Engineering T cell receptor fusion proteins using nonviral CRISPR/Cas9 genome editing for cancer immunotherapy.

Manufacture of chimeric antigen receptor (CAR)-T cells usually involves the use of viral delivery systems to achieve high transgene expression. However, it can be costly and may result in random integration of the CAR into the genome, creating several disadvantages including variation in transgene expression, functional gene silencing and potential oncogenic transformation. Here, we optimized the method of nonviral, CRISPR/Cas9 genome editing using large donor DNA delivery, knocked-in an anti-tumor single chain variable fragment (scFv) into the N-terminus of CD3ε and efficiently generated fusion protein (FP) T cells. These cells displayed FP integration within the TCR/CD3 complex, lower variability in gene expression compared to CAR-T cells and good cell expansion after transfection. CD3ε FP T cells were predominantly CD8+ effector memory T cells, and exhibited anti-tumor activity in vitro and in vivo. Dual targeting FP T cells were also generated through the incorporation of scFvs into other CD3 subunits and CD28. Compared to viral-based methods, this method serves as an alternative and versatile way of generating T cells with tumor-targeting receptors for cancer immunotherapy.

Wire-driven flexible manipulator with constrained spherical joints for minimally invasive surgery.

PURPOSE: One of the main factors that affect the rigidity of flexible robots is the twist deformation because of the external force exerted on the end effector. Another important factor that affects accuracy is the fact that such robots do not have a constant curvature. The conventional kinematic model assumes that the curvature is constant; however, in reality, it is not. To improve the rigidity and accuracy of flexible robots used in minimally invasive surgery via preventing the twist deformation while ensuring a constant curvature, we propose a novel flexible manipulator with ball-constrained spherical (BCS) joints and a spring. METHODS: The BCS joints are used to prevent the twist deformation in the flexible robot. The joints have two degrees of freedom (DOFs), which limit the rotation about the axial direction. The rotation is limited because the ball that is inserted into a BCS joint can move only along the ball guide. To obtain a constant curvature, springs are installed among the BCS joints. The springs receive the uniform compression force generated among the joints, thus achieving a constant curvature. The proposed BCS joint is designed based on the diameter of the forceps, desired workspace, and desired bending angle. RESULTS: To evaluate the proposed mechanism, three experiments were performed using a 20-mm-diameter prototype consisting of 13 BCS joints with a two-DOF motion. The experimental results showed that the prototype can realize a constant curvature with a mean error of 0.21°, which can support up to 5 N with no apparent twist deformation. CONCLUSIONS: We developed a flexible manipulator with BCS joints for minimally invasive surgery. The proposed mechanism is anticipated to help prevent the twist deformation of the robot and realize a constant curvature. Accordingly, it is expected that rigidity is improved to ensure accuracy.

Hydrogen Production by Steam Reforming of Liquefied Natural Gas (LNG) Over Nickel-Phosphorus-Alumina Xerogel Catalyst Prepared by a Carbon-Templating Epoxide-Driven Sol-Gel Method.

A nickel-phosphorus-alumina xerogel catalyst was prepared by a carbon-templating epoxide-driven sol-gel method (denoted as CNPA catalyst), and it was applied to the hydrogen production by steam reforming of liquefied natural gas (LNG). For comparison, a nickel-phosphorus-alumina xerogel catalyst was also prepared by a similar method in the absence of carbon template (denoted as NPA catalyst). The effect of carbon template addition on the physicochemical properties and catalytic activities of the catalysts in the steam reforming of LNG was investigated. Both CNPA and NPA catalysts showed excellent textural properties with well-developed mesoporous structure. However, CNPA catalyst retained a more reducible nickel aluminate phase than NPA catalyst. XRD analysis of the reduced CNPA and NPA catalysts revealed that nickel sintering on the CNPA catalyst was suppressed compared to that on the NPA catalyst. From H2-TPD and CH4-TPD measurements of the reduced CNPA and NPA catalysts, it was also revealed that CNPA catalyst with large amount of hydrogen uptake and strong hydrogen-binding sites showed larger amount of methane adsorption than NPA catalyst. In the hydrogen production by steam reforming of LNG, CNPA catalyst with large methane adsorption capacity showed a better catalytic activity than NPA catalyst.