Autocrine, Not Paracrine, Interferon-Gamma Gene Delivery Enhances Ex Vivo Antigen-Specific Cytotoxic T Lymphocyte Stimulation and Killing

The adoptive transfer of antigen-specific cytotoxic T lymphocytes (CTL) shows promise in the treatment of cancer and infectious diseases. We utilize adeno-associated virus-(AAV-) based antigen gene-loaded dendritic cells (DCs) to stimulate such antigen-specific CTL. Yet further improvements in CTL stimulation and killing may result by gene delivery of various Th1-response interferons/cytokines, such as interferon γ (IFN-γ), as the delivered gene can continuously produce that interferon. However which immune cell type should optimally express IFN-γ is unclear as the phenotypes of both DC and T cells are enhanced by it. Here, we used AAV to compare and contrast IFN-γ gene delivery into DC or T cells, and versus the addition of exogenous IFN-γ, for stimulating carcinoembryonic antigen-(CEA-) specific CTL. It was found that AAV/IFN-γ delivery into T cells (autocrine) resulted in T cell populations with the highest CD8(+)/CD4(+) ratio, highest IFN-γ(+)/IL-4(+) ratio, highest CD69(+),CD8(+) levels, and lowest CD4(+)/CD25(+) levels, all consistent with the strongest Th1 response. Most importantly, AAV/IFN-γ transduction of T cells resulted in antigen-specific T cell populations with the highest killing capabilities, 49% above other treatments. These data strongly suggest that AAV/IFN-γ autocrine gene delivery into T cells is worthy of further study towards maximizing the generation of antigen-specific anticancer CTL killers

PCR-Based Seamless Genome Editing with High Efficiency and Fidelity in <i>Escherichia coli</i>

Efficiency and fidelity are the key obstacles for genome editing toolboxes. In the present study, a PCR-based tandem repeat assisted genome editing (TRAGE) method with high efficiency and fidelity was developed. The design of TRAGE is based on the mechanism of repair of spontaneous double-strand breakage (DSB) via replication fork reactivation. First, cat-sacB cassette flanked by tandem repeat sequence was integrated into target site in chromosome assisted by Red enzymes. Then, for the excision of the cat-sacB cassette, only subculturing is needed. The developed method was successfully applied for seamlessly deleting, substituting and inserting targeted genes using PCR products. The effects of different manipulations including sucrose addition time, subculture times in LB with sucrose and stages of inoculation on the efficiency were investigated. With our recommended procedure, seamless excision of cat-sacB cassette can be realized in 48 h efficiently. We believe that the developed method has great potential for seamless genome editing in E. coli

Multiobjective Optimization of Tool Geometric Parameters Using Genetic Algorithm

Tool geometric parameters have a huge impact on tool wear. Up to now, there are only a few researches on tool geometric parameters and optimization, and the single objective function of parameter optimization used by researchers during high-speed machining (HSM) mainly is the minimum cutting force. However, the elevated cutting temperature also greatly affects tool wear due to the numerous cutting heat generation. Thus, to reduce tool wear, it is the most fundamental approach to taking into account the comprehensive control of the cutting force and cutting temperature because they are the two most important physical quantities in metal cutting processes. This work proposes a new optimization idea of the cutting-tool’s multi geometric parameters (three main parameters: rake angle, clearance angle, and cutting edge radius) with two objective functions (the cutting force and the temperature). Based on the response surface method (RSM), we have established the modified functional relation models of the influence of tool geometric parameters on the cutting force and temperature according to the finite element simulation results in high-speed cutting of Ti6Al4V. Then the models are solved by using a genetic algorithm, and the optimal tool geometric parameters values that can concurrently control the two objectives in their minimum values are obtained. The advantages lie in the strategy of the separate models of the cutting force and cutting temperature owing to their different dimensions and the solution of the models through giving the cutting force and cutting temperature different weight coefficients. The optimal results are verified by experiments, which shows that the optimal tool geometric parameters are very effective and vital for ensuring both the cutting force and the cutting temperature not too high. This work is of great significance to the cutting tool design theory and its manufacturing for reducing tool wear

The Influence of Bit Edge Shape Parameters on Bone Drilling Force Based on Finite Element Analysis

Bone drilling is a common surgery procedure. The drill bit shape directly affects the drilling force. Excessive drilling force may cause bone damage. In this work, on the premise of analyzing and comparing several finite element method (FEM) simulation results for drill bit of 5 mm in diameter commonly used in medical practice, a combination of drilling speed and feed rates which can minimize the drilling force for drilling parameters is determined. Then, the effects of the drill bit shape parameters including helix angle, point angle and edge radius on the drilling force are simulated by using the obtained drilling parameters, and after validation the FEM analysis results show that their variation trend is the same as the experimental one. Then, the optimum bit structure parameters are obtained through the following research: (1) the prediction model of the relationship between drill edge parameters and drilling force is established based on the result of FEM of the drilling process; (2) A particle swarm optimization algorithm is used to obtain the optimal matching parameters of the bit structure; (3) The priority order of the influence of the parameters of the bit on the drilling force is analyzed. The results show that the order of the influence is: the edge radius is the largest, the point angle is the second, and the helix angle is the smallest. The optimum combination of bit structure is that point angle, helix angle and edge radius are 95&deg;, 35&deg;, and 0.02 mm, respectively