Development of a universal antibiotic resistance screening system for efficient enrichment of C-to-G and A-to-G base editing.

To expand the scope of genomic editing, a C-to-G transversion-based editor called CGBE has been developed for precise single-nucleotide genomic editing. However, limited editing efficiency and product purity have hindered the development and application of CGBE. In this study, we introduced the Puromycin-Resistance Screening System, referred to as CGBE/ABE-PRSS, to select genetically modified cells via the CGBE or ABE editors. The CGBE/ABE-PRSS system significantly improves the enrichment efficiency of CGBE- or ABE-modified cells, showing enhancements of up to 59.6 % compared with the controls. Our findings indicate that the CGBE/ABE-PRSS, when driven by the CMV promoter, results in a higher enrichment of edited cells compared to the CAG and EF1α promoters. Furthermore, we demonstrate that this system is compatible with different versions of both CGBE and ABE, enabling various cell species and simultaneous multiplexed genome editing without any detectable random off-targets. In conclusion, our developed CGBE/ABE-PRSS system facilitates the selection of edited cells and holds promise in both basic engineering and gene therapy applications.

Elastic porous microspheres/extracellular matrix hydrogel injectable composites releasing dual bio-factors enable tissue regeneration.

Injectable biomaterials have garnered increasing attention for their potential and beneficial applications in minimally invasive surgical procedures and tissue regeneration. Extracellular matrix (ECM) hydrogels and porous synthetic polymer microspheres can be prepared for injectable administration to achieve in situ tissue regeneration. However, the rapid degradation of ECM hydrogels and the poor injectability and biological inertness of most polymeric microspheres limit their pro-regenerative capabilities. Here, we develop a biomaterial system consisting of elastic porous poly(l-lactide-co-ε-caprolactone) (PLCL) microspheres mixed with ECM hydrogels as injectable composites with interleukin-4 (IL-4) and insulin-like growth factor-1 (IGF-1) dual-release functionality. The developed multifunctional composites have favorable injectability and biocompatibility, and regulate the behavior of macrophages and myogenic cells following injection into muscle tissue. The elicited promotive effects on tissue regeneration are evidenced by enhanced neomusle formation, vascularization, and neuralization at 2-months post-implantation in a male rat model of volumetric muscle loss. Our developed system provides a promising strategy for engineering bioactive injectable composites that demonstrates desirable properties for clinical use and holds translational potential for application as a minimally invasive and pro-regenerative implant material in multiple types of surgical procedures.

Establishing a Predictive Model to Predict Survival of de Novo Metastatic HER2-Low Breast Cancer: A National Cancer Database Analysis.

BACKGROUND: Breast cancer with low human epidermal growth factor receptor (HER2) expression is increasingly considered as a distinct subtype which consists of types of HER2 immunohistochemistry (IHC) 1+ and HER2 IHC 2+/in-situ hybridization (ISH)-negative. We aim to assess the survival difference between HER2 IHC 1+ and HER2 IHC 2+/ISH-negative breast cancer patients with metastasis at presentation and construct a prognostic nomogram for HER2-low patients. METHOD: Patients diagnosed with de novo metastatic HER2-low breast cancer from 2010 to 2015 were included and analyzed using the National Cancer Database (NCDB). Cox proportional hazards regression model and Kaplan-Meier (KM) method were used for survival analysis. Nomograms were built to predict survival. RESULT: A total of 7897 patients were included in the final analysis, among which 5458 (69.1%) patients were HER2 IHC 1+ and 2439 (30.9%) were HER2 IHC 2+/ISH-negative. Although the Kaplan-Meier survival analysis showed difference in survival, this survival difference was lost in the multivariate Cox analysis (multivariate: HR (hazard ratio) = 0.97; 95% CI (confidence interval) [0.92-1.03]). A prognostic nomogram was successfully constructed for individually predicting the long-term survival rate of HER2-low patients, which exhibited an acceptable predictive capability in training (C index: 0.719) and validation cohort (C index: 0.706). This nomogram could easily divide patients into high and low-risk subgroups with distinct prognoses. CONCLUSIONS: Our data suggest no statistical survival differences between HER2 1+ and HER2 2+ breast cancer. Additionally, a nomogram was constructed with an acceptable capacity to individually predict the long-term outcome of HER2-low metastatic breast cancer patients.

Lumbar spine segmentation method based on deep learning.

Aiming at the difficulties of lumbar vertebrae segmentation in computed tomography (CT) images, we propose an automatic lumbar vertebrae segmentation method based on deep learning. The method mainly includes two parts: lumbar vertebra positioning and lumbar vertebrae segmentation. First of all, we propose a lumbar spine localization network of Unet network, which can directly locate the lumbar spine part in the image. Then, we propose a three-dimensional XUnet lumbar vertebrae segmentation method to achieve automatic lumbar vertebrae segmentation. The method proposed in this paper was validated on the lumbar spine CT images on the public dataset VerSe 2020 and our hospital dataset. Through qualitative comparison and quantitative analysis, the experimental results show that the method proposed in this paper can obtain good lumbar vertebrae segmentation performance, which can be further applied to detection of spinal anomalies and surgical treatment.

Cytosine base editors induce off-target mutations and adverse phenotypic effects in transgenic mice.

Base editors have been reported to induce off-target mutations in cultured cells, mouse embryos and rice, but their long-term effects in vivo remain unknown. Here, we develop a Systematic evaluation Approach For gene Editing tools by Transgenic mIce (SAFETI), and evaluate the off-target effects of BE3, high fidelity version of CBE (YE1-BE3-FNLS) and ABE (ABE7.10F148A) in ~400 transgenic mice over 15 months. Whole-genome sequence analysis reveals BE3 expression generated de novo mutations in the offspring of transgenic mice. RNA-seq analysis reveals both BE3 and YE1-BE3-FNLS induce transcriptome-wide SNVs, and the numbers of RNA SNVs are positively correlated with CBE expression levels across various tissues. By contrast, ABE7.10F148A shows no detectable off-target DNA or RNA SNVs. Notably, we observe abnormal phenotypes including obesity and developmental delay in mice with permanent genomic BE3 overexpression during long-time monitoring, elucidating a potentially overlooked aspect of side effects of BE3 in vivo.

Synergistic antimicrobial activity of a novel cationic micelle L/D2 and imipenem against multidrug-resistant Acinetobacter baumannii.

Background: The multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) has become one of the most important pathogens of nosocomial infection due to widespread use of broad spectrum antimicrobial drugs and immunosuppressor therapy. As polymyxins resistance emerges, developing novel effective antibacterial agents capable of overcoming multidrug resistance is urgently needed. Methods: In this study, biodegradable triblock copolymers of polyethylene glycol (PEG), guanidinium-functionalized polycarbonate and polylactide, PEG-PGC20-PLLA20 (L2) and PEG-PGC20-PDLA20 (D2), were utilized as antibacterial agents. Results: The copolymers self-assemble into micellar nanoparticles (L/D2), and exhibit broad-spectrum antibacterial activity against 20 clinically isolated multidrug-resistant A. baumannii strains. L/D2 had more rapid killing kinetics than conventional antibiotics imipenem and ceftazidime, and exhibited potent anti-biofilm activity. Repeated use of L/D2 did not induce drug resistance. From scanning electron microscopy and nucleic acid release analyses, L/D2 showed membrane-lytic mechanism. We also demonstrated that L/D2 was synergistically active with imipenem against MDR A. baumannii strains. Additionally, strong synergistic antibacterial activity was also observed for the combined use of L/D2 and imipenem in a MDR A. baumannii abdominal infection mouse model. Conclusions: Therefore, the combination of L/D2 and imipenem might be an alternative option for the prevention of nosocomial infection caused by A. baumannii.

80MAP17 promotes the tumorigenesis of papillary thyroid carcinoma by reducing the stability of p53.

Background: Papillary thyroid cancer (PTC) is an endocrine malignancy whose incidence has increased rapidly worldwide. MAP17 (PDZKIP1) is a small protein related to tumor progression. The aim of this study was to investigate the role of MAP17 in PTC and the underlying molecular mechanism. Methods: Bioinformatics, Western blotting and immunohistochemistry were used to analyze the expression of MAP17 in PTC. The gene transcription was measured by qPCR. Cell viability was determined by CCK8 assay. Cell growth was measured by clonal formation assay. Cell apoptosis was measured by TUNEL. Wound healing assay and transwell assay were used to measure the mobility of cells. The expression of E-cadherin and N-cadherin was determined by immunofluorescence. The effect of MAP17 on tumor growth was determined in animal experiments. Results: The results showed that MAP17 was up-regulated in PTC, which significantly promoted the growth and motility of PTC cells, but inhibited cell apoptosis. Besides, overexpression of MAP17 accelerated cycloheximide (CHX, a protein synthesis inhibitor)-induced p53 degradation, while low expression of MAP17 slowed down CHX-induced p53 degradation, suggesting that MAP17 can regulate p53 stability. Notably, NUMB exhibited an opposite effect on P53 stability. Interestingly, p53 overexpression reversed the effects of MAP17 overexpression on cell viability, motility, and apoptosis, indicating that p53 was involved in the progression of PTC. In vivo studies have shown that tumor growth was positively correlated with MAP17 expression and negatively correlated with p53 expression. Conclusion: Our findings revealed that MAP17 exhibited carcinogenic effects through interacting with NUMB to reduce the stability of p53, demonstrating that MAP17 may serve as a potential prognostic biomarker for PTC treatment.

Impact of prior cancer history on survival of patients with gastric cancer.

BACKGROUND: Patients with prior cancer history are commonly excluded from clinical trial. However, the impact of prior cancer on survival of patients with gastric cancer remains largely unknown. The aim of this study was to evaluate the prevalence of prior cancer and assess its impact on survival of patients diagnosed with gastric cancer. METHODS: Patients with gastric cancer as the primary or second primary malignancies diagnosed from 2004 to 2010 were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Propensity score matching (PSM) was conducted to balance baseline characteristics. Kaplan-Meier method, multivariate Cox proportional hazard model, and multivariate competing risk model were performed for survival analysis. RESULTS: A total of 28,795 eligible patients with gastric cancer were included, of whom 2695 (9.35%) had a history of prior cancer. Prostate (35%), breast (12%), colon (8%), and urinary bladder (7%) malignancies were the most common prior cancer types. Patients with prior cancer history had slightly inferior overall survival (AHR = 1.06; 95% CI [1.00-1.12]; P = 0.043) but superior gastric cancer-specific survival (AHR = 0.82; 95% CI [0.76-0.88]; P < 0.001) compared with those without prior cancer. The subgroup analysis determined that a prior cancer history did not adversely affect gastric patients' clinical outcomes, except in those with prior cancer diagnosed within one year, at distant stage, or originating from lung and bronchus. CONCLUSION: A substantial proportion of gastric cancer patients with a history of prior cancer had non-inferior clinical outcome to those without prior cancer. These patients should be considered in clinical trials.

Improved Transcatheter aortic valve implantation for aortic regurgitation using a new-type stent: the first preclinical experience.

BACKGROUND: In this study, we sought to evaluate the feasibility of improved transcatheter aortic valve implantation (TAVI) in noncalcified aortic valve by using the novel concept of double-layer ChenValve prosthesis. TAVI was initially considered as an alternative treatment for high-risk patients with aortic stenosis. However, non noncalcified aortic valve disease was considered as a contraindication to TAVI. METHODS: ChenValve prosthesis, which consisted of a self-expanding Nitinol ring, a balloon-expandable cobalt-chromium alloy stent and a biological valve, was implanted at the desired position under fluoroscopic guidance in a transapical approach through a 20F sheath in 10 goats. Aortic angiography was performed to measure the diameter of the aotic annulus and assess the performance of the artificial valve. The ultrasound was used to evaluate the regurgitation or paravalvular leakage and trans-prosthetic vascular flow velocity postoperatively. The aortogram and transthoracic echocardiography were applied to observe whether the valve stent was implanted at the desired position. RESULTS: ChenValve prosthesis was successfully transppical implanted in all animals. The aortogram and transthoracic echocardiography performed immediately after implantation revealed that the valve stent was implanted at the desired position. There was no significant paravalvular leakage, obstruction of coronary artery ostia, stent malpositioning or dislodgement occurred. CONCLUSIONS: This preliminary trial with the novel double-layer ChenValve prosthesis demonstrated the feasibility of improved TAVI in noncalcified aortic valve. The mechanism of Nitinol ring-guided locating the aortic sinus enables us to anatomically correct position the artifact valve. This improved strategy seems to make the TAVI process more safe and repeatable in noncalcified aortic valve.

Comparison of Distal Transradial Access in Anatomic Snuffbox Versus Transradial Access for Coronary Angiography.

BACKGROUND: To compare distal transradial access (dTRA) in the anatomic snuffbox and conventional transradial access (cTRA) for coronary angiography. METHODS: Eighty cases that underwent coronary angiography were selected at The 903 Hospital of PLA in China from April 2017 to March 2018. Patients in the dTRA group underwent distal transradial access, and patients in the cTRA group received conventional transradial access. Puncture success rate, fluoroscopy time, and hemostasis time were determined. RESULTS: The puncture success ratio was 85% in the dTRA group and 100% in the cTRA group (P < .05). The fluoroscopy time was 36 minutes and 19 min minutes in the dTRA group and cTRA group (P < .05), respectively. The compression hemostasis time after operation was 120 minutes and 240 minutes in the dTRA group and cTRA group (P < .05), respectively. The complication rate in the cTRA group was 7.5%, while there was no complication in the dTRA group. In addition, there also was no significant difference between the two groups in the rate of complications (cTRA = 7.5%, dTRA = 0.0%, P > .05). CONCLUSION: Distal transradial access in the anatomical snuffbox for coronary angiogram is a potential alternative to conventional radial arterial access.

[Evaluation of osteonecrosis of the femoral head treated with percutaneous retaining catheterization].

OBJECTIVE: To evaluate the efficacies of treating osteonecrosis of the femoral head (ONFH) with percutaneous retaining catheterization. METHODS: A total of 85 patients (106 hips) of ONFH underwent percutaneous retaining catheterization with self-made instrument. There were stage I (n = 32, 40 hips), stage II (n = 41, 50 hips), stage III (n = 7, 9 hips) and stage IV (n = 5, 7 hips) by Ficat and Arlet classification. The selection criteria include bone marrow edema (BME) of the femoral head and neck and joint fluid on magnetic resonance imaging (MRI).The percent method(%) of Orthopedics Branch of Association of Chinese Medical Association was used for evaluating the efficacies of ONFH with retained femoral head. A follow-up period of 2-10 years was conducted with plain radiograph.The standard was as follows: >90, excellent; 75-89, good; 60-74, passable; <60, bad. The combination of excellent and good denoted obvious effect rate; passable for valid rate; bad for invalid rate.The historical core decompression was used as control group.And χ(2) test was used for statistical analysis (P < 0.05). RESULTS: The obvious effect rate was 88%(35/40), 74%(37/50), 22%(2/9), 14%(1/7), the valid rate 7%(3/40), 12% (6/50) , 22% (2/9), 14%(1/7) and the invalid rate 5%(2/40), 14% (7/50) , 56% (5/9), 72% (5/7) respectively in Ficat I, II, III and IV stage. No statistically significant differences existed between Ficat I and II stage (P > 0.05) .But there were statistically significant differences in other groups (P < 0.01) . CONCLUSION: Percutaneous retaining catheterization is efficaciously treating ONFH in Ficat I and II stage and it may also be used for ONFH in Ficat III and IV stage.

[Clinical significance of venous return disturbance in patients with osteonecrosis of femoral head].

OBJECTIVE: To evaluate the correlation of venous return disturbance (VRD), bone marrow edema (BME) and the amount of joint fluid with clinical symptoms in osteonecrosis of the femoral head (ONFH). METHODS: Twenty patients (25 hips) with ONFH proved by pathology were parallelly examined by 1.5 T MR and medullograph. RESULT: In total 25 hips VRD was observed in 16 hips with medullograph and 13 hips with the clinical symptoms (P <0.05). BME was found in 17 with MRI (P <0.01), and the was found in 15 hips with MRI (Spearman=0.949). VRD were found in 16 hydrarthrus. CONCLUSION: ONFH patients with clinical symptoms have VRD, BME and the hydrarthrus. The medullograph is of value in monitoring the development of ONFH.