Non-equilibrium behaviour in coacervate-based protocells under electric-field-induced excitation

Although numerous strategies are now available to generate rudimentary forms of synthetic cell-like entities, minimal progress has been made in the sustained excitation of artificial protocells under non-equilibrium conditions. Here we demonstrate that the electric field energization of coacervate microdroplets comprising polylysine and short single strands of DNA generates membrane-free protocells with complex, dynamical behaviours. By confining the droplets within a microfluidic channel and applying a range of electric field strengths, we produce protocells that exhibit repetitive cycles of vacuolarization, dynamical fluctuations in size and shape, chaotic growth and fusion, spontaneous ejection and sequestration of matter, directional capture of solute molecules, and pulsed enhancement of enzyme cascade reactions. Our results highlight new opportunities for the study of non-equilibrium phenomena in synthetic protocells, provide a strategy for inducing complex behaviour in electrostatically assembled soft matter microsystems and illustrate how dynamical properties can be activated and sustained in microcompartmentalized media.</p

A versatile approach to multiple gene RNA interference using microRNA-based short hairpin RNAs

Background: Effective and stable knockdown of multiple gene targets by RNA interference is often necessary to overcome isoform redundancy, but it remains a technical challenge when working with intractable cell systems. Results: We have developed a flexible platform using RNA polymerase II promoter-driven expression of microRNA-like short hairpin RNAs which permits robust depletion of multiple target genes from a single transcript. Recombination-based subcloning permits expression of multi-shRNA transcripts from a comprehensive range of plasmid or viral vectors. Retroviral delivery of transcripts targeting isoforms of cAMP-dependent protein kinase in the RAW264.7 murine macrophage cell line emphasizes the utility of this approach and provides insight to cAMP-dependent transcription. Conclusion: We demonstrate functional consequences of depleting multiple endogenous target genes using miR-shRNAs, and highlight the versatility of the described vector platform for multiple target gene knockdown in mammalian cells

Evolutionary transition between invertebrates and vertebrates via methylation reprogramming in embryogenesis

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Xu, X., Li, G., Li, C., Zhang, J., Wang, Q., Simmons, D. K., Chen, X., Wijesena, N., Zhu, W., Wang, Z., Wang, Z., Ju, B., Ci, W., Lu, X., Yu, D., Wang, Q., Aluru, N., Oliveri, P., Zhang, Y. E., Martindale, M. Q., & Liu, J. Evolutionary transition between invertebrates and vertebrates via methylation reprogramming in embryogenesis. National Science Review, 6(5), (2019):993-1003, doi:10.1093/nsr/nwz064.Major evolutionary transitions are enigmas, and the most notable enigma is between invertebrates and vertebrates, with numerous spectacular innovations. To search for the molecular connections involved, we asked whether global epigenetic changes may offer a clue by surveying the inheritance and reprogramming of parental DNA methylation across metazoans. We focused on gametes and early embryos, where the methylomes are known to evolve divergently between fish and mammals. Here, we find that methylome reprogramming during embryogenesis occurs neither in pre-bilaterians such as cnidarians nor in protostomes such as insects, but clearly presents in deuterostomes such as echinoderms and invertebrate chordates, and then becomes more evident in vertebrates. Functional association analysis suggests that DNA methylation reprogramming is associated with development, reproduction and adaptive immunity for vertebrates, but not for invertebrates. Interestingly, the single HOX cluster of invertebrates maintains unmethylated status in all stages examined. In contrast, the multiple HOX clusters show dramatic dynamics of DNA methylation during vertebrate embryogenesis. Notably, the methylation dynamics of HOX clusters are associated with their spatiotemporal expression in mammals. Our study reveals that DNA methylation reprogramming has evolved dramatically during animal evolution, especially after the evolutionary transitions from invertebrates to vertebrates, and then to mammals.This work was supported by the National Key Research and Development Program of China (2018YFC1003303), the Strategic Priority Research Program of the CAS (XDB13040200), the National Natural Science Foundation of China (91519306, 31425015), the Youth Innovation Promotion Association of the CAS and the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SMC016)

The Alliance for Cellular Signaling Plasmid Collection: A Flexible Resource for Protein Localization Studies and Signaling Pathway Analysis

Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines

Exploring the mechanism of aloe-emodin in the treatment of liver cancer through network pharmacology and cell experiments

Objective: Aloe-emodin (AE) is an anthraquinone compound extracted from the rhizome of the natural plant rhubarb. Initially, it was shown that AE exerts an anti-inflammatory effect. Further studies revealed its antitumor activity against various types of cancer. However, the mechanisms underlying these properties remain unclear. Based on network pharmacology and molecular docking, this study investigated the molecular mechanism of AE in the treatment of hepatocellular carcinoma (HCC), and evaluated its therapeutic effect through in vitro experiments.Methods: CTD, Pharmmapper, SuperPred and TargetNet were the databases to obtain potential drug-related targets. DisGenet, GeneCards, OMIM and TTD were used to identify potential disease-related targets. Intersection genes for drugs and diseases were obtained through the Venn diagram. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of intersecting genes were conducted by the website of Bioinformatics. Intersection genes were introduced into STRING to construct a protein-protein interaction network, while the Cytoscape3.9.1 software was used to visualize and analyze the core targets. AutoDock4.2.6 was utilized to achieve molecular docking between drug and core targets. In vitro experiments investigated the therapeutic effects and related mechanisms of AE.Results: 63 overlapped genes were obtained and GO analysis generated 3,646 entries by these 63 intersecting genes. KEGG analysis mainly involved apoptosis, proteoglycans in cancer, TNF signaling pathway, TP53 signaling pathway, PI3K-AKT signaling pathway, etc. AKT1, EGFR, ESR1, TP53, and SRC have been identified as core targets because the binding energies of them between aloe-emodin were less than -5 kcal/Mol.The mRNA and protein expression, prognosis, mutation status, and immune infiltration related to core targets were further revealed. The involvement of AKT1 and EGFR, as well as the key target of the PI3K-AKT signaling pathway, indicated the importance of this signaling pathway in the treatment of HCC using AE. The results of the Cell Counting Kit-8 assay and flow analysis demonstrated the therapeutic effect of AE. The downregulation of EGFR, PI3KR1, AKT1, and BCL2 in mRNA expression and PI3KR1, AKT,p-AKT in protein expression confirmed our hypothesis.Conclusion: Based on network pharmacology and molecular docking, our study initially showed that AE exerted a therapeutic effect on HCC by modulating multiple signaling pathways. Various analyses confirmed the antiproliferative activity and pro-apoptotic effect of AE on HCC through the PI3K-AKT signaling pathway. This study revealed the therapeutic mechanism of AE in the treatment of HCC through a novel approach, providing a theoretical basis for the clinical application of AE

Reprogramming histone modification patterns to coordinate gene expression in early zebrafish embryos

Abstract Background Multicellular organisms require precise gene regulation during ontogeny, and epigenetic modifications, such as DNA methylation and histone modification, facilitate this precise regulation. The conservative reprogramming patterns of DNA methylation in vertebrates have been well described. However, knowledge of how histone modifications are passed on from gametes to early embryos is limited, and whether histone modification reprogramming is conserved is not clear. Results We profiled H3K4me3/H3K27me3 modifications in gametes and early embryos in zebrafish and found that the patterns in gene promoter regions have been largely set to either co-occupied or active states in gametes and then passed on to early embryos. Co-occupied states are partially maintained, while active states are largely restored to nearly match the sperm’s pattern prior to zygotic genome activation (ZGA). However, repressive H3K27me3 modifications in promoter regions are largely discarded in early embryos. Prior to ZGA, patterns of genes that initialize ZGA are converted to nonrepressive states to coordinate gene expression. Moreover, promoter peaks that mark stage-specific genes are hypermethylated, and histone modifications in these regions are erased independently of DNA methylation reprogramming. Furthermore, comparative analysis revealed that the functions of co-occupied and active genes passed on from gametes are conserved in vertebrates. Gene age preferences by co-occupied and active histone modifications are also confirmed in vertebrates. Conclusions Our data provide fundamental resources for understanding H3K4me3/H3K27me3 modifications in early zebrafish embryos. The data also reveal that the reprogramming progress of histone modifications is conserved in vertebrates and coordinates with gene expression during ZGA

Numerical Simulation Analysis of Dual-Beam Laser Welding of Tailored Blanks with Different Thicknesses

In order to meet the requirements of alignment capability, gap tolerance and welding track control, dual-beam laser welding has been put forward to weld tailored blanks with different thicknesses. The arrangement mode and power ratio of dual-beam laser determine the heat transfer and temperature field distribution, and then influence the weld profile and weld quality. In this study, based on consideration of the temperature dependence of material physical properties, convection and radiation heat transfer, and material latent heat, the finite element model was developed. The model validated was conducted to compare with experimental results and it showed good agreement. The arrangement mode and power ratio of dual-beam laser welding were compared and optimized based on the consideration of the weld width on top surface and gap tolerance. Finally, the temperature field characteristics with the preferable process were analyzed in detail according to the numerical simulation. The calculated results indicated the weld width is mainly controlled by the laser irradiation on the thin plate, and dual-beam laser welding of tailored blanks has great advantages on improving heat transfer, weld profile and gap tolerance, which helps to reduce welding defects and enhance welding quality

Experimental Investigation on Electric Current-Aided Laser Stake Welding of Aluminum Alloy T-Joints

In the present study, aluminum alloy T-joints were welded using the laser stake-welding process. In order to improve the welding quality of the T-joints, an external electric current was used to aid the laser stake-welding process. The effects of the process parameters on the weld morphology, mechanical properties, and microstructure of the welded joints were analyzed and discussed in detail. The results indicate that the aided electric current should be no greater than a certain maximum value. Upon increasing the aided electric current, the weld width at the skin and stringer faying surface obviously increased, but there was an insignificant change in the penetration depth. Furthermore, the electric current and pressing force should be chosen to produce an expected weld width at the faying surface, whereas the laser power and welding speed should be primarily considered to obtain an optimal penetration depth. The tensile shear specimens failed across the faying surface or failed in the weld zone of the skin. The specimens that failed in the weld of the skin could resist a higher tensile shear load compared with specimens that failed across the faying surface. The microstructural observations and microhardness results demonstrated that the tensile shear load capacity of the aluminum alloy welded T-joint was mainly determined by the weld width at the faying surface

Mountainous village relocation planning with 3D GIS virtual imaging space model and neural network

China’s mountainous village relocation planning faces the problems of extensive survey ranges and high travel costs, and current planning still depends on traditional field visits and census data survey method. To improve efficiency and control costs, digital survey methods are proposed to support efficient data collection and produce data with accurate spatial reference . This research discusses a specific survey method for relocation planning using virtual imaging space modeling based on 3D GIS combined with neural network algorithm. A digital village model of a pilot village is established by oblique photography and WEBGIS technology, while the improved Mask R-CNN algorithm is adopted to identify and classify different buildings in the village. A relocation plan including the number of buildings in each class and the reclamation area is provided and the cost and value of the plan is fully evaluated. The results demonstrate that compared with the traditional survey method, the proposed method of digital survey can better address the issue of remote mountain areas, including provide more accurate, complete and sustainable information, make the surveying and planning more convenient, efficient and smart and improve the objectivity of village relocation planning