A sensitive chemiluminescence(CL) method was developed for determining melamine in urine and plasma samples based on the fact that melamine can remarkably enhance the chemiluminescence of Luminol-K3 Fe(CN)6 system in alkaline medium.The determination conditions were optimized.Under optimum conditions,the chemiluminescence intensity had a good linear relationship with melamine in the range of 9.0 × 10 97.0 × 10 6 g/mL with a correlation coefficient of 0.9992.The detection limits (3σ) were 3.54 ng/mL for urine sample and 6.58 ng/mL for plasma sample.The average recoveries of melamine were 102.6％ for urine sample and 95.1％ for plasma sample.Melamine in samples was extracted with liquid-liquid extraction procedures and the assay results coincided very well with that determined with flow injection chemiluminescence method.The method provides a reproducible and stable approach for sensitive detection and quantification of melamine in urine and plasma samples.

Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAalpha1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAalpha1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAalpha1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2-4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.
Animals ; Base Sequence ; Chiroptera ; genetics ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Pichia ; genetics ; metabolism ; Plasminogen Activators ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Sequence Analysis, DNA

ObjectiveTo investigate the condition of infants with urolithiasis caused by melaminetainted powdered formula at one-year follow-up. Methods Eighty-one young children with melamine-induced urolithiasis were followed up,which included urinalysis,renal-function tests,urinary tests for biochemical markers of renal glomerular and tubular function,and ultrasonography.Eighty-one age-matched healthy infants without exposure to melamine-contaminated formulas were enrolled as controls. Results Fifty-one of the patients were male and 30 female,with a mean age of 26 months (range 13 -48 months).The 81 children were divided into 2 groups according to treatment protocols.Group 1 received conservative treatment ( n =54 ) and Group 2 received surgical treatment ( n =27 ).In Group 1,3 patients (5.6％) had stone-residual:1 girl had calculus of 0.4 cm in diameter in the left kidney and 2 girls had calculus of 0.3 cm in diameter in the right kidney.In Group 2,2 patients (7.4％) had stone-residual,1 boy had crystallization of 0.2 cm in diameter in the right kidney and 1 boy had calculus of 1.5 cm in diameter in the left kidney,positive stone was confirmed by X-ray and accepted percutaneous nephrolithotomy.Urinary microalbumin levels were significantly increased in the children with melamine-induced urolithiasis compared with the control group (P ＜0.01 ).Blood urea nitrogen and serum creatinine were within the normal range with no significant difference between the 2 groups. ConclusionsAt one-year follow-up most of the children exposed to melamine-tainted infant formula in both treatment groups had returned to a healthy state,suggesting a satisfactory outcome for both treatment options.However,surgery is recommended in patients with stoneresidual ＞ 10 mm.

Objective To investigate the effect of local injection of rat adipose-derived mesenchymal stem cells (ADSCs)on the phenotype of macrophages in skin wound.Methods The cryopreserved primary SD rat ADSCs were resuscitated and then sub-cultured.ADSCs of the third generation were used in the experiments.Thirty six SD rats were divided into ADSCs group (n =18) and control group (n =18) by random numbers table method.The full thickness skin wounds were established on bothsides of the spine.After the model establishment 0.2 ml ADSCs suspension labeled by live cell stain Chloromethylbenzamido derivatives of 1,l'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate (CM-Dil) with the concentration of 5 × 106/ml was subcutaneously annularly injected in the skin wound of SD rats in ADSCs group.The SD rats in control group were given 0.2 ml serum-free Dulbecco modified Eagle medium (DMEM).On 3,7,and 14 days after injury,six rats were selected from each group to measure the wound area and healing rate.The healed wound tissues were harvested to observe the morphology by HE staining.The expressions of interleukin (IL)-10 were detected by immunohistochemistry staining.The double-positive expressions of CD68 and inducible nitric oxide synthase (iNOS) (M1 type macrophages) and of CD68 and arginase-1 (Arg-1) (M2 type macrophages) were detected by immunofluorescent staining.The distribution of CM-Dil-labeled ADSCs in healed wound tissue 14 days after injury was observed by inverted fluorescence microscope.Results (1) At day 3 after injury,the wound areas in two groups were covered with crust and surrounding redness,and the wound healing rates were slightly different;at day 7 after injury,the wound area of ADSCs group was significantly smaller than that of control group,and the healing speed and rate of ADSCs group was significantly higher than that of control group (P ＜ 0.01);at day 14 after injury,the healing rate of ADSCs group was nearly 99％ (P ＜ 0.01),and the healing skin tissue texture of ADSCs group was better than that of control group.(2) At day 3 after injury,there were a large number of inflammatory cells and disorganized collagen fiber in the wound areas of two groups;at day 7 after injury,the inflammatory cells infiltration reduced in ADSCs group compared with control group,and the collagen fiber arrangement in control group was in disorder;at day 14 after injury,the inflammatory cells in both groups obviously decreased,and ADSCs group had more new vessels and more orderly arrangement of collagen fiber than control group.CM Dil labeled ADSCs were seen in the healing wound tissue in ADSCs group.(3) At day 3 after injury,there was little difference in M1 type macrophage distribution in the two groups;ADSCs group had more M2 type macrophage cells than control group significantly (P ＜0.01);the expression of IL 10 in ADSCs group was not high,which did not differ from that of control group;at days 7 and 14 after injury,ADSCs group has fewer M1 type macrophage cells,more M2 type macrophage cells,and higher expressions of IL-10 than control group (P ＜ 0.01).Conclusion The ADSCs trasplantation can promote the change from M1 type to M2 type macrophages,facilitating wound regeneration and healing.

Objective To investigate the effects of Pixu Yihao Fang (PXYHF, No.1 Formula for Spleen Deficiency) on the expression of Glucose Transporter (GLUT1)protein (GLUT1) and mRNA in small intestine tissue of FD spleen-deficient rats and functional dyspepsia (FD) the intervention of Pixu I Fang.Methods 70 10-day-old male SD rat pups were randomly divided into normal control group (n=10), FD model group (single model, n=10), and spleen-deficient FD model group (double model, n=50).The normal control group received intragastric administration of 0.2 mL of 2% sucrose, while the single and double model groups were given 0.2 mL of 0.1 % iodoacetamide (IA)IA in 2 % sucrose, for 6 days.6 weeks after birth consecutive days, the Modified multiple platform method (MMPM) was also applied to establish double model for 14 days after week 6.These spleen-deficient FD rats were randomly divided into double model control group, domperidone group, PXYHF low-dose, medium-dose and high-dose dose groups, and were orally gavaged with 10 mL/(kg·d) of distilled water, 3.125 mg/(kg·d) of domperidone, 1.275, 2.55, 5.1 g/(kg·d) of PXYHF respectively for 14 days.Immunohistochemistry, Western-blot and RT-PCR methods were used to measure the GLUT1 protein expression in liver tissues.Results Compared with the normal group, average optical density of GLUT1 protein in small intestinal mucosal tissue, the expression of GLUT1 protein and GLUT1 mRNA in small intestinal tissue of double model group were decreased (P<0.05, P<0.01);Compared with double model group, average optical density of GLUT1 protein in small intestinal mucosa tissue, expression of GLUT1 protein and GLUT1 mRNA in small intestinal tissue of low-dose PXYHF were increased (P<0.01).Conclusion The expression of GLUT1 gene and protein decreased in the FD rats with spleen deficiency, and Pixu Yihao Fang could increase the expression of GLUT1 gene and protein.

Objective To investigate the intervention of Pixu Fang 1 (Spleen-deficiency Formula 1) to the protein and mRNA expressions of myosin light chain (MLC) in gastric tissue in rats with functional dyspepsia (FD)-spleen deficiency pattern(SDP).Methods SD rats (n =60,aged 7 d) were randomly divided into normal group (n =10) and FD-SDP model group (n =50).The normal group was orally given 2％ sugar solution and FD-SDP model group was given 0.1％ iodoacetamide sugar solution in a dose of O.2 mL each rat a day for 6 d.The FD-SDP model group was fed with normal forage until rats aged 6 weeks and additionally given modified multiple platform method (MMPM) continuously for 14 d.After modeling,FD-SDP model group was divided randomly into model group (distilled water,10 mL/kg · d),domperidone group (3.125 mg/kg · d),low-dose Pixu Fang 1 group (low-dose group,1.275 g/ kg · d),mid-dose Pixu Fang 1 group (mid-dose group,2.550 g/kg · d) and high-dose Pixu Fang 1 group (high-dose group,5.100 g/kg · d,each n =10),and all groups were given orally corresponding medicinal for 14 d.The protein and mRNA expressions of MLC in gastric tissue were detected by using immunohistochemistry technique,Western blotting and reverse transcription-polymerase chain reaction (RT-PCR).Results Compared with normal group,MLC protein expression decreased in model group detected by immunohistochemistry technique and Western blotting,and MLC mRNA expression decreased in model group detected by RT-PCR (P ＜0.01).Compared with model group,MLC protein expression increased in low-dose group,mid-dose group and high-dose group detected by immunohistochemistry technique (P ＜ 0.05,P ＜ 0.01).MLC protein expression increased in mid-dose group and high-dose group detected by Western blotting (P ＜ 0.O1).MLC mRNA expression increased in high-dose group detected RT-PCR (P ＜ 0.01).Conclusion There are decreases of protein and mRNA expressions of MLC in FD-SDP rats,and Pixu Fang 1 can up-regulate these expressions.

Objective:To analyze and compare the association between different obesity-related indices and vitamin D deficiency in middle-aged and elderly population dwelled in Lanzhou city.Methods:From May, 2011 to September, 2012, middle-aged and elderly individuals with complete baseline data were included via randomly cluster sampling from 3 communities in Lanzhou. The subjects were divided into 4 subgroups by vitamin D levels and various obesity-related indices were compared across subgroups with the same gender. The relationship between the obesity-related indices and the severity of vitamin D deficiency was analyzed using Spearman correlation analysis, and the effects of different obesity-related indices on the severity of vitamin D deficiency was analyzed using multivariate logistic regression analysis.Results:A total of 9 437 residents were included. The overall prevalence of vitamin D deficiency was 97.7%. Compared with the group with lower vitamin D level, participants in the group with higher vitamin D level showed evidently lower body mass index (BMI), waist circumference (WC), lipid accumulation product (LAP), visceral adiposity index (VAI) and triglyceride/ high density lipoprotein cholesterol (TG/HDL-C) ratio in the total population and females, while only WC, LAP, VAI and TG/HDL-C in the males (all P<0.05). Spearman correlation analysis showed that BMI, WC, LAP, VAI and TG/HDL-C were positively correlated with the severity of vitamin D deficiency in the total population and the females, while only LAP, VAI and TG/HDL-C in the males (all P<0.05) . Multivariate logistic regression analysis showed that higher levels of these obesity related indices were correlated with more severe vitamin D deficiency in the total population and the females, while only higher LAP, VAI and TG/HDL-C in the males (all P<0.05). The effects of higher LAP was the most prominant in the total population ,the females and the males. Conclusion:Various obesity phenotypes are closely related to vitamin D deficiency in middle-aged and elderly women, while only visceral obesity and abnormal lipid metabolism are related to vitamin D deficiency in middle-aged and elderly men, with LAP being the most important influencing factor.

The ticks feed large amount of blood from their hosts and transmit pathogens to the victims. The salivary gland plays an important role in the blood feeding. When the female ticks are near engorgement, the salivary gland gradually loses its functions and begins to rapidly degenerate. In this study, data-independent acquisition quantitative proteomics was used to study changes in the phosphorylation modification of proteins during salivary gland degeneration in Haemaphysalis longicornis. In this quantitative study, 400 phosphorylated proteins and 850 phosphorylation modification sites were identified. Trough RNA interference experiments, we found that among the proteins with changes in phosphorylation, apoptosis-promoting Hippo protein played a role in salivary gland degeneration.

OBJECTIVE: To detect metabolic changes of bilateral frontal lobe in patients with multiple system atrophy (MSA) and cognitive dysfunction by 1H-proton magnetic resonance spectroscopy (1H-MRS).
 METHODS: N-acetylaspartate (NAA)/creatine(Cr), choline (Cho)/Cr, myoinositol (mI)/Cr in three sides of frontal lobe were detected by 1H-MRS in 48 healthy controls, 23 patients with MSA and cognitive dysfunction and 19 patients with MSA but without cognitive dysfunction.
 RESULTS: NAA/Cr of bilateral frontal lobes in patients with MSA and cognitive dysfunction was significantly decreased compared with MSA patients without cognitive dysfunction and healthy controls (P<0.05). mI/Cr of right frontal lobes was significantly increased in patients with MSA and cognitive dysfunction compared with healthy controls (P<0.05). There was a negative correlation between NAA/Cr of bilateral frontal lobes and duration while a positive correlation between NAA/Cr of bilateral frontal lobes and MoCA score in patients with MSA and cognitive dysfunction.
 CONCLUSION There is a decrease in NAA/Cr and an increase in mI/Cr in frontal lobes in patients with MSA and cognitive dysfunction, which may be associated with cognitive dysfunction in MSA patients.
Aspartic Acid ; analogs & derivatives ; metabolism ; Choline ; metabolism ; Cognition Disorders ; physiopathology ; Creatine ; metabolism ; Frontal Lobe ; metabolism ; Humans ; Inositol ; metabolism ; Multiple System Atrophy ; physiopathology ; Proton Magnetic Resonance Spectroscopy

【Objective】 To share the technical key points and experience of transvesical robot-assisted radical prostatectomy (TvRARP). 【Methods】 The clinical data of 13 patients with prostate cancer (PCa) receiving TvRARP during Nov.2021 and May 2022 were collected. The operation time, estimated blood loss, blood transfusion rate, catheter removal time, postoperative length of hospital stay, immediate urinary continence rate, postoperative IIEF-5 score and perioperative complications were evaluated. 【Results】 The operation time was (142±39) min, estimated intraoperative blood loss was (76±40) mL, and no transfusion was needed. The median postoperative IIEF-5 score was 16 (12-22), hospital stay 3 (2-5)days, and catheter removal time 7(5-14)days. Of all 13 patients, 12(92.3%) achieved immediate urinary continence at the removal of catheter. There were no postoperative complications of Clavien Ⅲ and above. Clavien Ⅰ-Ⅱ complications were observed in 4 patients (30.8%). 【Conclusion】 TvRARP is feasible and safe for selected patients with clinically localized PCa, which can ensure promising postoperative urinary continence and preserve erectile functional.