The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Background:Single port laparoscopic surgery (SPLS) is an innovative approach that is rapidly gaining recognition worldwide. The aim of this study was to determine the feasibility and safety of SPLS compared to conventional laparoscopic surgery for the treatment of benign adnexal masses.Methods:In total, 99 patients who underwent SPLS for benign adnexal masses between December 2013 and March 2015 were compared to a nonrandomized control group comprising 104 conventional laparoscopic adnexal surgeries that were performed during the same period. We retrospectively analyzed multiple clinical characteristics and operative outcomes of all the patients, including age, body mass index, size and pathological type of ovarian mass, operative time, estimated blood loss (EBL), duration of postoperative hospital stay, etc.Results:No significant difference was observed between the two groups regarding preoperative baseline characteristics. However, the pathological results between the two groups were found to be slightly different. The most common pathological type in the SPLS group was mature cystic teratoma, whereas endometrioma was more commonly seen in the control group. Otherwise, the two groups had comparable surgical outcomes, including the median operation time (51 min vs. 52 min, P = 0.909), the median decreased level of hemoglobin from preoperation to postoperation day 3 (10 g/L vs. 10 g/L, P = 0.795), and the median duration of postoperative hospital stay (3 days vs. 3 days, P = 0.168). In SPLS groups, the median EBL and the anal exsufflation time were significantly less than those of the conventional group (5 ml vs. 10 ml, P < 0.001; 10 h vs. 22 h, P < 0.001).Conclusions:SPLS is a feasible and safe approach for the treatment of benign adnexal masses. Further study is required to better determine whether SPLS has significant benefits compared to conventional techniques.

ObjectiveTo assess the effects of 17β-estradiol (E2) on proliferation, apoptosis, and protein expressions of fibroblasts at different concentrations and time intervals to reveal the mechanism of E2 in the treatment of pelvic organ prolapse (POP).Study DesignThe uterosacral ligament fibroblasts were collected from seven POP patients for primary culture of fibroblasts. The culture media containing 0, 10-6, 10-7, 10-8, and 10-9 mol/L E2 were used for 24, 48, 72, and 96 h.Main Outcome MeasuresThe cells were collected for cell counting kit-8 (CCK-8), apoptosis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western blotting assays.ResultsCompared with the control group, in the values of fibroblasts cultured in 10-8 mol/L E2 for 72 h, the proliferation, mRNA and protein expression of Mitofusin-2 (Mfn2) separately increased (P &lt; 0.05), decreased (P&lt;0.001) and decreased (P&lt;0.001). However, the expression level of procollagen 1A1/1A2/3A1 and cyclinD1 markedly increased (P&lt;0.001, all), which was consistent with the results of protein level. What’s more, the expression of estrogen receptor α(ERα), estrogen receptor β(ERβ) and G protein-coupled receptor 30(GPR30) were significantly increased in 10-8 mol/L E2 group.ConclusionsE2 can inhibit the progress of POP by inhibiting the expression level of Mfn2, as well as promoting expression of procollagens and proliferation of fibroblasts. This effect is time- and concentration-dependent. Only when the estrogen concentration reaches 10-8 mol/L, the therapeutic effect is the greatest after 72 h.

Adhesion to the gastrointestinal tract is considered to be important for bifidobacteria to colonize the human gut and exert their probiotic effects. Some cell surface proteins of bifidobacteria, known as adhesins, play critical roles in the binding to host cells or the extracellular matrix (ECM). To elucidate the mechanisms associated with the adhesion of Bifidobacterium longum BBMN68, a centenarian originated potential probiotic, PSORTdb was employed to identify putative extracellular localized proteins in the B. longum BBMN68. Of the 560 predicted extracellular proteins, 21 were further identified as putative adhesion proteins using the conserved domain database of NCBI, and four were successfully overexpressed in the heterologous host, Lactococcus lactis NZ9000. Notably, a recombinant strain expressing FimM showed a significantly increased adhesive affinity for both HT-29 and mucus-secreting LS174T goblet cells (2.2- and 5.4-fold higher than that of the control strain, respectively). Amino acid sequence alignment showed that FimM is a major pilin subunit protein containing a Cna-B type domain and a C-terminal LPKTG sequence. However, in silico analysis of the fimM-coding cluster revealed that BBMN68_RS10200, encoding a pilus-specific class C sortase, was a pseudogene, indicating that FimM may function as a surface adhesin that cannot polymerize into a pili-like structure. Immunogold electron microscopy results further confirmed that FimM localized to the surface of L. lactis NZfimM and B. longum BBMN68 but did not assemble into pilus filaments. Moreover, the adhesive affinity of L. lactis NZfimM to fibronectin, fibrinogen, and mucin were 3.8-, 2.1-, and 3.1-fold higher than that of the control. The affinity of FimM for its attachment receptors was further verified through an inhibition assay using anti-FimM antibodies. In addition, homologs of FimM were found in Bifidobacterium bifidum 85B, Bifidobacterium gallinarum CACC 514, and 23 other B. longum strains by sequence similarity analysis using BLASTP. Our results suggested that FimM is a novel surface adhesin that is mainly present in B. longum strains.

Bacterial vaginosis (BV) is a common infectious disease of the reproductive tract. Metronidazole treatment, as the first line of treatment, frequently fails at recovery of the microbiome.