Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml−1 Dexloaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10−8 M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quanti!ed, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be bene!cial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation.This study was supported by the Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes (SFRH/BD/21786/2005) and by the Canon Foundation in Europe. We wish to thank P.B. Malafaya for the technical support during the micro-CT analyses, and to Materialise for providing the Mimics software. This work was also supported by the European Union funded STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283)

A power efficiency improvement technique for a bi-directional dual active bridge DC-DC converter at light load

Recently, the bi-directional dc-dc converter has been focused on because of the huge demand for diversification of power supply network including battery. The dual active bridge (DAB) dc-dc converter is one of the most popular circuits for bi-directional applications because of its simple structure. However, power efficiency at light load is the intrinsic problem of a bi-directional DAB DC-DC converter. In this paper, the simple solution with digital operation for the problem is proposed and experiments are performed with 1kW system. This method can reduce a switching surge without other circuits such as snubber and improve power efficiency at light load. Therefore it can reduce loss of switching surge and, improve power efficiency. From the results, 37% maximum power efficiency improvement at light load is confirmed. Furthermore, this method is capable of control in the conventional method in the heavy load range. Consequently, it is possible to reduce the switching surge and realize high power efficiency in a wide load range.5th Annual IEEE Energy Conversion Congress and Exhibition, ECCE 2013; Denver, CO; United States; 15 September 2013 through 19 September 2013; Category numberCFP13ECD-US

A static characteristic analysis of proposed bi-directional dual active bridge DC-DC converter

Recently, the power supply network with energy storage devices such as battery has been focused. This network topology uses bi-directional isolated DC-DC converter of low or medium capacity is required for the diversification of power supply network. The dual active bridge (DAB) DC-DC converter is one of the effective bi-directional isolated DC-DC converters. However, the circuit has some instinct problems such as degradation of power efficiency and the occurrence of the surge in light-load operation. In this paper, we have been done a static characteristic analysis and highly power-efficient technique for DAB DC-DC Converter at light load. Also the analysis results and the proposed technique are verified with some experimental results.7th International Power Electronics Conference, IPEC-Hiroshima - ECCE Asia 2014; Hiroshima; Japan; 18 May 2014 through 21 May 201

Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion

タンパク質の抗体ラベリング技術を改良し、構造解析をアシスト --電子顕微鏡やX線結晶解析による構造決定を加速化--. 京都大学プレスリリース. 2021-04-20.Antibody labeling has been conducted extensively for structure determination using both X-ray crystallography and electron microscopy (EM). However, establishing target-specific antibodies is a prerequisite for applying antibody-assisted structural analysis. To expand the applicability of this strategy, an alternative method has been developed to prepare an antibody complex by inserting an exogenous epitope into the target. It has already been demonstrated that the Fab of the NZ-1 monoclonal antibody can form a stable complex with a target containing a PA12 tag as an inserted epitope. Nevertheless, it was also found that complex formation through the inserted PA12 tag inevitably caused structural changes around the insertion site on the target. Here, an attempt was made to improve the tag-insertion method, and it was consequently discovered that an alternate tag (PA14) could replace various loops on the target without inducing large structural changes. Crystallographic analysis demonstrated that the inserted PA14 tag adopts a loop-like conformation with closed ends in the antigen-binding pocket of the NZ-1 Fab. Due to proximity of the termini in the bound conformation, the more optimal PA14 tag had only a minor impact on the target structure. In fact, the PA14 tag could also be inserted into a sterically hindered loop for labeling. Molecular-dynamics simulations also showed a rigid structure for the target regardless of PA14 insertion and complex formation with the NZ-1 Fab. Using this improved labeling technique, negative-stain EM was performed on a bacterial site-2 protease, which enabled an approximation of the domain arrangement based on the docking mode of the NZ-1 Fab

Switching surge reduction of a bi-directional dual active bridge DC-DC converter with a digital operation

Recently, the bi-directional dc-dc converter has been focused on because of the huge demand for diversification of power supply network including battery. The dual active bridge (DAB) dc-dc converter is one of the most popular circuits for bi-directional applications because of its simple structure. However, power efficiency at light load is the intrinsic problem of a bi-directional DAB DC-DC converter. In this paper, the simple solution with digital operation for the problem is proposed and experiments are performed with 1kW system. This method can reduce a switching surge without other circuits such as snubber and improve power efficiency at light load. Therefore it can reduce loss of switching surge, and improve power efficiency. From the results, 37% maximum power efficiency improvement at light load is confirmed. Furthermore, this method is capable for control in the conventional method in the heavy load range. Consequently, it is possible to reduce the switching surge and realize high power efficiency in a wide load range.2013 15th European Conference on Power Electronics and Applications, EPE 2013; Lille; France; 2 September 2013 through 6 September 201

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/ poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml!1 Dexloaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.The authors thank the funds provided by Portuguese Foundation for Science and Technology (FCT) through POCTI and FEDER programmes including project ProteoLight (PTDC/FIS/68517/2006). This work was also carried out with the support of the European Union funded STREP Project HIPPOCRATES (NMP3-CF-2003-505758) and European NOE EXPERTISSUES (NMP3-CT-2004-500283). The funding provided by Canon Foundation in Europe is gratefully acknowledged

Approach for growth of high-quality and large protein crystals

Three crystallization methods, including crystallization in the presence of a semi-solid agarose gel, top-seeded solution growth (TSSG) and a large-scale hanging-drop method, have previously been presented. In this study, crystallization has been further evaluated in the presence of a semi-solid agarose gel by crystallizing additional proteins. A novel crystallization method combining TSSG and the large-scale hanging-drop method has also been developed

維持期虚血性心疾患患者及び健常中高年者におけるゴルフ打球中の心拍数

PURPOSE: To evaluate the effect of golf swing on the heart rate level in patients with chronic ischemic heart disease (IHD) and healthy adults. METHODS: Study subjects were 7 male patients with IHD (mean age 59.3±8.7 years) and 7 age-matched, healthy male golfers. AIl subjects underwent golf swing at driving range, during which heart rate and electrocardiogram were monitored via telemetry system. Blood pressure was taken immediately after each golf swing. RESULTS: The average heart rate value for driver shot in the group of patients with IHD was significantly higher (116.1±10.8 bpm, p<0.01) compared to patting. Delta heart rate was calculated from difference between resting heart rate and peak heart rate during golf swing. Delta heart rate for driver shot in the group of IHD patients and healthy controls were significantly higher (32.7±12.5 bpm, p<0.01) compared to patting. CONCLUSIONS: In cardiovascular patients and healthy adults, heart rate of golf swing indicated severe increasing even in driving range

Structural insights into the G protein selectivity revealed by the human EP3-Gi signaling complex

熱、炎症などに関与するプロスタグランジン受容体EP3シグナリング複合体の可視化 --緑内障、高眼圧症治療薬の合理的設計に貢献--. 京都大学プレスリリース. 2022-09-15.Prostaglandin receptors have been implicated in a wide range of functions, including inflammation, immune response, reproduction, and cancer. Our group has previously determined the crystal structure of the active-like EP3 bound to its endogenous agonist, prostaglandin E₂. Here, we present the single-particle cryoelectron microscopy (cryo-EM) structure of the human EP3-Gi signaling complex at a resolution of 3.4 Å. The structure reveals the binding mode of Gi to EP3 and the structural changes induced in EP3 by Gi binding. In addition, we compare the structure of the EP3-Gi complex with other subtypes of prostaglandin receptors (EP2 and EP4) bound to Gs that have been previously reported and examine the differences in amino acid composition at the receptor-G protein interface. Mutational analysis reveals that the selectivity of the G protein depends on specific amino acid residues in the second intracellular loop and TM5

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

細胞膜の中ではたらく特殊なタンパク質分解酵素の構造を解明 --細菌感染症の新たな治療法の開発へ期待--. 京都大学プレスリリース. 2022-08-25.Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including Escherichia coli RseP. Structure-based chemical modification and cross-linking experiments indicated that the RseP domains surrounding the active center undergo conformational changes to expose the substrate-binding site, suggesting that RseP has a gating mechanism to regulate substrate entry. Furthermore, mutational analysis suggests that a conserved electrostatic linkage between the transmembrane and peripheral membrane-associated domains mediates the conformational changes. In vivo cleavage assays also support that the substrate transmembrane helix is unwound by strand addition to the intramembrane β sheet of RseP and is clamped by a conserved asparagine residue at the active center for efficient cleavage. This mechanism underlying the substrate binding, i.e., unwinding and clamping, appears common across distinct families of intramembrane proteases that cleave transmembrane segments