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.

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.

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.

Child ; Humans ; Neurology ; Pediatrics

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

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

Objective To construct recombinant lentivirus silence vector aiming at rictor gene in mTORC2 specific protein, and to investigate its regulation on mTORC2/SGK1 signal pathway and the effect on pulmonary alveolar epithelial sodium ion chan-nel,as well as the role in acute respiratory distress syndrome(ARDS)and acute lung injury.Methods The interfering vector plas-mid and empty vector plasmid of target gene rictor were constructed,which and the lentivirus packaging system were co-transfected to 293T cells.The viral supernatant was collected,centrifuged,concentrated and purified for obtaining recombinant lentivirus.The virus titer was detected and the virus was infected to A549 cells.Stable cell lines were screened.RT-PCR was used to confirm the silencing situation of target gene rictor.The expression situation of various signal indexes in this pathway was detected by PCR and Western blot.Results The recombinant lentivirus of silence gene rictor was successfully constructed and transfected to A549 cell for obtaining stable cell lines.Compared with blank and control groups,the mRNA levels of rictor,downstream SGK1 andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased (P <0.05 ).Meanwhile,the protein levels of rictor,downstream SGK1,P-SGK andα-,β-andγ-ENaC in the shRNA-rictor group were significantly decreased compared with the other two groups(P<0.05).Conclusion Silence rictor gene has the obvious regulation effect on mTORC2/SGK1 signal pathway,meanwhile affects the expression of pulmonary alveolar epithelial cellular α-,β-and γ-ENaC at gene and protein level.It is speculated that mTORC2/SGK1 may be an important signal pathway for regulating the clearance capacity of pulmonary alveolar epithelial cells on pulmonary alveolar fluid and simultaneously affecting the pulmonary edema formation.

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.