ObjectiveTo investigate the mechanism by which Mingshi prescription regulates the retinal melanopsin-dopamine （Opn4-DA） axis in myopic mice to inhibit endoplasmic reticulum （ER） stress in the retina and sclera， thereby delaying axial elongation associated with myopia. MethodsSixty 4-week-old male SPF-grade C57BL/6J mice were randomly divided into a normal group， a form-deprived myopia group （FDM group）， an intrinsically photosensitive retinal ganglion cells ablation group （ipRGCs group）， a Mingshi Prescription group （MSF group， 5.2 g·kg^-1）， and an ipRGCs + MSF group （5.2 g·kg^-1）. Except for the normal group， all other groups underwent FDM modeling. Additionally， the ipRGCs and ipRGCs + MSF groups received retinal ipRGC ablation. Three weeks after modeling， the MSF and ipRGCs + MSF groups were administered Mingshi prescription via continuous gavage for six weeks. After refraction and axial length were measured in all mice， eyeballs were collected along with retinal and scleral tissues. Pathological and morphological changes in the retina， choroid， and sclera were observed using periodic acid-Schiff （PAS） staining. Western blot was employed to detect the relative protein expression levels of dopamine D1 receptor （DRD1）， C/EBP homologous protein （CHOP）， and glucose-regulated protein 78 （GRP78） in the retina， and CHOP and GRP78 in the sclera. Real-time PCR was used to detect the relative mRNA expression of Opn4， CHOP， and GRP78 in the retina， and CHOP and GRP78 in the sclera. Immunofluorescence staining （IF） was performed to detect the expression of Opn4 and DRD1 in retinal tissues. ResultsCompared with the normal group， the FDM group showed a significant myopic shift in refraction （P<0.05） and a significant increase in axial length （P<0.05）. The retinal layers were thinner， the number of ganglion cells was reduced， and collagen fibers in the sclera were loosely arranged with evident gaps. Opn4 and DRD1 protein and mRNA expression in the retina were significantly decreased （P<0.05）， while CHOP and GRP78 protein and mRNA expression in both retinal and scleral tissues were significantly increased （P<0.05）. Compared with the FDM group， the ipRGCs group exhibited further increases in myopic refraction and axial length （P<0.05）， more pronounced thinning and looseness in the retinal， choroidal， and scleral layers， lower expression of Opn4 and DRD1 protein and mRNA in the retina （P<0.05）， and higher expression of CHOP and GRP78 protein and mRNA in the retina and sclera （P<0.05）. Compared with the FDM group， the MSF group showed significantly reduced refractive error and axial length （P<0.05）， with improved cellular number， arrangement， and thickness in ocular tissues， increased Opn4 and DRD1 protein and mRNA expression in the retina （P<0.05）， and reduced CHOP and GRP78 protein and mRNA expression in both retina and sclera （P<0.05）. Similarly， the ipRGCs + MSF group showed significant improvements in terms of the above items compared with the ipRGCs group （P<0.05）. ConclusionMingshi Prescription delays myopic axial elongation and refractive progression by regulating the Opn4-DA axis in the retina of myopic mice， thereby inhibiting ER stress in the retina and sclera. This intervention promotes Qi and blood nourishment of the eyes， softens the fascia， and restores ocular rhythm.

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′s 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, and follow-up was conducted after the baby was born. This study has been approved by the Ethics Committee of the Women and Children′s Hospital of Ningbo University (Ethics No. EC2023-094). Results:In total 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 samples, PGT with LRS can directly construct the SNP-based haplotype while avoiding interference from pseudogenes, providing an effective strategy for PGT.

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 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′s 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, and follow-up was conducted after the baby was born. This study has been approved by the Ethics Committee of the Women and Children′s Hospital of Ningbo University (Ethics No. EC2023-094). Results:In total 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 samples, PGT with LRS can directly construct the SNP-based haplotype while avoiding interference from pseudogenes, providing an effective strategy for PGT.

Ameloblastoma (AM) is a benign odontogenic tumor with unknown etiology. It is prone to recurrence and has a potential for malignant transformation. Patients often show high rates of relapse after curettage, or suffer from structural and functional damage of jaw after partial resection. Whole-genome sequencing data revealed that BRAF mutations and SMO mutations were common and likely to be mutually exclusive in AM. It was also reported that BRAF inhibitors were effective in several patients carrying BRAF V600E mutation. However, reliable preclinical models are urgently needed for exploring targeted therapy as it′s so difficult to conduct large clinical trials in this tumor. Patient-derived cell models in vitro and xenograft models in vivo are frequently used preclinical models. In fact, benign tumor cells generally showed a finite proliferative capacity in two-dimensional culture, and most likely, they could exhibit altered cellular phenotype after immortalization. Moreover, this benign tumor presented low chances of subcutaneous engraftment in nude mice. Accordingly, humanized mouse xenograft model needs more exploration. Yet, it is worth mentioning that a three-dimensional organoid model presents a high potential in culturing stem-cell-like epithelial cells in AM, and it would further be used in recapitulating corresponding tumors and developing targeted medicines. In this paper, we review research progress in preclinical models and the genetic variations of AM, and raise drug screening prospect of the current organoid models, which may pave the way for the possible personalized medicine in AM.

Long non-coding RNA (lncRNA) is a class of highly conserved transcript with a length of more than 200 nucleotides, which is of great significance for the occurrence, development, diagnosis and treatment of malignant tumors. The abnormal expression of linc01410 in malignant tumors can affect the occurrence and development of malignant tumors by regulating the biological processes such as proliferation, migration and epithelial-mesenchymal transformation of malignant tumor cells, acting on related signaling pathways such as nuclear factor-κB and Notch or through exosome pathways.

Objective:To investigate the factors affecting phenotypes in the patients of methylmalonic acidemia combined with homocysteinemia cblC type with MMACHC c. 609G>A homologous variant. Methods:A retrospective study on the clinical manifestations, complications, treatment, and outcome in 164patients of cblC type with MMACHC c. 609G>A homologous variant was conducted.The patients were diagnosed by biochemical and genetic analysisfrom January 1998 to December 2020. Results:Among the 164 patients, 2 cases were prenatally diagnosed and began treatment after birth. They are 3 and 12 years old with normal physical and mental development. Twenty-one cases were diagnosed by newborn screening. Among them, 15 cases had with normal development. They were treated fromthe age of two weeks at the asymptomatic period. Six cases began treatment aged 1 to 3 months after onset. Their development was delayed. One hundred and forty-one cases were clinically diagnosed. Their onset age ranges from a few minutes after birth to 6 years old. 110 cases had early-onset (78.0%). 31 cases had late-onset (22.0%). Five of them died. 24 patients lost to follow-up. Of the 141 clinically diagnosed patients, 130 (92.2%) with psychomotor retardation, 69 (48.9%) with epilepsy, 39 (27.7%) with anemia, 30 (21.3%) had visual impairment, 27 (19.1%) had hydrocephalus, 26 (18.4%) had feeding difficulties, 7 (5.0%) with liver damage, and 5 (3.5%) with metabolic syndrome. The frequency of hydrocephalus and seizures was significantly higher in the early-onset group. The urinary methylmalonic acid increased significantly in the patients with epilepsy. During the long-term follow-up, the level of plasma total homocysteine in the seizure-uncontrolled group was significantly higher than that in the seizure-controlled group, the difference had a statistical significance ( P<0.05). Conclusion:Most of the patients with MMACHC c. 609G>A homozygous variant had early-onset disease, with a high mortality and disability rate. If not treated in time, it will lead to neurological damage, resulting in epilepsy, mental retardation, hydrocephalus, and multiple organ damage. Pre-symptomatic diagnosis and treatment are crucial to prevent irreversible neurological damage. Neonatal screening and prenatal diagnosis are important to improve the outcome of the patients.

Objective:To determine the effect of miR-122-5p on microglia polarization, apoptosis and inflammation after traumatic brain injury (TBI).Methods:A mouse model and an in vitro TBI model were established. Astrocytes were stimulated to synthesize and release exosomes by brain extracts. microRNA microarray analysis was used to analyze the significantly altered microRNAs. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was applied to detect the expression of miR-122-5p in the in vivo and in vitro TBI model. TUNEL, immunofluorescence, and Western blot were performed to detect the effects of miR-122-5p inhibitors on microglia apoptosis, microglia M1/M2 phenotype transformation and the activation of NLRP3 inflammasome pathway and the phosphorylation of NF-κB after TBI.Results:The results of microRNA microarray analysis showed that 83 miRNAs were downregulated significantly (altered more than 2 folds, P < 0.05), among which miR-122-5p was significantly down-regulated ( P < 0.01). Expression of miR-122-5p was significantly decreased in the in vivo and in vitro TBI model [(1.00±0.00) vs. (0.41±0.15), P < 0.001; (1.00±0.00) vs. (0.34±0.07), P < 0.001]. TUNEL and immunofluorescence showed that miR-122-5p inhibitor significantly alleviated microglia apoptosis[(8.03±1.30) vs. (3.17±0.34), P < 0.001] and promoted microglia M1→M2 phenotype transformation ,M1 phenotype polarization was reduced [(56.96±13.70) vs. (34.70±3.47), P =0.002] and M2 phenotype polarization was increased [(30.46±3.67) vs. (40.74±2.49), P =0.005]. Western blot showed that NLRP3 inflammasome activation was inhibited and NF-κB phosphorylation was decreased when miR-122-5p was downregulated[(0.77±0.10) vs. (0.51±0.11), P =0.02; (0.73±0.08) vs. (0.50±0.07), P =0.003]. Conclusions:miR-122-5p is downregulated in microglia and exosomes secreted by astrocytes after TBI. miR-122-5p inhibitor can attenuate the microglia inflammatory response after TBI by inhibiting the activation of NLRP3 inflammasome pathway and the phosphorylation of NF-κB, promoting the microglia M1→M2 phenotypic transformation and reducing microglia apoptosis, thereby reducing the microglia inflammatory injury after TBI.

Transcatheter aortic valve replacement (TAVR) has become the preferred treatment for severe aortic stenosis. The localization and anchor of many transcatheter heart valves available in the clinic today are dependent on the calcific aortic valve leaflet of patients. We reported here a successful case of transapical aortic valve implantation with Ken-Valve heart valve in an 82-year-old male patient with pure severe aortic regurgitation without native valve calcium. Postoperative follow-up (3 months after the surgery) showed that the cardiac function significantly improved. The echocardiography indicated that the Ken-Valve prosthesis worked well without perivalvular regurgitation. The short-term clinical effect was satisfactory. The Ken-Valve with three position anchors is proved to be suitable for the treatment of pure aortic regurgitation.

Transcatheter aortic valve replacement (TAVR) for severe aortic stenosis is growing rapidly. The use of new heart valves prosthesis has improved surgical safety and efficacy. This report described a 72-year-old male patient with severe aortic stenosis combined with severe aortic regurgitation, who was evaluated at moderate-high risk of surgery and received a transapical TAVR using the Ken-Valve heart valve. The transcatheter operation time was 8 min, and the blood loss was 50 mL. The tracheal intubation was removed immediately after the surgery. Transesophageal echocardiography on the 4th postoperative day showed that the aortic valve leaflets worked well, and there was no valve orifice and paravalvular leakage. The patient was discharged on the 5th day after the surgery without complications. Transapical TAVR using Ken-Valve was an easy surgical procedure for aortic valve disease, and had short operation time.