The interaction of several small-molecule alkaloids,namely coptisine,jateorhizine,epiberberine,berberrubine,columbamine,fangchinoline and tetrandrine,with guanine-guanine-adenine(GGA)triplex DNA was investigated by mass spectrometry(MS),ultraviolet-visible(UV-vis)spectroscopy,fluorescence spectroscopy,and circular dichroism(CD)spectroscopy.The effect of the 7 kinds of metal ions on the stability of GGA triplex DNA was investigated by direct infusion electrospray ionization mass spectrometry(ESI-MS),and the binding degree was highest when Ni2+was employed.Among the 7 kinds of alkaloids,tetrandrine had the highest affinity to the GGA triplex.All the data unequivocally accommodated diverse mechanisms for interaction between the isoquinoline alkaloids and the GGA DNA triplexes.At least,electrostatic attraction and intercalative-binding modes were observed in the study.Interestingly,UV melting denaturation assay revealed that the two isoquinoline alkaloids including columbamine and berberrubine enhanced the stability of the GGA triplex structure.The UV-melting curve profile could not accurately determine the stabilizing effect of small molecules on the structure of GGA triplex DNA.Further detection by UV-vis spectroscopy showed that epiberberine could stabilize the structure of GGA triplex DNA,but other small molecules couldn't stabilize the structure of GGA triplex DNA.The mode of interaction was further investigated,and firstly,fluorescence spectroscopy was applied to construct the ethidium bromide(EB)-GGA triplex DNA system,and then the reactions of small molecule alkaloids at different concentrations with EB-GGA triplex DNA system were studied.And it was found that epiberberine,fangchinoline and tetrandrine small molecules could replace the EB and insert to bind GGA triplex DNA.Finally,the circular dichroism spectroscopy was used to investigate the binding mode,and the negative CD signal was found.The investigation provided new insights into the interaction of isoquinoline alkaloids with GGA triplex DNA.

Objective: To explore the drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacter freundii. Methods: A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital, Ningbo Medical Center. Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment, respectively. The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System. The complete genome sequence was obtained by "third-generation" sequencing methods, and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN, RefSeq, ConservedDomains, ResFinder, Isfinder, etc. Results: The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group, and contained bla_KPC-2 and conjugative transfer (type Ⅳ secretory system, T4SS) genes, which could induce conjugative transfer. A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI, which were from Citrobacter freundii, and the rest were from Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Raoultella planticola and other bacteria, and were broad-host-range plasmids. The sequence comparative analysis of all 6 of the novel plasmid from Citrobacter freundii showed that the structure of the novel plasmid had certain conserved property, with Tn6296 variant structure carrying bla_KPC-2, and plasmid pCF1807-3 had both repA_pNY2385-KPC and repA_IncX8. Conclusion: The pNY2385-KPC type plasmids in Citrobacter freundii carried bla_KPC-2 resistance gene, which were divided into two subtypes: repA_pNY2385-KPC single replicator and repA_pNY2385-KPC/repA_IncX8 complex replicator, belonging to broad-host-range plasmids. And as a mobile genetic element, the plasmids promote the spread of bla_KPC-2.
Humans ; Citrobacter freundii/genetics* ; RNA, Ribosomal, 16S/genetics* ; Emergency Service, Hospital ; Escherichia coli ; Genomics

Objective: To explore the drug resistance mechanism and gene structure characteristics of a carbapenemase-producing novel incompatibility group plasmid pNY2385-KPC from Citrobacter freundii. Methods: A multi-drug resistant strain was obtained from urine samples of patients with fever in the emergency ward of Li Huili Hospital, Ningbo Medical Center. Bacterial species was preliminary identified and finally confirmed by 16S rRNA gene amplification and the average nucleotide identity alignment, respectively. The minimum inhibitory concentrations of the antimicrobial agents were determined by VITEK 2 Compact System. The complete genome sequence was obtained by "third-generation" sequencing methods, and then detailed annotation of gene function and comparative genomic analysis of plasmid structure were carried out by BLASTP/BLASTN, RefSeq, ConservedDomains, ResFinder, Isfinder, etc. Results: The pNY2385-KPC carried by citrobacter freundii NY2385 belonged a novel incompatibility group, and contained bla_KPC-2 and conjugative transfer (type Ⅳ secretory system, T4SS) genes, which could induce conjugative transfer. A total of 15 plasmids of the same type as pNY2385-KPC were retrieved by NCBI, which were from Citrobacter freundii, and the rest were from Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Raoultella planticola and other bacteria, and were broad-host-range plasmids. The sequence comparative analysis of all 6 of the novel plasmid from Citrobacter freundii showed that the structure of the novel plasmid had certain conserved property, with Tn6296 variant structure carrying bla_KPC-2, and plasmid pCF1807-3 had both repA_pNY2385-KPC and repA_IncX8. Conclusion: The pNY2385-KPC type plasmids in Citrobacter freundii carried bla_KPC-2 resistance gene, which were divided into two subtypes: repA_pNY2385-KPC single replicator and repA_pNY2385-KPC/repA_IncX8 complex replicator, belonging to broad-host-range plasmids. And as a mobile genetic element, the plasmids promote the spread of bla_KPC-2.
Humans ; Citrobacter freundii/genetics* ; RNA, Ribosomal, 16S/genetics* ; Emergency Service, Hospital ; Escherichia coli ; Genomics

A total of 15 batches of the substance reference of Guizhi Jia Gegen Decoction(GZGGD) were prepared and the characteristic fingerprints of them were established. Furthermore, the similarity of the fingerprints and peak attributes were explored. The extraction rate, and the content and the transfer rate ranges of the index components, puerarin, paeoniflorin, liquiritin, and ammonium glycyrrhizate were determined for the analysis of the quality value transfer. The result demonstrated that the fingerprints of the 15 batches of the samples showed high similarity(>0.99). A total of 15 characteristic peaks were identified from the fingerprints, with 10 for Puerariae Lobatae Radix, 1 for Cinnamomi Ramulus, 2 for Paeoniae Radix Alba, and 2 for Glycyrrhizae Radix et Rhizoma. The content of puerarin was 11.05-18.35 mg·g~(-1) and the average transfer rate was 21.27%-39.49%. The corresponding figures were 7.95-10.90 mg·g~(-1) and 23.28%-43.23% for paeoniflorin, 3.25-4.95 mg·g~(-1) and 32.31%-61.27% for ammonium glycyrrhizate, and 3.65-5.80 mg·g~(-1) and 14.57%-27.05% for liquiritin. The extraction rate of the 15 batches of samples was in the range of 16.85%-21.78%. In this paper, the quality value transfer of the substance reference of GZGGD was analyzed based on characteristic fingerprint, content of index components, and the extraction rate. This study is expected to lay a basis for the quality control and further development of GZGGD.
Ammonium Compounds ; Benchmarking ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Paeonia

Objective To analyze the dynamic changes of schistosomiasis in Poyang County of Jiangxi Province from 2004 to 2020, so as to provide insight into the development of the schistosomiasis elimination strategy. Methods Schistosomiasis control data were captured from Poyang County from 2004 to 2020, and the epidemiological data of schistosomiasis were collected from national schistosomiasis surveillance sites in Poyang County from 2005 to 2020. The endemic status of schistosomiasis was analyzed in Poyang County from 2004 to 2020, and a Joinpoint regression analysis was performed to investigate the trends of schistosomiasis in Poyang County from 2004 to 2020. Results The sero-prevalence and egg-prevalence of human Schistosoma japonicum infections reduced from 24.39% (24 976/102 397) and 4.53% (259/5 721) in 2004 to 5.37% (2 421/45 100) [annual percent change (APC) = average annual percent change (AAPC) = −8.64%] and 0 (0/3 963) in 2020 (APC = AAPC = −32.07%) in Poyang County, and the trends were both significant (both P < 0.01). The sero-prevalence of S. japonicum infections reduced from 1.21% (294/24 332) in bovines in 2013 to 0.58% (35/5 999) in 2020 in Poyang County, with one turning point (AAPC = −8.20%, P > 0.05). There were no townships or villages with emerging snail habitats in Poyang County from 2004 to 2020, and there were three turning points of trend in the proportion of snail areas detected in total snail areas (AAPC = −2.30%, P > 0.01). The sero-prevalence and adjusted prevalence of S. japonicum infections reduced from 60.82% (742/1 220) and 10.16% (124/1 220) in local residents in 2005 to 5.73% (70/1 221) and 0 in 2020 in national schistosomiasis surveillance sites of Poyang County, and the trends for sero-prevalence (APC = AAPC = 17.47%, P < 0.01) and adjusted prevalence of S. japonicum infections (APC = AAPC = −44.92%, P < 0.01) were both statistically significant. S. japonicum infections were identified in 10 (2005) and 2 local livestock (2007), with prevalence of 10.00% (10/100) and 13.33% (2/15), respectively, and S. japonicum infections were detected in snails in 2008 and 2009; however, no positive samples of mixed O. hupensis were detected by loop-mediated isothermal amplification. Conclusions The endemic situation of schistosomiasis control had remarkably reduced in Poyang County from 2004 to 2020; however, there are still challenges for consolidating schistosomiasis control achievements and even elimination of schistosomiasis.

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*

OBJECTIVE: To explore the clinical characteristics and genetic findings of patients with infantile intrahepatic cholestasis. METHODS: The clinical data were collected in children who were admitted to the Department of Gastroenterology in Children's Hospital, Capital Institute of Pediatrics from June 2017 to June 2019 and were suspected of inherited metabolic diseases. Next generation sequencing based on target gene panel was used for gene analysis in these children. Sanger sequencing technology was used to verify the genes of the members in this family. RESULTS: Forty patients were enrolled. Pathogenic gene variants were identified in 13 patients (32%), including CONCLUSIONS The etiology of infantile intrahepatic cholestasis is complex. Next generation sequencing is helpful in the diagnosis of infantile intrahepatic cholestasis.
Alagille Syndrome/genetics* ; Child ; Cholestasis, Intrahepatic/genetics* ; Citrullinemia ; Genetic Testing ; High-Throughput Nucleotide Sequencing ; Humans ; Mitochondrial Membrane Transport Proteins ; Mutation

For sexual reproduction, oocytes are mammalian female germ cells that provide the majority of maternal genetic material for early stage embryo production and development. Early stage embryos begin the process of multicellular organism formation through cell differentiation. Studies on mammalian female germ cells (oocytes) not only reveal its unique physiological characteristics, but also help understand the mechanism involved in cell differentiation of other cell types. However, because it is difficult to culture in vitro, our understanding of the function of oocytes and early stage embryos remains very limited. Gene editing or manipulation is one of the most commonly used method, which is also useful in the field of gametes study. In this review, we summarized the principles, advantages and disadvantages of techniques, which include conditional knockout, RNA interference, Morpholino, Trim-Away and antibody-mediated inhibition of protein function, currently used for gene manipulation in oocytes and early stage embryos. We also discuss the issues the investigators need to consider. Finally, we highlight the future directions for gene manipulation or editing in female germ cells and early stage embryos.

Objective:To investigate constituents containing phenolic hydroxyl or carboxylic acid （excluding diarylheptanoids） from Curcumae Rhizoma and Sparganii Rhizoma herbal pair and single herb. Method:Multiple chromatographic separation techniques， including silica gel，MCI gel and Sephadex LH-20 gel， were employed to isolate and purify the compounds. Their structures were identified by means of the nuclear magnetic resonance （NMR），mass spectrometry （MS） and physicochemical properties. Constituents were quickly analyzed by UPLC-LTQ-Orbitrap-MSⁿ，and scanned in positive and negative ion modes. Result:These compounds were determined as trans-p-hydroxycinnamic acid（1），vanillic acid（2），protocatechuic acid（3），fumalic acid（4），succinic acid（5），succinic acid monomethyl ester（6），docosanoic acid（7），azelaic acid（8） and p-hydroxybenzaldehyde（9）. Forty compounds were speculated by comparing mass spectrometry data，retention times of some compounds and reference materials，including 14 phenols and 26 organic acids. Among the compounds of herbal pair，8 phenols and 22 organic acids in Sparganii Rhizoma as well as 11 phenols and 13 organic acids in Curcumae Rhizoma were identified. Cleavage pathways of main compounds were described. Conclusion:There are abundant phenols and organic acids in Curcumae Rhizoma and Sparganii Rhizoma herbal pair and single herb. The results enrich pharmacodynamic material basis of Curcumae Rhizoma and Sparganii Rhizoma herbal pair.