PTHrP participates in the bone destruction of middle ear cholesteatoma via promoting macrophage differentiation into osteoclasts induced by RANKL. / PTHrP促进RANKLè¯±å¯¼å·¨å™¬ç»†èƒžåˆ†åŒ–ä¸ºç ´éª¨ç»†èƒžå‚ä¸Žä¸­è€³èƒ†è„‚ç˜¤éª¨ç ´å.

OBJECTIVES: Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. METHODS: A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. RESULTS: Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. CONCLUSIONS: PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

m6A modification of lncRNA in middle ear cholesteatoma. / 中耳胆脂瘤中lncRNA的m6A修饰.

OBJECTIVES: Middle ear cholesteatoma is a non-tumorous condition that typically leads to hearing loss, bone destruction, and other severe complications. Despite surgery being the primary treatment, the recurrence rate remains high. Therefore, exploring the molecular mechanisms underlying cholesteatoma is crucial for discovering new therapeutic approaches. This study aims to explore the involvement of N6-methyladenosine (m6A) methylation in long non-coding RNAs (lncRNAs) in the biological functions and related pathways of middle ear cholesteatoma. METHODS: The m6A modification patterns of lncRNA in middle ear cholesteatoma tissues (n=5) and normal post-auricular skin tissues (n=5) were analyzed using an lncRNA m6A transcriptome microarray. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to identify potential biological functions and signaling pathways involved in the pathogenesis of middle ear cholesteatoma. Methylated RNA immunoprecipitation (MeRIP)-PCR was used to validate the m6A modifications in cholesteatoma and normal skin tissues. RESULTS: Compared with normal skin tissues, 1 525 lncRNAs were differentially methylated in middle ear cholesteatoma tissues, with 1 048 showing hypermethylation and 477 showing hypomethylation [fold change (FC)≥3 or <1/3, P<0.05]. GO enrichment analysis indicated that hypermethylated lncRNAs were involved in protein phosphatase inhibitor activity, neuron-neuron synapse, and regulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activity. Hypomethylated lncRNAs were associated with mRNA methyltransferase activity, secretory granule membrane, and mRNA methylation. KEGG analysis revealed that hypermethylated lncRNAs were mainly associated with 5 pathways: the Hedgehog signaling pathway, viral protein interaction with cytokines and cytokine receptors, mitogen-activated protein kinase (MAPK) signaling pathway, cytokine-cytokine receptor interaction, and adrenergic signaling in cardiomyocytes. Hypomethylated lncRNAs were mainly involved in 4 pathways: Renal cell carcinoma, tumor necrosis factor signaling pathway, transcriptional misregulation in cancer, and cytokine-cytokine receptor interaction. Additionally, MeRIP-PCR confirmed the changes in m6A methylation levels in NR_033339, NR_122111, NR_130744, and NR_026800, consistent with microarray analysis. Real-time PCR also confirmed the significant upregulation of MAPK1 and NF-κB, key genes in the MAPK signaling pathway. CONCLUSIONS: This study reveals the m6A modification patterns of lncRNAs in middle ear cholesteatoma, suggests a direction for further research into the role of lncRNA m6A modification in the etiology of cholesteatoma. The findings provide potential therapeutic targets for the treatment of middle ear cholesteatoma.

Clinical characteristics of patients diagnosed with bilateral sudden sensorineural hearing loss.

This study investigated the etiology, clinical features, and prognosis of patients diagnosed with bilateral sudden sensorineural hearing loss (BSSNHL). The clinical data of 100 patients with bilateral sudden hearing loss as a chief complaint treated at Xiangya Second Hospital of Central South University between January 2010 and August 2022, including clinical characteristics, audiometric data, and prognosis, were retrospectively analyzed. These 100 cases accounted for 8.09% (100/1235) of all patients admitted for sudden sensorineural hearing loss (SSNHL) during the same period. Of these, 71 were simultaneous cases and 29 were sequential cases of BSSNHL. Among the 200 ears analyzed in this study, 13, 36, 57, and 94 had mild, moderate, severe, and profound sensorineural hearing loss, respectively. The overall effective rate after comprehensive treatment was 32%, with significant differences in efficacy and prognosis among different degrees of hearing loss (p < 0.05). Comorbidities of hypertension (24 cases), diabetes (14 cases), and coronary heart disease (9 cases) significantly impacted therapeutic efficacy and prognosis in patients with BSSNHL (p < 0.05). Compared to unilateral SSNHL, BSSNHL exhibits distinctive characteristics.

N6-methyladenosine methylation analysis of circRNAs in acquired middle ear cholesteatoma.

Introduction: Middle ear cholesteatoma is a chronic middle ear disease characterized by severe hearing loss and adjacent bone erosion, resulting in numerous complications. This study sought to identify pathways involved in N6-methyladenosine (m6A) modification of circRNA in middle ear cholesteatoma. Methods: A m6A circRNA epitranscriptomic microarray analysis was performed in middle ear cholesteatoma tissues (n = 5) and normal post-auricular skin samples (n = 5). Bioinformatics analyses subsequently explored the biological functions (Gene Ontology, GO) and signaling pathways (Kyoto Encyclopedia of Genes and Genomes, KEGG) underlying middle ear cholesteatoma pathogenesis. Methylated RNA immunoprecipitation qPCR (MeRIP-qPCR) was performed to verify the presence of circRNAs with m6A modifications in middle ear cholesteatoma and normal skin samples. Results: Microarray analysis identified 3,755 circRNAs as significantly differentially modified by m6A methylation in middle ear cholesteatoma compared with the normal post-auricular skin. Among these, 3,742 were hypermethylated (FC ≥ 2, FDR < 0.05) and 13 were hypomethylated (FC ≤ 1/2, FDR < 0.05). GO analysis terms with the highest enrichment score were localization, cytoplasm, and ATP-dependent activity for biological processes, cellular components, and molecular functions respectively. Of the eight hypermethylated circRNA pathways, RNA degradation pathway has the highest enrichment score. Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway was hypomethylated. To validate the microarray analysis, we conducted MeRIP-qPCR to assess the methylation levels of five specific m6A-modified circRNAs: hsa_circRNA_061554, hsa_circRNA_001454, hsa_circRNA_031526, hsa_circRNA_100833, and hsa_circRNA_022382. The validation was highly consistent with the findings from the microarray analysis. Conclusion: Our study firstly presents m6A modification patterns of circRNAs in middle ear cholesteatoma. This finding suggests a direction for circRNA m6A modification research in the etiology of cholesteatoma and provides potential therapeutic targets for the treatment of middle ear cholesteatoma.

Case report: A novel mutation in the EYA1 gene in a child with branchiootic syndrome with secretory otitis media and bilateral vestibular hypofunction.

Branchiootic syndrome (BOS) is a rare, autosomal dominant syndrome characterized by malformations of the ear associated with hearing loss, second branchial arch anomalies, and the absence of renal anomalies. Herein, we report the case of an 8-year-old male patient with BOS. The proband also experiences mixed conductive and sensorineural hearing loss in the right ear, and severe-to-profound sensorineural hearing loss in the left ear. Preauricular pits, branchial fistulae, and cochlear hypoplasia were present bilaterally. Type III cup-shaped ear, and external auditory canal stenosis were detected in the right ear. Lateral semicircular canal-vestibule dysplasia was detected in the left ear. Moreover, the patient had unilateral secretory otitis media (SOM) in the right ear and bilateral vestibular hypofunction (VH), which has not been reported in previous studies. The patient's hearing on the right side was restored to nearly normal after myringotomy. Whole exome sequencing identified a novel frameshift mutation in EYA1 (NM_000503.6): c.1697_1698delinT [p.(Lys566IlefsTer73)] in the proband, which was defined a "pathogenic" mutation according to American College of Medical Genetics and Genomics guidelines. This is the first report of a child presenting with BOS, SOM and VH, which expands the known clinical manifestations of this syndrome. We also observed a novel EYA1 gene mutation in this patient with BOS, which enriches the mutation map and provides a reference for genetic diagnosis of this syndrome.

Mitochondrial stress response in drug-induced liver injury.

Drug-induced liver injury (DILI) caused by the ingestion of medications, herbs, chemicals or dietary supplements, is a clinically widespread health problem. The underlying mechanism of DILI is the formation of reactive metabolites, which trigger mitochondrial oxidative stress and the opening of mitochondrial permeability transition (MPT) pores through direct toxicity or immune response, leading to cell inflammation, apoptosis, and necrosis. Traditionally, mitochondria play an indispensable role in maintaining the physiological and biochemical functions of cells by producing ATP and mediating intracellular signal transduction; drugs can typically stimulate the mitochondria and, in the case of sustained stress, can eventually cause impairment of mitochondrial function and metabolic activity. Meanwhile, the mitochondrial stress response, as an adaptive protective mechanism, occurs when mitochondrial homeostasis is threatened. In this review, we summarize the relevant frontier researches of the protective effects of mitochondrial stress response in DILI as well as the potential related mechanisms, thus providing some thoughts for the clinical treatment of DILI.

Involvement of SIRT1 in amelioration of schistosomiasis-induced hepatic fibrosis by genistein.

Previous study revealed that genistein alleviate the extent of hepatic fibrosis in schistosomiasis-infected mice, however, the potential mechanism is still incomplete. Present study was, therefore, carried out to investigate the underlying mechanism of ameliorating schistosomiasis-induced hepatic fibrosis by genistein. α-smooth muscle actin (α-SMA) expression, as a critical fibrotic marker, was markedly upregulated in Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis, and gradually inhibited by genistein administration in infected mice. Contrary to the changes of α-SMA expression, hepatic SIRT1 expression and activity was greatly inhibited in mice upon S. japonicum infection, and the repression was reversed in liver tissues after receiving 25 mg/kg genistein. 50 mg/kg genistein treatment gave rise to the higher SIRT1 expression and activity than that of the control group. In hepatic stellate cells (HSCs), genistein (5, 10, 20 µM) treatment resulted in the increases of SIRT1 expression and activity in concentration-dependent manner. Moreover, to mimic the fibrogenesis in vivo, macrophage was treated with soluble egg antigen (SEA) to obtain macrophage-conditioned medium (MφCM), which was used to stimulate HSCs. Intriguingly, SIRT1 overexpression decreased fibrosis associated gene expression in HSCs exposed to MφCM or not. Additionally, MφCM gave rise to high levels of α-SMA and p-Smad3 and the increments were reversed upon genistein treatment in HSCs. Furthermore, EX527, SIRT1 specific inhibitor, abrogated the inhibitory effects of genistein on HSCs activation. Together, the results support the notion that the strong elevation of SIRT1 expression and activity may represent a potential mechanism of protection against schistosomiasis-induced hepatic fibrosis by genistein.

Water-soluble thermoresponsive α-helical polypeptide with an upper critical solution temperature: synthesis, characterization, and thermoresponsive phase transition behaviors.

Water-soluble polypeptides bearing 1-alkylimidazolium (methyl or n-butyl) and various counter-anions (i.e., Cl(-), I(-) or BF4 (-)) are prepared by ring-opening polymerization of γ-4-chloromethylbenzyl-l-glutamate-based N-carboxyanhydride (3), post-polymerization of poly(γ-4-chloromethylbenzyl-l-glutamate) (4), and ion-exchange reaction. Circular dichroism (CD) analysis reveals that the resulting polypeptides adopt an α-helical conformation in water with a fractional helicity in the range of 30%-56% at 20 °C and exhibit good conformational stability against temperature variations. The polypeptides exhibit lower critical solution temperature (LCST)-type or upper critical solution temperature (UCST)-type transitions in organic solvents or in water. The UCST-type transition temperature (Tpt ) in water is independent on the molecular weight, yet it decreases upon addition of NaCl and increases upon addition of NaI or NaBF4, suggesting a mainly electrostatic interaction mechanism.

One-pot synthesis of molecular bottle-brush functionalized single-walled carbon nanotubes with superior dispersibility in water.

Molecular bottle-brush functionalized single-walled carbon nanotubes (SWCNTs) with superior dispersibility in water are prepared by a one-pot synthetic methodology. Elongating the main-chain and side-chain length of molecular bottle-brushes can further increase SWCNT dispersibility. They show significant enhancement of SWCNT dispersibility up to four times higher than those of linear molecular functionalized SWCNTs.