Osteoprotective effect of denosumab on patients with chronic kidney disease / 中华肾脏病杂志

Osteoporosis is a comorbidity or complication of chronic kidney disease (CKD). Low bone mineral density, osteoporosis, and fractures are risk factors of poor prognosis in patients with CKD. Active prevention and treatment of osteoporosis is essential for patients with CKD. Denosumab is a fully human monoclonal antibody that targets receptor activator of nuclear factor-κB ligand (RANKL) and is not metabolized or excreted by the kidney. Clinical studies have shown that the use of denosumab in CKD (including dialysis and renal transplantation) patients with osteoporosis, can increase bone mineral density and reduce the incidence of fractures with acceptable safety. As hypocalcemia has been observed in patients with CKD, close monitoring and early supplementation of calcium and vitamin D are required before denosumab initiation, and serum calcium levels need to be monitored during treatment. The review summarized the published literature of denosumab in bone protection of CKD.

Silencing of Myh3 Gene by siRNA Inhibits Glycolysis in C2C12 Cells / 中国生物化学与分子生物学报

The Myh3 (myosin heavy chain 3) gene is a marker gene of muscle cell differentiation and regulates the utilization of energy in muscle cells, but whether it affects the glycolysis process of muscle cells in different states is rarely reported. In this paper, the expression patterns of Myh3 and glycolysis-related genes Pkm (M-type pyruvate kinse), Prkag3 (protein kinase adenosine monophosphate-activated γ3-subunit), and Gsk3β (glycogen synthase kinase-3β) were studied by the qRT-PCR (quantitative-Real-Time-PCR) method using C2C12 cells at different stages of myoblast and adipogenic differentiation as models. It was found that in the process of myoblast differentiation of C2C12 cells, the relative expression trends of Myh3 and glycolysis genes Prkag3 and Pkm were basically the same, and the relative expression levels first increased, reached the peak on the second day of differentiation, and then decreased; glycogen synthase the expression trend of the inhibitory gene Gsk3β was relatively stable. In the process of adipogenic differentiation of C2C12 cells, the relative expression trend of Myh3 and glycolysis genes Prkag3 and Pkm remained basically the same, and the relative expression gradually increased, reaching the highest value on the 8th day of differentiation; glycogen synthase inhibitory gene Gsk3β expression remained stable. In the myogenic differentiation state of C2C12 cells, qRT-PCR and Western blotting were used to detect the effects of interfering Myh3 on the mRNA and protein expressions of glycolysis-related genes Pkm, Prkag3, and Gsk3β. The results showed that after interfering with Myh3, the mRNA expressions of glycolysis genes Pkm and Prkag3 were significantly decreased (P 0.05). The protein levels of Myh3 and Pkm were significantly lower than those in the blank group and NC group. Under the adipogenic differentiation state of C2C12 cells, after interfering with Myh3, the mRNA levels of glycogen synthase inhibitor Gsk3β and glycolysis gene Prkag3 were significantly increased (P<0.01), and the mRNA level of glycolysis gene Pkm was decreased; the protein levels of Myh3 and Pkm in the Myh3 interference group were also lower than those in the blank group and NC group. Based on the above studies, there are significant differences in the levels of glycolysis in C2C12 cells in the myogenic and adipogenic states, and the expression patterns of Myh3 and glycolysis genes are similar. Further results showed that Myh3 suppression could inhibit the glycolysis of C2C12 cells in the myogenic state without affecting the glycogen synthesis. Unlike in the myogenic state, interfering expression of Myh3 in the adipogenic state of C2C12 cells inhibited both glycogen synthesis and glycolysis.

Clinical Predictors of Outcome in Patients with Anti-neutrophil Cytoplasmic Autoantibody-related Renal Vasculitis: Experiences from a Single-center / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Primary anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) is a chronic autoimmune disease associated with multisystem dysfunction. Renal involvement is common and closely associated with outcome. The purpose of this study was to investigate the clinical determinants of mortality of patients with AAV-related renal injury in the first 2 years after diagnosis in a single West Chinese center.</p><p><b>METHODS</b>Demographic and laboratory parameters of 123 consecutive patients with AAV-related renal injury diagnosed in Renal Division and Institute of Nephrology, Sichuan Provincial People's Hospital between 2004 and 2012 were collected retrospectively. All patients were followed up for 2 years after diagnosis. Survivors were compared with nonsurvivors to identify the clinical baseline variables associated with mortality. Multivariate Cox regression model was used to determine the independent predictors of mortality.</p><p><b>RESULTS</b>Of the 123 patients, 46 (37.4%) died by the end of 2 years after diagnosis, with 41 (33.3%) patients dying within the first 12 months. In comparison with the survivors, Birmingham Vasculitis Activity Score (BVAS), the incidence of pulmonary hemorrhage and digestive system (DS) involvement, serum creatinine, and erythrocyte sedimentation rate were significantly higher in nonsurvivors, whereas lymphocyte counts, hemoglobin, and complement 3 (C3) were significantly lower. Renal replacement therapy was more common in nonsurvivors. High BVAS (hazard ratio [HR] = 1.058, 95% confidence interval [CI]: 1.002-1.117; P = 0.042), pulmonary hemorrhage (HR = 1.970, 95% CI: 1.033-3.757; P = 0.04), DS involvement (HR = 2.911, 95% CI: 1.212-6.911; P = 0.017), and serum creatinine >400 μmol/L (HR = 2.910, 95% CI: 1.271-6.664; P = 0.012) were independent predictors of death in patients with AAV-related renal injury.</p><p><b>CONCLUSIONS</b>Patients with AAV-related renal injury have high early mortality. Those with high BVAS (particularly with pulmonary or DS involvement) and serious renal dysfunction should receive aggressive therapy and careful monitoring to reduce the occurrence of adverse events and improve prognosis.</p>

Construction of Ischemic Cardiomyopathy Model via Ligation of Left Anterior Descending Artery by using Diannan Miniporcine / 昆明医科大学学报

Objective To construct a large animal model of ischemic heart disease induced by acute myocardial infarction (AMI) . Methods Male Diannan Mini-pigs were selected (n=8), weight 20 ± 4 Kg. After general anesthesia and endotracheal intubation, blood pressure and electrocardiograph were monitored. Parasternal incision was taken and thoracotomyv was performed at 4th-to-5th intercostalgap. Pericardium was opened and left anterior descending artery was ligated. Echocardiography was performed at baseline, postoperative 28 and 60 day. Euthanasia and heart explanted was conducted at 60 day of experimental pig. After gross examination of the heart, the histological examination was used to assess myocardial infarction by using H&E staining. Results All of the pigs were presented with ventricular arrhythmias in varying degrees after ligation of the left anterior descending artery, of whom 2 died of refractory ventricular fibrillation, the mortality was 25%. Echocardiography assessment showed the loss left ventricular function, the thinning of left ventricular apical and the enlargement of left ventricule. Histological examination revealed that myocardial fibrosis and fibrous scar were formed in myocardial infarction area. Conclusion The ligation of left anterior descending artery through thoracotomy under direct vision is a reliable strategy for construction of AMI porcine model and the experimental porcine can develop ischemic cardiac dysfunction progressively.

Low level of 25 (OH)D predict mortality in maintenance hemodialysis patients / 重庆医学

Objective To analyze the relationship between the level of 25 (OH )D and mortality in maintenance hemodialysis (M HD) patients .Methods This study was a prospective cohort study .We enrolled 156 M HD patients of Sichuan people′s hospital dialysis center in July of 2010 .The patients were divided into three groups according to the level of 25(OH)D .The three groups were normal(25(OH)D>30 ng/mL) ,insufficient(15 ng/mL<25(OH)D≤30 ng/mL) and deficient(25(OH)D≤15 ng/mL) re‐spectively .All the patients were follow‐up 40 months ;the end point was all‐cause and cardiovascular death .Results After follow‐up 40 months ,there were 26 deaths (16 .7% ) and 13 cardiovascular deaths among the 156 cases .There were 15 deaths (30 .6% ) in in‐sufficient group ,among which there were nine cardiovascular deaths ;there were eight deaths (11 .6% ) in deficient group ,among which there were three cardiovascular deaths ;there were three deaths (7 .9% ) in normal group ,among which there was one cardio‐vascular death .There was statistically significance either between all‐cause and cardiovascular mortality of deficient and normal group or between deficient and insufficient group (P<0 .05) .The Kaplan‐Meier curve analysis showed 25(OH)D≤15 ng/mL was the independent risk factor of the all‐cause and cardiovascular mortality(P<0 .05) .Cox regression showed 25(OH)D≤15 ng/mL was the independent risk factor of the all‐cause mortality in crude analysis (RR=4 .43 ,95% CI:1 .28-15 .32 ,P<0 .05) and adjus‐ted analysis (RR=4 .92 ,95% CI 1 .23-19 .66 ,P<0 .05) .Cox regression showed 25(OH)D≤15 ng/mL was the risk factor of the cardiovascular mortality in crude analysis(RR=8 .12 ,95% CI:1 .04 -64 .15 ,P=0 .047) .Conclusion 25(OH)D≤15 ng/mL was the risk factor and predictor of the all‐cause and cardiovascular mortality in M HD patients .

Clinical Analysis of 142 Cases of Coronary Artery Bypass Grafting under Extracorporeal Circulation / 昆明医科大学学报

Objective To summarize the clinical experience, operation indicators and curative effect of 142 cases of coronary artery bypass grafting (CABG) surgery. Methods We retrospectively analyzed the clinical data of 142 cases of CABG from January 2012 to July 2013 in the Department of Cardiovascular Surgery of Affiliated Yan 'an Hospital of Kunming Medical University. The patients included 92 cases of male, 50 cases of female, aged 24 to 78 years old, average (59.5±5.8) gears. Surgery was performed within the thoracic artery on the left of the left anterior descending branch and right coronary artery and convolution using great saphenous vein arterial anastomosis, treatment of valvular disease and aneurysm at same period,the average bypass 3.89/person. Results Two patients died after the operation,the motality rate was 1.41%. Postoperative complications included low cardiac arrhythmia, cardiac,renal insufficiency, respiratory failure, cerebral infarction/cerebral hemorrhage, open chest again stop bleeding,poor healing of incision, the incidence was 11.2%. The hospital total cost was (68.9±23.4) thousand yuan per patient,the postoperative follow-up time was (8.9±8.9) months,the follow-up rate was 96%,the postoperative patients were reviewed by echocardiography,suggesting left ventricular end-diastolic diameter and left ventricular ejection fraction were improved by different degrees. Conclusion Application of the mulitivessel transplantation methods can reduce the surgical mortality improve the cardiac function and decrease the hospital costs of patients.

Seed cells for tissue-engineered heart valves:source and application / 中国组织工程研究

BACKGROUND:Application of mechanical valve and bioprosthetic valve replacement is an effective means for the treatment of end-stage valve disease. However, their clinical application is constrained by multiple factors. Tissue-engineered heart valves with biological activity hold the potential to overcome the defect of the mechanical valves and bioprosthetic valves. Choosing appropriate cells for cellseeding is a significant aspect for the research of tissue-engineered heart valves. A growing number of somatic cells and stem cells have been used for construction of tissue-engineered heart valves, however, not yet achieve the desired result. OBJECTIVE:To review the studies on seed cells for tissue-engineered heart valves and the cellseeding methods in vitro on the basis of the cellular components of heart valves. METHODS:First author searched PubMed and Wanfang database with computer for related articles published from January 2000 to December 2012. The keywords were“tissue engineering, heart valves, cel”. Preferred articles concerned seed cells of tissue-engineered heart valves with the relevance and authority. A total of 39 articles published in the prestigious journals were reviewed. RESULTS AND CONCLUSION:Endothelial cells and interstitial cells were mainly valvular cellular components. In the early stage, endothelial cells and fibroblasts were used to construct tissue-engineered heart valves. With deep stem cellresearch, the application of pulsatile bioreactor cultivation mesenchymal stem cells holds a great potential to produce tissue-engineered heart valves.

Effects of retinol binding protein 4 knockdown on the PI3K/Akt pathways in porcine adipocytes / 生物工程学报

Retinol-binding protein 4 (RBP4) is adipocyte-derived secreted adipokines and elevated RBP4 expression level was closely related to insulin resistance and type II diabetes mellitus. However, the exact mechanisms are unknown. To clarify the mechanism, RBP4 lentivirus particles were packaged to infect porcine preadipocytes. Then porcine preadipocytes were activated by insulin or induced model of insulin resistance. RBP4 interference efficiency and the gene expression of each treatment groups in PI3K/Akt pathways were examined by QRT-PCR and Western blotting. The result shows that RBP4 mRNA and protein expressions were suppressed more than 60% (P < 0.01). Furthermore, no matter under insulin stimulation or insulin resistance, RBP4 knockdown significantly increased the mRNA expressions of AKT2, PI3K, GLUT4 and IRS1 compared with the control. The protein phosphorylate levels of AKT2, PI3K, IRS1 arised, meanwhile enhanced the AKT2, PI3K, GLUT4 total protein expressions. Collectively, knockdown of RBP4 increased the insulin sensitivity through upregulated PI3K/Akt pathways related factors' expression and phosphorylation in porcine adipocytes. This research will provide a new idea to treat insulin resistance related diseases.

Long-term efficacy of calcitonin and bisphosphonates on renal osteopathy in maintenance hemodialysis patients / 中华肾脏病杂志

Objective To observe the efficacy of ealcitonin and bisphosphonates on renal osteopathy of maintenance hemodialysis (MHD)patients. Methods Forty-three MHD patients were raindomly divided into two groups: A group and B group. All the patients were routinely received oral calcium carbonate 1.0 g tid and calcitriol 0.25 μg qd. Calcitonin (20U) hypodermic injection was given three times a week additionally during hemodialysis in A group. Patients in B group received bisphosphonates 70 mg once a week based on the therapy of A group. Serum levels of intact parathyroid hormone (iPTH), calcium, phosphorus, alkaline phosphatase (AKP), bone mass density (BMD) of lumbar spine and femoral neck, and the degree of bone ache (visual analogue scale, VAS) were assessed before the therapy and 3, 6 and 12 months after treatment. The adverse reactions were recorded during treatment. Results The levels of AKP and iPTH in both two groups decreased significantly after treatment. The above values of pre-treatment and 12 months after treatment were as follows: AKP(U/L)of A group 244.05±41.99 and 148.35±27.71,of B group 245.60±40.86 and 143.40±28.03;PTH(ng/L) of A group 697.5±119.7 and 267.4±45.9,of B group 708.2±120.3 and 277.6±41.9 (all P<0.05). While the levels of calcium and phosphorus did not change obviously during treatment (P>0.05). BMD was not improved at 3, 6 mouths and became better at 12 mouths after treatment. As compared to pre-treatment, BMD of lumbar spine(g/cm2) in A group was 1.062±0.223 vs 1.202±0.251 ,in B group 1.033±0.152 vs 1.189±0.225; BMD of femoral neck (g/cm2)in A group was 0.993±0.108 vs 1.067±0.095,in B group 0.947±0.083 vs 1.018 ±0.217 (all P<0.05). The scores of VAS also decreased significantly at 3, 6, 12 months after treatment(P<0.05). No severe adverse reaction was found during the treatment. Conclusions Utilization of calcitonin and combination with bisphosphonates during bemodialysis can effectively preserve the BMD and prevent bone loss in MHD patients and is well tolerated. No significant difference of therapeutic effect is observed between using ealcitonin or combination with bisphosphonates.

High output of a Trametes laccase in Pichia pastoris and characterization of recombinant enzymes / 生物工程学报

A laccase gene (lacD) from the basidiomycete Trametes sp. 420 was heterologously expressed in Pichia pastoris in two ways, resulting in two recombinant enzymes of rLacDx with native N-terminus and rLacDe with eight additional amino acid residues at N-terminus. The yields of rLacDx and rLacDe in shaken-flask cultures after an 18-day growth were 1.21 x 10(5) u/L and 7.38 x 10(4) u/L, respectively, as determined with 2,2'-azinobis(3-ethylbenzothia-zoline- 6-sulfonic acid) (ABTS) as substrate. The yield of rLacDx was further increased to 2.39 x 10(5) u/L under high-density fermentation while the production process was decreased to 7.5 days. In addition, rLacDx and rLacDe exhibited similar enzymatic characters in oxidizing substrate guaiacol, and were stable at 50 degrees C and at a pH range from 3 to 10. However, the specific activity of rLacDx (1761 u/mg) for ABTS was higher than that of rLacDe (1122 u/mg), and the apparent Km value of rLacDx (427 microM) was less than that of rLacDe (604 microM).

Studies on production, purification and partial characteristics of the extracellular laccase from Armilliria mellea / 生物工程学报

The production conditions of extracellular laccase from Armilliria mellea and the characteristics of the enzyme were studied. The experiment proved that initial pH5.5 of the culture medium and temperature at 25 degrees C were favorable for laccase synthesis. As carbon resources, cellobiose and raffinose were better in terms of productivity than maltose, sorbose and galactose. Organic nitrogen source was more suitable for Armilliria mellea to synthesize laccase than inorganic nitrogen source. Peat extract (PE) enhanced notably the yield of laccase; the maximal yield was 7 times as much as that of the control when PE concentration was 50%. Three isozymes were detected in culture supernatant named A, B and C respectively after their mobility on PAGE. After concentrated by (NH4)2SO4 precipitation, laccase A was further purified to homogeneity by preparative native PAGE and anion exchange column chromatography. The native enzyme was a single polypeptide with a molecular mass of approximately 59 kD estimated by SDS-PAGE, while 58 kD by gel filtration chromatography under non-denaturing conditions. Determined by IEF its isoelectric point was 4.0. The optimal pH value and temperature were 5.6 and 60 degrees C respectively in catalytic reaction of oxidizing guaiacol. At 60 degrees C and 65 degrees C, half-lives of laccase A were 45 min and 36.8 min, respectively. Enzyme activity was inhibited with 100 mmol/L Cl-, but was activated with 1 mmol/L SO4(2-). However, if the concentration of NaN3 was only 1 mmol/L, laccase A lost its activity completely. 10 mmol/L EDTA had no effect on laccase A, while 1 mmol/L Cu2+ could enhance its activity. Laccase A showed a good stability when the pH of the buffer varied from 5.2 to 7.2. Using guaiacol as the substrate, the Km was 1.026 mmol/L and the Vmax was 5 mumol/(min.mg); using ABTS instead, the Km was 0.22 mmol/L and Vmax was 69 mumol/(min.mg).

PRELIMINARY STUDIES ON LACCASE PRODUCTION BY A WHITE-ROT FUNGUS AH28-2 / 微生物学通报

A novel white-rot fungus AH28-2,which was isolated from 224 fungi samples,ability to produce effectively laccase by induction.Several factors influencing laccase production were investigated.The optimum conditions were as follows:the 300mL Erlenmeyer flask containing 150mL of liquid medium was inoculated with 7.5mL of mycelial fragments and the medium was supplied with lignin at a concentration of 0.1%(initial pH8.5).The cultures was incubated at 28℃ on rotary shaker(150r/min) for 4～5 days.The maximum enzyme activity was 20184IU/L.