Nitrocellulose membrane based urinary protein preservation method is simple, fast and economic, but its advantage over the traditionally used acetone precipitation method is still unclear. In this work, we prepared urinary proteins by the two methods by LC-MS/MS. Then we used protein spectra counts to assess the reproducibility of the two methods. Proteins identified by the two methods were almost the same in number, spectral count distribution and distribution of coefficients of variation value. In conclusion, nitrocellulose membrane method is generally the same as acetone precipitation method. It can be used for large scale preservation of clinical urine samples.
Acetone ; Chromatography, Liquid ; Collodion ; Humans ; Mass Spectrometry ; Proteins ; isolation & purification ; Reproducibility of Results ; Tandem Mass Spectrometry ; Urine ; chemistry

To compare two enrichment and preservation methods of urinary proteins, stored in polyvinylidene difluoride (PVDF) membrane (Urimem) or direct freezing, we examined the differences between the two methods in time, space, costs of supplies and electricity, degree of protein degradation and convenience of the sample handling. The urimem method is superior in the storage space, the cost of electricity and the clinical convenience compared to the direct freezing method. However, the direct freezing method is superior in the time and the cost of supplies to the urimem method. The enrichment and preservation of urinary proteins using urimem have more cost-effective benefits compared to those of the direct freezing method.
Cost-Benefit Analysis ; Freezing ; Humans ; Polyvinyls ; Preservation, Biological ; methods ; Proteins ; chemistry ; Urine ; chemistry

Objective To investigate the effects of lentivirus-mediated heat shock protein 70 (HSP70) gene on voltage-gated calcium channels on the cell membrane of pheochromocytoma cell 12 (PC 12 cells) induced by ischemic/hypoxia and its mechanisms.Methods PC12 cells at logarithmic phase were collected,which were challenged with hypoxia for 6 hours and reoxygenation for 12 hours to stimulate the nerve cells suffered ischemia/reperfusion pathological process in vitro.PC12 cells were divided into non-infection group,infected by lentivirus containing green fluorescent protein (GFP) without HSP70 gene lentivirus control group (GFP lentivirus control group),and infected by lentivirus containing HSP70 and GFP gene recombined lentiviral infection group (HSP70 recombined lentiviral infection group).Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to determine mRNA and protein expressions of L-type voltage-gated Ca2+ channel subunits cav1.2 and cav1.3,receptor gated channel subunits NR1 and NR2,and Na+/Ca2+ exchange (NCX) in PC12 cells.Results After being challenged with hypoxia/reoxygenation,the mRNA and protein expressions of cav1.2,NR1 and NR2 in the PC12 cells were significantly lower in HSP70 recombined lentiviral infection group than those of GFP lentivirus control group and non-infection group [cav1.2 mRNA (2-△△Ct):3.13 ± 0.46 vs.5.12 ± 0.52,5.13 ± 0.66;NR1 mRNA (2-△△△Ct):1.61 ± 0.44 vs.3.23 ±0.82,3.31 ±0.78;NR2 mRNA (2-△△Ct):2.09±0.41 vs.3.91 ±0.64,3.88±0.62;cav1.2 protein (gray value):2.82±0.39 vs.3.98±0.23,3.96±0.24;NR1 protein (gray value):1.84±0.35 vs.2.79±0.21,2.86±0.23;NR2 protein (gray value):0.87±0.24 vs.1.57±0.31,1.33±0.44;all P ＜ 0.01].But there were no statistical differences in the mRNA and protein expressions of cav1.2,NR1 and NR2 between GFP lentivirus control group and non-infection group (all P ＞ 0.01).There were no statistical differences in the mRNA and protein expressions of cav1.3 and NCX among non-infection group,GFP lentivirus control group and HSP70 recombined lentiviral infection group [cav1.3 mRNA (2-△△Ct):4.82 ± 0.32,4.72 ± 0.36,4.82 ± 0.29;NCX mRNA (2-△△Ct):3.49 ± 0.78,3.47 ± 0.71,3.56 ± 0.65;cav 1.3 protein (gray value):2.63±0.40,2.64±0.39,2.68±0.39;NCX protein (gray value):3.27±0.48,3.34±0.48,3.31 ±0.42;all P ＞ 0.01].Conclusion Exogenous HSP70 affects the expression of L-type voltage-gated Ca2+ channel subunit cav1.2 and receptor gated channel subunits NR1 and NR2,which may protect PC12 cells from the injury caused by ischemic/hypoxia.

Objective To explore the effect of electroacupuncture at Quchi (LI11), Zusanli (ST36) on differentiation of neural stem cells after cerebral ischemia-reperfusion in rats. Methods Thirty-six male Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and electroacupuncture group (n=12). The latter two groups were occluded the left middle cerebral arteries for 90 min-utes and reperfused. The electroacupuncture group received electroacupuncture at Quchi and Zusanli acupoints for 21 days. They were evalu-ated with modified Neurological Severity Scores 7, 14 and 21 days after electroacupuncture. Their infarct volumes were tested with MRI T2WI 21 days after electroacupuncture, while the differentiation of neural stem cells was observed with double-immunopositive BrdU/Dcx and BrdU/NeuN. Results Compared with the model group, the neurological deficits score improved in the electroacupuncture group in all the time points (P<0.05). The infarct volumes decreased in the electroacupuncture group (P<0.05), with less number of BrdU+/Dcx+cells in subventricular zone (P<0.001) and more number of BrdU+/NeuN+ cells in peri-infarct cortex (P<0.001) 21 days after electroacupuncture. Conclusion Electroacupuncture at Quchi and Zusanli acupoints can improve neurological function and decrease the infarct volumes in rats after cerebral ischemia-reperfusion, which may be associated with promoting differentiation of neural stem cells to neurons.

Objective:To investigate the clinical manifestations and distribution characteristics of dry eye patients with ocular discomfort symptoms in Jilin Province, and to analyze the relationship between the risk factors associated with dry eye and its severity. Methods:The secondary or tertiary hospitals in Jilin Province were randomly selected and used as screening bases from July 2014 to August 2015.1 173 people initiative to the hospital for eye examinations after publicity were selected.Questionnaire was used to collect the subjective symptoms of dry eye.The breakup time(BUT) of tear film, corneal fluorescein staining, meibomian gland and tear secretion were examined and the detection rate and risk factors of dry eye of the dry eye patients with different clinical characteristics were analyzed.Results: A total of 1 122 people was actually surveyed, 896 individuals were diagnosed as dry eye, and the prevalence rate was 79.8%. The prevalence rate of the females was higher than of the males(χ2 =4.070,P<0.05).The prevalence of dry eye between different ages was statistically significant(χ2 =61.547,P<0.05).The multivariate Logistic regression analysis results showed that the age ≥40 years (40-49 years,OR =6.313,95% CI: 3.498-11.393;50-59 years,OR =6.919,95% CI: 3.876-12.351;60-69 years,OR =5.175,95% CI: 2.650-10.104;over 70 years,OR =9.508,95% CI: 3.608-25.061), moderate grade of meibomian gland dysfunction(MGD) (OR =2.123,95% CI: 1.186-3.803), and the patients with rheumatoid arthritis (OR =2.186,95% CI: 1.098-4.353) and eye surgery (OR =3.692,95% CI: 1.204-11.323) were the risk factors for dry eye. While the occupation of farmer was a protective factor for dry eye (OR =0.351, 95% CI: 0.135-0.917).Conclusion:Age, occupation, MGD grade, rheumatoid arthritis and eye surgery history affect the occurrence of dry eye to a certain extent. So enough attention and appropriate health guidance should be given to reduce the incidence of dry eye.

Biomarker is the measurable change associated with a physiological or pathophysiolog-ical process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F;hydro-chlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were col-lected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the criteria of P 6 0.05, a fold change P2, a spectral count P5, and false positive rate (FDR) 61%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC-MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics.

Objective To explore the effect of electroacupuncture on activation of microglia in peri-infarct cortex after cerebral isch-emia-reperfusion in rats. Methods 36 male Sprague-Dawley rats were randomly divided into sham group (n=12), model group (n=12) and electroacupuncture group (n=12). The latter two groups were occluded the left middle cerebral arteries with modified Longa's method for 2 hours and reperfused. The electroacupuncture group received electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints for 3 days. The nerve cell damage in peri-infarct cortex was observed with HE staining, while the expression of ED1 was determined with immunohisto-chemical staining, and the expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and IL-6 were determined with Western blotting. Results The neurological deficits score improved significantly in the electroacupuncture group (P<0.05), with less nerve cell dam-age, less number of ED1 positive microglia (P<0.05) and less levels of TNF-α, IL-1βand IL-6 (P<0.05), compared with the model group. Conclusion The electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints can protect brain from ischemia-reperfusion injury, which might be associated with inhibiting the microglial activation and proinflammatory response in peri-infarct cortex.

Objective The prevalence of obesity is constantly increasing. Multiple metabolic complications are related to obesity, including type 2 diabetes mellitus and non-alcholic fatty liver disease(NAFLD). Our study aimed to investigate the prevalence of obesity comorbidities and its association with BMI. Methods 765 individuals who visited the multidisciplinary clinic for obesity in Peking University First Hospital from 2015, Jun. to 2018, Sept. were enrolled in this study. The height, body weight, waist circumference, hip circumference were measured during the first visit. Body adipose percentage and basal metabolic rate were recorded. Questionnaires for daily food intake, comorbidity, and lifestyle were recorded. Fasting insulin, C peptide, glucose, HbA1C , uric acid, liver enzymes and lipid profile were measured. Statistical analysis was performed using SPSS 16. 0, and P<0. 05 was considered as statistical significant. Results Daily energy intake was higher in obesity group [ obese vs non-obese, (2136.6±739.4vs1905.7±468.4)kcal/d,P=0.046].Hypertension,NAFLDandgoutriskincreasedsignificantly in obesity group (obese vs non-obese, 36.0％vs 24.5％, P=0.02;76.5％ vs 60.6％, P<0.01;6.9％ vs 1.8％, P=0.04, respectively) . Family history of obesity and diabetes increased in obesity group ( obese vs non-obese, 64.5％vs 53.6％, P=0.03;47.4％vs 37.3％, P=0.048). Fasting insulin and C-peptide levels were higher in obesity group [obese vs non-obese, (24.8 ± 15.3 vs 13.6 ± 9.5)μIU/ml, P<0.01;(3.72 ± 1.40 vs 2.70 ± 1.16)μIU/ml, P<0.01). Liver enzymes increased significantly in obesity group [obese vs non-obese, (47.2±45.4 vs 23.3±21.4)U/L, P<0.01; ( 30. 4 ± 24. 0 vs 19. 9 ± 8. 5 ) U/L, P=0. 001 ] . Conclusions Obesity population had higher risk of hypertension, NAFLD and gout. Fasting insulin, C-peptide, liver enzymes, and UA also increased significantly in these patients. It is critically important to those obese individuals for regular screening of NAFLD and diabetes mellitus.

Objective To study the association between reasons for smoking and results related to successful smoking cessation. Methods A community-based case-control study was conducted with 642 male adults recognized as successful spontaneous smoking quitters being the subject cases while another 700 male adults who had failed to quit smoking were served as the controls. Russell Reason for Smoking Questionnaire(RRSQ)was used to investigate the reasons for smoking. Dominance logistic regression was performed to determine the relative importance of reasons for starting smoking and the endpoint of successful quit smoking. Results After adjusting the potential confounders of age,age of smoking initiation,marital status,profession,and education,the adjusted means of sedative and stimulation of RRSQ in the successful quitters were significant lower than that in the failed quitters. Results from the dominance logistic regression showed that the first two important factors for smoking cessation were sedative and stimulation of RRSQ,followed by psychological image,automatic,hand-mouth activity,indulgence,and addiction. Conclusion This study demonstrated that factors as dependence-addiction showed more important impacts than the social-psychological ones on the endpoint of successful smoking cessation.

Objective To analyze the characteristics of alcohol-related brain damage according to 18F-fluorodeoxy-glucose (18F-FDG) position emission tomography-computer tomography (PET-CT).Methods Excessive alcohol consumption patients accepted 18F-FDG PET-CT in our hospital from August 2016 to November 2018 were chosen as excessive alcohol consumption group and non-drinking patients accepted 18F-FDG PET-CT in our hospital at the same time were chosen as non-drinking group according to 1:4 ratio.The 18F-FDG PET-CT imaging data were analyzed;the characteristics of abnormal changed rate of CT sequences after stratification by age and the relations of alcohol consumption with abnormal CT sequences and PET sequences were compared between the two groups.Results A total of 52 patients with excessive alcohol consumption and 200 non-drinking patients were chosen.Among patients aged 45-59 years,the abnormal changed rate of CT sequences in the excessive alcohol consumption group was significantly higher than that in the non-drinking group (P＜0.05).In the CT sequences,33 patients in the 2 groups presented abnormalities,mainly including cerebral atrophy and lacunar infarction.In the PET sequences,62 patients in the two groups presented abnormalities,mainly manifesting as reduced metabolism level of bilateral frontal and parietal lobes.Among the 52 patients from excessive alcohol consumption group,those with abnormal CT sequences were older,had longer drinking history and had higher total drinking amount than those with normal CT sequences,and the differences were statistically significant (P＜0.05).As compared with those in patients with normal PET sequences,those with abnormal PET sequences had higher weekly and total alcohol consumption,and longer drinking history,and the differences were statistically significant (P＜0.05).Conclusion Excessive alcohol consumption is an important risk factor for structural damages in middle-aged men,which can cause a decrease of glucose metabolism level in the frontal and parietal lobes.