A histidine-rich fusion tag enables real-time monitoring of recombinant protein expression by Pauly reaction-based colorimetric assay.

Commercially available recombinant expression systems always use fusion tags to facilitate target protein purification and SDS-PAGE analysis followed by Coomassie Brilliant Blue (CBB) staining is the classical method to validate the expression level of target protein, which is time-consuming, although not very laborious. Previously, we found that a histidine-rich elastin-like polypeptide (HRELP) tag could make its fusion proteins being quickly and specifically stained with Pauly's reagent. In this study, we designed a Pauly reaction-based colorimetric assay to real-time monitoring of the expression level of recombinant protein tagged HRELP and found that the absorption value of post-induction E. coli cells stained with Pauly's reagent correlated well with both the band intensity of the target protein from Pauly's reagent-stained and CBB-stained gels. Moreover, we found the colorimetric assay could also be helpful to roughly estimate the expression efficiency by using a poly-histidine-tagged protein, which has only 1.17% histidine residue. In our opinion, Pauly reaction-based colorimetric assay could significantly shorten the time to validate the over-expression of recombinant protein tagged with either HRELP or poly-histidine. And HRELP seemed to be an ideal fusion tag for it can not only facilitate protein purification but also simplify protein detection.

The novel STAT3 inhibitor WZ-2-033 causes regression of human triple-negative breast cancer and gastric cancer xenografts.

Hyperactive signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in human triple-negative breast cancer (TNBC) and gastric cancer, leading to uncontrolled tumor growth, resistance to chemotherapy, and poor prognosis. Thus, inhibition of STAT3 signaling is a promising therapeutic approach for both TNBC and gastric cancer, which have high incidences and mortality and limited effective therapeutic approaches. Here, we report a small molecule, WZ-2-033, capable of inhibiting STAT3 activation and dimerization and STAT3-related malignant transformation. We present in vitro evidence from surface plasmon resonance analysis that WZ-2-033 interacts with the STAT3 protein and from confocal imaging that WZ-2-033 disrupts HA-STAT3 and Flag-STAT3 dimerization in intact cells. WZ-2-033 suppresses STAT3-DNA-binding activity but has no effect on STAT5-DNA binding. WZ-2-033 inhibits the phosphorylation and nuclear accumulation of pY705-STAT3 and consequently suppresses STAT3-dependent transcriptional activity and the expression of STAT3 downstream genes. Moreover, WZ-2-033 significantly inhibited the proliferation, colony survival, migration, and invasion of TNBC cells and gastric cancer cells with aberrant STAT3 activation. Furthermore, administration of WZ-2-033 in vivo induced a significant antitumor response in mouse models of TNBC and gastric cancer that correlated with the inhibition of constitutively active STAT3 and the suppression of known STAT3 downstream genes. Thus, our study provides a novel STAT3 inhibitor with significant antitumor activity in human TNBC and gastric cancer harboring persistently active STAT3.

[A preliminary investigation of relationship between serum apelin level and pulmonary artery pressure in children with congenital heart disease].

OBJECTIVE: To preliminarily investigate the relationship between serum apelin level and pulmonary artery pressure in children with congenital heart disease. METHODS: One hundred and twenty-six children with congenital heart disease undergoing surgical treatment were enrolled as subjects. The serum level of apelin was determined before surgery and at 7 days after surgery. The ratio of pulmonary artery systolic pressure to aortic systolic pressure (Pp/Ps) was calculated before extracorporeal circulation. According to the Pp/Ps value, patients were classified into non-pulmonary arterial hypertension (PAH) group, mild PAH group, moderate PAH group, and severe PAH group. Pulmonary artery mean pressure was estimated by echocardiography at 7 days after surgery. RESULTS: The non-PAH group had the highest serum level of apelin before and after surgery, followed by the mild PAH group, moderate PAH group, and severe PAH group (P<0.05). All groups had significantly increased serum levels of apelin at 7 days after surgery (P<0.05). The serum level of apelin was negatively correlated with pulmonary artery pressure before surgery (r=-0.51, P<0.05) and at 7 days after surgery (r=-0.54, P<0.05). CONCLUSIONS: The decrease in serum apelin level is associated with the development of pulmonary hypertension in children with congenital heart disease. The significance of serum apelin in predicting the development and degree of pulmonary hypertension in children with congenital heart disease deserves further studies.

[Protective effect of cold autologous blood cardioplegic solution on the heart of infants with cyanotic congenital heart disease].

OBJECTIVE: To study the protective effect of cold autologous blood cardioplegic solution on the heart of infants with cyanotic congenital heart disease (CCHD). METHODS: Ninety-six infants with CCHD who underwent cardiopulmonary bypass (CPB) were randomly and equally divided into three groups: histidine-tryptophan-ketoglutarate (HTK) solution, cold non-autologous blood cardioplegic solution, and cold autologous blood cardioplegic solution. The right auricular tissues were taken before aortic cross-clamping and at 30 minutes after aortic declamping, and ATP level and energy charge (EC) in the myocardium were measured. Venous blood was collected before and immediately after CPB, and the serum levels of creatine kinase (CK)-MB and cardiac troponin I (cTnI) were measured. The clinical parameters, such as the re-beat time and re-beat rate during CPB, cardiac index, dependence on positive inotropic agents, and left ventricular ejection fraction (LVEF) at 2 hours after CPB, the incidence rate of arrhythmia within 24 hours after CPB, and postoperative complications and mortality, were recorded. RESULTS: At 30 minutes after aortic declamping, the three groups showed significantly decreased ATP and EC levels (P<0.05), and the cold autologous blood group had significantly higher ATP and EC levels than the other two groups (P<0.05). Immediately after CPB, the three groups showed significantly increased serum levels of CK-MB and cTnI (P<0.05), and the cold autologous blood group had significantly lower serum levels of CK-MB and cTnI than the other two groups (P<0.05). The cold autologous blood group had significantly better outcomes than the other two groups in terms of the re-beat time during CPB and the dependence on positive inotropic agents and LVEF at 2 hours after CPB (P<0.05). CONCLUSIONS: Cold autologous blood cardioplegic solution is superior to HTK and cold non-autologous blood cardioplegic solutions in preserving myocardial energy and reducing myocardial injury in infants with CCHD who undergo CPB, thus providing a better protective effect on the heart.

[One-stage complete correction of 52 cases infantile aortic coarctation or interrupted aortic arch associated with intracardiac anomalies].

OBJECTIVE: To sum up one-stage complete correction of infantile aortic coarctation (CoA) or interrupted aortic arch (IAA) associated with intracardiac anomalies through median sternotomy. METHODS: The clinical data of 52 infants with CoA or IAA associated with intracardiac anomalies from May 2004 to March 2010 was analyzed. There were 32 male and 20 female, aged from 25 d to 7 months with a mean of (2.03 ± 0.15) months, weighted from 2.5 to 8.0 kg with a mean of (3.9 ± 0.5) kg. All of intracardiac defect were corrected by self-arcula cordisand. Forty cases with CoA were underwent by operative techniques, including resection with end to side anastomosis, extended end to side anastomosis (n = 34), and vertical incision and cross joint (n = 3). Three cases of pseudo-CoA were cut and ductus arteriosus or ligamentum arteriosus and dissected arch. Twelve cases of IAA were underwent by extended end to side anastomosis. RESULTS: The time of cardiopulmonary bypass was (98 ± 41) min, and all patients hemorrhaged (78 ± 13) ml during operation. One case of IAA associated with double outlet right ventricle died after 43 d post-operation because of left bronchial stenosis. The other patients were in good condition. The rate of aneurysm formation was 11% in 1 to 6 years' follow-up. CONCLUSIONS: One-stage complete correction of infantile CoA or IAA associated with intracardiac anomalies through median sternotomy yields excellent intermediate surgical results. This operative approach is beneficial, not only with shorten period of therapy and loss operative cost.

The number and function of circulating endothelial progenitor cells in patients with Kawasaki disease.

Kawasaki disease (KD) is associated with coronary artery injury. Studies have shown that the endothelial progenitor cell (EPC) participates in the process of arterial repair. Data have been reported that the number of EPC increased significantly in the subacute phase of KD. However, until now, there are no data about the functions of EPC in KD patients. The present study was designed to further investigate the number and functions of EPC in KD. Ten KD patients in the acute phase and ten healthy volunteers were recruited and attributed to the KD group and control group, respectively. The circulating CD34/kinase insert domain-containing receptor double positive cells were evaluated in the two groups using flow cytometry. In vitro assays were used to measure the functions of EPC, including proliferation, adhesion, and migration activities. The plasma levels of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and high sensitivity C-reactive protein (hs-CRP) were also assessed in both groups. The number of EPC in the KD group was significantly higher than that of the control group (0.021 +/- 0.007% vs. 0.014 +/- 0.003%, P < 0.05). The migratory response of EPC was significantly decreased in the KD group, compared with that of the control group (5.50 +/- 1.78 vs. 3.40 +/- 1.35 cells/high power field, P < 0.01). Similarly, the proliferative and adhesive activities of EPC in the KD group were also decreased (0.47 +/- 0.08 vs. 0.66 +/- 0.07, P < 0.01; 6.5 +/- 2.12 vs. 11.2 +/- 2.04 cells/high power field, P < 0.01). The plasma NO, TNF-alpha, and hs-CRP levels in the KD group were higher than those of the control group (54.10 +/- 11.78 vs. 38.80 +/- 11.10 mumol/l, P < 0.01; 48.20 +/- 7.42 vs. 37.00 +/- 11.12 pg/ml, P < 0.05; 87.10 +/- 30.18 vs. 5.30 +/- 3.37 mg/l, P < 0.01). The number of circulating EPC positively correlated with the level of NO (r = 0.92, P < 0.001), and the functions of EPC negatively correlated with the levels of TNF-alpha and hs-CRP, respectively. In Kawasaki disease, the number of EPC was enhanced and the functions of EPC were attenuated. The two-way regulation of circulating EPC in KD patients may be associated with the disorders of cytokines or messengers in KD patients.

[A series of observation on the expression of TGF-beta1 in the lung of nitrofen-induced congenital diaphragmatic hernia rat model].

OBJECTIVE: To access the expression of transforming growth factor beta1 (TGF-beta1) in the lung of Nitrofen-induced congenital diaphragmatic hernia rat model. METHODS: Twelve timed-pregnant Sprague-Dawley rats were randomly divided into two groups, namely control group and CDH group on day 9.5 of gestation. Each rat in the CDH group was given 125 mg of Nitrofen (dissolved in seed fat) by gavage. Each rat in the control group was given the same dose of single oil. On day 16 of gestation, the two groups mentioned above were divided into three subgroups, and fetuses were delivered by cesarean section respectively on day 16, 18 and 21 of gestation. After the fetuses were checked for diaphragmatic hernia, lung tissue weight (LW) and body weight (BW) of each fetus on gestational day 21 were recorded. Lung histologic evaluations were made with microscope and TGF-beta1 immunohistochemistry staining were performed with image analyzing. RESULTS: At day 16 of gestation, no visible diaphragm closure was observed in all fetuses. Diaphragmatic hernia was observed in 32 of the 44 rat fetuses of the CDH groups on day 18 and day 21 of gestation (72.7%). Lw/Bw of the 21d subgroups of CDH group were lower than those of corresponding control group (P < 0.01). Observed under the microscope, the lungs of fetuses in CDH groups showed marked hypoplasia. The expression of TGF-beta1 was detected in cytoplasmic, without definite expression in nuclear. It was significantly stronger that the expression of TGF-beta1 was in the lungs of the CDH group than that of the control group (P < 0.01). CONCLUSIONS: Nitrofen interferes with lung development in early stage of the fetal before the diaphragm developed. TGF-beta1 would be one of the important factors which lead to pulmonary hypoplasia.

[Non-thoracoscopic Nuss procedure for correction operation of pectus excavatum ].

OBJECTIVE: To review the experience in correction operation of pectus excavatum with non-thoracoscopic Nuss procedure. METHODS: From September 2005 to August 2007, 108 patients with pectus excavatum were surgically corrected by non-thoracoscopic Nuss procedure. There were 91 male patients and 17 female patients. The age was from 2 years and 10 months old to 25 years old with an average of 7 years and 9 months old. The Haller indexes were from 3.6 to 10.1 before the operation. RESULTS: The operation in all patients had been performed successfully without any severe complications. The average time of operation was 40 minutes. The average bleeding volume during procedure was 10 ml. Uneventful recovery was achieved in all the cases. Excellent outcome was obtained in the follow-up of 2 months to 21 months in 92 patients. CONCLUSIONS: Non-thoracoscopic Nuss procedure for correction of pectus excavatum is safe and effective. It is unnecessary to perform the procedure into thoracic cavity so that there is less trauma and shorter time for the operation.

[Effect of hemin on the lung development and pulmonary arterial structural remodeling in congenital diaphragmatic hernia: experiment with rats].

OBJECTIVE: To assay the effects of prenatal hemin therapy on pulmonary hypoplasia (PH) and pulmonary arterial structural remodeling in congenital diaphragmatic hernia (CDH). METHODS: Six pregnant female SD rats were randomly divided into 3 equal groups: control group, undergoing gastric perfusion of olive oil once on day 9.5 and intraperitoneal injection of normal saline on days 11-14; CDH group, undergoing gastric perfusion of nitrofen 125 mg once on day 9.5 and intraperitoneal injection of normal saline on days 11-14; and hemin group, undergoing gastric perfusion of nitrofen 125 mg once on day 9.5 and intraperitoneal injection of hemin 15 mg x kg(-1) x d(-1) on days 11-14. On the gestational day 21 caesarean section was performed to take out the fetuses to undergo histological examination and image analysis. RESULTS: CDH were detected in 28 of the 44 (63.6%) fetuses from the 2 groups receiving nitrofen. The lungs of all CDH group fetuses were hypoplastic, and the fetuses of the hemin group showed improved lung development. The right lung/body weight ratio and pulmonary alveolar area ratio (PAA%) of the hemin group were (16.6 +/- 1.0) mg/g and (45 +/- 6)% respectively, both significantly higher than those of the CDH group [(14.6 +/- 1.7) mg/g and (28 +/- 6)% respectively, P = 0.03 and P < 0.01]. The alveolar septum area ratio (ASA%) of the hemin group was (44 +/- 6)%, significantly lower than that of the CDH group [(64 +/- 8)%, P < 0. 01]. The media thickness percentages (MT%) of pre-acinar artery (PAPA) and intra-acinar artery (IAPA) of the fetuses of the hemin group were (21.2 +/- 2.2)% and (18.2 +/- 2.1)% respectively, both significantly lower than those of the CDH group [(24.3 +/- 4.0)% and (21.9 +/- 3.9)% respectively, both P < 0.05], which were significantly higher than those of the control group [(20.0 +/- 2.4)% and (17.2 +/- 2.3)% respectively, both P < 0.01]. The component ratio of nonmuscularized artery (NMA) in the IAPA level of the hemin group was (78.2 +/- 3.0)%, significantly higher than that of the CDH group [(72.8 +/- 3.2)%, P = 0.001]. CONCLUSION: PH and pulmonary arterial structural remodeling are present in CDH. Although prenatal administration of hemin cannot prevent the genesis of CDH in rats, it may improve the pulmonary development, inhibit medial hypertrophy, and reduce the percentage of muscularized IAPA.

[Effect of tetrandrine on lung development: experiment of nitrofen-induced congenital diaphragmatic hernia fetal rat model].

OBJECTIVE: To assess the efficacy of prenatal administration of tetrandrine (TET) on pulmonary hypoplasia in the nitrofen-induced congenital diaphragmatic hernia (CDH) fetal rat model. METHODS: Six timed-pregnant female Sprague-Dawley rats were randomly divided into 3 equal groups: CDH group (receiving gavage of nitrofen 125 mg dissolved in seed fat on day 9.5), and TET group (receiving gavage of nitrofen 125 mg on day 9.5 and then gavage of TET 30 mg/kg on days 11.5 - 14.5), and control group (given the same dose of peanut oil on day 9.5 and the same dose of normal saline on days 11.5 - 14.5). The fetuses were delivered by cesarean section on day 21 to undergo light microscopy and electron microscopy. The numbers of type II pneumocytes were recorded and compared. RESULTS: CDH were detected in 32 of the 41 fetuses from the CDH and TET groups with a teratogenic rate of 78%, however, without a significant difference between the CDH and TET groups (P = 0.645). Microscopy showed significant lung hypoplasia in both histologic structure and cellular structure in the CDH group; however the lung development of the TET group was improved in comparison to the CDH group. There was no significant difference in numbers of type II pneumocytes among the 3 groups (P = 0.779). CONCLUSION: Prenatal administration of TET can improve the lung development of CDH rats in both histological structure and cellular structure. This may provide a new idea for the clinical treatment of CDH.