In this study we used two types of cell cultures, i.e., anchorage-dependent basket and full suspension batch cultures of sTNFRII-gAD-expressing CHO cells in the CelliGen 310 bioreactor (7.5 L) to compare their yields in order to optimize the culturing conditions for efficient expression of sTNFRII-gAD fusion protein consisting of soluble tumor necrosis factor receptor II and globular domain of adiponectin. The anchorage-dependent basket culture was performed in 4L 10% serum-containing medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 3 days, and then switched to 4 L serum-free LK021 medium to continue the culture for 4 days. The full suspension batch culture was carried out in the 4 L serum-free LK021 medium with the final inoculating concentration of 3 x 10(5) to 4 x 10(5) cells/mL of sTNFRII-gAD-expressing CHO cells for 7 days. The culturing conditions were monitored in real-time to maintain pH and dissolved oxygen stability through the whole process. The supernatants were collected by centrifuge, and the protein was concentrated through Pellicon flow ultrafiltration system and then purified by DEAE anion exchange. The results showed that the yields of sTNFRII-gAD fusion protein were 8.0 mg/L with 95% purity and 7.5 mg/L with 98% purity in the anchorage-dependent basket and the full suspension batch cultures, respectively. The study provided the framework for the pilot production of sTNFRII-gAD fusion protein.
Adiponectin ; biosynthesis ; genetics ; Animals ; Bioreactors ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Receptors, Tumor Necrosis Factor, Type II ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

Objective : To investigate the cytotoxicity of slightly acidic electrolyzed water (SAEW) on oral keratinocyte monolayers. Methods: TR146 human keratinocyte monolayers were exposed to SAEW pretreated with bovine serum albumin（BSA）. It was divided into 4 groups, BSA 0 mg/mL (SAEW stock solutsion), BSA 0.5 mg/mL, BSA 1 mg/mL and BSA 2 mg/mL. The relative growth rate (RGR) was measured using a CCK-8 assay at 1 min, 5 min, 15 min, 30 min and 1 h, and the survival rate was measured using a Trypan Blue exclusion assay at 1 h. Results: The CCK-8 assay showed significantly different OD values in the SAEW and negative control groups at different times and FAC concentrations (P<0.05). With increasing FAC concentrations and observation times, the RGR in the SAEW group decreased, and the SAEW showed moderate to severe cytotoxic effects. The OD values in the BSA (0.5~2 mg/mL)-pretreated SAEW and negative control groups were not significantly different at different times or FAC concentrations (P > 0.05); the RGRs of the BSA-pretreated SAEW group all approached 100%, and no cytotoxic effects were observed in the BSA-pretreated SAEW group. The Trypan Blue exclusion assay showed significantly different survival rates in the SAEW and negative control groups at different FAC concentrations (P < 0.05). As the FAC concentration increased, the survival rate in the SAEW group decreased, and SAEW showed moderate to severe cytotoxic effects. The survival rates in the BSA-pretreated SAEW and negative control groups were not significantly different at different FAC concentrations (P > 0.05); the survival rates in the BSA-pretreated SAEW group all approached 100%, and no cytotoxic effects were observed. Conclusion SAEW showed no adverse effects on the viability of dental oral keratinocyte monolayers in vitro in the presence of BSA at concentrations equivalent to that of protein in saliva.

Objective:To investigate the value of systemic inflammatory response syndrome (SIRS), pediatric sequential organ failure assessment (pSOFA) and pediatric critical illness score (PCIS) in predicting mortality of pediatric sepsis in pediatric intensive care units (PICU) from Southwest China.Methods:This was a prospective multicenter observational study. A total of 447 children with sepsis admitted to 12 PICU in Southwest China from April 2022 to March 2023 were enrolled. Based on the prognosis, the patients were divided into survival group and non-survival group. The physiological parameters of SIRS, pSOFA and PCIS were recorded and scored within 24 h after PICU admission. The general clinical data and some laboratory results were recorded. The area under the curve (AUC) of the receiver operating characteristic curve was used to compare the predictive value of SIRS, pSOFA and PCIS in mortality of pediatric sepsis.Results:Amongst 447 children with sepsis, 260 patients were male and 187 patients were female, aged 2.5 (0.8, 7.0) years, 405 patients were in the survival group and 42 patients were in the non-survival group. 418 patients (93.5%) met the criteria of SIRS, and 440 patients (98.4%) met the criteria of pSOFA≥2. There was no significant difference in the number of items meeting the SIRS criteria between the survival group and the non-survival group (3(2, 4) vs. 3(3, 4) points, Z=1.30, P=0.192). The pSOFA score of the non-survival group was significantly higher than that of the survival group (9(6, 12) vs. 4(3, 7) points, Z=6.56, P<0.001), and the PCIS score was significantly lower than that of the survival group (72(68, 81) vs. 82(76, 88) points, Z=5.90, P<0.001). The predictive value of pSOFA (AUC=0.82) and PCIS (AUC=0.78) for sepsis mortality was significantly higher than that of SIRS (AUC=0.56) ( Z=6.59, 4.23, both P<0.001). There was no significant difference between pSOFA and PCIS ( Z=1.35, P=0.176). Platelet count, procalcitonin, lactic acid, albumin, creatinine, total bilirubin, activated partial thromboplastin time, prothrombin time and international normalized ratio were all able to predict mortality of sepsis to a certain degree (AUC=0.64, 0.68, 0.80, 0.64, 0.68, 0.60, 0.77, 0.75, 0.76, all P<0.05). Conclusion:Compared with SIRS, both pSOFA and PCIS had better predictive value in the mortality of pediatric sepsis in PICU.