Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.
CRISPR-Cas Systems/genetics* ; Gene Editing/methods* ; RNA, Guide, CRISPR-Cas Systems/genetics* ; Plasmids/genetics*

Objective:To summarize and analyze the clinical features of patients with inflammatory bowel disease (IBD) accompanied by axial spondyloarthritis (ax-SpA) .Methods:A descriptive case series study was conducted. Consecutive IBD patients with ax-SpA admitted to Peking Union Medical College Hospital from January 2012 to December 2023 were enrolled, and the clinical data were collected and analyzed descriptively.Results:A total of 21 IBD patients with ax-SpA were enrolled, including 14 males (66.7%) and 7 females (33.3%). The median age at IBD diagnosis was 29 (24, 42) years. Among them, 10 had ulcerative colitis (UC) and 11 had Crohn's disease (CD). In 11 patients (52.4%), ax-SpA was diagnosed before IBD, while in 7 patients (33.3%), IBD was diagnosed before ax-SpA; the remaining 3 patients (14.3%) were diagnosed simultaneously. Thirteen patients (61.9%) had other extraintestinal manifestations, with 12 involving mucocutaneous manifestations. Nineteen patients including 9 UC and 10 CD were followed up for a median duration of 34 (12, 57) months. Among UC patients, 5 (55.6%) were escalated from conventional therapy to biologics or small-molecule drugs, and 6 (66.7%) developed opportunistic infections. Among CD patients, 3 (30.0%) were escalated to biologic therapy, and 2 (20.0%) developed opportunistic infections. Three patients (15.8%) underwent intestinal resection surgery.Conclusions:IBD patients with ax-SpA are predominantly male, with a similar proportion of UC and CD. There is no clear sequential pattern in the diagnosis timing of IBD and ax-SpA. These patients often present with other extraintestinal manifestations of IBD, primarily mucocutaneous manifestations.

Objective:To investigate the incidence and influencing factors of optic disc changes in children and adolescents with high myopia.Methods:A clinical cross-sectional study. A total of 162 children and adolescents with high myopia (162 eyes) who visited Department of Ophthalmology, Northern Jiangsu People's Hospital Affiliated to Yangzhou University from January to April 2025 were included in this study. Myopia refractive error ≥6.00 D and/or axial length (AL) ≥26 mm. All participants underwent best-corrected visual acuity, refraction, fundus color photography, swept-source optical coherence tomography (SS-OCT), and AL measurement. Subfoveal choroidal thickness (ChT) was measured within 1 mm using SS-OCT. Optic disc changes assessed included tilt, rotation, peripapillary atrophy (PPA), and peripapillary hyperreflective ovoid mass-like structures (PHOMS). The patients were divided into the children group (4-11 years old) and the adolescents group (12-18 years old) based on age, with 63 (38.9%, 63/162) and 99 (61.1%, 99/162) cases respectively. The incidence of ocular features and optic disc morphology changes in the two groups was compared and observed. According to the myopia diopter, the patients were divided into the high diopter long axial group (myopia diopter ≥6.00 D, AL≥26 mm) and the low diopter long axial group (myopia diopter <6.00 D, AL≥26 mm), with 85 (52.5%, 85/162) and 77 (47.5%, 77/162) eyes respectively. The incidence of optic disc morphological changes in the two groups was compared and observed. The comparison of quantitative data between groups was conducted using the Mann-Whitney U test. Multivariate logistic regression was used to analyze the correlations between PPA, optic disc tilt, PHOMS occurrence and gender, age, diopter, AL, and ChT. Results:Among the 162 patients, 103 were male and 59 were female. Age was 12 (10.5, 13.5) years old. Among the 162 eyes, the optic disc morphology changed in 152 eyes (93.8%, 152/162). Among them, the PPA, optic disc tilt, PHOMS, and optic disc rotation were 148 (91.4%, 148/162), 95 (58.6%, 95/162), 62 (38.3%, 62/162), and 35 (21.6%, 35/162) eyes respectively. Myopic macular degeneration in 137 eyes. There were 56 eyes with peripheral retinopathy. There was no statistically significant difference in myopia diopter, AL and ChT between the children group and the adolescent group ( Z=-1.201, -1.934, ?0.761; P=0.230, 0.053, 0.447). There was no statistically significant difference in the incidences of PPA, optic disc tilt and optic disc rotation ( χ2=0.293, 2.618, 0.398; P>0.05). There was no statistically significant difference in the incidence of optic disc morphological changes between the low diopter long axial group and the high diopter long axial group ( χ2=0.000, P>0.05). The results of multivariate logistic regression analysis showed that the thinner the ChT, the higher the risk of PPA [odds ratio (OR) =0.98, 95% confidence interval ( CI) 0.97-0.99, P<0.001]. Female ( OR=2.3, 95% CI 1.04-5.07, P=0.039), older age ( OR=1.17, 95% CI 1.01-1.37, P=0.043), thinner ChT ( OR=0.99, 95% CI 0.99-1.00, P=0.012), the higher the risk of optic disc tilt. The older the age, the higher the risk of developing PHOMS ( OR=1.22, 95% CI 1.06-1.40, P=0.006). Conclusions:Optic disc morphology changes may be the most common fundus alterations in children and adolescents with high myopia. The influencing factors of optic disc morphological changes (including PPA, optic disc tilt, and PHOMS) are female sex, advanced age, and ChT thinning.

Objective:To summarize and analyze the clinical features of patients with inflammatory bowel disease (IBD) accompanied by axial spondyloarthritis (ax-SpA) .Methods:A descriptive case series study was conducted. Consecutive IBD patients with ax-SpA admitted to Peking Union Medical College Hospital from January 2012 to December 2023 were enrolled, and the clinical data were collected and analyzed descriptively.Results:A total of 21 IBD patients with ax-SpA were enrolled, including 14 males (66.7%) and 7 females (33.3%). The median age at IBD diagnosis was 29 (24, 42) years. Among them, 10 had ulcerative colitis (UC) and 11 had Crohn's disease (CD). In 11 patients (52.4%), ax-SpA was diagnosed before IBD, while in 7 patients (33.3%), IBD was diagnosed before ax-SpA; the remaining 3 patients (14.3%) were diagnosed simultaneously. Thirteen patients (61.9%) had other extraintestinal manifestations, with 12 involving mucocutaneous manifestations. Nineteen patients including 9 UC and 10 CD were followed up for a median duration of 34 (12, 57) months. Among UC patients, 5 (55.6%) were escalated from conventional therapy to biologics or small-molecule drugs, and 6 (66.7%) developed opportunistic infections. Among CD patients, 3 (30.0%) were escalated to biologic therapy, and 2 (20.0%) developed opportunistic infections. Three patients (15.8%) underwent intestinal resection surgery.Conclusions:IBD patients with ax-SpA are predominantly male, with a similar proportion of UC and CD. There is no clear sequential pattern in the diagnosis timing of IBD and ax-SpA. These patients often present with other extraintestinal manifestations of IBD, primarily mucocutaneous manifestations.

Objective:To investigate the incidence and influencing factors of optic disc changes in children and adolescents with high myopia.Methods:A clinical cross-sectional study. A total of 162 children and adolescents with high myopia (162 eyes) who visited Department of Ophthalmology, Northern Jiangsu People's Hospital Affiliated to Yangzhou University from January to April 2025 were included in this study. Myopia refractive error ≥6.00 D and/or axial length (AL) ≥26 mm. All participants underwent best-corrected visual acuity, refraction, fundus color photography, swept-source optical coherence tomography (SS-OCT), and AL measurement. Subfoveal choroidal thickness (ChT) was measured within 1 mm using SS-OCT. Optic disc changes assessed included tilt, rotation, peripapillary atrophy (PPA), and peripapillary hyperreflective ovoid mass-like structures (PHOMS). The patients were divided into the children group (4-11 years old) and the adolescents group (12-18 years old) based on age, with 63 (38.9%, 63/162) and 99 (61.1%, 99/162) cases respectively. The incidence of ocular features and optic disc morphology changes in the two groups was compared and observed. According to the myopia diopter, the patients were divided into the high diopter long axial group (myopia diopter ≥6.00 D, AL≥26 mm) and the low diopter long axial group (myopia diopter <6.00 D, AL≥26 mm), with 85 (52.5%, 85/162) and 77 (47.5%, 77/162) eyes respectively. The incidence of optic disc morphological changes in the two groups was compared and observed. The comparison of quantitative data between groups was conducted using the Mann-Whitney U test. Multivariate logistic regression was used to analyze the correlations between PPA, optic disc tilt, PHOMS occurrence and gender, age, diopter, AL, and ChT. Results:Among the 162 patients, 103 were male and 59 were female. Age was 12 (10.5, 13.5) years old. Among the 162 eyes, the optic disc morphology changed in 152 eyes (93.8%, 152/162). Among them, the PPA, optic disc tilt, PHOMS, and optic disc rotation were 148 (91.4%, 148/162), 95 (58.6%, 95/162), 62 (38.3%, 62/162), and 35 (21.6%, 35/162) eyes respectively. Myopic macular degeneration in 137 eyes. There were 56 eyes with peripheral retinopathy. There was no statistically significant difference in myopia diopter, AL and ChT between the children group and the adolescent group ( Z=-1.201, -1.934, ?0.761; P=0.230, 0.053, 0.447). There was no statistically significant difference in the incidences of PPA, optic disc tilt and optic disc rotation ( χ2=0.293, 2.618, 0.398; P>0.05). There was no statistically significant difference in the incidence of optic disc morphological changes between the low diopter long axial group and the high diopter long axial group ( χ2=0.000, P>0.05). The results of multivariate logistic regression analysis showed that the thinner the ChT, the higher the risk of PPA [odds ratio (OR) =0.98, 95% confidence interval ( CI) 0.97-0.99, P<0.001]. Female ( OR=2.3, 95% CI 1.04-5.07, P=0.039), older age ( OR=1.17, 95% CI 1.01-1.37, P=0.043), thinner ChT ( OR=0.99, 95% CI 0.99-1.00, P=0.012), the higher the risk of optic disc tilt. The older the age, the higher the risk of developing PHOMS ( OR=1.22, 95% CI 1.06-1.40, P=0.006). Conclusions:Optic disc morphology changes may be the most common fundus alterations in children and adolescents with high myopia. The influencing factors of optic disc morphological changes (including PPA, optic disc tilt, and PHOMS) are female sex, advanced age, and ChT thinning.

Objective:To analyze the influential factors of blurred vision after general anesthesia.Methods:The clinical data of 997 patients who underwent elective general anesthesia at The No. 1 People's Hospital of Pinghu from September 2022 to May 2023 were retrospectively analyzed. The data collected included age, sex, body mass index, American Society of Anesthesiologists classification, history of hypertension, history of diabetes, operation duration (specifically whether it exceeded 3 hours), surgical position (whether the patient was in the supine position), operating room temperature, use of penehyclidine hydrochloride, use of muscle relaxant antagonists, use of atropine, blood pressure (specifically whether it was ≥ 30% of the baseline value), fluid input, blood loss, and use of pneumoperitoneum. Collinearity diagnosis and univariate logistic regression analysis were conducted to select factors with statistical significance. Subsequently, multivariate logistic regression analysis was performed.Results:Univariate and multivariate logistic regression analyses showed that age > 65 years ( OR = 1.47, 95% CI: 1.01-2.15, P = 0.043), surgical position (non-supine position) ( OR = 1.54, 95% CI: 1.06-2.25, P = 0.025), operation time exceeding 3 hours ( OR = 1.76, 95% CI: 1.05-2.94, P = 0.031), and the use of penehyclidine hydrochloride ( OR = 4.91, 95% CI: 3.35-7.21, P < 0.001) were identified as factors contributing to postoperative blurred vision in patients undergoing general anesthesia. Conclusion:Factors contributing to postoperative blurred vision in patients undergoing general anesthesia include age > 65 years, the use of penehyclidine hydrochloride during surgery, operation time exceeding 3 hours, and non-supine surgical position. Clinically, it is essential to implement early and effective preoperative education, enhance intraoperative nursing quality, and optimize preoperative medication for general anesthesia to reduce the incidence of blurred vision after surgery.

AIM: To investigate the effects of hypobaric hypoxia in plateau on tear indexes and related anatomical structures in rabbits.METHODS: A total of 18 healthy New Zealand rabbits were selected and randomly divided into plateau group and control group, with 9 rabbits(18 eyes)in each group. The plateau group was housed in the Simulated Climate Cabin for Special Environment of Northwest of China, simulating hypobaric hypoxia at an altitude of 6 000 m. The control group was housed in a clean animal room with atmospheric pressure and oxygen. Changes in the tear meniscus height and non-invasive tear break-up time were detected by using RHCT-1 corneal topographer dry eye comprehensive analysis system, changes in tear secretion was measured by Schirmer Ⅰ test, before intervention and on the 3, 7 d, 2 and 4 wk. Meanwhile, the changes in tear composition before and after intervention in the plateau environment were analyzed using Raman Spectroscopy. The histopathological changes of the lower lid conjunctiva, cornea, lacrimal gland, and Hardarian gland were observed by hematoxylin-eosin(HE)staining after 4 wk of intervention, and the expression of mucin 5AC(MUC5AC)in conjunctiva was detected by immunohistochemistry.RESULTS: Compared with the control group, Schirmer Ⅰ test, tear meniscus height, first and average non-invasive tear break-up time in the plateau group decreased significantly since 3 d, and the difference was significant with the extension of observation time(P<0.05). The above indexes increased from 2 wk. After 4 wk of intervention, the protein and lipid content of the tear composition of rabbits in the plateau group increased, and the nucleic acid content decreased compared with the pre-intervention period. Compared with the control group, rabbits in the plateau group showed thickening of corneal stromal edema, an increase in the number of conjunctival cup cells, increase in the level of expression of MUC5AC, an increase in the level of expression of MUC5AC, an atrophy and flattening of cytoplasm in lacrimal epithelial cells, an enlargement of glandular lumen, and no obvious destructive changes in the Hardarian glands.CONCLUSION: Acute plateau environment can destroy the homeostasis of rabbit ocular surface, so that the tear secretion and the tear film stability decreases, but within a certain period of time, rabbits undergo compensation with the habituation to the hypobaric hypoxia environment, which can increase the tear secretion to a certain extent and restore the tear film stability.

ObjectiveTo analyze the research status and trends in low-level occupational benzene exposure. Methods Articles on low-level occupational benzene exposure from Chinese and English journals from January 1st, 2000, to December 31th, 2022 were retrieved using the Web of Science and the China National Knowledge Infrastructure, and a bibliometric analysis was conducted. Results A total of 327 articles were included in the analysis, comprising 216 English articles and 111 Chinese articles. i) The number of articles published in English fluctuates greatly over the years, without a trend of continuous growth or decline. Authors from 359 research institutions in 45 countries and regions have published relevant English articles in 97 kinds of journals, involving 281 grants from 226 foundations. The top three countries in terms of articles amount were the United States, Italy, and China, with 81, 46, and 43 papers, respectively. The English articles mainly focused on mechanistic research at the genetic level, such as hematotoxicity, oxidative stress, and DNA damage. ii) The number of Chinese articles increased gradually after 2012, with the growth peak in 2017. Authors from 127 research institutions in 26 provinces, autonomous regions, and municipalities published Chinese articles in 51 kinds of journals, involving 154 grants from 78 foundations. Chinese articles tended to focus on benzene-induced hematotoxicity and occupational health damage. Conclusion Most studies on low-level occupational benzene exposure were conducted in China, the United States and Italy, focused on hematotoxicity. Monitoring international research topics and hotspots of the field has certain reference value for related research in China.

Frailty is a geriatric syndrome associated with aging, which is characterized by a decrease in the ability to stress and maintain homeostasis after the loss of physiological reserve function, resulting in an increased risk of falls, disability, and even death.Cognitive impairment is a disorder of cognitive function due to a variety of causes, between normal aging and dementia.In the early stages, frailty is a certain reversibility.As the core of physical frailty, sarcopenia has been a research hotspot in recent years.With the deep research on sarcopenia, researchers have found that is closely related to cognitive disorder.Muscle-brain axis may play a role in sarcopenia and cognitive impairment.This article illustrates its possible mechanisms at the crossroad of sarcopenia and cognitive disorder, and to provide reference for exploring new targets for intervention of sarcopenia and cognitive impairment.

Objective:To explore the impact of problem-inquiry teaching based on the concept of science-technology-engineering-arts-mathematics (STEAM) on the critical thinking and practical abilities of clinical interns in neurology.Methods:The 194 clinical interns majoring in clinical medicine and enrolled in 2020 were selected as subjects. The 98 interns from classes 1-4 were included in the traditional group with traditional teaching, and the 96 clinical interns from classes 5-8 were included in the teaching reform group with problem-inquiry teaching based on the STEAM concept. After the internship, the assessment scores (basic theoretical knowledge, professional theoretical knowledge, classroom performance), critical thinking, practical ability, and teaching satisfaction were compared between the two groups of interns.Results:The assessment scores of the teaching reform group [(40.97±3.26) for basic theoretical knowledge, (42.12±2.95) for professional theoretical knowledge, and (38.47±2.78) for classroom performance] were significantly higher than those of the traditional group [(38.14±3.07), (40.33±2.83), and (35.89±2.71)] (all P<0.05). After the internship, the California Critical Thinking Disposition Inventory-Chinese Version scores of both groups of clinical interns increased, and the score was higher in the teaching reform group than in the traditional group. The Leicester Assessment Package score (82.49±6.71), case writing score (85.63±5.34), and teaching satisfaction (91.67%) of the teaching reform group were significantly higher than those of the traditional group [(77.35±6.23), (82.57±5.12), and 79.59%] (all P<0.05). Conclusions:Problem-inquiry teaching based on the STEAM concept can improve the theoretical knowledge level of clinical interns in neurology, promote their active speaking, enhance their critical thinking and practical abilities, and increase teaching satisfaction.