Objective To establish and verify an imaged capillary isoelectric focusing(iCIEF)method for analyzing the charge heterogeneity of recombinant adeno-associated virus(rAAV)capsid proteins,in order to provide a reliable detection method for characterization research,quality control and process stability monitoring of rAAV products. Methods After denaturation,the sample was analyzed by iCIEF using UV fluorescence detection imaging mode,with injection time of 55 s,voltage of 1 500 V,focusing for 1 min,and then voltage adjusted to 3 000 V for another 10 min. The excitation wavelength was set at 280 nm,and the exposure time of emission fluorescence detection was 80 s. Taking rAAV 5 empty particles(rAAV-E)as the research object,the specificity,precision,linear range,accuracy and limit of quantitation(LOQ)of the method were verified. The established method was used to detect the charge heterogeneity of rAAV 5 full particles(rAAV-F).Results The rAAV-E sample showed two main peaks(Peak1 and Peak2)between pH 6. 9 and pH 7. 1,and no virus capsid protein peak was detected in the blank control. In the six repeated detections,the average pI values of the two main peaks Peak1 and Peak2 of rAAV-E sample were 6. 91 and 7. 04 respectively with both RSDs of 0. 14%,and the average proportions of peak area were 35. 6% and 55. 8%,with RSDs of 1. 1% and 0. 4%,respectively. The RSDs of peak area proportions of peak 1 and peak 2 of rAAV-E sample at three time points was 3. 45% and 3. 38%,and the RSDs of pI were 0. 10%and 0. 11%,respectively. In the range of(4. 08-6. 12)× 1012vp/mL,the rAAV-E concentration showed a good linear relationship with the average peak area of the main peaks,and the linear regression equation was y = 54 888 x-76 556,R~2 = 0. 977. The recovery rates of the the main peak content in rAAV-E samples at 80%,100% and 120% expected concentrations were all in the range of 80%-120%. The LOQ was 1. 02 × 10~(12)vp/mL for Peak 1,and 0. 76 × 10~(12)vp/mL for Peak 2. In addition to Peak 1 and Peak 2,obvious extra peaks were observed in the acidic region(pI < 5. 85)of rAAV-F sample,which was the main characteristic of rAAV-F compared with rAAV-E,and also indicated that rAAV-F had complex charge heterogeneity distribution. Conclusion The established iCIEF method has good specificity,precision and accuracy,and can be used to analyze the charge heterogeneity of rAAV capsid proteins.

Alzheimer's disease( AD) , a neurodegenerative disease, can result in memory loss, cognitive and behavioral deficits. The pathological hallmarkes are β amyloid plaques and neurofibrillary tangles which lead loss of neurons in brain. As the extension of the central nervous system, retina has a similar tissue anatomy with central nervous system. The β amyloid plaques have also been detected in retina of AD. Furthermore, according to eye examinations of AD patients, we have found the loss of retinal ganglion cells, the attenuation of retinal nerve fiber layer thickness, the smaller changes of macula lutea, the decline of vascular density and so on. And then, there occurs the visual field loss and the decline of contrast sensitivity and so on in AD patients. Thus, the retina has occurred nerve degenerative changes in AD. Meanwhile, there has been proved that the retina nerve degeneration is even earlier than senile plaques formation in brain. In addition, curcumin, a natural and safe fluorescent dye, can be used to label β amyloid plaques in retina. The above suggests that retina can be a window for the early diagnosis of AD.

Most of the clinical teachers in medical colleges have not received training in the teaching profession.This article explores the post-job education system of the clinical teachers in our hospital,aiming at improving the quality of medical education.

OBJECTIVE:To prepare nifuratel vaginal effervescent tablets(NVET)and establish a method for its quality control.METHODS:The NVET was prepared using acid or alkali effervescent by principal ingredient-separated granulation.The content of the principal ingredient was determined by UV spectrophotometry.RESULTS:The preparation was white or off-white colored tablet and its property was up to the standard stated in Chinese Pharmacopeia(2005 Edition)in identification and test.The linear concentration range of nifuratel was 100～400 ?g?mL-1(r=0.999 6)with an average recovery rate of 101.1%(RSD=0.62%,n=6).CONCLUSION:The preparation process for NVET is simple,and its quality is stable and controllable.

Costs and Cost Analysis ; Dentists ; Family Practice ; Humans ; Physician-Patient Relations ; Private Practice ; economics ; standards ; Social Values

Child ; Humans ; Neurology ; Pediatrics

Acquired Immunodeficiency Syndrome ; blood ; complications ; diagnosis ; Antibodies, Viral ; blood ; Child ; Cough ; complications ; Fatal Outcome ; Fever ; complications ; HIV-1 ; immunology ; Humans ; Male

Objective To explore the anti-inflammatory mechanisms of purslane by evaluating its effects on the expression of tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule 1 (ICAM-1).Methods A model of inflammation was developed in 45 mice by painting xylene to the auricle of the right ears,which were then divided into 3 groups to receive no treatment (negative control group),be topically treated with the extraction of purslane from South Korea (positive control group) or Yunnan province (experimental group).Fifteen mice receiving no sensitization nor treatment served as the blank control group.Two hours after the single topical treatment,skin tissue samples were obtained from the site of experimental inflammation and subjected to pathological examination by using hematoxylin and eosin (HE) staining.Immunohistochemistry was performed to quantify the expression of TNF-α and ICAM-1 in the tissue samples.Results Pathological examination showed blood vessels and a small quantity of lymphocytes in murine dermis of the blank control group as well as loose and edematous dermis infiltrated with massive lymphocytes in the negative control group.However,there was only mild edema and perivascular infiltration with some inflammatory cells in the dermis of the positive control group and experimental group.Neither TNF-α nor ICAM-1 was expressed in the skin tissue of the blank control group,but an intense expression was observed for TNF-α in the vascular endothelial cell membrane and for ICAM-1 in the vascular endothelial cell membrane and lymphocyte membrane in the negative control group,which was significantly downregulated by the purslane from South Korea in the positive control group and by the purslane from Yunnan province in the experimental group (all P ＜ 0.01).Rank sum test showed a statistical difference in the expression level of TNF-α and ICAM-1 between the blank control group and experimental group (both P ＜0.01).Conclusion The purslane from Yunnan province may counteract inflammation by affecting the expression of TNF-α and ICAM-1.

EC-SOD inclusion bodies was refolded on column using metal (Ni) affinity chromatography, based on the metal-binding property of His-tag. The effect of protein amount, urea removal speed and temperature on refolding was observed. We compared the different efficiency purified with Ni-sepharose and Heprain-sepharose affinity chromatography, and studied the stability of the refolded proteins. The results indicate that the inclusion bodies can be renatured with Ni-sepharose affinity chromatography. The increase of the protein amount and urea removal rate , the lower of the renaturation efficiency. Higher temperature was benefit to protein renaturation. Both the Ni-sepharose and Heprain-sepharose affinity column can be used to purified the refolded proteins, but purified by Heprain-sepharose affinity column the protein had higher activity. The activity of renatured protein was stable in 10 ℃～50℃,when pH10 its stability was lower significantly. In denaturating solution the stability of renatured protein was low.

Objective To observe the influence of atorvastatin on the arteria carotis atheromatous plaque and blood lipid in old people. Methods 57 hospitalized patients who had cerebral infarction or transient ischemic attack(TIA) were examined for atheromatous plaque by the color Doppler. They were administered atorvastatin 20 mg/d for three months. The changes of diameter of arteria carotis, rate of flow and size of plaque were measured be-fore and after treatment. Results The diameter of arteria carotis was expanded [left ( 0.99 ± 0. 11 ) era, right (0.98 ± 0.08 ) cm vs left (0.94 ± 0.09 ) cm, right ( 0.95 ± 0.07 ) cm, P < 0.05], the hemorrheology was improved, the size of plaque was diminished[left(0.57±0.20)cm2, right (0.54± 0.18 )cm2 vs left (0.86±0. 17 ) cm2 and right (0.82 ±0. 16 )cm2, P < 0. 05], TC, TG, LDL-C were lowered and HDL-C was elevated with statistical signifi-cance ( P < 0. 05). Conclusions The atorvastatin has positive therapeutical effect in diminishing the size of plaque, adjusting blood lipid and improving hemorrheology.