The marine biological source of mineral drugs recorded in Chinese Pharmacopoeia (2010 version) mainly including pearl, nacre, clam shell, common oyster shell, ark shell, cuttle bone, and sea-ear shell are widely used in clinical. Calcium carbonate and a small amount of protein are the main components in this type of drugs. In this paper, a systematical and comparable study were carried out by determination of calcium carbonate by EDTA titration method, the crystal of calcium carbonate by X-Ray powder diffraction and the total amino acids (TAAs) of the hydrolyzed samples by ultraviolet spectrophotometry method. As a result, the crystal structure is calcite for common oyster shell, mixture of calcite and aragonite for nacre and sea-ear shell, aragonite for the other drugs. The content of calcium carbonate ranged from 86% to 96%. Cuttle bone has the highest amount of TAAs among the seven drugs which reached 1.7% while clam shell has the lowest content of 0.16% on average. In conclusion, an effective method was developed for the quality control of marine mineral drugs by comprehensive analysis of calcium carbonate and TAAs in the seven marine mineral drugs.
Amino Acids ; analysis ; chemistry ; Animal Shells ; chemistry ; Animals ; Calcium Carbonate ; analysis ; chemistry ; Crystallization ; Edetic Acid ; chemistry ; Mollusca ; chemistry ; classification ; Pharmaceutical Preparations ; analysis ; chemistry ; standards ; Quality Control ; Reproducibility of Results ; Seawater ; Species Specificity ; Spectrophotometry, Ultraviolet ; X-Ray Diffraction

To construct knockout vectors containing ampicillin resistant gene and partial sequence of hyaluronidase gene(Hyl)so that Hyl can be knock out by transforming the plasmid into Streptococcus zoopidemics mutans.First,partial sequence of Hyl(Hyl-1)was cloned into the vector of pMD19-T by using DNA of Streptococcus zoopidemics as template,and then a knockout vector pMD19T-SA was constructed,in which Hyl-1 gene was disrupted by inserting ampicillin resistant gene(Amp)from reverse PCR.As expected,the vector was proved to be consisted of Hyl-1-Amp-Hyl-1-pMD19-T.Thereafter,DNA fragment of Hyl-1-Amp-Hyl-1 was subcloned into pBR322 vector,the resulting construct was then checked by PCR and restriction analysis for the proper configuration of the knockout vector pBR322-SA.Both of the knockout vectors were used to transform Streptococcus zoopidemics and one recombinant was obtained in result.From results of PCR and Hyl activity assay,it was indicated that in the recombinant the Hyl gene was disrupted completely.

OBJECTIVETo investigate the functional relations between the putative proteins YpCD1.08, YpCD1.09, YpCD1.16 encoded in pCD1 plasmid of Yersinia pestis and its type III secretion system (T3SS).

METHODSMutants of YpCD1.08, YpCD1.09, YpCD1.16 were constructed using λ-Red recombinant system. The growth curves of the mutant strains cultivated in TMH medium with or without calcium at 26 °C and 37 °C were determined to analyze the low calcium response phenotype. The transcription levels of ΔYpCD1.08, ΔYpCD1.09, ΔYpCD1.16 in Yersinia pestis and the dependence to temperature were determined using real time RT-PCR after cultivation at 26 °C and 37 °C and extraction of RNA. A β-lactamases reporter system was adopted to study the influence of these genes on the translocation of effector YopE of T3SS.

RESULTSWhen grown in TMH medium without calcium at 26 °C and 37 °C, the growth curve of the YpCD1.08, YpCD1.09, YpCD1.16 mutants were similar to that of the wild-type strain, indicating that the low calcium response of all the mutants were normal. The ratios of YpCD1.08, YpCD1.09, YpCD1.16 gene transcriptional level at 37 °C and 26 °C were 2.3 ± 0.3, 2.3 ± 0.5 and 3.2 ± 0.7, respectively, indicating that these genes were transcribed in Yersinia pestis and their transcription regulations showed a temperature-dependence that was consistent with the well established temperature-dependent expression of Yersinia T3SS genes. The β-lactamases reporter assays demonstrated that ΔYpCD1.08 could translocate much higher level of YopE into HeLa cells, since that the light intensity ratio of 477/520 nm at 140 min was 2.5, whereas it was 1.8 for the wild-type strain, and the values in ΔYpCD1.09 and ΔYpCD1.16 were similar to the wild-type strain.

CONCLUSIONYpCD1.08, YpCD1.09, YpCD1.16 gene are likely to be the new members of T3SS, and the putative protein YpCD1.08 could play some roles in YopE secretion and translocation.

Bacterial Outer Membrane Proteins ; secretion ; Bacterial Secretion Systems ; genetics ; Genes, Bacterial ; Plasmids ; Protein Interaction Mapping ; Yersinia pestis ; genetics ; metabolism ; pathogenicity

OBJECTIVETo obtain information on viral molecular structural and evolutionary characteristics, we conducted the SZ2010422 full-length genomic analysis.

METHODSPrimers were designed by New Orleans full sequence, SZ2010422 full genome was amplified by RT-PCR, the whole genome sequence and the capsid domain amino acid sites was analysised after cloned and sequenced.

RESULTSThe genome of G II-4 Norovirus SZ2010422 strain was consist of 7559 bp, it revealed three ORFs composites of the whole genome, ORF1 (5100 bp), ORF2 (1623 bp), ORF3 (807 bp) respectively, ORF1 and ORF2 had 19 nucleotide overlap. By evolutionary comparative analysis found SZ2010422 genomic nucleotide sequences with reference strains of G II-4 New Orleans1805 strains the highest homology with a total length of homology was 99.3%, of ORF1 (99.5%), ORF2 (99.2%), ORF3 (98.6%). Phylogenetic analyses showed SZ2010422 belonging to G II-4 New Orleans variant. Date of 541 amino acid analyses showed: New Orleans variant strains of popular sites: aa310N or K, --> S aa341D --> of N, aa359T--> S, aa396H --> P, aa460H --> Y.

CONCLUSIONNorovirus SZ2010422 belonged to the G II-4 New Orleans variant. In This study, SZ2010422 full sequence can be used not only as a full-length NoV variant sequence standard for future comparison studies, but also as useful material for the public health field by enabling the diagnosis, vaccine development, and prediction of new emerging variants. Noroviruses; Genes; Sequence analysis

China ; Genome, Viral ; Norovirus ; classification ; genetics ; Open Reading Frames ; Phylogeny ; Sequence Analysis, DNA

OBJECTIVETo explore the influence of 5'UTR tandem repeat and 3'UTR 6 bp deletion polymorphism of thymidylate synthase (TS) gene on the development and lymphatic metastases of esophageal squamous-cell carcinoma (ESCC).

METHODSPeripheral leucocyte DNA was extracted from 232 ESCC patients and 348 age- and sex-matched healthy controls. TS 5'UTR and 3'UTR genotyping in all study subjects was performed by PCR fragment analysis and PCR-RFLP analysis, respectively.

RESULTSThe distribution of TS 5'UTR and 3'UTR variants in ESCC patients was not significantly different from that in healthy controls. However, individuals with 6 bp+/6 bp+ and 3R/3R genotypes significantly reduced the risk to ESCC development compared to those with other genotype combinations (adjusted OR = 0.32, 95% CI = 0.08-0.92). In addition, the frequency of 2R/3R genotype in ESCC patients with lymphatic metastases (40%) was significantly higher than that in lymph node negative cases (14.7%) (chi(2) = 10.11, P = 0.001). Compared to 3R/3R genotype, the 2R/3R genotype significantly increased the risk of lymphatic metastases in ESCC (adjusted OR = 3.68, 95% CI = 1.54-8.93).

CONCLUSIONThe genotyping of TS 5'UTR and 3'UTR polymorphisms might be used as a stratification maker for predicting susceptibility to ESCC. The TS 5'UTR 2R/3R genotype might be a candidate molecular marker to predict the potential of lymphatic metastases in ESCC.

Biomarkers, Tumor ; Carcinoma, Squamous Cell ; genetics ; secondary ; Esophageal Neoplasms ; genetics ; pathology ; Genotype ; Humans ; Lymphatic Metastasis ; Polymorphism, Genetic ; Tandem Repeat Sequences ; genetics ; Thymidylate Synthase ; genetics

Objective:To investigate the prognosis of childhood adrenoleukodystrophy (ALD) with cognitive disorder after haploidentical allogenic hematopoietic stem cell transplantation (haplo-HSCT), and to identify risk factors affecting the prognosis.Methods:It was a single-center retrospective study involving 31 ALD children receiving haplo-HSCT in Peking University People′s Hospital from January 2014 to October 2022.Survival analysis was performed by Kaplan-Meier method. Cox regression analysis was performed to identify risk factors for the prognosis of childhood ALD following haplo-HSCT. Results:Among the 31 children with ALD, 1 case died of cardiogenic shock during the transplantation, and the remaining had a successful haplo-HSCT.Ten children with ALD had cognitive disorder before haplo-HSCT, including 3 cases with the minimal LOES score ≥10 points and 8 cases with the Neurologic Function Score (NFS)>0 point before haplo-HSCT.Six children had major functional disability (MFD) and 2 cases died due to progression of ALD after haplo-HSCT.Twenty children did not have cognitive disorder before haplo-HSCT, of whom 3 cases had the LOES score≥10 points and 6 cases had NFS>0 before haplo-HSCT.Four children had MFD and 2 cases died due to progression of ALD after haplo-HSCT.For ALD patients without cognitive disorder after haplo-HSCT, the 3-year and 5-year survival rate were 100.0% and 72.9%, respectively, and the 5-year MFD-free survival was 61.6%.For ALD patients with cognitive disorder after haplo-HSCT, the 3-year survival rate was 83.3%.Compared with ALD patients with the LOES score<10 points before haplo-HSCT, those with the LOES score≥10 points had 9.243 times the risk of developing MFD after haplo-HSCT ( P=0.024, 95% CI: 1.332-64.127). Compared with ALD patients without cognitive disorder before haplo-HSCT, ALD patients with cognitive disorder had 9.749 times the risk of developing MFD after haplo-HSCT ( P=0.023, 95% CI: 1.358-66.148). Conclusions:Cognitive disorder and LOES score≥10 points before haplo-HSCT are risk factors for developing MFD in children with ALD following haplo-HSCT.

Objective:To explore the effect and mechanism of esculentoside on lipopolysaccharide-induced mastitis in mice. Method:Female BALB/c mice were randomly divided into control group, model group, dexamethasone group (DEX, 5.0 mg·kg^-1) and esculentoside group (50, 25, 12.5 mg·kg^-1). The mastitis model of postpartum female mice (BALB/c) induced by lipopolysaccharide (LPS) was used to analyze the pathological conditions of breast tissue and the activity of recombinant myeloperoxidase（MPO）, factor content and oxidative stress level. Western blot was used to evaluate the effect of esculentoside on Toll-like receptor4 (TLR4)/nuclear transcription factor (NF-κB) signaling pathway proteins. Result:Compared with the normal group, the breast tissue of the model group had typical mastitis changes, such as hyperemia and congestion, the level of MPO increased, and the expression levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) increased significantly (P<0.01). Compared with LPS model group, esculentoside groups could significantly improve the inflammatory damage of mammary gland tissue, reduce the secretion of neutrophils and the activity of MPO, the expression levels of pro-inflammatory factors TNF-α, IL-1β and IL-6 were also significantly down-regulated, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were increased, while the level of malondialdehyde (MDA) was decreased, and the activation of TLR4/NF-κB signaling pathway was inhibited by esculentoside（P<0.05，P<0.01）. Conclusion:Esculentoside have a protective effect on lipopolysaccharide-induced mastitis in mice, which may be related to the inhibition of TLR4/NF-κB signaling pathway protein expression.