Objective: To investigate the correlation between color parameters (L^*, a^*, b^*, Eab^*) and the contents of reducing sugars, total polysaccharides, total oligosaccharides, as well as four saccharides (fructose, glucose, sucrose, and kestose) during the nine cycles of steaming and sun-drying processing of Polygonatum kingianum, and to preliminarily explore the optimal processing duration.
Methods: The color changes were objectively evaluated using a colorimeter. The anthrone-sulfuric acid method was employed to determine total polysaccharides and oligosaccharides. The 3,5-dinitrosalicylic acid (DNS) colorimetric method was used to measure total reducing sugar content. High-performance liquid chromatography coupled with charged aerosol detection (HPLC-CAD) was applied for quantitative analysis of fructose, glucose, sucrose, and kestose. Multivariate statistical analysis was conducted to assess samples from different processing stages.
Results: Significant variations in color and component contents were observed across processing stages. The herbal pieces progressively darkened with increased processing cycles: brightness (L^*) and total color difference (Eab^*) initially decreased then stabilized, while a^* (red-green) and b^* (yellow-blue) values first increased then declined. Total polysaccharides and oligosaccharides showed overall decreasing trends, whereas reducing sugars initially increased before stabilizing. Fructose and glucose levels rose continuously, while sucrose and kestose decreased progressively, becoming undetectable after five cycles.
Conclusion: The chromatic alteration and saccharide composition of P. kingianum showed significant correlation with processing duration. Both total color difference (Eab^*) and reducing sugar content stabilized after four processing cycles (12 hours), suggesting that four cycles of steaming and sun-drying may represent the optimal processing duration.

Accurate timing of myelination is crucial for the proper functioning of the central nervous system. Here, we identified a de novo heterozygous mutation in TMEM63A (c.1894G>A; p. Ala632Thr) in a 7-year-old boy exhibiting hypomyelination. A Ca²⁺ influx assay suggested that this is a loss-of-function mutation. To explore how TMEM63A deficiency causes hypomyelination, we generated Tmem63a knockout mice. Genetic deletion of TMEM63A resulted in hypomyelination at postnatal day 14 (P14) arising from impaired differentiation of oligodendrocyte precursor cells (OPCs). Notably, the myelin dysplasia was transient, returning to normal levels by P28. Primary cultures of Tmem63a^-/- OPCs presented delayed differentiation. Lentivirus-based expression of TMEM63A but not TMEM63A_A632T rescued the differentiation of Tmem63a^-/- OPCs in vitro and myelination in Tmem63a^-/- mice. These data thus support the conclusion that the mutation in TMEM63A is the pathogenesis of the hypomyelination in the patient. Our study further demonstrated that TMEM63A-mediated Ca²⁺ influx plays critical roles in the early development of myelin and oligodendrocyte differentiation.
Animals ; Cell Differentiation/physiology* ; Oligodendroglia/metabolism* ; Mice, Knockout ; Mice ; Male ; Myelin Sheath/metabolism* ; Humans ; Child ; Cells, Cultured ; Oligodendrocyte Precursor Cells/metabolism*

The angiogenic response is essential for the repair of ischemic brain tissue. Integrin α6 (Itga6) expression has been shown to increase under hypoxic conditions and is expressed exclusively in vascular structures; however, its role in post-ischemic angiogenesis remains poorly understood. In this study, we demonstrate that mice with endothelial cell-specific knockout of Itga6 exhibit reduced neovascularization, reduced pericyte coverage on microvessels, and accelerated breakdown of microvascular integrity in the peri-infarct area. In vitro, endothelial cells with ITGA6 knockdown display reduced proliferation, migration, and tube-formation. Mechanistically, we demonstrated that ITGA6 regulates post-stroke angiogenesis through the PI3K/Akt-eNOS-VEGFA axis. Importantly, the specific overexpression of Itga6 in endothelial cells significantly enhanced neovascularization and enhanced the integrity of microvessels, leading to improved functional recovery. Our results suggest that endothelial cell Itga6 plays a crucial role in key steps of post-stroke angiogenesis, and may represent a promising therapeutic target for promoting recovery after stroke.
Animals ; Nitric Oxide Synthase Type III/metabolism* ; Mice ; Proto-Oncogene Proteins c-akt/metabolism* ; Integrin alpha6/genetics* ; Endothelial Cells/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Stroke/pathology* ; Vascular Remodeling/physiology* ; Vascular Endothelial Growth Factor A/metabolism* ; Mice, Knockout ; Signal Transduction/physiology* ; Mice, Inbred C57BL ; Male ; Neovascularization, Physiologic/physiology*

This paper explores the impacts of accelerated rehabilitation protocols following anterior cruciate ligament reconstruction(ACLR)on bone tunnel enlargement(BTE).While accelerated rehabilitation can shorten the recovery time and improve the knee function,it may increase the risk of BTE.In the early rehabilitation phase after ACLR,excessive early weight-bearing and rapid progression of knee flexion angles should be avoided,along with the proper use of braces.Continuous passive motion is not recommended in the early phase post-ACLR to prevent potential effects on BTE.Further research is needed to investigate the mechanisms of BTE and develop more effective rehabilitation strategies.This will help to select appropriate rehabilitation protocols for patients and balance functional recovery with the risk of BTE,thereby reducing the revision rate and improving postoperative outcomes.
Humans ; Anterior Cruciate Ligament Reconstruction/rehabilitation*

Objective:To develop a validation method for microbial targeted next generation sequencing (tNGS) detection for respiratorypathogens, and to evaluate the performance of the pathogen-targeted high-throughput sequencing test implemented in local hospital.Methods:Cross-sectional study. A total of 14 patients with severe pulmonary infections were admitted to Huangshi Central Hospital from December 2023 to January 2024. Samples were collected as follows:Bronchoalveolar lavage fluid (BALF) samples ( n=7) subjected to culture, fluorescent PCR, and tNGS testing. Sputum samples ( n=2) analyzed via sputum culture, fluorescent PCR, and tNGS. Throat swab samples ( n=5) tested using fluorescent PCR-capillary electrophoresis and tNGS. Reference samples were prepared using representative species such as Influenza A virus, Adenovirus C, Klebsiella pneumoniae, Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans. Jurkat cells at different concentrations were used as a source of human cells. Traditional detection methods such as fluorescent PCR-capillary electrophoresis and culture methods were used as reference methods. The detection performance of tNGS was evaluated by assessing the detection limit, precision, human cell impact, stability, cross-reactivity, and accuracy of metagenomic next-generation sequencing for pathogen detection. Results:The detection limits for Klebsiella pneumoniae, Human Adenovirus C, and Influenza A virus were 2×10 2 copies/ml, and for Aspergillus fumigatus, Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans, the detection limits were 4×10 2 copies/ml. The consistency rate of repeated detection results for all pathogens in the reference samples was 100%. The impact assessment experiment of human cells showed that when the concentration of Jurkat cells reached 1×10 6 cells/ml, Influenza A virus, Adenovirus C, Klebsiella pneumoniae, and Aspergillus fumigatus could all be detected. Stability experiments showed that there was no significant change in the number of pathogen sequences after the specimens were stored at 4 ℃ and -20 ℃ for 1 day, 4 days, and 7 days, respectively. Cross-reactivity experiments showed that when the concentration ratios of Staphylococcus aureus, Staphylococcus epidermidis, Candida parapsilosis, and Candida albicans were (5∶1∶1∶5), (1∶5∶5∶1), and (1∶1∶1∶1), respectively, the detection rate of closely related microbial species was 3/3. Accuracy assessment showed that the accuracy of 19 clinical specimens was 18/19 cases. Conclusion:Compared with traditional detection methods as the reference, tNGS demonstrates high sensitivity and a high positive concordance rate, underscoring its significant clinical value in the detection of respiratory pathogens.

Objective:To explore the efficacy of non-invasive prenatal testing (NIPT) of fetal free DNA in maternal peripheral blood in fetuses with increased nuchal translucency (NT).Methods:A retrospective analysis was conducted on 1 184 singleton pregnant women that underwent chromosomal microarray analysis (CMA) at Nanjing Drum Tower Hospital, Nanjing University Medical School from June 2014 to December 2022 due to fetal increased NT (≥3.0 mm). These subjects were categorized based on whether the increased NT was accompanied by other high-risk factors into isolated increased NT without advanced maternal age (further subdivided into 3.0 mm≤NT<3.5 mm, 3.5 mm≤NT<4.0 mm, and NT≥4.0 mm subgroups), isolated increased NT with advanced maternal age, increased NT with nasal bone abnormalities, increased NT with other soft markers, and increased NT with structural abnormalities groups. Assuming the sensitivity and specificity of NIPT and expanded NIPT at this center were both 100%, genomic abnormalities outside the detection range of NIPT or expanded NIPT were termed as residual risk of NIPT or expanded NIPT. Chi-square test and Bonferroni correction were used to compare the residual risks of NIPT and expanded NIPT among the three subgroups of isolated increased NT without advanced maternal age group. Results:(1) In the group of isolated increased NT without advanced maternal age: For the 3.0 mm≤NT<3.5 mm subgroup (329 cases), 19 abnormalities were detected by CMA [12 cases of chromosome aneuploidy, seven cases of pathogenic copy number variation (pCNV)], with residual risks of NIPT and expanded NIPT both at 2.1% (7/329). For the 3.5 mm≤NT<4.0 mm subgroup (173 cases), 29 abnormalities were detected by CMA (17 cases of chromosome aneuploidy, nine cases of pCNV, three cases of chromosome unbalanced translocation), with residual risks of NIPT at 8.1% (14/173) and expanded NIPT at 7.5% (13/173). For the NT≥4.0 mm subgroup (270 cases), CMA detected abnormalities in 70 cases (50 cases of chromosome aneuploidy, 16 cases of pCNV, three cases of unbalanced translocations, and one case of sex chromosome abnormality combined with pCNV). The residual risk of NIPT was 12.2% (33/270), and the residual risk of expanded NIPT was 7.0% (19/270). The residual risks of NIPT and expanded NIPT in the 3.0 mm≤NT<3.5 mm subgroup were lower than those in the 3.5 mm≤NT<4.0 mm and NT≥4.0 mm subgroups (Bonferroni correction, all P<0.017). (2) In the group of 92 cases with isolated increased NT and advanced maternal age, CMA detected abnormalities in 36 cases (29 cases of chromosome aneuploidy, five cases of pCNV, one case of trisomy 21 combined with sex chromosome abnormality, and one case of trisomy 18 combined with sex chromosome abnormality). The residual risk of NIPT was 7.6% (7/92), and that of expanded NIPT was 5.4% (5/92). (3) In the group of 49 cases with increased NT combined with nasal bone abnormalities, CMA detected abnormalities in 24 cases (23 cases of chromosome aneuploidy and one case of pCNV). The residual risks of NIPT and expanded NIPT were both 2.0% (1/49). (4) In the group of 26 cases with increased NT combined with other soft markers, CMA detected abnormalities in nine cases (six cases of chromosome aneuploidy, one case of pCNV, and two cases of chromosome unbalanced translocations). The residual risks of NIPT and expanded NIPT were both 11.5% (3/26). (5) In the group of 245 cases with increased NT combined with structural abnormalities, CMA detected abnormalities in 121 cases (107 cases of chromosome aneuploidy, seven cases of pCNV, four cases of chromosome unbalanced translocations, one case of trisomy 21 combined with trisomy 20, and two cases of trisomy 18 combined with sex chromosome abnormalities). The residual risk of NIPT was 16.7% (41/245), and that of expanded NIPT was 4.1% (10/245). Conclusions:For isolated NT≥3.5 mm or NT≥3.0 mm combined with other high-risk factors, chorionic villus sampling in early pregnancy can be recommended, advancing the timing of prenatal diagnosis from the second trimester to the first trimester. For fetuses with isolated 3.0 mm≤NT<3.5 mm, the 2.1% residual risk of chromosomal abnormalities should be fully informed during counseling, even if the risk of NIPT is low.

OBJECTIVE: To explore the genotype-phenotype relationship in a child with Opitz G/BBB syndrome (OS) with mild clinical phenotype. METHODS: A child with motor developmental delay as the initial symptom admitted to Xi'an Children's Hospital on June 10, 2021 was selected for this study. Clinical data were collected, and peripheral blood samples were obtained from the child and his mother. Whole exome sequencing (WES) was performed to identify genetic variant in the child. Candidate variant were verified by Sanger sequencing to assess inheritance patterns and pathogenicity. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (WB) analyses were conducted to evaluate the effects of the variant on mRNA and protein expression, respectively, using recombinant expression plasmids generated in vitro. This study was approved by the Medical Ethics Committee of Xi'an Children's Hospital (Ethics No. 20240045). RESULTS: The child, a 9-month-and-7-day-old boy, presented with a low nasal bridge, hypertelorism, and difficulty sitting independently. Echocardiography revealed an atrial septal defect. WES identified a homozygous variant in the MIDI gene, c.1483C>T (p.R495X), which was confirmed by Sanger sequencing and found to be inherited from the mother.Recombinant expression plasmids were successfully constructed. RT-qPCR analysis showed that the variant significantly reduced MIDI gene mRNA expression, while WB results indicated that the variant led to the production of a truncated protein. CONCLUSION The mild clinical phenotype of OS in this child may be attributed to the mRNA degradation escape mechanism induced by the nonsense variant c.1483C>T (p.R495X) in the MIDI gene. These findings provide valuable diagnostic insights for this pedigree and contribute to the understanding of the genotype-phenotype correlation in OS.
Humans ; Male ; Infant ; Transcription Factors/metabolism* ; Microtubule Proteins/genetics* ; Craniosynostoses/genetics* ; Hypospadias/genetics* ; Codon, Nonsense/genetics* ; RNA, Messenger/metabolism* ; Female ; RNA Stability/genetics* ; Phenotype ; Nuclear Proteins/genetics* ; Ubiquitin-Protein Ligases ; Esophagus/abnormalities* ; Hypertelorism

OBJECTIVE: To investigate the clinical phenotype and genetic etiology of a patient with tall stature and primary amenorrhea presenting with 47,XYY Disorder of sex development (DSD). METHODS: A female patient presenting with "tall stature and primary amenorrhea" at Nanjing Drum Tower Hospital in July 2024 was selected as the study subject. A retrospective study design was employed to collect the patient's clinical data. Peripheral venous blood sample was collected. Following the extraction of genomic DNA, genetic testing was performed including chromosomal karyotyping analysis, copy number variation sequencing (CNV-seq), multiplex PCR for the AZF regions and sex-determining genes Y (SRY), and whole-exome sequencing (WES). Candidate variants were validated by Sanger sequencing and classified for pathogenicity based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). This study was approved by the Medical Ethics Committee of Nanjing Drum Tower Hospital (Ethics No.: 2022-451-01). RESULTS: The patient had a height of 188 cm and a body weight of 50 kg, in addition with infantile uterus, absent ovaries, and primary amenorrhea. G-banded karyotyping analysis of peripheral blood sample revealed 47,XYY. CNV-seq indicated Seq[GRCh37]Yp11.32q12×2. No deletion was detected in the AZF regions of Y chromosome, and SRY was positive. WES identified a heterozygous c.86C>A (p.Thr29Lys) variant of the NR5A1 gene, leading to substitution of threonine with lysine at position 29 of the encoded protein. Sanger sequencing confirmed the presence of the variant. According to the ACMG guidelines, this variant was classified as variant of uncertain significance (VUS) with supporting evidence (PS3_Moderate+PM5+PP3+PM2_Supporting+PS4_Supporting). Reviewing the nearly 60 years of previously reported cases, all 7 documented 47,XYY DSD patients were assigned a female social gender and presented with abnormal gonadal and external genitalia development. Among them, 5 cases underwent SRY testing, all of which were positive. Only 1 case underwent whole-exome sequencing (WES), but no pathogenic or likely pathogenic variants were identified. CONCLUSION This DSD patient presented with the clinical features of tall stature and primary amenorrhea. The NR5A1 gene variant c.86C>A (p.Thr29Lys) probably underlay the Disorder of sex development in this patient. Above finding has enriched the spectrum of pathogenic variants of the NR5A1 gene.
Humans ; Female ; Steroidogenic Factor 1/genetics* ; DNA Copy Number Variations/genetics* ; XYY Karyotype/genetics* ; Karyotyping ; Retrospective Studies ; Phenotype ; Sex Chromosome Disorders of Sex Development/genetics* ; Sex Chromosome Disorders

OBJECTIVE: To explore the genetic etiology of a Chinese pedigree affected with Primary ciliary dyskinesia (PCD). METHODS: A child who presented at the ENT Department of Zhengzhou University Children's Hospital in March 2024 due to secretory otitis media, chronic sinusitis, adenoid hypertrophy, dextrocardia, and bronchiectasis was selected as study subject. Relevant clinical data were collected. Peripheral blood samples from the child and her family members were collected. Following DNA extraction, whole exome sequencing was carried out. Candidate variants were validated by Sanger sequencing, and the correlation between the variants and phenotype was analyzed. This study was approved by the Medical Ethics Committee of the Hospital (Ethics No.: 2024-K-135). RESULTS: The child and her elder siblings exhibited similar clinical manifestations including recurrent cough, secretory otitis media, chronic sinusitis, tracheobronchitis, and pneumonia. The child also presented with bronchiectasis and visceral situs inversus. Genetic testing results indicated that the child and her elder siblings had all harbored compound heterozygous variants of the DNAH11 gene, namely c.3000 1G>A and c.5775C>G (p.Tyr1925*), which were respectively inherited from their phenotypically normal parents. Both variants can affect mRNA splicing and protein translation integrity. Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic. It was predicted that they may jointly lead to a functional defect in axonemal dynein, resulting in the phenotype of PCD, conforming to an autosomal recessive inheritance. CONCLUSION The compound heterozygous variants c.3000 1G>A and c.5775C>G (p.Tyr1925*) of the DNAH11 gene probably underlay the pathogenesis of PCD in this pedigree. The same variant in different individuals may lead to different clinical phenotypes, which has reflected significant heterogeneity in genetic background and clinical phenotype. Above findings have enriched the mutational spectrum of PCD gene and have important implications for the accurate diagnosis, treatment, prognosis, and genetic counseling.
Humans ; Pedigree ; Female ; Axonemal Dyneins/genetics* ; Male ; Child ; Asian People/genetics* ; Kartagener Syndrome/genetics* ; Mutation ; Phenotype ; China ; Adult ; East Asian People

Objective:To explore the genotype-phenotype relationship in a child with Opitz G/BBB syndrome (OS) with mild clinical phenotype.Methods:A child with motor developmental delay as the initial symptom admitted to Xi ′an Children′s Hospital on June 10, 2021 was selected for this study. Clinical data were collected, and peripheral blood samples were obtained from the child and his mother. Whole exome sequencing (WES) was performed to identify genetic variant in the child. Candidate variant were verified by Sanger sequencing to assess inheritance patterns and pathogenicity. Real-time fluorescence quantitative PCR (RT-qPCR) and Western blot (WB) analyses were conducted to evaluate the effects of the variant on mRNA and protein expression, respectively, using recombinant expression plasmids generated in vitro. This study was approved by the Medical Ethics Committee of Xi′an Children′s Hospital (Ethics No. 20240045).Results:① The child, a 9-month-and-7-day-old boy, presented with a low nasal bridge, hypertelorism, and difficulty sitting independently. Echocardiography revealed an atrial septal defect. ② WES identified a homozygous variant in the MIDI gene, c. 1483C>T (p.R495X), which was confirmed by Sanger sequencing and found to be inherited from the mother.③ Recombinant expression plasmids were successfully constructed. RT-qPCR analysis showed that the variant significantly reduced MIDI gene mRNA expression, while WB results indicated that the variant led to the production of a truncated protein. Conclusion:The mild clinical phenotype of OS in this child may be attributed to the mRNA degradation escape mechanism induced by the nonsense variant c. 1483C>T(p.R495X) in the MIDI gene. These findings provide valuable diagnostic insights for this pedigree and contribute to the understanding of the genotype-phenotype correlation in OS.