OBJECTIVE: To analyze the clinical phenotype and pathogenic mechanism of the ARSL gene variant in a fetus with nasal bone aplasia. METHODS: A 34-year-old pregnant woman who attended Quzhou Maternal and Child Health Care Hospital on January 3, 2023 was selected as the study subject. Whole exome sequencing (WES) was performed on the fetus. Bioinformatics analysis was carried out to identify and prioritize candidate gene variants, followed by Sanger sequencing for familial validation. A mutant plasmid expression vector was constructed and subsequently transfected into HEK293T cells to preliminarily investigate the pathogenetic mechanism of the identified variant. Additionally, a comprehensive review of literature was conducted to systematically summarize the associated clinical phenotypes. This study was approved by the Medical Ethics Committee of Quzhou Maternal and Child Health Care Hospital (Ethics No.: KY-2023-11). RESULTS: WES revealed that the fetus harbored a c.827del (p.L276Rfs*48) variant of the ARSL gene, for which its mother was heterozygous. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic(PVS1+PM2_Supporting). In vitro cellular function studies demonstrated that this variant can result in a substantial decrease in the expression of mutant mRNA, thereby preventing the production of normal ARSL protein. Clinical phenotypes resulting from ARSL gene variants exhibited considerable diversity, with nasal hypoplasia being the most common manifestation. CONCLUSION The c.827del (p.L276Rfs*48) variant of the ARSL gene can lead to degradation of mRNA via the nonsense-mediated mRNA decay pathway, resulting in reduced levels of ARSL protein. The pathogenetic mechanism underlying the ARSL gene variant may be associated with its haploinsufficiency effect.
Humans ; Female ; Pregnancy ; Adult ; Frameshift Mutation ; HEK293 Cells ; Nasal Bone/abnormalities* ; Fetus/abnormalities* ; Exome Sequencing

OBJECTIVE: To analyze the functional impact of a rare heterozygous variant of SOS1 gene (c.283G>A, p.E95K) identified in a fetus with cervical cystic hygroma and to explore its association with the disease phenotype. METHODS: A pedigree analysis was carried out to evaluate the co-segregation of the variant with the disease phenotype. Bioinformatic tools were employed to assess the conservation, protein structure and stability. Functional validation was conducted on HEK293T cells using fluorescence quantitative reverse transcription-PCR and Western blotting to measure the expression of SOS1 and phosphorylation levels of extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase. A literature review of previously reported disease-associated SOS1 variants was also carried out. This study has been approved by the Medical Ethics Committee of West China Second University Hospital, Sichuan University (Ethics No.: 201940). RESULTS: The variant was inherited from the husband of the woman with distinctive facial features and has co-segregated with the phenotype. Bioinformatics analysis indicated that the variant is located in a highly conserved region, and that p.E95K could disrupt key amino acid interactions and protein stability. Multiple bioinformatic predictions consistently suggested the pathogenicity of this variant. Functional assays demonstrated reduced SOS1 protein expression and decreased ERK phosphorylation. CONCLUSION This study has revealed the functional impact of the SOS1 c.283G>A (p.E95K) variant, suggesting that it may contribute to the developmental phenotypes through a haploinsufficiency mechanism.
Humans ; SOS1 Protein/chemistry* ; Female ; HEK293 Cells ; Male ; Pedigree ; Phenotype ; Adult

OBJECTIVE

The aim of this study was to qualitatively review the effects of genotoxicity and cytotoxicity on buccal mucosal epithelial cells after cone beam computed tomography (CBCT) exposure focusing on DNA damage and cell changes.

A literature search was carried out in PubMed, Wiley, Google Scholar, and Semantic Scholar for articles published in the last five years. In vivo studies that analyzed the DNA damage and cell changes on buccal mucosal epithelial cells, before and several days after CBCT exposure were included in this review. This review was prepared according to the PRISMA checklist for systematic review and the risk of bias was assessed using the Quality Assessment of Diagnostic Accuracy Studies tool.

A total of four studies were included in this review. The risk of bias analysis showed that all studies had generally good methodological quality. All the studies used buccal epithelial cells to analyze micronucleus (MN) as a parameter for DNA damage (genotoxicity), three of the studies also analyzed cytotoxicity using pyknotic nucleus and three studies analyzed karyolysis and karyorrhexis. All the studies consistently reported a significant increase in MN frequency, and cytotoxic effect were more evident before and 10-15 days after CBCT exposure.

This study demonstrated a significant impact on DNA and cell damage in oral mucosal cells following CBCT examination. The effect of ionizing radiation from CBCT has a more pronounced impact on cell damage than DNA damage.

OBJECTIVES: To analyze the differential expression and biological functions of circRNAs in the anterior lens capsules of high myopic patients with cataract and their pathogenic roles in the development of this condition. METHODS: Anterior lens capsule specimens were collected intraoperatively from 36 patients with age-related cataract (ARC) and 36 high myopic patients with cataract. Among these, 18 specimens from each group were selected for whole transcriptome sequencing and biological analysis, and the remaining 36 specimens were used for validation of circPDGFRA, circFOXJ3, hsa_circ_0004767, hsa_circ_0007528, ciCRIM1, circMAN1A2, circSLC5A3, and circPTK2 expressions using RT-qPCR. hsa_circ_0007528 was selected for cell experiments to examine its effects on proliferation, migration, and apoptosis of lens epithelial cells (LECs). RESULTS: A total of 16 192 circRNAs were detected in the specimens from both groups, among which 62 circRNAs were differentially expressed (29 upregulated and 33 downregulated). GO and KEGG analyses revealed that the differentially expressed circRNAs were primarily localized in the cytoplasm, nucleoplasm, and endoplasmic reticulum, and were involved in signaling pathways associated with Gap junction and the PI3K-Akt, NF-κB, Jak-STAT, HIF-1, and MAPK signaling pathways. The ceRNA network predicted multiple target genes. RT-qPCR validation results were consistent with the sequencing data. In the LECs, upregulation of hsa_circ_0007528 significantly inhibited cell proliferation and migration and obviously promoted cell apoptosis. CONCLUSIONS The expression profile of circRNAs in the anterior lens capsule of high myopic patients with cataract differs from that of ARC patients. Upregulation of hsa_circ_0007528 inhibits LEC proliferation and migration and promotes cell apoptosis.
Humans ; Cataract/complications* ; RNA, Circular ; Myopia/genetics* ; Apoptosis ; Cell Proliferation ; Epithelial Cells ; Cell Movement ; Anterior Capsule of the Lens/metabolism* ; Male ; Female

OBJECTIVES: To investigate the mechanism through which pinostrobin (PSB) alleviates dextran sulfate sodium (DSS)-induced colitis in mice. METHODS: C57BL/6 mice were randomized into control group, DSS model group, and PSB intervention (30, 60, and 120 mg/kg) groups. Colitis severity of the mice was assessed by examining body weight changes, disease activity index (DAI), colon length, and histopathology. The expressions of tight junction proteins ZO-1 and claudin-1 in the colon tissues were examined using immunofluorescence staining, and macrophage infiltration and polarization were analyzed with flow cytometry. ELISA and RT-qPCR were used for detecting the expressions of inflammatory factors (TNF‑α and IL-6) and chemokines (CCL₂, CXCL₁₀, and CX₃CL₁) in the colon tissues, and PI3K/AKT phosphorylation levels were analyzed with Western blotting. In cultured Caco-2 and RAW264.7 cells, the effect of PSB on CCL₂-mediated macrophage migration was assessed using Transwell assay. Network pharmacology analysis was performed to predict the key pathways that mediate the therapeutic effect of PSB. RESULTS: In DSS-induced mouse models, PSB at 60 mg/kg optimally alleviated colitis, shown by reduced weight loss and DAI scores and increased colon length. PSB treatment significantly upregulated ZO-1 and claudin-1 expressions in the colon tissues, inhibited colonic macrophage infiltration, and promoted the shift of macrophage polarization from M1 to M2 type. In cultured intestinal epithelial cells, PSB significantly inhibited PI3K/AKT phosphorylation and suppressed chemokine CCL₂ expression. PSB treatment obviously blocked CCL₂-mediated macrophage migration of RAW264.7 cells, which could be reversed by exogenous CCL₂. Network pharmacology analysis and rescue experiments confirmed PI3K/AKT and CCL₂ signaling as the core targets of PSB. CONCLUSIONS PSB alleviates DSS-induced colitis in mice by targeting intestinal epithelial PI3K/AKT signaling, reducing CCL₂ secretion, and blocking macrophage chemotaxis and migration, highlighting the potential of PSB as a novel natural compound for treatment of inflammatory bowel disease.
Animals ; Mice ; Mice, Inbred C57BL ; Phosphatidylinositol 3-Kinases/metabolism* ; Colitis/drug therapy* ; Dextran Sulfate ; Proto-Oncogene Proteins c-akt/metabolism* ; Macrophages ; Chemokine CCL2/metabolism* ; Humans ; Signal Transduction/drug effects* ; Caco-2 Cells ; RAW 264.7 Cells ; Epithelial Cells/drug effects* ; Intestinal Mucosa/metabolism*

OBJECTIVES: To investigate the effect of hypaphorine (HYP) on Crohn's disease (CD)‑like colitis in mice and its molecular mechanism. METHODS: Thirty male C57BL/6J mice were equally randomized into WT, TNBS, and HYP groups, and in the latter two groups, mouse models of CD-like colitis were established using TNBS with daily gavage of 15 mg/kg HYP or an equivalent volume of saline. The treatment efficacy was evaluated by assessing the disease activity index (DAI), body weight changes, colon length and histopathology. The effect of HYP was also tested in a LPS-stimulated Caco-2 cell model mimicking intestinal inflammation by evaluating inflammatory responses and barrier function of the cells using qRT-PCR and immunofluorescence staining. GO and KEGG analyses were conducted to explore the therapeutic mechanism of HYP, which was validated in both the cell and mouse models using Western blotting. RESULTS: In the mouse models of CD-like colitis, HYP intervention obviously alleviated colitis as shown by significantly reduced body weight loss, colon shortening, DAI and inflammation scores, and expressions of pro-inflammatory factors in the colon tissues. HYP treatment also significantly increased the TEER values, reduced bacterial translocation to the mesenteric lymph nodes, liver, and spleen, lowered serum levels of I-FABP and FITC-dextran, increased the number of colonic tissue cup cells, and upregulated colonic expressions of MUC2 and tight junction proteins (claudin-1 and ZO-1) in the mouse models. In LPS-stimulated Caco-2 cells, HYP treatment significantly inhibited the expressions of pro-inflammatory factors and increased the expressions of tight junction proteins. Western blotting showed that HYP downregulated the expressions of the key proteins in the TLR4/MyD88 signaling pathway in both the in vitro and in vivo models. CONCLUSIONS HYP alleviates CD-like colitis in mice possibly by suppressing intestinal epithelial inflammation and improving gut barrier function.
Animals ; Male ; Mice, Inbred C57BL ; Crohn Disease/drug therapy* ; Mice ; Humans ; Caco-2 Cells ; Intestinal Mucosa/metabolism* ; Colitis/drug therapy* ; Disease Models, Animal ; Inflammation ; Toll-Like Receptor 4/metabolism* ; Myeloid Differentiation Factor 88/metabolism* ; Intestinal Barrier Function

OBJECTIVES: To investigate the therapeutic effect of the natural compound niranthin on Crohn's disease-like colitis in mice and explore the underlying molecular mechanisms. METHODS: In a mouse model of colitis induced by 2,4,6-trinitro-benzenesulfonic acid (TNBS), the therapeutic effect of niranthin was evaluated by observing the changes in body weight, disease activity index (DAI), and colon length of the mice. The levels of inflammatory cytokines (IL-6, IL-1β, TNF-α, IL-17A and IL-10) in the intestinal mucosal tissue were detected using ELISA and quantitative real-time PCR (qRT-PCR). TUNEL staining and Western blotting were used to assess intestinal epithelial cell apoptosis and the expressions of Bcl-2 and Bax. The expression levels of tight junction proteins (ZO-1 and claudin-1) and the activation of the p38/JNK signaling pathway were investigated using Western blotting, and diprovocim intervention experiments were conducted to explore the molecular regulatory mechanism of niranthin. RESULTS: Niranthin treatment significantly increased body weight of TNBS-treated mice, lowered the DAI and histological inflammation scores, and increased colon length of the mice. The niranthin-treated mouse models showed obviously reduced protein and mRNA levels of IL-6, IL-1β, IL-17A, and TNF-α and upregulated expression of IL-10 in the colon tissue. TUNEL staining and Western blotting demonstrated that niranthin significantly inhibited intestinal epithelial cell apoptosis and activated the anti-apoptotic pathway in the mouse models. Niranthin treatment obviously upregulated the expression levels of ZO-1 and claudin-1 and downregulated the phosphorylation levels of p38 and JNK in the colon tissues of the mice. Diprovocim intervention obviously attenuated the inactivation of the p38/JNK signaling pathway induced by niranthin in the mouse models. CONCLUSIONS Niranthin ameliorates TNBS-induced Crohn's disease-like colitis in mice by inhibiting intestinal epithelial cell apoptosis and protecting the integrity of the intestinal barrier via regulating the activation of the p38/JNK signaling pathway.
Animals ; Apoptosis/drug effects* ; Mice ; Intestinal Mucosa/drug effects* ; Crohn Disease/drug therapy* ; MAP Kinase Signaling System/drug effects* ; Epithelial Cells/drug effects* ; Disease Models, Animal ; Signal Transduction/drug effects* ; p38 Mitogen-Activated Protein Kinases/metabolism* ; Male

The KCNQ potassium channels play a crucial role in modulating neural excitability, and their dysfunction is closely associated with epileptic disorders. While variants in KCNQ2 have been extensively studied, KCNQ3-related disorders have rarely been reported. With advances in next-generation sequencing technologies, an increasing number of cases of KCNQ3-related disorders have been identified. However, the correlation between genotype and phenotype remains poorly understood. In this study, we established a variant library consisting of 24 missense mutations in KCNQ3 and introduced these mutations into three different template types: KCNQ3, KCNQ3-A315T (Q3*), and KCNQ3-KCNQ2 tandem (Q3-Q2). We then analyzed the effects of these mutations on the KCNQ3 channel function using patch-clamp recording. The most informative parameter across all three backgrounds was the current density of the mutant channels. The current density patterns in the Q3* and Q3-Q2 backgrounds were similar, with most mutations resulting in an almost complete loss of function (LOF), they were concentrated in the pore-forming domain of KCNQ3. In contrast, mutations in the voltage-sensing domain or C-terminus did not show significant differences from the wild-type channel. Interestingly, these LOF mutations were typically associated with self-limited familial neonatal epilepsy, while neurodevelopmental disorders (NDD) were more closely associated with mutations that did not significantly differ from the wild-type. V_1/2, another important parameter of the electrophysiological properties, could not be accurately determined in the majority of KCNQ3 mutations due to its nearly complete LOF in the Q3* and Q3-Q2 backgrounds. Intriguingly, the V_1/2 of functional mutations were primarily leftward shifted, indicating a gain-of-function (GOF) effect, which was typically associated with NDD. In addition to previously reported mutations, we identified G553R as a novel GOF mutation. In the co-transfection background, parameters such as V_1/2 could be determined, but the dysfunctional effects of these mutations were mitigated by the co-expression of wild-type KCNQ3 and KCNQ2 subunits, resulting in no significant differences between most mutations and the wild-type channel. Furthermore, we applied KCNQ modulators to reverse the electrophysiological abnormalities caused by KCNQ3 variants. The LOF mutations were reversed by the application of Pynegabine (HN37), a KCNQ opener, while the GOF mutation responded well to Amitriptyline (AMI), a KCNQ inhibitor. These findings provide essential insights into the pathogenic mechanisms underlying KCNQ3-related disorders and may inform clinical decision-making.
KCNQ3 Potassium Channel/genetics* ; Humans ; Mutation, Missense/genetics* ; KCNQ2 Potassium Channel/genetics* ; Patch-Clamp Techniques ; HEK293 Cells ; Animals ; Phenylenediamines/pharmacology* ; Carbamates

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes, thereby hindering a comprehensive understanding of their interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using a key oral pathobiont, Fusobacterium nucleatum, as the primary test bed, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. In addition, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. Finally, we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont, Porphyromonas gingivalis. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research.
Coculture Techniques/methods* ; Humans ; Fusobacterium nucleatum/physiology* ; Gingiva/microbiology* ; Keratinocytes/microbiology* ; Host Microbial Interactions ; Mouth/microbiology* ; Host-Pathogen Interactions ; Epithelial Cells/microbiology* ; Cells, Cultured ; Porphyromonas gingivalis

BACKGROUND: Mitochondria, which harbor their own genome (mtDNA), have attracted attention due to the potential of mtDNA copy number (mtDNA-CN) as an indicator of mitochondrial dysfunction. Although mtDNA-CN has been proposed as a simple and accessible biomarker for metabolic disorders such as metabolic dysfunction-associated steatotic liver disease, the underlying mechanisms and the causal relationship remain insufficiently elucidated. In this investigation, we combined longitudinal epidemiological data, animal studies, and in vitro assays to elucidate the potential causal relationship between reduced mtDNA-CN and the development of steatotic liver disease (SLD). METHODS: We conducted a longitudinal study using data from a health examination cohort initiated in 1981 in Yakumo, Hokkaido, Japan. Data from examinations performed in 2015 and 2022 were analyzed, focusing on 76 subjects without SLD at baseline (2015) to assess the association between baseline mtDNA-CN and subsequent risk of SLD development. In addition, 28-day-old SD rats were fed ad libitum on a 45% high-fat diet and dissected at 2 and 8 weeks of age. Blood and liver mtDNA-CN were measured and compared at each feeding period. Additionally, in vitro experiments were performed using HepG2 cells treated with mitochondrial function inhibitors to induce mtDNA-CN depletion and to examine its impact on intracellular lipid accumulation. RESULTS: Epidemiological analysis showed that the subjects with low mtDNA-CN had a significantly higher odds ratio for developing SLD compared to high (odds ratio [95% confidence interval]: 4.93 [1.08-22.50]). Analysis of the animal model showed that 8 weeks of high-fat diet led to the development of fatty liver and a significant decrease in mtDNA-CN. A further 2 weeks of high-fat diet consumption resulted in a significant decrease in hepatic mtDNA-CN, despite the absence of fatty liver development, and a similar trend was observed for blood. Complementary in vitro experiments revealed that pharmacologically induced mitochondrial dysfunction led to a significant reduction in mtDNA-CN and was associated with increases in intracellular lipid accumulation in HepG2 cells. CONCLUSIONS Our findings suggest that reduced mtDNA-CN may contribute causally to SLD development and could serve as a convenient, noninvasive biomarker for early detection and risk assessment.
Animals ; DNA, Mitochondrial/genetics* ; Humans ; Male ; DNA Copy Number Variations ; Female ; Fatty Liver/blood* ; Rats ; Middle Aged ; Longitudinal Studies ; Rats, Sprague-Dawley ; Adult ; Japan/epidemiology* ; Aged ; Biomarkers/blood* ; Hep G2 Cells ; Diet, High-Fat/adverse effects*