Induced pluripotent stem cell‐derived myeloid cells expressing OX40 ligand amplify antigen‐specific T cells in advanced melanoma

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune‐related adverse events have been reported. Therefore, more effective and antigen‐specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS‐ML). In this study, we generated OX40L‐overexpressing iPS‐ML (iPS‐ML‐Zsgreen‐OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS‐ML‐Zsgreen‐OX40L suppressed the progression of B16‐BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)‐expressing B16 melanoma (MO4). The number of antigen‐specific CD8⁺ T cells was higher in spleen cells treated with OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L than in those without OX40L. The OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L significantly increased the number of tumor‐infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS‐ML in the clinical applications, iPS‐ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS‐ML‐Zsgreen‐OX40L therapy might be a new method for antigen‐specific cancer immunotherapy.     

不同降水条件下科尔沁沙地南缘疏林草地樟子松针叶δ13C和叶性状特征

通过比较不同自然降水年份(极端干旱和极端湿润)19年生疏林草地樟子松的针叶δ¹³C、比叶面积和干物质含量,结合土壤含水量和地下水埋深,探讨了极端降水对樟子松水分利用的影响.结果表明: 干旱年份(2009)樟子松林土壤含水量显著低于湿润年份(2010),但樟子松当年生针叶的δ¹³C在两年间没有显著差异,且两年相同月份间亦无显著差异;干旱年份当年生针叶的比叶面积显著低于湿润年份,而不同年份间干物质含量的差异不显著.在两种极端降水条件下,樟子松的水分利用效率没有明显变化,主要通过改变当年生针叶的比叶面积来适应降水量的变化.对于地下水埋深高于3.0 m的疏林草地樟子松人工林生态系统,极端干旱不会严重影响樟子松的存活和生长.     

Sex-specific reaction norms to intraspecific larval competition in the mosquito Aedes aegypti

As the relationship between a given life‐history trait and fitness is not necessarily the same for the two sexes, an ‘intersexual ontogenetic conflict’ may arise. We analysed the phenotypic reaction to intraspecific larval competition of the mosquito, Aedes aegypti, asking: (i) Do both sexes pay the cost of competition with the same life‐history traits and are they equal competitors? (ii) Is there a specific cost of competition beyond sharing food resources? We found that competition incurs a specific cost that was expressed differently by the two sexes. Indeed, each sex maintained the more important life‐history trait(s) for their fitness (developmental time for males and body weight and size for females) at the expense of other traits, thus minimizing the effects of competition on their fitness. The competition exerted by females was estimated as being more intense, probably linked with the greater importance of body size for their fitness.     

苦楝果实活性物质研究

本文报道苦楝果实中的生物活性物质。发现果实的抽提物和单体化合物不仅具有防治作物害虫的作用,而且,还对植物有生长调节作用。通过生物活性鉴定结果表明:果实种仁的水抽提物对水稻早期萌动、发芽与秧苗的生长均有促进作用。其中,处理稻种第2天(48h),其发芽率比对照高出24.67％～42.86％;第4天秧苗的根长在1cm以上占56.5％～65.0％,而对照组只有15.5％。尤其是抽提液浓度在6000ppm时处理的稻种,测得生长10d的秧苗总长为对照的120.27％,胚根长为对照的121.54％;以及叶片中的叶绿素总含量比对照高8.5％。还证明了从果实中提取分离的几种三萜类化合物和在果实中发现的一种新化合物对苯二甲酸二甲酯,均对菜青虫有拒食和胃毒活性。     

Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling

The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.     

Histidyl-tRNA synthetase urzymes: Class I and II aminoacyl tRNA synthetase urzymes have comparable catalytic activities for cognate amino acid activation

Four minimal (119-145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine activation as fusion proteins and after TEV cleavage, using the (32)PP(i) exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (～0.1-0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered K(m) values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ～10(9) times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate amino acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.     

A quantitative real-time PCR method for in planta monitoring of Phytophthora infestans growth

Aim: To develop a real‐time PCR‐based strategy for the detection of Paenibacillus larvae vegetative cells and spores to improve the diagnosis and the screening of American foulbrood (AFB), the most harmful pathology of honeybee brood. Methods and Results: A real‐time PCR that allowed selective identification and quantification of P. larvae 16S rRNA sequence was developed. Using standard samples quantified by flow cytometry, detection limits of 37·5 vegetative cells ml^?1 and 10 spores ml^?1 were determined. Compared to spread plate method, this real‐time PCR‐based strategy allowed, in only 2 h, the detection of P. larvae in contaminated honeys. No false‐positive results were obtained. Moreover, its detection limit was 100 times lower than that of the culture method (2 vs 200 spores g^?1 of honey). Conclusion: A rapid, selective, with low detection limit, sensitive and specific method to detect and quantify vegetative cells and spores of P. larvae is now available. Significance and Impact of Study: In addition to honey samples, this real‐time PCR‐based strategy may be also applied to confirm AFB diagnosis in honeybee brood and to screen other apiary supplies and products (bees, pollen, wax), thus broadening the control of AFB spreading.     

The PDZ-GEF Gef26 regulates synapse development and function via FasII and Rap1 at the Drosophila neuromuscular junction

Guanine nucleotide exchange factors (GEFs) are essential for small G proteins to activate their downstream signaling pathways, which are involved in morphogenesis, cell adhesion, and migration. Mutants of Gef26, a PDZ-GEF (PDZ domain-containing guanine nucleotide exchange factor) in Drosophila, exhibit strong defects in wings, eyes, and the reproductive and nervous systems. However, the precise roles of Gef26 in development remain unclear. In the present study, we analyzed the role of Gef26 in synaptic development and function. We identified significant decreases in bouton number and branch length at larval neuromuscular junctions (NMJs) in Gef26 mutants, and these defects were fully rescued by restoring Gef26 expression, indicating that Gef26 plays an important role in NMJ morphogenesis. In addition to the observed defects in NMJ morphology, electrophysiological analyses revealed functional defects at NMJs, and locomotor deficiency appeared in Gef26 mutant larvae. Furthermore, Gef26 regulated NMJ morphogenesis by regulating the level of synaptic Fasciclin II (FasII), a well-studied cell adhesion molecule that functions in NMJ development and remodeling. Finally, our data demonstrate that Gef26-specific small G protein Rap1 worked downstream of Gef26 to regulate the level of FasII at NMJs, possibly through a βPS integrin-mediated signaling pathway. Taken together, our findings define a novel role of Gef26 in regulating NMJ development and function.