was detected using qRT-PCR. AQP5 expression was knocked down in HBV-related Huh7-1.3 and HepG2.2.15 cells using small interfering RNA (siRNA) technology. Down-regulation was confirmed using real-time PCR and Western blot analysis. Effects of AQP5 down-regulation on the proliferation and apoptosis were assessed. Dual luciferase reporter gene assay, Western blot and qRT-PCR were employed to evaluate the effect of miR-325-3p on the luciferase activity and expression of AQP5. Moreover, miR-325-3p mimic-induced changes in cellular proliferation and apoptosis were detected through CCK-8 assay, BrdU assay, flow cytometry analysis and ELISA. RESULTS: In this study, the expression of AQP5 was up-regulated in human HBV-HCC tissue, Huh7-1.3 and HepG2.2.15 cells. Knockdown of AQP5 significantly inhibited the proliferation and promoted apoptosis of HBV-HCC cells. Next, miR-325-3p was obviously down-regulated in HBV-HCC. In concordance with this, MiR-325-3p directly targeted AQP5, and reduced both mRNA and protein levels of AQP5, which promoted cell proliferation and suppressed cell apoptosis in HCC cells. Overexpression of miR-325-3p dramatically inhibited cell proliferation and induced cell apoptosis. CONCLUSIONS: Our findings clearly demonstrated that introduction of miR-325-3p inhibited proliferation and induced apoptosis of Huh7-1.3 and HepG2.2.15 cells by directly decreasing AQP5 expression, and that silencing AQP5 expression was essential for the pro-apoptotic effect of miR-325-3p overexpression on Huh7-1.3 and HepG2.2.15 cells. It is beneficial to gain insight into the mechanism of HBV infection and pathophysiology of HBV-related HCC.

FoxO3a inhibiting expression of EPS8 to prevent progression of NSCLC: A new negative loop of EGFR signaling.

BACKGROUND: The resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKI) is a major challenge in the treatment of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms behind resistance is therefore an important issue. Here we assessed the role of EGFR pathway substrate 8 (EPS8) and Forkhead box O 3a (FoxO3a) as potentially valuable targets in the resistance of NSCLC . METHODS: The expression levels of EPS8 and FoxO3a in patients with NSCLC (n = 75) were examined by immunohistochemistry staining, while in cells were detected by qPCR and western blot. The effects of EPS8 and FoxO3a on resistance, migration and invasion, cell cycle arrest were detected by MTT, transwell and flow cytometry, respectively. Chromatin immunoprecipitation and luciferase reporter assays were performed to determine the mechanisms of EPS8 expression and FoxO3a regulation. FINDINGS: We observed that the expression of EPS8 inversely correlated with FoxO3a in NSCLC cell lines and NSCLC patients. FoxO3a levels were significantly decreased in tumor tissues compared with para-carcinoma tissues, while EPS8 is opposite. Besides, they play reverse roles in the resistance to gefitinib, the migration and invasion abilities, the cell cycle arrest in vitro and the tumor growth in vivo. Mechanistically, FoxO3a inhibits EPS8 levels by directly binding its gene promoter and they form a negative loop in EGFR pathway. INTERPRETATION: Targeting FoxO3a and EPS8 in EGFR signaling pathway prevents the progression of NSCLC, which implied that the negative loop they formed could served as a therapeutic target for overcoming resistance in NSCLC. FUNDS: National Natural Science Foundation of China, Science and Technology Project of Henan, Outstanding Young Talent Research Fund of Zhengzhou University and the National Scholarship Fund.

Structural and Functional Analysis of UGT92G6 Suggests an Evolutionary Link Between Mono- and Disaccharide Glycoside-Forming Transferases.

Glycosylation mediated by UDP-dependent glycosyltransferase (UGT) is one of the most common reactions for the biosynthesis of small molecule glycosides. As glycosides have various biological roles, we characterized UGT genes from grapevine (Vitis vinifera). In silico analysis of VvUGT genes that were highly expressed in leaves identified UGT92G6 which showed sequence similarity to both monosaccharide and disaccharide glucoside-forming transferases. The recombinant UGT92G6 glucosylated phenolics, among them caffeic acid, carvacrol, eugenol and raspberry ketone, and also accepted geranyl glucoside and citronellyl glucoside. Thus, UGT92G6 formed mono- and diglucosides in vitro from distinct compounds. The enzyme specificity constant Vmax/Km ratios indicated that UGT92G6 exhibited the highest specificity towards caffeic acid, producing almost equal amounts of the 3- and 4-O-glucoside. Transient overexpression of UGT92G6 in Nicotiana benthamiana leaves confirmed the production of caffeoyl glucoside; however, the level of geranyl diglucoside was not elevated upon overexpression of UGT92G6, even after co-expression of genes encoding geraniol synthase and geraniol UGT to provide sufficient precursor. Comparative sequence and 3-D structure analysis identified a sequence motif characteristic for monoglucoside-forming UGTs in UGT92G6, suggesting an evolutionary link between mono- and disaccharide glycoside UGTs. Thus, UGT92G6 functions as a mono- and diglucosyltransferase in vitro, but acts as a caffeoyl glucoside UGT in N. benthamiana.

[Cytogenetic and clinical study of myeloid leukemia].

OBJECTIVE: To explore the clinical cytogenetic features and prognosis of myeloid leukemia patients. METHODS: Bone marrow direct method and/or 24h culture without phytohaemagglutimin(PHA) were used to prepare the chromosomes and karyotype analysis was performed with R-banding and G-banding techniques. RESULTS: Among 420 patients with acute myeloid leukemia (AML), 223 cases were found to exhibit clonal chromosome abnormalities, accounted for 53.1%. t(8; 21), t(15; 17), inv(16)and del(11) were specifically associated with M2b, M3, M4Eo and M5 respectively. Out of 158 patients with chronic myeloid leukemia (CML), 96.8% (153/158) were found to exhibit clonal chromosome abnormalities. T(9;22) was specifically associated with CML and some cases of M0, M1 and M2. In these myeloid leukemia cases, there were 18 cases (AML 13 cases, CML 15 cases) without clonal chromosome abnormalities, accounted for 3.1% (18/578) and this phenomenon agreed with the diagnose of clinical signs, marrow morphology and immunology incompletely. CONCLUSION: Karyotype analysis was not only helpful to the diagnose and differential diagnose of myeloid leukemia, but also an important standard of the remission, relapse and therapeutic effect of myeloid leukemia. Chromosome analysis can be made exactly with the probe and FISH technique on the basic of chromosome karyotype analysis.