Exosomes Isolated From Human Umbilical Cord Mesenchymal Stem Cells Alleviate Neuroinflammation and Reduce Amyloid-Beta Deposition by Modulating Microglial Activation in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease characterized by excessive accumulation of the amyloid-β peptide (Aβ) in the brain, which has been considered to mediate the neuroinflammation process. Microglial activation is the main component of neuroimmunoregulation. In recent years, exosomes isolated from human umbilical cord mesenchymal stem cells (hucMSC-exosomes) have been demonstrated to mimic the therapeutic effects of hucMSCs in many inflammation-related diseases. In this study, exosomes from the supernatant of hucMSCs were injected into AD mouse models. We observed that hucMSC-exosomes injection could repair cognitive disfunctions and help to clear Aβ deposition in these mice. Moreover, we found that hucMSC-exosomes injection could modulate the activation of microglia in brains of the mice to alleviated neuroinflammation. The levels of pro-inflammatory cytokines in peripheral blood and brains of mice were increased and the levels of anti-inflammatory cytokines were decreased. We also treated BV2 cells with hucMSC-exosomes in culture medium. HucMSC-exosomes also had inflammatory regulating effects to alternatively activate microglia and modulate the levels of inflammatory cytokines in vitro.     

The P680 Difference Spectrum Induced by Continuous Light in Malaxis monophyllos Chloroplasts

In chloroplasts from M. monophyllos (L.) Sw., the difference spectrum of light-induced P680 which was measured under physiological temperature and neutral pH and in the presence of potassium ferricyanide was obtained by steady-state method. In contrast under the same conditions, the P680 signal was not observed in spinach chloroplasts. Either preilumination with white light or long duration treatment with potassium ferricyanide caused marked similar changes in the difference spectrum of M. monophyllos chloroplasts, i.e. besides the bleaching bands at wavelength near 680 mm and 440 nm were the same as the control, the remarkable bleaching bands at about 660 nm and 422 nm appeared. These results were discussed briefly.     

Influenza Virus Directly Infects Human Natural Killer Cells and Induces Cell Apoptosis

Influenza is an acute respiratory viral disease that is transmitted in the first few days of infection. Evasion of host innate immune defenses, including natural killer (NK) cells, is important for the virus''s success as a pathogen of humans and other animals. NK cells encounter influenza viruses within the microenvironment of infected cells and are important for host innate immunity during influenza virus infection. It is therefore important to investigate the direct effects of influenza virus on NK cells. In this study, we demonstrated for the first time that influenza virus directly infects and replicates in primary human NK cells. Viral entry into NK cells was mediated by both clathrin- and caveolin-dependent endocytosis rather than through macropinocytosis and was dependent on the sialic acids on cell surfaces. In addition, influenza virus infection induced a marked apoptosis of NK cells. Our findings suggest that influenza virus can directly target and kill NK cells, a potential novel strategy of influenza virus to evade the NK cell innate immune defense that is likely to facilitate viral transmission and may also contribute to virus pathogenesis.Influenza is an acute respiratory virus infection that continues to pose endemic, zoonotic, and pandemic threats to human health, with significant morbidity and mortality (17). At the early phase of viral infection, innate immunity plays important roles in host defense by limiting viral replication and helping to initiate an adaptive immune response. Natural killer (NK) cells are key effector cells in innate immunity and play a critical role in the first line of host defense against acute viral infections by directly destroying infected cells without the need for prior antigen stimulation (7, 20). As influenza illness and virus transmission usually occur in the first few days of infection, the virus has to devise strategies to evade host innate immune responses, including NK cell immunity (15, 21).NK cells can recognize and kill influenza virus-infected cells (2, 10, 23); to counteract this killing, however, influenza virus has developed an escape strategy that inhibits NK cell cytotoxicity by increasing the binding of two inhibitory receptors to the infected cells after infection (1). The individuals with complete NK cell deficiency developed life-threatening varicella zoster virus and cytomegalovirus infection, but no severe influenza virus infection occurred (30, 40). Indeed, the interaction between human NK cells and influenza virus remains poorly understood. After influenza virus infection, respiratory epithelial cells release inflammatory chemokines that recruit NK cells to the site of infection (12). As a lytic virus, numerous influenza virus particles are released from the infected epithelia and macrophages (5, 9, 33). In the infected microenvironment, NK cells undoubtedly encounter these infective virus particles. It is therefore important to investigate the direct interaction of NK cells with influenza virus. Patients with severe influenza virus infection were shown to have diminished NK cells in peripheral blood and an almost complete absence of pulmonary NK cells, together with marked apoptosis (13, 42). During influenza virus infection in mice, a transient increase of NK cytotoxicity is followed by a marked decrease in NK cell activity, with a virus dose-dependent effect (8, 28). These data suggest that influenza virus may directly target NK cells as part of its immunoevasion strategies. However, no reports of the direct effects of influenza virus on human NK cells have so far been available.In this study, we demonstrated that influenza virus infects and replicates in primary human NK cells. Viral infection was dependent on sialic acids on the cells. The entry was mediated by both clathrin- and caveolin-dependent endocytosis rather than macropinocytosis. Influenza virus infection induced a marked apoptosis of NK cells, which contributed to reduced NK cell cytotoxicity. This, to the best of our knowledge, is the first paper to demonstrate that influenza virus can directly infect NK cells and induce cell apoptosis. These findings suggest that influenza virus may have developed a novel strategy to evade NK cell innate immune defenses, which is likely to facilitate viral transmission and may also contribute to virus pathogenesis.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

腾冲嗜热厌氧杆菌Cas2原核表达及生物信息学分析

为研究CRISPR/Cas系统及其相关蛋白Cas2(TTE2657)在腾冲嗜热厌氧杆菌热适应中的作用,应用PCR技术构建了原核重组质粒pET-28a::cas2,并在大肠埃希菌BL21表达Cas2蛋白;结合生物信息学软件对cas2编码蛋白的基本理化性质、氨基酸同源性、空间结构及蛋白质相互作用网络进行预测和分析。结果显示,成功构建了原核表达载体pET-28a::cas2并在大肠埃希菌BL21中得到表达,Cas2分子质量大小为9.9 ku,主要以可溶性形式存在;qRT-PCR显示cas2 mRNA在60℃和75℃高表达;生物信息学分析显示cas2基因其完整的ORF全长264 bp,编码88个氨基酸,其中Ile(14)、Ser(14)、Phe (12)含量较高,等电点为9.31,不存在跨膜结构。其蛋白质二级空间结构以α-螺旋、无规则卷曲、β-折叠为主,蛋白互作预测网络显示Cas2与Cas3、Cas5、Cas7等其家族大部分蛋白存在相互作用。进化树分析显示腾冲嗜热厌氧杆菌cas2基因与厌氧菌芽胞杆菌B7M1同源性最高(39.5%)。腾冲嗜热厌氧杆菌cas2编码蛋白是一种亲水性蛋白,在原核系统能高效表达。本研究为嗜热蛋白质的热稳定性机制的研究提供参考。