Recent advances in genome editing, especially CRISPR-Cas nucleases, have revolutionized both laboratory research and clinical therapeutics. CRISPR-Cas nucleases, together with the DNA damage repair pathway in cells, enable both genetic diversification by classical non-homologous end joining (c-NHEJ) and precise genome modification by homology-based repair (HBR). Genome editing in zygotes is a convenient way to edit the germline, paving the way for animal disease model generation, as well as human embryo genome editing therapy for some life-threatening and incurable diseases. HBR efficiency is highly dependent on the DNA donor that is utilized as a repair template. Here, we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA donor. Moreover, we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian embryos. Finally, we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.
Animals ; CRISPR-Cas Systems/genetics* ; DNA/genetics* ; Embryo, Mammalian/metabolism* ; Endonucleases/metabolism* ; Gene Editing ; Mammals/metabolism*

Objective:To observe preventive effect of intestinal stent against anastomotic leakage after rectal cancer operation.Methods:A retrospective cohort study was carried out. Clinical data of 107 patients with low rectal cancer undergoing laparoscopic radical resection from January 2015 to August 2019 were retrospectively analyzed. Intestinal stent was placed intraoperatively in 48 cases and was not placed in 59 cases. Postoperative Wexner score for anal function and incidence of anastomotic leakage were compared between patients with and without intstinal stent.Results:There was no significant differences in age, distance between tumor and the anal verge, operative time and postoperative Wexner score for anal function between the two groups (all P>0.05). After a month of follow-up, the incidence of anastomotic leakage was 16.9% (10/59) in the non-stent group, while no anastomotic leakage was found in the stent group ( P=0.002). Conclusion:Placement of intestinal stent can effectively prevent anastomotic leakage after low rectal cancer surgery.

Objective:To observe preventive effect of intestinal stent against anastomotic leakage after rectal cancer operation.Methods:A retrospective cohort study was carried out. Clinical data of 107 patients with low rectal cancer undergoing laparoscopic radical resection from January 2015 to August 2019 were retrospectively analyzed. Intestinal stent was placed intraoperatively in 48 cases and was not placed in 59 cases. Postoperative Wexner score for anal function and incidence of anastomotic leakage were compared between patients with and without intstinal stent.Results:There was no significant differences in age, distance between tumor and the anal verge, operative time and postoperative Wexner score for anal function between the two groups (all P>0.05). After a month of follow-up, the incidence of anastomotic leakage was 16.9% (10/59) in the non-stent group, while no anastomotic leakage was found in the stent group ( P=0.002). Conclusion:Placement of intestinal stent can effectively prevent anastomotic leakage after low rectal cancer surgery.

While the majority of all human cancers counteract telomere shortening by expressing telomerase, ~15% of all cancers maintain telomere length by a telomerase-independent mechanism known as alternative lengthening of telomeres (ALT). Here, we show that high load of intrinsic DNA damage is present in ALT cancer cells, leading to apoptosis stress by activating p53-independent, but JNK/c-Myc-dependent apoptotic pathway. Notably, ALT cells expressing wild-type p53 show much lower apoptosis than p53-deficient ALT cells. Mechanistically, we find that intrinsic DNA damage in ALT cells induces low level of p53 that is insufficient to initiate the transcription of apoptosis-related genes, but is sufficient to stimulate the expression of key components of mTORC2 (mTOR and Rictor), which in turn leads to phosphorylation of AKT. Activated AKT (p-AKT) thereby stimulates downstream anti-apoptotic events. Therefore, p53 and AKT are the key factors that suppress spontaneous apoptosis in ALT cells. Indeed, inhibition of p53 or AKT selectively induces rapid death of ALT cells in vitro, and p53 inhibitor severely suppresses the growth of ALT-cell xenograft tumors in mice. These findings reveal a previously unrecognized function of p53 in anti-apoptosis and identify that the inhibition of p53 or AKT has a potential as therapeutics for specifically targeting ALT cancers.

Protein nitration and nitrosylation are essential post-translational modifications (PTMs) involved in many fundamental cellular processes. Recent studies have revealed that excessive levels of nitration and nitrosylation in some critical proteins are linked to numerous chronic diseases. Therefore, the identification of substrates that undergo such modifications in a site-specific manner is an important research topic in the community and will provide candidates for targeted therapy. In this study, we aimed to develop a computational tool for predicting nitration and nitrosylation sites in proteins. We first constructed four types of encoding features, including positional amino acid distributions, sequence contextual dependencies, physicochemical properties, and position-specific scoring features, to represent the modified residues. Based on these encoding features, we established a predictor called DeepNitro using deep learning methods for predicting protein nitration and nitrosylation. Using n-fold cross-validation, our evaluation shows great AUC values for DeepNitro, 0.65 for tyrosine nitration, 0.80 for tryptophan nitration, and 0.70 for cysteine nitrosylation, respectively, demonstrating the robustness and reliability of our tool. Also, when tested in the independent dataset, DeepNitro is substantially superior to other similar tools with a 7%-42% improvement in the prediction performance. Taken together, the application of deep learning method and novel encoding schemes, especially the position-specific scoring feature, greatly improves the accuracy of nitration and nitrosylation site prediction and may facilitate the prediction of other PTM sites. DeepNitro is implemented in JAVA and PHP and is freely available for academic research at http://deepnitro.renlab.org.
Amino Acid Sequence ; Amino Acids ; metabolism ; Deep Learning ; Humans ; Internet ; Neural Networks (Computer) ; Nitrosation ; Proteins ; chemistry ; metabolism ; Reproducibility of Results ; Software

Objective To study the application value of endoscopic thyroidectomy via total membrane dissection. Methods One hundred and sixteen patients with benign thyroid nodules were given axillo-breast approach endoscopic thyroidectomy via total membrane dissection from January 2014 to December 2016. Results Endoscopic thyroidectomy was completed in all the patients without conversion to open surgery.The operation time was 45-125 min[mean,(65.6 ±36.7)min],the blood loss was 10-220 ml[mean,(43.2 ±22.7)ml],the postoperative drainage volume was 25-305 ml[mean,(95.3 ±53.8)ml], the postoperative drainage time was 2-4 d[mean,(2.3 ±0.7)d)],and the length of hospital stay was 3-6 d[mean,(4.1 ±1.1)d].Short-term twitch occurred in 1 case and seroma occurred in 2 cases.No other complications such as hoarseness,bucking or asphyxia occurred in this series. Conclusion Endoscopic thyroidectomy via total membrane dissection can effectively avoid the injury of recurrent laryngeal nerve and parathyroid gland.

Objective To compare the surgical effect of gasless endoscopic thyroidectomy and CO2- insufflation endoscopic thyroidectomy, and evaluate the safety and applicability of gasless endoscopic thyroidectomy. Methods A prospective randomized controlled study was carried out.Sixty patients who were scheduled for bilateral thyroid surgery under endoscope were divided into 2 groups by sortition method: gasless group (30 patients, treated with gasless endoscopic thyroidectomy) and CO2-insufflation group (30 patients, treated with CO2-insufflation endoscopic thyroidectomy). The data of arterial partial pressure of carbon dioxide (PaCO2) and pH value before operation and 60 min after operation were detected. The operation time, intraoperative bleeding, time of putting the drain, hospitalization time after operation and postoperative complications were recorded. Results All the 60 patients completed endoscopic surgery,and no case was converted to the conventional procedure.There were no statistical differences in PaCO2and pH value before operation between 2 groups(P>0.05).The PaCO260 min after beginning of operation in gasless group was significantly lower than that in CO2-insufflation group:(36.43 ± 1.98)mmHg(1 mmHg=0.133 kPa)vs.(37.93 ± 3.27)mmHg,the pH value 60 min after beginning of operation was significantly higher than that in CO2-insufflation group:7.42 ± 0.02 vs. 7.37 ± 0.01, and there were statistical differences (P<0.05 or <0.01). There were no statistical difference in operation time, intraoperative bleeding, time of putting the drain, hospitalization time after operation and incidence of hoarseness between 2 groups (P>0.05). There were no complications related with trachea, parathyroid gland and superior laryngeal nerve in 2 groups. The incidence of CO2retention related complications in gasless group was significantly lower than that in CO2-insufflation group: 6.7% (2/30) vs. 43.3% (13/30), and there was statistical difference (P<0.01). Conclusions The modified spring suspension gasless endoscopic thyroidectomy not only acquires the equivalent surgical effect and indication,compared with the CO2-insufflation endoscopic surgery,but also is safer and has lower incidence rate of CO2retention related complications.

A retrospective study was conducted based on the clinical data of 42 patients of portal hypertension and splenomegaly who underwent laparoscopic splenectomy.The patients were divided into two groups including pedicle priority group and conventional group by different operative method.The operation time and intraoperative blood loss in the pedicle priority group were significantly lower than those in the conventional group (both P ＜ 0.05),and there was no statistically significant difference on the conversion rate of laparotomy,active time postoperation,exhaust time,postoperative hospitalization stay and the incidence of complications (all P ＞ 0.05).Priority processing for splenic pedicle has obvious advantages in laparoscopic splenectomy for portal hypertension and splenomegaly,and it could reduce the difficulty of operation,shorten the operation time and reduce bleeding.