Objective:To observe the therapeutic efficacy and prognosis of daratumumab combined with chemotherapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT) followed by daratumumab and lenalidomide maintenance treatment for primary plasma cell leukemia (PCL).Methods:The clinical data of a patient with primary PCL admitted to the First People's Hospital of Yunnan Province in January 2020 were retrospectively analyzed, and relevant literatures were reviewed.Results:The patient was diagnosed with primary PCL and treated with daratumumab combined with BD (bortezomib + dexamethasone) for 1 course and BCDD (bortezomib + cyclophosphamide + liposomaldoxorubicin + dexamethasone) for two courses. The patient was treated with daratumumab combined with allo-HSCT after complete remission. The donor cells were successfully implanted and the chimerism rate of donor cells was 94.36% without acute graft-versus-host disease reaction. And then the patient received intermittent maintenance therapy of daratumumab combined with low dose lenalidomide after transplantation, and the current remission period after transplantation reached 4 months.Conclusions:Daratumumab combined with chemotherapy bridging to allo-HSCT followed by daratumumab and lenalidomide may improve the prognosis of primary PCL and prolong survival time.

Objective To investigate the DNA staining efficiencies of 4 kinds of nucleic acid fluorescent dyes,including EB,SYBR Green Ⅰ,Gold View and AO,in single-cell gel electrophoresis (SCGE) assay,and the possibility to use a new nucleic acid fluorescent dye instead of EB.Methods The peripheral blood lymphocytes from healthy individuals were isolated and treated with 0,20,40,60 and 80 μg/mL of H2O2,respectively.Then,the DNA damages of lymphocytes were detected by the neutral SCGE assay.The DNA was stained with EB,SYBR Green Ⅰ,Gold View and AO dyes,respectively,and the staining results were observed and compared under a fluorescence microscope.In addition,the percentages of tail DNA (％ Tail DNA) from different staining methods were analyzed and compared.Results The results of SCGE showed that SYBR Green Ⅰ,Gold View and AO staining could well reflect the DNA damages of lymphocytes,and that the optimal concentrations for SYBR Green Ⅰ,Gold View and AO were 1 ×-5 ×,2 ×-5 × and 2-5 μg/mL,respectively.The regression coefficients for EB,SYBR Green Ⅰ,Gold View and AO were 2.71,2.81,2.73 and 2.75,respectively,which indicated that there was consistent dyeing effect between them.The results of ％ Tail DNA were stable with in 48 hours,and there was no significant difference between the 4 fluoresent dyes (P ＞ O.05).The inter-assay coefficients of variation (CVs) of SYBR Green Ⅰ,Gold View and AO were 6.92％,7.10％ and 8.25％,respectively,which were superior to that of EB (8.35％).The intra-assay CVs of SYBR Green Ⅰ and Gold View were 3.07％ and 2.74％,respectively,which were superior to that of EB (3.59％).Conclusion SYBR Green Ⅰ,Gold View and AO may be used for the nucleic acid fluorescent staining,and especially Gold View is more suitable for instead of EB in SCGE.

Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P ＜ 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] ％,[7.84 ± 0.68] ％,[8.40 ± 0.50] ％ and [14.03 ± 1.03] ％) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] ％,F =35.879,P ＜ 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P ＜ 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P ＜ 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P ＜0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.

Objective: To investigate the efficacy and safety of IA regimen which contains idarubicin (IDA) 8 mg/m², 10 mg/m² or 12 mg/m² as induction chemotherapy for adult patients with de-novo acute myeloid leukemia (AML) . Methods: A total of 1 215 newly diagnosed adult AML patients, ranging from May 2011 to March 2015 in the First Affiliated Hospital of Soochow University and other 36 clinical blood centers in China were enrolled in the multicenter, single-blind, non-randomized, clinical controlled study. To compare the response rate of complete remission (CR) , adverse events between different dose idarubicin combined with cytarabine (100 mg/m²) as induction chemotherapy in newly diagnosed patients of adult AML. Results: Of 1 207 evaluable AML patients were assigned to this analysis of CR rate. The CR rates of IDA 8 mg/m² group, IDA 10 mg/m² group and IDA 12 mg/m² group were 73.6% (215/292) , 84.1% (662/787) and 86.7% (111/128) , respectively (P<0.001) . After adjusted for age, blast ratio of bone marrow, FAB classification and risk stratification, the odds ratios (95% CI) of IDA 10 mg/m² group and IDA 12 mg/m² group were 0.49 (0.34-0.70) and 0.36 (0.18-0.71) , as compared with the IDA 8 mg/m² group (P<0.001, P=0.003) . In the intermediate and favorable groups, CR rates was 76.5% (163/213) , 86.9% (506/582) and 86.1% (68/79) in different doses of IDA (P=0.007) . Interestingly, IA regimen with IDA 10 mg/m² was the only beneficial factor affecting CR in this group after adjusted for age, blast ratio of bone marrow and FAB classification[OR=0.47 (95% CI 0.31-0.71) , P<0.001]. CR rates in adverse group was 50.0% (18/36) , 60.6% (43/71) and 81.8% (18/22) respectively (P=0.089) . However, the odds ratios (95% CI) of IDA 12 mg/m² when compared with the IDA 8 mg/m² was 0.22 (0.06-0.80) , after adjusted for age, blast ratio of bone marrow and FAB classification. The median time (days) of neutrophil count less than 0.5×10⁹/L in IDA 8 mg/m² group, IDA 10 mg/m² group and IDA 12 mg/m² group were 14 (11-18) , 15 (11-20) and 18 (14-22) , respectively (P=0.012) and of platelet count lower than 20×10⁹/L were 14 (7-17) , 15 (11-20) and 17 (15-21) , respectively (P=0.001) . The incidences of lung infection in the three groups were 9.8%, 13.5% and 25.2%, respectively (P<0.001) . Conclusions For young adult patients (aged 18-60 years) with AML in China, intensifying induction therapy with idarubicin 10 mg/m² is clinically superior to IDA 8 mg/m² and IDA 12 mg/m² in favorable intermediate AML subgroup. However, idarubicin 12 mg/m² is more suitable to adverse AML subgroup.

Although efficient nonsurgical transfer of embryos in mice would provide many advantages over a surgical method, the low success rate of nonsurgical transfer has hampered its acceptance and use. Here, a plastic catheter was used to mimic embryo transfer process and then the transfer efficiency was evaluated by intrauterine trypan blue dye dispersion. Also 3.5-day blastocysts from natural pregnant mice were transferred through cervix into uterine horns. The results show that 70.9% of CD-1 mouse 3.5-day blastocysts transferred into unilateral uterine horns of pseudopregnant 2.5-day recipients can be developed to live newborns, and an efficient mouse nonsurgical embryo transfer technique was established. The technique was simple, rapid, inexpensive, unlikely to get contaminated, ethical and do not need specialized apparatus, and can completely replace surgical embryo transfer techniques. Moreover, the mouse nonsurgical embryo transfer technique provides a research model for human and other large animal embryo transfer.
Animals ; Blastocyst ; Embryo Transfer ; methods ; veterinary ; Female ; Mice ; Pregnancy

Bacterial two-hybrid system is a newly developed method for studying protein-protein interactions. However, in our studies of the interaction of regulatory proteins in Streptomyces, it was found that the bacterial two-hybrid system is not sensitive enough by the blue-and-white selection on X-gal plate. To overcome this drawback, the reason of false positive clone was firstly determined, which was the disturbance of other direct or indirect regulation on lacZ promoter. Then the disturbance was diluted by introducing multicopy lacZ promoter, which drive another reporter gene gfp. By such design, the sensitivity of the modified bacterial two-hybrid system was significantly inproved and the two different reporters also help to decrease the rate of the false positive clones. Further the evaluation of the modifiedd bacterial two-hybrid system indicated that the sensitivity was significantly improved.
Bacteria ; Genes, Reporter ; Promoter Regions, Genetic ; Protein Interaction Mapping ; methods ; Two-Hybrid System Techniques

Gene expression exhibits temporal and spatial patterns to response environmental changes and growth cycle. Gene expression is under strict control at different levels among which control at transcription level is the predominant mode, especially in prokaryotes. In this review, we summarized the new developments of methods used in transcriptional studies, including modifications and improvements of the classic methods, such as gel-shift assay, DNA foot printing, and in vivo reporter system. In addition, we introduced examples to apply new methods, such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize protein-DNA, ligand-protein, and ligand-protein-DNA interactions. The collection of these methods and their application could guide and accelerate relevant studies.
Calorimetry ; DNA Footprinting ; Gene Expression ; Ligands ; Proteins ; Surface Plasmon Resonance ; Transcription, Genetic

Penicillin expandase, also known as deacetoxycephalosporin C synthase (DAOCS), is an essential enzyme involved in cephalosporin C biosynthesis. To evaluate the catalytic behaviors of penicillin expandase under high penicillin G concentration and to identify mutants suitable for industrial applications, the specific activities of wild-type DAOCS and several mutants with increased activities toward penicillin G were determined by HPLC under high penicillin G concentrations. Their specific activity profiles were compared with theoretical predictions by different catalytic dynamics models. We evaluated the specific activities of wild-type DAOCS and previous reported high-activity mutants H4, H5, H6 and H7 at concentrations ranging from 5.6 to 500 mmol/L penicillin G. The specific activities of wild-type DAOCS and mutant H4 increased as penicillin G concentration increased, but decreased when concentrations of substrate go above 200 mmol/L. Other mutants H5, H6 and H7 showed more complex behaviors under high concentration of penicillin G. Among all tested enzymes, mutant H6 showed the highest activity when concentration of penicillin G is above 100 mmol/L. Our results revealed that the substrate inhibition to wild-type DAOCS' by penicillin G is noncompetitive. Other DAOCS mutants showed more complex trends in their specific activities at high concentration of penicillin G (>100 mmol/L), indicating more complex substrate inhibition mechanism might exist. The substrate inhibition and activity of DAOCS mutants at high penicillin G concentration provide important insight to help select proper mutants for industrial application.
Catalysis ; Intramolecular Transferases ; genetics ; Mutation ; Penicillin G ; pharmacology ; Penicillin-Binding Proteins ; genetics ; Streptomyces ; enzymology ; genetics

Streptomycetes produce many antibiotics and are important model microorgansims for scientific research and antibiotic production. Metabolomics is an emerging technological platform to analyze low molecular weight metabolites in a given organism qualitatively and quantitatively. Compared to other Omics platform, metabolomics has greater advantage in monitoring metabolic flux distribution and thus identifying key metabolites related to target metabolic pathway. The present work aims at establishing a rapid, accurate sample preparation protocol for metabolomics analysis in streptomycetes. In the present work, several sample preparation steps, including cell quenching time, cell separation method, conditions for metabolite extraction and metabolite derivatization were optimized. Then, the metabolic profiles of Streptomyces coelicolor during different growth stages were analyzed by GC-MS. The optimal sample preparation conditions were as follows: time of low-temperature quenching 4 min, cell separation by fast filtration, time of freeze-thaw 45 s/3 min and the conditions of metabolite derivatization at 40 degrees C for 90 min. By using this optimized protocol, 103 metabolites were finally identified from a sample of S. coelicolor, which distribute in central metabolic pathways (glycolysis, pentose phosphate pathway and citrate cycle), amino acid, fatty acid, nucleotide metabolic pathways, etc. By comparing the temporal profiles of these metabolites, the amino acid and fatty acid metabolic pathways were found to stay at a high level during stationary phase, therefore, these pathways may play an important role during the transition between the primary and secondary metabolism. An optimized protocol of sample preparation was established and applied for metabolomics analysis of S. coelicolor, 103 metabolites were identified. The temporal profiles of metabolites reveal amino acid and fatty acid metabolic pathways may play an important role in the transition from primary to secondary metabolism in S. coelicolor.
Gas Chromatography-Mass Spectrometry ; Metabolic Networks and Pathways ; Metabolome ; Metabolomics ; methods ; Streptomyces coelicolor ; metabolism

Spinosad represents a new class of insecticides produced by Saccharopolyspora spinosa. To understand the transcription of the spinosad biosynthetic gene cluster, two promoter detection plasmids based on different reporter genes were constructed and used to detect 9 promoters in the spinosad biosynthetic gene cluster. In addition, the temporal transcriptional profiles of the corresponding genes controlled by the 9 promoters, together with 4 genes outside of the spinosad cluster but are required for the synthesis of sugars in spinosad, were examined by real-time PCR. The results indicate that the 9 spinosad biosynthetic genes were highly expressed at the stationary phase, which coincides with the accumulation of spinosad in the fermentation broth. Of particular note is that the transcription of the 4 sugar synthetic genes showed higher level at the exponential phase, suggesting the expression of sugar synthetic genes is not correlated with the spinosad synthetic genes. The data suggest that spinosad biosynthesis could be improved by engineering the expression pattern of the sugar synthetic genes that lie outside the spinosad gene cluster.
Drug Combinations ; Insecticides ; metabolism ; Macrolides ; metabolism ; Multigene Family ; Plasmids ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Saccharopolyspora ; genetics ; metabolism