Objective To establish a double reporter transgenic mice for monitoring Cre recombinase activity. Methods ZAP DNA fragment with lacZ and human alkaline phosphatase (hAP) gene was microinjected into the male pronucleus of 554 fertilized eggs from C57BL/6 mice. The founder mice and their progeny were screened for integration of transgene into the mouse genome by PCR and Southern blotting. The expression of lacZ transgene at early embryos from F1 generation mice was analyzed by X gal staining. Results A total of 398 survival ZAP DNA injected fertilized eggs were transfered to the oviducts of 21 pseudopregnant recipient mice. Of the 21 recipient mice, 13 became pregnancy and gave birth to 68 offspring mice. The zygote survival rate and birth rate were 71% (398/554) and 17% (68/398), respectively. Of the 68 offspring mice, 9 mice (5 males and 4 females) were identified by PCR and Southern blot analysis. Total integration rate and efficiency of transgene was 13% (9/68) and 1.6% (9/554), respectively. Nine mice as the founders were back crossed to set up F1 generation with other inbred C57BL/6 mice. Out of 9 transgenic mice, transmission of reporter gene in F1 offspring mice followed Mendelian rules, but the expression of lacZ protein was detected at the early embryonic stage (13.5 days postcoitum) in only 3 mice. Conclusion A double reporter transgenic mice for monitoring Cre recombinase activity is established.

The phenylacetone monooxygenase, isolated from Thermobifida fusca, mainly catalyzes Baeyer-Villiger oxidation reaction towards aromatic compounds. Met446 plays a vital role in catalytic promiscuity, based on the structure and function of phenylacetone monooxygenase. Mutation in Met446 locus can offer enzyme new catalytic feature to activate C-H bond, oxidizing indole to finally generate indigo and indirubin, but the yield was only 1.89 mg/L. In order to further improve the biosynthesis efficiency of the whole-cell catalyst, metabolic engineering was applied to change glucose metabolism pathway of Escherichia coli. Blocking glucose isomerase gene pgi led to pentose phosphate pathway instead of the glycolytic pathway to become the major metabolic pathways of glucose, which provided more cofactor NADPH needed in enzymatic oxidation of indole. Engineering the host E. coli led to synthesis of indigo and indirubin efficiency further increased to 25 mg/L. Combination of protein and metabolic engineering to design efficient whole-cell catalysts not only improves the synthesis of indigo and indirubin, but also provides a novel strategy for whole-cell catalyst development.
Escherichia coli ; genetics ; metabolism ; Glucose ; metabolism ; Indigo Carmine ; metabolism ; Indoles ; metabolism ; Industrial Microbiology ; methods ; Metabolic Engineering ; Metabolic Networks and Pathways ; Protein Engineering

Objective To investigate the influence of simvastatin treatment on Toll-like receptor 4 (TLR4) in monocytes of peripheral blood in patients with sepsis and severe sepsis and its significance. Methods A prospective randomized controlled trial was conducted. 106 patients with sepsis and 92 patients with severe sepsis admitted to Department of Critical Care Medicine of Henan Provincial People's Hospital from August 2013 to June 2015 were enrolled. These two groups of patients were randomized into conventional treatment group and simvastatin group. All patients received treatment according to the 2012 International Sepsis Treatment Guidelines, including anti-infection drugs, nutritional support, and palliative treatment, and the patients with severe sepsis were given early goal-directed therapy (EGDT). The patients in simvastatin group received simvastatin 40 mg daily orally for at least 15 days. The peripheral blood was collected and the monocytes were isolated at 1, 5, 10, 15 days after intensive care unit (ICU) admission. TLR4 expression on the surface of TLR4/CD14+ double positive monocytes was determined by flow cytometry, and adverse reaction was observed during treatment. Results TLR4 expression on the surface of monocytes showed a tendency of decreasing with prolongation of simvastatin treatment in the simvastatin group in patients with sepsis (n = 59) or severe sepsis (n = 54). However, in patients with sepsis, TLR4 level was significantly decreased from 10 days in simvastatin group as compared with that of conventional therapy group (n = 47), and it was decreased up to 15 days [mean fluorescence intensity (MFI): 21 (19, 28) vs. 27 (25, 33) at 10 days, Z = 2.198, P = 0.021; 16 (15, 21) vs. 26 (23, 34) at 15 days, Z = 4.611, P = 0.002]. In patients with severe sepsis, there was no significant difference in TLR4 level at different time points between simvastatin group and conventional treatment group (n = 38) [MFI: 55 (52, 63) vs. 56 (48, 65) at 1 day, Z = 0.313, P = 0.692; 47 (42, 56) vs. 49 (41, 58) at 5 days, Z = 0.827, P = 0.533; 40 (35, 42) vs. 42 (37, 45) at 10 days, Z = 1.012, P = 0.301; 33 (30, 38) vs. 38 (35, 41) at 15 days, Z = 0.539, P = 0.571]. No adverse reaction related with simvastatin was found during treatment in patients with sepsis or severe sepsis. Conclusions Statins could significantly down-regulate the TLR4 expression on peripheral blood monocytes in septic patients, while it showed no significant influence on TLR4 expression in patients with severe sepsis. A different effect of statins on TLR4 expression and the downstream inflammation process in sepsis and severe sepsis patients might partially explain the discrepancy in previous reports about the therapeutic effect of statins therapy in sepsis and severe sepsis patients.

Blood C-peptide,first-void fasting urinary C-peptide/creatinine ratio ( UCPCR ),second-void fasting UCPCR,and 24 h urinary C-peptide (UCP) were determined in 90 type 2 diabetics and 30 health volunteers.The results showed that first-void fasting UCPCR and second-void fasting UCPCR were positively related to 24 h UCP and the index of islet β-cell function( all P＜0.01 ).

Objective To discuss the health education effect of a self-made health education video for patients with hepatic carcinoma who are receiving transcatheter arterial chemoembolization (TACE)therapy.Methods Based on the needs of patients,a TACE health education video was designed and produced,which was input into the tablet personal computer,such as iPad,to let patients watch.A total of 451 patients with liver cancer,who were admitted to the Department of Intervention,First Affiliated Hospital of Henan University of Technology and Science,China,to receive TACE during the period from June 2014 to June 2016,were included in this study.By using the random number table method,the patients were divided into the observation group (n=226) and the control group (n=225).The self-made health education video together with regular education was used to educate the patients of the observation group,while routine oral and written education was executed for the patients of the control group.The cognition to TACE-related knowledge,the compliance with postoperative treatment and nursing,and the patient's satisfaction were assessed and the results were compared between the two groups.Results The cognition in the following three aspects,including TACE-related knowledge,perioperative education and discharge education,in the patients of the observation group was significantly higher than that in the patients of the control group (P＜0.05).The compliance with postoperative treatment and nursing in the patients of the observation group was remarkably higher than that in the patients of the control group (P＜0.01).The patient's satisfaction in the patients of the observation group was strikingly higher than that in the patients of the control group (P＜0.01).Conclusion Health education by using a self-made health education video can improve liver cancer patient's cognition to TACE as well as the patient's compliance with postoperative treatment and nursing.The health education in the form of watching video can significantly improve health education effect and the satisfaction of the patients.Thus,it is worthy of clinical application.

OBJECTIVE: To verify the accuracy of oxygen concentration (FiO₂) of modified oxygen treatment with Venturi and humidity system. METHODS: Patients just after ventilator weaning and before the removal of tracheal intubation/tracheotomy tube, who admitted to the intensive care unit (ICU) of Henan Provincial People's Hospital from May 1st to December 15th in 2017, were enrolled. All patients were given a modified oxygen treatment with Venturi and humidity system, and the oxygen flow rate (Flow) of the Venturi device and the oretical value of FiO₂ were adjusted according to the patient's condition. Patients were divided into five groups based on doctor's orders: Flow 3 L/min FiO₂ 0.24, Flow 3 L/min FiO₂ 0.26, Flow 6 L/min FiO₂ 0.28, Flow 6 L/min FiO₂ 0.30, Flow 9 L/min FiO₂ 0.35. The value of FiO₂ at the inhalation end of patients of each group was measured by TSI airflow analyzer, and the consistency between the measured value of FiO₂ at the inhalation end and the FiO₂ marked value of Venturi was compared and analyzed. RESULTS: When the FiO₂ theoretical value of Venturi were adjusted to 0.24, 0.26, 0.28, 0.30, and 0.35, the measured values of FiO₂ at the inhalation end of patients were 0.38±0.05, 0.38±0.05, 0.40±0.04, 0.41±0.04, and 0.77±0.11, respectively, which were all significantly higher than the theoretical value of FiO₂ (all P < 0.01). The difference between the measured value of FiO₂ at the inhalation side and the FiO₂ value of the Venturi annotated and the difference rate were both "V"-shaped, both of which decreased with the increase in theoretical value of FiO₂ to a Flow of 9 L/min and a theoretical value of FiO₂ 0.35, the accuracy was the worst, with the FiO₂ difference of 0.42±0.11, and the FiO₂ difference rate of (121.6±36.5)%. CONCLUSIONS There is a difference between the measured value and the theoretical value of FiO₂ at the inhalation end of the modified Venturi oxygen therapy humidification system, which needs to be paid attention to during clinical oxygen therapy.
Humans ; Humidity ; Oxygen/analysis* ; Oxygen Inhalation Therapy ; Respiration, Artificial ; Ventilator Weaning

Objective To investigate the effects of Shenqi Tiaoshen Formula(SQTSF)for alleviating airway inflammation in rats with both chronic obstructive pulmonary disease(COPD)and lung-kidney qi deficiency syndrome and explore its therapeutic mechanism.Methods Forty-eight SD rats were randomly divided into control group,model group,low-,medium-,and high-dose SQTSF groups,and aminophylline(APL)group.In all but the control group,rat models of COPD with lung-kidney qi deficiency syndrome were established and treated with saline,SQTSF or APL via daily gavage as indicated(starting from day 30).The rats were observed for changes in body weight,grip strength,lung function,lung pathology,inflammatory cytokines in bronchoalveolar lavage fluid(BALF),oxidative stress levels,iron ion metabolism,cellular and mitochondrial ultrastructural changes in the lung tissue,and expressions of Nrf2/SLC7A11/GPX4 signaling pathway and ferroptosis-related proteins.Results The rats in the model group exhibited obvious symptoms of lung-kidney qi deficiency syndrome with significantly decreased body weight,grip strength,and lung function parameters.Examination of the lung tissue revealed showed significant inflammatory cell infiltration and emphysema with obvious bronchial,perivascular,and alveolar inflammation and alveolar destruction,significantly increased IL-1β,TNF-α,IL-6,and IL-13 levels in BALF,and elevated pulmonary oxidative stress levels and Fe2+and total iron ion concentrations.The rat models also showed characteristic ultrastructural changes of ferroptosis in the lung tissue cells under transmission electron microscope and significantly decreased Nrf2,GPX4,and SLC7A11 and increased ACSL4 expressions in the lung tissue.Treatment with SQTSF significantly improved these pathological changes in the rat models with a better effect than APL.Conclusion SQTSF can effectively improve airway inflammation and oxidative stress in COPD rats with lung-kidney qi deficiency possibly by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway.

Objective To investigate the effects of Shenqi Tiaoshen Formula(SQTSF)for alleviating airway inflammation in rats with both chronic obstructive pulmonary disease(COPD)and lung-kidney qi deficiency syndrome and explore its therapeutic mechanism.Methods Forty-eight SD rats were randomly divided into control group,model group,low-,medium-,and high-dose SQTSF groups,and aminophylline(APL)group.In all but the control group,rat models of COPD with lung-kidney qi deficiency syndrome were established and treated with saline,SQTSF or APL via daily gavage as indicated(starting from day 30).The rats were observed for changes in body weight,grip strength,lung function,lung pathology,inflammatory cytokines in bronchoalveolar lavage fluid(BALF),oxidative stress levels,iron ion metabolism,cellular and mitochondrial ultrastructural changes in the lung tissue,and expressions of Nrf2/SLC7A11/GPX4 signaling pathway and ferroptosis-related proteins.Results The rats in the model group exhibited obvious symptoms of lung-kidney qi deficiency syndrome with significantly decreased body weight,grip strength,and lung function parameters.Examination of the lung tissue revealed showed significant inflammatory cell infiltration and emphysema with obvious bronchial,perivascular,and alveolar inflammation and alveolar destruction,significantly increased IL-1β,TNF-α,IL-6,and IL-13 levels in BALF,and elevated pulmonary oxidative stress levels and Fe2+and total iron ion concentrations.The rat models also showed characteristic ultrastructural changes of ferroptosis in the lung tissue cells under transmission electron microscope and significantly decreased Nrf2,GPX4,and SLC7A11 and increased ACSL4 expressions in the lung tissue.Treatment with SQTSF significantly improved these pathological changes in the rat models with a better effect than APL.Conclusion SQTSF can effectively improve airway inflammation and oxidative stress in COPD rats with lung-kidney qi deficiency possibly by inhibiting ferroptosis via regulating the Nrf2/SLC7A11/GPX4 signaling pathway.

ObjectiveTo establish and evaluate a chronic obstructive pulmonary disease （COPD） model with lung-spleen qi deficiency. MethodA rat model mimicking COPD with lung-spleen qi deficiency was established by the combination of cigarette smoking and intratracheal instillation of lipopolysaccharide （LPS） along with gavage of Sennae Folium infusion. Forty male SPF-grade SD rats were randomly assigned to blank， model， and low- （L-FXY）， medium- （M-FXY）， and high-dose （H-FXY） Sennae Folium infusion groups. Other groups except the blank group were exposed to daily cigarette smoke， with LPS administrated via intratracheal instillation on the 1st and 14th days. On the 28th day of modeling， the L-FXY， M-FXY， and H-FXY groups were administrated with Sennae Folium infusion at 5， 10， and 20 g·kg^-1， respectively， and at 4 ℃ for three weeks. The modeling lasted for 49 days. The general conditions （body mass， food intake， fecal water content， and anal temperature） and behaviors （grip strength test and tail suspension test） of rats in different groups were examined. The lung function， lung histopathology， D-xylose， amylase， and gastrin levels in the serum， interleukin（IL）-1β and IL-6 levels in the alveolar lavage fluid， levels of T-lymphocyte subsets （CD4⁺， CD8⁺， and CD4⁺/CD8⁺） in the peripheral blood， and thymus and spleen indices were measured. ResultTwo rats died in the H-FXY group. Compared with the blank group， both the M-FXY and H-FXY groups exhibited reduced body mass and food intake （P<0.01） and increased fecal water content （P<0.01）. The anal temperature in the H-FXY group was lower than that in the blank group （P<0.01）. The grip strength decreased in the modeling groups compared with the blank group （P<0.01）， and the duration of immobility in the tail suspension test increased in the M-FXY and H-FXY groups （P<0.05， P<0.01）. Compared with the blank group， the modeling groups showed reduced 0.3 second forced expiratory volume （FEV_0.3）， FEV_0.3/forced vital capacity （FVC）（P<0.01）， thickening of bronchial walls， proliferation of goblet cells， and the presence of emphysematous changes. In terms of gastrointestinal function， the M-FXY and H-FXY groups had lower levels of D-xylose， gastrin， and α-amylase than the blank group （P<0.01）. Regarding the immune and inflammatory indices， the M-FXY and H-FXY groups showed lower thymus and spleen indices than the blank group （P<0.01）. Compared with the blank group， the modeling groups presented lowered CD4⁺ level （P<0.01） and CD4⁺/CD8⁺ ratio （P<0.05， P<0.01） in the peripheral blood and elevated levels of IL-1β and IL-6 in the alveolar lavage fluid （P<0.01） than the blank group. ConclusionA model of COPD with lung-spleen Qi deficiency was established through the combination of daily cigarette smoke， intratracheal instillation with LPS， and gavage of Sennae Folium infusion. The comprehensive evaluation results suggested medium-dose （10 g·kg^-1） Sennae Folium infusion for gavage during the modeling of COPD with lung-spleen Qi deficiency.

Objective: To verify the accuracy of oxygen concentration (FiO₂) of modified oxygen treatment with Venturi and humidity system. Methods: Patients just after ventilator weaning and before the removal of tracheal intubation/tracheotomy tube, who admitted to the intensive care unit (ICU) of Henan Provincial People's Hospital from May 1st to December 15th in 2017, were enrolled. All patients were given a modified oxygen treatment with Venturi and humidity system, and the oxygen flow rate (Flow) of the Venturi device and the oretical value of FiO₂ were adjusted according to the patient's condition. Patients were divided into five groups based on doctor's orders: Flow 3 L/min FiO₂ 0.24, Flow 3 L/min FiO₂ 0.26, Flow 6 L/min FiO₂ 0.28, Flow 6 L/min FiO₂ 0.30, Flow 9 L/min FiO₂ 0.35. The value of FiO₂ at the inhalation end of patients of each group was measured by TSI airflow analyzer, and the consistency between the measured value of FiO₂ at the inhalation end and the FiO₂ marked value of Venturi was compared and analyzed. Results: When the FiO₂ theoretical value of Venturi were adjusted to 0.24, 0.26, 0.28, 0.30, and 0.35, the measured values of FiO₂ at the inhalation end of patients were 0.38±0.05, 0.38±0.05, 0.40±0.04, 0.41±0.04, and 0.77±0.11, respectively, which were all significantly higher than the theoretical value of FiO₂ (all P < 0.01). The difference between the measured value of FiO₂ at the inhalation side and the FiO₂ value of the Venturi annotated and the difference rate were both "V"-shaped, both of which decreased with the increase in theoretical value of FiO₂ to a Flow of 9 L/min and a theoretical value of FiO₂ 0.35, the accuracy was the worst, with the FiO₂ difference of 0.42±0.11, and the FiO₂ difference rate of (121.6±36.5)%. Conclusion There is a difference between the measured value and the theoretical value of FiO₂ at the inhalation end of the modified Venturi oxygen therapy humidification system, which needs to be paid attention to during clinical oxygen therapy.