Aqueous two phase system(ATPS) provides a gentle, non-denaturing separation environment for proteins, enzymes. While high-speed countercurrent chromatography (HSCCC) is a liquid-liquid partition chromatography which uses centrifugal force to hold the stationary phase and facilities the mobile phase partitioning through the stationary phase, it can produce high separation efficiency with large sample loading capacity. However, the ordinary HSCCC apparatus (Type J ) fails to retention a satisfactory stationary phase of ATPS because of its high viscosity and low interfacial tension. Nevertheless, the later designed cross-axis planetary centrifuge system(X-CPC) can produce a greater lateral force field and enhances significantly the retention of the ATPS stationary phase. A review of the application of these CCC techniques with ATPS in the separation of proteins was given. Meanwhile, new techniques such as pH-peak focusing CCC and dye-ligand affinity CCC and some new CCC column design for improvement of separation efficiency and retention of ATPS stationary phase are introduced.

Objective To investigate the effect of mixed application of lecture-based learning and flipped classroom and problem-based learning in microbiology teaching. Methods The five-year program students of Grade 2013 and Grade 2014 were randomly divided into two groups, and the experimental group (48 students in the teaching reform class) carried out the blended teaching, while the contrast group (48 students of parallel class) carried on the traditional teaching. After the lecture, the teaching effect of the hybrid teaching method and traditional teaching method was analyzed after the theoretical and the experi-mental operation test were adopted, and the four part questionnaire surveys including the integrated use of knowledge, active classroom atmosphere, innovation ability and teaching satisfaction were proceeded in each group. The data of each group was analyzed by t test analysis with SPSS 19.0 respectively. Results The theory test scores of experimental group was (90.16±3.14), which was higher than the control group (82.33± 4.21). The difference between them was very significant (P=0.000). Survey results showed that the integrated use of knowledge, active classroom atmosphere, innovation ability and teaching satisfaction were higher than traditional group. The difference was statistically considered significant (P<0.01). Conclusion Hybrid teaching method is very good for training and development of students' comprehensive quality and ability. It has important significance in improving the students' score.

Animals ; Calcitonin Gene-Related Peptide ; blood ; Electric Stimulation ; Endothelin-1 ; blood ; Female ; Heart Arrest ; blood ; Male ; Rabbits ; Resuscitation ; methods

Animals ; Death, Sudden, Cardiac ; Disease Models, Animal ; Rabbits

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.
Amino Acid Sequence ; Enterovirus D, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Sequence Alignment ; Viral Proteins ; chemistry ; genetics ; metabolism

Background Neurofilament 200 (NF200) is an indirect indicator of the specific distribution of axons.It reflects the condition of the maintenance of neuronal morphology.Whether NF200 is involved in the damage of the visual pathway after retinal ischemia reperfusion injury (RIRI) should be further examined.Objective The present study was to investigate the expression of NF200 in retinal ganglion cells (RGCs),lateral geniculate nucleus (LGN),superior colliculus and visual cortex following RIRI.Methods Forty SD rats were randomized into the RIRI 1-,2-,3-,4-,6-,8-week groups,sham operation group and control group.Acute intraocular hypertension was induced in the right eye by anterior chamber perfusion of normal saline solution for 60 minutes to elevate the intraocular pressure to 110 mmHg.The animals were sacrificed at different time points for the preparation of the retina,LGN,superior colliculus and visual cortex sections.The expression of NF200 in RGCs,LGN,superior colliculus and visual cortex was assayed by immunohistochemistry.Results The expression level (A value) of NF200 in the RGCs was significantly different among the 8 groups after reperfusion (F =78.855,P =0.000),and that in the 1-week group was significantly lower than in the control group (t =36.563,P＜0.01).In the RIRI 1-week group,the expression of NF200 in the contralateral LGN in the experimental group was significantly lower than that in the control group (t =6.483,P＜0.01).In the 4-week group and 6-week group,the expression of NF200 in the contralateral LGN was significantly higher than that in the control group (t =2.904,4.313,P＜0.01).One week after RIRI,the expression of NF200 in contralateral superior celliculus in the experimental group was significantly lower than that in the control group (t =2.966,P＜0.05),and in the 2-week group,the expression of NF200 in the contralateral superior colliculus was significantly higher than that in the control group (t =7.397,P＜0.01).In the 2-week group,3-week group and 4-week group,the expression of NF200 in bilateral visual cortex was significantly higher in the experimental group than that in the control group (contralateral ∶ t =18.728,18.213,15.088,P＜0.01 ; ipsilateral ∶ t =8.690,5.704,7.805,P＜0.01).Conclusions RIRI can induce axonal damage of RGCs,contralateral LGN,superior colliculus and bilateral visual cortex neurons.

Through morphological observation, HE staining, TRAP staining and toluidine blue staining of bone resorption pits to identify osteoclasts which obtained by 1α, 25-(OH)2 VitD3 inducing rabbit bone marrow cells. Three indicators-TRAP staining, TRAP enzyme activity detecting and the number and area of bone resorption pits were adapted to detect the effect of Sargentodoxae caulis on the activity of osteoclasts. Culturing MC3T3-E1 Subclong 14 cells and detecting the effect of S. caulis on differentiation and proliferation of them by MTT and detecting the alkaline phosphatase in cells. The results show that all of the low, middle and high doses of water and alcohol extracts of S. caulis have significant inhibition on osteoclast differentiation and bone resorption ability in a dose-dependent manner. The low and middle doses of water and alcohol extracts of S. caulis can stimulate differentiation and proliferation of MC3T3-ElSubclone 14 cells, which indicates S. caulis can prevent osteoporosis and the function could be achieved by inhibiting osteoclast activity and promoting the proliferation and differentiation of osteoblasts.
Animals ; Bone Resorption ; drug therapy ; physiopathology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Mice ; Osteoclasts ; cytology ; drug effects ; Rabbits

Objective To improve the prognosis among patients with respiratory failure poisoned by organophosphorus pesticide though analyzing and discussing the emergency measures and treatment effects. Methods From January 2014 to December 2016, 86 patients with respiratory failure caused by severe organophosphorus pesticide poisoning were received in the Emergency Department in Nanchong Central Hospital.The patients were numbered according to the order of the treatment and randomly divided into control group and observation group with 43 cases in each group.All patients were treated with routine emergency measures (gastric lavage and endotracheal intubation, rehydration, and symptomatic treatment).The control group was treated with pralidoxime chloride injection and injection of atropine detoxification while the observation group was given pralidoxime chloride combined with Penehyclidine Hydrochloride Injection detoxification.We compared the alleviation of clinical symptoms and the changes of cholinesterase (CHE) and respiratory function (respiratory frequency, Pa02, Pa02/Fi02) between the two groups of patients 10 min and 30 min after administration. The statistics of atropinization time, blood purification treatment rate, tracheotomy rate, hospital mortality,complications and treatment time were recorded. Results No statistic significance was observed among the two groups of patients in gender,age, weight,body mass index (BMI),the severity of poisoning,types of organophosphorus drugs and blood cholinesterase (CHE) at the first visit (P＞0.05).Blood CHE was effectively improved among the two groups 10 min and 30 min after the treatment and significantly higher CHE was seen in the observation group compared to the control group (P＜0.05) . The overall clinical symptom rate was lower 10min and 30min after the treatment (P＜ 0.05), and the clinical symptom rate of the observation group was lower than that of the control group (P＜0.05) . The respiratory function indexes (respiratory rate, Pa02, Pa02/Fi02) were significantly improved 10 min and 30 min after the treatment in both groups compared with those before the treatment (P＜ 0.05) .The blood purification treatment rate, tracheotomy rate, complication rate, hospitalization time and atropine time of the observation group were significantly lower than those of the control group (P＜ 0.05) . No significant difference was found in mortality rate between the two groups (P＞0.05).Conclusion After giving effective respiration and circulation support, administration of penehyclidine hydrochloride combined with pralidoxime chloride detoxification can effectively restore the cholinesterase activity among patients with organophosphorus pesticide poisoning and improve respiratory function and prognosis, whichshows a high clinical value.

Objective To observe the protective effects of Diazoxide (DZ) on myocardial ischemia and reperfusion (I/R) in non-diabetic rats with stressed hyperglycemia and to explore its possible mechanism. Methods The stressed hyperglycemia (SHG) myocardical I/R model was prepared by ligation of the left anterior descending branch of the coronary artery for 30 minutes and reperfusion for 120 minutes on the healthy adult Sprague-Dawley (SD) rats. Blood sugar was required up to 10 mmol/L in the qualified animal model after ischemia for 30 minutes. The 48 successful model rats were randomly divided into 4 groups (12 in each group): I/R group, low, medium and high dose DZ treated group (LIPO group, MIPO group, HIPO group). Sham-operated group (sham group) was only threaded without deligation. I/R group, LIPO group, MIPO group and HIPO group were challenged to 0.1% dimethyl sulfoxide (DMSO), DZ (0.1% DMSO dissolved) 4, 7, 10 mg/kg for 2 mL, respectively after ischemia for 25 minutes. Hemodynamics indicators were continuously monitored. After reperfusion for 120 minutes, blood glucose, serum creatine kinase (CK) concentration and lactate dehydrogenase (LDH) activity were detected, myocardial infarction area was analyzed by triphenyltetrazolium chloride (TTC) staining, myocardial ultrastructure was observed by electron microscope, expressions of phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) were detected by Western Blot. Results Compared with sham group, I/R group had an elevated blood glucose, decreased heart rate (HR), systolic diastolic dysfunction, increased myocardial enzymes. Obvious necrosis of myocardium, myocardial tissue edema, mitochondria swelling, cristae, disappearing glycogen granules were observed under electron microscope with TTC staining. After reperfusion for 120 minutes, comparing with I/R group, blood glucose of HIPO group was significantly increased (mmol/L: 16.93±3.22 vs. 14.65±3.61, P < 0.05); the maximum rate of left ventricle internal pressure drop (-dp/dt max) of LIPO group was improved (mmHg/s: -1 055±16 vs. -982±10, P < 0.05) and the infarct size was evidently shrunk [(32.45±3.54)% vs. (41.30±3.21)%, P < 0.05]; left ventricular systolic pressure (LVSP) of MIPO group and HIPO group [LVSP (mmHg, 1 mmHg = 0.133 kPa): 60±2, 74±4 vs. 54±4], left ventricular end-diastolic pressure [LVEDP (mmHg): 24.6±1.5, 18.9±1.3 vs. 27.9±1.6], the maximum rate of left ventricle internal pressure were increased [+dp/dt max (mmHg/s): 1 049±37, 1 262±75 vs. 975±17], and -dp/dt max (mmHg/s: -1 068±21, -1 321±63 vs. -982±10) were improved in different degrees (all P < 0.05); CK (kU/L: 10.7±0.5, 11.0±1.3 vs. 12.9±1.0), LDH (kU/L: 6.8±0.2, 7.8±0.1 vs. 8.8±0.1) was evidently decreased (all P < 0.05), infarct size was smaller [(31.24±2.45)%, (30.81±2.68)% vs. (41.3±3.21)%, all P < 0.05], electron microscope showed that the myocardial injury was repaired. After reperfusion for 120 minutes, compared with sham group, expressions of p-Akt and p-GSK-3β in I/R group have obviously reduced (grey value: 0 vs. 0.187±0.018, 0.110±0.045 vs. 0.200±0.081, both P < 0.05). Compared with I/R group, expressions of p-Akt in HIPO group and p-GSK-3β in LIPO group, MIPO group and HIPO group were obviously increased (grey value: 0.101±0.009 vs. 0; 0.180±0.057, 0.270±0.062, 0.280±0.039 vs. 0.110±0.045, all P < 0.05). But there were significant increase in MIPO group and HIPO group. There was no significant difference in HR among different treatment groups. Conclusions I/R with SHG can significantly inhibit the activity of PI3K/Akt-GSK-3β signaling pathways, middle and high dose of DZ has a protective effect on I/R myocardium complicating with SHG, and middle dose will not lead to evident increase of blood glucose; DZ may act on GSK-3β through PI3K/Akt-GSK-3β signaling pathways, phosphorylate it and inhibit its activity, so as to develop the cardioprotective effect.

Adolescent ; Child ; Child, Preschool ; Complement System Proteins ; analysis ; Female ; Humans ; Immunoglobulins ; analysis ; Infant ; Male ; Pneumonia, Mycoplasma ; immunology