Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA.Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers.A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments.Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively.HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor.Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups.Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression.ResultsRestriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully.The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+mmu-miR-30b, pmirGLO+mmu-miR-30b inhibitor, pmirGLO-mFoxO3+mmu-miR-30b, pmirGLO-mFoxO3+mmu-miR-30b+mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively.Compared with pmirGLO+mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+mmu-miR-30b group (P<0.05), but showed no significant change in pmirGLO-mFoxO3 mt+mmu-miR-30b group (P>0.05).In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05).Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05).Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b.There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA.

Objective To evaluate expression of inducible nitric oxide synthase (iNOS) in human abdominal aortic aneurysm (AAA) and to identify its initiation in the oxidative vascular injury. Methods This study included 22 AAA patients and 10 cadaveric normal abdominal aorta. In situ hybridization and immunofluorenscent staining were used to localize iNOS messenger RNA (mRNA) and protein. Double staining with a combination of in situ hybridization and immunofluorenscent staining was used to simultaneously demonstrate iNOS mRNA expression and its cellular localization. The presence of end-product of oxidative injury induced by iNOS was indirectly assessed with immunofluorenscent staining by anti-nitrotyrosine antibody, its cellular localization were assessed by double immunofluorenscent staining. Results In situ hybridization and immunohistochemistry confirmed the presence of iNOS in media and adventitia of AAA in all 22 patients. Specific cell markers identified iNOS mRNA-positive cells were T and B lymphocytes, macrophages, and smooth muscle cells. Positive immunostaining for nitrotyrosine was present in macrophages and smooth muscle cells. Normal abdominal aorta demonstrated virtually no iNOS or nitrotyrosine expression. Conclusion Stimulated expression of iNOS is associated with degeneration of AAA in human beings leading to oxidative tissue and cellular injury in AAA.

Objective To investigate the effects of dexamethasone on absorption of lung edema in rabbits with seawater drowning-induced acute lung injury.Methods Seawater(4 ml/kg body weight)was instilled into the lower trachea of ventilated and anesthetized rabbits.These rabbits were assigned randomly to receive intravenous injection of 1 mg/ kg body weight of dexamethasone(dexamethasone group,DG)or 2 ml of normal saline(control group,CG).Lung edema was measured by extravascular lung water index(EVLWI)using a gravimetric method.Three hours after treatment, epithelial Na~+ channel subunit-?(?-ENaC)mRNA and Na~+/K~+-adenosine triphosphatase subunit-?l(NKA-?l) protein abundances in lung tissues were respectively measured by reverse transcriptase-polymerase chain reaction and Western blotting,and NKA activity was measured by monitoring the release of inorganic phosphate(Pi)from adenosine triphosphate(ATP).Results The DG's EVLWI was significantly lower than the CG's[(0.508?0.089)vs.(0.648?0.102),P＜0.05)],but the DG's NKA activity,?-ENaC mRNA and NKA-?l protein abundances were significantly higher than the CG's,correspondingly(P＜0.05).Conclusions With up-regulation of the NKA activity and expressions of?-ENaC and NKA-?l,dexamethasone treatment could promote the absorption of lung edema in rabbits with seawater drowing-induced acute lung injury.

Objective To explore the effect of heme oxygenase-1 (HO-1) on expressions of hypoxia inducing factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF)and regeneration of hepatic vascular plexus after orthotopic liver transplantation ischemia-reperfusion injury in rats.Methods Theexperimental study was conducted.According to the random number table,240 SD rats were divided into the 3 groups,80 rats in each group.Empty virus group:rats were transfected with the empty virus.Induced group:rats were transfected with HO-1 overexpression adenovirus.Inhibited group:rats were transfected with HO-1 RNAi adenovirus.Rats were made pairs (1 ∶ 1) and established rat liver transplantation model according to two cuffs method.Rats with less weight and with heavier weight were respectively chosen as donor rats and recipient rats,and then recieved tail intravenous injection of adenovirus at 24 hours before operation.(1) Detection of transfection efficiency of adenovirus before operation:HO-1 expression of liver tissue of rats in each group was detected by Western blot at 12 and 24 hours after injection of adenovirus.(2) Liver function test of recipient rats after liver transplantation:liver functions of recipient rats [alanine transaminase (ALT),aspartate transaminase (AST),alkaline phosphatase (ALP),gamma-glutamyl transferase (GGT)] were detected at l,3,7 and 14 days postoperatively.(3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation:paraffin sections of recipient rats in the 3 groups at postoperative 1 and 14 days were stained by HE staining and observed by light microscope,and were evaluated by Suzuki damage score standard.(4) Relative expressions of HIF-1α,VEGF and HO-1 in liver tissue of recipient rats were detected by Western blot.(5) Von Willebrand factor (vWF) in liver tissue of recipient rats at 14 days postoperatively was detected by immunofluorescence staining and small vessels were counted.Measurement data with normal distribution were represented as x ±s.Comparison between groups was analyzed by the independent-sample t test,comparison among groups was done using one-way ANOVA,and pairwise comparison was analyzed by the LSD test.Results (1) Detection of transfection efficiency of adenovirus before operation:the relative expression of HO-1 of liver tissue of rats at 12 and 24 hours preoperatively after injection of adenovirus was 1.08±0.16 and 1.08±0.26 in the empty virus group,1.18±0.21 and 1.39±0.19 in the induced group,0.87±0.26 and 0.57±0.12 in the inhibited group,respectively,with statistically significant differences in different time points (F =4.232,36.513,P＜ 0.05).(2) Liver function test of recipient rats after liver transplantation:level of ALT at 3 days postoperatively in the empty virus group,induced group and inhibited group was (504±67)U/L,(438±47)U/L and (490±39)U/L,with a statistically significant difference (F=3.517,P＜0.05).Levels of ALT,AST and ALP at 7 days posto-peratively were (443±49) U/L,(430± 34) U/L,(455± 38) U/L in the empty virus group and (382± 49) U/L,(372±50) U/L,(394±25) U/L in the induced group and (493±44) U/L,(455±62) U/L,(470±72) U/L in the inhibited group,respectively,with statistically significant differences (F =10.950,5.667,5.398,P＜0.05).Levels of ALT,AST,ALP and GGT at 14 days postoperatively were (394±46)U/L,(361 ±68)U/L,(417 ±17)U/L,(4.5±1.1)U/L in the empty virus group and (283±47) U/L,(288±60) U/L,(332±46) U/L,(2.5±0.5) U/L in the induced group and (446± 43) U/L,(422± 51) U/L,(423± 63) U/L,(4.3 ± 1.3) U/L in the inhibited group,respectively,with statistically significant differences (F=26.906,9.924,8.013,9.279,P＜ 0.05).(3) Pathological histology of liver tissue and injury scores of recipient rats in the 3 groups after liver transplantation:liver cell swelling,loose cytoplasm and a varying quantity of inflammatory cell infiltration in the portal regions in the liver tissue of 3 groups were observed at 1 day postoperatively.A few inflammatory cell infiltrations in the portal regions,basically normal liver cell arrangement and a slightly swelling of liver cell were found in the empty virus group at 14 days postoperatively.Reduced liver cell swelling and basically normal structure of liver lobule were observed in the induced group.There were small patchy or focal necrosis of liver cell,masses of inflammatory cell infiltration in the portal regions and damage of bile duct in the inhibited group.Suzuki score at 1 day postoperatively in the empty virus group,induced group and inhibited group were respectively 6.7± 1.7,6.1 ± 1.2 and 7.6± 1.3,with no statistically significant difference (F=2.257,P＞0.05).Suzuki score at 14day postoperatively in the empty virus group,induced group and inhibited group were respectively 4.0±0.8,2.9± 0.8 and 5.1± 1.4,with a statistically significant difference (F=9.776,P＜0.05).(4) Western blot results:the relative expressions of HIF-1α and VEGF (43 KD) in liver tissue of recipient rats at 1 day postoperatively were 0.21±0.10,0.30±0.12 in the empty virus group and 0.23±0.09,0.34±0.14 in the induced group and 0.17± 0.06,0.29±0.11 in the inhibited group,respectively,with no statistically significant difference (F =0.902,0.410,P＞0.05).The relative expressions of VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 1 day postoperatively were 1.21 ±0.25,0.55±0.12 in the empty virus group and 2.13±0.40,0.72±0.12 in the induced group and 0.91±0.22,0.26±0.07 in the inhibited group,respectively,with statistically significant differences (F=35.158,39.082,P ＜ 0.05).The relative expressions of HIF-1α,VEGF (43 KD),VEGF (24 KD) and HO-1 in liver tissue of recipient rats at 7 days postoperatively were 0.49±0.22,0.46±0.13,0.98± 0.37,0.98±0.37 in the empty virus group and 0.83±0.26,0.63±0.19,1.60±0.33,1.49±0.46 in the induced group and 0.24±0.09,0.30±0.12,0.64±0.18,0.75±0.26 in the inhibited group,respectively,with statistically significant differences (F=16.853,10.021,20.756,8.156,P＜0.05).(5) Immunofluorescence staining results:number of small vessels at 14 days postoperatively in the empty virus group,induced group and inhibited group was respectively 7.9±2.0,10.6± 1.9 and 7.6 ± 1.9,with a statistically significant difference (F=5.921,P＜0.05).Conclusion HO-1 could promote expressions of HIF-1α and VEGF in liver tissue after liver transplantation ischemia-reperfusion injury and regeneration of intrahepatic vascular plexus,and it also alleviate bile duct ischemia-reperfusion injury after liver transplantation.

OBJECTIVETo study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.

METHODSUltraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3'-end of HCV negative-strand RNA. Competition experiment was used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.

RESULTSA cellular protein of 45 kD (p45) was found to bind specifically to the 3'-end of HCV negative-strand RNA by UV cross-linking. Nonhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3'-end of HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3'-end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201.

CONCLUSIONThe cellular protein p45 could specifically bind to the secondary structure of the 3'-end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.

Binding Sites ; Hepacivirus ; genetics ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; metabolism ; RNA-Binding Proteins ; analysis ; metabolism ; Virus Replication

Objective:To explore the effects of Alpha-2-macroglobulin-rich serum (A2MRS) on knee post-traumatic osteoarthritis (PTOA).Methods:The knee PTOA models were constructed by transection of the anterior cruciate ligament (ACL) in 80 SD male rats, aged 2 months and weighing from 250 to 300 g, which were randomized into 4 groups ( n=20): a high dose group (A2MRS containing 20 μg/μL A2M administered), a low dose group (A2MRS containing 10 μg/μL A2M administered), a positive control group (normal saline administered), and a blank control group (the knee joint cut pseudooperatively and normal saline administered). HE, toluidine blue staining, safranine O staining, modified Mankin scoring and Osteoarthritis Research Society International (OARSI) scoring were conducted to evaluate and compare the therapeutic effects of A2MRS on the knee PTOA among the 4 groups. Results:The rat cartilage was thinner with patchy and cracked surface, and the chondrocytes were reduced and distributed unevenly in the positive control group, compared with the blank control group. The modified Mankin score (3.89±0.93) and OARSI score (10.05±0.72) in the positive control group were significantly higher than those in the blank control group (0.67±0.07 and 3.10±0.29) ( P<0.05). The rat cartilage was thicker with basically complete and crack-free surface, and the chondrocytes were increased and distributed more evenly in the high dose group and the low dose group, compared with the positive control group. The modified Mankin scores (1.33±0.50 and 1.56±0.53) and OARSI scores (6.30±0.64 and 4.75±0.66) in the high dose group and the low dose group were significantly lower than those in the positive control group ( P<0.05). However, there were no such differences between the high dose group and the low dose group ( P>0.05). Conclusion:A2MRS effectively delays the pathological process of knee PTOA.

Objective To explore the expression and significance of vitamin D (VitD) in patients with rheumatoid arthritis (RA),and analyze the relationship between its expression and clinical indicators.Methods Clin-ical parameters and laboratory examinations of RA cases (n=250) were collected.Clinical parameters included were gender,age,disease course,swollen joints number,tenderness joints number,visual analog pain score (VAS),disease activity score (DAS)28 score.Laboratory examinations included erythrocyte sedimentation rate (ESR),C-reactive protein (CRP),rheumatoid factor (RF) and anti-cyclic citrullinated peptide (CCP) antibody,antinuclear (ANA) antibody,antikeratin (AKA) antibody,anti-perinuclear factor (APF),anti-mutated citrullinated vimentin (MCV),antibody and anti-6-glucose phosphate isomerase (GPI) antibody,lymphocyte subsets in the peripheral blood and lymphocyte subsets of CD4+T cells.The level of 25-(OH)-Vit-D and clinical parameters,laboratory examinations were analyzed retrospectively.One-way ANOVA and KruskalWallis test were used for comparison among the groups;and the correlation analysis was performed by Pearson and Spearman rank correlation analysis.Results ① The level of 25-(OH) D in RA patients was significantly lower than that in healthy controls (t=11.676,P＜0.01).② According to 25-(OH)D level,RA patients were divided into the deficiency group,insufficient group and normal group,the tender joints count (x2=17.793,P＜0.001),the number of swollen joints (x2=12.635,P=0.002),ESR (F=6.330,P=0.002),VAS score (F=5.095,P=0.007,DAS28 (F=4.990,P=0.008) were different significantly amorg the three groups.③RF (x2=6.742,P=0.034) and anti-CCP antibody (x2=6.836,P=0.033) were different significantly among the three groups and the level of 25-(OH) D was negatively correlated with RF (r=-0.202,P=0.001),anti-CCP antibody (r=-0.220,P＜0.01),anti-MCV antibody (r=-0.109,P=0.002) and AKA (r=-0.215,P=0.001).④ The level of 25-(OH) D in the RF (t=-2.715,P=0.007),anti-CCP antibody (t=-2.03,P=0.044),AKA (t=-2.108,P=0.036) negative group was significantly higher than that in patients with antibody positive group.⑤ The level of Th1 (IFN-γ) cells (F=3.259,P=0.043) and Treg (CD4+CD25+Foxp3+) cells (F=4.342,P=0.031) were significantly different among the three groups and the level of 25-(OH) D was positively correlated with Treg (CD4+CD25+Foxp3+) cells (r=0.146,P=0.025).Conclusion Vitamin D is generally deficient in RA patients,which is significantly correlated with disease activity,RF,anti-CCP antibody,anti-MCV antibody,AKA and Th1,Treg cells.It is suggested that vitamin D may play an important role in the immunological pathogenesis and disease progression of RA.

Objective To explore the expression and their significance of peripheral Th17 cells and regulatory T cells (Tregs) in idiopathic inflammatory myopathy,and analyze the relationship between the expression and clinical indicators,imaging and pathological changes.Methods Clinical data,laboratory tests,imaging and pathological changes of IIM cases (n=85) and healthy controls (n=70) were enrolled.Clinical data included the classification,age,gender,course of the disease;laboratory tests including erythrocyte sedimentation rate (ESR),C-reactive protein (CRP),creatine kinase (CK),creatine kinase isoenzyme-MB (CKMB),lactate dehydrogenase (LDH),hydroxybutyrate dehydrogenase (HBDH).The level of peripheral Th17,Treg cells and clinical indicators,laboratory tests,imaging and pathological changes were analyzed retrospectively.Since the data was disregarded from the normal distribution,the median four quantile method was used for statistical description.Two samples were compared with Mann-Whitney U test,and the correlation between variables was Spearman rank correlation analysis.Results ①) The levels of Th17 cells in the case group was not significantly different from that in the control group [6.18(3.42,13.65) cell/μl vs 7.42(5.02,11.13) cell/μl,P＞0.05],the levels of Treg cells in patients was significantly lower than that in the control group [21.25(12.48,35.67) cell/μl vs 36.95(30.37,47.12) cell/μl,P＜0.05],the ratio of Th17/Treg was also significantly higher than that in the control group [0.31(0.21,0.47) vs 0.18(0.14,0.31),P＜0.05].② Peripheral Treg cells levels were not correlated with ESR,CRP,CK-MB,LDH and HBDH (P＞0.05).Peripheral Treg cells levels were negatively correlated with CRP (r=-0.279,P＜0.05),but no correlated with ESR,CK-MB,LDH and HBDH (P＞0.05).③ According to the involvement of important organs,patients were classified into two groups:organ involvement group and non-organ involvement group.The levels of Treg cells in the organ involvement group was fewer than that in non-organ involvement group [16.54(8.84,27.34) cell/ul vs 24.87(14.44,43.37) cell/ul,P＜0.05],and the ratio of Th17/Treg in the organ involvement group was significantly higher than that in non-organ involvement group [0.41(0.29,0.68) vs 0.29(0.19,0.39),P＜0.05].④) Peripheral Th17 cells levels in patients with skeletal muscle inflammatory edema was significantly higher than that of non-inflammatory edema patients [10.70 (4.11,14.51) cell/μl vs 3.10 (1.27,5.15) cell/μl,Z=-2.460,P＜0.05].⑤ The levels of Th17,Treg cells and ratio of Th17/Treg did not correlate with pathological features of inflammatory infiltration (P＞0.05).Conclusion The absolute number of peripheral Treg cells decreases significantly in IIM,and correlates with CRP.Patients with organ involvement have fewer Treg cells,and there is imbalance between Th17 and Treg.When muscle MRI presents with inflammatory edema,patients may have high level of Th17 cells.Our results suggest that Treg cells may play an important role in the pathogenesis of IIM.

Electrocardiogram (ECG) signals are easily disturbed by internal and external noise, and its morphological characteristics show significant variations for different patients. Even for the same patient, its characteristics are variable under different temporal and physical conditions. Therefore, ECG signal detection and recognition for the heart disease real-time monitoring and diagnosis are still difficult. Based on this, a wavelet self-adaptive threshold denoising combined with deep residual convolutional neural network algorithm was proposed for multiclass arrhythmias recognition. ECG signal filtering was implemented using wavelet adaptive threshold technology. A 20-layer convolutional neural network (CNN) containing multiple residual blocks, namely deep residual convolutional neural network (DR-CNN), was designed for recognition of five types of arrhythmia signals. The DR-CNN constructed by residual block local neural network units alleviated the difficulty of deep network convergence, the difficulty in tuning and so on. It also overcame the degradation problem of the traditional CNN when the network depth was increasing. Furthermore, the batch normalization of each convolution layer improved its convergence. Following the recommendations of the Association for the Advancements of Medical Instrumentation (AAMI), experimental results based on 94 091 2-lead heart beats from the MIT-BIH arrhythmia benchmark database demonstrated that our proposed method achieved the average detection accuracy of 99.034 9%, 99.498 0% and 99.334 7% for multiclass classification, ventricular ectopic beat (Veb) and supra-Veb (Sveb) recognition, respectively. Using the same platform and database, experimental results showed that under the comparable network complexity, our proposed method significantly improved the recognition accuracy, sensitivity and specificity compared to the traditional deep learning networks, such as deep Multilayer Perceptron (MLP), CNN, etc. The DR-CNN algorithm improves the accuracy of the arrhythmia intelligent diagnosis. If it is combined with wearable equipment, internet of things and wireless communication technology, the prevention, monitoring and diagnosis of heart disease can be extended to out-of-hospital scenarios, such as families and nursing homes. Therefore, it will improve the cure rate, and effectively save the medical resources.
Algorithms ; Arrhythmias, Cardiac ; diagnosis ; Electrocardiography ; Heart Rate ; Humans ; Neural Networks (Computer) ; Sensitivity and Specificity