The efficient production of L-glutamate is dependent on the product's rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on L-glutamate synthesis and transport in SCgGC7, a constitutive L-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of L-glutamate producers. Next, temperature-sensitive L-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for L-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of L-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the L-glutamate excretion and metabolic flux for both L-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of L-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high L-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for L-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.
Corynebacterium glutamicum/genetics* ; Glutamic Acid/biosynthesis* ; Fermentation ; Metabolic Engineering ; Bacterial Proteins/metabolism* ; Bioreactors/microbiology* ; Gene Knockout Techniques

Objective:To investigate the efficacy and prognosis of CLAG±DAC (Clofarabine, Cytarabine, G-CSF±Decitabine) chemotherapy in patients with relapsed/refractory acute myeloid leukemia (R/R AML) .Methods:Continuous cases of R/R AML treated with the CLAG+DAC protocol or CLAG alone at the First Affiliated Hospital of Soochow University from January 2017 to December 2021 were retrospectively analyzed. The baseline characteristics, individual treatment regimen, treatment effect, disease progression, and survival status of patients were recorded. The factors influencing the efficacy of the CLAG±DAC chemotherapy regimens were analyzed, and the overall survival (OS) time after reinduction was calculated using the Kaplan-Meier method.Results:This study included a total of 53 patients, with 33 male patients and an average age of 40.6 years. Thirty-three patients achieved complete remission (CR+CRi) of the disease after the CLAG±DAC chemotherapy regimen and six patients achieved partial remission (PR), while 14 did not. Thirty-two patients eventually underwent hematopoietic stem cell transplantation, and the median OS of the patients was 55.9 months until follow-up. Patients with disease remission after the application of the CLAG±DAC chemotherapy had a significantly longer survival time than those without remission ( P<0.001). The results of the multifactorial analysis have revealed that combined DAC ( OR=4.60, 95% CI 1.14-23.5, P=0.04) and DNMT3A mutation ( OR=0.14, 95% CI 0.01-0.89, P=0.05) were the factors influencing the efficacy of the CLAG±DAC chemotherapy regimen. The remission rate was relatively higher in patients with R/R AML combined with FLT3-ITD mutation by applying the DAC+CLAG regimen ( OR=10.84, 95% CI 1.48-288.50, P=0.04) . Conclusion:The CLAG±DAC regimen is considered effective in patients with R/R AML, whereas decitabine combined with the CLAG regimen is more suitable for patients with R/R AML combined with FLT3-ITD mutation.

Objective To examine the value ofprocalcitonin (PCT) in differential diagnosis of bloodstream infections (BSI) caused by coagulase-negative Staphylococcus (CNS) from contamination in Department of Hematology.Methods One hundred and fifty-six patients with bloodstream or intravenous catheter-related CNS infection were included in this study.The patients were treated in Department of Hematology,the First Affiliated Hospital of Soochow University during the period from January 2013 to December 2015.The patients were divided into CNS bloodstream infection group (n=66) and blood culture contamination group (n=90).The two groups were compared in terms of sex,age,diagnosis,length of hospital stay,duration of neutropenia,neutrophil count and lymphocyte count,peak fever,C-reactive protein (CRP) and PCT.The receiver operating characteristic (ROC) curve was plotted with SPSS 21.0 software to analyze the value of PCT in differential diagnosis.Results Age,sex,clinical diagnosis,length of hospital stay,duration of neutropenia,neutrophil count and lymphocyte count did not show significant difference between bloodstream infection group and contamination group (P＞0.05),while peak fever (P＜0.001),CRP (P=0.002) and PCT (P=0.018) were significantly higher in bloodstream infection group than in contamination group.ROC analysis indicated that PCT provided optimal discrimination between these two groups at cnt-offvalue of 0.374 μg/L,with sensitivity of 54.5％ and specificity of 94.4％.The area under the curve (AUC) was 0.830±0.032 (95％ CI:0.767-0.893,P＜0.001).Conclusions PCT may be a good marker for differentiating CNS bloodstream infection from contamination with higher specificity than the commonly used marker CRP.This finding may help clinicians reduce the overuse of antibiotics.

Objective: To investigate the efficacy of sequential treatment with first-line administration of second-generation tyrosine kinase inhibitors (TKI) and first-generation TKI (imatinib) in patients with Ph⁺ acute lymphoblastic leukemia (Ph⁺ ALL) followed by allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Retrospective analysis of clinical features and prognosis of 76 newly diagnosed Ph ⁺ALL patients from June 2011 to December 2015 treated by allo-HSCT combined with first-line administration of second-generation or first-generation TKI was performed and the efficacy compared. Results: Of 76 Ph⁺ ALL patients, first-generation TKI was administered in 57 cases, second-generation TKI in 19 cases, including 10 cases of nilotinib and 9 cases of dasatinib. There was no significant difference in age, WBC counts, additional chromosomal abnormalities, time form diagnosis to transplantation, transplantation type, conditioning regimen or TKI initiation time between the two groups. Complete remission (CR) rates at the fourth week of induction therapy in first-generation TKI group and second-generation TKI group was 93.0% and 94.7% (P=1.000), respectively. Major molecular response (MMR, BCR-ABL/ABL reduce 3 log) rates meanwhile were 46.0% and 40.0% (χ²=0.169, P=0.681). Relapse rates before transplantation were 14.0% and 10.5% (P=1.000). MMR rates before transplantation were 54.4% and 68.2% (χ²=1.152, P=0.283). The 2-year overall survival (OS) rates of first-generation and second-generation TKI group were 62.0% and 94.7% (χ²=5.765, P=0.016), 2-year event-free survival (EFS) rates were 46.3% and 84.2% (χ²=5.644, P=0.018), respectively. Univariate analysis showed that second-generation TKI could improve OS (HR=0.126, 95%CI 0.017-0.939, P=0.043). Multiple factors analysis showed that second-generation TKI (HR=0.267, 95%CI 0.081-0.873, P=0.029) and MMR before transplantation (HR=0.496, 95%CI 0.254-0.968, P=0.040) were good independent prognostic factors of EFS. Conclusions There was significant difference in the efficacy of second-generation TKI and first-generation TKI for Ph⁺ ALL patients treated by allo-HSCT. First-line administration of second-generation TKI showed better efficacy than that of first-generation TKI for Ph⁺ ALL patients.

Objective: To explore the efficacy and safety of chimeric antigen receptor T (CAR-T) cells in the treatment of central nervous system leukemia (CNSL). Methods: Two leukemia patients with CNSL were treated with CD19-CAR-T cells. The process and results of the entire treatment is reported and related literature review is conducted. Results: The patients were diagnosed as acute myeloid leukemia (AML)-M₂ with B lymphoid antigen expression and B cell acute lymphoblastic leukemia(B-ALL) by morphology and immunophenotype assay. The immunophenotype was consistent with the abnormal manifestations of AML-M₂ and B-ALL. Their clinical manifestations and laboratory tests met the diagnostic criteria of CNSL. The diagnosis was clear and the two patients were treated with CD19-CAR-T cell immunotherapy. Central nervous system symptoms were relieved. The imaging abnormalities of patient one has disappeared but cytokines release syndrome (CRS) occurred during the treatment. Cerebrospinal fluid of patient two was negative and no obvious CRS reaction was found. Conclusions CAR-T cell immunotherapy is likely to induce the remission of CNSL and improve the prognosis.

Objective: To investigate the efficacy of first-line administration of generic dasatinib or first-generation TKI (imatinib) in patients with Philadelphia chromosome positive acute lymphoblastic leukemia (Ph⁺ ALL) treated by hematopoietic stem cell transplantation (HSCT). Methods: Clinical features and prognoses of 63 newly diagnosed Ph⁺ ALL patients from Jan 2014 to June 2017 treated by HSCT combined with first-line administration of generic dasatinib or imatinib were retrospective analyzed. Results: Of 63 Ph⁺ ALL patients, 31 cases were administered generic dasatinib, and the other 32 ones imatinib. Complete remission (CR) rates at the fourth week of induction therapy in generic dasatinib and imatinib groups were 96.8% and 93.8% (P=1.000) , respectively. Meanwhile major molecular response (MMR; BCR-ABL/ABL reduce 3log) rates were 41.9% and 43.8% (χ²=0.021, P=0.884), respectively. Relapse rates before transplantation were 6.5% and 12.5% (P=0.672), respectively. MMR rates before HSCT were 83.9% and 68.8% (χ²=1.985, P=0.159), respectively. The 20-monthes overall survival (OS) rates of generic dasatinib and imatinib groups were 95.5% and 76.5% (χ²=0.990, P=0.320) respectively; 20-monthes event-free survival (EFS) rates were 93.5% and 61.4% (χ²=5.926, P=0.015), respectively. Statistically significant differences of EFS were reached. Multiple factors analysis showed that generic dasatinib (HR=0.201, 95% CI 0.045-0.896, P=0.035) and MMR before transplantation (HR=0.344, 95% CI 0.124-0.956, CI=0.041) could improve EFS. Conclusions First-line administration of generic dasatinib could improve EFS for Ph⁺ALL patients treated by HSCT when compered with imatinib.

OBJECTIVETo clarify the clinical, cytogenetical and molecular characteristics and prognosis of Ph(+) ALL patients with ABL kinase domain mutations (ABL-KDMs), and to evaluate the therapeutic value of allogeneic hematopoietic stem cell transplantation (allo-HSCT) combined with tyrosine kinase inhibitor (TKI) in these patients.

METHODSRetrospective analysis of clinical features, molecular genetic characteristics, mutation distribution and prognosis of newly diagnosed Ph(+) ALL patients with ABL-KDMs from February 2010 to August 2014 were performed, and the efficacy of treatment regimen of allo-HSCT combined with different TKIs was compared.

RESULTSOf 88 Ph(+) ALL patients during maintenance treatment stage for ABL-KDMs monitoring, mutation was detected in 42 patients with median time of 8 months from diagnosis to mutation occurrence. The median age of mutation group was 40-year-old, older than that of non-mutation group (32.5-year-old) (P=0.023). The incidence of complex chromosome abnormality of mutation group was higher than that of non-mutation group (P=0.043), with alternations in chromosome 7, 5 and +Ph more frequently observed. There were 21 types of mutation at 18 locations detected, with T315I mutation ranking the top followed by E255K/V, Y253H/F and E459K. Mutation group featured no significant difference in complete remission (CR) rate in contrast to nonmutation group, but was remarkably lower in major molecular remission (MMR) rate than non-mutation group. The 2 year and 5 year overall survival rate of mutation group was 45.4% and 35.0% respectively, much shorter than that of non-mutation group (67.8% and 63.3%), (P=0.047). The median survival of patients with T315I and E255K/V was 19 and 10 months, significantly shorter than that of patients with other mutations. Among the 42 patients with mutations, 14 underwent allo-HSCT, and the median survival was 29 months, longer than that of patients received chemotherapy alone (17 months) (P=0.024). Fourteen allo-HSCT patients were given nilotinib or dasatinib at the time of mutation occurrence, and there was no significant difference in the overall survival in contrast to patients who continue to take imatinib.

CONCLUSIONSABL kinase domain mutations are closely related to the older age and high genomic instability in the newly diagnosed Ph(+) ALL patients. Mutation types showed diversity and complexity, which remarkably affected patients' prognosis and survival. T315I and E255K mutations account for more than half of all cases, characterized by a less favorable prognosis. Currently, allo-HSCT is the only method that has the potential of elongating life expectancy, but the utility of second-generation TKI during relapse does not necessarily have an edge on survival over imatinib.

Chromosome Aberrations ; Dasatinib ; therapeutic use ; Hematopoietic Stem Cell Transplantation ; Humans ; Imatinib Mesylate ; therapeutic use ; Mutation ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; genetics ; Prognosis ; Protein Kinase Inhibitors ; therapeutic use ; Proto-Oncogene Proteins c-abl ; genetics ; Pyrimidines ; therapeutic use ; Remission Induction ; Retrospective Studies ; Survival Rate

OBJECTIVETo investigate the effects of ursolic acid on T cell lymphoma cell lines-Hut-78 cells and its mechanism.

METHODSInhibition of Hut-78 cells proliferation by ursolic acid at different concentration (10, 20, 40 and 80 μmol/L) for different incubation time (4, 12, 24, 48 and 72 h)was examined by MTT method, and early apoptosis by flow cytometry. The protein expressions of p65, p50, p52 and p100, and caspase-8, caspase-3 and caspase-9 were detected by Western blot. VEGF and COX-2 mRNA expressions were measured by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSIt was showed that ursolic acid inhibited proliferation of Hut-78 cells (P<0.05). Apoptosis of Hut-78 cells was induced by 10, 20, 40 and 80 μmol/L ursolic acid treatment (P<0.01). Likewise, expression of p65 and p50 proteins were down-regulated by ursolic acid treatment (10, 20, 40 and 80 μmol/L) (P<0.01), but there was no significant change in the expression of p52 and p100. Moreover, ursolic acid could up-regulate expression of caspase-8, caspase-3 and caspase-9 protein (P<0.01). RT-PCR examination showed that VEGF and COX-2 mRNA expression decreased by ursolic acid treatment.

CONCLUSIONInhibition of Hut-78 cells proliferation may be related to ursolic acid induced apoptosis through h death receptors and mitochondrial pathways. NF-κB classical signal pathway may be one of its mechanisms, and VEGF and cox-2 may also be involved.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphoma, T-Cell ; NF-kappa B ; Signal Transduction ; Triterpenes

OBJECTIVETo explore the clinical and laboratory characteristics in favor of the diagnosis of Ph/BCR-ABL positive acute myeloid leukemia (Ph/BCR-ABL⁺ AML).

METHODSRetrospectively analyzed the clinical and laboratory characteristics of 12 Ph/BCR-ABL⁺ AML cases from Feb, 2006 to Dec, 2013, with classic myeloid blast crisis of chronic myeloid leukemia (CML-MBC) as control, and followed-up the survival in these two cohorts of patients.

RESULTSThe median age of 12 Ph/BCR-ABL⁺ AML was 27.5 years, 10 cases (83.3%) showed non/mild splenomegaly, and mainly comprised of M₂ and M₄ subtypes according to FAB classification. The median number of basophils and megakaryocytes in peripheral blood and bone marrow was lower than that of CML-CBC patients. All the cases expressed myeloid antigens, 8 cases (66.7%) expressed CD34, 11 cases were detected with t(9;22), 5 cases (45.5%) with additional chromosomal abnormalities, including 1 case of inv(16). All the cases had BCR-ABL transcripts at diagnosis:3(25.0%) cases were e1a2 type and the remaining was b2a2/b3a2type, among which 1 case coexpressed CBFβ-MYH11. Two out of 6 cases existed AML-like mutations:1 case of CEBPA and the other of FLT3-TKD. For all the patients, 7 cases achieved complete remission (CR), including 6 out of 7 cases receiving induction chemotherapy combined with tyrosine kinase inhibitor (TKI) achieved CR, and 1 out of 3 cases receiving chemotherapy alone achieved CR. The median overall survival was 16.5 months, that of allo-HSCT group was 33.5 months, which was higher than that of non-HSCT group (5.5 months).

CONCLUSIONThe expression of e1a2 type BCR-ABL, the coexpression of fusion genes which were more common in AML, the existence of AML-like mutations were all indications of a de novo Ph/BCR-ABL⁺ AML. Low induction CR rate and short survival of Ph/BCR-ABL⁺ AML implied that chemotherapy combined with TKI and followed by allo-HSCT in CR was the only effective way to improve their survival.

Adult ; Blast Crisis ; Chromosome Aberrations ; Fusion Proteins, bcr-abl ; Humans ; Leukemia, Myeloid, Acute ; Oncogene Proteins, Fusion ; Protein Kinase Inhibitors ; Retrospective Studies

OBJECTIVETo investigate the mechanism of di-2-ethylhexyl phthalate (DEHP) and cypermethrin (CYP) inducing gonadal dysgenesis in prepubertal male rats.

METHODSA total of 40 healthy 3-week-old specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into four groups: control group (corn oil), DEHP group (500 mg/kg, dissolved in corn oil), CYP group (80 mg/kg, dissolved in corn oil), and combined exposure group (exposed to 500 mg/kg DEHP and 80 mg/kg CYP, dissolved in corn oil). Rats were treated by gavage administration once a day for 30 days. Twenty-four hours after the last exposure, the animals were sacrificed. The body weight and the wet weight of testis were determined, and the weight coefficient of testis was calculated. Radioimmunoassay was used to determine serum testosterone level. Ultrastructural-level histopathological changes of the testis were examined by transmission electron microscopy. The mRNA and protein expression of follicle stimulating hormone receptor (FSHR), androgen binding protein (ABP), inhibin beta-B (INHBB) and vimentin (VIM) were analyzed by real-time PCR and Western blot, respectively. Factorial design analysis of variance was used to compare differences between groups; interaction diagrams were used to determine the interaction between DEHP and CYP.

RESULTSCompared with those of the control group, the testis weights and testis coefficients of the DEHP, CYP, and combined exposure groups significantly decreased by 39.3-59.2%and 19.7-58.6%, respectively, and all exposure groups showed significant reductions in serum level of testosterone, ranging from 49.1% to 62.7% (P < 0.05 or P < 0.01). And all the exposure groups showed different levels of ultrastructural damages in the testes. Compared with that in the control group, the mRNA expression of FSHR, ABP, INHBB, and VIMin the DEHP group was down-regulated by 1.72, 2.64, 2.83 and 1.79 times, and their protein levels were significantly reduced by 65.2%, 53.7%, 70.1%, and 51.9% (P < 0.05 or P < 0.01). Significant decreases in mRNA expression of ABP (down 1.72 times) and INHBB (down 2.06 times) were observed in the CYP group, and their protein levels decreased by 38.3% and 49.7%, respectively (P < 0.05). The combined exposure to both DEHP and CYP resulted in big decreases in the mRNA levels of FSHR (down 1.62 times), ABP (down 2.00 times), INHBB (down 2.35 times), and VIM (down 1.54 times) and protein levels of FSHR (down 52.1%), INHBB (down 53.9%), and VIM (down 58.8%) (P < 0.05). Factorial design analysis of variance showed that the combination of two substances had an antagonistic effect on the expression of ABP and INHBB (P < 0.05).

CONCLUSIONDEHP and CYP, alone or combined, can lead to gonadal dysgenesis in prepubertal male rats. Both of them can disrupt functional mRNA and protein expression in Sertoli cells to certain levels. The combination of DEHP and CYP shows antagonistic effects, and DEHP has a stronger reproductive toxicity than CYP.

Animals ; Diethylhexyl Phthalate ; toxicity ; Gonadal Dysgenesis ; chemically induced ; Male ; Pyrethrins ; toxicity ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; metabolism ; Testis ; cytology ; drug effects