Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.Tsukamoto T., Sakai E., Nishimae F., et al. Efficient generation of adenovirus vectors carrying the Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas)12a system by suppressing Cas12a expression in packaging cells. Journal of Biotechnology 304, 1 (2019); https://doi.org/10.1016/j.jbiotec.2019.08.004

Autonomic nervous alterations associated with daily level of fatigue

<p>Abstract</p> <p>Background</p> <p>Fatigue is a common symptom in both sick and healthy people. We examined autonomic nervous alterations associated with fatigue to clarify the mechanisms underlying fatigue.</p> <p>Methods</p> <p>The study group consisted of 19 healthy participants who performed a 2-back test for 30 min as a fatigue-inducing mental task session. Before and after the session, they completed the advanced trail making test (ATMT) for 30 min for mental fatigue evaluation, subjective scales to measure fatigue sensation, and underwent electrocardiography to allow assessment of autonomic nerve activities.</p> <p>Results</p> <p>After the fatigue-inducing task, the total error counts on the ATMT tended to increase (<it>P </it>= 0.076); the ATMT for total trial counts (<it>P </it>= 0.001), the subjective level of fatigue (<it>P </it>< 0.001), and the % low-frequency power (%LF) (<it>P </it>= 0.035) increased significantly; and the % high-frequency power (%HF) decreased compared with before the fatigue-inducing task although this did not reach the statistical significance (<it>P </it>= 0.170). Although LF measured in absolute units did not change significantly before and after the fatigue-inducing task (<it>P </it>= 0.771), and HF measured in absolute units decreased after the task (<it>P </it>= 0.020). The %LF and LF/HF ratio were positively associated with the daily level of fatigue evaluated using Chalder's fatigue scale. In addition, %HF was negatively associated with the fatigue score.</p> <p>Conclusions</p> <p>Increased sympathetic activity and decreased parasympathetic activity may be characteristic features of both acute and daily levels of fatigue. Our findings provide new perspectives on the mechanisms underlying fatigue.</p

Accuracy assessment methods of tissue marker clip placement after 11-gauge vacuum-assisted stereotactic breast biopsy: comparison of measurements using direct and conventional methods

BACKGROUND:　 The objective of the study was to compare direct measurement with a conventional method for evaluation of clip placement in stereotactic vacuum-assisted breast biopsy (ST-VAB) and to evaluate the accuracy of clip placement using the direct method.　 METHODS:　 Accuracy of clip placement was assessed by measuring the distance from a residual calcification of a targeted calcification clustered to a clip on a mammogram after ST-VAB. Distances in the craniocaudal (CC) and mediolateral oblique (MLO) views were measured in 28 subjects with mammograms recorded twice or more after ST-VAB. The difference in the distance between the first and second measurements was defined as the reproducibility and was compared with that from a conventional method using a mask system with overlap of transparent film on the mammogram. The 3D clip-to-calcification distance was measured using the direct method in 71 subjects.　 RESULTS:　 The reproducibility of the direct method was higher than that of the conventional method in CC and MLO views (P = 0.002, P < 0.001). The median 3D clip-to-calcification distance was 2.8 mm, with an interquartile range of 2.0-4.8 mm and a range of 1.1-36.3 mm.　 CONCLUSION:　 The direct method used in this study was more accurate than the conventional method, and gave a median 3D distance of 2.8 mm between the calcification and clip