Hepatic stellate cells (HSCs) are the primary fibrogenic cells in the liver, and their activation plays a crucial role in the development and progression of hepatic fibrosis. Here, we report that retinoid X receptor-alpha (RXRα), a unique member of the nuclear receptor superfamily, is a key modulator of HSC activation and liver fibrosis. RXRα exerts its effects by modulating calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ)-mediated activation of AMP-activated protein kinase-alpha (AMPKα). In addition, we demonstrate that K-80003, which binds RXRα by a unique mechanism, effectively suppresses HSC activation, proliferation, and migration, thereby inhibiting liver fibrosis in the CCl₄ and amylin liver NASH (AMLN) diet animal models. The effect is mediated by AMPKα activation, promoting mitophagy in HSCs. Mechanistically, K-80003 activates AMPKα by inducing RXRα to form condensates with CaMKKβ and AMPKα via a two-phase process. The formation of RXRα condensates is driven by its N-terminal intrinsic disorder region and requires phosphorylation by CaMKKβ. Our results reveal a crucial role of RXRα in liver fibrosis regulation through modulating mitochondrial activities in HSCs. Furthermore, they suggest that K-80003 and related RXRα modulators hold promise as therapeutic agents for fibrosis-related diseases.

Objective To explore the underlying mechanisms by which time-restricted feeding(TRF)attenuates dextran sodium sulfate(DSS)-induced colitis in mice via modulation of regenerating islet-derived protein 3 gamma(Reg3γ)expression and gut microbiota.Methods Six-week-old C57BL/6 mice were stratified by body weight and randomized into ad libitum feeding(AL)and TRF groups(n=7).The AL mice were given unrestricted food access,whereas the TRF mice were allowed feeding only during 00:00 and 08:00 daily,for totally 4 weeks.Mouse colitis model was induced at the fourth week by adding 2.5%dextran sodium sulfate(DSS)in drinking water for 6 d.Disease severity and the effects of TRF were assessed with disease activity index(DAI)scoring,colon length measurement,HE staining and histopathological scoring,and mRNA expression levels of regenerating islet-derived 3 gamma(Reg3g)and inflammatory cytokines in colonic tissues.Another 14 mice were randomized into AL plus antibiotic cocktail(AL+ABX)and TRF plus antibiotic cocktail(TRF+ABX)groups,with 7 animals in each group.ABX was administered to deplete gut microbiota and evaluate the microbiota dependence of TRF in attenuating colitis.Fecal samples from AL and TRF mice were analyzed by 16S ribosomal RNA sequencing(16S rRNA-seq),and serum lipopolysaccharide(LPS)level was measured.The colonic epithelial cells were collected for RNA-seq.Results After modeling,the AL mice exhibited typical colitis symptoms,such as weight loss,diarrhea,and hematochezia.TRF intervention significantly attenuated these above symptoms,with lower DAI scores from day 4 post-modeling(P<0.001),reduced colon shortening(P<0.01),preserved tissue architecture,and decreased inflammation.RT-qPCR analysis showed that TRF down-regulated colonic mRNA expression levels of Reg3g and pro-inflammatory cytokines(IL-1 β,IL-6,TNF-α)(P<0.05)while up-regulated that of anti-inflammatory factor IL-10(P<0.000 1)when compared with the corresponding levels in AL mice.ABX treatment led no significant differences between the AL+ABX and TRF+ABX groups in term of DAI score,colon length,or histopathology.Obviously down-regulated Reg3g was observed in the TRF+ABX group than the AL+ABX group(P<0.05),whereas L-1β,IL-6,TNF-α and IL-10 showed no notable changes.16S rRNA-seq revealed that TRF markedly reshaped gut microbiota composition,with increased Gram-positive bacterial abundance,reduced Gram-negative bacteria,with concomitant lower serum LPS level(P<0.001).RNA-seq also indicated significant suppression of NF-κB and other inflammation-related signaling pathways in the TRF group.Conclusion TRF attenuates DSS-induced colitis in mice by downr-egulating Reg3γ expression,reshaping gut microbiota,and reducing serum LPS level,and thereby suppressing NF-κB-mediated inflammatory signaling pathways.

OBJECTIVE: To provide preimplantation genetic testing (PGT) for a patient with Incontinentia pigmenti (IP) due to IKBKG gene variant but without family samples through construction of single nucleotide polymorphism (SNP)-based haplotype by Long-read sequencing (LRS) technology. METHODS: A female IP patient with a heterozygous IKBKG c.1167dup variant but without family genetic data who sought genetic counseling at Women and Children' Hospital of Ningbo University in November 2021 was selected as the study subject. The IKBKG gene has a highly homologous pseudogene IKBKGP1. Genomic DNA was extracted from peripheral blood samples from the couple, and LRS was used to obtain informative SNP loci flanking the variant locus, enabling the construction of SNP haplotype with a long segment spanning from the non-homologous region of IKBKG to the variant site. Trophoblast cells were biopsied from blastocysts fertilized through intracytoplasmic sperm injection, and next-generation sequencing (NGS) was used to determine the SNP information of the embryos. Linkage analysis with the parental SNP haplotypes was conducted to detect the carrier status of the embryos and exclude chromosomal aneuploidies. Sanger sequencing was carried out to validate the result. A euploid embryo without the pathogenic variant was selected for transfer. Prenatal diagnosis was carried out by amniocentesis at mid-trimester to verify the result of PGT tests, and follow-up was conducted after the baby was born. This study has been approved by the Ethics Committee of Women and Children's Hospital of Ningbo University (Ethics No. EC2023-094). RESULTS: A total of seven blastocysts were tested, and PGT results indicated that two embryos were euploid and did not carry the pathogenic variant. One euploid embryo was transferred, which resulted in a singleton pregnancy. Amniocentesis at 24 weeks of gestation confirmed that the status of fetal IKBKG gene, and its chromosomal status was consistent with the PGT results. A healthy male infant was born at 38+6 weeks of gestation. CONCLUSION For IP patients with de novo mutation or without family genetic samples, PGT with LRS can directly construct the SNP-based haplotype while avoiding interference from pseudogenes, providing an effective strategy for PGT.
Female ; Humans ; Male ; Pregnancy ; Genetic Testing/methods* ; Haplotypes/genetics* ; High-Throughput Nucleotide Sequencing/methods* ; I-kappa B Kinase/genetics* ; Incontinentia Pigmenti/diagnosis* ; Polymorphism, Single Nucleotide/genetics* ; Preimplantation Diagnosis/methods* ; Infant, Newborn

OBJECTIVE: To investigate the clinical phenotypes and genetic variant characteristics in children with epilepsy. METHODS: A total of 91 children with epilepsy admitted to the Women's and Children's Hospital Affiliated to Ningbo University from July 2021 to October 2022 were selected as the study subjects. Peripheral blood samples were collected from the children for whole exome sequencing. Candidate genetic variants were validated by Sanger sequencing and copy number variation sequencing (CNV-seq). The clinical phenotypes and treatment outcomes of the children with epilepsy were followed up, and an analysis of the relationship between genotype and phenotype was conducted. This study was approved by the Women's and Children's Hospital Affiliated to Ningbo University (Ethics No.: EC2020-048). RESULTS: Among the 91 children with epilepsy, 21 cases (23.08%, 21/91) were found to carry pathogenic or likely pathogenic variants. Of these, 18 cases had involved single base variant or insertional deletion, while 3 cases involved copy number variations. The gene with the highest detection rate was PRRT2 (38.10%, 8/21). Among the children with genetic variants, 47.62% (10/21) had onset during infancy, with 8 diagnosed with Benign familial infantile epilepsy (BFIE), 8 with Developmental epileptic encephalopathy (DEE), and 3 with Epileptic encephalopathy (EE). One case of Dravet syndrome (DS) and one case of Infantile spasms (IS) were also noted. The clinical manifestations of children were diverse and primarily included generalized tonic-clonic seizures and focal seizures. Among them, 52.38% (11/21) had exhibited cluster seizures, 23.81% (5/21) showed fever sensitivity, and 14.29% (3/21) experienced status epilepticus. After pharmacological treatment, 42.86% (9/21) of children had achieved complete seizure control, while 61.90% (13/21) had intellectual disability and 19.05% (4/21) had co-morbid autism spectrum disorder. CONCLUSION Pathogenic or likely pathogenic variants were identified in 23.08% of the pediatric epilepsy cases, with the PRRT2 gene being the most frequently involved. Among children carrying genetic variants, 47.62% had seizure onset during infancy. Genetic factors are an important cause of epilepsy, and early genetic testing may facilitate precise diagnosis, treatment, and prognostic evaluation.
Humans ; Female ; Male ; Epilepsy/genetics* ; Child, Preschool ; Child ; Phenotype ; Genotype ; DNA Copy Number Variations/genetics* ; Infant ; Membrane Proteins/genetics* ; Nerve Tissue Proteins/genetics* ; Adolescent ; Exome Sequencing

Objective:To explore the molecular basis for a Chinese pedigree affected with Achromatopsia (ACHM).Methods:A pedigree with ACHM which was admitted to the Women and Children′s Hospital of Ningbo University on April 14, 2023 was selected as the study subject. Whole exome sequencing (WES) was carried out for the proband. Candidate variants were verified by Sanger sequencing and bioinformatic analysis. Related literature was reviewed, and clinical and genetic features of Chinese patients with ACHM due to variants of CNGA3 gene were summarized. This study was approved by the Women and Children′s Hospital of Ningbo University (Ethics No. EC2020-048). Results:WES revealed that the proband and his younger brother had both harbored compound heterozygous variants of the CNGA3 gene, namely c. 1190G>T (p.Gly397Val) and c. 2013del (p.Gly672ValfsTer69), which were respectively inherited from their mother and father. The c. 1190G>T was a known pathogenic variant, while the c.2013del was unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c. 2013del variant was predicted to be likely pathogenic (PM2_Supporting+ PVS1_Moderate+ PM3+ PP4). Literature review has identified 41 CNGA3 gene variants among 43 patients from 38 pedigrees, most of which were missense variants and had located in exon 8. Most patients were males, with nystagmus, photophobia, amblyopia and other symptoms during infancy/childhood as the main clinical manifestations, and there was a lack of genotype-phenotype correlation. Conclusion:The c. 1190G>T (p.Gly397Val) and c. 2013del (p.Gly672ValfsTer69) variants of the CNGA3 gene probably underlay the ACHM in the proband. Discovery of the c. 2013del variant has enriched the mutational spectrum of the CNGA3 gene and provided a basis for genetic counseling and reproduction guidance for this pedigree.

Objective:To investigate the clinical characteristics and prognostic factors of extracranial malignant rhabdoid tumors (eMRTs) in children.Methods:In this retrospective case series study, a retrospective analysis was conducted on clinical data of 21 eMRT patients admitted to Children′s Hospital of Nanjing Medical University from April 2018 to January 2023 and followed up until October 30, 2023.Patients were grouped according to their gender, age, tumor origin site, clinical staging, initial lactate dehydrogenase (LDH) level, extent of tumor resection, chemotherapy regimen, and radiotherapy.The Kaplan-Meier method was used to calculate the 2-year progression-free survival rate (PFS) and overall survival rate (OS) of the patients, and the Cox regression model was used to analyze the prognostic factors.Results:Among the 21 patients with eMRTs, there were 7 males and 14 females, with the age of onset of 24 (3-138) months.Immunohistochemistry showed that all tumor tissues of the patients did not secrete integrase interactor 1 (INI-1).Among them, 13 cases originated from the kidney, and 8 cases originated from extrarenal non-central sites.At the time of diagnosis, there were 4 cases in clinical stages Ⅰ-Ⅱ, 17 cases in stage Ⅲ-Ⅳ.Thirteen patients underwent complete tumor resection surgery, 7 underwent partial resection, and 1 only underwent biopsy.Among the 13 cases of renal rhabdoid tumors, 8 cases were treated with the AVDC (Epirubicin, Vincristine, Actinomycin D, Cyclophosphamide)/ICE (Ifosfamide, Carboplatin, Etoposide) regimen, and 5 cases were treated with the protocol for nephroblastoma; among the 8 cases of extrarenal non-central rhabdoid tumors, 5 cases were treated with the AVDC/ICE regimen, and 3 cases were treated with the commonly used protocol for soft tissue sarcoma.Thirteen patients received radiotherapy.One patient received consolidation therapy with autologous stem cell transplantation following chemotherapy and radiotherapy.As of October 2023, there were 14 survivors and 7 deaths.The overall 2-year PFS and OS were 56%(95% CI: 35.7%-88.5%) and 62%(95% CI: 43.2%-89.4%), respectively.Among the patients who received the AVDC/ICE alternating chemotherapy regimen, the 2-year PFS and OS were 73%(95% CI: 47.0%-100.0%) and 79% (95% CI: 56.4%-100.0%), respectively.Univariate Cox regression analysis showed that complete tumor resection, the AVDC/ICE alternating chemotherapy, and radiotherapy were associated with a better prognosis in children (all P≤0.05).Multivariate Cox regression analysis showed that whether to receive radiotherapy was an independent risk factor affecting the overall survival in children. Conclusions:eMRTs are more common in infants and young children, with high malignancy and invasiveness.There is currently no standard treatment.Complete tumor resection combined with the AVDC/ICE alternating chemotherapy and radiotherapy may improve the prognosis of children with eMRTs.

OBJECTIVE: To analyze the clinical manifestation and genetic basis for four children with delayed onset Ornithine transcarbamylase deficiency (OTCD). METHODS: Clinical data of four children with OTCD admitted to the Children's Hospital of the First Affiliated Hospital of Zhengzhou University from January 2020 to April 2021 were reviewed. Peripheral blood samples of the children and their parents were collected and subjected to whole exome sequencing (WES). Bioinformatic analysis and Sanger sequencing verification were carried out to verify the candidate variants. Impact of the candidate variants on the protein structure was also predicted. RESULTS: The clinical manifestations of the four children included vomiting, convulsion and disturbance of consciousness. WES revealed that the child 1 was heterozygous for a c.421C>T (p.R141X) variant in exon 5, children 2 and 3 were hemizygous for a c.119G>A (p.R40H) variant in exon 2, and child 4 was hemizygous for a c.607T>A (p.S203T) variant in exon 5 of the OTC gene. Among these, the c.607T>A variant was unreported previously and predicted to be pathogenic (PM1+PM2_Supporting+PP3+PP4). Bioinformatic analysis has predicted that the variant may result in breakage of hydrogen bonds and alter the protein structure and function. Sanger sequencing confirmed that the variants in children 2 to 4 have derived from their mothers. CONCLUSION The pathogenic variants of the OTC gene probably underlay the delayed OTCD in 4 children. The discovery of the c.607T>A variant has enriched the mutational spectrum of the OTC gene.
Child ; Humans ; Ornithine Carbamoyltransferase Deficiency Disease/genetics* ; Exons ; Seizures ; Computational Biology ; Heterozygote

OBJECTIVE: To explore the types of NF1 gene variants and clinical characteristics among patients with Neurofibromatosis type I (NF1). METHODS: Clinical data of 12 patients diagnosed at Ningbo Women and Children's Hospital between December 2019 and May 2022 were retrospectively analyzed. The probands and their family members were subjected to high-throughput sequencing, and candidate variants were verified by Sanger sequencing and chromosome microarray analysis. RESULTS: The 12 patients had ranged from 4 months to 27 years old, with a male-to-female ratio of 2 : 1. Cafè-au-lait spots were found in all patients. 83.3% of them also had axillary and/or inguinal freckling, 58.3% had neurofibromas, and 16.7% had congenital pseudarthrosis of the tibia. Five types of NF1 gene variants were identified in the patients, including 5 nonsense variants, 4 frameshift variants, 1 missense variant, 1 splice variant, 1 large deletion involving the whole gene. Six patients were found to harbor de novo variants, 2 had inherited the variants from their parents, and 4 were not verified for their parental origin. The c.3379del (p.Thr1127Glnfs*15) and c.6628_6629del (p.Glu2210Thrfs*10) variants were unreported in literature and databases. CONCLUSION Most NF1 patients may present with Cafè-au-lait spots initially and are due to pathogenic variant of the NF1 gene. High-throughput sequencing can efficiently identify such variants among the patients and enable the definite diagnosis.
Child ; Humans ; Female ; Male ; Neurofibromatosis 1/diagnosis* ; Cafe-au-Lait Spots/diagnosis* ; Genes, Neurofibromatosis 1 ; Retrospective Studies ; Frameshift Mutation

Triple-negative breast cancer (TNBC) represents a distinct subtype of breast cancer, characterized by unique clinical traits including early lung metastasis, elevated recurrence rates, and diminished survival prospects. Owing to the lack of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression, concrete therapeutic targets remain elusive, thereby confining available clinical treatment methods. In the context of advanced TNBC, chemotherapy remains the predominant therapeutic approach. In recent years, with the in-depth study of tumor microenvironment, new immunotherapy targets have been discovered one after another. Thus, immunotherapy-based combined therapy strategies have brought new hope in patients with advanced TNBC.

With the advances in understanding the relationships among biomaterials, the immune system and the skeletal system, the host responses elicited by implanted biomaterials can be balanced by properly designing material characteristics from the perspective of osteoimmunology. The immunoregulatory properties of bone tissue engineering scaffolds provide advantages for inducing macrophages from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype and promoting osseointegration. Hydrogels are increasingly a focus in bone tissue engineering, and the immune response can be affected by different compositions of hydrogels, such as the sources, concentration, molecular weight, coupling with fibronectin, and the addition of cross-linking agents. Different physicochemical properties of modified hydrogels can trigger different host immune responses, modified by using soft photolithography to fabricate micropatterned hydrogels, adding enzyme-sensitive sequences, ester bonds and dynamic covalent chemistry to prevent rapid or slow degradation of the hydrogels, and adding porogens and 3D printing to modify the hydrogels with macroporous interconnective pore structures, soft and injectable hydrogels, etc. These optimized hydrogels can reduce proinflammatory factors, promote M2 macrophage polarization, and minimize foreign body reactions to evoke bone regeneration. However, the mechanism underlying the bone immune response is still poorly understood, and further study of the effects of hydrogels with different physicochemical properties on immune regulation is needed.