Study on the Compositional Analysis, Extraction Process, and Hemostatic and Anti-Inflammatory Activities of Cirsium japonicum Fisch. ex DC.-Cirsium setosum (Willd.) MB Extracts.

Cirsium japonicum Fisch. ex DC. (CF) and Cirsium setosum (Willd.) MB (CS) are commonly used clinically to stop bleeding and eliminate carbuncles. Still, CF is mainly used for treating inflammation, while CS favors hemostasis. Therefore, the present study used UHPLC-MS to analyze the main chemical constituents in CF-CS extract. We optimized the extraction process using single-factor experiments and response surface methodology. Afterward, the hemostatic and anti-inflammatory effects of CF-CS extract were investigated by determining the clotting time in vitro, the bleeding time of rabbit trauma, and the induction of rabbit inflammation using xylene and lipopolysaccharide. The study of hemostatic and anti-inflammatory effects showed that the CF-CS, CF, and CS extract groups could significantly shorten the coagulation time and bleeding time of rabbits compared with the blank group (p < 0.01); compared with the model group, it could dramatically inhibit xylene-induced ear swelling in rabbits and the content of TNF-α, IL-6, and IL-1ß in the serum of rabbits (p < 0.01). The results showed that combined CF and CS synergistically increased efficacy. CF-CS solved the problem of the single hemostatic and anti-inflammatory efficacy of a single drug, which provided a new idea for the research and development of natural hemostatic and anti-inflammatory medicines.

Mechanism of action of Sambucus williamsii Hance var. miquelii in the treatment of osteoporosis analyzed by UHPLC-HRMS/MS combined network pharmacology and experimental validation.

Sambucus williamsii Hance var. miquelii(SWH) is a precious wild Chinese herb whose fruit, rhizome, leaves and root bark can be used as medicine. Sambucus Linn has pharmacological effects such as anti-osteoporosis, promoting fracture healing, anti-viral and anti-inflammatory. In this study, the main chemical components of the alcoholic extracts from SWH were rapidly identified by ultra-high performance liquid chromatography-quadrupole orbit trap high-resolution mass spectrometry (UHPLC- HRMS MS), and a total of 42 compounds were characterized from the alcoholic extracts of SWH. The results of network pharmacological validation showed that kaempferol, quercetin, luteolin, isorhamnetin and morroniside were the main active components, and KEGG enrichment demonstrated that SWH mainly affected the signaling pathways such as PI3K-Akt, TNF and FoxO by modulating the related targets such as AKT1, PIK3R1, EGFR, RELA SRC and PTGS2. The molecular docking results showed binding solid activity between the main active components of SWH and the targets. The network pharmacology was validated by establishing an animal model of osteoporosis (OP) in rats by gavage administration of vitamin A acid. The results of the pharmacological experiments showed that SWH could improve the degree of bone loss in the femur of osteoporotic rats, increase the number of trabeculae and decrease trabeculae porosity, up-regulate the Ca and P content in the serum of OP rats, down-regulate the scope of ALP and BGP in the serum, and promote the calcification of the bone matrix, and then exert the anti-OP efficacy. In this study, network pharmacology and pharmacological experiments verified the pharmacological mechanism of SWH in anti-OP rats. This provides a theoretical basis for the research and development of anti-OP drugs and a reference for the application of other traditional Chinese medicines in treating OP diseases.

Genetic analysis of isolated methylmalonic acidemia in Henan, China: c.1663G>A variant of MMUT prevalent in the Henan population.

BACKGROUND: Methylmalonic acidemia (MMA) is the most common organic acidemia in China, and isolated MMA accounts for approximately 30 % of all types of MMA. Common variants of the MMUT gene vary greatly around the world. The present study aims to determine the high-frequency and novel genetic variants of the MMUT gene in the Henan population of China and evaluate the prognosis of patients carrying the c.1663G>A (p.Ala555Thr) variant. METHODS: We performed next-generation sequencing for 41 patients with isolated MMA screened by tandem mass spectrometry (MS/MS) and analysed the genetic results. We also evaluated the prognosis of patients with the c.1663G>A variant. We used Jalview software for multispecies sequence alignment and Missense3D and DynaMut to predict the protein function of the detected novel variants. RESULTS: A total of 43 variants from 41 patients with isolated MMA were detected, of which c.1663G>A (14.63 %), c.729_730insTT (10.98 %), and c.1106G>A (8.53 %) are high-frequency variants of the MMUT gene in the Henan population. The patients carrying the c.1663G>A variant tended to be responsive to vitamin B12, have a low mortality rate. We also identified 5 novel variants (c.479C>T, c.811G>C, c.965T>A, c.1142G>A and c.1667C>T). CONCLUSION: The rare variant c.1663G>A is prevalent in the Henan population, and infants with this variant tend to have good prognosis. Our findings, especially novel variants, will help broaden the spectrum of genetic variants and facilitate clinical diagnosis and genetic counselling for affected families.

Compound heterozygous variants of THG1L result in autosomal recessive cerebellar ataxia.

tRNA-histidine guanyltransferase 1-like protein (THG1L), located in the mitochondria, plays a crucial role in the tRNA maturation process. Dysfunction of THG1L results in abnormal mitochondrial tRNA modification and neurodevelopmental disorders. To date, few studies have focused on THG1L-related cerebellar ataxia. Whole-exome sequencing revealed compound heterozygous variants NM_017872.5: [c.224A > G]; [c.369-8T > G] in THG1L in a 6-year-old boy with moderate cerebellar ataxia. The variant c.224A > G was demonstrated to downregulate its RNA and protein expression, and c.369-8 T > G resulted in a 7 bp insertion before exon 3. Our case expanded the gene variation and clinical spectrum of THG1L-related cerebellar ataxia.

Allelic phenotype prediction of phenylketonuria based on the machine learning method.

BACKGROUND: Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Our study aimed to predict the phenotype using the allelic genotype. METHODS: A total of 1291 PKU patients with 623 various variants were used as the training dataset for predicting allelic phenotypes. We designed a common machine learning framework to predict allelic genotypes associated with the phenotype. RESULTS: We identified 235 different mutations and 623 various allelic genotypes. The features extracted from the structure of mutations and graph properties of the PKU network to predict the phenotype of PKU were named PPML (PKU phenotype predicted by machine learning). The phenotype of PKU was classified into three different categories: classical PKU (cPKU), mild PKU (mPKU) and mild hyperphenylalaninemia (MHP). Three hub nodes (c.728G>A for cPKU, c.721 for mPKU and c.158G>A for HPA) were used as each classification center, and 5 node attributes were extracted from the network graph for machine learning training features. The area under the ROC curve was AUC = 0.832 for cPKU, AUC = 0.678 for mPKU and AUC = 0.874 for MHP. This suggests that PPML is a powerful method to predict allelic phenotypes in PKU and can be used for genetic counseling of PKU families. CONCLUSIONS: The web version of PPML predicts PKU allele classification supported by applicable real cases and prediction results. It is an online database that can be used for PKU phenotype prediction http://www.bioinfogenetics.info/PPML/ .

Identification of phenylketonuria patient genotypes using single-gene full-length sequencing.

BACKGROUND: Phenylketonuria (PKU) is a common, autosomal recessive inborn error of metabolism caused by PAH gene variants. After routine genetic analysis methods were applied, approximately 5% of PKU patients were still not diagnosed with a definite genotype. METHODS: In this study, for the first time, we identified PKU patients with unknown genotypes via single-gene full-length sequencing. RESULTS: The detection rate of PKU genotype increased from 94.6 to 99.4%, an increase of approximately 5%. The variants c.1199 + 502A > T and 1065 + 241C > A were found at a high frequency in Chinese PKU patients. CONCLUSION: Our study suggest that single-gene full-length sequencing is a rapid, efficient and cost-effective tool to improve the genotype detection rate of PKU patients. Moreover, we provides additional case data to support pathogenicity of deep intronic variants in PAH.

A Novel Frameshift Variant of the MITF Gene in a Chinese Family with Waardenburg Syndrome Type 2.

Waardenburg syndrome (WS) is a rare genetic disorder characterized by varying combinations of sensorineural hearing loss and abnormal pigmentation involving the hair, skin and iris. WS is classified into 4 subtypes (WS1-WS4) based on additional symptoms. WS2 is characterized by the absence of additional symptoms and is mainly attributed to variants in the microphthalmia-associated transcription factor (MITF) gene. We detected a novel frameshift variant c.1025_1032delGGAACAAG (NM_198159) of MITF in 5 patients with WS2 from the same Chinese family by using targeted next-generation sequencing and Sanger sequencing. Phenotypic and genotypic analyses of the family members suggested that this novel variants was pathogenic. Our finding expands the spectrum of MITF variants.

Pathogenic variants identified by whole-exome sequencing in 43 patients with epilepsy.

BACKGROUND: Epilepsy is a group of neurological disorders characterized by recurrent epileptic seizures. Epilepsy is affected by many factors, approximately 20-30% of cases are caused by acquired conditions, but in the remaining cases, genetic factors play an important role. Early establishment of a specific diagnosis is important to treat and manage this disease. METHODS: In this study, we have recruited 43 epileptic encephalopathy patients and the molecular genetic analysis of those children was performed by whole-exome sequencing (WES). RESULTS: Fourteen patients (32.6%, 14/43) had positive genetic diagnoses, including fifteen mutations in fourteen genes. The overall diagnostic yield was 32.6%. A total of 9 patients were diagnosed as pathogenic mutations, including 4 variants had been reported as pathogenic previously and 6 novel variants that had not been reported previously. Therefore, WES heralds promise as a tool for clinical diagnosis of patients with genetic disease. CONCLUSION: Early establishment of a specific diagnosis, on the one hand, is necessary for providing an accurate prognosis and recurrence risk as well as optimizing management and treatment options. On the other hand, to unveil the genetic architecture of epilepsy, it is of vital importance to investigate the phenotypic and genetic complexity of epilepsy.

Variant analysis of PEX11B gene from a family with peroxisome biogenesis disorder 14B by whole exome sequencing.

BACKGROUND: Peroxisome biogenesis disorder 14B (PBD14B) is an autosomal recessive peroxisome biogenesis disorder characterized clinically by mild intellectual disability, congenital cataracts, progressive hearing loss, and polyneuropathy peroxisome biogenesis disorders are genetically heterogeneous group of disorders caused by biallelic mutations in peroxin (PEX) genes. METHODOLOGY/LABORATORY EXAMINATION: DNA of the family was extracted and sequenced by whole exome sequencing. The results were validated with Sanger sequencing analyzed with Bioinformatics software. RESULTS: Sequencing result showed that the patient has carried a homozygous variant of c.277C>T of the PEX11B gene. The patient's brother has carried a homozygous variant of c.277C>T of the PEX11B gene and their variants of c.277C>T of the PEX11B gene were inherited, respectively, from his mother and father. DISCUSSION AND CONCLUSION: The homozygous variant of c.277C>T of the PEX11B gene probably underlie the disease in this child and her brother.

Identification of a novel splicing mutation in the SLC25A13 gene from a patient with NICCD: a case report.

BACKGROUND: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is an autosomal recessive disorder and one of the most common inherent causes of cholestatic jaundice in Asian infants. Mutations in the SLC25A13 gene, which encodes citrin protein expressed in the liver, have been identified as the genetic cause for NICCD. CASE PRESENTATION: Here, we report a 4-month-old female with clinical features including jaundice, hyperbilirubinemia, hyperlactacidemia, and abnormal liver function. The patient was diagnosed with NICCD by differential diagnosis using genetic analysis. Mutations in 60 jaundice-related genes were tested by using amplicon sequencing, which was performed on an Ion S5XL genetic analyzer. A compound heterozygous mutation in the SLC25A13 gene was identified, consisting of a known deletion SLC25A13:c.852_855delTATG and a novel splicing mutation SLC25A13:c.1841 + 3_1841 + 4delAA. Sanger sequencing for the proband and her parents was performed to validate the result and reveal the source of mutations. CONCLUSION: A compound heterozygous mutation in the SLC25A13 gene was identified in a 4-month-old female patient with NICCD. Our data suggest that amplicon sequencing is a helpful tool for the differential diagnosis of inherited diseases with similar symptoms. Further studies of the mutation spectrum of neonatal jaundice in China are warranted.

Analysis of the accuracy of Z-scores of non-invasive prenatal testing for fetal Trisomies 13, 18, and 21 that employs the ion proton semiconductor sequencing platform.

BACKGROUND: Non-invasive prenatal testing (NIPT) is frequently being used to screen for trisomies 13, 18 and 21 for prenatal diagnosis. However, NIPT performs poorly when compared with invasive testing and thus should not be used to diagnose trisomies. The result of NIPT for an individual woman in most genome-wide methods is calculated as a Z-score. The aim of this study was to assess the correlation between Z-scores of NIPT results and the accuracy of non-invasive prenatal testing. RESULTS: We evaluated 108 pregnant women with positive NIPT results, which were validated through karyotype analysis of amniotic fluid puncture by means of sequencing, bioinformatics analysis, and follow-up. Utilizing the ion proton semiconductor sequencing platform, we report a performance evaluation of NIPT-positive results at Third Affiliated Hospital of Zhengzhou University of Henan Province, China, by classifying Z-scores as 3 ≤ Z<5, 5 ≤ Z < 9 and Z ≥ 9. The findings indicate that positive NIPT results at Z ≥ 9 have a higher accuracy compared with positive NIPT results at 5 ≤ Z < 9 and 3 ≤ Z<5. CONCLUSIONS: The findings show that Z-scores of NIPT results are closely related to the accuracy of non-invasive prenatal testing. However, false-positive NIPT results at 3 ≤ Z<5 may occur due to confined placental mosaicism (CPM).