Objective: To study the expression and prognosis of DHX32 in pancreatic cancer and observe the biological function of DHX32 in pancreatic cancer cell line SW1990 . To investigate the clinical significance and prognostic value of DHX32 in pancreatic cancer , and predict the possible related mechanism. Methods : GEPIA database was used to analyze the expression and prognosis of DHX32 in pancreatic cancer , and TCGA database was used to analyze the relationship between DHX32 mRNA expression level and clinicopathological features and prognosis of patients with pancreatic cancer. The effects of DHX32 on the proliferation , invasion and migration of SW1990 pancreatic cancer cells were studied by EdU immunofluorescence assay , Transwell assay and scratch assay. GSEA was used to predict DHX32 the possible related signaling pathways involved with DHX32 in pancreatic cancer. Results: The results of GEPIA database analysis showed that the expression level of DHX32 in pancreatic cancer tumor tissues was significantly higher than that in normal pancreatic tissues ( P < 0. 05 ) . Meanwhile , the prognosis of pancreatic cancer patients with high DHX32 expression was worse than that of patients with low DHX32 expression (P < 0. 001) . Cox regression analysis showed that DHX32 mRNA expression level and M stage were independent risk factors for prognosis of pancreatic cancer patients (all P < 0. 05) . EdU immunofluorescence assay , Transwell assay and scratch assay confirmed that the overexpression of DHX32 promoted the proliferation , invasion and migration of pancreatic cancer cells (all P < 0. 001) , while silencing DHX32 inhibited the proliferation , invasion and migration of pancreatic cancer cells ( all P < 0. 001) . The results of GSEA enrichment analysis showed that DHX32 was enriched in four signaling pathways : hematopoietic cell lineage , neuroactive ligand receptor interaction , primary immune deficiency and spliceosome (all P < 0. 05) . Conclusion DHX32 is highly expressed in pancreatic cancer. It promotes the proliferation , invasion and metastasis of pancreatic cancer cells , and is closely related to the poor prognosis of pancreatic cancer patients .

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×10¹⁰ IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.
Humans ; HEK293 Cells ; Genetic Vectors/genetics* ; Batch Cell Culture Techniques ; Bioreactors ; Perfusion

Objective To investigate the expression of Member RAS Oncogene Family(RAB10)in pancreatic cancer(PAAD)and its effects on the proliferation,migration,invasion and apoptosis of SW1990 cells(human pancreatic cancer cells).Methods The expression of RAB1 0 mRNA in PAAD tissues wasanalyzed by the cancer gene database GEPIA(Gene Expression Profiling Interactive Analysis)and TCGA(The Cancer Genome Atlas).Cox regression analysis was used to detect relationship between RAB10 mRNA expression and the prognosis of pan-creatic cancer patients.We targeted small interfering RNA(R4B10-siRNA)targeting RAB10 as the silence group,and constructed an overexpression plasmid(RAB10-OE)for overexpression of RAB10 as the overexpression group.The effects of silencing and overexpressionweredetected by Q-PCR;protein expression levelsweredetected by West-ern blot.EdUcellproliferation test,wound healing test,Transwelltestand flow cytometry test were used to determine the effects of RAB10 on the proliferation,migration,invasion and apoptosis of SW1990 pancreatic cancer cells.Re-sults RAB10 mRNA expression in PAAD tissues was higher than that innormal pancreatic tissues(P＜0.05).The results of EdUcellproliferation testshowed that the proliferation rate of SW1990 cells in the RAB10-OE group was higher thanthat in the control group,and the proliferation rate of SW1990 cells in the RAB10-siRNA group was lower than that inthe control group(P＜0.05).The results of the Transwell test and wound healing test showed that the invasion rate and mobility rate of RAB10-OE group were higher thanthose of the control group,and the mobility and invasion rate of RAB10-siRNA group were lower than those of the control group(P＜0.05).The re-sults of flow cytometry test showed that the apoptosis rate was lower in the RAB10-OE group than the control group,and the apoptosis rate in the RAB10-siRNA group was higher than the control group(P＜0.05).The median sur-vival time of RAB10 high expression group was significantly lower than that of RAB10 low expression group(P＜0.05).Cox regression analysis showed that clinical grade,T stage,M stage and RAB10 mRNA expression were re-lated to survival and prognosis of pancreatic cancerpatients(P＜0.05).Multivariate Cox regression analysis showed that the expression level of RAB10 mRNA was the independent risk factor affecting the prognosis of pancre-atic cancer patients(P＜0.05).Conclusion RAB10 is highly expressed in PAAD tissues and RAB10 can pro-mote the proliferation of pancreatic cancer cells,accelerate the ability to invade and migrate,and inhibit the apop-tosis of pancreatic cancer cells.RAB10 is an independent risk factor for survival prognosis in patients with pancreat-ic cancer.

Objective : To investigate the expression and prognosis of protein regulator of cytokinesis 1 ( PRC1) in pancreatic carcinoma tissues．Moreover，to explore the effects of PRC1 on the biological functions of pancreatic carcinoma cell line SW1990 and its related mechanisms． Methods: The GEPIA database was used to analyze the expression difference of PRC1 in pancreatic carcinoma tissues and normal pancreatic tissues．Overexpression and interference of PRC1 were achieved by Lipofectamine 3000 transfection plasmid or shRNA method．Then CCK-8 assay，Transwell assay and flow cytometry were used to detect the proliferation level，invasion ability and apoptosis of the SW1990 cells，respectively．The pancreatic carcinoma data were collected from the Cancer Genome Atlas (TCGA) database．The correlation between expression level of PRC1 and clinicopathological features of pancreatic carcinoma was analyzed．The STRING database was used to analyze the network of proteins interacting with PRC1 . Gene set enrichment analysis ( GSEA) was used to predict the possible signal pathways of PRC1 in pancreatic car- cinoma. Results: GEPIA database results showed that PRC1 expression in pancreatic carcinoma tissue was higher than that in normal pancreatic tissue (P＜0．05) ．The results of CCK-8 assay，Transwell assay and flow cytometry showed that PRC1 overexpression significantly enhanced SW1990 cell proliferation，invasion and inhibited apoptosis (P＜0. 01) ．Whereas PRC1 interference significantly inhibited SW1990 cell proliferation，invasion and enhanced apoptosis (P＜0. 01) ．TCGA database data analysis identified PRC1 mRNA expression level and M stage were independent risk factors affecting the prognosis of pancreatic carcinoma (P＜0. 05) ．STRING database showed that there was an interaction between PRC1 and PLK1 and so on．GSEA research results showed that the PRC1 mRNA high expression samples were enriched into P53 signaling pathway and so on (P＜0. 05) ． Conclusion PRC1 is highly expressed in pancreatic carcinoma，and it is associated with proliferation，invasion，apoptosis and prognosis of pancreatic carcinoma．Moreover，it plays an important role in pancreatic carcinoma by regulating interacting proteins PLK1 and activating P53 signaling pathways.

Objective : To investigate the effects of actin like 6A (ACTL6A) knockdown on the proliferation, apop⁃ tosis, migration and invasion of SW1990 cells in pancreatic cancer. Methods : The Oncomine database was used to analyze the expression of ACTL6A mRNA in the tissues of pancreatic cancer and normal pancreas. The plasmid of knockdown ACTL6A and siRNA negative control were established and transfected into SW1990 cell line as siRNA⁃ACTL6A group and siRNA⁃NC group. CCK⁃8, cell apoptosis experiment, Wound healing and Transwell assay were used to determine the effects of ACTL6A knockdown on the proliferation, apoptosis, migration and invasion of SW1990 cells. GSEA predicted a possible pathway regulated by ACTL6A in pancreatic cancer. T⁃test was used between the two groups. Results : The expression of ACTL6A in pancreatic cancer tissues was higher than that in normal pancreatic tissues ( P < 0. 05 ) . The results of CCK⁃8 assay showed that the absorbance of siRNA⁃ACTL6A group at 24 and 48 h were lower than those in the siRNA⁃NC group, and the difference was statistically significant ( t = 5. 840, 8. 454, P < 0. 01) . The results of Wound healing assay and Transwell assay showed that the healing rate and the number of invasive cells in siRNA⁃ACTL6A group were both lower than those in the siRNA⁃NC group. The difference was statistically significant ( t = 3. 960,4. 464, P < 0. 05), but the apoptosis rate of siRNA⁃ACTL6A group was significantly higher than that of the siRNA⁃NC group, and the difference was statistically significant( t = 12. 192, P < 0. 001) . GSEA results showed that the group with high expression of ACTL6A mRNA was up⁃regulated in cell cycle, nucleotide excision repair, base excision repair, DNA replication, pathways in cancer, NOTCH signaling pathway and other related gene sets(P < 0. 05) . These pathways were activated when the expression of ACTL6A was up⁃regulated. Conclusion ACTL6A is highly expressed in pancreatic cancer tissues. ACTL6A knockdown promotes the cell apoptosis of SW1990 cells, and inhibits proliferation, invasion and migration of SW1990 cells. The mechanism of the occurrence and development of ACTL6A in pancreatic cancer is attributed to the activation of cell cycle, nucleotide excision repair, base excision repair, DNA replication, pathways in cancer, NOTCH signaling pathway.

Objective : To investigate the expression and prognosis of (progestin and adipoQ receptor family member 4 , PAQR4) in hepatocellular carcinoma (HCC) and its effect on the proliferation , invasion , migration and ap⁃ optosis of HepG2 cells. Methods : The HCC data in the cancer genome atlas(TCGA) database was used to analyze the expression of PAQR4 mRNA in the tissues of HCC and its prognostic significance. HepG2 cell lines of pcDNA3. 1 ⁃PAQR4 experimental group and pcDNA3. 1 ⁃vector control group were established. CCK⁃8 assay was used to detect the effect of overexpression PAQR4 on the proliferation of HepG2 cells. The scratch assay and Transwell assay were used to detect the effects of PAQR4 overexpression on the migration and invasion of HepG2 cells. PI and Annexin V double staining experiment was used to observe the effect of PAQR4 overexpression on HepG2 cell apoptosis. Results : TCGA database data analysis results showed that the expression of PAQR4 mRNA in HCC tissues was higher than that in adjacent tissues ( P < 0. 05 ) , and the overall survival rate of HCC patients with PAQR4 mRNA high expression group was lower than that of low expression group (P = 0. 012) . Univariate regression analysis showed that PAQR4 mRNA expression level(HR : 1. 104 , 95% CI: 1. 051 - 1. 160 , P < 0. 001) , Tstage(HR:1.816,95% C1:1.442 -2.287,P<0.001),M stage( HR:3.924,95% CI:1.230 -12.519,P= 0. 021), pathological staging(HR:1.879, 95% C1: 1.466 -2.408, P<0. 001 ) had a significant impact on the prognosis of HCC patients. Multivariate regression analysis showed that PAQR4 mRNA expression level ( HR :1. 396 , 95% CI: 1. 081 - 1. 804 , P = 0. 011) was an independent risk factor of the prognosis of HCC patients. The results of CCK⁃8 assay , scratch assay and Transwell assay showed that the proliferation , migration , invasion and invasion ability of HepG2 cells in the experimental group were significantly improved compared with the control group (P < 0. 05) . Overexpression PAQR4 could inhibit HepG2 cell apoptosis(P < 0. 05) . Conclusion PAQR4 is significantly up⁃regulated in HCC tissue and is related to the prognosis of the patients. Overexpression PAQR4 promotes the proliferation , invasion and migration of HepG2 cells , and inhibits HepG2 cells apoptosis. PAQR4 maybe a new marker for the diagnosis and prognosis of HCC.

Objective To establish a simultaneous detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsules by TLC-SERS. Methods The samples to be measured were placed on the silica gel plate, and the thin layer was developed with petroleum ether-ethyl acetate (3:5). Location detection was carried out under 254 nm ultraviolet lamp. Nano-silver colloidal solution was sprayed at each spot of separation according to the optimum conditions, and then qualitative identification was done by TLC-SERS. Results A method for simultaneous determination of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added in Yinhuang soft capsule by TLC-SERS was established. And the minimum detection limits of three chemical constituents were determined. Conclusion TLC-SERS was a rapid, accurate and sensitive method for the simultaneous and rapid detection of diclofenac sodium, fenbufen and rotundine hydrochloride illegally added into Yinhuang soft capsule.

We constructed bicistronic expression system containing AH6 promoter, 5' UTR and its fore 38 bp sequence from Corynebacterium glutamicum, followed by a conserved Shine-Dalgarno (SD) sequence for xylanase expression. The two major secretory pathways signal peptide in C. glutamicum, Tat (CgR0949) and Sec (CspB) dependent signal peptide were added before xylanase for its secretion. Fed-batch cultivation was done in a 5 L jar for high-level xylanase secretion. The enzyme properties of the purified xylanase were then studied, including the effect of temperature and pH on its activity. The xylanase could be secreted into the culture supernatant when the Sec-dependent signal peptide CspB was used, but none was detected when CgR0949 was used. The secretory production level of xylanase in a flask was 486.2 U/mL and become 1 648.7 U/mL when in a 5 L jar, which was 3.4 fold as in the flask. The optimal pH and temperature of xylanase were pH 4.5 and 45 ℃, respectively. Its activity was 80% of initial activity after pretreatment at 4 ℃ for 24 h at pH 4-11, 95% after incubation below 50 ℃ for 15 min, and 20% when the temperature above 60 ℃. The xylanase could be efficiently secreted into the culture medium by C. glutamicum using its own genetic elements, and the secretion level could be improved through large-scale fed-batch cultivation. This bicistronic expression system can provide a useful tool for heterologous proteins secretion in C. glutamicum. In addition, the catalyze activity of xylanase could be further improved by enzyme properties study.
Corynebacterium glutamicum ; Promoter Regions, Genetic ; Protein Sorting Signals ; Protein Transport

Objective:To study parameters of MRI image in different tissue differentiation,cell type of primary liver cancer.Methods:The pathological results and MRI data of regeneration nodules (27),hepatocellular carcinoma (HCC) (81 total;15,highly differentiated;40,moderately differentiated;26,low-grade differentiation),intrahepatic cholangiocarcinoma (ICC) (20) were retrospectively analyzed and compared To compare the difference of ADC values,strengthen degree among the regeneration nodules,HCC and ICC,and among HCC tissue differentiation.Results:Most cases of primary liver cancer can be accurately diagnosed by conventional MRI combined with LAVA.there are statistically significant differences of ADC values among regenerative nodules,HCC,ICC (P＜0.01),and among highly,moderately,poorly differentiated HCC groups (P＜0.01).But there is not actual clinical significance of the ADC values between moderately and poorly HCC.there is no statistically significant difference of the ADC values between highly differentiated HCC and ICC (P=0.27).Conclusion:Conventional MRI combining with DWI,LAVA can help distinguish the primary liver cancer differentiation degree and the cell type.

Objective To develop a rapid method for the detection of toxic adulterant (diethylene glycol) in pharmaceuti-cal excipient (glycerol) .Method The detection sensitivity of Raman/near infrared (NIR) spectroscopy combined with moving window correlation coefficient (MWCC) method was evaluated .Results The detection sensitivity of Raman spectroscopy was superior to that of NIR spectroscopy and with the assistance of MWCC method ;the sensitivity had been further improved . Conclusion Raman spectroscopy has the potential to become the effective method in the on-site detection of toxic adulterant in pharmaceutical excipient .