Objective To clone fliH, fliⅠ, fliY and fliN genes that encoding flagellum-associated proteins of L. interrogans for construction of their prokaryotic expression systems, and to determine the loca- tions of Flirt, FliⅠ, FIiY and FIiN. Methods The fliH, fliⅠ, fliY andfliN genes were amplified by PCR and sequenced after T-A cloning. Prokaryotie expression systems of the target genes were constructed subsequently. Expression of target recombinant proteins were demonstrated by SDS-PAGE and BioRad Gel Image Analyzer, and Ni-NTA affinity chromatography was performed to extract the target expression prod- ucts. Rabbits were subcutaneously immunized with the four recombinant proteins respectively to obtain anti- sera. ELISA was performed to measure the titers of antisera and Western blot assay was used to determine the immunobinding abilities among the antisera and their antigens. Immunoelectron microscopy was selected to locate the position of FliH, FliⅠ, FliY and FIiN. Results Segments of fliH, fliⅠ, fliY andfliN genes with 924, 1365, 1065 and 318 bp in size were successfully obtained by PCR. Similarities of nucleotide and puta- tive amino acid sequences from the four genes were 100% compared with the reported sequences. The con- structed prokaryotic systems efficiently expressed rFliH, rFliⅠ, rFliY and rFliN with the outputs of approxi- mate 20% of the total bacterial proteins. The rabbits immunized by rFliH, rFliⅠ, rFliY or rFliN could pro- duce antibody. The antisera had the titers above 1:100 000, and could recognize the corresponding recombi- nant proteins and membrane proteins of L interrogans to display positive Western hybridization bands. Flirt, FliⅠ, FliY and FliN were found to distribute on the external surface of inner envelope, the internal surface of outer envelope or the interspaces between the two layers of L. interrogans envelope. Conclusion The pro- karyotic expression systems was successfully constructed in this study, which could efficiently express flagel-lum-associated proteins FliH, FliⅠ, FliY and FliN of L. interrogans. The antisera with high titers to recognize their protein antigens were also obtained. Flagellum-associated proteins Flirt, FliⅠ, FIiY and FIiN are the inner envelope proteins and/or outer envelope proteins of L. interrogans.

Objective To evaluate upfront common bile duct suturing against T-tube drainage after exploration in the treatment of common bile duct stone.Methods 253 cases of extrahepatic bile duct stones treated at our department from 2008 June to 2012 January were randomly divided into primary suture group and T tube drainage group,by t test or analysis of variance independent sample comparison.Results All operations were successful.Postoperative bile leakage was observed in 2 patients in group A and 4 in group B respectively(P ＞ 0.05),there was no reoperations in the two groups.In group B retrograde biliary tract infection developed in one and was cured by biliary tract flush combined with antibiotics administration.The abdominal drainage was bile tainted fluid about 5-10 ml a day in bile leakage cases in both groups and healed itself in 3-4 days without fever,jaundice symptoms.There was no other severe complications such as pancreatitis.The postoperative biliary complication rate (P =0.802),operative time (P =0.137),intraoperative blood loss (P =0.069) and liver function recovery(ALT P =0.087,AST P =0.752,TBIL P =0.459,DBIL P =0.217,ALP P =0.576,GGT P =0.362) was not significantly different between the two groups.In group A postoperative flatus passing (P =0.037),postoperative fluid volume (P =0.008),postoperative hospital stay(P =0.015) were better than that in T-tube drainage group.At 3 to 12 months follow-up,no patients were found to have residual stones and biliary stricture in group A and group B.Conclusions With the definite indication and proficient surgical technology,primary suture of common bile duct after exploration is a safe and effective way after treatment of choledocholithiasis.

Humans ; Lung ; Lung Diseases ; rehabilitation ; Pneumoconiosis ; rehabilitation ; Respiratory Therapy

AIM To investigate whether ATP sensitive pota ss ium channels are involved in the protective effects of ischemic preconditioning on spinal cord in rabbits. METHODS Twenty seven male New Zealands white rabbits were randomly assigned to 3 groups (9 in each group):ischemia gro up(IC)、ischemic preconditioning group (IPC) and glibenclamide + ischemic precon ditioning group(G+I). In IC group, spinal cord ischemia was induced by an infrar enal aorta clamping for 20 min; IPC group underwent a 6 min ischemic preconditio ning followed by 30 min of reperfusion before the 20 min clamping; G+I group wer e administered glibenclamide (an ATP sensitive potassium channel blocker, 2 mg? kg -1 )intravenously 20 min before the ischemic preconditioning. Neurologic function was scored at 8,12,24 and 48 h after reperfusion. All animals were sa crificed at 48 h after reperfusion and the spinal cords (L 5～7 ) were remov ed for histopathologic study. RESULTS The neurologic function sco res in IPC group at each observe interval were higher than those in IC group and G+I group (P

AIM: To evaluate the protective effect of rapid phase of ischemic preconditioning against spinal cord ischemic injury in rabbits. METHODS: Thirty six male New Zealands white rabbits were randomly assigned to 3 groups (12 in each group): ischemia and reperfusion injury group (IR group), ischemic preconditioning + IR group (IPC+IR group) and sham operation group (sham). In IR group, spinal cord ischemia was induced by an infrarenal aorta clamping for 20 min; The rabbits in IPC+IR group underwent a 6 min ischemic preconditioning followed by 30 min of reperfusion before the 20 min clamping; The rabbits in sham group underwent the same procedures as the IR group except for infrarental aortic unclamping. Neurologic status was scored at 8, 12, 24 and 48 h after reperfusion. All animals were sacrificed at 48 h after reperfusion and the spinal cords (L_(5-7)) were removed for histopathologic study and determination of the activity of Na~+, K~+-ATPase. RESULTS: The neurologic function scores in sham group and IPC+IR group at each observation interval were higher than those in IR group (P

Objective To explore the effectivity and feasibility of uindirectional barbed suture for primary closure of common bile duct on Laparoscopic Exploration of Common Bile Duct Stones, Methods From January 2013 to August 2015,109 cases of primary closure of common bile duct after Laparoscopic Exploration of Common Bile Duct Stones were performed in this hospital.The characteristics of these cases were retrospectively comparatively analyzed.Results Conventional braided Sutures in 68 cases(group A),barbed Suture Devices in 41 cases (group B).There was no difference in postoperative hospital stay,intraoperative blood loss and complication rate between the two groups.Differences in bile duct incision suture time and operative time between the two groups were significantly different.Conclusion The application of uindirectional barbed suture for primary closure of common bile duct using barbed suture after Laparoscopic Exploration is a safe and effective way after treatment of choledocholithiasis.This method cuold reduce the difficulty of operation,and shorten the operation time and the learning curve.

Objective:To develop couples′ communication quality scale for gynecological cancer patients and test its reliability and validity in accordance with Chinese cultural background.Methods:The scale was initially formed by literature review and Delphi expert consultation. From May to November in 2021, the scale was initially formed by literature review and Delphi expert consultation. A cross-sectional survey of 360 gynecologic cancer patients in Qilu Hospital, Shandong University was conducted from May to August 2021 using a convenience sampling method, and after pretesting, item analysis and exploratory factor analysis were used to screen the scale items. After the formal scale was formed, 385 gynecological cancer patients from Qilu Hospital, Shandong University were conveniently selected for formal testing from September to November 2021, and the reliability and validity of the scale was tested.Results:The formal couples′ communication quality scale for gynecological cancer patients was composed of 34 items from 5 dimensions of "self-disclosure", "perceived response", "stress coping", "normal creation" and "constructive action", with a cumulative variance contribution rate of 68.181%. The Cronbach α coefficient of the scale was 0.949, the split-half reliability coefficient was 0.766, the retest reliability coefficient was 0.898, and the criterion validity coefficient was 0.696. The model′s χ2/ df was 1.778, root mean square error of approximation was 0.047, comparative fit index was 0.956, incremental fix index was 0.956, Tucker-Lewis index was 0.952, normal of fit index was 0.905. Conclusions:The scale can be used to evaluate the quality of couples′ communication among gynecological cancer patients in Chinese context with good reliability and validity.

OBJECTIVETo analyze the quantity and size distribution of 24-hour urinary extracellular vesicles (uEVs) from healthy adults.

METHODSThe 24-hour uEVs from 9 healthy adults were isolated by hydrostatic filtration dialysis (HFD). The effectiveness of uEVs enrichment was evaluated using Western blotting and transmission electron microscopy (TEM). The quantity and size distribution of the uEVs was analyzed with BCA protein quantification, TEM, and nanoparticle tracking analysis (NTA).

RESULTSuEVs with different sizes and morphologies were observed under TEM. Western blotting confirmed the expression of TSG101 in all the uEV fractions from the 9 donors, ranging from 132.50 to 760.70 ng/mL. NTA results showed that the number of 24-hour uEVs amount ranged from 3.56 × 10¹² particles to 5.12 × 10¹² particles, with a CV of 14.23%. The proportion of the vesicles with a diameter <40 nm was 0.04%-0.69% with a number range of (1.80-26.49)× 10⁹ particles; the proportion of vesicles with a diameter of 40-100 nm (which is consistent with the size of exosomes)was 22.07%-42.08% with a number range of (1.00-1.77)× 10¹² particles. The proportion of vesicles with a diameter of 100-1000 nm (consistent with the size of microvesicles) was 57.88%-77.85% with a number range of (2.09-3.86)× 10¹² particles.

CONCLUSIONThe established HFD method allows efficient and convenient isolation of uEVs from a large amount of urine samples. The 24-hour uEVs from healthy adults show narrow differences between individuals and thus can be an ideal source of samples for relevant studies.

Adult ; Blotting, Western ; Cell-Derived Microparticles ; Exosomes ; Extracellular Vesicles ; Humans ; Microscopy, Electron, Transmission ; Nanoparticles ; Urine

OBJECTIVETo investigate the value of serum IgA/C3 ratio in the diagnosis of IgA nephropathy and explore its relationship with the clinicopathological features of the patients.

METHODSSixty-six patients with IgA nephropathy, 111 with other glomerular diseases, and 40 healthy control subjects without kidney disease were tested for serum IgA and C3 levels using CRM470 adjusted standardized immune turbidimetric method, and the IgA/C3 ratio was calculated. According to Oxford and Lee's classification criteria, we analyzed the pathological grades of the renal biopsy samples from patients with IgA nephropathy. The ROC curve was used to assess the value of serum IgA and IgA/C3 ratio in predicting IgA nephropathy.

RESULTSPatients with IgA nephropathy had an elevated serum IgA/C3 ratio than those with other glomerular diseases and the control subjects, with an area under the ROC curve of 0.776. An elevated serum IgA/C3 ratio was not found to significantly correlate with the pathological grade of renal biopsy samples in patients with IgA nephropathy.

CONCLUSIONIn the absence of renal biopsy findings, serum IgA/C3 ratio can help in the diagnosis of IgA nephropathy.

Biopsy ; Case-Control Studies ; Complement C3 ; analysis ; Glomerulonephritis, IGA ; blood ; diagnosis ; Humans ; Immunoglobulin A ; blood ; Kidney ; pathology

Objective:To identify the genetic variation in a mucopolysaccharidosis type Ⅱ(MPS Ⅱ)family, and conduct a functional study of iduronate-2-sulfatase(IDS): c.323A>C.Methods:A five-generation MPS Ⅱ family of 83 individuals including 4 patients from northern China was collected. Urine mucopolysaccharide and Alder-Reilly body were tested to assist the clinical diagnosis of MPS Ⅱ. IDS enzyme activity was detected on core family members. By the whole exome sequencing of a MPS Ⅱ patient in this family and bioinformatics analysis, the variant was screened and further identified by PCR-Sanger sequencing. Finally, to validate the function of the variant in vitro, the wild-type IDS overexpression plasmid(pCMV-hIDS-WT)and the IDS overexpression plasmid carrying the mutation site(pCMV-hIDS-c.323A>C)were transfected into COS-7 cells and the IDS activity was detected. Results:The proband(Ⅳ3)and Ⅳ4 were diagnosed as MPS Ⅱ by urine mucopolysaccharide, Alder-Reilly body, and IDS enzyme activity tests. Ⅳ3, Ⅳ4, Ⅲ19, and Ⅲ32 were determined to carry IDS: c.323A>C missense variant through the whole-exome sequencing, and diagnosed as MPS Ⅱ. Meanwhile, Ⅱ2, Ⅱ4, Ⅱ8, Ⅱ12, Ⅱ14, Ⅲ5, Ⅲ7, Ⅳ14 in the MPS Ⅱ family carried IDS: c.323A>C missense variant, and were excluded as MPS Ⅱ. The in vitro experiment in COS-7 cells showed that the missense mutation led to a significant decrease in IDS enzyme activity. Conclusion:The variant IDS: c.323A>C: p.Y108S significantly decreases the activity of IDS enzyme in vivo and in vitro, and it is identified as a pathogenic variant for MPS Ⅱ.