Explore the vast range of titles available.

Methicillin-resistant Staphylococcus aureus (MRSA) exemplifies high-level antibiotic resistance in this major human pathogen. Its resistance to chloramphenicol is majorly conferred by enzymatic inactivation via chloramphenicol acetyltransferases (CATs). This modification sterically blocks the antibiotic’s ribosomal binding and thus neutralizes its inhibitory potency. Although CATs have been structurally studied across diverse bacteria species, the structures of S. aureus CATs (saCATs) have remained uncharacterized. To address this gap and elucidate species-specific resistance mechanisms, we determined the first high-resolution crystal structure of saCAT1, the prototypical saCAT enzyme. Structural analysis delineates the active site architecture and reveals the molecular basis for substrate recognition of both chloramphenicol and fusidic acid (FA). Further enzymatic assays demonstrated that the K m value against chloramphenicol is 16.9 µM, and the K i value of the inhibitor FA is 83.7 µM, indicating that the inhibitory capacity of FA is relatively limited. These findings provide an essential structural framework for understanding chloramphenicol resistance in S. aureus and facilitate the rational design of novel antimicrobial strategies to combat multidrug-resistant pathogens.

Menaquinone-7 (MK-7) is an important bioactive form of vitamin K that inhibits vascular calcification, maintains systemic calcium homeostasis, demonstrates high bioavailability, and possesses an extended plasma half-life. naturally possesses the complete biosynthetic pathway for MK-7, benefits from a well-characterized genetic background and advanced genome-editing tools, and is regarded as a safe and efficient microbial chassis for industrial MK-7 fermentation. This review summarizes recent advances in the metabolic engineering of for high-level MK-7 production, highlights pathway analyses, and discusses engineering strategies targeting four key modules: substrate utilization, secretion, spore/biofilm formation, and oxidative-stress defense.

2-Phenylethanol (2-PE), an aromatic alcohol with a rose-like fragrance, is widely used in the food, pharmaceutical, and high-end cosmetic industries. In this study, a high-yield 2-PE-producing strain was isolated and identified as Saccharomyces cerevisiae based on morphological characterization and taxonomic identification. Fermentation medium components (carbon and nitrogen sources) were optimized through single-factor experiments in shaking flasks, and fermentation medium with 40 g/L glucose, 5 g/L malt extract, 1.75 g/L corn steep liquor, 2.5 g/L yeast extract, 5 g/L malt extract, 1.75 g/L corn steep liquor was considered suitable for 2-PE production. RT-qPCR results indicated that corn steep liquor activates expression of genes related to the shikimate pathway and Ehrlich pathway (pha2, aro4, aro8, and aro9), thereby promoting the synthesis of 2-PE through these pathways. Excess yeast extract inhibited the expression of aro8 and aro9, while enhancing the expression of tdh3 and adh2, thus promoting the de novo synthesis of 2-PE. Furthermore, fermentation in a 5 L bioreactor was applied to investigate the effects of feeding strategies, inoculum proportion, and pH on 2-PE production. With a pH of 5.5 and10% inoculum proportion, the supplementation of the substrate L-Phe led to a 2-PE production of 4.81 g/L after 24 h of fermentation. Finally, in situ product recovery (ISPR) techniques was applied to alleviate 2-PE cytotoxicity, achieving a production of 6.41 g/L. This process offers a promising strategy for producing 2-PE efficiently and naturally, paving the way for further industrial applications in food, pharmaceutical, and cosmetic sectors.

Traceless protein cleavage is a significant challenge in intein application, as most common inteins studied today are not both active and promiscuous. In this study, the intein gp41‐1 is engineered, which demonstrates the most efficient traceless cleavage reported to date and shows high compatibility to 1st amino acid. The evidence provided for the first time is that the unique THN motif, which is prevalent in class 3 inteins, is essential for achieving high‐efficiency traceless C‐terminal cleavage. The hydrogen bond between the hydroxyl group of Thr123 and the main chain of His124 is suggested to be indispensable for stabilizing the THN motif to separate Asp107 (the limiting factor for C‐cleavage) from Asn125 and the C‐extein residues from the active sites, which jointly lead to the highest traceless C‐cleavage activity. Both cleavage data and molecular dynamics (MD) simulations results demonstrate that mutating Thr123 greatly disturbed the THN motif, leading to inactivity. These findings reveal a pivotal motif for intein traceless cleavage efficiency, providing valuable insights for designing inteins with enhanced traceless C‐terminal cleavage capabilities in future applications. THN is identified as the key motif to facilitate the rapid traceless cleavage of gp41‐1. The deflection of THN motif not only avoids the interaction between C‐exteins and active sites, but also separates Asp107 away from Asn125. In addition, a flexible block F contributes to faster C‐cleavage. The unique THN offers a new insight for identifying high active inteins.

Protein sequence design has been widely applied in rational protein engineering and increasing the design accuracy and efficiency is highly desired. Here we present ProDESIGN-LE, an accurate and efficient design approach, which adopts a concise but informative representation of residue's local environment and trains a transformer to select an appropriate residue at a position from its local environment. ProDESIGN-LE iteratively applies the transformer on the positions in the target structure, eventually acquiring a designed sequence with all residues fitting well with their local environments. ProDESIGN-LE designed sequences for 68 naturally occurring and 129 hallucinated proteins within 20 seconds per protein on average, and the predicted structures from the designed sequences perfectly resemble the target structures with state-of-the-art average TM-score exceeding 0.80. We further experimentally validated ProDESIGN-LE by designing five sequences for an enzyme, chloramphenicol O-acetyltransferase type III (CAT III), and recombinantly expressing the proteins in E. coli. Of these proteins, three exhibited excellent solubility, and one yielded monomeric species with circular dichroism spectra consistent with the natural CAT III protein. Competing Interest Statement The authors have declared no competing interest. Footnotes * None

Background Macrophages play a crucial role in the development of cardiac fibrosis (CF). Although our previous studies have shown that glycogen metabolism plays an important role in macrophage inflammatory phenotype, the role and mechanism of modifying macrophage phenotype by regulating glycogen metabolism and thereby improving CF have not been reported. Methods Here, we took glycogen synthetase kinase 3β (GSK3β) as the target and used its inhibitor NaW to enhance macrophage glycogen metabolism, transform M2 phenotype into anti-fibrotic M1 phenotype, inhibit fibroblast activation into myofibroblasts, and ultimately achieve the purpose of CF treatment. Results NaW increases the pH of macrophage lysosome through transmembrane protein 175 (TMEM175) and caused the release of Ca 2+ through the lysosomal Ca 2+ channel mucolipin-2 (Mcoln2). At the same time, the released Ca 2+  activates TFEB, which promotes glucose uptake by M2 and further enhances glycogen metabolism. NaW transforms the M2 phenotype into the anti-fibrotic M1 phenotype, inhibits fibroblasts from activating myofibroblasts, and ultimately achieves the purpose of treating CF. Conclusion Our data indicate the possibility of modifying macrophage phenotype by regulating macrophage glycogen metabolism, suggesting a potential macrophage-based immunotherapy against CF. Graphical Abstract

The involvement of gut microbiota in calcific aortic valve disease (CAVD) pathogenesis remains underexplored. Here, we provide evidence for a strong association between the gut microbiota and CAVD development. ApoE−/− mice were stratified into easy‐ and difficult‐ to calcify groups using neural network and cluster analyses, and subsequent faecal transplantation and dirty cage sharing experiments demonstrated that the microbiota from difficult‐to‐calcify mice significantly ameliorated CAVD. 16S rRNA sequencing revealed that reduced abundance of Faecalibacterium prausnitzii (F. prausnitzii) was significantly associated with increased calcification severity. Association analysis identified F. prausnitzii‐derived butyric acid as a key anti‐calcific metabolite. These findings were validated in a clinical cohort (25 CAVD patients vs. 25 controls), where serum butyric acid levels inversely correlated with disease severity. Functional experiments showed that butyric acid effectively hindered osteogenic differentiation in human aortic valve interstitial cells (hVICs) and attenuated CAVD progression in mice. Isotope labeling and 13C flux analyses confirmed that butyric acid produced in the intestine can reach heart tissue, where it reshapes glycolysis by specifically modifying GAPDH. Mechanistically, butyric acid‐induced butyrylation (Kbu) at lysine 263 of GAPDH competitively inhibited lactylation (Kla) at the same site, thereby counteracting glycolysis‐driven calcification. These findings uncover a novel mechanism through which F. prausnitzii and its metabolite butyric acid contribute to the preservation of valve function in CAVD, highlighting the gut microbiota‐metabolite‐glycolysis axis as a promising therapeutic target. Multi‐omics sequencing and correlation analysis identified the beneficial role of Faecalibacterium prausnitzii (F. prausnitzii)‐derived butyric acid (BA) as a key metabolite in the restoration of valve function in calcific aortic valve disease (CAVD). The therapeutic efficacy of BA in attenuating CAVD progression was confirmed in vitro, ex vivo, and in vivo. Subsequent mechanistic investigations revealed that BA reshape glycolysis through site‐specific inhibition of lactylation at the Lys‐263 residue of GAPDH, which is mediated by competitive inhibition of butyrylation at the same site. Highlights The inseparable relationship between the gut microbiota and calcific aortic valve disease development. Faecalibacterium prausnitzii (F. prausnitzii)‐derived butyric acid (BA) played an important role in anti‐calcification functions. BA‐derived butyrylation (Kbu) blocked lactylation (Kla) at the same site by occupying the GAPDH 263 lysine. The gut microbial‐metabolite‐epigenetic modification pathway represents a promising therapeutic target for calcific aortic valve disease (CAVD).

Purpose To construct a gadoxetic acid-enhanced MRI (EOB-MRI) -based multivariable model to predict Ki-67 expression levels in hepatocellular carcinoma (HCC) using LI-RADS v2018 imaging features. Methods A total of 121 patients with HCC who underwent EOB-MRI were enrolled in this study. The patients were divided into three groups according to Ki-67 cut-offs: Ki-67 ≥ 20% ( n  = 86) vs. Ki-67 < 20% ( n  = 35); Ki-67 ≥ 30% ( n  = 73) vs. Ki-67 < 30% ( n  = 48); Ki-67 ≥ 50% ( n  = 45) vs. Ki-67 < 50% ( n  = 76). MRI features were analyzed to be associated with high Ki-67 expression using logistic regression to construct multivariable models. The performance characteristic of the models for the prediction of high Ki-67 expression was assessed using receiver operating characteristic curves. Results The presence of mosaic architecture ( p  = 0.045), the presence of infiltrative appearance ( p  = 0.039), and the absence of targetoid hepatobiliary phase (HBP,  p  = 0.035) were independent differential factors for the prediction of high Ki-67 status (≥ 50% vs. < 50%) in HCC patients, while no features could predict high Ki-67 status with thresholds of 20% (≥ 20% vs. < 20%) and 30% (≥ 30% vs. < 30%) ( p  > 0.05). Four models were constructed including model A (mosaic architecture and infiltrated appearance), model B (mosaic architecture and targetoid HBP), model C (infiltrated appearance and targetoid HBP), and model D (mosaic architecture, infiltrated appearance and targetoid HBP). The model D yielded better diagnostic performance than the model C (0.776 vs. 0.669, p  = 0.002), but a comparable AUC than model A (0.776 vs. 0.781, p  = 0.855) and model B (0.776 vs. 0.746, p  = 0.076). Conclusions Mosaic architecture, infiltrated appearance and targetoid HBP were sensitive imaging features for predicting Ki-67 index ≥ 50% and EOB-MRI model based on LI-RADS v2018 features may be an effective imaging approach for the risk stratification of patients with HCC before surgery.

Aims: Calcific aortic valve disease (CAVD) is a chronic cardiovascular disease with high morbidity that lacks effective pharmacotherapeutics. As a natural flavonoid extracted from Ampelopsis grossedentata, dihydromyricetin (DHM) has been shown to be effective in protecting against atherosclerosis; yet, the therapeutic role of DHM in CAVD remains poorly understood. Herein, we aimed to clarify the therapeutic implications of DHM in CAVD and the underlying molecular mechanisms in human valvular interstitial cells (hVICs). Methods and Results: The protein levels of two known osteogenesis-specific genes (alkaline phosphatase, ALP; runt-related transcription factor 2, Runx2) and calcified nodule formation in hVICs were detected by Western blot and Alizarin Red staining, respectively. The results showed that DHM markedly ameliorated osteogenic induction medium (OM)–induced osteogenic differentiation of hVICs, as evidenced by downregulation of ALP and Runx2 expression and decreased calcium deposition. The SwissTargetPrediction database was used to identify the potential AVC-associated direct protein target of DHM. Protein–protein interaction (PPI) analysis revealed that c-KIT, a tyrosine-protein kinase, can act as a credible protein target of DHM, as evidenced by molecular docking. Mechanistically, DHM-mediated inhibition of c-KIT phosphorylation drove interleukin-6 (IL-6) downregulation in CAVD, thereby ameliorating OM-induced osteogenic differentiation of hVICs and aortic valve calcification progression. Conclusion: DHM ameliorates osteogenic differentiation of hVICs by blocking the phosphorylation of c-KIT, thus reducing IL-6 expression in CAVD. DHM could be a viable therapeutic supplement to impede CAVD.

Doxorubicin (DOX) is the most effective chemotherapeutic for breast cancer, but it is usually associated with severe cardiotoxicity. Further investigation to alleviate its side effects is essential. The present study investigated the mechanism of the cross-organ communication between tumors and the heart and potential intervention targets. Morphological bubble-like protrusions were observed in both adult murine ventricular cardiomyocytes (AMVCs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) cocultured with breast cancer cells (BCCs), along with elevated expression of pyroptosis-related proteins. Exosomes (EXOs) from DOX-treated BCCs aggravated DOX-induced cardiotoxicity (DOXIC) in an orthotopic mouse model of breast cancer. Blocking miRNAs by knocking down Rab27a or inhibiting the release of EXOs in cancer tissue by Dicer enzyme knockout attenuated this additional injury effect. Exosomal miRNA sequencing revealed that miR-216a-5p is especially upregulated in EXOs from DOX-induced BCCs. Mechanistically, miR-216a-5p was upregulated by enhanced transcription mediated by DOX-induced AMP-dependent transcription factor 3 (ATF3) and packaged into EXOs by splicing factor 3b subunit 4 (SF3B4) in BCCs. Itchy E3 ubiquitin-protein ligase (ITCH) was identified as a novel downstream target mRNA of miR-216a-5p. ITCH negatively mediated thioredoxin-interacting protein (TXNIP) ubiquitination to activate the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome pathway, ultimately leading to cardiomyocyte pyroptosis. Our findings revealed novel cross-organ pathogenic communication between breast cancer and the heart through the exosomal miR-216a-5p-mediated ITCH/TXNIP/NLRP3 pathway, which drives cardiomyocyte pyroptosis. These findings suggest that targeting myocardial miR-216a-5p or blocking harmful EXOs from breast cancer is a potential therapeutic strategy for alleviating DOXIC.