Objective: Scutellarin in Shuganning injection has potential allergenicity and is prone to causing anaphylactoid reactions. There is an urgent need to establish a simple and rapid chromatographic quantitative method to achieve the limit control of scutellarin in Shuganning injection.
Methods: For scutellarin, HPLC method was used in the quantitative analysis using a Shim-pack GIS C₁₈ (2.1 mm×150 mm, 5 μm) column. The mobile phase was a blend of 0.1% trifluoroacetic acid in water and acetonitrile, and gradient elution progress was applied for the whole process. The flow rate was 0.3 mL·min^-1 and the column temperature was maintained at 37 ℃. The detecting wavelength was 320 nm.
Results: The linear regression equation was y=51 456x-27 352, the correlation coefficient r=0.999 8, namely the calibration curve was linear in the range of 1.02-102.00 μg·mL^-1; the average recovery was 103.76%; the detection and quantification limit for determining scutellarin was 0.16 μg·mL^-1 and 0.57 μg·mL^-1, respectively; the injection sample was stable at room temperature for 48 h.
Conclusion: This method is easy to operate, reproducible and has high accuracy, thus can be used for quality control of Shuganning injection.

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 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.

Objective:To discuss the effects of bone marrow-derived mesenchymal stem cells(BMSCs)transplantation on mitophagy in the vascular dementia(VaD)rats through reactive oxygen species(ROS)/nuclear factor erythroid 2-related factor 2(Nrf2)signaling,and to clarify its mechanism.Methods:Forty-five male adult SD rats were randomly divided into sham operation group,model group,unloaded group,BMSCs group,and MSCs+ML385(Nrf2 inhibitor)group(combination group),and there were 9 rats in each group.After intraperitoneal anesthesia,the VaD models were established in all groups except sham operation group.Morris water maze test was used to detect the learning and memory abilities of the rats in various groups;HE staining was used to observe the histopathological morphology of brain tissue of the rats in various groups;Nissl staining was used to observe the changes of Nissl bodies in hippocampus region of brain tissue of the rats in various groups;transmission electron microscope was used to observe the ultrastructure of hippocampus region of the rats in various groups;fluorescence probe method was used to detect the ROS levels in hippocampus neurons in various groups;Western blotting method was used to detect the expression levels of Nrf2,heme oxygenase-1(HO-1),PTEN-induced putative kinase 1(PINK1),parkin RBR E3 ubiquitin protein ligase(Parkin),Beclin-1,ubiquitin-binding protein p62(P62),and microtubule-associated protein 1A/1B-light chain 3(LC3-Ⅱ/LC3-Ⅰ)ratio in brain tissue of the rats in various groups.Results:The Morris water maze results showed that compared with sham operation group,the escape latency of the rats in model group was significantly increased(P<0.01),while the number of crossing time and residence time were significantly decreased(P<0.01).Compared with model group,the escape latency of the rats in BMSCs group was significantly decreased(P<0.01),while the number of crossing time and residence time were significantly increased(P<0.01).Compared with BMSCs group,the escape latency of the rats in combination group was significantly increased(P<0.01),while the number of crossing time and residence time were significantly decreased(P<0.01).The HE staining results showed that hippocampus neurons of the rats in sham operation group were normal in quantity and morphology,with uniform staining and clear structure.Compared with sham operation group,the hippocampus tissue of the rats in model group showed sparse arrangement,disordered structure,reduced neuronal quantity,varied morphology,uneven staining,nuclear pyknosis,and partial neuronal necrosis.Compared with model group,the neuronal damage of the rats in hippocampus regio in BMSCs group was alleviated,with restored morphology and improved neuronal loss.Compared with BMSCs group,the neurons of the rats in hippocampus region in combination group showed irregular morphology,disordered structure,unclear cell boundaries,uneven staining,and nuclear pyknosis.The Nissl staining results showed that the hippocampal neurons in sham operation group were tightly arranged with intact morphology,obvious nucleoli,and abundant darkly stained Nissl bodies.Compared with sham operation group,the neurons in hippocampus region of the rats in model group showed pyknosis,vacuolization,and sparse Nissl bodies.Compared with model group,the BMSCs group showed reduced neuronal pyknosis,relatively intact morphology,and increased Nissl bodies.Compared with BMSCs group,the combination group showed neuronal pyknosis,loss of morphological integrity,and fragmented Nissl bodies.The transmission electron microscope results showed that mitochondria in sham operation group exhibited oval shape with intact double-membrane structure and cristae.Compared with sham operation group,the mitochondria in model group showed swelling,disrupted membranes,broken cristae,and numerous autophagosomes.Compared with model group,the BMSCs group showed improved mitochondrial structure and reduced autophagosomes.Compared with BMSCs group,the combination group showed swollen mitochondria,disrupted membranes,broken cristae,and visible autophagosomes.The fluorescence probe results showed that compared with sham operation group,the ROS levels in the hippocampus neurons in brain tissue of the rats in model group were significantly increased(P<0.01);compared with model group,the ROS levels in hippocampus neurons in brain tissue of the rats in BMSCs group were significantly decreased(P<0.01);compared with BMSCs group,the ROS levels in hippocampus neurons in brain tissue of the rats in combination group were significantly increased(P<0.01).The Western blotting results showed that compared with sham operation group,the expression levels of Nrf2 and HO-1 proteins in brain tissue of the rats in model group were significantly decreased(P<0.01);compared with model group,the expression levels of Nrf2 and HO-1 proteins in brain tissue of the rats in BMSCs group were significantly increased(P<0.01);compared with BMSCs group,the expression levels of Nrf2 and HO-1 proteins in brain tissue of the rats in combination group were significantly decreased(P<0.01);compared with sham operation group,the expression levels of Parkin,PINK1,and Beclin-1 proteins,and LC3-Ⅱ/LC3-Ⅰ ratio of the rats in model group were significantly increased(P<0.01),while the expression level of P62 protein was significantly decreased(P<0.01);compared with model group,the expression levels of Parkin,PINK1,and Beclin-1 proteins,as well as the LC3-Ⅱ/LC3-Ⅰ ratio,of the rats in BMSCs group were significantly decreased(P<0.01),while the expression level of P62 protein was significantly increased(P<0.01);compared with BMSCs group,the expression levels of Parkin,PINK1,and Beclin-1 proteins,as well as the LC3-Ⅱ/LC3-Ⅰ ratio,of the rats in combination group were significantly increased(P<0.01),while the expression level of P62 protein was significantly decreased(P<0.01).Conclusion:BMSCs can alleviate the hippocampal neuronal pathological changes and improve cognitive function in the VaD rats,and its mechanism may be related to the regulation of ROS/Nrf2 signaling pathway to inhibit mitophagy.

Objective To explore the role of pseudogene AC106872.1 in maintaining the self-renewal capacity of human embryonic stem cells(hESCs).Methods AC106872.1 was knocked out in hESCs and knockout effi-ciency was validated by PCR and agarose gel electrophoresis.The colony formation of hESCs was assessed through colony formation assays and alkaline phosphatase(AP)staining.The expression level of pluripotency and differ-entiation marker genes was analyzed by qPCR and flow cytometry.RNA sequencing(RNA-seq)was performed to assess transcriptomic changes upon AC106872.1 knockout.Results Knockout of AC106872.1 significantly in-hibited the colony formation of hESCs(P＜0.05).The expression level of pluripotency marker genes was signifi-cantly reduced(P＜0.000 1),while the expression of differentiation marker genes was markedly increased(P＜0.000 1).Conclusions The pseudogene AC 1 06872.1 plays a crucial role in maintaining human embryonic stem cell self-renewal through regulation of pluripotency genes expression.

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.

Background::The conversion of adenosine (A) to inosine (I) through deamination is the prevailing form of RNA editing, impacting numerous nuclear and cytoplasmic transcripts across various eukaryotic species. Millions of high-confidence RNA editing sites have been identified and integrated into various RNA databases, providing a convenient platform for the rapid identification of key drivers of cancer and potential therapeutic targets. However, the available database for integration of RNA editing in hematopoietic cells and hematopoietic malignancies is still lacking.Methods::We downloaded RNA sequencing (RNA-seq) data of 29 leukemia patients and 19 healthy donors from National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database, and RNA-seq data of 12 mouse hematopoietic cell populations obtained from our previous research were also used. We performed sequence alignment, identified RNA editing sites, and obtained characteristic editing sites related to normal hematopoietic development and abnormal editing sites associated with hematologic diseases.Results::We established a new database, "REDH", represents RNA editome in hematopoietic differentiation and malignancy. REDH is a curated database of associations between RNA editome and hematopoiesis. REDH integrates 30,796 editing sites from 12 murine adult hematopoietic cell populations and systematically characterizes more than 400,000 edited events in malignant hematopoietic samples from 48 cohorts (human). Through the Differentiation, Disease, Enrichment, and knowledge modules, each A-to-I editing site is systematically integrated, including its distribution throughout the genome, its clinical information (human sample), and functional editing sites under physiological and pathological conditions. Furthermore, REDH compares the similarities and differences of editing sites between different hematologic malignancies and healthy control.Conclusions::REDH is accessible at http://www.redhdatabase.com/. This user-friendly database would aid in understanding the mechanisms of RNA editing in hematopoietic differentiation and malignancies. It provides a set of data related to the maintenance of hematopoietic homeostasis and identifying potential therapeutic targets in malignancies.

Objective To observe the effects of Ditan Yizhi Decoction on learning and memory ability,structure of hippocampal tissue,neuronal morphology of hippocampus,and the expression of endoplasmic reticulum autophagy-related protein FAM134B in hippocampal tissue;To explore the mechanism of its therapeutic effect on vascular dementia.Methods Totally 32 SD rats were randomly divided into sham-operation group,model group,donepezil group and Ditan Yizhi Decoction group,with 8 rats in each group.The model group,donepezil group and Ditan Yizhi Decoction group were prepared with a modified permanent ligation method of bilateral common carotid arteries to create a rat model of vascular dementia,the common carotid artery was separated in the sham-operation group,but not ligated.After modeling,the donepezil group was given donepezil hydrochloride,Ditan Yizhi Decoction group was given Ditan Yizhi Decoction,and the sham-operation group and model group were given equal volume of distilled water for gavage for 4 consecutive weeks.Morris water maze experiment was used to evaluate the learning and memory ability,HE staining and Nissl staining were used to observe the morphological changes of hippocampus,ultrastructure of hippocampal neurons was observed using transmission electron microscopy,Western blot was used to detect the protein expression of FAM134B and p-FAM134B in hippocampal tissue.Results Compared with the sham-operation group,the escape latency period was prolonged of the rats in model group,and the number of crossing the original platform and the duration of stay in the target quadrant was reduced(P<0.01),the gap between neurons in CA1 region of the hippocampus increased,the cell morphology was irregular,the boundaries were blurred,the neurons shrinked,the Nissl bodies dissolved and broke,the number decreased,the endoplasmic reticulum arrangement was scattered,mitochondria swelled and deformed,and the expressions of FAM134B and p-FAM134B protein in hippocampal tissue increased(P<0.01).Compared with the model group,the escape latency period of rats in donepezil group and Ditan Yizhi Decoction group were significantly shortened,and the number of crossing the original platform and the duration of stay in the target quadrant were increased(P<0.01),the morphology and quantity of neurons in CA1 region of the hippocampus were more regular,with a decrease in neuronal pyknosis,an increase in the number of Nissl bodies,and a reduction in dissolution and fragmentation,the swelling and deformation of the endoplasmic reticulum were restored,and the expression of FAM134B and p-FAM134B protein in hippocampal tissue increased(P<0.01).Moreover,the effects of Ditan Yizhi Decoction group were better than those of the donepezil group(P<0.01).Conclusion Ditan Yizhi Decoction can improve the learning and memory ability and the morphology of neurons in vascular dementia rats.The mechanism may related to increasing the expression and phosphorylation of FAM134B protein,thereby promoting endoplasmic reticulum autophagy.

To explore screening tools for children with autism spectrum disorder (ASD), which are convenient for primary hospitals, it can provide basic data for formulating ASD prevention policies. This was a cross-sectional study by cluster sampling. Huyi District and Xincheng District were extracted for investigation in Xi′an City. From July 2021 to September 2022, all children aged from 3 months to 36 months who live in the two districts were subjected to primary screening. The child care physician used the routine screening tool "warning signs checklist for screening psychological, behavioral and developmental problems of children" and cartoon pictures of "early high-risk warning signs of autism", the children who were positive in the initial screening were referred to the district level maternal and child health hospital for re-screening, and those who were positive in the re-screening were referred to Xi ′an Children′s Hospital for diagnosis. The results showed that a total of 17 905 children aged from 3 months to 36 months were initially screened in the two districts, including 10 588 children aged from 18 months to 36 months, 50 children who were positive in the initial screening and 50 children who were re-screened. 23 children (18 boys and 5 girls) were diagnosed with ASD. The prevalence rate of ASD in children was 2.17‰ (95% confidence interval:1.29‰-3.06‰). 42 children were positive for "warning signs checklist" at the preliminary screening, and 19 were confirmed as ASD. 27 children were positive for "cartoon pictures" in the preliminary screening, and 23 were confirmed with ASD. The "cartoon pictures" in the preliminary screening and diagnosis of consistent rate was higher than the "warning signs checklist", two kinds of screening methods comparison were statistically significant difference in the odds of consistent (χ 2=11.01, P=0.001). In conclusion, relying on the three-level network of maternal and child health care, it is conducive to the whole process management of screening and diagnosis of children with ASD, and to guide the formulation of prevention policies. The cartoon pictures of "early high-risk warning signs of autism" can assist the identification of children with ASD based on the "warning signs checklist", which is simple, effective and suitable for promotion in the community health care.

Objective:To develop and evaluate a rapid and sensitive point-of-care chemiluminescent assay(POC-CLIA)for β-human chorionic gonadotropin(β-HCG).Methods:POC-CLIA was constructed based on alkaline phosphatase(Alp)-AMPPD lumi-nescence system and magnetic particles(Mps)carrier.Performance of POC-CLIA,including sensitivity,precision,accuracy,linear dilution,specificity,stability,hook effect and clinical application were evaluated.Results:Detection limit of β-HCG was 0.71 mU/ml,linear detection range was 0.710～1.092×104 mU/ml,and was no hook effect up to 1.7×105 mU/ml.Intra and inter batch coefficients of variation were less than 10％,and could be stored stably at 37℃ for 10 days.Accuracy deviation was within±10％,so results were reliable.There was no cross-reactivity between interfering substances and anti-β-HCG antibdies.For detecting β-HCG in 100 clinical serum samples,results were highly correlated with those that were tested by clinical standard methods(R2=0.997 0).Turnaround time for single sample was less than 15 min and throughput could reach 200 T/h.Conclusion:This method is adequate that can be widely used in grassroots communities to help large-scale screening of pregnancy and related diseases.