One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.     

英国国家公园内住宅建设管控政策研究

住宅是社区管理的重要物质载体,住宅建设管控是国家自然保护地体系建设的重要部分,英国国家公园内住宅建设管控的经验能为我国自然保护地社区管理带来新思路,有助于解决我国自然保护地人口聚居的问题.梳理英国国家公园内住宅建设管控背景,引介英国国家公园内住宅建设管控政策,借鉴强制管制结合柔性引导的管控经验,丰富我国自然保护地体系建设中住宅建设管控的思路.     

Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and β-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins.     

弧焊机器人焊接系统的研制与应用

本文介绍弧焊机器人焊接系统的研制及应用。该系统与示教再现型机器人配套,适于CO_2焊接。它采用高性能晶闸管式弧焊电源和推拉丝式送丝机,通过接口盒与机器人控制器通讯,能检测多种故障和报警。经反复试验和生产现场使用,证明该系统性能优良,工作可靠,焊接质量高,成本低,可实现国产化,替代进口产品。     

Structural and transport effects of doping perfluorosulfonic acid polymers with the heteropoly acids, H3PW12O40 or H4SiW12O40

A perfluorosulfonic acid (PFSA) polymer with pendant side chain -O(CF₂)₄SO₃H was doped with the heteropoly acids (HPAs), H₃PW₁₂O₄₀ and H₄SiW₁₂O₄₀. Infrared spectroscopy revealed a strong interaction between the HPA and the PFSA ionomer. Modes associated with the peripheral bonds of the HPA were shifted to lower wave numbers when doped into PFSA membranes. Small-angle X-ray scattering (SAXS) measurements showed the presence of large crystallites of HPA in the membrane with d spacings of ca. 10 Å, close to the lattice spacing observed in bulk HPA crystals. Under wet conditions the HPA was more dispersed and constrained the size of the sulfonic acid clusters to 20 Å at a 5 wt% HPA doping level, the same as in the vacuum treated ionomer samples. Under conditions of minimum hydration the HPA decreased the E_a for the self-diffusion of water from 27 to 15 kJ mol⁻¹. The reverse trend was seen under 100% RH conditions. Proton conductivity measurements showed improved proton conductivity of the HPA doped PFSAs at a constant dew point of 80 °C for all temperatures up to 120 °C and at all relative hummidities up to 80%. The activation energy for proton conduction generally was lower than for the undoped materials at RH ≤80%. Significantly the E_a was 1/2 that of the undoped material at RHs of 40 and 60%. A practical proton conductivity of 113 mS cm⁻¹ was observed at 100 °C and 80% RH.     

大分子相容剂在填充聚烯烃中的应用

综述了国内外大分子相容剂在填充聚烯烃塑料中的应用，以及大分子相容剂改性填充聚烯烃塑料的物理与力学性能；指出大分子相容剂用于填充塑料，能改善高分子基体与填料间界面粘结，提高填充塑料复合材料的物理与力学性能，比传统有机偶联剂更具特点。     

A technique for extracting behavioral sequence patterns from GPS recorded data

The mobile wireless market has been attracting many customers. Technically, the paradigm of anytime-anywhere connectivity raises previously unthinkable challenges, including the management of million of mobile customers, their profiles, the profiles-based selective information dissemination, and server-side computing infrastructure design issues to support such a large pool of users automatically and intelligently. In this paper, we propose a data mining technique for discovering frequent behavioral patterns from a collection of trajectories gathered by Global Positioning System. Although the search space for spatiotemporal knowledge is extremely challenging, imposing spatial and temporal constraints on spatiotemporal sequences makes the computation feasible. Specifically, the mined patterns are incorporated with synthetic constraints, namely spatiotemporal sequence length restriction, minimum and maximum timing gap between events, time window of occurrence of the whole pattern, inclusion or exclusion event constraints, and frequent movement patterns predictive of one ore more classes. The algorithm for mining all frequent constrained patterns is named cAllMOP. Moreover, to control the density of pattern regions a clustering algorithm is exploited. The proposed method is efficient and scalable. Its efficiency is better than that of the previous algorithms AllMOP and GSP with respect to the compactness of discovered knowledge, execution time, and memory requirement.     

Peer-to-peer bichromatic reverse nearest neighbours in mobile ad-hoc networks

The increasing use of mobile communications has raised many issues of decision support and resource allocation. A crucial problem is how to solve queries of Reverse Nearest Neighbour (RNN). An RNN query returns all objects that consider the query object as their nearest neighbour. Existing methods mostly rely on a centralised base station. However, mobile P2P systems offer many benefits, including self-organisation, fault-tolerance and load-balancing. In this study, we propose and evaluate 3 distinct P2P algorithms focusing on bichromatic RNN queries, in which mobile query peers and static objects of interest are of two different categories, based on a time-out mechanism and a boundary polygon around the mobile query peers. The Brute-Force Search Algorithm provides a naive approach to exploit shared information among peers whereas two other Boundary Search Algorithms filter a number of peers involved in query processing. The algorithms are evaluated in the MiXiM simulation framework with both real and synthetic datasets. The results show the practical feasibility of the P2P approach for solving bichromatic RNN queries for mobile networks.     

聚丙烯熔融特性与MDSC条件的相关性

用调制式差示扫描量热法研究了聚丙烯(PP)升温过程熔融特性与线性升温速率（β)、温度调制振幅（AT)和调制周期(p)的相关性。实验结果表明，在低β时，总热流部分PP熔融呈现双峰；β加快时，呈现单熔融峰。随β加快，不可逆热流部分熔融热(△H)增加；可逆热流部分出现明显的放热峰。随p延长，总热流部分和不可逆热流部分的熔融峰温(Tp)基本不变，起始熔融温度(Ton)降低；但△H前者不变，后者增加；可逆热流部分从吸热变成放热，放热峰温移向高温。总热流和不可逆热流部分的Tp和Ton不受AT的影响，但随AT增大△H前者不变，后者降低。