Journal of advanced research最新文献

Introduction: Excessive osteoclastogenesis is a key driver of inflammatory bone loss. Suppressing osteoclastogenesis has always been considered essential for the treatment of inflammatory bone loss. N-acetyltransferase 10 (NAT10) is the sole enzyme responsible for N4-acetylcytidine (ac4C) modification of mRNA, and is involved in cell development. However, its role in osteoclastogenesis and inflammatory bone loss remained elusive.

Objectives: We aimed to clarify the regulatory mechanism of NAT10 and ac4C modification in osteoclastogenesis and inflammatory bone loss.

Methods: NAT10 expression and ac4C modification during osteoclastogenesis were determined by quantitative real-time PCR (qPCR), western blotting, dot blot and immunofluorescent staining, and the effect of NAT10 inhibition on osteoclast differentiation in vitro was measured by the tartrate-resistant acid phosphatase staining, podosome belts staining assay and bone resorption pit assay. Then, acRIP-qPCR and NAT10RIP-qPCR, ac4C site prediction, mRNA decay assay and luciferase reporter assay were performed to further study the underlying mechanisms. At last, mice models of inflammatory bone loss were applied to verify the therapeutic effect of NAT10 inhibition in vivo.

Results: NAT10 expression was upregulated during osteoclast differentiation and highly expressed in alveolar bone osteoclasts from periodontitis mice. Inhibition of NAT10 notably reduced osteoclast differentiation in vitro, as indicated by great reduction of tartrated resistant acid phosphatse positive multinuclear cells, osteoclast-specific gene expression, F-actin ring formation and bone resorption capacity. Mechanistically, NAT10 catalyzed ac4C modification of Fos (encoding AP-1 component c-Fos) mRNA and maintained its stabilization. Besides, NAT10 promoted MAPK signaling pathway and thereby activated AP-1 (c-Fos/c-Jun) transcription for osteoclastogenesis. Therapeutically, administration of Remodelin, the specific inhibitor of NAT10, remarkably impeded the ligature-induced alveolar bone loss and lipopolysaccharide-induced inflammatory calvarial osteolysis.

Conclusions: Our study demonstrated that NAT10-mediated ac4C modification is an important epigenetic regulation of osteoclast differentiation and proposed a promising therapeutic target for inflammatory bone loss.

Introduction: Ochratoxins (OTs) are worldwide regulated mycotoxins contaminating a variety of food-environment and agro-environment. Several Aspergillus and Pencillium species synthesize OTs from a six-gene biosynthetic gene cluster (BGC) to produce the highly toxic final product OTA. Although many studies on OTA-degrading enzymes were performed, high efficiency enzymes with strong stability are extremely needed, and the OTA degrading mechanism is poorly understood.

Objectives: The study aimed to explore the OT-degradation enzyme and investigate its degradation mechanisms in Metarhizium, which contain an OT biosynthetic gene cluster.

Methods: Phylogenomic relationship combined with RNA expression analysis were used to explore the distribution of OT BGC in fungi. Bioactivity-guided isolation and protein mass spectrometry were conducted to trace the degrading enzymes in Metarhizium spp., and the enzymes were heterologously expressed in E. coli and verified by in vitro assays. Structure prediction and point mutation were performed to reveal the catalytic mechanism of MbAmh1.

Results: Beyond Aspergillus and Pencillium species, three species of the distant phylogenetic taxon Metarhizium contain an expressed OT-like BGC but lack an otaD gene. Unexpectedly, no OT BGC products were found in some Metarhizium species. Instead, Metarhizium metabolized both OTA and OTB to their non-toxic degradation products. This activity of M. brunneum was attributed to an intracellular hydrolase MbAmh1, which was tracked by bioactivity-guided proteomic analysis combined with in vitro reaction. Recombinant MbAmh1 (5 μg/mL) completely degraded 1 μg/mL OTA within 3 min, demonstrating a strong degrading ability towards OTA. Additionally, MbAmh1 showed considerable temperature adaptability ranging from 30 to 70 °C and acidic pH stability ranging from 4.0 to 7.0. Identification of active sites supported the crucial role of metal iron for this enzymatic reaction.

Conclusion: These findings reveal different patterns of OT synthesis in fungi and provide a potential OTA degrading enzyme for industrial applications.

Introduction: Ultra-high static magnetic fields (SMFs) have unique advantages in improving medical and academic research. However, the research on the early embryo exposure of ultra-high SMFs is minimal, extensive exploration is indispensable in living organisms.

Objectives: The present study was aimed to study the effects of ultra-high SMFs on the early embryonic division and development of Caenorhabditis elegans (C. elegans).

Methods: Early adult parents containing fertilized eggs in vivo were exposed to SMFs at intensities ranging from 4 T to 27 T. The number of mitotic cells in the reproductive glands of the P0 worms, early embryonic cell spindle localization, embryo hatching and the reproductive as well as developmental indicators of F1 and F2 nematodes were examined as endpoints.

Results: Our results indicated that ultra-high SMFs has no obvious effect on the germ cell cycle, while 14 T and 27 T SMFs significantly increased the proportion of multi-polar spindle formation in early embryonic cells, and reduced the developmental rate and lifespan of C. elegans exposed at the embryonic stage. Spindle abnormalities of early embryonic cells, as well as the down-regulation of genes related to asymmetric embryonic division and the abnormal expression of the non-muscle myosin NMY-2 in the division grooves played a critical role in the slowing down of embryonic development induced by ultra-high SMFs.

Conclusions: This study provided novel information and a new sight for evaluating the biosafety assessment by exposure to ultra-high SMFs at the early embryonic stage in vivo.

Introduction: Exaggerated neutrophil recruitment and activation are the major features of pathological alterations in periodontitis, in which neutrophil extracellular traps (NETs) are considered to be responsible for inflammatory periodontal lesions. Despite the critical role of NETs in the development and progression of periodontitis, their specific functions and mechanisms remain unclear.

Objectives: To demonstrate the important functions and specific mechanisms of NETs involved in periodontal immunopathology.

Methods: We performed single-cell RNA sequencing on gingival tissues from both healthy individuals and patients diagnosed with periodontitis. High-dimensional weighted gene co-expression network analysis and pseudotime analysis were then applied to characterize the heterogeneity of neutrophils. Animal models of periodontitis were treated with NETs inhibitors to investigate the effects of NETs in severe periodontitis. Additionally, we established a periodontitis prediction model based on NETs-related genes using six types of machine learning methods. Cell-cell communication analysis was used to identify ligand-receptor pairs among the major cell groups within the immune microenvironment.

Results: We constructed a single-cell atlas of the periodontal microenvironment and obtained nine major cell populations. We further identified a NETs-related subgroup (NrNeu) in neutrophils. An in vivo inhibition experiment confirmed the involvement of NETs in gingival inflammatory infiltration and alveolar bone absorption in severe periodontitis. We further screened three key NETs-related genes (PTGS2, MME and SLC2A3) and verified that they have the potential to predict periodontitis. Moreover, our findings revealed that gingival fibroblasts had the most interactions with NrNeu and that they might facilitate the production of NETs through the MIF-CD74/CXCR4 axis in periodontitis.

Conclusion: This study highlights the pathogenic role of NETs in periodontal immunity and elucidates the specific regulatory relationship by which gingival fibroblasts activate NETs, which provides new insights into the clinical diagnosis and treatment of periodontitis.

Introduction: Genetically modified (GM) crops have been widely cultivated across the world and the development of rapid, ultrasensitive, visual multiplex detection platforms that are suitable for field deployment is critical for GM organism regulation.

Objective: In this study, we developed a novel one-pot system, termed MR-DCA (Multiplex RPA and Dual CRISPR assay), for the simultaneous detection of CaMV35S and NOS genetic targets in GM crops. This innovative approach combined Multiplex RPA (recombinase polymerase amplification) with the Dual CRISPR (clustered regularly interspaced short palindromic repeat) assay technique, to provide a streamlined and efficient method for GM crop detection.

Methods: The RPA reaction used for amplification CaMV35S and NOS targets was contained in the tube base, while the dual CRISPR enzymes were placed in the tube cap. Following centrifugation, the dual CRISPR (Cas13a/Cas12a) detection system was initiated. Fluorescence visualization was used to measure CaMV35S through the FAM channel and NOS through the HEX channel. When using lateral flow strips, CaMV35S was detected using rabbit anti-digoxin (blue line), whilst NOS was identified using anti-mouse FITC (red line). Line intensity was quantified using Image J and depicted graphically.

Results: Detection of the targets was completed in 35 min, with a limit of detection as low as 20 copies. In addition, two analysis systems were developed and they performed well in the MR-DCA assay. In an analysis of 24 blind samples from GM crops with a wide genomic range, MR-DCA gave consistent results with the quantitative PCR method, which indicated high accuracy, applicability and semi-quantitative ability.

Conclusion: The development of MR-DCA represents a significant advancement in the field of GM detection, offering a rapid, sensitive and portable method for multiple target detection that can be used in resource-limited environments.

Introduction: Nav1.6 is closely related to the pathology of Alzheimer's Disease (AD), and astrocytes have recently been identified as a significant source of β-amyloid (Aβ). However, little is known about the connection between Nav1.6 and astrocyte-derived Aβ.

Objective: This study explored the crucial role of Nav1.6 in mediated astrocyte-derived Aβ in AD and knockdown astrocytic Nav1.6 alleviates AD progression by promoting autophagy and lysosome-APP fusion.

Methods: A mouse model for astrocytic Nav1.6 knockdown was constructed to study the effects of astrocytic Nav1.6 on amyloidosis. The role of astrocytic Nav1.6 on autophagy and lysosome-APP(amyloid precursor protein) fusion was used by transmission electron microscope, immunostaining, western blot and patch clamp. Glial cell activation was detected using immunostaining. Neuroplasticity and neural network were assessed using patch-clamp, Golgi stain and EEG recording. Behavioral experiments were performed to evaluate cognitive defects.

Results: Astrocytic Nav1.6 knockdown reduces amyloidosis, alleviates glial cell activation and morphological complexity, improves neuroplasticity and abnormal neural networks, as well as promotes learning and memory abilities in APP/PS1 mice. Astrocytic Nav1.6 knockdown reduces itself-derived Aβ by promoting lysosome- APP fusion, which is related to attenuating reverse Na⁺-Ca²⁺ exchange current thus reducing intracellular Ca²⁺ to facilitate autophagic through AKT/mTOR/ULK pathway.

Conclusion: Our findings unveil the crucial role of astrocyte-specific Nav1.6 in reducing astrocyte-derived Aβ, highlighting its potential as a cell-specific target for modulating AD progression.

Introduction: Insulin resistance (IR) is associated with multiple pathological features. Although p53- or TRIB3-orchestrated IR is extensively studied in adipose tissue and liver, the role of p53-TRIB3 axis in myocardial IR remains unknown, and more importantly target-directed therapies of myocardial IR are missing.

Objectives: Considering the beneficial effects of sulforaphane (SFN) on cardiovascular health, it is of particular interest to explore whether SFN protects against myocardial IR with a focus on the regulatory role of p53-TRIB3 axis.

Methods: Mouse models including cardiac specific p53-overexpressing transgenic (p53-cTg) mice and Trib3 knockout (Trib3-KO) mice, combined with primary cardiomyocytes treated with p53 activator (nutlin-3a) and inhibitor (pifithrin-α, PFT-α), or transfected with p53-shRNA and Trib3-shRNA, followed by multiple molecular biological methodologies, were used to investigate the role of p53-TRIB3 axis in SFN actions on myocardial IR.

Results: Here, we report that knockdown of p53 rescued cardiac insulin-stimulated AKT phosphorylation, while up-regulation of p53 by nutlin-3a or p53-cTg mice blunted insulin sensitivity in cardiomyocytes under diabetic conditions. Diabetic attenuation of AKT-mediated cardiac insulin signaling was markedly reversed by SFN in p53-Tg^fl/fl mice, but not in p53-cTg mice. Importantly, we identified TRIB3 was elevated in p53-cTg diabetic mice, and confirmed the physical interaction between p53 and TRIB3. Trib3-KO diabetic mice displayed improved insulin sensitivity in the heart. More specifically, the AMPKα-triggered CHOP phosphorylation and degradation were essential for p53 on the transcriptional regulation of Trib3.

Conclusion: Overall, these results indicate that inhibiting the p53-TRIB3 pathway by SFN plays an unsuspected key role in the improvement of myocardial IR, which may be a promising strategy for attenuating diabetic cardiomyopathy (DCM) in diabetic patients.

Introduction: Globally, colorectal cancer (CRC) is the third most common type of cancer, and its treatment frequently includes the utilization of drugs based on antibodies and small molecules. The development of CRC has been linked to various signaling pathways, with the Wnt/β-catenin pathway identified as a key target for intervention.

Objectives: We have explored the impact of imidazopyridine-tethered chalcone-C (CHL-C) in CRC models.

Methods: To determine the influence of CHL-C on apoptosis and autophagy, Western blot analysis, annexin V assay, cell cycle analysis, acridine orange staining, and immunocytochemistry were performed. Next, the activation of the Wnt/β-catenin signaling pathway and the anti-cancer effects of CHL-C in vivo were examined in an orthotopic HCT-116 mouse model.

Results: We describe the synthesis and biological assessment of the CHL series as inhibitors of the viability of HCT-116, SW480, HT-29, HCT-15, and SNU-C2A CRC cell lines. Further biological evaluations showed that CHL-C induced apoptosis and autophagy in down-regulated β-catenin, Wnt3a, FZD-1, Axin-1, and p-GSK-3β (Ser9), and up-regulated p-GSK3β (Tyr216) and β-TrCP. In-depth analysis using structure-based bioinformatics showed that CHL-C strongly binds to β-catenin, with a binding affinity comparable to that of ICG-001, a well-known β-catenin inhibitor. Additionally, our in vivo research showed that CHL-C markedly inhibited tumor growth and triggered the activation of both apoptosis and autophagy in tumor tissues.

Conclusion: CHL-C is capable of inducing apoptosis and autophagy by influencing the Wnt/β-catenin signaling pathway.

Introduction: One of the methods for pain management involves the use of local anesthesia, which numbs sensations in specific body regions while maintaining consciousness.

Objectives: Considering the certain limitations (e.g., pain, the requirement of skilled professionals, or slow passive diffusion) of conventional delivery methods of local anesthetics, developing alternative strategies that offer minimally invasive yet therapeutically effective delivery systems is of great concern for ophthalmic regional anesthesia.

Methods and results: In this study, a rapidly dissolving cambered microneedle (MNs) patch, composed of poly(vinylpyrrolidone) (PVP) and hyaluronic acid (HA) and served as a delivery system for lidocaine (Lido) in local anesthesia, was developed taking inspiration from the mosquito proboscis's ability to extract blood unnoticed. The lidocaine-containing MNs patch (MNs@Lido) consisted of 25 microneedles with a four-pronged cone structure (height: 500 μm, base width: 275 μm), arranged in a concentric circle pattern on the patch, and displays excellent dissolubility for effective drug delivery of Lido. After confirming good cytocompatibility, MNs@Lido was found to possess adequate rigidity to penetrate the cornea without causing any subsequent injury, and the created corneal pinhole channels completely self-healed within 24 h. Interestingly, MNs@Lido exhibited effective analgesic effects for local anesthesia on both heel skin and eyeball, with the sustained anesthetic effect lasting for at least 30 min.

Conclusions: These findings indicate that the mosquito proboscis-inspired cambered MNs patch provides rapid and painless local anesthesia, overcoming the limitations of conventional delivery methods of local anesthetics, thus opening up new possibilities in the treatment of ophthalmic diseases.

Introduction: Diarrhea is the primary dose-limiting side effect of capecitabine(Cap) hindering its clinical application, but the mechanism is unclear. Clarifying this mechanism may enhance the patient compliance and improve the treatment outcome.

Objectives: To assess if the endogenous metabolic profile could prodict the diarrhea induced by Cap and explore and validate underlying mechanisms.

Methods: Untargeted and targeted bile acids(BAs) metabolomics were performed to analyzed the metabolic profile of baseline samples from colorectal cancer(CRC) patients and the association with the diarrhea induced by Cap was assessed. The toxicity of BAs and Cap and its metabolites alone or their combinations to the human normal intestinal epithelial cell(HIEC) was assessed, and the key genes that mediated the BAs-enhanced toxicity of Cap were discovered by RNA-seq and then validated. A mouse model with high exposure levels of BAs was constructed and then treated with Cap to verify the Cap-induced diarrhea enhanced by BAs.

Results: The baseline endogenous metabolic profile showed obviously difference between diarrhea and non-diarrhea CRC patients, and the differential metabolites mainly enriched in BAs metabolism; the deoxycholic acid(DCA) and lithocholic acid(LCA) were selected to be the key BAs that enhanced the toxicity of Cap metabolite 5-FU to the HIEC cell; the DCA and LCA could inhibit the Wnt/β-catenin signaling pathway, which then suppressed the P-glycoprotein and increased the exposure level of 5-FU in the HIEC cell. The results of animal experiment verified that the excessive DCA and LCA could aggravate the Cap-induced diarrhea through inhibiting Wnt/β-catenin-P-glycoprotein pathway.

Conclusions: The disordered BAs metabolic profile showed close relationship with diarrhea induced by Cap, and excessive DCA and LCA were proved to be the key BAs, which could aggravate the Cap-induced diarrhea through inhibiting Wnt/β-catenin-P-glycoprotein pathway.