Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses

Failure mode of laser welds in lap‐shear specimens of high strength low alloy (HSLA) steel sheets

In this paper, the failure mode of laser welds in lap‐shear specimens of non‐galvanized SAE J2340 300Y high strength low alloy steel sheets under quasi‐static loading conditions is examined based on experimental observations and finite element analyses. Laser welded lap‐shear specimens with reduced cross sections were made. Optical micrographs of the cross sections of the welds in the specimens before and after tests are examined to understand the microstructure and failure mode of the welds. Micro‐hardness tests were also conducted to provide an assessment of the mechanical properties in the base metal, heat‐affected and fusion zones. The micrographs indicate that the weld failure appears to be initiated from the base metal near the boundary of the base metal and the heat‐affected zone at a distance away from the pre‐existing crack tip, and the specimens fail due to the necking/shear of the lower left load carrying sheets. Finite element analyses based on non‐homogenous multi‐zone material models were conducted to model the ductile necking/shear failure and to obtain the J integral solutions for the pre‐existing cracks. The results of the finite element analyses are used to explain the ductile failure initiation sites and the necking/shear of the lower left load carrying sheets. The J integral solutions obtained from the finite element analyses based on the 3‐zone finite element model indicate that the J integral for the pre‐existing cracks at the failure loads are low compared to the fracture toughness and the specimens should fail in a plastic collapse or necking/shear mode. The effects of the sheet thickness on the failure mode were then investigated for laser welds with a fixed ratio of the weld width to the thickness. For the given non‐homogenous material model, the J integral solutions appear to be scaled by the sheet thickness. With consideration of the plastic collapse failure mode and fracture initiation failure mode, a critical thickness can be obtained for the transition of the plastic collapse or necking/shear failure mode to the fracture initiation failure mode. Finally, the failure load is expressed as a function of the sheet thickness according to the governing equations based on the two failure modes. The results demonstrate that the failure mode of welds of thin sheets depends on the sheet thickness, ductility of the base metal and fracture toughness of the heat‐affected zone. Therefore, failure criteria based on either the plastic collapse failure mode or the fracture initiation failure mode should be used cautiously for welds of thin sheets.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90305/1/j.1460-2695.2011.01609.x.pd

Ontology-based Fuzzy Markup Language Agent for Student and Robot Co-Learning

An intelligent robot agent based on domain ontology, machine learning mechanism, and Fuzzy Markup Language (FML) for students and robot co-learning is presented in this paper. The machine-human co-learning model is established to help various students learn the mathematical concepts based on their learning ability and performance. Meanwhile, the robot acts as a teacher's assistant to co-learn with children in the class. The FML-based knowledge base and rule base are embedded in the robot so that the teachers can get feedback from the robot on whether students make progress or not. Next, we inferred students' learning performance based on learning content's difficulty and students' ability, concentration level, as well as teamwork sprit in the class. Experimental results show that learning with the robot is helpful for disadvantaged and below-basic children. Moreover, the accuracy of the intelligent FML-based agent for student learning is increased after machine learning mechanism.Comment: This paper is submitted to IEEE WCCI 2018 Conference for revie

Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts

Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction

Macrophage activation increases the invasive properties of hepatoma cells by destabilization of the adherens junction

AbstractTumor-associated macrophages play an important role in tumor progression, but whether they exert a tumor-progressive effect remains controversial. Here, we demonstrated that activated macrophage-conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial-mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial-cadherin (E-cadherin) and β-catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E-cadherin and β-catenin in the cell membrane. Importantly, levels of β-catenin-associated E-cadherin were also decreased. Following RAW/AMCM treatment, enhanced activation of c-Src was seen prior to increased tyrosine phosphorylation of β-catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E-cadherin and β-catenin. AMCMs obtained from human THP-1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E-cadherin and β-catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor-progressive effect on HepG2 cells which involves the c-Src- and EGFR-dependent signaling cascades