Intrauterine adhesions (IUA), characterized by pathological endometrial fibrosis, represent a significant clinical challenge impairing female reproductive health and fertility. MicroRNA-29b (MiR-29b) serves as a negative regulatory factor within the fibrosis-related transforming growth factor-beta (TGF-beta)/Smad signaling pathway. In this study, we employed extracellular vesicles (EVs) as vehicles for the delivery of miR-29b to enhance its therapeutic efficacy in the treatment of IUA. Using HEK-293T cells overexpressing miR-29b (29b-OE) as a source of EVs, EVs overloaded with miR-29b (EV29b) were enriched with miR-29b via the endosomal sorting complex required for transport-dependent pathway associated with tetraspanin-mediated endocytosis. Notably, treatment with EV29b significantly promoted endometrial regeneration. Mechanistically, EV29b was found to target the 3 ' untranslated region (3 ' UTR) of TGFB2, thereby inhibiting the TGF-beta/Smad signaling pathway and promoting repair of endometrial damage in both cultured endometrial stromal cells (EndoSCs) and a rat model of IUA. Collectively, our findings indicate that miR-29b, when effectively sorted and loaded into EVs, can substantially mitigate endometrial fibrosis by suppressing the TGF-beta/Smad signaling pathway, thereby presenting a novel therapeutic strategy for the treatment of IUA.

Introduction Lupus nephritis (LN) exhibits substantial clinical and pathological heterogeneity. We aimed to define proteomics-based molecular subtypes of LN and identify candidate biomarkers for subtype discrimination. Methods We analysed formalin-fixed paraffin-embedded (FFPE) renal biopsy specimens from 292 patients with biopsy-proven LN from four tertiary hospitals using data-independent acquisition (DIA)-liquid chromatography–tandem mass spectrometry (LC-MS/MS) proteomics. Molecular subtypes were identified by non-negative matrix factorisation. Differential proteins, functional enrichment, immune pathway activity, protein–protein interaction networks and subtype-associated clinical/pathological features were evaluated. Extreme Gradient Boosting (XGBoost) with SHapley Additive exPlanations (SHAP) and Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression were used to identify key subtype-related features and derive a protein panel distinguishing proliferative (class III/IV) from membranous (class V) LN. Results Two stable molecular subtypes were identified, with 1002 differential proteins between them. Subtype_2 was enriched for interferon-related innate immunity, complement activation, phagocytosis-endocytosis-lysosome pathways and ribosome biogenesis/RNA metabolism, whereas Subtype_1 was characterised by keratinisation and epithelial structural remodelling. Subtype_2 was associated with higher serum creatinine, lower estimated glomerular filtration rate and higher chronicity index. APOL1 showed discriminatory value between subtypes, and serum ELISA demonstrated a consistent pattern with FFPE proteomic findings. A five-protein LASSO panel achieved an area under the curve of approximately 0.76 for distinguishing class III/IV from class V LN. Conclusion DIA-based proteomic profiling of FFPE renal biopsies identifies biologically and clinically relevant LN molecular subtypes and may support tissue-informed classification and risk stratification.

Investigating the correlation between micronucleus formation and male infertility has the potential to improve clinical diagnosis and deepen our understanding of pathological progression. Our study enrolled 2252 male patients whose semen was analyzed from March 2023 to July 2023. Their clinical data, including semen parameters and age, were also collected. Genetic analysis was used to determine whether the sex chromosome involved in male infertility was abnormal (including the increase, deletion, and translocation of the X and Y chromosomes), and subsequent semen analysis was conducted for clinical grouping purposes. The participants were categorized into five groups: normozoospermia, asthenozoospermia, oligozoospermia, oligoasthenozoospermia, and azoospermia. Patients were randomly selected for further study; 41 patients with normozoospermia were included in the control group and 117 patients with non-normozoospermia were included in the study group according to the proportions of all enrolled patients. Cytokinesis-block micronucleus (CBMN) screening was conducted through peripheral blood. Statistical analysis was used to determine the differences in micronuclei (MNi) among the groups and the relationships between MNi and clinical data. There was a significant increase in MNi in infertile men, including those with azoospermia, compared with normozoospermic patients, but there was no significant difference between the genetic and nongenetic groups in azoospermic men. The presence of MNi was associated with sperm concentration, progressive sperm motility, immotile spermatozoa, malformed spermatozoa, total sperm count, and total sperm motility. This study underscores the potential utility of MNi as a diagnostic tool and highlights the need for further research to elucidate the underlying mechanisms of male infertility.

BackgroundEffective intrauterine treatments for placental-mediated fetal growth restriction (FGR) remain limited, necessitating reliable protein biomarkers for early diagnosis and management.MethodsIn this study, we analyzed differential protein expression in peripheral blood plasma samples from 44 placental-mediated FGR patients and 44 normal pregnant women using the Olink-Explore-384-Inflammation panel. The analysis identified significant differences in protein expression levels, followed by enrichment analyses to explore the underlying biological mechanisms. Protein-protein interaction (PPI) network analysis and Least Absolute Shrinkage and Selection Operator (LASSO) modeling were used to identify key proteins as potential biomarkers.ResultsWe identified 225 proteins with significantly altered expression between FGR patients and normal pregnancies. Proteins such as Placental Growth Factor (PGF) and Hepatocyte Growth Factor (HGF) were previously found to be strongly associated with FGR. In addition, we discovered novel proteins potentially associated with FGR, including ESM1 and TIMP3. Enrichment analyses revealed that several pathways, including placental dysfunction, inflammatory responses, and oxidative stress, may play crucial roles in FGR pathophysiology. PPI network analysis further identified key proteins such as ANGPT2, CD40, and HGF, as potentially linked to FGR. LASSO modeling validated PGF and ESM1 as important biomarkers. Additionally, integrating a multi-protein panel with blood flow disruption analysis significantly improved diagnostic accuracy.ConclusionOur findings provide valuable insights into the molecular mechanisms of FGR, identifying key proteins as potential biomarkers. The multi-protein panel model offers a promising tool for early screening and diagnosis of FGR.

BACKGROUND:Single atrium and/or single ventricle (SA and/or SV) are severe congenital heart malformations linked to gene mutations, though the underlying genetic mechanisms remain unclear. This study seeks to elucidate these mechanisms. METHODS:Trio-whole exome sequencing (trio-WES) was performed to identify de novo mutations in 36 familial SA/SV cases. Identified genes were analyzed using Gene Ontology (GO), KEGG pathways, and protein-protein interaction (PPI) analyses. Pathogenicity predictions were performed using established databases, and key de novo mutations were validated via Sanger sequencing. Zebrafish morpholino (MO) knockdown experiments were utilized to assess the functional impact of candidate genes. RESULTS:Trio-WES analysis identified 183 candidate genes harboring de novo single nucleotide variants (SNVs), with 24 genes linked to congenital heart disease (CHD) and enriched in the heart morphogenesis signaling pathway. Screening through a deleterious variants database revealed ten de novo SNVs, eight of which were confirmed by Sanger sequencing. Subsequent screening using mouse, zebrafish, and cardiac single-cell databases highlighted two genes: CORIN and TTLL10. Zebrafish knockdown experiments showed cardiac defects and altered heart rate, supporting their involvement in SA/SV development. CONCLUSIONS:Our findings suggest that mutations in de novo genes play a critical role in cardiac development, offering insights into SA/SV mechanisms and potential diagnostic targets.

γ-globin genes (HBG1 and HBG2) are usually expressed during fetal life, and almost no expression after birth. Therefore, the reactivation of HBG is a key target for the treatment of hemoglobinopathy. ZFP64 is a C2H2 type zinc finger transcription factor, which has been shown to play an important role in the maintenance of gene expression in mixed lineage leukemia, and other C2H2 type zinc finger transcription factors (such as ZFP410 and ZFP644) have been shown to regulate the expression of fetal hemoglobin (HbF) in thalassemia. This study aims to investigate the effect of ZFP64 on mRNA expression of HBG. We performed bioinformatics analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks to identify genes and transcription factors associated with ZFP64. ZFP64 was knocked out in K562 and HUDEP-2 cell lines by CRISPR-Cas9 electroporation, and the transcription levels of ZFP64, HBB and HBG were analyzed. In undifferentiated and 7-day differentiated HUDEP-2 cells, knocking down ZFP64 resulted in a 1.5-fold and 2.5-fold increase in HBG mRNA expression, respectively (p < 0.05). These findings suggest that ZFP64 is a potential regulator of HBG expression and warrants further investigation as a therapeutic target in hemoglobinopathies.

IntroductionEpilepsy is a complex neurodegenerative disorder characterized by recurrent seizures, which poses significant challenges for clinical management and therapeutic development. Recent advances in stem cell biology have enabled the use of patient-specific induced pluripotent stem cells (iPSCs) as a novel in vitro model for studying human diseases, including neurological disorders. In particular, iPSCs offer a promising platform for investigating the molecular mechanisms of epilepsy and facilitating personalized medicine.MethodsIn this study, we generated iPSC lines from individuals diagnosed with epilepsy who carry a novel mutation in the CLCNKB gene. These patient-derived iPSCs were reprogrammed from somatic cells and subsequently characterized. To explore the molecular alterations associated with epilepsy, we performed transcriptomic profiling using RNA sequencing (RNA-seq). Differential gene expression analysis was conducted to compare the patient-derived iPSCs with control iPSC lines.ResultsThe RNA-seq analysis revealed a set of differentially expressed genes in epilepsy-derived iPSCs, with both upregulated and downregulated genes identified relative to controls. Several of these genes have previously been implicated in epilepsy-related phenotypes, as supported by published literature. This suggests a potential role for these genes in the pathophysiology of epilepsy linked to CLCNKB mutations.DiscussionThe successful establishment of CLCNKB-mutant patient-specific iPSC lines and their transcriptomic characterization provide a valuable tool for studying the molecular basis of epilepsy. These findings underscore the potential of iPSC-based models to advance our understanding of disease mechanisms and support the development of novel diagnostic and therapeutic strategies. Moreover, this study demonstrates the applicability of iPSCs for epilepsy research and their promise in the field of personalized medicine.

Background: Preeclampsia (PE) is a pregnancy-specific disease and hypertensive disorder with a multifactorial pathogenesis involving complex molecular regulatory networks. Recent studies highlight the critical role of non-coding RNAs, particularly miRNAs and lncRNAs, in PE development. This study investigates the molecular interaction between miR-7151-5p and the lncRNA KCNQ1OT1 and their functional contributions to PE pathogenesis. Methods: An integrative approach combining RNAhybrid-based bioinformatics, dual-luciferase reporter assays, qRT-PCR, Transwell migration and invasion assays, and RNA sequencing was employed to characterize the binding between miR-7151-5p and KCNQ1OT1 and assess their influence on trophoblast cell function and gene expression. Results: A bioinformatic analysis predicted a stable binding site between miR-7151-5p and KCNQ1OT1 (minimum free energy: –37.3 kcal/mol). The dual-luciferase reporter assay demonstrated that miR-7151-5p directly targets KCNQ1OT1, leading to suppressed transcriptional activity. In HTR8/SVneo cells, miR-7151-5p overexpression significantly downregulated both KCNQ1OT1 and Notch1 mRNA, whereas its inhibition showed no significant changes, suggesting additional regulatory mechanisms of Notch1 expression. Transwell assays indicated that miR-7151-5p overexpression suppressed trophoblast cell migration and invasion, whereas its inhibition enhanced these cellular behaviors. RNA-seq analysis further revealed that miR-7151-5p overexpression altered key signaling pathways, notably the TGF-β pathway, and significantly modulates PE-associated genes, including PLAC1, ANGPTL6, HIRA, GLA, HSF1, and BAG6. Conclusions: The regulatory effect of miR-7151-5p on KCNQ1OT1, along with its influence on trophoblast cell dynamics via Notch1 and TGF-β signaling pathways, highlights its role in PE pathogenesis and supports its potential as a biomarker in early PE screening.

β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are common genetic disorders remaining significant global health challenges due to their associated morbidity and mortality. Increasing fetal hemoglobin (HbF) levels has emerged as a promising therapeutic strategy for these disorders. In this study, we report Myc-associated zinc finger (MAZ) as an indirect repressor of γ-globin (HBG) expression in human erythroid cells. Depletion of MAZ in HUDEP-2 and patient-derived β-thalassemia cells leads to significant inductions of both HBG mRNA and protein levels, resulting in increased HbF percentages and HbF + erythroid cells. We demonstrate that MAZ occupies at the promoter of MYB, a well-known HBG repressor. MAZ depletion reduced MYB levels. Restoration of MYB re-silenced the HBG levels in MAZ depleted cells. Our findings uncover the MAZ-MYB axis in HBG regulation, highlighting MAZ as a potential target to enhance HbF levels in patients with hemoglobin disorders.

Introduction Dent disease type I is a rare X-linked recessive renal tubular disease resulting from pathogenic variants in the CLCN5 gene. Due to the rarity of Dent disease type I and the diversity of its phenotypes, its clinical diagnosis is complex and poses a challenge to clinicians. Methods A foetus and a child from a 36-year-old pregnant woman with a birth history of abnormal children were enrolled in this study. Pregnant women undergo amniocentesis for prenatal diagnosis at the gestational age of 12 + 3 weeks. Chromosomal microarray (CMA) analysis and whole-exome sequencing (WES) were employed to investigate the chromosomal copy number and single gene variants. Literature retrieval and data analysis were performed for genotype and phenotype collection analysis. Results No chromosomal abnormalities or CNVs were detected in the entire family through karyotype and familial CMA analyses. WES identified a nonsense pathogenic variant in CLCN5 of the X chromosome, c.1942 C > T (exon 11, NM_000084), which was inherited from his mother, who exhibited regular clinical features. Conclusion This study suggests that children with low-molecular-weight proteinuria and hypercalciuria should undergo prompt genetic testing to exclude Dent disease.

Human myeloid leukemia cells (such as K562) could be used for the study of erythropoiesis, and mature erythroid markers and globins could be induced during leukemia cell differentiation; however, the pathways involved are different compared with those of hematopoietic stem cells (HSCs). We identified the differentially expressed genes (DEGs) of K562 cells and HSCs associated with stem cells and erythroid differentiation. Furthermore, we showed that hemin-induced differentiation of K562 cells could be induced by serum starvation or treatment with the tyrosine kinase inhibitor saracatinib. However, erythroid differentiation of HSCs was inhibited by the deprivation of the important serum component erythropoietin (EPO) or treatment with saracatinib. Finally, we found that the mRNA expression of K562 cells and HSCs was different during saracatinib-treated erythroid differentiation, and the DEGs of K562 cells and HSCs associated with tyrosine-protein kinase were identified. These findings elucidated the cellular phenomenon of saracatinib induction during erythroid differentiation of K562 cells and HSCs, and the potential mechanism is the different mRNA expression profile of tyrosine-protein kinase in K562 cells and HSCs.

β-thalassemia (β-thal) is the most common monogenic disorder caused by various mutations in the human hemoglobin β (HBB) gene and affecting millions of people worldwide. Electroporation of Cas9 and single-guide RNA (sgRNA)–ribonucleoprotein (RNP) complex-mediated gene targeting in patient-derived hematopoietic stem cells (HSCs), followed by autologous transplantation, holds the promise to cure patients lacking a compatible bone marrow donor. In this study, a universal gene correction method was devised to achieve in situ correction of most types of HBB mutations by using validated CRISPR/sgRNA–RNP complexes and recombinant adeno-associated viral 6 (rAAV6) donor-mediated homology-directed repair (HDR) in HSCs. The gene-edited HSCs exhibited multi-lineage formation abilities, and the expression of β-globin transcripts was restored in differentiated erythroid cells. The method was applied to efficiently correct different mutations in β-thal patient-derived HSCs, and the edited HSCs retained the ability to engraft into the bone marrow of immunodeficient NOD-scid-IL2Rg−/− (NSI) mice. This study provides an efficient and safe approach for targeting HSCs by HDR at the HBB locus, which provides a potential therapeutic approach for treating other types of monogenic diseases in patient-specific HSCs.

Amniotic fluid derived mesenchymal stem cells (AFMSCs), shed along the fetal development, exhibit superior multipotency and immunomodulatory properties compared to MSCs derived from other somatic tissues (e.g., bone marrow and fat). However, AFMSCs display heterogeneity due to source ambiguity, making them an underutilized stem cells source for translational clinical trials. Consequently, there is an urgent need to identify a method to purify the AFMSCs for clinical use. We found that the AFMSCs can be categorized into three distinct groups: kidney-specific AFMSCs (AFMSCs-K), lung-specific AFMSCs (AFMSCs-L), and AFMSCs with an undefined tissue source (AFMSCs-X). This classification was based on tissue-specific gene expression pattern of single cell colony. Additionally, we observed that AFMSCs-X, a minority population within the AFMSCs, exhibited the highest multipotency, proliferation, resistance to senescence and immuno-modulation. Our results showed that AFMSCs-X significantly improved survival rates and reduced bacterial colony forming units (CFU) in cecal ligation and puncture (CLP)-induced septic mice. Therefore, our study introduces a novel classification method to enhance the consistency and efficacy of AFMSCs. These subpopulations, originating from different tissue source, may offer a valuable and innovative resource of cells for regenerative medicine purposes.

Virus receptors determine the tissue tropism of viruses and have a certain relationship with the clinical outcomes caused by viral infection, which is of great importance for the identification of virus receptors to understand the infection mechanism of viruses and to develop entry inhibitor. Proximity labeling (PL) is a new technique for studying protein-protein interactions, but it has not yet been applied to the identification of virus receptors or co-receptors. Here, we attempt to identify co-receptor of SARS-CoV-2 by employing TurboID-catalyzed PL. The membrane protein angiotensin-converting enzyme 2 (ACE2) was employed as a bait and conjugated to TurboID, and a A549 cell line with stable expression of ACE2-TurboID was constructed. SARS-CoV-2 pseudovirus were incubated with ACE2-TurboID stably expressed cell lines in the presence of biotin and ATP, which could initiate the catalytic activity of TurboID and tag adjacent endogenous proteins with biotin. Subsequently, the biotinylated proteins were harvested and identified by mass spectrometry. We identified a membrane protein, AXL, that has been functionally shown to mediate SARS-CoV-2 entry into host cells. Our data suggest that PL could be used to identify co-receptors for virus entry.

Spinocerebellar ataxia type 3 (SCA3) is a genetic degeneration disease of the nervous system with ataxia as the main clinical manifestation, and the most frequent subtype of SCA3 is known to be caused by CAG repeat expansions of more than 55 units in ATXN3. In this study, we used peripheral blood mononuclear cells (PBMCs) from a volunteer with 14/63 CAG repeats in ATXN3 to generate induced pluripotent stem cells (iPSCs), which will be a good model for studying SCA3.

Recurrent proximal 16p11.2 deletion (16p11.2del) is a risk factor for diverse neurodevelopmental disorders with incomplete penetrance and variable expressivity. Although investigation with human induced pluripotent stem cell models has confirmed disruption of neuronal development in 16p11.2del neuronal cells, which genes are responsible for abnormal cellular phenotypes and what determines the penetrance of neurodevelopmental abnormalities are unknown. We performed haplotype phasing of the 16p11.2 region in a 16p11.2del neurodevelopmental disorders cohort and generated human induced pluripotent stem cells for two 16p11.2del families with distinct residual haplotypes and variable neurodevelopmental disorder phenotypes. Using transcriptomic profiles and cellular phenotypes of the human induced pluripotent stem cell-differentiated cortex neuronal cells, we revealed MAPK3 to be a contributor to dysfunction in multiple pathways related to early neuronal development, with altered soma and electrophysiological properties in mature neuronal cells. Notably, MAPK3 expression in 16p11.2del neuronal cells varied on the basis of a 132 kb 58 single nucleotide polymorphism (SNP) residual haplotype, with the version composed entirely of minor alleles associated with reduced MAPK3 expression. Ten SNPs on the residual haplotype were mapped to enhancers of MAPK3. We functionally validated six of these SNPs by luciferase assay, implicating them in the residual haplotype-specific differences in MAPK3 expression via cis-regulation. Finally, the analysis of three different cohorts of 16p11.2del subjects showed that this minor residual haplotype is associated with neurodevelopmental disorder phenotypes in 16p11.2del carriers.

Liver iron overload is a common and serious organ injury in β -thalassemia patients. Ferroptosis has been shown to play a crucial role in the pathological injury in iron overloaded hepatocytes. In our study, we focused on the protective effects of baicalein, a natural, active flavone extracted from an herb used in traditional Chinese medicine against RSL3-induced ferroptosis in hepatocytes and high iron diet (HID) induced liver iron-overload in a murine β 654 -thalassemia model. In vitro , the effects of baicalein on RSL3-induced hepatocyte ferroptosis were examined by testing ferroptosis related genes, protein, and the GSH, MDA, iron ion level by RT-PCR, Western blot and the commercial kits respectively. The anti-iron overload injury effects of baicalein were assessed in a β 654 -thalassemia mouse model of high iron diet induced liver injury by the same method In vitro experiments. Here, we found that baicalein could reverse cell ferroptosis in hepatocytes treated with RSL3. Importantly, changes in the iron ion content, and MDA and GSH levels in β 654 -thalassemia mouse livers were significantly restored by baicalein. Mechanistically, baicalein may activated the NRF2 antioxidant pathway and increased the expression of GPX4 in vivo and In vitro . We concluded that baicalein is a potential therapeutic drug for the treatment of iron overload in β -thalassemia.

目的:了解广州地区育龄人群的地中海贫血(简称:地贫)基因携带率及基因型分布特征,分析地贫筛查的漏检和误诊原因,为育龄地贫基因携带者进行人工辅助受孕提供依据.方法:收集2019年1月到2021年12月在我院生殖医学科就诊并同时进行地贫筛查及地贫基因检测的育龄患者31455例,分析其地贫相关实验室检查结果,包括红细胞参数、红细胞渗透脆性、血红蛋白电泳及地贫基因检测,计算地贫基因携带率,并通过比较筛查和基因检测结果找出漏筛和误诊病例.结果:共检出育龄地贫基因携带者4455例,地贫基因携带率为14.16％.其中,α-地中海贫血3365例,常见的基因型有--SEA/αα、-α3.7/αα和-α4.2/αα;β-地中海贫血914例,常见的基因型有βCD41-42(-TCCT)/βN、βIVS-2-654(C→T)/βN和β-28(A-G)/βN;α-合并β-地中海贫血176例,最常见的基因型是βCD41-42(-TCCT)/βN/--SEA/αα.漏筛病例有731例,多为静止型α-地贫.误诊病例有4784例,其中有2701例误诊病例其红细胞渗透脆性和红细胞参数均正常,仅因HbA2＜2.5％且HbA＞97.5％被误诊为携带地贫基因.结论:广州地区的育龄人群具有较高的地贫基因携带率,静止型α-地贫容易在筛查中被漏检.