Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Crystallization and preliminary X-ray structure analysis of pigeon egg-white lysozyme.

Calcium binding lysozyme from pigeon egg-white was crystallized by the hanging drop vapor diffusion technique using ammonium sulphate as a precipitant. The crystals belong to the orthorhombic system, space group P2(1)2(1)2(1), and have unit cell dimensions of a = 34.2 A, b = 34.8 A, and c = 99.4 A. One asymmetric unit contains one molecule of the pigeon lysozyme. The crystals diffract X-rays at least to 2.0 A resolution and are suitable for high resolution structure analysis. The diffraction data up to 3.0 A resolution were collected with a diffraction image processor, DIP100, using a Fuji imaging plate as an area detector. The structure was solved by the molecular replacement technique and refined to an R factor of 0.216. Least-squares fitting of the main-chains of pigeon egg-white lysozyme with those of chicken egg-white lysozyme and baboon alpha-lactalbumin showed that the main-chain folding of pigeon lysozyme is more similar to that of chicken lysozyme than that of alpha-lactalbumin. The largest differences between the pigeon and chicken lysozymes are in the surface loop regions.     

Cytoskeletal changes correlated with the loss of neuronal polarity in axotomized lamprey central neurons

Axotomy within 500 μm of the soma (close axotomy) causes identified neurons (anterior bulbar cells or ABCs) in the lamprey hindbrain to lose their normal polarity and regenerate axons ectopically from dendritic tips, while axotomy at more distal sites (distant axotomy) results in orthotopic axonal regeneration from the axon stump. We performed immunocytochemical, electron microscopic and in situ hybridization analyses comparing ABCs subjected to close and distant axotomy to elucidate the mechanism by which neuronal polarity is lost. We show that polarity loss in ABCs is selectively and invariably preceded and accompained by the following cellular changes: (1) a loss of many dendritic microtubules and their replacement with neurofilaments, (2) a loss of immunostaining for acetylated tubulin in the soma and proximal dendrites, and (3) an increase of immunostaining for phosphorylated neurofilaments in the distal dendrites. We also show that these changes do not depend on either the upregulation or spatial redistribution of neurofilament message, and thus must involve changes in the routing of neurofilament protein within axotomized ABCs. We conclude that close axotomy causes dendrites to undergo axonlike changes in the mechanisms that govern the somatofugal transport of neurofilament protein, and suggest that these changes require the reorganization of dendritic microtubules. We also suggest that the bulbous morphology and lack of f-actin in the tips of all regenerating sprouts supports the possibility that axonal regeneration in the lamprey CNS does not involve actin-mediated "pulling" of growth cones, but depends instead on the generation of internal extrusive forces.     

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

NF-κB p65的敲减加重结肠上皮HCT116细胞氧化损伤

炎症细胞诱导的活性氧类生成和肠道氧化应激与慢性炎症性肠疾病以及结直肠肿瘤的发病密切相关。NF-κB信号通路参与氧化应激反应以及在结直肠炎症和肿瘤发生中的作用还并不完全清楚。本研究将化学合成一对编码小干扰RNA 序列、靶向人NF-κB 基因的长60 bp寡核苷酸链定向克隆至pSUPER小干扰RNA表达载体中,通过单酶切、双酶切及测序证实重组RNA干扰载体构建成功. 将构建成功的质粒转染至结肠上皮细胞HCT116中敲减p65,分别采用Western blot方法检测NF-κB p65蛋白表达水平,（3-(4,5-二甲基噻唑-2)-3,5-二苯基四氮唑溴盐（MTT）方法检测细胞存活情况. 结果显示,pSUPER-NF-κB p65载体可特异性下调NF-κB p65蛋白表达;下调p65表达可导致过氧化氢诱导的HCT116内活性氧类物质生成增高,存活细胞数目显著减少,氧化损伤加重。研究表明,在人结肠上皮细胞内NF-κB p65通路的抑制显著加重了结肠上皮细胞氧化损伤情况.     

Halogen Engineering for Operationally Stable Perovskite Solar Cells via Sequential Deposition

The performance of perovskite solar cells (PSCs) relies on the synthesis method and chemical composition of the perovskite materials. So far, PSCs that have adopted two‐step sequential deposited perovskite with the state‐of‐art composition (FAPbI₃)_1?x(MAPbBr₃)_x (x < 0.05) have achieved record power conversion efficiency (PCE), while their one‐step antisolvent dripping counterparts with typical composition Cs_0.05FA_0.81MA_0.14Pb(I_0.85Br_0.15)₃ with more bromine have exhibited much better long‐term operational stability. Thus, halogen engineering that aims to elevate bromine content in sequential deposited perovskite film would push operational stability of PSCs toward that of antisolvent dripping deposited perovskite materials. Here, a Br‐rich seeding growth method is devised and perovskite seed solution with high bromine content is introduced into a PbI₂ precursor, leading to bromine incorporation in the resulting perovskite film. Photovoltaic devices fabricated by Br‐rich seeding growth method exhibit a PCE of 21.5%, similar to 21.6% for PSCs having lower bromine content. Whereas, the operational stability of PSCs with higher bromine content is significantly enhanced, with over 80% of initial PCE retained after 500 h tracking at maximum power point under 1‐sun illumination. This work highlights the vital importance of halogen composition for the operational stability of PSCs, and introduces an effective way to incorporate bromine into mixed‐cation‐halide perovskite film via sequential deposition method.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.