Derivation of Mouse Haploid Trophoblast Stem Cells

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Membrane fusogenic lysine type lipid assemblies possess enhanced NLRP3 inflammasome activation potency

Lysine (K) type cationic lipid with a propyl spacer and ditetradecyl hydrophobic moieties composing liposomes, K3C14, previously studied for gene delivery, were reported to activate the NLRP3 inflammasomes in human macrophages via the conventional phagolysosomal pathway. In this study, K3C16, a propyl spacer bearing lysine type lipids with dihexadecyl moieties (an extension of two hydrocarbon tail length) were compared with K3C14 as liposomes. Such a small change in tail length did not alter the physical properties such as size distribution, zeta potential and polydispersity index (PDI). The NLRP3 activation potency of K3C16 was shown to be 1.5-fold higher. Yet, the toxicity was minimal, whereas K3C14 has shown to cause significant cell death after 24 h incubation. Even in the presence of endocytosis inhibitors, cytochalasin D or dynasore, K3C16 continued to activate the NLRP3 inflammasomes and to induce IL-1β release. To our surprise, K3C16 liposomes were confirmed to fuse with the plasma membrane of human macrophages and CHO-K1 cells. It is demonstrated that the change in hydrophobic tail length by two hydrocarbons drastically changed a cellular entry route and potency in activating the NLRP3 inflammasomes.     

Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K⁺ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K⁺, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.     

Melatonin inhibits Gram-negative pathogens by targeting citrate synthase

Bacterial infections caused by Gram-negative pathogens represent a growing burden for public health worldwide. Despite the urgent need for new antibiotics that effectively fight against pathogenic bacteria, very few compounds are currently under development or approved in the clinical setting. Repurposing compounds for other uses offers a productive strategy for the development of new antibiotics. Here we report that the multifaceted melatonin effectively improves survival rates of mice and decreases bacterial loads in the lung during infection. Mechanistically, melatonin specifically inhibits the activity of citrate synthase of Gram-negative pathogens through directly binding to the R300, D363, and H265 sites, particularly for the notorious Pasteurella multocida. These findings highlight that usage of melatonin is a feasible and alternative therapy to tackle the increasing threat of Gram-negative pathogen infections via disrupting metabolic flux of bacteria.

     

Opposing effects of plant growth regulators via clonal integration on apical and basal performance in alligator weed

植物生长调节剂通过克隆整合对空心莲子草顶端和基部生长的不同作用 入侵植物不仅对全球生物多样性造成了巨大的威胁,同时也严重影响了农业生产与粮食安全。克隆整合使得相连植株进行资源共享,能促进入侵植物的生长从而获得优势。然而,入侵杂草 在植物调节剂(plant growth regulators, PGRs)影响下的克隆整合作用则很少有报道。PGRs被广泛应用于 农作物生产上,并能通过土壤淋溶、侵蚀和径流作用,影响分布在作物附近的农田杂草的生长。本 研究采用两种PGRs赤霉素(gibberellins, GA)和多效唑(paclobutrazol,PAC)处理恶性入侵杂草空心莲子草 (Alternanthera philoxeroides)基端,并保持或者通过剪切达到控制基端与顶端的连通,从而探究克隆整合作用在空心莲子草响应两种农业常用PGRs中的作用。研究结果表明,GA和PAC对空心莲子草生长的作用相反。GA通过克隆整合作用显著促进顶端植株的地上生长。相反地,PAC显著抑制基端和顶端的地 上生长,但是能够通过克隆整合作用显著促进基端和顶端的地下生长。这些研究结果解释了克隆整合作用能促进PGRs对空心莲子草生长的促进作用,这很可能是外来杂草能够成功入侵人为干扰较多的农业生态系统的重要原因之一。     

Arbuscular mycorrhizal fungi protect a subtropical tree species exposed to simulated acid rain by accelerating photosynthetic ability,antioxidant enzymes and osmolyte accumulation

丛枝菌根真菌对其宿主光合能力、抗氧化酶和渗透物质积累的促进作用 及其抗酸雨机制的探讨 酸雨在中国南方发生频繁,对亚热带树种生长具有明显抑制作用。以往研究表明,丛枝菌根真菌(AM真菌)可以缓解酸雨对宿主植物的胁迫效应。榉树(Zelkova serrata)为中国南方主要经济树种之一,其如何与共生AM真菌协同、增强其抗酸雨胁迫的能力是本项研究所要探讨的关键科学问题。通过温室控制实验,将榉树幼苗随机接受4个水平的AM真菌接种处理(接种灭菌菌种;单独接种Rhizophagus intraradices;单独接种Diversispora versiformis;接种这两种菌种的混合菌种)和3个pH水平(pH2.5、pH4.0和pH5.6)的硫酸型酸雨和硝酸型酸雨处理组成的12个处理组合,同时测定其生长、光合性能、抗氧化酶、渗透调节和土壤酶的响应格局。研究发现酸雨处理显著降低了非菌根榉树幼苗的总干重、总叶绿素含量、叶片净光合速率和可溶性蛋白的含量;接种AM真菌,特别是接种混合菌种,显著提高了强酸胁迫下榉树幼苗的总干重、光合性能、丙二醛、过氧化物酶、超氧化物歧化酶、可溶性蛋白和根系酸性磷酸酶活性。此外,菌根效应依赖于AM真菌的种类和酸胁迫的梯度。本研究 结果表明,AM真菌对榉树幼苗抗酸胁迫的调控作用主要源于调节宿主植株光合能力、抗氧化酶和渗透物质的积累。榉树与其共生AM真菌在应对酸胁迫上协同机制的解析为该树种在中国南方酸雨区的栽培提供理论基础、具有重要的实践指导意义。     

Preparation and application of freeze-dried platelets

Clinical platelet infusion is primarily used to prevent or stop bleeding, but can also have a role in treating infections or promoting wound healing. The demand for platelets has increased in recent years. However, as platelets can only be stored for short periods, there is a substantial loss due to the products reaching their expiry date. Platelet lyophilization is a particularly valuable and important research field. The purpose of studying the freeze-drying preservation of platelets is to realize the long-term preservation of platelets at room temperature. It is very possible to prepare qualified freeze-dried platelets. However, there are still problems that have not been solved in the process of platelet lyophilization. This review mainly summarizes research progress in the preparation and application of freeze-dried platelets.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

Generation of chimeric HBc proteins with epitopes in E.coli: formation of virus-like particles and a potent inducer of antigen-specific cytotoxic immune response and anti-tumor effect in vivo

The major aim of the project was to develop the virus-like particles (VLPs) displaying single or multi-epitope of hepatocellular carcinomas (HCC) in Escherichia coli and to evaluate the effect on inducing Ag-specific CD8(+) T cell response and antitumor efficacy as candidate vaccines. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCC epitopes. Four HCC epitopes MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1 (158-166aa) or AFP2 (542-550aa) were fused to the 3' terminus of the truncated HBV core gene, respectively, or conjunctively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete "mature" VLPs. E. coli-derived truncated HBc(1-144) chimeric protein self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. We also showed that they could be internalized and presented by DCs in vitro. Additionally, DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-gamma secretion by responding T cells compared with peptid-pulsed DCs. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in significantly higher survival rate of immunized animals. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCC-specific immunogen.