Phosphorylation of USP27X by GSK3ß maintains the stability and oncogenic functions of CBX2.

Chromobox protein homolog 2 (CBX2) exerts a multifaceted impact on the progression of aggressive cancers. The proteasome-dependent pathway is crucial for modulating CBX2 regulation, while the specific regulatory roles and mechanisms of deubiquitinating enzymes targeting CBX2 remain poorly understood. Mass spectrometry analysis identified ubiquitin-specific peptidase 27X (USP27X) as a deubiquitinating enzyme that targets CBX2. Overexpression of USP27X significantly enhances CBX2 levels by promoting deubiquitination, while deficiency of USP27X leads to CBX2 degradation, thereby inhibiting tumorigenesis. Furthermore, it has been revealed that glycogen synthase kinase 3 beta (GSK3ß) can directly bind to and phosphorylate USP27X, thereby enhancing the interaction between USP27X and CBX2 and leading to further stabilization of the CBX2 protein. Clinically, the co-expression of high levels of USP27X and CBX2 in breast cancer tissues is indicative of a poor prognosis for patients with this disease. These findings collectively underscore the critical regulatory role played by USP27X in modulating CBX2, thereby establishing the GSK3ß-USP27X-CBX2 axis as a pivotal driver of malignant progression in breast cancer.

QingXiaoWuWei decoction alleviates methicillin-resistant Staphylococcus aureus-induced pneumonia in mice by regulating metabolic remodeling and macrophage gene expression network via the microbiota-short-chain fatty acids axis.

IMPORTANCE: Methicillin-resistant Staphylococcus aureus (MRSA) colonizes the upper respiratory airways and is resistant to antibiotics. MRSA is a frequently acquired infection in hospital and community settings, including cases of MRSA-induced pneumonia. Multidrug-resistant Staphylococcus aureus and the limited efficacy of antibiotics necessitate alternative strategies for preventing or treating the infection. QingXiaoWuWei decoction (QXWWD) protects against both gut microbiota dysbiosis and MRSA-induced pneumonia. Furthermore, the QXWWD-regulated metabolic remodeling and macrophage gene expression network contribute to its protective effects through the microbiota-short-chain fatty acid axis. The results of this study suggest that QXWWD and its pharmacodynamic compounds might have the potential to prevent and treat pulmonary infections, especially those caused by multidrug-resistant organisms. Our study provides a theoretical basis for the future treatment of pulmonary infectious diseases by manipulating gut microbiota and their metabolites via traditional Chinese medicine.

[Rapid analysis of serum components and metabolites of Sanzi San by high performance liguid chromatography-quadrupole/ electrostatic field orbitrap high resolution mass spectrometry].

Sanzi San, a Mongolian medicine, comprises three herbs: Terminalia chebula, Melia toosendan, and Gardenia jasminoides. Clinically, Sanzi San is administered orally and distributed via blood to the action site, which implies that the absorption, distribution, metabolism, and excretion (ADME) are closely related to the pharmacological action and curative effect. Therefore, possible explanations for the material basis of Sanzi San were explored in this study preliminarily. A strategy based on serum pharmacochemistry was first applied to explore the absorbed bioactive components and metabolites of Sanzi San. Wistar rats were randomly divided into normal and dosing groups, which were provided with the Sanzi San's water extract for three days. Then, the rat's blood samples were obtained from their abdomiral aorta using a sterile blood collection tube after administering the medicine. The blood samples were then centrifuged at 3500 r/min for 10 min to obtain the serum samples. A practical method based on high performance liquid chromatography coupled with quadrupole and electrostatic field orbitrap high resolution mass spectrometry (HPLC-Q/Orbitrap HRMS) was developed to screen and analyze numerous bioactive components and metabolites adsorbed in the serum of the dosing rats after oral administration of the Sanzi San's water extract. Chromatographic separation was achieved on a SHIMADZU GIST C18 chromatographic column (150 mm×4.6 mm, 5 µm). The temperature of the column was maintained at 30 ℃. The flow rate was 0.5 mL/min, and the injection volume was 10 µL. The mobile phase comprised an aqueous solution of 0.1% formic acid and methanol under gradient elution. A heated electrospray ion (HESI) source was used with positive and negative ion scanning modes. To rapidly screen out and identify the absorbed bioactive components and metabolites of Sanzi San in the rat serum samples, a simple three-step approach was developed. First, the known components in Sanzi San were listed systematically by exploring various databases, such as the Web of Science, PubMed, and Chinese National Knowledge Infrastructure. In addition, relevant information on drug biotransformation and the characteristic fragmentation patterns of parent compounds were summarized. Second, the absorbed components and metabolites were ascertained using the Xcalibur 3.0 software. Based on the information related to the parent compound's structure, the software could be used to identify the unique peaks by comparing the chromatograms of the normal and dosing samples. Consequently, the total ion chromatograms of serum samples were established. Finally, the Compound Discover 3.0 software was used to predict the metabolic pathways and fragmentation of the absorbed compounds. Using this approach, 55 compounds were characterized, including 41 prototype components and 14 metabolites. The main prototype components in the serum sample were tannins, iridoids, and phenolic acids. The details of these compounds have been summarized and presented. Regarding the absorbed bioactive components and metabolites in the serum samples of rats administered with Sanzi San, phase Ⅰ and phase Ⅱ biochemical reactions were involved in the biotransformation pathways. The phase Ⅰ reaction modified the components and created sites for the phase Ⅱ reaction, involving reduction and hydrolysis. The phase Ⅱ reaction coupled groups to existing conjugation sites, including glucuronide to glucuronic acid, sulfate, and methyl. MS/MS spectra indicated that methylation, demethylation, and dehydroxylation are the metabolic pathways of procyanidins. Additionally, glucuronidation, deglucosidation, hydration, and demethylation are the metabolic pathways of iridoids in Sanzi San. This study comprehensively analyzed the components of the Sanzi San's water extract absorbed in the rat's serum. Our results revealed information regarding the pharmacodynamic substances and the major pathways involved in the ADME of Sanzi San. Further, potential medicinal ingredients for the pharmacological effects and clinical use of Sanzi San were explored at the serum pharmacochemistry level.

Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy.

BACKGROUND: miR-15a and miR-16-1 have been identified as tumor suppressor genes in prostate cancer, but their safe and effective delivery to target cells is key to the successful use of this therapeutic strategy. RNA aptamer A10 has been used as a ligand, targeting prostate cancer cells that express prostate-specific membrane antigen (PSMA). Compared with A10, the binding of the second-generation RNA aptamer, A10-3.2, to PSMA is more efficient. METHODS: A10-3.2 was investigated as a PSMA-targeting ligand in the design of a polyamidoamine (PAMAM)-based microRNA (miR-15a and miR-16-1) vector to prostate cancer cells. Using polyethyleneglycol (PEG) as a spacer, PAMAM was conjugated to aptamer (PAMAM-PEG-APT) and used as a vehicle for miRNA target delivery. RESULTS: Luciferase assays of pGL-3 expression against PC3 (PSMA(-)) and LNCaP (PSMA(+)) cells demonstrated that the transfection efficiency of the synthesized DNA/PAMAM-PEG-APT complex was higher than that of the DNA/PAMAM-PEG complex. In addition, cell viability assays of LNCaP (PSMA(+)) cells showed that, with a N/P ratio of 15:1, the IC(50) value of miRNA/PAMAM-PEG-APT was approximately 4.7-fold lower than that of miRNA/PAMAM-PEG. CONCLUSION: This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of prostate cancer cells.