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.

OBJECTIVE: To explore the pathogenic mechanism of a child with Waardenburg syndrome type 4C due to a c.661_664dup (p.P222Lfs*60) variant of SOX10 gene through in vitro experiments. METHODS: A child diagnosed at the Handan First Hospital was selected as the study subject. Clinical data of the child was collected. Peripheral blood samples were collected from the child and his parents. Following extraction of genomic DNA, trio-whole exome sequencing was carried out. Pathogenicity of candidate variant was determined by bioinformatic analysis and reference to the guidelines from the American College of Medical Genetics and Genomics (ACMG). Candidate variant was verified by Sanger sequencing. Expression plasmids of wild-type SOX10 and the c.661_664dup (p.P222Lfs*60) variant were constructed and transiently transfected into 293T cells to determine the expression at the RNA and protein levels. The 293T cells transiently transfected with the wild-type/mutant SOX10 were treated with 10 ug/mL cycloheximide (CHX) for 0, 4, 8, 24 h, respectively, and the degradation rate of target protein was detected by Western blotting assay. This study has been approved by the Ethics Committee of Handan First Hospital (Ethics No. HDYY-LW-25053). RESULTS: The child was found to harbor a heterozygous c.661_664dup (p.P222Lfs*60) variant of the SOX10 gene, which was unreported previously. The variant did not significantly alter the expression of SOX10 at the mRNA level but the protein level. After the CHX treatment, the degradation of mutant SOX10 protein had slowed down. CONCLUSION The mutant SOX10 may affect the expression of downstream genes by affecting the degradation rate of its protein product.
Humans ; HEK293 Cells ; Mutation ; SOXE Transcription Factors/metabolism* ; Waardenburg Syndrome/genetics* ; Child

OBJECTIVE: To investigate the molecular mechanism for a family with Hereditary coagulation factor Ⅻ (FⅫ) deficiency. METHODS: The proband, a 63-year-old female, was admitted to the First Affiliated Hospital of Wenzhou Medical University in August 2024 for lumbar disc herniation. Coagulation tests, including prothrombin time (PT), activated partial thromboplastin time (APTT), and FⅫ activity (FⅫ:C), were carried out for the proband and her family members (9 individuals from three generations) using a one-stage clotting assay. The level of FⅫ antigen (FⅫ:Ag) was determined with an Enzyme-linked immunosorbent assay (ELISA). Sanger sequencing was conducted to identify potential variants in the F12 gene. Multiple in silico tools were used to predict the conservation, hydrophobicity, and structural impact of the identified variants. Recombinant expression plasmids were constructed and transiently transfected into HEK293T cells. The recombinant FⅫ protein was analyzed using Western blotting (WB) and ELISA. This study was approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Ethics No.: KY2022-R193). RESULTS: The proband showed a markedly prolonged APTT (160.3 s) and significantly decreased FⅫ:C (2%) and FⅫ:Ag (5%) levels. Analysis of the F12 gene sequence revealed a 46C/T genotype in the promoter region, a heterozygous c.1457G>A (p.Cys467Tyr) missense variant in exon 12, and a heterozygous c.1561G>A (p.Glu502Lys) missense variant in exon 13. Bioinformatic analysis showed that the p.Cys467 is highly conserved across various species, and the p.Cys467Tyr variant may affect local structural stability of the FⅫ protein. The p.Cys467Tyr variant had no effect on the transcription of the F12 gene. However, the variant has significantly decreased the FⅫ:Ag levels and FⅫ protein expression in the cell culture supernatant compared to the wild-type expression vector, while in the cell lysate, it is higher than the wild-type expression vector. In other words, the p.Cys467Tyr variant has probably caused a secretion defect of FⅫ protein. CONCLUSION The 46C/T genotype, the heterozygous p.Cys467Tyr missense variant, and the heterozygous p.Glu502Lys missense variant are associated with reduced plasma FⅫ levels in this pedigree. The p.Cys467Tyr variant, which was unreported previously, did not affect the synthesis of FⅫ but may have resulted in a secretion defect.
Humans ; Female ; Middle Aged ; Mutation, Missense ; Pedigree ; HEK293 Cells ; Male ; Factor XII/genetics* ; Adult ; Factor XII Deficiency/genetics*

Objective This study investigated the regulatory effect of high mobility group protein B1 (HMGB1) in the peripheral blood of patients with primary immune thrombocytopenia (ITP) on myeloid dendritic cells (mDC) and Th17/regulatory T cells (Treg) balance. Methods The study enrolled 30 newly diagnosed ITP patients and 30 healthy controls.Flow cytometry was used to measure the proportion of mDC, Th17, and Treg cells in the peripheral blood of ITP patients and healthy controls. ELISA was conducted to quantify the serum levels of HMGB1, interleukin 6 (IL-6), IL-23, IL-17, and transforming growth factor β(TGF-β). The mRNA levels of retinoic acid-related orphan receptor γt(RORγt) and forehead box P3(FOXP3) were detected by real-time PCR. The correlation between the abovementioned cells, cytokines, and platelet count was assessed using Pearson linear correlation analysis. Results The proportion of Th17 cells and the expression levels of HMGB1, IL-6, IL-23, IL-17 and the level of RORγt mRNA in the peripheral blood of ITP patients were higher than those in healthy controls. However, the Treg cell proportion and TGF-β level were lower in ITP patients than those in healthy controls. In patients with ITP, the proportion of mDC and the level of FOXP3 mRNA did not show significant changes. The proportion of mDC cells was significantly correlated with the expression of IL-6 and IL-23. Moreover, the expression of HMGB1 showed a significant correlation with the expression of mDC, IL-6, IL-23, RORγt mRNA, and IL-17. Notably, both the proportion of mDC cells and the expression of HMGB1 were negatively correlated with platelet count. Conclusion The high expression of HMGB1 in peripheral blood of ITP patients may induce Th17/Treg imbalance by promoting the maturation of mDC and affecting the secretion of cytokines, thereby potentially playing a role in the immunological mechanism of ITP.
Humans ; Th17 Cells/cytology* ; HMGB1 Protein/genetics* ; T-Lymphocytes, Regulatory/cytology* ; Female ; Male ; Dendritic Cells/metabolism* ; Adult ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic/genetics* ; Nuclear Receptor Subfamily 1, Group F, Member 3/genetics* ; Young Adult ; Interleukin-23/blood* ; Interleukin-17/blood* ; Interleukin-6/blood* ; Forkhead Transcription Factors/genetics* ; Myeloid Cells/cytology* ; Aged

Objective To investigate the impact of Qizhi Jiangtang Capsule (QZJT) on renal damage in diabetic nephropathy (DN) mice via NOD like receptors family pyrin domain containing 3/caspase-1/ Gasdermin D (NLRP3/caspase-1/GSDMD) signaling pathway. Methods Mice were randomly allocated into six experimental groups: a normal control group (NC), a diabetic nephropathy model group (DN), a low-dose QZJT treatment group (L-QZJT), a high-dose QZJT treatment group (H-QZJT), a positive control group administered Shenqi Jiangtang Granules (SQJT), and an ML385 group (treated with an inhibitor of nuclear factor erythroid 2-related factor 2, Nrf2). Upon successful model induction, therapeutic interventions were commenced. Renal function impairment in the mice was evaluated through quantification of fasting blood glucose (FBG), 24-hour urinary albumin (UAlb), serum creatinine (SCr), blood urea nitrogen (BUN), and the kidney-to-body mass ratio (K/B). Renal tissue pathology was evaluated using HE and PAS staining. Serum levels of inflammatory cytokines IL-1β and IL-18 were quantified by ELISA. Levels of podocyte markers and proteins involved in relevant pathways were assessed using Western blot analysis. Results Compared with the NC group, FBG, 24 h UAlb, SCr, and BUN were increased in the DN group, and the K/B mass ratio was also increased. In contrast, compared with the DN group, FBG, 24 h UAlb, SCr, and BUN in both the low-dose (L-QZJT) and high-dose Quanzhou Jintang (H-QZJT) groups were decreased, and the K/B mass ratio was decreased as well. The therapeutic efficacy of H-QZJT was comparable to that of Shenqi Jiangtang Granules. QZJT ameliorated renal histopathological injury in DN mouse, increased the protein levels of Nephrin (a podocyte marker), and decreased the protein levels of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), pro-caspase-1, and GSDMD-N. After ML385 treatment, renal cells exhibited swelling and morphological changes, the inflammatory infiltrate area was enlarged, the protein levels of NLRP3, ASC, pro-caspase-1, and GSDMD-N were up-regulated, and the levels of IL-1β and IL-18 were increased. Conclusion QZJT may inhibit podocyte pyroptosis by acting on the Nrf2 to regulate the NLRP3/caspase-1/GSDMD pathway, thus improving renal damage in DN mouse.
Animals ; Diabetic Nephropathies/pathology* ; Podocytes/pathology* ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Pyroptosis/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Caspase 1/genetics* ; Signal Transduction/drug effects* ; Mice ; Phosphate-Binding Proteins/genetics* ; Male ; Intracellular Signaling Peptides and Proteins/metabolism* ; Mice, Inbred C57BL ; Kidney/pathology* ; Gasdermins

Objective To explore the effects of miR-373 and Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signaling pathways on the M2 polarization of tumor associated macrophages (TAM) in rectal cancer. Methods THP-1 cells were induced into M0/M1/M2 macrophages, M0 macrophages were cocultured with Caco-2 cells to obtain TAM, Flow cytometry was used to detect the expression of CD86 and CD206, Real-time quantitative qPCR and Western blot were used to detect miR-373, inducible nitric oxide synthase (iNOS), toll-like receptor 4 (TLR-4), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), arginase 1 (Arg1), chitinase 3-like 1 (Ym1), resistin like α (Fizz1), IL-10 mRNA and protein levels. TAM were transfected and divided into overexpressing miR-373 group (miR-373-TAM) and control group (miR-NC-TAM), overexpressing miR-373+JAK2-TAM group (miR-373 combined with JAK2-TAM) and control group (miR-373 combined with NC-TAM), and then cocultured with Caco-2 cells. Flow cytometry was used to detect the expression of CD206 in TAM; Real-time quantitative PCR and Western blot were used to detect miR-373, Arg1, Ym1, Fizz1, IL-10, JAK2, STAT6 mRNA and protein levels in TAM; CCK-8 assay, colony formation assay, and Transwell assay were used to detect the proliferation, migration, and invasion ability of Caco-2 cells. Thirty nude mice were randomly divided into Caco-2 cells group, Caco-2 cells combined with miR-NC-TAM group, and Caco-2 cells combined with miR-373-TAM group, with 10 mice in each group. Rats in each group were subcutaneously injected with pure Caco-2 cells, Caco-2 cells combined with TAM, and Caco-2 cells combined with TAM overexpressing miR-373. After 4 weeks of cell inoculation, immunofluorescence staining was used to detect F4/80⁺CD206⁺cells level in tumor tissue; Real-time quantitative PCR and Western blot were used to detect miR-373, JAK2, STAT6, Arg1, Ym1, Fizz1, IL-10 mRNA and protein levels in tumor tissues. Results TAM tended to M2 polarization. After overexpression of miR-373, miR-373 level in TAM was increased, while Arg1, Ym1, Fizz1, IL-10, JAK2, STAT6 mRNA and protein levels were decreased, proliferation, migration, invasion ability of Caco-2 cells were decreased; Overexpression of JAK2 could partially reverse the effect of overexpression of miR-373 on the M2 polarization of TAM and proliferation, migration, invasion ability of Caco-2 cells. TAM could promote tumor growth; Overexpression of miR-373 could inhibit tumor growth and inhibit M2 polarization of TAM. Conclusion miR-373 could inhibit M2 polarization of TAM in rectal cancer, and miR-373 might inhibit proliferation and metastasis of rectal cancer cells by regulating the JAK2/STAT6 pathway.
MicroRNAs/metabolism* ; Humans ; STAT6 Transcription Factor/genetics* ; Signal Transduction/genetics* ; Animals ; Janus Kinase 2/genetics* ; Mice ; Tumor-Associated Macrophages/metabolism* ; Rectal Neoplasms/pathology* ; Caco-2 Cells ; Mice, Nude ; THP-1 Cells ; Mice, Inbred BALB C ; Cell Polarity ; Male

Objective To investigate the effects of exosomes (Exo) derived from high glucose-stimulated glomerular mesangial cells (GMC) on the kidneys of C57BL/6 mice and the intervention mechanism of Tongluo Yishen Formula (TLYSF). Methods The rat GMC were divided into a normal glucose group (NG, with 5.6 mmol/L glucose) and a high glucose group (HG, with 30 mmol/L glucose). After 24 hours of culture, the supernatant was collected, and exosomes were extracted using the ultracentrifugation method. The exosomes were then identified by transmission electron microscopy and Western blot analysis. Male C57BL/6 mice were divided into three groups: NO-Exo group, NG-Exo group, and HG-Exo group. These groups were respectively administered tail vein injections of PBS buffer, exosomes derived from GMC cultured in normal glucose, and exosomes derived from GMC cultured in high glucose, three times a week for a total of 8 weeks. After 8 weeks, the mice in the HG-Exo group were randomly divided into three subgroups: the HG-Exo group [gavaged with saline], the HG-Exo+TLYSF group [gavaged with TLYSF at 34.32 g/(kg.d)], and the HG-Exo + VAL group [gavaged with valsartan suspension at 10.4 mg/(kg.d)], and the intervention lasted for 4 weeks. Urinary microalbumin (mALb), urinary N-acetyl-β-D-aminoglucosidase (NAG), glycated hemoglobin (HbA1c), serum creatinine (Scr) and urea nitrogen (BUN) were detected. Transmission electron microscopy was used to observe the ultrastructure of renal tissues. TUNEL was used to detect the DNA damage of renal tissue cells. Immunofluorescence was used to detect the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and wilms tumor 1(WT-1). RT-PCR was used to detect the mRNA levels of NLRP3, cysteinyl aspartate-specific proteinase 1 (caspase-1), interleukin-1 beta (IL-1β), miR-200c-3p and miR-148a-3p. Western Blot was employed to detect the protein expression of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1 and IL-1β. Results Compared with the NG-Exo group, mice in the HG-Exo group exhibited significantly increased levels of mALb, urinary NAG, Scr and BUN. Transmission electron microscopy revealed ruptured podocyte membranes and swollen mitochondria. The positive rate of cells stained by the TUNEL increased, with elevated optical density of NLRP3 and decreased optical density of WT-1. Additionally, there was a significant increase in the level of NLRP3, caspase-1, IL-1β mRNA, as well as miR-200c-3p and miR-148a-3p. The protein expression of NLRP3, ASC, caspase-1, and IL-1β also increased. Compared with HG-Exo group, mice in the HG-Exo+TLYSF group showed decreased levels of mALb, urinary NAG, Scr, and BUN. The podocyte membranes were relatively intact, and mitochondrial damage was alleviated. The positive rate of cells stained by the TUNEL decreased, along with a reduction in the optical density of NLRP3 and an increase in the optical density of WT-1. Furthermore, the mRNA expression levels of NLRP3, caspase-1, IL-1β, miR-200c-3p, and miR-148a-3p were all downregulated to varying degrees. The protein expression levels of NLRP3, ASC, caspase-1, and IL-1β also decreased. Conclusion Exosomes derived from GMC stimulated by high glucose can damage the kidneys of mice and induce podocyte pyroptosis. TLYSF may ameliorate podocyte pyroptosis by downregulating the expression of exosomal miR-200c-3p and miR-148a-3p and inhibiting the activation of the NLRP3/ASC/caspase-1 pathway.
Animals ; Exosomes/ultrastructure* ; Glucose/pharmacology* ; Male ; Podocytes/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Mice, Inbred C57BL ; Mice ; Mesangial Cells/metabolism* ; Pyroptosis/drug effects* ; Rats ; MicroRNAs/genetics*

Objective To observe the effect of m⁶A methylation regulation on Notch1 pathway on the homing of BMSCs in asthma, and the intervention study of traditional Chinese medicine compound Yanghe Pingchuan Granules. Methods Rat bone mesenchymal stem cells(BMSC)and bronchial epithelial cells were cocultured. The extracted cells were divided into: bronchial epithelial cell group, asthma bronchial epithelial cell+mesenchymal stem cell co-culture group (co-culture group), co-culture cell+normal serum group, coculture cell+serum containing optimal drug group, siRNA FTO+normal serum group, siRNA FTO-NC+normal serum group, and siRNA FTO+serum containing optimal drug group. The vitality and cell cycle changes of co-cultured cells were detected. The level and markers of homing BMSC were detected by immunofluorescence staining. The expression of Notch1 pathway related genes were detected by qRT-PCR. The expression of Notch1 pathway related proteins were detected by Western blot. Results Compared with bronchial epithelial cell group, the co-cultured cell group showed an increase in the homing level of BMSCs and the expression of C-X-C motif chemokine receptor 4 (CXCR4), stromal cell-derived factor 1 (SDF-1), Notch1, transcription factor recombination signal binding protein-J (RBP-J), and hairy enhancer of split 1 (Hes1) proteins. Compared with the co-cultured cell group and co-cultured cell+normal serum group, the co-cultured cell+serum containing optimal drug group showed an increase in the homing level of BMSCs and the expressions of CXCR4 and SDF-1, while the protein and mRNA levels of Notch1 and Hes1 decreased. Compared with the siRNA FTO-NC+normal serum group, the siRNA FTO+normal serum group showed an increase in the levels of Notch1, activated Notch1, RBP-J, Hes1 protein, and cell viability, while the level of homing BMSC decreased. Compared with siRNA FTO+normal serum group, the levels of Notch1, RBP-J mRNA, activated Notch1, and Hes1 protein decreased, while the level of homing BMSCs increased in siRNA FTO+serum containing optimal drug group. The levels of Notch1, RBP-J, and Hes1 mRNA were reduced in the co-cultured cells+serum containing optimal drug group. Compared with siRNA FTO+serum containing optimal drug group, the expressions of Notch1, activated Notch1, RBP-J, Hes1 protein and cell viability decreased, while the level of homing BMSCs increased in the co-cultured cells+serum containing optimal drug group. Conclusion Yanghe Pingchuan Granules may promote the homing of BMSCs in asthma and alleviate asthma inflammation by upregulating the expression of FTO and inhibiting the expression of downstream genes in the Notch1 signaling pathway.
Animals ; Receptor, Notch1/genetics* ; Mesenchymal Stem Cells/cytology* ; Asthma/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Rats ; Coculture Techniques ; Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics* ; Epithelial Cells/metabolism* ; Rats, Sprague-Dawley ; Cells, Cultured ; Male