Platycodon D protects human nasal epithelial cells from pyroptosis through the Nrf2/HO-1/ROS signaling cascade in chronic rhinosinusitis.

BACKGROUND: Pyroptosis has been demonstrated being closely associated with the inflammatory progression in chronic rhinosinusitis (CRS). However, platycodon D (PLD) has emerged as a key anti-inflammatory mediator in the inflammatory progression of various respiratory diseases. This study aims at investigating whether PLD could reduce inflammatory progression of CRS by inhibiting pyroptosis. METHODS: Nasal mucosal tissues from patients with CRS and the control group (simple nasal septal deviation) were analyzed for morphological difference using hematoxylin & eosin staining and for the expression of pyroptosis-related makers by immunofluorescence (IF). Human nasal epithelial cells (HNEpCs) were cultured and co-stimulated with lipopolysaccharide (LPS)/adenosine triphosphate (ATP) to construct an in vitro cellular model simulating CRS. After pretreatment with PLD, EthD-I staining, TUNEL staining, transmission electron microscopy (TEM), and GSDMD-NT detection were performed to evaluate pyroptosis markers. The NLRP3 inflammasome was detected by IF and western blotting (WB). Reactive oxygen species (ROS) were detected by H2DCFDA staining, and mitochondrial membrane potential was evaluated by JC-1 staining. Mitochondrial morphology and structure were observed using TEM. The Nrf2/HO-1 antioxidant signaling pathway was detected using WB. RESULTS: The nasal mucosa structure of patients with CRS exhibited significant damage, with a marked increase in the expression of pyroptosis-related proteins compared with the control group. LPS/ATP co-stimulation resulted in an increased expression of IL-18 and IL-1ß in HNEpCs, causing significant damage to nuclear and cell membranes, GSDMD-NT accumulation around the cell membrane, and intracellular NLRP3 inflammasome activation. Furthermore, it led to increased ROS expression, significantly decreased mitochondrial membrane potential, and damaged mitochondrial structure. However, pretreatment with PLD significantly reversed the aforementioned trends and activated the Nrf2/HO-1 antioxidant signaling pathway. CONCLUSIONS: The results of this study confirm that NLRP3-mediated pyroptosis plays a crucial role in the pathological process of nasal mucosal impairment in patients with CRS. PLD inhibits NLRP3-mediated pyroptosis, preventing inflammatory damage in HNEpCs of patients with CRS by activating the Nrf2/HO-1 antioxidant signaling pathway, which in turn reduces ROS production and ameliorates mitochondrial damage.

RNF115 aggravates tumor progression through regulation of CDK10 degradation in thyroid carcinoma.

BACKGROUND: RING Finger Protein 115 (RNF115), a notable E3 ligase, is known to modulate tumorigenesis and metastasis. In our investigation, we endeavor to unravel the putative function and inherent mechanism through which RNF115 influences the evolution of thyroid carcinoma (THCA). METHODS: We analyzed RNF115 expression in THCA using the Cancer Genome Atlas (TCGA) database. The influence of RNF115 on the progression of THCA was evaluated using both in vitro and in vivo experimental approaches. The protein regulated by RNF115 was identified through bioinformatics analysis, and its biological significance was further explored. RESULTS: In both THCA tissues and cells, RNF115 showed elevated expression levels. Enhanced expression of RNF115 fostered cell proliferation, tumor growth, and the exacerbation of epithelial-mesenchymal transition (EMT) in THCA, while also promoting tumor lung metastasis. Bioinformatics analysis identified cyclin-dependent kinase 10 (CDK10) as a downstream target of RNF115, which was found to be ubiquitinated and degraded by RNF115 in THCA cells. Functionally, overexpression of CDK10 was found to counteract the promotion of malignant phenotype in THCA induced by RNF115. From a mechanistic perspective, RNF115 activated the Raf-1 pathway and enhanced cancer cell cycle progression by degrading CDK10 in THCA cells. CONCLUSION: RNF115 triggers cell proliferation, EMT, and tumor metastasis by ubiquitinating and degrading CDK10. The regulation of the Raf-1 pathway and cell cycle progression in THCA may be profoundly influenced by this process.

Xiaoqinglong decoction improves allergic rhinitis by inhibiting NLRP3-mediated pyroptosis in BALB/C mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoqinglong decoction (XQLD), first recorded in Shang Han Lun, is a traditional Chinese medicine prescribed for the treatment of allergic rhinitis (AR). XQLD alleviates the clinical symptoms of AR by inhibiting the occurrence of an inflammatory response, but the specific regulatory mechanism remains unclear. AIM OF THE STUDY: NLRP3-mediated pyroptosis is closely related to AR pathogenesis. Hence, this study aimed to explore the potential role of NLRP3-mediated pyroptosis pathway in the AR-associated pharmacological mechanism of XQLD. MATERIALS AND METHODS: BALB/C mice models of AR was established by using ovalbumin (OVA) and aluminum hydroxide sensitization. After intragastric administration of different dosages of XQLD, nasal allergic symptoms were observed. The expression of OVA-sIgE and Th2 inflammatory factors (IL-4, IL-5, and IL-13) in serum was detected by ELISA. The histopathological morphology and expression of inflammatory factors in nasal mucosa along with pyroptosis were investigated. Molecular docking was performed to analyze the binding of representative compounds of XQLD with NLRP3. Activation of the NLRP3 inflammasome was detected by immunofluorescence and western blotting. RESULTS: XQLD significantly improved the nasal allergic symptoms of mice, reduced the degree of goblet cell proliferation, mast cell infiltration, and collagen fiber hyperplasia in nasal mucosa. Meanwhile, it could downregulate the expression of Th2 inflammatory factors (IL-4, IL-5, and IL-13) in serum and nasal mucosa. XQLD significantly reduced the number of GSDMD and TUNEL double-positive cells and IL-1ß and IL-18 expression. Molecular docking confirmed that seven representative compounds of XQLD had good binding properties with NLRP3 and were able to inhibit the activation of the NLRP3 inflammasome. CONCLUSIONS: The representative compounds of XQLD might inhibit pyroptosis in nasal mucosa mediated by the NLRP3 inflammasome to helping the recovery of AR, which provides a new modern pharmacological proof for XQLD to treat AR.

Diagnosis and Treatment Strategy of Nasogenic Olfactory Dysfunction.

Since the global outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a symptom of the onset of SARS-CoV-2, olfactory dysfunction (OD), has attracted tremendous attention. OD is not only a negative factor for quality of life but also an independent hazard and early biomarker for various diseases, such as Parkinson's and Huntington's diseases. Therefore, early identification and treatment of OD in patients are critical. Many etiological factors are responsible for OD based on current opinions. Sniffin'Sticks are recommended to identify the initial position (central or peripheral) for OD when treating patients clinically. It is worth emphasizing that the olfactory region in nasal cavity is recognized as the primary and critical olfactory receptor. Many nasal diseases, such as those with traumatic, obstructive and inflammatory causes, can lead to OD. The key question is no refined diagnosis or treatment strategy for nasogenic OD currently. This study summarizes the differences in medical history, symptoms, auxiliary examination, treatment and prognosis of different types of nasogenic OD by analyzing the current studies. We propose using olfactory training after 4-6 weeks of initial treatment for nasogenic OD patients with no significant improvement in olfaction. We hope that our research can provide valuable clinical guidance by systematically summarizing the clinical characteristics of nasogenic OD.

LncRNA TRG-AS1 inhibits bone metastasis of breast cancer by the miR-877-5p/WISP2 axis.

TRG-AS1 has been proved to inhibit cancer progression, whereas its effect on bone metastases of breast cancer is unknown. In this study, we determined breast cancer patients with disease free survival is longer in breast cancer patients with high TRG-AS1 expression. Moreover, TRG-AS1 was downregulated in breast cancer tissues and even lower in bone metastatic tumor tissues. Compared with parental breast cancer cell MDA-MB-231, TRG-AS1 expression was downregulated in MDA-MB-231-BO cells with strong bone-metastatic characteristics. Next, the binding sites of miR-877-5p on TRG-AS1 and WISP2 mRNA were predicted and result showed that miR-877-5p could bind to 3'UTR of TRG-AS1 and WISP2. Subsequently, BMMs and MC3T3-E1 cells were cultured in the conditioned media of MDA-MB-231 BO cells transfected with TRG-AS1 overexpression vector, shRNA and/or miR-877-5p mimics or inhibitor and/or overexpression vector and small interfering RNA of WISP2. TRG-AS1 silencing or miR-877-5p overexpression promoted MDA-MB-231 BO cell proliferation and invasion. TRG-AS1 overexpressing reduced TRAP positive cells, decreased TRAP, Cathepsin K, c-Fos, NFATc1 and AREG expression in BMMs, and promoted OPG, Runx2 and Bglap2 expression, and decreased RANKL expression in MC3T3-E1 cells. Silencing WISP2 rescued the effect of TRG-AS1 on BMMs and MC3T3-E1 cells. In vivo results showed that tumor volumes significantly decreased in mice injected with LV-TRG-AS1 transfected MDA-MB-231 cells. TRG-AS1 knockdown markedly reduced the number of TRAP+ cells and the percentage of Ki-67+ cells and decreased E-cadherin expression in xenograft tumor mice. In summary, TRG-AS1 acts an endogenous RNA, inhibited breast cancer bone metastasis by competitively binding with miR-877-5p to upregulate WISP2 expression.

Impairment mechanism of nasal mucosa after radiotherapy for nasopharyngeal carcinoma.

The nasal mucosa, which performs the crucial functions of filtering, humidifying and temperature regulation, is one of the most vulnerable areas of nasopharyngeal carcinoma (NPC) patients after radiotherapy (RT). Following RT, NPC patients experience a series of pathological changes in the nasal mucosa, ultimately leading to physiological dysfunction of the nasal epithelium. This article systematically reviews the clinical and pathological manifestations of RT-related nasal damage in NPC patients and summarizes the potential mechanism of damage to the human nasal epithelium by RT. Finally, we outline the current mechanistic models of nasal epithelial alterations after RT in NPC patients and provide additional information to extend the in-depth study on the impairment mechanisms of the nasal mucosa resulting from RT. We also describe the relationship between structural and functional alterations in the nasal mucosa after RT to help mitigate and treat this damage and provide insights informing future clinical and fundamental investigations.

Genome-Wide Determination of Gene Essentiality by Transposon Insertion Sequencing in Yeast Pichia pastoris.

In many prokaryotes but limited eukaryotic species, the combination of transposon mutagenesis and high-throughput sequencing has greatly accelerated the identification of essential genes. Here we successfully applied this technique to the methylotrophic yeast Pichia pastoris and classified its conditionally essential/non-essential gene sets. Firstly, we showed that two DNA transposons, TcBuster and Sleeping beauty, had high transposition activities in P. pastoris. By merging their insertion libraries and performing Tn-seq, we identified a total of 202,858 unique insertions under glucose supported growth condition. We then developed a machine learning method to classify the 5,040 annotated genes into putatively essential, putatively non-essential, ambig1 and ambig2 groups, and validated the accuracy of this classification model. Besides, Tn-seq was also performed under methanol supported growth condition and methanol specific essential genes were identified. The comparison of conditionally essential genes between glucose and methanol supported growth conditions helped to reveal potential novel targets involved in methanol metabolism and signaling. Our findings suggest that transposon mutagenesis and Tn-seq could be applied in the methylotrophic yeast Pichia pastoris to classify conditionally essential/non-essential gene sets. Our work also shows that determining gene essentiality under different culture conditions could help to screen for novel functional components specifically involved in methanol metabolism.

PiggyBac transposon-mediated mutagenesis and application in yeast Komagataella phaffii.

OBJECTIVE: Around one-fourth of the Komagataella phaffii genes encode hypothetical proteins with unknown functions. However, lack of powerful tools for genetic screening in K. phaffii significantly limits the functional analysis of these unknown genes. Transposon mutagenesis has been utilized as an insertional mutagenesis tool in many other organisms and would be extremely valuable if it could be applied in K. phaffii. RESULTS: In this study, we investigated in K. phaffii the transposition activity and efficiency of piggyBac (PB) transposon, a DNA transposon from the cabbage looper moth Trichoplusia ni through the integrated-plasmid system. We also designed a binary-plasmid system which could generate stable mutants. Finally we evaluated the quality of this mutagenesis system by a simple screening for functional genes involved in K. phaffii carbon catabolite repression. CONCLUSIONS: Our results demonstrate that PB-mediated mutagenesis could be a feasible and useful tool for functional gene screening in K. phaffii.

Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris.

Pichia pastoris expression system has been widely used in recombinant protein production. So far the majority of heterologous proteins are expressed by methanol inducible promoter PAOX1 and constitutive promoter PGAP . The use of other promoters is rather limited. Here we selected 16 potentially efficient and regulatory promoter candidates based on the RNA-seq and RNA folding free energy ΔG data. GFP and recombinant amylase were inserted after these promoters to reveal their strength and efficiency under different carbon sources and culture scales. Two novel promoters were successfully identified and could possibly be applied in recombinant protein expression: the methanol-inducible promoter P0547 and the constitutive promoter P0472 .

miR­483­5p promotes growth, invasion and self­renewal of gastric cancer stem cells by Wnt/ß­catenin signaling.

Gastric carcinoma (GC) ranks as the second most common cause of cancer­associated mortality worldwide. Emerging evidence has suggested a potential novel therapeutic strategy based on the ability of cancer stem cells (CSCs) to trigger tumorigenesis. MicroRNAs (miRNAs) have previously been implicated in CSC formation and regulation of their functional characteristics. In the current study, a significant upregulation of miR­483­5p levels was demonstrated in spheroid body­forming cells (P<0.01) by reverse transcription­quantitative polymerase chain reaction, which were isolated from the MKN­45 gastric cancer cell line and possessed gastric CSC (GCSC) properties. An MTT assay demonstrated that overexpression of miR­483­5p by transfection with miR­483­5p mimics significantly increased cell proliferation and Annexin V­propidium iodide staining indicated the suppression of cell apoptosis, suggesting that miR­483­5p has an important function in GCSC growth. Notably, Transwell and sphere formation assays demonstrated that miR­483­5p elevation promoted GCSC invasion and cell self­renewal ability, respectively. Further western blotting assays demonstrated that miR­483­5p upregulation induced an increase in the protein expression levels of ß­catenin and its downstream target molecules, including cyclin D1, Bcl­2 and matrix metalloproteinase 2, indicating that miR­483­5p activates Wnt/ß­catenin signaling. Inhibition of this pathway by ß­catenin small interfering RNA transfection attenuated the miR­483­5p­induced effects on cell growth, invasion and self­renewal. These results demonstrate that miR­483­5p may act as an oncogene to promote the development of GC by regulating GCSC growth, invasion and self­renewal via the Wnt/ß­catenin signaling pathway. Thus, the present study suggests that miR­483­5p may be a promising therapeutic target against GC.

PpNrg1 is a transcriptional repressor for glucose and glycerol repression of AOX1 promoter in methylotrophic yeast Pichia pastoris.

OBJECTIVE: The regulator in glycerol repression of Pichia pastoris AOX1 promoter (P AOX1 ) is still unclear. RESULTS: A Cys2His2 zinc finger transcriptional repressor PpNrg1 localized to nucleus and participated in the repression of P AOX1 in P. pastoris in glucose and glycerol. Quantitative real-time PCR revealed that PpNrg1 repressed expression of numerous genes involved in methanol utilization and peroxisome biogenesis in 0.02 % glucose and 1 % (v/v) glycerol. Electrophoretic mobility shift assay and DNase I footprinting assay revealed that PpNrg1 bound to five sites of P AOX1 , including two binding sites of PpMxr1, which is an indispensable activator of P AOX1 in P. pastoris. CONCLUSION: Transcriptional repressor PpNrg1 suppresses P AOX1 in glucose and glycerol by directly binding to five sites of P AOX1 , including two binding sites of transcriptional activator PpMxr1.

[The effect of modified bone morphogenetic protein-2 polylactic acid nanospheres sustained-release system on promoting mandibular defect repair].

OBJECTIVE: To investigate the effect of modified bone morphogenetic protein-2 polylactic acid nanospheres (BMP-2-PLA-Ns) sustained-release system on rabbit mandibular defect repair. METHODS: The polylactic acid s nanospheres (PLA-Ns) and BMP-2-PLA-Ns were prepared by ultrasonic emulsification after graft polymerization. Forty-five rabbits were randomly divided into 3 groups: Blank group, PLA-Ns gel group(control group), and BMP-2-PLA-Ns gel group (experimental group). The rabbit mandibular defect models were established. The defect area of control group was implanted with PLA-Ns gel, meanwhile, the experimental group was implanted with BMP-2-PLA-Ns gel, the blank group experienced no special handling. Rabbits were killed in 1, 2, 4 weeks after operation and the iconography, hematine eosin(HE) staining and PCNA immunohistochemistry were used to detect the reparative effect on rabbit mandible defects. RESULTS: Image observation showed that bone defect repair in the experimental group was well and the shadow was not obvious. Better repair effect was seen compared with the control group and blank group. HE staining showed that the experimental group and the control group had a large number of neovascularization and secondary callus formation, callus in experimental group was obviously higher than that of control group and blank group. Immunohistochemical observation showed that the experimental group's PCNA positive chondrocytes were more than those in the control group and the blank group in the first 2 weeks; all groups of PCNA positive cells were rare in the fourth week, PCNA positive expression rate of the fourth week was lower than that of the first 2 weeks. CONCLUSION: The modified BMP-2-PLA-Ns sustained-release system promotes mandibular defect repair obviously.

miR-518b is down-regulated, and involved in cell proliferation and invasion by targeting Rap1b in esophageal squamous cell carcinoma.

MicroRNAs (miRNAs) represent a class of small non-coding RNAs that regulate gene expression at the post-transcriptional levels. Recent studies show that miRNAs may function as oncogenes or tumor suppressor genes. In this study, we demonstrated that miR-518b was down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and correlated with metastasis and survival. miR-518b suppressed the proliferation by inducing apoptosis and repressed the invasion in ESCC cells, but had no effect on the cell cycle. Furthermore, Rap1b was revealed to be directly regulated by miR-518b. These findings indicate that miR-518b may function as a tumor suppressor by targeting Rap1b in the development of ESCC and has important clinical and prognostic value.