Embryo Research ; Humans

Cell membrane affinity chromatography has been widely applied in membrane protein (MP)-targeted drug screening and interaction analysis. However, in current methods, the MP sources are derived from cell lines or recombinant protein expression, which are time-consuming for cell culture or purification, and also difficult to ensure the purity and consistent orientation of MPs in the chromatographic stationary phase. In this study, a novel in situ synthesis membrane protein affinity chromatography (iSMAC) method was developed utilizing cell-free protein expression (CFE) and covalent immobilized affinity chromatography, which achieved efficient in situ synthesis and unidirectional insertion of MPs into liposomes in the stationary phase. The advantages of iSMAC are: 1) There is no need to culture cells or prepare recombinant proteins; 2) Specific and purified MPs with stable and controllable content can be obtained within 2 h; 3) MPs maintain the transmembrane structure and a consistent orientation in the chromatographic stationary phase; 4) The flexible and personalized construction of cDNAs makes it possible to analyze drug binding sites. iSMAC was successfully applied to screen PDGFRβ inhibitors from Salvia miltiorrhiza and Schisandra chinensis. Micro columns prepared by in-situ synthesis maintain satisfactory analysis activity within 72 h. Two new PDGFRβ inhibitors, salvianolic acid B and gomisin D, were screened out with K _D values of 13.44 and 7.39 μmol/L, respectively. In vitro experiments confirmed that the two compounds decreased α-SMA and collagen Ӏ mRNA levels raised by TGF-β in HSC-T6 cells through regulating the phosphorylation of p38, AKT and ERK. In vivo, Sal B could also attenuate CCl₄-induced liver fibrosis by downregulating PDGFRβ downstream related protein levels. The iSMAC method can be applied to other general MPs, and provides a practical approach for the rapid preparation of MP-immobilized or other biological solid-phase materials.

Astragali Radix(AR)is a clinically used herbal medicine with multiple immunomodulatory activities that can strengthen the activity and cytotoxicity of natural killer(NK)cells.However,owing to the complexity of its composition,the specific active ingredients in AR that act on NK cells are not clear yet.Cell membrane chromatography(CMC)is mainly used to screen the active ingredients in a complex system of herbal medicines.In this study,a new comprehensive two-dimensional(2D)NK-92MI CMC/C18 column/time-of-flight mass spectrometry(TOFMS)system was established to screen for potential NK cell acti-vators.To obtain a higher column efficiency,3-mercaptopropyltrimethoxysilane-modified silica was synthesized to prepare the NK-92MI CMC column.In total,nine components in AR were screened from this system,which could be washed out from the NK-92MI/CMC column after 10 min,and they showed good affinity for NK-92MI/CMC column.Two representative active compounds of AR,isoastragaloside Ⅰ and astragaloside Ⅳ,promoted the killing effect of NK cells on K562 cells in a dose-dependent manner.It can thus suggest that isoastragaloside Ⅰ and astragaloside Ⅳ are the main immunomodulatory compo-nents of AR.This comprehensive 2D NK-92MI CMC analytical system is a practical method for screening immune cell activators from other herbal medicines with immunomodulatory effects.

Therapeutic drug monitoring(TDM)has played an important role in clinical medicine for precise dosing.Currently,chromatographic technology and immunoassay detection are widely used in TDM and have met most of the needs of clinical drug therapy.However,some problems still exist in practical appli-cations,such as complicated operation and the influence of endogenous substances.Surface plasmon resonance(SPR)has been applied to detect the concentrations of small molecules,including pesticide residues in crops and antibiotics in milk,which indicates its potential for in vivo drug detection.In this study,a new SPR-based biosensor for detecting chloramphenicol(CAP)in blood samples was developed and validated using methodological verification,including precision,accuracy,matrix effect,and extraction recovery rate,and compared with the classic ultra-performance liquid chromatography-ultraviolet(UPLC-UV)method.The detection range of SPR was 0.1-50 ng/mL and the limit of detec-tion was 0.099±0.023 ng/mL,which was lower than that of UPLC-UV.The intra-day and inter-day ac-curacies of SPR were 98％-114％and 110％-122％,which met the analysis requirement.The results show that the SPR biosensor is identical to UPLC-UV in the detection of CAP in rat blood samples;moreover,the SPR biosensor has better sensitivity.Therefore,the present study shows that SPR technology can be used for the detection of small molecules in the blood samples and has the potential to become a method for therapeutic drug monitoring.

Purpose: Whether craniospinal irradiation (CSI) could be replaced by limited-field radiation in non-metastatic bifocal germinoma remains controversial. We addressed the issue based on the data from our series and the literature. Methods: Data from 49 patients diagnosed with non-metastatic bifocal germinoma at our hospital during the last 10 years were collected. The Pediatric Quality of Life Inventory 4.0 was used to evaluate health-related quality of life (HRQOL). Additionally, 81 patients identified from the literature were also analyzed independently. Results: In our cohort, 34 patients had tumors in the sellar/suprasellar (S/SS) plus pineal gland (PG) regions and 15 in the S/SS plus basal ganglia/thalamus (BG/T) regions. The median follow-up period was 52 months (range, 10 to 134 months). Our survival analysis showed that patients treated with CSI (n=12) or whole-brain radiotherapy (WBRT; n=34) had comparable disease-free survival (DFS; p=0.540), but better DFS than those treated with focal radiotherapy (FR; n=3, p=0.016). All 81 patients from the literature had tumors in the S/SS+PG regions. Relapses were documented in 4/45 patients treated with FR, 2/17 treated with whole-ventricle irradiation, 0/4 treated with WBRT, and 1/15 treated with CSI. Survival analysis did not reveal DFS differences between the types of radiation field (p=0.785). HRQOL analysis (n=44) in our cohort found that, compared with S/SS+PG germinoma, patients with BG/T involvement had significantly lower scores in social and school domains. However, HRQOL difference between patients treated with CSI and those not treated with CSI was not significant. Conclusion In patients with non-metastatic bifocal germinoma, it is rational that CSI could be replaced by limited-field radiation. HRQOL in patients with BG/T involvement was poorer.

Objective To observe the effects of dopamine in different doses on hemodynamics and cerebral oxygen metabolism in the early stage of post-resuscitation in rabbit with cardiac arrest.Methods Healthy adult rabbits were randomly(random number) divided into 4 groups according to the different doses of dopamine administration:control group (CG),low dose group (LG),medium dose group (MG),high dose group (HG),(n=15 in each group).Ventricular fibrillation (VF) was induced by electricity and cardiopulmonary resuscitation (CPR) was performed subsequently as the experiment designed.When 10 rabbits with restoration of spontaneous circulation (ROSC) were got each group,it was enough for experiment carried out.Cardiac output (CO),mean arterial pressure (MAP),heart rate (HR),systemic vascular resistance index (SVRI) and the cerebral local tissue blood oxygen saturation (TOI) were observed at 0 min,15 min,30 min,60 min,120 min after ROSC.The animals were sacrificed at 120 min after ROSC,brain tissues were harvested for study by using HE staining.Repeated measure analysis of variance was used to determine the statistical significance among the four groups at different intervals.Multi-group quantitative data was analyzed by one way ANOVA and then further by LSD test for multiple comparisons.Chi-square test or Fisher's exact probabilities was applied for multi-group binomial classification variable.Log-rank test was used for comparisons of survival curves in four groups.A twotailed value of P＜0.05 was considered statistically significant.Results There were no differences in the rate of ROSC among groups.Compared with CG and LG,ROSC time was shorter in MG (277±15 vs.190±12,P＜0.01;252±16 vs.190±12,P=0.016) with higher 120 min survival rate (20％ vs.90％,x2=9.899,P=0.005;30％ vs.90％,x2=7.5,P=0.02).CO was higher in MG than that in other groups at all given intervals in the early stage of post-resuscitation(P＜0.05).MAP levels were significantly higher in MG and HG compared with CG and LG at given intervals 15 min after ROSC (P＜0.05).SVRI was significantly higher in HG than that in other groups at all given intervals after ROSC (P＜0.05).Compared with other groups,the TOI levels in MG were significantly higher than that in other groups at 15 min and 30 min after ROSC (P＜0.05).The median survival time in MG and HG was significantly longer than that in CG and LG.The number of cellular necrosis in MG (28.4±1.0) was significantly fewer than that in other groups (CG 41.2±1.5;LG 41.0±2.0;HG 39.6±1.9) (P＜0.01).Conclusion The moderate dose of dopamine might maintain MAP and CO at a higher level meeting the cerebral oxygen metabolism in the early stage of post-resuscitation from cardiac arrest in rabbits.

Objective To explore the clinical value of the monitoring of electronic cardiac index (CI) in the evaluation of neonatal congenital heart disease complicated with heart failure. Methods Sixty neonates with congenital heart disease treated in neonatal department from March 1, 2016 to December 30, 2016 were selected, and divided into severe group (n=11), moderate group (n=15), mild group (n=34), and no heart failure group (n=10) according to the modified Ross heart failure score. CI was measured by electronic force measurement. Left ventricular ejection fraction (LVEF) and pulmonary arterial pressure (PAP) were measured by echocardiography. Venous blood sampling was collected to detect the N-terminal type B brain natriuretic peptide (NT-proBNP). Results The neonates in the severe group were mainly under 2-week-old, while those in the mild group and the moderate group were more than 2-week-old. The differences of CI, LVEF, NT-proBNP, and PAP among the groups were statistically different. The CI and LVEF values were lowest in the severe group, followed by moderate group and mild group, and the highest in no heart failure group. The NT-proBNP and PAP values were the highest in the severe group, followed by moderate group and mild group, and the lowest in no heart failure group. Correlation analysis showed that CI was positively correlated with LVEF (r=0.845, P<0.001), and negatively correlated with NT-proBNP (r=-0.886, P<0.001); CI and PAP were weakly negatively correlated (r=-0.595, P<0.001). Conclusions CI reflects the degree of heart failure to some extent and has some clinical value.

According to the Chinese Pharmacopoeia 2015, only processed Aconitum tubers can be clinically applied, and the effect of processing is unclear. This research aimed to explore the effect of processing on cardiac efficacy of alkaloids in Aconitum tubers. First, the chemical ingredients in unprocessed and processed Aconitum tubers were identified and compared by using high performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) and multivariate pattern recognition methods. Then the representative alkaloids in Aconitum tubers, aconitine, benzoylaconine, and aconine, which belong to diester-diterpenoid alkaloids, monoester-diterpenoid alkaloids, and amine-diterpenoid alkaloids, respectively, were selected for further validation of attenuated mechanism. Subsequent pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats were used to validate the effect of processing on cardiac functions. After processing the Aconitum tubers, it was found that the contents of diester-diterpenoid alkaloids were reduced, and those of monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids were increased, suggesting that diesterditerpenoid alkaloids were transformed into monoester-diterpenoid alkaloids and amine-diterpenoid alkaloids. Through further decocting the aconitine in boiling water, it was confirmed that the three alkaloids could be progressively transformed. Pharmacological experiments with aconitine, benzoylaconine, and aconine in SD rats showed that aconitine at a dose of 0.01 mg/kg and aconine at a dose of 10 mg/kg enhanced the cardiac function, while benzoylaconine at a dose of 2 mg/kg weakened the cardiac function. The effect of processing is attributed to the transformation of the most toxic diester-diterpenoid alkaloids into less toxic monoesterditerpenoid alkaloids and amine-diterpenoid alkaloids.