Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Child ; China/epidemiology* ; Cryptorchidism/genetics* ; Disorders of Sex Development/genetics* ; Female ; Genital Diseases, Male ; Genotype ; Humans ; Hypospadias/genetics* ; Male ; Membrane Proteins/genetics* ; Penis/abnormalities* ; Phenotype ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics*

Rhizome rot disease is one of the main disease of planted Polygonatum kingianum. In this study, six strains of pathogenic fungus was isolated from P. kingianum samples with rhizome rot disease collected from six counties in Yunnan province. Its pathogenicity was confirmed by inoculation to healthy P. kingianum rhizome according to Koch's postulates. The colonies of the isolated fungi on potato dextrose agar(PDA) were orange with abundant crescentic conidia which were eseptate with a mean size of 19. 3-24. 9 μm×5. 2-5. 9 μm and a L/W ratio of 3. 4-4. 5. There was an oil ball in the center of the conidium. It's easy to see setae on PDA colony.The phylogenetic tree based on ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2 sequences by maximum likelihood(ML) method indicated that the pathogenic fungus for P. kingianum rhizome rot disease was clustered into the clade of Colletotrichum spaethianum species complex, and was close to C. spaethianum. However, there were some differences in morphological and genetic characteristics between the pathogenic fungus and C. spaethianum. Therefore, the pathogenic fungus for rhizome rot disease of P. kingianum was identified as a new Colletotrichum species named C. kingianum. The disease spreads primarily due to the plantation of infected seedlings of P. kingianum. It is necessary to choose healthy seedlings and take rigorous disinfection measures for the disease prevention.
China ; Colletotrichum/genetics* ; Phylogeny ; Polygonatum ; Rhizome

Wolfberry has important unique medical values as well as edible and commerce values. In this paper,we analyze the characteristics and problems of international trade of wolfberry based on the customs data between 2008 and 2017. During periods of these ten years,the wolfberry was mainly exported with a small proportions of imports. The total export volume increased steadily,reached 82 182. 08 tons and 696. 622 million dollars respectively. Wolfberry came from 31 provinces/autonomous regions and exported to 105 countries and regions through 21 ports. Most of the total exports of wolfberry flew to markets of Asia and Europe,the Ningxia autonomous region was the major export province. Large amount of wolfberry exported through Tianjin port. Compared with the export volume,the import is almost negligible,mainly coming from North Korea,almost all through Changchun port,Jilin province to enter the domestic market. There is a situation of"import of domestic goods". To enhance the international competitiveness of wolf berry industry,we must rely on the fundamental research of wolfberry,speed up the standardization process,strengthen the scientific and technological innovation in wolfberry products,improve the added value and profit of wolfberry.
Asia ; China ; Commerce ; Europe ; Lycium

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
China ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; RUNX1 Translocation Partner 1 Protein ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; WT1 Proteins

Chorismate synthase(CS, EC:4.2.3.5) catalyses 5-enolpyruvy-shikimate-3-phosphate to form chorismate, which is the essential enzyme for chorismate biosynthesis in organisms. The amino acid sequences of CS from 79 species of higher plants were reported in GenBank at present. 125 amino acid sequences of CS from Baphicacanthus cusia and other 78 species of plants were predicted and analyzed by using various bioinformatics software, including the composition of amino acid sequences, signal peptide, leader peptide, hydrophobic/hydrophilic, transmembrane structure, coiled-coil domain, protein secondary structure, tertiary structure and functional domains. The phylogenetic tree of CS protein family was constructed and divided into eight groups by phylogenetic analysis. The homology comparison indicated that B. cusia shared a high homology with several plants such as Sesamum indicum, Nicotiana tabacum, Solanum tuberosum and so on. The open reading frame(ORF) of all samples is about 1 300 bp, the molecular weight is about 50 kDa, the isoelectric point(pI) is 5.0-8.0 which illustrated that CS protein is slightly basic. The ORF of CS we cloned in B. cusia is 1 326 bp, the amino acid residues are 442, the molecular weight is 47 kDa and pI is 8.11. The CS in B.cusia showed obvious hydrophobicity area and hydrophilicity area, no signal peptide, and may exists transmembrane structure areas. The main secondary structures of CS protein are random coil and Alpha helix, also contain three main structural domains which are an active structural domain, a PLN02754 conserved domain and a FMN binding site. The acquired information in this study would provide certain scientific basis for further study on structure-activity relationship and structure modification of CS in plants in the future.
Acanthaceae ; enzymology ; Amino Acid Sequence ; Computational Biology ; Phosphorus-Oxygen Lyases ; chemistry ; Phylogeny ; Plant Proteins ; chemistry ; Protein Structure, Secondary

Andrographolide is a main active ingredient in traditional Chinese medicine Andrographis paniculata，with a variety of pharmacological activity，widely used in clinical practice. However its biosynthetic pathway has not been resolved. Cytochrome P450 reductase provides electrons for CYP450 and plays an important role in the CYP450 catalytic process. In this study，the coding sequence of A. paniculata CPR was screened and cloned by homologous alignment，named ApCPR4. The ApCPR4 protein was obtained by prokaryotic expression. After isolation and purification，the enzyme activity was identified . The results showed that ApCPR4 could reduce the cytochrome c and ferricyanide in NADPH-dependent manner. In order to verify its function，ApCPR4 and CYP76AH1 were co-transformed into yeast engineering bacteria. The results showed that ApCPR4 could help CYP76AH1 catalyze the formation of rustols in yeast. Real-time quantitative PCR results showed that the expression of ApCPR4 increased gradually in leaves treated with methyl jasmonate (MeJA). The expression pattern was consistent with the trend of induction and accumulation of andrographolide by MeJA，suggesting that ApCPR4 was associated with biosynthesis of andrographolide.
Acetates ; Andrographis ; enzymology ; genetics ; Biosynthetic Pathways ; Cloning, Molecular ; Cyclopentanes ; Diterpenes ; metabolism ; NADPH-Ferrihemoprotein Reductase ; genetics ; Oxylipins ; Plant Leaves ; enzymology ; Plant Proteins ; genetics

Protein complexes are involved in the synthesis of multiple secondary metabolites in plants, and their separation is essential to elucidate plant secondary metabolism and improve in vitro catalytic efficiency. In this study, the transgenic hairy roots of CYP76AH1, a key enzyme of tanshinone synthesis pathway, was constructed and the transgenic hairy roots of Danshen overexpressing CYP76AH1 protein were screened by Western blotting and used as a tissue culture material for the subsequent extraction of protein complex in tanshinone synthesis pathway. By optimizing the type and concentration of the detergent in the protein extraction buffer, the buffer containing 0.5% Triton X-100 was selected as the best extraction buffer, and a relatively large amount of soluble CYP76AH1 protein was isolated. This study lays the foundation for the further separation and purification of protein complexes interacting with CYP76AH1, and provides the idea for deep analysis of tanshinone metabolic pathway.

OBJECTIVETo investigate the comparability of bcr-abl (P210) transcript levels detected in different hospitals.

METHODSTen hospitals in China took part in the four times of sample exchange and comparisons from April, 2010 to August, 2011. The exchange samples were prepared by Peking University People's Hospital. Firstly, the BCR-ABL (P210)(+) cells from a newly diagnosed chronic myeloid leukemia patient were 10-fold serially diluted by BCR-ABL (P210)(-) cells and they covered 4 magnitudes. Then, TRIzol reagents were thoroughly mixed with cells in each tube. Every 12 samples (three samples per magnitude) were sent to the other 9 hospitals. The cell number of each sample was 8×10(6). The detection of bcr-abl transcript levels by real-time quantitative PCR were performed in every hospital according to their own protocols. Conversion factors (CF) were calculated using regression equation.

RESULTSDifferences in bcr-abl transcript levels did exist among results of 10 hospitals in each comparison. In general, the results of the most of hospitals were in line with the dilutions of cells. CF of every hospital fluctuated. Three hospitals had relatively stable CF, and their ranges were 2.8 - 5.2, 1.2 - 2.8 and 2.2 - 6.8, respectively; two hospitals had unstable CF with ranges 0.76 - 7.0 and 2.1 - 18.7; three hospitals couldn't be calculated CF one or two times because of the significant deviation of the results from the actually bcr-abl transcript levels, and their ranges of CF which could be calculated were 1.9 - 19.2, 3.6 - 7.6 and 0.18 - 14.7; One hospital only had two CF (3.3 and 5.0) because of the replacement of an important reagent during the period of comparisons.

CONCLUSIONSComparability of bcr-abl (P210) transcript levels between different hospitals could be achieved through CF which acquired by sample exchange and comparison. The stable and reliable detection system is the premise to acquire correct CF.

Bone Marrow Cells ; China ; Fusion Proteins, bcr-abl ; genetics ; isolation & purification ; Hospitals ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; diagnosis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the role of interleukin (IL) 21 alone, IL12 alone, and IL21 plus IL12 for inducing the antitumor activity of peripheral blood mononuclear cells (PBMCs) in patients with endometrial cancer.

METHODSPBMCs were isolated from peripheral blood in patients with endometrial cancer in vitro, and kept the culture with low-level IL2. IL2-stimulated PBMCs were cocultured under different conditions (with anti-IL21 antibody, IL21 alone, IL12 alone, or IL21 plus IL12) for 72 h. The cytotoxicity of PBMCs was then examined by lactate dehydrogenase(LDH) released assay. CD4(+) CD25(+) FOXP3(+) regulatory (Treg) cell and CD4(+) IL17A(+) T-helper (Th17) cell proportion were determined with flow cytometry. Cell proliferation and apoptosis were measured by cell counting kit-8(CCK-8)assay and flow cytometry, respectively.

RESULTSIn comparison to control group, both IL21 and IL12 significantly enhanced the cytotoxicity of PBMCs. The IL21 plus IL12 group had superior effect to IL21 alone and IL12 alone. IL21 and IL12 significantly decreased the percentages of Treg cells and the rate of PBMCs apoptosis. IL21 or IL12 had no significant effect on the differentiation of Th17 cells and the proliferation of PBMCs.

CONCLUSIONSIL21 and IL12 can enhance the cytotoxicity of PBMCs in patients with endometrial cancer, which can be further strengthened with treatment of IL21 plus IL12. Such effects may be achieved by inhibiting the differentiation of Treg cells and the apoptosis of PBMCs, but not by the differentiation of Th17 cell.

Adult ; Aged ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endometrial Neoplasms ; immunology ; pathology ; Female ; Humans ; Interleukin-12 ; pharmacology ; Interleukins ; pharmacology ; Leukocytes, Mononuclear ; drug effects ; immunology ; pathology ; Middle Aged ; T-Lymphocytes, Regulatory ; immunology

OBJECTIVETo investigate the effect of DNA methylation in combination with histone deacetylase inhibitor on transcription regulation of Ras associated domain family gene 1(RASSF1A) tumor suppressor gene and the molecular biological behaviors in U266 cells.

METHODSThe U266 cells were treated with different doses of 5-Aza-2'-deoxycytidine (5-Aza-CdR) and Valproate (VPA) each alone or in combination. Methylation-specific PCR (MSP) was used to detect CpG island methylation in RASSF1A promoter. Quantitative real-time reverse transcription polymerase chain reaction (RQ-PCR) was used to examine the expression of RASSF1A gene in U266 cells. MTT was used for cell proliferation. Cell apoptosis and cell cycle were analyzed by flow cytometry.

RESULTSThe methylation of RASSF1A gene promoter was detected in U266 cells, while there was little RASSF1A gene expressing in the control group. The demethylation effect could be detected in the 5-Aza-CdR treated and combined treatment groups but no in the VPA group. The expression level of RASSF1A was induced by 5-Aza-CdR in a concentration-dependent manner while VPA had no such effect. The expression level of RASSF1A mRNA was increased significantly in the combined treatment group. Higher growth inhibition and apoptosis effects were found in 5-Aza-CdR and VPA combination group than that in 5-Aza-CdR or VPA alone group (P < 0.05). After treatment with 5-Aza-CdR or VPA alone for 72 h, more cells were arrested in G(0)/G(1) phase as conpared with control group (P < 0.05), and even more cells were so arrested in combined treatment group (P < 0.05).

CONCLUSIONDNA methylation and histone deacetylase inhibitor can synergistically induce demethylation of the RASSF1A gene, re-express RASSF1A gene silenced in U266 cells, inhibit the proliferation of U266 cells and induce cell apoptosis.

Cell Line, Tumor ; CpG Islands ; DNA Methylation ; Gene Expression ; Histone Deacetylase Inhibitors ; Humans ; Promoter Regions, Genetic