Early Detection of Molecular Residual Disease and Risk Stratification for Children with Acute Myeloid Leukemia via Circulating Tumor DNA.

PURPOSE: Patient-tailored minimal residual disease (MRD) monitoring based on circulating tumor DNA (ctDNA) sequencing of leukemia-specific mutations enables early detection of relapse for pre-emptive treatment, but its utilization in pediatric acute myelogenous leukemia (AML) is scarce. Thus, we aim to examine the role of ctDNA as a prognostic biomarker in monitoring response to the treatment of pediatric AML. EXPERIMENTAL DESIGN: A prospective longitudinal study with 50 children with AML was launched, and sequential bone marrow (BM) and matched plasma samples were collected. The concordance of mutations by next-generation sequencing-based BM-DNA and ctDNA was evaluated. In addition, progression-free survival (PFS) and overall survival (OS) were estimated. RESULTS: In 195 sample pairs from 50 patients, the concordance of leukemia-specific mutations between ctDNA and BM-DNA was 92.8%. Patients with undetectable ctDNA were linked to improved OS and PFS versus detectable ctDNA in the last sampling (both P < 0.001). Patients who cleared their ctDNA post three cycles of treatment had similar PFS compared with persistently negative ctDNA (P = 0.728). In addition, patients with >3 log reduction but without clearance in ctDNA were associated with an improved PFS as were patients with ctDNA clearance (P = 0.564). CONCLUSIONS: Thus, ctDNA-based MRD monitoring appears to be a promising option to complement the overall assessment of pediatric patients with AML, wherein patients with continuous ctDNA negativity have the option for treatment de-escalation in subsequent therapy. Importantly, patients with >3 log reduction but without clearance in ctDNA may not require an aggressive treatment plan due to improved survival, but this needs further study to delineate.

Preparation of DES lignin-chitosan aerogel and its adsorption performance for dyes, catechin and epicatechin.

Herein, DES lignin was obtained by pretreatment of grapevine with a deep eutectic solvent (ChCl-LA). A novel chitosan-DES lignin composite aerogel material (CS-LIG aerogel) was prepared to adsorb methylene blue (MB), Congo red (CR), catechin (C), and epicatechin (EC). The CS-LIG aerogel was systematically characterized by modern technological instruments. It was demonstrated that the DES lignin was successfully incorporated and had an important effect on the morphological structure and adsorption of dyes and natural products in the aerogel. The adsorption kinetic models for both adsorbed CR and MB are pseudo-second-order models. Adsorption isotherms followed Langmuir for the adsorption of CR and Freundlich for the adsorption of MB. The π-π interaction and hydrogen bonding of DES lignin aromatic groups in CS-LIG aerogels were responsible for the adsorption of C and EC with 86.42 % and 90.85 % removal rates, respectively. This study opens a new avenue for the high-value utilization of DES lignin and the preparation of chitosan-based composites for the adsorption of dyes and purification of natural products.

Acidic and alkaline deep eutectic solvents (DESs) pretreatment of grapevine: Component analysis, characterization, lignin structural analysis, and antioxidant properties.

Deep eutectic solvents (DESs) have been extensively applied to pretreat lignocellulose; however, comparative research on acidic and alkaline DES pretreatment is relatively lacking. Herein, pretreatment of grapevine agricultural by-products with seven DESs were compared in terms of removal of lignin and hemicellulose and component analysis of the pretreated residues. Among the tested DESs, both acidic choline chloride-lactic (CHCl-LA), and alkaline potassium carbonate-ethylene glycol (K2CO3-EG) were effective in delignification. Thereafter, the CHCl-LA and K2CO3-EG extracted lignin was compared by analyzing their physicochemical structure changes and antioxidant properties. The results showed that the thermal stability, molecular weight, and phenol hydroxyl percentage of CHCl-LA lignin were inferior to K2CO3-EG lignin. It was found that the high antioxidant activity of K2CO3-EG lignin was mainly attributed to the abundant phenol hydroxyl, guaiacyl (G), and para-hydroxy-phenyl (H). By comparing acidic and alkaline DES pretreatments and their lignin nuances in biorefining, novel insights are derived for the scheduling and selection of DES for lignocellulosic pretreatment.

[Clinical features and prognosis of juvenile myelomonocytic leukemia: an analysis of 63 cases]. / 63ä¾‹å¹¼å¹´åž‹ç²’å•æ ¸ç»†èƒžç™½è¡€ç— çš„ä¸´åºŠç‰¹å¾ä¸Žé¢„åŽåˆ†æž.

OBJECTIVES: To investigate the clinical features of juvenile myelomonocytic leukemia (JMML) and their association with prognosis. METHODS: Clinical and prognosis data were collected from the children with JMML who were admitted from January 2008 to December 2016, and the influencing factors for prognosis were analyzed. RESULTS: A total of 63 children with JMML were included, with a median age of onset of 25 months and a male/female ratio of 3.2∶1. JMML genetic testing was performed for 54 children, and PTPN11 mutation was the most common mutation and was observed in 23 children (43%), among whom 19 had PTPN11 mutation alone and 4 had compound PTPN11 mutation, followed by NRAS mutation observed in 14 children (26%), among whom 12 had NRAS mutation alone and 2 had compound NRAS mutation. The 5-year overall survival (OS) rate was only 22%±10% in these children with JMML. Of the 63 children, 13 (21%) underwent hematopoietic stem cell transplantation (HSCT). The HSCT group had a significantly higher 5-year OS rate than the non-HSCT group (46%±14% vs 29%±7%, P<0.05). There was no significant difference in the 5-year OS rate between the children without PTPN11 gene mutation and those with PTPN11 gene mutation (30%±14% vs 27%±10%, P>0.05). The Cox proportional-hazards regression model analysis showed that platelet count <40×109/L at diagnosis was an influencing factor for 5-year OS rate in children with JMML (P<0.05). CONCLUSIONS: The PTPN11 gene was the most common mutant gene in JMML. Platelet count at diagnosis is associated with the prognosis in children with JMML. HSCT can improve the prognosis of children with JMML.

An open time-series simulated dataset covering various accidents for nuclear power plants.

Nuclear energy plays an important role in global energy supply, especially as a key low-carbon source of power. However, safe operation is very critical in nuclear power plants (NPPs). Given the significant impact of human-caused errors on three serious nuclear accidents in history, artificial intelligence (AI) has increasingly been used in assisting operators with regard to making various decisions. In particular, data-driven AI algorithms have been used to identify the presence of accidents and their root causes. However, there is a lack of an open NPP accident dataset for measuring the performance of various algorithms, which is very challenging. This paper presents a first-of-its-kind open dataset created using PCTRAN, a pre-developed and widely used simulator for NPPs. The dataset, namely nuclear power plant accident data (NPPAD), basically covers the common types of accidents in typical pressurised water reactor NPPs, and it contains time-series data on the status or actions of various subsystems, accident types, and severity information. Moreover, the dataset incorporates other simulation data (e.g., radionuclide data) for conducting research beyond accident diagnosis.

Improved outcome of children with relapsed/refractory acute myeloid leukemia by addition of cladribine to re-induction chemotherapy.

BACKGROUND: The preferred salvage treatment for children with relapsed/refractory acute myeloid leukemia (R/R-AML) remains unclear. The combination of cladribine/Ara-C/granulocyte-colony stimulating factor and mitoxantrone (CLAG-M) shown promising results in adult R/R-AML. We aim to investigate the efficacy and safety of CLAG-M versus mitoxantrone/etoposide/cytarabine (MEC) or idarubicin/etoposide/cytarabine (IEC) in R/R-AML children. METHODS: Fifty-five R/R-AML children were analyzed. The overall response rate (ORR), overall survival (OS), and progression-free survival (PFS) at 3-year were documented. Karyotype or mutations status were summarized as different risk groups. RESULTS: The ORR was achieved in 80% (16/20) and 51% (18/35) of patients after one-cycle of CLAG-M and MEC/IEC treatment (p < 0.001). The CLAG-M group's OS (66.8% ± 16.2% vs. 40.4% ± 10.9%, p = 0.019) and PFS (52.6% ± 13.7% vs. 34.9% ± 9.1%, p = 0.036) at 3-year was significantly higher than the MEC/IEC group. In high-risk patients, 33.3% experienced progression of disease (PD) and 22.2% dead in CLAG-M group, while 50% experienced PD and 43.8% dead in MEC/IEC. When it comes to low-risk group, none of them in CLAG-M experienced PD or death, while up to 50% of patients received MEC/IEC suffered PD, and all of them died eventually. Similar results were also found in the intermediate-risk group. Surprisingly, the presence of FLT3-ITD was associated with poor outcome in both groups. The most common adverse events were hematologic toxicities, and the incidence was similar in both group. CONCLUSIONS: CLAG-M group demonstrated effective palliation along with acceptable toxicity in R/R-AML patients. However, patients with FLT3-ITD may benefit less from CLAG-M, owing to higher PD rate and all-cause mortality than other patients.

[The Correlation of Minimal Residual Disease with Prognosis in TCF3-PBX1+ Acute Lymphoblastic Leukemia in Children].

OBJECTIVE: To investigate the consistency between FCM and PCR on the detecting of MRD in TCF3-PBX1+ ALL, and to investigate the prognosis value of these 2 methods. METHODS: 55 cases of paediatric TCF3-PBX1+ ALL patients from April 2008 to April 2015 were enrolled and analyzed. The FCM and PCR was used to detect the MRD in 239 bone marrow samples of 55 patients. All statistical analyses were carried out by using SPSS software version 16. RESULTS: Among the 55 children with TCF3-PBX1+ ALL, there were 30 male and 25 female. The median age was 5 (1-14) years. 20 patients relapsed during follow-up. The MRD results from PCR and FCM showed a strong correlation between both methods (K=0.774, P<0.001). There was no significant difference in 5-years DFS and OS between the patients in PCR+ and PCR- groups on day 15 or day 33. The 5 year DFS rate between the patients in FCM- and FCM+ was 63.9%±7.0% and 0; the 5 year OS rate was 66.5%±7.9% and 0. Combined with the result of FCM and PCR, at the d 33 of treatment, the 5-year DFS rate in FCM-/PCR- and single positive group was 65.4%±7.2% and 25.0%±15.3% (P<0.01). CONCLUSION: The detection result of MRD in TCF3-PBX1 detect by FCM and PCR shows better consistency. MRD positivity detected by FCM at the end of induction therapy (day 33) predicts a high risk of relapse in TCF3-PBX1 ALL patients.

[Pharmacokinetics and pharmacodynamics of pegylated recombinant human granulocyte colony-stimulating factor in children with acute lymphoblastic leukemia: a prospective control trial].

OBJECTIVE: To study the pharmacokinetic characteristics, clinical effect, and safety of pegylated recombinant human granulocyte colony-stimulating factor (PEG-rhG-CSF) in children with acute lymphoblastic leukemia (ALL). METHODS: A prospective study was performed on children with ALL who cyclophosphamide, cytarabine, and 6-mercaptopurine were used for consolidation therapy. PEG-rhG-CSF (PEG-rhG-CSF group) or rhG-CSF (rhG-CSF group) was injected after chemotherapy. The plasma concentration of PEG-rhG-CSF was measured, and clinical outcome and safety were observed for both groups. RESULTS: A total of 17 children with ALL were enrolled, with 9 children in the PEG-rhG-CSF group and 8 children in the rhG-CSF group. In the PEG-rhG-CSF group, the peak concentration of PEG-rhG-CSF was 348.2 ng/mL (range 114.7-552.0 ng/mL), the time to peak was 48 hours (range 12-72 hours), and the half life was 14.1 hours (range 11.1-18.1 hours). The plasma concentration curve of PEG-rhG-CSF was consistent with the mechanism of neutrophil-mediated clearance. Compared with the rhG-CSF group, the PEG-rhG-CSF group had a significantly shorter median time to absolute neutrophil count (ANC) recovery (P<0.05). There were no significant differences between the two groups in ANC nadir, incidence rate of febrile neutropenia, duration of grade IV neutropenia, incidence rate of infection, and length of hospital stay. No bone pain or muscle soreness was observed in either group (P>0.05). CONCLUSIONS: The pharmacokinetic characteristics of PEG-rhG-CSF in children with ALL receiving consolidation chemotherapy are consistent with the mechanism of neutrophil-mediated clearance, with a short half life and fast recovery of ANC, and there are no significant differences in safety between PEG-rhG-CSF and rhG-CSF.

[Clinical effect of two different regimens in treatment of acute lymphoblastic leukemia children with bone marrow recurrence].

OBJECTIVE: To study the short-term effect of two different re-induction regimens in the treatment of acute lymphoblastic leukemia (ALL) children with bone marrow recurrence. METHODS: A retrospective analysis was performed for 57 ALL children with bone marrow recurrence. According to their treatment regimen, they were divided into two groups: VMDP (vincristine + mitoxantrone + dexamethasone + PEG-asparaginase; n=42) and VIDP (vincristine + idarubicin + dexamethasone + PEG-asparaginase; n=15). The two groups were compared in terms of complete response rate and incidence rate of adverse reactions. RESULTS: There was no significant difference in complete response rate between the VMDP and VIDP groups (74% vs 73%, P>0.05). All children experienced grade ≥3 hematological adverse events. The VMDP group had a significantly lower chemotherapy-related mortality rate than the VIDP group (P<0.05). There was no significant difference in the incidence rate of infection between the two groups (P>0.05). CONCLUSIONS: For ALL children with bone marrow recurrence, both re-induction regimens can achieve a relatively high complete response rate, and VMDP regimen has a lower chemotherapy-related mortality rate and can thus be used as an option for re-induction in ALL children with bone marrow recurrence.

[Association of cerebrospinal fluid status with prognosis in children with acute lymphoblastic leukemia].

OBJECTIVE: To study the clinical features of central nervous system infiltration-positive (CNSI+) children with acute lymphoblastic leukemia (ALL) based on flow cytometry, as well as the association of such clinical features with prognosis. METHODS: A retrospective analysis was performed for the clinical data of 66 CNSI+ children with ALL treated from April 2008 to June 2013. Clinical features, laboratory examination results and prognosis were compared between the children in different chemotherapy stages (induction stage and consolidation/maintenance stage). RESULTS: Among the 66 CNSI+ children, 50 were in the induction stage and 16 in the consolidation/maintenance stage. Compared with the CNSI+ children in the induction stage, the CNSI+ children in the consolidation/maintenance stage had a significantly higher proportion of children with the genes associated with good prognosis based on the results of molecular biology (P<0.05), as well as a significantly higher recurrence rate (P<0.05). Recurrence was observed in 21 CNSI+ ALL children, among whom 10 were in the induction stage and 11 were in the consolidation/maintenance stage. Compared with the children experiencing recurrence in the induction stage, the children experiencing recurrence in the consolidation/maintenance stage had a significantly higher proportion of children with recurrence of the central nervous system and bone marrow (P<0.05), as well as significantly higher proportion of biochemical positive rate of cerebrospinal fluid (P<0.05). The children in the induction stage had a significantly higher recurrence-free survival rate than those in the consolidation/maintenance stage (P<0.001), while there was no significant difference in overall survival rate between the two groups (P>0.05). CONCLUSIONS: In children with ALL, CNSI+ has a marked effect on recurrence-free survival rate in different chemotherapy stages, but has no obvious effect on overall survival rate. CNSI+ patients in the consolidation/maintenance stage have a higher recurrence.

SOCS1 Regulates the Immunomodulatory Roles of MSCs on B Cells.

BACKGROUND AND OBJECTIVES: The effective use of MSCs for the treatment of some B cell-mediated immune diseases is quite limited. The main reason is that the immunomodulatory effects of mesenchymal stem cells (MSCs) on B cells are unclear, and their underlying mechanisms have not been fully explored. METHODS AND RESULTS: By co-culturing B cells with MSCs without (MSC/CTLsh) or with suppressor of cytokine signaling 1 (SOCS1) knockdown (MSC/SOCS1sh), we found that MSCs inhibited B cell proliferation, activation and terminal differentiation. Remarkably, the highest inhibition of B cell proliferation was observed in MSC/SOCS1sh co-culture. Besides, MSC/SOCS1sh reversed the inhibitory effect of MSCs in the last stage of B cell differentiation. However, MSC/SOCS1sh had no effect on inhibiting B cell activation by MSCs. We also showed that IgA+ B cell production was significantly higher in MSC/SOCS1sh than in MSC/CTLsh, although no difference was observed when both MSCs co-cultures were compared to isolated B cells. In addition, MSCs increased PGE2 production after TNF-α/IFN-γ stimulation, with the highest increase observed in MSC/SOCS1sh co-culture. CONCLUSIONS: Our results highlighted the role of SOCS1 as an important new mediator in the regulation of B cell function by MSCs. Therefore, these data may help to develop new treatments for B cell-mediated immune diseases.

Prognostic stratification of molecularly and clinically distinct subgroup in children with acute monocytic leukemia.

BACKGROUND: The prognosis of children with acute monocytic leukemia (AML-M5) remains unsatisfactory and the risk profile is still controversial. We aim to investigate the prognostic value of clinical and cytogenetic features and propose a new risk stratification in AML-M5 children. METHODS: We included 132 children with AML-M5. Overall survival (OS) and progression-free survival (PFS) were documented. Cox regression was performed to evaluate the potential risk factors of prognosis. RESULTS: The 5-year-OS was 46.0% (95% confidence intervals, 41.6%-50.4%) in all patients. There was significantly lower OS in the age ≤ 3 years old (P = .009) and hyperleukocytosis (P < .001). The FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) and MLL-rearrangement carriers were associated with fewer survivors in all patients (37.1% and 36.7%) and chemotherapy-only group (19.0% and 35.0%). Notably, the number of survivor with MLL-rearrangement did not increase in hematopoietic stem cell transplant (HSCT) group. According to the Cox regression analysis, HSCT was a significantly favorable factor (P = .001), while hyperleukocytosis, age ≤ 3 years old, and BM blast ≥ 70% adversely affected the OS in all patients (all P < .05). Additionally, FLT3-ITD was a risk factor for OS in the chemotherapy-only group (P = .023), while hyperleukocytosis and age ≤ 3 years independently contributed to poor PFS (both P < .05). In comparison to the standard-risk group, significant poorer outcome was found in the high-risk group (both P < .005). CONCLUSIONS: We propose that AML-M5 children with any of MLL-rearrangement, FLT3-ITD, hyperleukocytosis, BM blast ≥ 70%, or age ≤ 3 years old are classified into the high-risk group, and HSCT is beneficial especially in patients with FLT3-ITD mutation, hyperleukocytosis, and age ≤ 3 years old. Importantly, the choice of HSCT should be made more carefully in children with MLL-rearrangement for its suboptimal performance.

Honokiol antagonizes doxorubicin­induced cardiomyocyte senescence by inhibiting TXNIP­mediated NLRP3 inflammasome activation.

Senescence of cardiomyocytes is considered a key factor for the occurrence of doxorubicin (Dox)­associated cardiomyopathy. The NOD­like receptor family pyrin domain­containing 3 (NLRP3) inflammasome is reported to be involved in the process of cellular senescence. Furthermore, thioredoxin­interactive protein (TXNIP) is required for NLRP3 inflammasome activation and is considered to be a key component in the regulation of the pathogenesis of senescence. Studies have demonstrated that pretreatment with honokiol (Hnk) can alleviate Dox­induced cardiotoxicity. However, the impact of Hnk on cardiomyocyte senescence elicited by Dox and the underlying mechanisms remain unclear. The present study demonstrated that Hnk was able to prevent Dox­induced senescence of H9c2 cardiomyocytes, indicated by decreased senescence­associated ß­galactosidase (SA­ß­gal) staining, as well as decreased expression of p16INK4A and p21. Hnk also inhibited TXNIP expression and NLRP3 inflammasome activation in Dox­stimulated H9c2 cardiomyocytes. When TXNIP expression was enforced by adenovirus­mediated gene overexpression, the NLRP3 inflammasome was activated, which led to inhibition of the anti­inflammation and anti­senescence effects of Hnk on H9c2 cardiomyocytes under Dox treatment. Furthermore, adenovirus­mediated TXNIP­silencing inhibited the NLRP3 inflammasome. Consistently, TXNIP knockdown enhanced the anti­inflammation and anti­senescence effects of Hnk on H9c2 cardiomyocytes under Dox stimulation. In summary, Hnk was found to be effective in protecting cardiomyocytes against Dox­stimulated senescence. This protective effect was mediated via the inhibition of TXNIP expression and the subsequent suppression of the NLRP3 inflammasome. These results demonstrated that Hnk may be of value as a cardioprotective drug by inhibiting cardiomyocyte senescence.

[Clinical features and prognosis of children with acute lymphoblastic leukemia and different platelet levels].

OBJECTIVE: To study the association of platelet level at diagnosis with prognosis in children with acute lymphoblastic leukemia (ALL). METHODS: A total of 892 children with ALL who underwent chemotherapy with the CCLG-ALL 2008 regimen were enrolled. According to the platelet count at diagnosis, these children were divided into normal platelet count group (platelet count ≥100×109/L; n=263) and thrombocytopenia group (platelet count <100×109/L; n=629). The thrombocytopenia group was further divided into (50- <100)×109/L (n=243), (20- <50)×109/L (n=263), and <20×109/L (n=123) subgroups. The association of clinical features (sex, age, immunophenotype, and molecular biology) with event-free survival (EFS) and overall survival (OS) was analyzed. RESULTS: Compared with the thrombocytopenia group, the normal platelet count group had significantly lower positive rate of MLL gene rearrangement and recurrence rate (P<0.05), as well as a significantly higher 10-year EFS rate (P<0.05). There was no significant difference in 10-year OS between the two groups (P>0.05). The normal platelet count group still had a significantly higher 10-year EFS rate than the thrombocytopenia group after the children with MLL gene rearrangement were excluded (P<0.05), and there was still no significant difference in 10-year OS between the two groups (P>0.05). The <20×109/L subgroup had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50- <100)×109/L subgroup, and the (20- <50)×109/L subgroup (P<0.05). After the children with MLL gene rearrangement were excluded, the <20×109/L subgroup still had significantly lower 10-year EFS and OS rates than the normal platelet count group, the (50-<100)×109/L subgroup, and the (20- <50)×109/L subgroup (P<0.05). CONCLUSIONS: ALL children with MLL gene rearrangement often have the clinical manifestation of thrombocytopenia. Platelet level at diagnosis is associated with the prognosis of ALL children. The children with normal platelet count have a low recurrence rate and good prognosis, and those with a platelet count of <20×109/L have the worst prognosis.

Prognostic Value of Neutrophil to Lymphocyte Ratio for In-hospital Mortality in Elderly Patients with Acute Myocardial Infarction.

Coronary artery disease (CAD) is a multifactorial disease in which inflammation plays a central role. This study aimed to investigate the association of inflammatory markers such as the neutrophil to lymphocyte ratio (NLR), the Global Registry of Acute Coronary Events (GRACE) score with in-hospital mortality of elderly patients with acute myocardial infarction (AMI) in an attempt to explore the prognostic value of these indices for elderly AMI patients. One thousand consecutive CAD patients were divided into two groups based on age 60. The laboratory and clinical characteristics were assessed retrospectively by reviewing the medical records. The NLR and GRACE score were calculated. In the elderly (≥60 years), patients with non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) had significantly higher NLR than did those with unstable angina (UA) and stable angina pectoris (SAP) (P<0.01). The NLR was considerably elevated in older AMI patients compared with their younger counterparts (<60 years) (P<0.05). In elderly AMI patients, the NLR was considerably higher in the high-risk group than in both the low-risk and medium-risk groups based on the GRACE score (P<0.05 and P<0.01, respectively), and the NLR was positively correlated with the GRACE score (r=0.322, P<0.001). Either the NLR level or the GRACE score was significantly higher in the death group than in the surviving group (P<0.05). By curve receiver operator characteristic curve (ROC) analysis, the optimal cut-off levels of 9.41 for NLR and 174 for GRACE score predicted in-hospital death [ROC area under the curve (AUC) 0.771 and 0.787, respectively, P<0.001]. It was concluded that an elevated NLR is a potential predictor of in-hospital mortality in elderly patients with AMI.

Nrf2 protects against diabetic dysfunction of endothelial progenitor cells via regulating cell senescence.

Diabetes is associated with an increased risk of cardiovascular disease. A decrease in the number and functionality of endothelial progenitor cells (EPCs) leads to reduced endothelial repair and the development of cardiovascular disease. The aim of the present study was to explore the effect and underlying mechanisms of nuclear factor erythroid 2­related factor 2 (Nrf2) on EPC dysfunction caused by diabetic mellitus. The biological functions of EPCs in streptozotocin­induced diabetic mice were evaluated, including migration, proliferation, angiogenesis and the secretion of vascular endothelial growth factor (VEGF), stromal­derived growth factor (SDF) and nitric oxide (NO). Oxidative stress levels in diabetic EPCs were also assessed by detecting intracellular reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). EPC senescence was evaluated by measuring p16 and b­gal expression and observing the senescence­associated secretory phenotype. In addition, the function of EPCs and level of oxidative stress were assessed following Nrf2 silencing or activation. Nrf2 silencing resulted in a decrease of EPC biological functions, accelerated cell senescence and increased oxidative stress, as indicated by ROS and MDA upregulation accompanied with decreased SOD activity. Furthermore, Nrf2 silencing inhibited migration, proliferation and secretion in EPCs, while it increased oxidative stress and cell senescence. Nrf2 activation protected diabetic EPCs against the effects of oxidative stress and cell senescence, ameliorating the biological dysfunction of EPCs derived from mice with diabetes. In conclusion, Nrf2 overexpression protected against oxidative stress­induced functional damage in EPCs derived from diabetic mice by regulating cell senescence.

Establishment of insect cell lines expressing green fluorescent protein on cell surface based on AcMNPV GP64 membrane fusion characteristic.

Displaying a protein on the surface of cells has been provided a very successful strategy to function research of exogenous proteins. Based on the membrane fusion characteristic of Autographa californica multiple nucleopolyhedrovirus envelope protein GP64, we amplified and cloned N-terminal signal peptide and C-terminal transmembrane domain as well as cytoplasmic tail domain of gp64 gene into vector pIZ/V5-His with multi-cloning sites to construct the cell surface expression vector pIZ/V5-gp64. To verify that the vector can be used to express proteins on the membrane of insect cells, a recombinant plasmid pIZ/V5-gp64-GFP was constructed by introducing the PCR amplified green fluorescent protein (GFP) gene and transfected into insect cell lines Sf9 and H5. The transected cells were screened with zeocin and cell cloning. PCR verification results showed that the GFP gene was successfully integrated into these cells. Green fluorescence in Sf9-GFP and H5-GFP cells was observed by using confocal laser scanning microscopy and immunofluorescence detection indicated that GFP protein was located on the cell membrane. Western blot results showed that a fusion protein GP64-GFP of about 40 kDa was expressed on the membrane of Sf9-GFP and H5-GFP cells. The expression system constructed in this paper can be used for localization and continuous expression of exogenous proteins on insect cell membrane.

[Association between clinical outcome and gene mutation in children with Fanconi anemia].

OBJECTIVE: To investigate the association between clinical outcome and gene mutations in children with Fanconi anemia (FA). METHODS: A retrospective analysis was performed for the clinical data of six children with the same severity of FA and receiving the same treatment. At first, single cell gel electrophoresis and chromosome breakage induced by mitomycin C were performed for diagnosis. Then the gene detection kit for congenital bone marrow failure diseases or complementation test was used for genotyping of FA. Finally the association between the clinical outcome at 3, 6, 9, or 12 months after treatment and gene mutation was analyzed. RESULTS: Of all the six FA children, five had FANCA type disease, and one had FANCM type disease; four children carried two or more FA gene mutations. Among the children with the same severity of FA, those with more FA mutations had a younger age of onset and poorer response to medication, and tended to progress to a severe type. CONCLUSIONS: Children carrying more than two FA mutations have a poor clinical outcome, and hematopoietic stem cell transplantation should be performed as soon as possible.

[Significance of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia without reproducible chromosomal abnormalities].

OBJECTIVE: To identify the incidence of PAX5 deletion in childhood B-lineage acute lymphoblastic leukemia (B-ALL) without reproducible chromosomal abnormalities and to investigate the association between PAX5 abnormalities and prognosis of ALL. METHODS: Multiplex ligation-dependent probe amplification was used to determine the copy numbers of PAX5 gene in children newly diagnosed with B-ALL without reproducible chromosomal abnormalities between April 2008 and April 2013 and controls (children with non-hematologic diseases or tumors). The patients were classifiied into deletion group and non-deletion group based on the presence of PAX5 deletion. RESULTS: Eighteen (21%) out of 86 children with B-ALL had PAX5 deletion. The deletion group had a significantly higher total white blood cell count at diagnosis than the non-deletion group (P=0.001). The Kaplan-Meier analysis demonstrated that the deletion group had a significantly lower disease-free survival (DFS) rate than the non-deletion group (0.69±0.12 vs 0.90±0.04; P=0.017), but there was no significant difference in the overall survival rate between the two groups (P=0.128). The Cox analysis showed that PAX5 deletion was a risk factor for DFS (P=0.03). CONCLUSIONS: PAX5 deletion is an independent risk factor for DFS in B-ALL children without reproducible chromosomal abnormalities.

[Detection of copy number variations in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia with multiplex ligation-dependent probe amplification].

OBJECTIVE: To investigate the application of multiplex ligation-dependent probe amplification (MLPA) in the detection of copy number variations (CNVs) in pediatric ETV6/RUNX1-positive acute lymphoblastic leukemia (ALL), to compare this method with conventional karyotype analysis and fluorescence in situ hybridization (FISH), and to evaluate the value of MLPA. METHODS: The clinical data of 95 children with ETV6/RUNX1-positive ALL who were treated from January 2006 to November 2012 were analyzed retrospectively, including clinical features, results of karyotype analysis, and results of FISH. CNVs were detected with MLPA. RESULTS: CNVs were detected in 73 (77%), and the median number of CNVs was 1 (range 0-6). The CNVs of EBF1, CDKN2A/2B, PAX5, ETV6, RB1, and BTG1 were detected in more than 10% of all the patients. The changes in the chromosome segments carrying the genes with CNVs detected by MLPA were not detected by conventional karyotype analysis. The coincidence rate between the CNVs in ETV6 gene detected by FISH and those detected by MLPA was 66%. CONCLUSIONS: MLPA is an efficient and convenient method to detect CNVs in children with ETV6/RUNX1-positive ALL.