Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects.

Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

[Notch1 mRNA and protein expression in human breast cancer and normal mammary gland tissues].

OBJECTIVE: To explore the Notch1 mRNA and protein expression in human breast cancers and normal mammary tissues, and their relationship with the clinical indicators of breast cancers were analyzed. METHODS: Notch1 gene of human breast invasive ductal carcinoma (IDC) and normal mammary gland tissues were amplified by RT-PCR, and the expression of Notch1 protein was detected by immunohistochemical Streptavidin-Biotin Complex (SP) stain in 60 IDC, 30 ductal carcinoma in situ (DCIS) and 60 normal mammary tissues. RESULTS: Notch1 gene of human IDC and normal mammary tissues both could express in a transcription level; the positive rates of Notch1 protein expression in normal mammary tissues and DCIS were 55% and 70%. Respectively, which did not differ statistically (P > 0.05), while the positive rate in IDC was 90%, significantly higher than that of the normal mammary tissues and DCIS (P < 0.05). The high expression of Notch1 protein in IDC correlate significantly with lymph node metastasis, pathological grades and TNM stages. CONCLUSIONS: Notch1 protein was over expressed in breast IDC. A high Notch1 protein expression is considered associating with the evolution and malignant transformation of the breast tumor. The expression of Notch1 gene maybe impact the effect of on the progression of breast cancers.

Applications of Ruthenium Complex in Tumor Diagnosis and Therapy.

Ruthenium complexes are a new generation of metal antitumor drugs that are currently of great interest in multidisciplinary research. In this review article, we introduce the applications of ruthenium complexes in the diagnosis and therapy of tumors. We focus on the actions of ruthenium complexes on DNA, mitochondria, and endoplasmic reticulum of cells, as well as signaling pathways that induce tumor cell apoptosis, autophagy, and inhibition of angiogenesis. Furthermore, we highlight the use of ruthenium complexes as specific tumor cell probes to dynamically monitor the active biological component of the microenvironment and as excellent photosensitizer, catalyst, and bioimaging agents for phototherapies that significantly enhance the diagnosis and therapeutic effect on tumors. Finally, the combinational use of ruthenium complexes with existing clinical antitumor drugs to synergistically treat tumors is discussed.

Effect of ultrasound on desorption kinetics of phenol from polymeric resin.

This work mainly involves the study of desorption kinetics of phenol from polymeric resins under the influence of an ultrasound field. A new phase equilibrium-kinetics model (PEKM), for estimation of diffusion coefficient was proposed, kinetic experiments of phenol desorption on NKA-II resin in the presence and the absence of ultrasound were separately conducted, and diffusion coefficients of phenol within an adsorbent particle were estimated by means of proposed PEKM. Results showed that the use of ultrasound could enhance the diffusion of phenol within the resin. The diffusion coefficient of phenol in the resin in an ultrasonic field increased by an order of magnitude in comparison with the diffusion coefficient in the absence of ultrasound. The more intense the ultrasonic field the larger was the diffusion coefficient.

Influence of cell physiological state on gene delivery to T lymphocytes by chimeric adenovirus Ad5F35.

Adoptive transfer of genetically-modified T cells is a promising approach for treatment of both human malignancies and viral infections. Due to its ability to efficiently infect lymphocytes, the chimeric adenovirus Ad5F35 is potentially useful as an immunotherapeutic for the genetic modification of T cells. In previous studies, it was found that the infection efficiency of Ad5F35 was significantly increased without enhanced expression of the viral receptor after T cell stimulation; however, little is known about the underlying mechanism. Nonetheless, cell physiology has long been thought to affect viral infection. Therefore, we aimed to uncover the physiologic changes responsible for the increased infection efficiency of Ad5F35 following T cell stimulation. Given the complexity of intracellular transport we analyzed viral binding, entry, and escape using a Jurkat T cell model and found that both cell membrane fluidity and endosomal escape of Ad5F35 were altered under different physiological states. This, in turn, resulted in differences in the amount of virus entering cells and reaching the cytoplasm. These results provide additional insight into the molecular mechanisms underlying Ad5F35 infection of T cells and consequently, will help further the clinical application of genetically-modified T cells for immunotherapy.

Quantum refrigeration cycles using spin-1/2 systems as the working substance.

The cycle model of a quantum refrigerator composed of two isothermal and two isomagnetic field processes is established. The working substance in the cycle consists of many noninteracting spin-1/2 systems. The performance of the cycle is investigated, based on the quantum master equation and semigroup approach. The general expressions of several important performance parameters, such as the coefficient of performance, cooling rate, and power input, are given. Especially, the case at high temperatures is analyzed in detail. The results obtained are further generalized and discussed, so that they may be directly used to describe the performance of the quantum refrigerator using spin-J systems as the working substance. Finally, the optimum characteristics of the quantum Carnot refrigerator are derived simply.