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Given its different target-binding affinity and varying residence time after DNA cleavage, the Cas9-sgRNA complex that remains bound to cleaved DNA may influence DNA double strand break (DSB) repair pathway choices, contributing to highly heterogeneous mutations in genome editing. Here, we found that DSB repair pathway choices vary significantly at different sites for Cas9-induced DSBs. Reduced target-binding affinity of Cas9-sgRNA promotes a bias toward classical NHEJ (c-NHEJ), but inactivation of c-NHEJ aggravates off-target activities. By dislodging Cas9-sgRNA from the cleaved target, local DNA replication suppresses c-NHEJ in favor of other DSB repair pathways by altering DSB configuration and repair timing. It also results in sister chromatid fusion, which may promote chromosomal breakage-fusion-bridge cycles. This suggests that varying Cas9-sgRNA target-binding affinity or residence duration modulate DSB repair pathway choices for Cas9-induced DSBs and widen the mutational spectra in CRISPR/Cas9 genome editing.

Background Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double strand breaks (DSBs) have to be exposed in order to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target-binding affinity and modulates its post-cleavage target-residence duration and exposure of Cas9-induced DSBs. This exposure by different mechanisms may initiate variable cellular DNA damage response, thus influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. Results In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical non-homologous end joining (c-NHEJ) is not even engaged at some sites. Weakening target interaction of Cas9-sgRNA promotes bias towards c-NHEJ, which is intrinsically accurate for Cas9-induced DSBs. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ however aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways whereas transcription has little effect on DSB repair pathway choices. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. Conclusions Target interaction of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Background: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X chromosome-linked recessive hereditary diseases. The mechanism is that the exon mutations of anti-myatrophy protein gene (Dystrophin gene) and lead to muscle dysfunction. Prenatal diagnosis can prevent the birth of children with defects and have good clinical significance. Methods: CMA and CNV-seq were used to detect the amniotic fluid after amniocentesis,. CNV-seq was used to detect spontaneous abortion tissue. The DMD gene mutations were found in 6 amniotic fluid samples and one spontaneous abortion sample. DMD gene mutations were confirmed by MLPA and new DMD mutations were found.Results: CMA found DMD mutations :1.Xp21.1, 75.5kb del (E52-53); 2.Xp21.2, 334.92kb dup (E61-79); 3.Xp21.2, 292.25kb dup (E58-74); 4.Xp21.1, 374.20 kb dup (E45-51). CNV-seq found DMD mutations: 5.X p21.2, E64-79 dup; 6.X p21.1, E1-7dup; 7.Xp21.1, E 44-52 del. Conclusions: 4 fetuses harboring DMD gene mutations were found by CMA, 2 fetuses and 1 induced abortion carrying DMD gene mutations was detected by CNV-seq. CMA/CNV-seq jointed with MLPA test can provide more comprehensive evidence for prenatal diagnosis.

Background：The clinical characteristics of second primary tumors in oral cavity after radiation was a special subtype of oral cancer and remained poorly studied. The purpose of the present study was to investigate the clinicopathological characteristics and prognostic factors of second primary oral squamous cell carcinoma (OSCC) after radiotherapy for head and neck cancer.Methods：The clinicopathological characteristics of 48 OSCC patients underwent radiotherapy for head and neck cancer were retrospectively analyzed by Kaplan-Meier survival analysis and Cox proportional hazards model, including gender, age, alcohol consumption, smoking, clinical stage, margin status, regional lymph node status, tumor differentiation and treatment mode.Results：The second primary OSCC mostly occurred on the tongue (18/48), bucca (12/48) and gingiva (10/48), and the 3- and 5-year overall survival (OS) was 60.3% and 39.4%, respectively. Margin status (p=0.001, log-rank=10.777) and extranodal extension (p=0.045, log-rank=4.017) were significantly associated with OS, while only margin status was found to be an independent prognostic factor of OS in the Cox proportional hazards model (p=0.003, HR=3.976).Conclusions： OSCC patients underwent radiotherapy for head and neck cancer show poor survival outcomes. Margin status is an independent prognostic factor of second primary OSCC, and the prognostic of second primary OSCC was not in accordance with sporadic OSCC.

Purpose: Intravertebral clefts (IVC) used to be considered as the unique characteristic of Kümmell’s disease (KD). However, accumulating literatures and our clinical findings identified the presence of IVC in acute osteoporotic vertebral compression fractures (OVCF) as well. Since the exact time for incidence of OVCF is usually difficult to recall for old people, it leads to challenging to differentiate IVC from KD and acute OVCF and begs for further investigation. Moreover, the treatment scheme of the two is not exactly the same. In order to identify the differences between IVC of KD and OVCF, we performed this study of radiological features.Materials and Methods: Seventy-nine patients with IVC were included in our study, with 44 cases in the KD group and 35 cases in the acute OVCF group. Radiological images, including X-ray, CT, and MRI, were collected for each group. Results: Consistent with previous reports, IVC was present in both groups. Intriguingly, we found several unique features that were only present in the KD group, including sclerosis of the cleft margin (95.5%, 42/44), ossification of the fractured vertebrae (100%, 44/44), ossification of the pedicle (31.8%, 14/44), double-line sign (27.3%, 12/44), stress fracture of the spinous process (13.6%, 6/44) and even formation of paravertebral callus (18.2%, 8/44).Conclusion: IVC could present in patients with both KD and acute OVCF, which leads to the challenges in the differentiation of clinical diagnosis. Due to the differences in the treatment, it is essential for surgeons to identify the differences among radiological features between the two. We found that marginal cleft sclerosis, vertebral and pedicle ossification, double-line sign, spinous process fracture, and formation of paravertebral callus are unique radiological features of KD and could be used for differentiation of KD from acute OVCF with IVC.

Background： Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator involved in various pathophysiological and inflammatory states.Accumulating line of evidence suggests a role for MIF in regulating bone metabolism and therefore a prime candidate for therapeutic targeting. In this study, we showed that Chicago sky blue 6B (CSB6B), an unique allosteric inhibitor of MIF catalytic and cytokine activity, suppresses RANKL-induced osteoclast and promotes osteogenesis in vitro via the inhibition of NF-κB signaling activationMethods: We examined the effects of CSB6B on osteoclast differentiation and bone resorption and the bone formation ability of osteoblasts in vitro . The effect of CSB6B on the NF-κB pathway was subsequently detected using western blotting and Co-IP. Finally, the model of mouse skull dissolution and ovarian severing were modeled and intraperitoneally injected with different doses of CSB6B to observe the anti-osteolytic and anti-osteoporosis effects of the drug in vivo.Results: In this study, we showed that Chicago sky blue 6B (CSB6B) suppresses RANKL-induced osteoclast and bone resorption in vitro via the inhibition of NF-κB signaling activation and promoting proteasome-mediated degradation of MIF. Consequently, the induction of NFATc1 was impaired resulting in downregulation of NFATc1-responsive osteoclast genes. We also demonstrated that CSB6B treatment enhanced primary calvarial osteoblast differentiation and bone mineralization in vitro via the suppression of NF-κB activation and upregulation of Runx expression. Using two murine models of osteolytic bone disorders, we further showed that administration of CSB6B protected mice against pathological inflammatoryc calvarial bone destruction induced by titanium particles mice as well as estrogen-deficiency induced bone loss as a result of ovariectomy.Conclusion: Together, as an MIF inhibitor, CSB6B can inhibit osteoclast differentiation and absorption function and enhance the mineralization of osteoblasts through the inhibition of NF-κB pathway. MIF is a prime target for therapeutic targeting for the treatment of osteolytic bone disorders and the MIF inhibitor CSB6B could be potential anti-osteoporosis drug.

Objective: This study aimed to assess the feasibility as well as the clinical and radiological outcomes of posterior percutaneous pedicle screw fixation (PPSF) combined with anterior monosegmental column reconstruction in unstable thoracolumbar burst fractures. Methods: From January 2011 to August 2017, thirty-five patients with unstable thoracolumbar burst fractures were enrolled in this study. The patients underwent posterior PPSF combined surgery with delayed anterior monosegmental reconstruction utilizing titanium mesh cages. Clinical outcomes, radiological parameters, and treatment-related complications were assessed. Results: The mean age of the patients was 44.8 years. The mean operative time and blood loss were 205 min and 560 ml, respectively. The mean follow-up period was 25.2 months. The Visual analog scale (VAS) pain score was significantly improved postoperatively, and the improvement was maintained until the final follow-up. The mean sagittal kyphosis was corrected from 16.3 preoperatively to 1.5 postoperatively, which increased slightly to 2.6 at the final follow-up. In 24 patients with neurologic dysfunction, 21 (87.5 %) patients had improvement after surgery. None obvious subsidence of the titanium mesh cage and none dislodgement, loosening or breakage of the instrumentation was observed in any patient during the follow-up period. Solid bony fusion was achieved in all patients. Conclusions: Combined posterior PPSF with delayed anterior monosegmental column reconstruction for unstable thoracolumbar burst fractures can produce good clinical and radiological outcomes.

Objective: To investigate the preliminary clinical and radiographic outcomes of oblique lateral interbody fusion (OLIF) combined with percutaneous transforaminal endoscopic discectomy (PTED) for the degenerative lumbar disease accompanied by prolapsed disc herniation.Methods: From March 2016 to December 2018, 15 patients with degenerative lumbar spondylolisthesis, lumbar spine instability or lumbar spinal stenosis accompanied by prolapsed disc herniation were underwent OLIF combined with PTED in our spine surgery center, including 6 males and 9 females, the mean age was 61.4±7.1 years. Clinical results, radiological parameters, and related complications were collected and analyzed.Results: All patients firstly received PTED with local anesthesia and then underwent OLIF with general anesthesia. All patients were followed up for an average of 14.6±3.7 months. Mean preoperative visual analog scale scores and Oswestry Disability Index scores were significantly improved postoperatively (P