IL-33-Dependent Endothelial Activation Contributes to Apoptosis and Renal Injury in Orientia tsutsugamushi-Infected Mice

Endothelial cells (EC) are the main target for Orientia tsutsugamushi infection and EC dysfunction is a hallmark of severe scrub typhus in patients. However, the molecular basis of EC dysfunction and its impact on infection outcome are poorly understood. We found that C57BL/6 mice that received a lethal dose of O. tsutsugamushi Karp strain had a significant increase in the expression of IL-33 and its receptor ST2L in the kidneys and liver, but a rapid reduction of IL-33 in the lungs. We also found exacerbated EC stress and activation in the kidneys of infected mice, as evidenced by elevated angiopoietin (Ang) 2/Ang1 ratio, increased endothelin 1 (ET-1) and endothelial nitric oxide synthase (eNOS) expression. Such responses were significantly attenuated in the IL-33^-/- mice. Importantly, IL-33^-/- mice also had markedly attenuated disease due to reduced EC stress and cellular apoptosis. To confirm the biological role of IL-33, we challenged wild-type (WT) mice with a sub-lethal dose of O. tsutsugamushi and gave mice recombinant IL-33 (rIL-33) every 2 days for 10 days. Exogenous IL-33 significantly increased disease severity and lethality, which correlated with increased EC stress and activation, increased CXCL1 and CXCL2 chemokines, but decreased anti-apoptotic gene BCL-2 in the kidneys. To further examine the role of EC stress, we infected human umbilical vein endothelial cells (HUVEC) in vitro. We found an infection dose-dependent increase in the expression of IL-33, ST2L soluble ST2 (sST2), and the Ang2/Ang1 ratio at 24 and 48 hours post-infection. This study indicates a pathogenic role of alarmin IL-33 in a murine model of scrub typhus and highlights infection-triggered EC damage and IL-33-mediated pathological changes during the course of Orientia infection.     

Aneuploid embryonic stem cells exhibit impaired differentiation and increased neoplastic potential

Aneuploidy leads to severe developmental defects in mammals and is also a hallmark of cancer. However, whether aneuploidy is a driving cause or a consequence of tumor formation remains controversial. Paradoxically, existing studies based on aneuploid yeast and mouse fibroblasts have shown that aneuploidy is usually detrimental to cellular fitness. Here, we examined the effects of aneuploidy on mouse embryonic stem (ES) cells by generating a series of cell lines that each carries an extra copy of single chromosomes, including trisomy 6, 8, 11, 12, or 15. Most of these aneuploid cell lines had rapid proliferation rates and enhanced colony formation efficiencies. They were less dependent on growth factors for self‐renewal and showed a reduced capacity to differentiate in vitro. Moreover, trisomic stem cells formed teratomas more efficiently, from which undifferentiated cells can be recovered. Further investigations demonstrated that co‐culture of wild‐type and aneuploid ES cells or supplementation with extracellular BMP4 rescues the differentiation defects of aneuploid ES cells.     

Proper autophagy is indispensable for angiogenesis during chick embryo development

People have known that autophagy plays a very important role in many physiological and pathological events. But the role of autophagy on embryonic angiogenesis still remains obscure. In this study, we demonstrated that Atg7, Atg8 and Beclin1 were expressed in the plexus vessels of angiogenesis at chick yolk sac membrane and chorioallantoic membrane. Interfering in autophagy with autophagy inducer or inhibitor could restrict the angiogenesis in vivo, which might be driven by the disorder of angiogenesis-related gene expressions, and also lead to embryonic hemorrhage, which was due to imperfection cell junctions in endothelial cells including abnormal expressions of tight junction, adheren junction and desmosome genes. Using HUVECs, we revealed that cell viability and migration ability changed with the alteration of cell autophagy exposed to RAPA or 3-MA. Interestingly, tube formation assay showed that HUVECs ability of tube formation altered with the change of Atg5, Atg7 and Atg8 manipulated by the transfection of their corresponding siRNA or plasmids. Moreover, the lost cell polarity labeled by F-actin and the absenced β-catenin in RAPA-treated and 3-MA-treated cell membrane implied intracellular cytoskeleton alteration was induced by the activation and depression of autophagy. Taken together, our current experimental data reveal that autophagy is really involved in regulating angiogenesis during embryo development.     

Predicting the functional motions of p97 using symmetric normal modes

p97 is a protein complex of the AAA+ family. Although functions of p97 are well understood, the mechanism by which p97 performs its unfolding activities remains unclear. In this work, we present a novel way of applying normal mode analysis to study this six‐fold symmetric molecular machine. By selecting normal modes that are axial symmetric and give the largest movements at D1 or D2 pore residues, we are able to predict the functional motions of p97, which are then validated by experimentally observed conformational changes. Our results shed light and provide new understandings on several key steps of the p97 functional process that were previously unclear or controversial, and thus are able to reconcile multiple previous findings. Specifically, our results reveal that (i) a venous valve‐like mechanism is used at D2 pore to ensure a one‐way exit‐only traffic of substrates; (ii) D1 pore remains shut during the functional process; (iii) the “swing‐up” motion of the N domain is closely coupled with the vertical motion of the D1 pore along the pore axis; (iv) because of the shut D1 pore and the one‐way traffic at D2 pore, it is highly likely that substrates enter the chamber through the gaps at the D1/D2 interface. The limited chamber volume inside p97 suggests that a substrate may be pulling out from D2 while at the same time being pulling in at the interface; (v) lastly, p97 uses a series of actions that alternate between twisting and pulling to remove the substrate. Proteins 2016; 84:1823–1835. © 2016 Wiley Periodicals, Inc.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

MCLPMDA: A novel method for miRNA‐disease association prediction based on matrix completion and label propagation

MiRNAs are a class of small non‐coding RNAs that are involved in the development and progression of various complex diseases. Great efforts have been made to discover potential associations between miRNAs and diseases recently. As experimental methods are in general expensive and time‐consuming, a large number of computational models have been developed to effectively predict reliable disease‐related miRNAs. However, the inherent noise and incompleteness in the existing biological datasets have inevitably limited the prediction accuracy of current computational models. To solve this issue, in this paper, we propose a novel method for miRNA‐disease association prediction based on matrix completion and label propagation. Specifically, our method first reconstructs a new miRNA/disease similarity matrix by matrix completion algorithm based on known experimentally verified miRNA‐disease associations and then utilizes the label propagation algorithm to reliably predict disease‐related miRNAs. As a result, MCLPMDA achieved comparable performance under different evaluation metrics and was capable of discovering greater number of true miRNA‐disease associations. Moreover, case study conducted on Breast Neoplasms further confirmed the prediction reliability of the proposed method. Taken together, the experimental results clearly demonstrated that MCLPMDA can serve as an effective and reliable tool for miRNA‐disease association prediction.