AIM:To investigate the effect of ankyrin repeat domain 49(ANKRD49)on epithelial-mesenchy-mal transition(EMT)in NCI-H1299 cells,and to explore its mechanism.METHODS:The ANKRD49 was over-ex-pressed in NCI-H1299 cells.The morphological changes of ANKRD49-overpressing NCI-H1299 cells were observed under microscope.The mRNA and protein expression levels of EMT-related markers[E-cadherin,transforming growth factor-β1(TGF-β1),vimentin and α-smooth muscle actin(α-SMA)]and EMT-related transcription factors(Snail1,Slug,Twist and ZEB1)were detected by RT-qPCR Western blot.Immunofluorescence staining was performed to observe the localiza-tion and expression of E-cadherin and vimentin in ANKRD49-overexpressing cells or control cells.Immunohistochemical method was performed to examine the levels of E-cadherin,α-SMA,Snail,Slug and ZEB1 in lung tissues of nude mice in-oculated with ANKRD49-overexpressing H1299 cells or control cells.RESULTS:Compared with the control group,the ANKRD49 overexpressing cells showed mesenchymal cell morphology(fusiform and less tight connections).RT-qPCR and Western blot results showed that the mRNA and protein levels of mesenchymal markers vimentin and α-SMA in ANKRD49 overexpressing cells were significantly higher than those in cells of control group,while the mRNA and protein levels of epithelial marker E-cadherin were lower than those in cells of control group.Compared with control group,the im-munofluorescence intensity of E-cadherin of H1299 cells decreased in after ANKRD49 overexpression,while that of vimen-tin increased significantly.Snail,Slug and ZEB1 expression were significantly elevated in ANKRD49 overexpressing cells compared with control group.The levels of E-cadherin in lung tissues of nude mice inoculated with ANKRD49-overexpressing H1299 cells declined,while the levels of α-SMA,Snail,Slug and ZEB1 increased compared with those in control mice.CONCLUSION:ANKRD49 promoted EMT of NCI-H1299 cells by increasing the expression of Snail1,Slug and ZEB1 and consequent downreguation of E-cadherin and upregulation of vimentin and α-SMA.

Abstract： Objective　To elucidate the antigenic antibody reaction of recombinant expression of non-structural protein 1 (NS1) of Japanese encephalitis (JE) virus with various mosquito-borne flaviviruses, including JE virus, and the antigenic antibody reaction of serum samples of patients infected with JE virus in acute stage. Methods　In this study, Escherichia coli prokaryotic expression vector (pET) system was used to recombinant express Japanese encephalitis virus NS1 gene. Western Blot assay was performed to detect the antibody responses of the recombinantly expressed protein against a variety of mosquito-transmitted flaviviruses, including JE virus, as well as antigen-antibody reactions of serum from patients with acute JE virus infection. Results　The NS1 gene expression product of JE virus (P3 strain) was in the form of an inclusion body, and the denatured and renatured expression product was displayed as a single band in the denatured gel (polyacrylamide gel electrophoresis, PAGE), with a molecular weight of about 45 000. The results of further antigen-antibody analysis showed that the antigen/antibody hybridization reaction of the expression product with polyclonal or monoclonal antibody of JE virus (mosquito isolates, encephalitis isolates) and serum samples of patients with acute JE virus infection could be completely consistent. The recombinant product showed negative antigen/antibody hybridization reactions with mosquito-transmitted flaviviruses, such as dengue virus and yellow fever virus polyclonal antibodies, but positive reactions with polyclonal antibodies to West Nile virus and Murray Valley encephalitis virus. Conclusions　In this study, the recombinant expression of the NS1 protein of JE virus was successfully obtained, and the antigen/antibody reaction between the recombinant protein and samples of patients infected with mosquito-borne flavivirus and JE virus was analyzed. The study results provide important basic data for elucidating the antigen-antibody reaction between the NS1 protein of JE virus and mosquito-borne flavivirus. The recombinant expression protein obtained in this study provides an important material basis for further research on the function of JE virus NS1 protein.

OBJECTIVE To establish the fingerp rint of decoction pi eces and dispensing granules of Gardenia jasminoides ，to determine the contents of 6 components，so as to evaluate its quality combined with chemical pattern recognition. METHODS High performance liquid chromatography （HPLC）was used. Using geniposide as the reference ，Similarity Evaluation System for Chromatographic Fingerprint of TCM （2012 edition）was used to draw the fingerprints of 20 batches of G. jasminoides decoction pieces and 10 batches of G. jasminoides dispensing granules. Similarity evaluation and common peaks identification were conducted. The same HPLC method was adopted to determine the contents of deacetyl asperulosidic acid methyl ester ，geniposide， picrocrocin，rutin，crocin-Ⅰ and crocin- Ⅱ. ORIGIN 9.1 software was used for hierarchical clustering analysis ，and SIMCA 16.0 software was used for principal component analysis （PCA） and partial least squares-discriminant analysis. The differential components affecting the quality of decoction pieces and dispensing granules were screened by taking the variable importance in projection（VIP）value＞1 as the standard. RESULTS There were 24 common peaks for both 20 batches of G. jasminoides decoction piece and 10 batches of G. jasminoides dispensing granules ；a total of 22 common peaks were found in the fingerprints of 30 batches of samples ，and the similarity was not lower than 0.96；six common peaks were identified ，i.e. deacetyl asperulosidic acid methyl ester （peak 2），geniposide（peak 6），picrocrocin（peak 9），rutin（peak 11），crocin-Ⅰ（peak 15），crocin-Ⅱ（peak 17）. Average contents of above 6 components in G. jasminoides decoction pieces were 1.04，57.00，1.30，1.03，9.63 and 0.99 mg/g， respectively；those of G. jasmin oides dispensing granules were 0.96，17.04，0.37，0.27，0.73 and 0.04 mg/g，respectively. PCA results showed that G. jasminoides decoction pieces and G. jasminoides dispensing granules were clustered into respective one category ，which was consistent with results of cluster analysis. There were 9 common peaks with VIP value ＞1， which were 16，14，3，17（crocin-Ⅱ），15（crocin-Ⅰ），18， 22， 2 （deacetyl asperulosidic acid methyl ester） and 21. CONCLUSIONS The estab lished fingerprint and content determination method are simple and reproducible. Combined with chemical pattern recognition ，it can be used to evaluate the quality of decoction pieces and dispensing granules of G. jasminoides . Nine corresponding components represented by peak 16 and so on are the differential components that affect the quality of them.

Objective:To evaluate the application of a medical image software (RadiAnt) in anatomical measurements and precision craniotomy via suboccipital retrosigmoid sinus approach.Methods:A total of 43 inpatients who underwent craniocerebral CT venography (CTV) in our hospital from June 2020 to June 2021 were selected for the study; the CTV data of 35 patients was used to measure the spatial relations between transverse sigmoid sinus junction (TSSJ) and asterion; the preoperative planning in suboccipital retrosigmoid sinus craniotomy with the software was performed in the left 8 patients. Craniotomy time (subjected to exposure of venous sinus margin), venous sinus injury and incidence of complications within 2 weeks of craniotomy in these 8 patients were recorded.Results:(1) Anatomic measurement: for the left side, TSSJ was located at (0.89±0.33) cm lateral and (0.63±0.46) cm inferior to the asterion, and their direct distance was (1.15±0.42) cm; TSSJ was located at (0.76±0.49) cm interior and (1.97±0.52) cm superior to the starting point of the mastoid notch, and their direct distance was (2.18±0.49) cm; about 29% asterion were located superior to the transverse sinus, 37% were located on the surface of the transverse sinus, and 34% were located inferior to the transverse sinus. For the right side, TSSJ was located at (0.88±0.39) cm lateral and (0.64±0.43) cm inferior to the asterion, and their direct distance was (1.12±0.54) cm; TSSJ was located at (0.74±0.40) cm interior and (1.93±0.45) cm superior to the starting point of the mastoid notch, and their direct distance was (2.16±0.43) cm; about 26% asterion were located superior to the transverse sinus, 40% were located on the surface of the transverse sinus, and 34% were located inferior to the transverse sinus. (2) Preoperative planning and surgeries: in these 8 patients, the key-hole was located at (0.96±0.49) cm lateral and (0.53±0.18) cm inferior to the asterion, and (0.46±0.35) cm interior and (1.76±0.47) superior to the starting point of mastoid notch. The interior of sigmoid sinus was located (0.13±0.51) cm interior and (0.21±0.46) cm superior to the starting point of mastoid notch. The inferior of the transverse sinus was located (2.17±0.45) cm interior and (0.53±0.35) cm inferior to the asterion. An accurate localization of the real position of TSSJ, inferior of transverse sinus and interior of sigmoid sinus was performed in all 8 surgical patients. The distance between the margin of the bone window and the interior of sigmoid sinus was (3.5±1.0) mm, and the craniotomy time was (25.7±4.1) min; no sinus injury was noted. Post-operative CT showed good reposition of the bone flaps and less bone defect. There was no cerebrospinal fluid leakage or subcutaneous effusion during the 2 weeks of follow-up.Conclusion:Anatomical measurements and preoperative planning can be quickly finished with low cost with Radiant ?, which provides an efficient and safe method for accurate craniotomy via suboccipital retrosigmoid approach.

Objective:To construct and evaluate a decision tree based on biomarkers for predicting severe acute kidney injury (AKI) in critical patients.Methods:A prospectively study was conducted. Critical patients who had been admitted to the department of critical care medicine of Xiaolan Hospital of Southern Medical University from January 2017 to June 2018 were enrolled. The clinical data of the patients were recorded, and the biomarkers, including serum cystatin C (sCys C) and urinary N-acetyl-β-D-glucosaminidase (uNAG) were established immediately after admission to intensive care unit (ICU), and the end points were recorded. The test cohort was established with patient data from January to December 2017. The decision tree classification and regression tree (CART) algorithm was used, and the best cut-off values of biomarkers were used as the decision node to construct a biomarker decision tree model for predicting severe AKI. The accuracy of the decision tree model was evaluated by the overall accuracy and the receiver operating characteristic (ROC) curve. The validation cohort, established on patient data from January to June 2018, was used to further validate the accuracy and predictive ability of the decision tree.Results:In test cohort, 263 patients were enrolled, of whom 57 developed severe AKI [defined as phase 2 and 3 of Kidney Disease: Improving Global Outcomes (KDIGO) criterion]. Compared with patients without severe AKI, severe AKI patients were older [years old: 64 (49, 74) vs. 52 (41, 66)], acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) score were higher [23 (19, 27) vs. 15 (11, 20)], the incidence of hypertension, diabetes and other basic diseases and sepsis were higher (64.9% vs. 40.3%, 28.1% vs. 10.7%, 63.2% vs. 29.6%), the levels of sCys C and uNAG were higher [sCys C (mg/L): 1.38 (1.12, 2.02) vs. 0.79 (0.67, 0.98), uNAG (U/mmol Cr): 5.91 (2.43, 10.68) vs. 2.72 (1.60, 3.90)], hospital mortality and 90-day mortality were higher (21.1% vs. 4.4%, 52.6% vs. 13.1%), the length of ICU stay was longer [days: 6.0 (4.0, 9.5) vs. 3.0 (1.0, 6.0)], and renal replacement therapy requirement was higher (22.8% vs. 1.9%), with statistically significant differences (all P < 0.05). ROC curve analysis showed that the areas under ROC curve (AUC) of sCys C and uNAG in predicting severe AKI were 0.857 [95% confidence interval (95% CI) was 0.809-0.897) ] and 0.735 (95% CI was 0.678-0.788), and the best cut-off values were 1.05 mg/L and 5.39 U/mmol Cr, respectively. The structure of the biomarker decision tree model constructed by biomarkers were intuitive. The overall accuracy in predicting severe AKI was 86.0%, and AUC was 0.905 (95% CI was 0.863-0.937), the sensitivity was 0.912, and the specificity was 0.796. In validation cohort of 130 patients, this decision tree yielded an excellent AUC of 0.909 (95% CI was 0.846-0.952), the sensitivity was 0.906, and the specificity was 0.816, with an overall accuracy of 81.0%. Conclusion:The decision tree model based on biomarkers for predicting severe AKI in critical patients is highly accurate, intuitive and executable, which is helpful for clinical judgment and decision.

Objective To establish the quality control ofBaiduyin syrup.Methods The TLC was used to identity Radix paeoniae rubra, Radix Scutellariae. The quantitative determination of baicalin and buddleoside was completed by HPLC.Results The spots on TLC plates were distinct and high resolution. Compared with the negative samples, the contrast medicinal materials or control products showed that there were no spots of the same color in the corresponding position. The linear ranges of baicalin and buddleoside were 0.2179-2.1790 μg (r2=0.999 9), 0.1319-1.3190 μg (r2=0.999 7). TheRSD were 1.51% and 2.01%. Conclusions The established quality control method is simple, accurate and reproducible, which can be used for the quality control ofBaiduyin syrup.

Objective To establish the quality standard of Fufang-Shiwu-Zhixue powder. Methods The microscopical identification was adopted to analyze charred typha pollen and cuttlebone. TLC was used to identity rhubarb and radix notoginseng. UV-HPLC was used to determine the contents of notoginsenosides R1, ginsenosides Rg 1, ginsenosides Rb1. Results Microscopic identifications shower the characteristics of harred typha pollen and cuttlebone. The identified characteristics of rhubarb and radix notoginseng by TLC were distinct and the spots were clear. Notoginsenosides R1, ginsenosides Rg 1, ginsenosides Rb1 showed good linearity in the range of 0.16-1.58 μg (r=0.9998), 0.58-5.81 μg (r=0.9999), 0.33-3.29 μg (r=1.0000), respervtively.The average recoveries were 98.51% (RSD=1.81%), 97.80% (RSD=2.04%), 98.22%(RSD=1.45%). Conclusions The method is accurate, simple and repeatable, which could be used for the quality control of Fufang-Shiwu-Zhixue powder.

The oral colon-specific drug delivery system (OCDDS) has gained more attention from investigators in recent years since it can increase local drug concentration and reduce dosage and side effects.The type of colonspecific drug delivery system consists of enzyme dependent,pH dependent,time dependent,pressure dependent system and CODEDSTM.The development of many new materials and technology is very important for the preparation of new type of precise positioning colon-specific preparation.This article summarizes the advances in excipients and technique for oral colon-specific drug delivery system in recent years.It may provide a reference and a basis for the researchers concerned.

Objective To explore the predictive ability of magnetic resonance spectroscopy (MRS) in overall survival (OS) and progression-free survival (PFS) of patients with glioblastoma multiforme (GBM) before,during,and 2 months after radiotherapy with concomitant/adjuvant temozolomide (TMZ).Methods GBM patients,admitted to our hospital from January 2011 to January 2016 and confirmed by pathology,were chosen in our study;all patients underwent postoperative three-dimensional conformal radiotherapy with concomitant/adjuvant TMZ.And 3D-MRS was performed before,during,and 2 months after radiotherapy,the levels of N-acetyl-aspartic acid (NAA),choline (Cho) and creatine (Cr),and ratios of Cho/NAA,Cho/Cr and NAA/Cr in the GBM/edge of surgery side and the normal brain tissues were observed.The survival curve,median overall survival (mOS) and median progression free survival (mPFS) of patients with standardized Cho decreased＜30％ and patients with standardized Cho decreased＞30％ 2 months after radiotherapy were compared.Results Twenty-one patients finished the scheduled MRS for 3 times.Until the end of our study,16 patients died and 5 survived.Standardized Cho gradually decreased before,during,and 2 months after radiotherapy (2.08±0.22,1.45 ±0.21 and 1.16±0.18),with significant differences (P＜0.05).Standardized Cho after radiotherapy was significantly decreased as compared with that before radiotherapy (P＜0.05).Ratios of Cho/NAA and Cho/Cr in the GBM/edge of surgery side were significantly higher than those in the normal brain tissues (P＜0.05),and ratio of NAA/Cr in the GBM/edge of surgery side was significantly lower than that in the normal brain tissues (P＜0.05).Ratio of Cho/NAA gradually decreased before,during,and 2 months after radiotherapy,with significant differences (P＜0.05).As compared with patients with standardized Cho decreased＜30％ 2 months after radiotherapy,patients with standardized Cho decreased＞30％ 2 months after radiotherapy had significantly decreased rates of OS and PFS,and statistically shorter mPFS and mOS (4.5 vs.13.5,10.9 vs.25.3,P＜0.05).Conclusion The changes of standardized Cho 2 months after radiotherapy have high prognostic significance for PFS and OS.

AIM: To observe the effect of receptor-interacting protein 2 (Rip2) overexpression on human pan-creatic cancer cell line Panc-1.METHODS: pEGFP-C2 and pEGFP-Rip2 plasmids were respectively transfected into the Panc-1 cells using JetPRIME reagent.The cells were divided into control group, pEGFP-C2 group and pEGFP-Rip2 group. The apoptosis in the cells was detected 48 h after transfection by flow cytometry.Rip2 level and the expression of apoptosis-related proteins, Bax, cytoplasmic cytochrome c ( Cyt-c) and Bcl-2, were analyzed by Western blot.The activity of caspase-3 was measured by colorimetric method.RESULTS: Rip2 protein expression significantly increased in the cells transfected with control and pEGFP-C2 plasmids.The apoptotic rate in pEGFP-Rip2 group was higher than that in control group and pEGFP-C2 group, whereas no significant difference of apoptotic rate was observed between control group and pEGFP-C2 group.The protein expression of Bax and cytoplasmic Cyt-c was remarkably increased and the protein expression of Bcl-2 was obviously decreased in pEGFP-Rip2 group as compared with control group and pEGFP-C2 group.The activity of caspase-3 in pEGFP-Rip2 group was obviously increased as compared with control group and pEGFP-C2 group.CON-CLUSION: Overexpression of Rip2 is able to induce apoptosis in the Panc-1 cells, and the mechanism may be related to the up-regulation of Bax and cytoplasmic Cyt-c protein expression, down-regulation of Bcl-2 protein expression and en-hancement of caspase-3 activity, thus activating intrinsic apoptotic pathway.