[Abstract] [Objective] To construct inducible immortalization gene vectors for transfection into primary cells, enabling the establishment of a conditionally immortalized cell line that support their sustained cultivation and proliferation in vitro. [Methods] Using gene homologous recombination technology, the coding sequences (CDS) of immortalization genes-including human telomerase reverse transcriptase (hTERT), simian virus 40 large T antigen (SV40LT), acute myeloid leukemia fusion genes NUP98-KDM5A (N/K) and CBFA2T3-GLIS2 (C/G), as well as the proto-oncogene KRAS were precisely inserted into the tetracycline (Tet)-inducible eukaryotic expression lentiviral vector pLV2-TRE3GS-EGFP-MCS-3×FLAG-hPGK-Tet-On-SV40-Neo and the transposon PB-TRE3G-3×FLAG-T2A-Puro-SV40-PA. Lentiviral packaging, cell transfection, mRNA expression analysis, Western blotting for protein detection, green fluorescent protein (GFP) visualization, and cell proliferation assays were conducted to evaluate transfection efficiency and assess the regulatory effects of Tet on gene expression in 293T and MEF cells. [Results] The Tet-inducible lentiviral vectors pLV2-Tet-SV40LT, pLV2-Tet-N/K, and pLV2-Tet-C/G, along with the transposon vectors PB-Tet-hTERT, PB-Tet-SV40LT, PB-Tet-N/K, PB-Tet-C/G, and PB-Tet-KRAS, were successfully constructed. In 293T cells, the expression levels of all target genes were upregulated after transfection. In MEF cells, the immortalizing functions of SV40LT and N/K were validated. By modulating Tet addition, cell proliferation levels were effectively regulated, leading to the successful establishment of conditionally immortalized pLV2-SV40LT-MEF and pLV2-N/K-MEF cell lines. [Conclusion] The construction of Tet-inducible immortalizing gene vectors provides a technical foundation for establishing conditionally immortalized primary cell lines, thereby facilitating research on the large-scale in vitro production and expansion of blood cells, such as erythrocytes and platelets.

BACKGROUND:De novo adipose regeneration induced by small extracellular vesicles has become a promising method for repairing soft tissue defects.However,due to different animal models and small extracellular vesicle application dosages,it is difficult to quantitatively compare the therapeutic effect of small extracellular vesicles from various sources on adipose regeneration. OBJECTIVE:To compare the regenerative effects of small extracellular vesicles derived from stem cells and small extracellular vesicles from tissue. METHODS:Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation.The particle number,particle size,morphology,and protein expression of small extracellular vesicles were identified by nanoparticle tracking analysis,transmission electron microscopy and western blot assay.A quantitative and evaluative subcutaneous model for adipose regeneration in C57 mice was established using a customized silicone tube.The regenerative effects of induced de novo adipose were compared by cell counting and hematoxylin-eosin staining. RESULTS AND CONCLUSION:(1)Small extracellular vesicles derived from adipose-derived stem cells and from adipose tissue were isolated by ultracentrifugation.Both small extracellular vesicles were round-shape in transmission electron microscopy with particle size between 50-200 nm,and abundant with the small extracellular vesicles marker protein CD81,CD63 and TSG101.(2)An equal number of small extracellular vesicles were mixed with matrigel in customized silicone tubes,implanted subcutaneously in the back of mice to establish a cell-free and quantifiable adipose regeneration model.(3)On days 3 and 7 after implantation,the results of cell counting and hematoxylin-eosin staining showed that both small extracellular vesicle groups recruited more host cells than the blank group,and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group.(4)4 weeks after implantation,hematoxylin-eosin staining of the contents in silicone tubes showed that small extracellular vesicles induced de novo adipose regeneration in vivo,and the small extracellular vesicles derived from adipose tissue group were superior to the small extracellular vesicles derived from adipose-derived stem cell group.The above results indicated that small extracellular vesicles derived from tissues have a superior effect on inducing de novo adipose regeneration compared to small extracellular vesicles derived from stem cells.

AIM: To explore the current status, research hotspots, and trends of global uveitis research to provide a theoretical basis and references for researchers in the field of uveitis, and promote further development in this area.METHODS: Relevant literatures on uveitis were retrieved from the China National Knowledge Infrastructure(CNKI)database, Wanfang database, and Web of Science core collection database since their establishment until 24 August 2023. The country/publishing institutions, research authors, high-frequency keywords, and burst keywords were visual analyzed by using software such as GraphPad Prism 9, CiteSpace 6.2. R2, and VOSviewer.RESULTS: Research teams for uveitis have been formed in various countries globally. The top three countries in terms of publications are the United States of America(7 585 papers), the United Kingdom(2 412 papers)and Germany(1 679 papers). The top three foreign institutions in terms of publications are Harvard University, Oregon Health & Science University, and Moorfields Eye Hospital, while the top three domestic institutions are Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, Chongqing Medical University, and Zhongshan Ophthalmic Center, Sun Yat-sen University. The analysis of high-frequency keywords and burst keywords in Chinese and English shows that research hotspots mainly focus on exploring pathogenesis and different treatment methods for uveitis. The research hotspots related to uveitis treatment are transitioning to molecular biology-related research topics, such as molecular biological signaling pathways(NF-κB signaling pathway with a strength value of 22.89), biological agents(adalimumab with a strength value of 32.21), and tumor necrosis factor(with a strength value of 48.44). Related research is also expanding to basic experiments on relevant rats.CONCLUSIONS: In recent years, the research hotspots and trends of global uveitis mainly focus on precise diagnosis, pathogenesis, and more effective treatment methods. It is important for more scholars to dedicate themselves to uveitis-related research in the future to make breakthroughs and progress in the field. More large-scale and multicenter clinical studies on uveitis can provide high-quality research evidence.

Objective:To report the results of national surveillance on the distribution and antimicrobial resistance profile of clinical Gram-negative bacteria isolates from bloodstream infections in China in 2022.Methods:The clinical isolates of Gram-negative bacteria from blood cultures in member hospitals of national bloodstream infection Bacterial Resistant Investigation Collaborative System（BRICS）were collected during January 2022 to December 2022. Antibiotic susceptibility tests were conducted by agar dilution or broth dilution methods recommended by Clinical and Laboratory Standards Institute（CLSI）. WHONET 5.6 and SPSS 25.0 software were used to analyze the data.Results:During the study period，9 035 strains of Gram-negative bacteria were collected from 51 hospitals，of which 7 895（87.4%）were Enterobacteriaceae and 1 140（12.6%）were non-fermenting bacteria. The top 5 bacterial species were Escherichia coli（ n=4 510，49.9%）， Klebsiella pneumoniae（ n=2 340，25.9%）， Pseudomonas aeruginosa（ n=534，5.9%）， Acinetobacter baumannii complex（ n=405，4.5%）and Enterobacter cloacae（ n=327，3.6%）. The ESBLs-producing rates in Escherichia coli， Klebsiella pneumoniae and Proteus spp. were 47.1%（2 095/4 452），21.0%（427/2 033）and 41.1%（58/141），respectively. The prevalence of carbapenem-resistant Escherichia coli（CREC）and carbapenem-resistant Klebsiella pneumoniae（CRKP）were 1.3%（58/4 510）and 13.1%（307/2 340）；62.1%（36/58）and 9.8%（30/307）of CREC and CRKP were resistant to ceftazidime/avibactam combination，respectively. The prevalence of carbapenem-resistant Acinetobacter baumannii（CRAB）complex was 59.5%（241/405），while less than 5% of Acinetobacter baumannii complex was resistant to tigecycline and polymyxin B. The prevalence of carbapenem-resistant Pseudomonas aeruginosa（CRPA）was 18.4%（98/534）. There were differences in the composition ratio of Gram-negative bacteria in bloodstream infections and the prevalence of main Gram-negative bacteria resistance among different regions，with statistically significant differences in the prevalence of CRKP and CRPA（ χ2=20.489 and 20.252， P<0.001）. The prevalence of CREC，CRKP，CRPA，CRAB，ESBLs-producing Escherichia coli and Klebsiella pneumoniae were higher in provinicial hospitals than those in municipal hospitals（ χ2=11.953，81.183，10.404，5.915，12.415 and 6.459， P<0.01 or <0.05），while the prevalence of CRPA was higher in economically developed regions（per capita GDP ≥ 92 059 Yuan）than that in economically less-developed regions（per capita GDP <92 059 Yuan）（ χ2=6.240， P=0.012）. Conclusions:The proportion of Gram-negative bacteria in bloodstream infections shows an increasing trend，and Escherichia coli is ranked in the top，while the trend of CRKP decreases continuously with time. Decreasing trends are noted in ESBLs-producing Escherichia coli and Klebsiella pneumoniae. Low prevalence of carbapenem resistance in Escherichia coli and high prevalence in CRAB complex have been observed. The composition ratio and antibacterial spectrum of bloodstream infections in different regions of China are slightly different，and the proportion of main drug resistant bacteria in provincial hospitals is higher than those in municipal hospitals.

Objective To elucidate the mechanisms of interface disruption between the actin filament and membrane of the cell pseudopodium that occurs during the breakage of the pseudopodium.Methods Time-lapse images of the behavior of actin filaments and membranes during the rupture process of cell pseudopodia were captured using the confocal microscopy.A theoretical model of the fracture of a cylindrical interface was developed to analyze the interface damage between the actin filament and the membrane during the breakage of the cell pseudopodium.Molecular dynamics simulations were employed to simulate the breaking process of the cell pseudopodium for comparison with the theoretical results.A finite element model considering the coupling of the tensile and torsional deformation of actin filaments was developed to simulate the torsional deformation of actin filaments under tension,both in the presence and absence of a membrane.Results The theoretical results indicated an exponential relationship between the critical load for the broken interface and crack length.The critical load increased with the interfacial strength.The effect of the fiber diameter on the critical load depended on the crack length,exhibiting different effects for small and large crack lengths.Finite element analysis suggested that the membrane substantially constrained torsional movement when the actin filament was extended.Conclusions This study revealed the breaking process of cell pseudopodia and the mechanical aspects underlying the disruption of the interface between the actin filament and the membrane.These results provide theoretical support for exploring cellular behaviors associated with pseudopodium breakage,such as the release of extracellular vesicles.

Objective:To investigate the current situation of falls in patients with amyotrophic lateral sclerosis (ALS) and analyze the influencing factors.Methods:A total of 110 ALS patients admitted to Department of Neurology of Peking University Third Hospital from February to April 2023 were selected as the research objects by the convenient sampling method. The Self-designed Patient General Data and Disease-related Questionnaire and Morse Fall Scale (MFS) were used for questionnaire investigation.Results:A total of 100 ALS patients were included, and the incidence of falls in ALS patients was 45.0% (45/100). Binomial Logistic regression analysis showed that gait abnormality, high risk of fall and abnormal muscle strength of lower extremity were risk factors for fall in ALS patients ( P<0.05) . Conclusions:Abnormal gait, high risk of falls and abnormal lower limb muscle strength are independent factors affecting falls in ALS patients, and they are important aspects in preventing and managing falls in ALS patients.

Patients who undergo sphincter-preserving surgery for rectal cancer may face intestinal dysfunction, psychological issues, and social problems. Patient self-management is of great significance for their recovery and quality of life. This paper mainly reviews the concept, evaluation tools, current situation, and intervention measures of self-management for patients after sphincter-preserving surgery for rectal cancer, in order to provide reference for clinical self-management education for those patients.

Objective:To explore the correlation between defecation disorders and diet in patients undergoing sphincter-preserving surgery for rectal cancer.Methods:This study was a cross-sectional survey. From 2021 to 2023, convenience sampling was used to select 159 patients with rectal cancer who underwent sphincter-preserving surgery at Peking University Third Hospital as participants. The General Information Questionnaire, Food Frequency Questionnaire, and Defecation Questionnaire were used for the survey.Results:The incidence of defecation disorders in 159 patients with rectal cancer after sphincter-preserving surgery was 74.2% (118/159), and the types of defecation disorders with high to low incidence were "frequent defecation (96/159, 60.4%) " "constipation (37/159, 23.3%) " "diarrhea (33/159, 20.8%) " and "fecal incontinence" (24/159, 15.1%). Diets were clustered into 9 categories (vegetables and fruits, carbohydrate staple foods, red meat foods, gas producing foods, dairy products, white meat foods, dessert foods, high-fat foods, and spicy and stimulating foods). Binomial Logistic regression analysis showed that red meat foods and gas producing foods were the influencing factors of frequent defecation ( P<0.05), red meat foods was the influencing factor of diarrhea ( P<0.05), and carbohydrate staple foods was the influencing factor of fecal incontinence ( P<0.05) . Conclusions:The incidence of defecation disorders in patients with rectal cancer after sphincter-preserving surgery is relatively high, and the intake of red meat foods, gas producing foods, and carbohydrate staple foods should be appropriately controlled. Clinical medical and nursing staff should pay close attention to the diet of elderly patients.

Objective:To explore the incidence of low anterior resection syndrome (LARS) in patients undergoing sphincter-preserving surgery for rectal cancer and analyze related factors.Methods:This study was a cross-sectional survey. From 2021 to 2023, convenience sampling was used to select 195 patients with rectal cancer who underwent sphincter-preserving surgery at Peking University Third Hospital as participants. The patients were surveyed using the General Information Questionnaire, LARS Scale, Visual Analog Score, and Chinese version of Insomnia Severity Index.Results:A total of 195 questionnaires were distributed, and 156 valid questionnaires were collected, with a valid response rate of 80.00% (156/195). The incidence of LARS in 156 rectal cancer patients with sphincter-preserving surgery was 36.54% (57/156), and the incidence of severe LARS was 15.38% (24/156). Binomial Logistic regression analyses showed that gender ( OR=0.445, P=0.034) and preoperative neoadjuvant chemoradiotherapy ( OR=6.343, P<0.01) were influencing factors for the occurrence of LARS in patients with rectal cancer after sphincter-preserving surgery. Preoperative neoadjuvant chemoradiotherapy ( OR=3.322, P=0.047) and intraoperative prophylactic stoma ( OR=4.855, P=0.035) were influencing factors for severe LARS. LARS score was positively correlated with anxiety score ( r=0.238, P=0.003) and total insomnia score ( r=0.168, P=0.036) in patients with rectal cancer who underwent sphincter-preserving surgery. Conclusions:Preoperative neoadjuvant chemoradiotherapy is an independent risk factor for postoperative LARS and severe LARS in rectal cancer patients undergoing sphincter-preserving surgery. LARS is correlated with patients' anxiety and insomnia.

Objective:To investigate the types and number of symptom clusters in patients undergoing sphincter-preserving surgery for rectal cancer and explore the influencing factors of these symptom clusters.Methods:Totally 192 patients who underwent sphincter-preserving surgery for rectal cancer in the Department of General Surgery at Peking University Third Hospital between January 2021 and January 2023 were selected by convenience sampling. A general information questionnaire, Post-sphincter-preserving Surgery Symptom Questionnaire, and the International Physical Activity Questionnaire-Short Form (IPAQ-SF) were used for data collection.Results:A total of 192 questionnaires were distributed, with 159 valid questionnaires returned. Exploratory factor analysis on 18 symptoms with an incidence rate of over 10.0% identified five primary symptom clusters: the psychological symptom cluster, increased defecation cluster, fatigue-pain cluster, sleep disturbance cluster, and constipation-related cluster. Logistic regression analysis showed that postoperative duration was an influencing factor for the psychological symptom cluster ( P<0.05) ; preoperative radiotherapy and postoperative duration were influencing factors for the increased defecation cluster ( P<0.05) ; postoperative chemotherapy was an influencing factor for the fatigue-pain cluster ( P<0.05) ; and weekly sedentary time was an influencing factor for the sleep disturbance cluster ( P<0.05) . Conclusions:Patients undergoing sphincter-preserving surgery for rectal cancer experience multiple symptom clusters. Preoperative radiotherapy and prolonged sedentary behavior increase the risk of symptom clusters, and different postoperative periods are associated with varying risks for different symptom clusters. However, physical activity levels do not appear to influence the occurrence of symptom clusters. Healthcare providers should implement targeted interventions based on the symptom clusters and their influencing factors to reduce the incidence of symptoms in patients.