Isolation of Aureimonas altamirensis from whole blood and whole genome sequence analysis / 中华微生物学和免疫学杂志

Objective:To understand the biological characteristics, identification methods, genome structure and clinical significance of a rare strain of Aureimonas altamirensis isolated from clinical blood sample. Methods:The culture and biochemical characteristics of a strain isolated from blood culture were observed. The routine biochemical identification methods, MALDI-TOF mass spectrometer and 16S rRNA gene sequencing were used to identify the isolate. A phylogenetic tree based on the 16S rRNA gene sequences of the isolate and related strains was constructed. The genome of the isolated strain was sequenced and assembled, and gene prediction and functional annotation were made using related software. Phylogenetic analysis based on 31 house-keeping genes and genome-wide average nucleotide identity (ANI) analysis were conducted between the isolate and other Aureimonas sp. strains.Results:The isolated bacteria were gram-negative bacillus positive for catalase, urease and oxidase. It grew slowly on blood plate and could not be reliably identified by automatic bacterial biochemical identification systems or MALDI-TOF mass spectrometry. Results of the 16S rRNA gene sequencing and phylogenetic tree showed that the 16S rRNA gene sequence of the isolated strain was highly homologous to the strains of Aureimonas altamirensis NML070722 and IARI-ABL-26 (GenBank accession number: EU442518.1 and KC581669.1) in GenBank, and the gene sequence similarity was 99.93%. The total genome (National Microbiology Data Center genome accession number: NMDC60043566) length was 4 332 458 bp and GC content was 65.14%. There were 4 088 protein-coding genes and functional gene annotation showed that functional genes were mainly enriched in protein, amino acid, carbohydrate transport and metabolic functional regions. Pathogenic gene analysis predicted two high reliable virulence factor genes, but no drug resistance genes. House-keeping gene phylogenetic tree analysis showed that this strain was highly homologous to Aureimonas altamirensis strains of DSM21988 and C2P003 (GenBank accession number: GCF 001463885.1 and GCF 000800175.1), but ANI analysis showed that its genome was significantly different from those of the two strains. Conclusions:A rare strain of Aureimonas altamirensis was isolated from clinical specimen in China. As the biological and genomic characteristics of Aureimonas altamirensis had not been fully recognized, it was difficult to be correctly identified by conventional methods. The pathogenicity of Aureimonas altamirensis to immunocompromised patients and the significance of isolation in clinical specimens might need more case studies.

Monte Carlo simulation study to optimize administration regimens of linezolid, teicoplanin and daptomycin for Staphylococcal bloodstream infections / 中华传染病杂志

Objective:To predict and evaluate the antibacterial efficacy of linezolid, teicoplanin and daptomycin against Staphylococci bloodstream infections with Monte Carlo simulation, and to optimize the clinical administration program. Methods:A total of 1 847 Staphylococci strains isolated from blood samples between January 2018 to December 2019 were collected with the help of the Blood Bacterial Resistant Investigation Collaborative System (BRICS). Minimum inhibitory concentrations (MIC) of linezolid and daptomycin were detected by broth dilution method, while MIC of teicoplanin were detected by agar dilution method. The dosage regimens of linezolid were 800 mg once daily, 500 mg once every 12 hours, 600 mg once every 12 hours and 600 mg once every eight hours. The dosage regimens of teicoplanin were 400 mg once every 12 hours, 600 mg once every 12 hours, 800 mg once every 12 hours, and 1 000 mg once every 12 hours. The dosage regimens of daptomycin were 4 mg·kg -1·d -1, 6 mg·kg -1·d -1, 8 mg·kg -1·d -1, 10 mg·kg -1·d -1and 12 mg·kg -1·d -1. The probability of target attainment (PTA) and cumulative fraction of response (CFR) of three different dosage regimens were calculated by Monte Carlo simulation. A dosage regimen with CFR≥90.0% was a reasonable choice for empirical antimicrobial therapy. Results:PTA of linezolid against Staphylococci when MIC≤0.500 mg/L at four dosage regimens (800 mg once daily, 500 mg once every 12 hours, 600 mg once every 12 hours and 600 mg once every eight hours) were all over 90.0%. When MIC was 1.000 mg/L, the PTA of linezolid against Staphylococci under the dosages of 500 mg once every 12 hours, 600 mg once every 12 hours and 600 mg once every eight hours were 92.2%, 96.6% and 97.6%, respectively. The CFR of the four dosage regimens of linezolid were 73.9%, 83.7%, 90.8% and 95.3%, respectively. When MIC≤1.000 mg/L, PTA of teicoplanin against Staphylococci were all 100.0% at four dosage regimens (400 mg once every 12 hours, 600 mg once every 12 hours, 800 mg once every 12 hours and 1 000 mg once every 12 hours). When MIC was 2.000 mg/L, the PTA of teicoplanin (800 mg once every 12 hours and 1 000 mg once every 12 hours) against Staphylococci were both 100.0%. The CFR of the four dosage regimens of teicoplanin were 90.8%, 92.8%, 93.5% and 94.6%, respectively. When MIC≤0.500 mg/L, PTA of daptomycin against Staphylococci under the five dosages of 4 mg·kg -1·d -1, 6 mg·kg -1·d -1, 8 mg·kg -1·d -1, 10 mg·kg -1·d -1 and 12 mg·kg -1·d -1 were all over 90.0%. When MIC was 1.000 mg/L, the PTA of daptomycin against Staphylococci under the three dosages of 8 mg·kg -1·d -1, 10 mg·kg -1·d -1 and 12 mg·kg -1·d -1were 96.9%, 100.0% and 100.0%, respectively. The CFR of the five dosage regimens of daptomycin against Staphylococci were 97.4%, 99.2%, 99.9%, 100.0% and 100.0%, respectively. Conclusions:Linezolid (600 mg once every 12 hours), teicoplanin (400 mg once every 12 hours) and daptomycin (4 mg·kg -1·d -1) can achieve satisfactory antibacterial activity for Staphylococci bloodstream infections.

MLVA genotyping characteristics of 13 strains of Brucella isolated from non-brucellosis epidemic area Lianyungang City of Jiangsu Province / 中华地方病学杂志

Objective:To study the genotyping characteristics of Brucella strains isolated from Lianyungang City (non-brucellosis epidemic area) of Jiangsu Province. Methods:Preliminary identification of 13 suspected strains of Brucella isolated from blood culture in Clinical Microbiology Laboratory of the First People's Hospital of Lianyungang City in 2018 was conducted; at the same time, the specific gene bcsp31 and insertion sequence IS-711 of Brucella were detected by quantitative real-time PCR (Real-time PCR), and the identification results were rechecked and typed. Multiple locus variable-number tandem repeat analysis (MLVA) was applied for genotyping, and the sequencing results were edited by Mega 4.0 software. Results:All the 13 strains were identified as Brucella by preliminary identification. Real-time PCR confirmed that all the 13 strains were Brucella melitensis. The results of MLVA showed that 13 strains of Brucella melitensis were divided into 12 genotypes and clustered in the "middle Mediterranean cluster". Among 13 strains of Brucella melitensis, 3 strains were biovar 1, 2 strains were biovar 2 and 8 strains were biovar 3. Conclusion:All the Brucella strains isolated from Lianyungang City are Brucella melitensis and the MLVA cluster is in the "middle Mediterranean cluster".

Screening and Identification of The Proteins Interacting With NLS-RARα Protein / 生物化学与生物物理进展

Acute promyelocytic leukemia (APL) is characterized by the generation of the prototypic promyelocytic leukemia-retinoicacid receptor alpha (PML-RARα), an oncogenic fusion protein due to chromosomal translocation. In a human myeloid cell line,PML-RARα is cleaved by neutrophil elastase (NE) to produce the mutational PML [nuclear localization signal (NLS) deletion andRARα (NLS-RARα, containing NLS of PML), both of which may play an important role in APL pathogenesis. The yeast two-hybridtechnique was used to screen the intracellular proteins interacting with NLS-RARα, which may be involved in NLS-RARα signaling. The NLS-RARα coding sequence was amplified by polymerase chain reaction method and was cloned into the bait plasmid pGBKT7vector, which, after the confirmation by sequencing, was transformed into yeast AH109 and the subsequent expression of bait plasmidwas proved by Western-blot. The transformed yeast AH109 was mated with yeast Y187 (containing leukemia cDNA library plasmidspACT2) in medium. Diploid yeast was plated on synthetic dropout nutrient medium containing X-α-gal for screening. After beingreintroduced into yeast AH109 and sequenced to verify the expression of ORF, eight positive colonies were obtained, among whichonecontaining JTV-1 was cloned. The interaction between NLS-RARα and JTV-1 was further supported by indirect immunofluorescence,GST pull-down and co-immunoprecipitation, respectively. These findings brought some new clues for the further exploration ofNLS-RARα signaling to APL.

Experimental study of K562 leukemia cells apoptosis induced by emodin / 重庆医科大学学报

Objective: To study the mechanism of K562 leukemia cells proliferation and apoptosis induced by emodin. Methods: Inhibitory effect of emodin on proliferation of K562 leukemia cells was assayed by MTT method. The morphologic changes of K562 cells were observed under microscop after Wight-Giemsa staining. Apoptosis was checked by Annexin V/PI. The changes of cell cycle and apoptosis were detected by flow cytometry. The activities of Caspase-3,8,9 were checked by chromatometry to analyze the apoptosis of K562 cells. Results: The proliferation of K562 leukemia cells was significantly inhibited by emodin. The IC50 of K562 cells inhibited with emodin for 24 h,48 h and 72 h were 80,50 and 40 ?mol/L respectively. Typical morphological changes of K562 cells were observed by microscopy. The proliferation of K562 cells was obviously inhibited in dose-dependent manner. In Annexin V/PI,emodin induced K562 cells toward apoptosis in dose-dependent manner(P

Construction and identification of bait expression vector of glucocorticoid receptor binding domain / 第三军医大学学报

Objective To construct the bait expression plasmid pGBKT7-GR of glucocorticoid receptor(GR)binding domain.Methods The fragments of GR binding domain was amplified by RT-PCR,and then was cloned into pMD18-T.After being verified by sequencing,it was subcloned into the bait expression vector pGBKT7.Then the bait vector pGBKT7-GR was transformed into AH109 yeast cells and the expression of the bait protein was analyzed by Western blot.Toxicity and self-activation of the bait protein were detected.Results GR binding domain was amplified and cloned into pMD18-T and pGBKT7 successfully.The bait vector was transformed into AH109 yeast cells successfully,without toxicity or self-activation.The expression of the bait protein was confirmed by Western blot.Conclusion The successful construction of bait expression vector of glucocorticoid receptor binding domain lays the foundation for constructing small molecule ligand yeast three-hybrid system.