Uremic serum-induced calcification of human aortic smooth muscle cells is a regulated process involving Klotho and RUNX2

© 2019 The Author(s). This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY).Vascular calcification (VC) is common in subjects with chronic kidney disease (CKD) and is associated with increased cardiovascular risk. It is an active process involving transdifferentiation of arterial smooth muscle cells (SMCs) into osteogenic phenotype. We investigated the ability of serum from CKD subjects to induce calcification in human SMCs in vitro (calcific potential of sera: CP), and associated changes in expression of Runt-related transcription factor 2 (RUNX2), SM22a, and Klotho. Sera from subjects with CKD (18 stage 3, 17 stage 4/5, and 29 stage 5D) and 20 controls were added to human cultured SMCs and CP quantified. The CP of CKD sera was greater (P>0.01) than that of controls, though not influenced by CKD stage. Modification of diet in renal disease estimated glomerular filtration rate (MDRD-4 eGFR) (P>0.001), serum phosphate (P=0.042), receptor activator of nuclear factor ?appa-B ligand (RANKL) (P=0.001), parathyroid hormone (PTH) (P=0.014), and high-density lipoprotein (HDL)/cholesterol ratio (P=0.026) were independent predictors of CP accounting for 45% of variation. Adding calcification buffer (CB: calcium chloride [7 mM] and β-glycerophosphate [7 mM]) increased the CP of control sera to approximate that of CKD sera. CP of CKD sera was unchanged. CKD sera increased RUNX2 expression (P>0.01) in human SMCs and decreased SM22a expression (P>0.05). Co-incubating control but not CKD serum with CB further increased RUNX2 expression (P>0.01). Both SM22a and Klotho expression decreased significantly (P>0.01) in the presence of CKD serum, and were virtually abolished with stage 5D sera. These findings support active regulation by CKD serum of in vitro VC by induction of RUNX2 and suppression of SM22a and Klotho.Peer reviewe

Расчет, проектирование и монтаж каркаса автостоянки открытого типа

Актуальность работы: в данной выпускной квалификационной работе выполняются расчет и проектирование каркаса автостоянки открытого типа. Целью работы является разработка технологии изготовления каркаса. Целью данного курсового проекта является изучение составных частей изделия, определение марки стали, выбор метода сварки, определение режимов сварки и сварочных материалов, нормализация операций, составление технологического процесса, расчет необходимого количества оборудования и работников.Relevance of work: in this final qualification work, the calculation and design of the open-type carcass frame are performed. The aim of the work is to develop a technology for manufacturing a carcass. The objectives of this course project is to study the component parts of a product, determine the grade of steel, choose a welding method, determine welding modes and welding materials, normalize operations, draw up a process, calculate the required amount of equipment and the number of workers

Copper effect on the protein composition of photosystem II

The definitive version is available at: http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-3054.2000.1100419.xWe provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μM CuCl2 (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB98-1632).Peer reviewe

Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins

Bone Biomarkers Help Grading Severity of Coronary Calcifications in Non Dialysis Chronic Kidney Disease Patients

BACKGROUND: Osteoprotegerin (OPG) and fibroblast growth factor-23 (FGF23) are recognized as strong risk factors of vascular calcifications in non dialysis chronic kidney disease (ND-CKD) patients. The aim of this study was to investigate the relationships between FGF23, OPG, and coronary artery calcifications (CAC) in this population and to attempt identification of the most powerful biomarker of CAC: FGF23? OPG? METHODOLOGY/PRINCIPAL FINDINGS: 195 ND-CKD patients (112 males/83 females, 70.8 [27.4-94.6] years) were enrolled in this cross-sectional study. All underwent chest multidetector computed tomography for CAC scoring. Vascular risk markers including FGF23 and OPG were measured. Logistic regression analyses were used to study the potential relationships between CAC and these markers. The fully adjusted-univariate analysis clearly showed high OPG (≥10.71 pmol/L) as the only variable significantly associated with moderate CAC ([100-400[) (OR = 2.73 [1.03;7.26]; p = 0.04). Such association failed to persist for CAC scoring higher than 400. Indeed, severe CAC was only associated with high phosphate fractional excretion (FEPO(4)) (≥38.71%) (OR = 5.47 [1.76;17.0]; p = 0.003) and high FGF23 (≥173.30 RU/mL) (OR = 5.40 [1.91;15.3]; p = 0.002). In addition, the risk to present severe CAC when FGF23 level was high was not significantly different when OPG was normal or high. Conversely, the risk to present moderate CAC when OPG level was high was not significantly different when FGF23 was normal or high. CONCLUSIONS: Our results strongly suggest that OPG is associated to moderate CAC while FGF23 rather represents a biomarker of severe CAC in ND-CKD patients

Analysis of cell cycle-related proteins in gastric intramucosal differentiated-type cancers based on mucin phenotypes: a novel hypothesis of early gastric carcinogenesis based on mucin phenotype

<p>Abstract</p> <p>Background</p> <p>Abnormalities of cell cycle regulators are common features in human cancers, and several of these factors are associated with the early development of gastric cancers. However, recent studies have shown that gastric cancer tumorigenesis was characterized by mucin expression. Thus, expression patterns of cell cycle-related proteins were investigated in the early phase of differentiated-type gastric cancers to ascertain any mechanistic relationships with mucin phenotypes.</p> <p>Methods</p> <p>Immunostaining for Cyclins D1, A, E, and p21, p27, p53 and β-catenin was used to examine impairments of the cell cycle in 190 gastric intramucosal differentiated-type cancers. Mucin phenotypes were determined by the expressions of MUC5AC, MUC6, MUC2 and CD10. A Ki-67 positive rate (PR) was also examined.</p> <p>Results</p> <p>Overexpressions of p53, cyclin D1 and cyclin A were significantly more frequent in a gastric phenotype than an intestinal phenotype. Cyclin A was overexpressed in a mixed phenotype compared with an intestinal phenotype, while p27 overexpression was more frequent in an intestinal phenotype than in a mixed phenotype. Reduction of p21 was a common feature of the gastric intramucosal differentiated-type cancers examined.</p> <p>Conclusions</p> <p>Our results suggest that the levels of some cell cycle regulators appear to be associated with mucin phenotypes of early gastric differentiated-type cancers.</p

Neutron reflection study of the adsorption of the phosphate surfactant NaDEHP onto alumina from water.

The adsorption of a phosphorus analogue of the surfactant AOT, sodium bis(2-ethylhexyl) phosphate (NaDEHP), at the water/alumina interface is described. The material is found to adsorb as an essentially water-free bilayer from neutron reflection measurements. This is similar to the behavior of AOT under comparable conditions, although AOT forms a thicker, more hydrated layer. The NaDEHP shows rather little variation with added salt, but a small thickening of the layer on increasing the pH, in contrast to the behavior of AOT.We thank BP plc and EPSRC for financial support for this work as well as the ISIS and ILL staff and scientists for the allocation of beam time and technical assistance with NR measurements. We also appreciate Chris Sporikou at Department of Chemistry, University of Cambridge, for help with the surfactant synthesis.This is the final version of the article. It first appeared at http://dx.doi.org/10.1021/la504837

Deletion of PKBα/Akt1 Affects Thymic Development

BACKGROUND: The thymus constitutes the primary lymphoid organ for the majority of T cells. The phosphatidyl-inositol 3 kinase (PI3K) signaling pathway is involved in lymphoid development. Defects in single components of this pathway prevent thymocytes from progressing beyond early T cell developmental stages. Protein kinase B (PKB) is the main effector of the PI3K pathway. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether PKB mediates PI3K signaling in the thymus, we characterized PKB knockout thymi. Our results reveal a significant thymic hypocellularity in PKBalpha(-/-) neonates and an accumulation of early thymocyte subsets in PKBalpha(-/-) adult mice. Using thymic grafting and fetal liver cell transfer experiments, the latter finding was specifically attributed to the lack of PKBalpha within the lymphoid component of the thymus. Microarray analyses show that the absence of PKBalpha in early thymocyte subsets modifies the expression of genes known to be involved in pre-TCR signaling, in T cell activation, and in the transduction of interferon-mediated signals. CONCLUSIONS/SIGNIFICANCE: This report highlights the specific requirements of PKBalpha for thymic development and opens up new prospects as to the mechanism downstream of PKBalpha in early thymocytes

Overexpression of Akt1 Enhances Adipogenesis and Leads to Lipoma Formation in Zebrafish

<div><h3>Background</h3><p>Obesity is a complex, multifactorial disorder influenced by the interaction of genetic, epigenetic, and environmental factors. Obesity increases the risk of contracting many chronic diseases or metabolic syndrome. Researchers have established several mammalian models of obesity to study its underlying mechanism. However, a lower vertebrate model for conveniently performing drug screening against obesity remains elusive. The specific aim of this study was to create a zebrafish obesity model by over expressing the insulin signaling hub of the <em>Akt1</em> gene.</p> <h3>Methodology/Principal Findings</h3><p><em>Skin oncogenic transformation screening shows that a stable zebrafish transgenic of Tg(krt4Hsa.myrAkt1</em>)^cy18 displays severely obese phenotypes at the adult stage. In Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18, the expression of exogenous human constitutively active Akt1 (myrAkt1) can activate endogenous downstream targets of mTOR, GSK-3α/β, and 70S6K. During the embryonic to larval transitory phase, the specific over expression of myrAkt1 in skin can promote hypertrophic and hyperplastic growth. From 21 hour post-fertilization (hpf) onwards, myrAkt1 transgene was ectopically expressed in several mesenchymal derived tissues. This may be the result of the integration position effect. Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18 caused a rapid increase of body weight, hyperplastic growth of adipocytes, abnormal accumulation of fat tissues, and blood glucose intolerance at the adult stage. Real-time RT-PCR analysis showed the majority of key genes on regulating adipogenesis, adipocytokine, and inflammation are highly upregulated in Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18. In contrast, the myogenesis- and skeletogenesis-related gene transcripts are significantly downregulated in Tg(<em>krt4:Hsa.myrAkt1</em>)^cy18, suggesting that excess adipocyte differentiation occurs at the expense of other mesenchymal derived tissues.</p> <h3>Conclusion/Significance</h3><p>Collectively, the findings of this study provide direct evidence that Akt1 signaling plays an important role in balancing normal levels of fat tissue in vivo. The obese zebrafish examined in this study could be a new powerful model to screen novel drugs for the treatment of human obesity.</p> </div