Protein liquid-liquid phase separation (LLPS), a pivotal phenomenon intricately linked to cellular processes, is regulated by various other proteins. However, there is still a lack of high-throughput methods for screening protein regulators of LLPS in target proteins. Here, we developed a CRISPR/Cas9-based screening method to identify protein phase separation regulators by integrating bimolecular fluorescence complementation (BiFC) and fluorescence-activated cell sorting (FACS). Using this newly developed method, we screened the RNA-binding proteins that regulate PABPN1 phase separation and identified the tumor suppressor QKI as a promoter of PABPN1 phase separation. Furthermore, QKI exhibits decreased expression levels and diminished nuclear localization in colorectal cancer cells, resulting in reduced PABPN1 phase separation, which, in turn, promotes alternative polyadenylation (APA), cell proliferation, and migration in colorectal cancer.
Humans ; Colorectal Neoplasms/genetics* ; RNA-Binding Proteins/genetics* ; Poly(A)-Binding Protein I/genetics* ; CRISPR-Cas Systems ; Flow Cytometry ; Cell Proliferation ; Cell Line, Tumor ; Cell Movement

Sustained inflammatory responses are closely related to various severe diseases, and inhibiting the excessive activation of inflammasomes and pyroptosis has significant implications for clinical treatment. Natural products have garnered considerable concern for the treatment of inflammation. Huanglian-Wumei decoction (HLWMD) is a classic prescription used for treating inflammatory diseases, but the necessity of their combination and the exact underlying anti-inflammatory mechanism have not yet been elucidated. Inspired by the supramolecular self-assembly strategy and natural drug compatibility theory, we successfully obtained berberine (BBR)-chlorogenic acid (CGA) supramolecular (BCS), which is an herbal pair from HLWMD. Using a series of characterization methods, we confirmed the self-assembly mechanism of BCS. BBR and CGA were self-assembled and stacked into amphiphilic spherical supramolecules in a 2:1 molar ratio, driven by electrostatic interactions, hydrophobic interactions, and π-π stacking; the hydrophilic fragments of CGA were outside, and the hydrophobic fragments of BBR were inside. This stacking pattern significantly improved the anti-inflammatory performance of BCS compared with that of single free molecules. Compared with free molecules, BCS significantly attenuated the release of multiple inflammatory mediators and lipopolysaccharide (LPS)-induced pyroptosis. Its anti-inflammatory mechanism is closely related to the inhibition of intracellular nuclear factor-kappaB (NF-κB) p65 phosphorylation and the noncanonical pyroptosis signalling pathway mediated by caspase-11.

Ketamine， a non-competitive N-methyl-D-aspartate （NMDA） receptor antagonist， has gained increasing attention for its rapid antidepressant effects in treatment-resistant depression （TRD） and suicidal ideation. Its mechanisms include NMDA receptor blockade， AMPA receptor activation， and upregulation of BDNF/mTOR signaling. Its enantiomers， esketamine and R-ketamine， as well as its metabolites， exhibit differences in efficacy and mechanisms. Intranasal esketamine has been approved in the Europe， United States， and China for TRD， demonstrating rapid efficacy in alleviating depressive symptoms and acute suicidal ideation. In contrast， R-ketamine demonstrates potential for longer-lasting effects and a improved safety profile. This review summarizes recent advances in the pharmacology， pharmacokinetics， and clinical applications of ketamine and its enantiomers in depressive disorders， bipolar disorder， substance use disorders， post-traumatic stress disorder and obsessive-compulsive disorder. Despite ketamine's multifaceted therapeutic potential， safety concerns such as dissociation， cognitive impairment， and abuse liability warrant continued attention.［Funded by Key Project of Natural Science Research for Universities of Anhui Provincial Education Department （number， 2023AH050647）； Anhui Provincial Department of Education Outstanding Young Talent Support Program Project 2022 （number， gxyqZD2022022）］

BACKGROUND:Eucommia ulmoides has a certain osteogenic effect,which can promote the proliferation and differentiation of osteoblasts.However,it is unclear whether Eucommia ulmoides has effects on alveolar bone formation and Wnt/β-Catenin signaling pathway. OBJECTIVE:To investigate the mechanism by which Eucommia ulmoides promotes alveolar bone formation in ovariectomized rats based on the Wnt/β-Catenin signaling pathway. METHODS:Sixty female Sprague-Dawley rats were selected and randomly divided into five groups:blank control group,sham-operation group,model group,low-dose group Eucommia ulmoides group,and high-dose Eucommia ulmoides group,with twelve rats in each group.Osteoporosis animal models were constructed by bilateral oophorectomy in the model group and the low-dose and high-dose Eucommia ulmoides groups.The sham-operation group underwent the same method to remove adipose tissue of equal mass around the bilateral ovaries.Three months after surgery,the low-and high-dose Eucommia ulmoides groups were given 2.1 g/kg/d and 4.2 g/kg/d Eucommia ulmoides by gavage,respectively.The sham-operation group and model group were given the same amount of physiological saline by gavage.After 12 weeks of drug intervention,the changes in alveolar bone mass of rats in each group were observed through Micro-CT;hematoxylin-eosin staining was used to observe the pathological structural changes of alveolar bone in rats;enzyme linked immunosorbent assay was used to detect the expression levels of alkaline phosphatase and osteocalcin in the serum of rats;western blot was used to detect the expression levels of β-Catenin and Frizzled9 receptor proteins in the alveolar bone of rats;and real-time fluorescence quantitative PCR was used to detect the expression of osteocalcin,Runt-related transcription factor 2(Runx2),alkaline phosphatase,β-catenin,and frizzled9 mRNAs in alveolar bone tissues of rats. RESULTS AND CONCLUSION:Compared with the blank control group,bone volume fraction,trabecular number,trabecular thickness,and bone mineral density were reduced in the model group(P＜0.05),and trabecular separation was elevated(P＜0.05).Pathological observation showed that the arrangement of trabeculae was disordered and irregular,the trabeculae were thinned or broken,and the marrow cavity was enlarged in the model group,with a significant reduction in bone volume;the level of alkaline phosphatase in the serum was increased(P＜0.05),and the level of osteocalcin was decreased(P＜0.05);mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were decreased(P＜0.05);protein expression of β-Catenin and Frizzled9 was decreased(P＜0.05).Compared with the model group,the low-and high-dose Eucommia ulmoides groups showed an increase in bone volume fraction,trabecular number,trabecular thickness,and bone mineral density(P＜0.05)and a decrease in trabecular separation(P＜0.05).In the low-and high-dose Eucommia ulmoides groups,bone trabeculae were slightly aligned and thickened,with a significant increase in bone mass.Compared with the model group,the serum level of alkaline phosphatase was reduced(P＜0.05)and the serum level of osteocalcin was elevated(P＜0.05)in the low-and high-dose Eucommia ulmoides groups.Compared with the model group,the mRNA expression of alkaline phosphatase,osteocalcin,Runx2,β-catenin,and frizzled9 were increased in the low-and high-dose Eucommia ulmoides groups(P＜0.05).Compared with the model group,the protein expression of Frizzled9 was increased in the low-dose Eucommia ulmoides group(P＜0.05),while the protein expression of β-Catenin and Frizzled9 was increased in the high-dose Eucommia ulmoides group(P＜0.05).Compared with the low-dose Eucommia ulmoides group,the high-dose Eucommia ulmoides group had a more significant improvement in the above indexes.To conclude,Eucommia ulmoides can effectively promote the alveolar bone formation,and its mechanism of action might be related to the activation of the Wnt/β-catenin signaling pathway.

ObjectiveTo investigate the effect of Homebox A6 （HOXA6） on the proliferation， invasion， metastasis， and apoptosis of HepG2 hepatoma cells and its association with the PI3K/AKT signaling pathway. MethodsHepG2 hepatoma cells were cultured， and HOXA6 overexpression plasmid and siRNA were constructed and transfected into cells. The cells were randomly divided into empty plasmid group， HOXA 6 overexpression group， siRNA negative control group， and siRNA HOXA6 interference group. CCK8 assay was used to measure cell proliferation， Transwell assay was used to observe cell invasion， and wound healing assay was used to observe cell migration （related proteins TIMP3， MMP9， and MMP3）. Flow cytometry was used to measure cell apoptosis （related proteins BAX and BCL2）， the BCA method was used to measure protein concentration， and Western Blot was used to measure the expression of related proteins. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the SNK-q test was used for further comparison between two groups. ResultsCompared with the empty plasmid group， HOXA6 overexpression significantly promoted the proliferation， invasion， and migration of HepG2 hepatoma cells （all P<0.001）， and there was a significant reduction in the protein expression of TIMP3 （P<0.001）， while there were significant increases in the expression levels of MMP9 and MMP3 （both P<0.001）. Compared with the siRNA negative control group， HOXA6 interference significantly inhibited the proliferation， invasion， and migration of HepG2 hepatoma cells （all P<0.001）， and there was a significant increase in the protein expression of TIMP3 （P<0.001）， while there were significant reductions in the expression levels of MMP9 and MMP3 （both P<0.001）. Flow cytometry showed that compared with the empty plasmid group， HOXA6 overexpression inhibited the apoptosis of HepG2 hepatoma cells （P<0.001）， with a significant reduction in the expression of the apoptosis-related protein BAX and a significant increase in the expression of BCL2 （both P<0.001）. Compared with siRNA negative control group， HOXA6 interference promoted the apoptosis of HepG2 hepatoma cells （P<0.001）， with a significant increase in the expression of BAX and a significant reduction in the expression of BCL2 （both P<0.001）. Compared with the empty plasmid group， the HOXA6 overexpression group had significantly higher ratios of p-AKT/AKT and p-PI3K/PI3K （both P<0.001）， and compared with the siRNA negative control group， the siRNA HOXA6 interference group had significantly lower ratios of p-AKT/AKT and p-PI3K/PI3K （both P<0.001）. ConclusionHOXA6 can promote the proliferation， invasion， and metastasis of HepG2 hepatoma cells and inhibit their apoptosis by activating the PI3K/AKT signaling pathway through phosphorylation.

Sustained inflammatory responses are closely related to various severe diseases,and inhibiting the excessive activation of inflammasomes and pyroptosis has significant implications for clinical treatment.Natural products have garnered considerable concern for the treatment of inflammation.Huanglian-Wumei decoction(HLWMD)is a classic prescription used for treating inflammatory diseases,but the necessity of their combination and the exact underlying anti-inflammatory mechanism have not yet been elucidated.Inspired by the supramolecular self-assembly strategy and natural drug compatibility theory,we successfully obtained berberine(BBR)-chlorogenic acid(CGA)supramolecular(BCS),which is an herbal pair from HLWMD.Using a series of characterization methods,we confirmed the self-assembly mechanism of BCS.BBR and CGA were self-assembled and stacked into amphiphilic spherical supra-molecules in a 2:1 molar ratio,driven by electrostatic interactions,hydrophobic interactions,and π-πstacking;the hydrophilic fragments of CGA were outside,and the hydrophobic fragments of BBR were inside.This stacking pattern significantly improved the anti-inflammatory performance of BCS compared with that of single free molecules.Compared with free molecules,BCS significantly attenuated the release of multiple inflammatory mediators and lipopolysaccharide(LPS)-induced pyroptosis.Its anti-inflammatory mechanism is closely related to the inhibition of intracellular nuclear factor-kappaB(NF-κB)p65 phosphorylation and the noncanonical pyroptosis signalling pathway mediated by caspase-11.

Objective:To analyze the present status and problems for management services in a public hospital, and explore the strategies to enhance the performance of hospital management services drawing on the experience of the reforms to streamline administration and delegate power, improve regulation, and upgrade services in the public service sector.Methods:In June 2023, a convenience sampling method was employed to select staff from all clinical departments, medical technology departments, and administrative units within a tertiary public hospital as survey participants. An anonymous online questionnaire was conducted to assess overall satisfaction with hospital management services, satisfaction with the " one-stop" service windows, satisfaction with individual administrative departments, and to gather suggestions for improving hospital management services. The Likert 5-level scoring method was used to evaluate the satisfaction. Suggestions and opinions were categorized and analyzed based on the three dimensions of the reforms to streamline administration and delegate power, improve regulation, and upgrade services.Results:A total of 962 valid questionnaires were collected. The overall satisfaction score for hospital management service was 4.40±0.89 points, with interdepartmental collaboration scoring the lowest score at 4.26±0.99 points. Information management, outpatient services, and research management processes were identified as areas requiring optimization, each mentioned over 100 times. Excessive and overly frequent inspection and evaluations, as well as documentation requirements were the most prominent manifestations of formalism and bureaucracy within administrative units. The average satisfaction score for the " one-stop" service windows was 4.59±0.68 points, and unclear service guidelines/processes and incomplete information provided by window staff were the primary causes of multiple return visits. The average satisfaction score of 32 administrative units was 4.53±0.09 points. A total of 322 valid suggestions were collected, with 13.35% concerning " streamline administration and delegate power", 28.58% addressing " improve regulation", and 58.07% focused on " upgrade services" .Conclusions:Staff at the hospital expressed overall satisfaction with the institution′s management services, but noted areas for improvement in departmental coordination, management of operational processes, and the implementation of the " one-stop service" initiative, and so on. Hospitals should optimize the following aspects: improving " upgrade services" as the primary task and important approach, focusing on building a service-oriented management culture; adhering to the " improve regulation", establishing and improving the modern hospital management system; deepening the " streamline administration and delegate power", transforming management styles and simplifying approval interventions.

ObjectiveThe U6 promoter is an essential element for driving sgRNA expression in the clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein 9（CRISPR/Cas9）gene editing system in dicotyledonous plants. Endogenous U6 promoters typically exhibit higher transcriptional activity， which can significantly improve gene editing efficiency. This study aims to identify endogenous U6 promoters in Artemisia annua to optimize its CRISPR/Cas9 gene editing system， which holds significant importance for its molecular breeding. MethodsOn the basis of the highly conserved U6 snRNA sequences in Arabidopsis thaliana， endogenous U6 promoters were screened in the A. annua genome. Expression vectors were constructed with candidate AaU6 promoter driving the firefly luciferase （LUC） reporter gene， and then transiently transformed into Nicotiana benthamiana. Transcriptional activities of the promoters were measured and compared by in vivo imaging and the Dual Luciferase Reporter assay. ResultsEight endogenous U6 promoters were successfully cloned from A. annua. Sequences alignment revealed that all these promoters contained the two conserved cis-acting elements， upstream sequence element （USE） and TATA-box， which affected their transcriptional activity. Dual-luciferase activity assays indicated that the transcriptional activities of AaU6-3， AaU6-1， and AaU6-5 were significantly higher than that of the Arabidopsis AtU6-26 promoter， with AaU6-3 exhibiting the highest activity. ConclusionThis study identified three endogenous AaU6 promoters with high transcriptional activity in A. annua， providing key functional elements for establishing an efficient gene editing system in A. annua. These findings will contribute to advancing precision molecular breeding and high-quality germplasm innovation in A. annua.

Objective:To conduct a scoping review of risk prediction models for the development of intraoperative hypothermia in patients undergoing cancer surgery to inform clinical nursing practice and future research.Methods:Relevant literature on constructing or validating intraoperative hypothermia risk prediction models for cancer surgery patients in five foreign language databases (PubMed, Embase, Web of Science, Cochrane Library, CINAHL) and four Chinese language databases (China National Knowledge Infrastructure, Wanfang, VIP, Chinese Biomedical Database) were searched from the time of library construction to June 1, 2024, extracted information on the applicable target, incidence of intraoperative hypothermia, methodology of model construction, predictors and performance, etc. The Prediction model Risk Of Bias Assessment Tool was used to evaluate the risk of bias of the studies, and the included literature was analyzed and discussed.Results:A total of 15 pieces of literature involving 18 models were included, with the study population focussing on patients undergoing surgery for colorectal cancer. The rate of intraoperative hypothermia ranged from 15.14% to 61.5%. Model construction methods included 2 types of Logistic regression models and machine learning, and model presentation was based on column-line plots. There were 8 predictors that appeared with a frequency of ≥5, including age, body mass index, operation time, anaesthesia time, operating room temperature, intraoperative rehydration volume, intraoperative bleeding volume, and heat preservation method.Conclusions:The performance of the included model was good, but the risk of bias was high for the predictors and the analysis part, and nursing staff should pay close attention to the risk factors of intraoperative hypothermia in patients undergoing cancer surgery, construct a risk prediction model with low bias and high applicability, and validate and improve the existing risk prediction model.

Objective:To investigate the death risk of gastric cancer patients with different primary sites.Methods:The data of 35 263 gastric cancer patients from 2004 to 2015 were extracted from of the National Cancer Institute the Surveillance, Epidemiology, and End Results (SEER) database. According to the recorded causes of death, the treatment outcomes were classified into 3 categories: death from gastric cancer, death from non-gastric cancer and others. All included patients were grouped by age, gender, race, region, and marital status. Statistical analysis was conducted by using R 4.2.1 software to compare the composition of patients with different treatment outcomes at 3-year, 5-year, and 10-year in each factor subgroup. Univariate Fine-Gray competing model was used to analyze the cumulative incidence of death at 3-year, 5-year, and 10-year in gastric cancer patients with different primary sites. The 5 factors mentioned above were included in the multivariate Fine-Gray competing model to analyze the factors influencing the risk of death from gastric cancer in the entire population at 3-year, 5-year, and 10-year and in gastric cancer patients with different primary sites for 10 years in each factor subgroup after adjusting for demographic differences.Results:Among the entire population, there were 13 392 cases of cardia, 2 198 cases of gastric fundus, 4 510 cases of gastric body, 8 394 cases of antrum, 1 154 cases of pylorus, 3 633 cases of lesser curvature, and 1,982 cases of greater curvature. There were statistically significant differences in the composition of 3-year, 5-year, and 10-year treatment outcomes including death from gastric cancer, non-gastric cancer and other outcomes of gastric cancer patients stratified by different age, gender, race, region, marital status, and primary sites of tumors among subgroups (all P < 0.001). Univariate Fine-Gray model analysis showed that the cumulative incidence of death from gastric cancer was 29.0%, 30.9% and 31.6%, respectively at 3-year,5-year and 10-year after the confirmed diagnosis in gastric cancer patients with primary sites in the cardia, which was all lower than that in those with primary site in the gastric fundus (44.5%, 46.8%, 47.7%), the gastric body (49.1%, 46.8%, 53.5%), the antrum (51.4%, 54.7%, 56.1%), the pylorus (53.6%, 57.8%, 59.8%), the lesser curvature (44.4%, 48.4%, 50.0%), and the greater curvature (42.4%, 45.0%, 46.4%). Multivariate Fine-Gray model analysis showed that the 3-year, 5-year, and 10-year mortality risks of gastric cancer patients with the primary site in the cardia were all lower than those of patients with the primary sites in other locations (all HR > 1, P < 0.001); taking the 10-year death from gastric cancer as an example, the death risks of gastric cancer patients with the primary site in the fundus ( HR = 1.74, 95% CI: 1.62-1.86), gastric body ( HR = 2.03, 95% CI: 1.93-2.14), gastric antrum ( HR = 2.13, 95% CI: 2.04-2.23), pylorus ( HR = 2.28, 95% CI: 2.11-2.47), lesser curvature ( HR = 1.76, 95% CI: 1.67-1.86), and greater curvature ( HR = 1.64, 95% CI: 1.53-1.76) were all higher than those of patients with primary site in the cardia (all P < 0.001). The results of subgroup multivariate Fine-Gray model analysis showed that there were no statistically significant differences in the 10-year death risk of gastric cancer between gastric cancer patients with other primary sites and patients with primary site in the cardia in the age group under 30 years (gastric fundus, gastric body, gastric antrum, lesser curvature, greater curvature), the black group (gastric fundus and lesser curvature) and other races group (gastric fundus, greater curvature and lesser curvature)(all P > 0.05); the results of other subgroups were the same as those of the entire population, namely, the 10-year risk of death from gastric cancer in patients with primary site in the cardia was lower than that in patients without primary site in the cardia (all HR > 1, P < 0.05). Conclusions:In SEER database, the patients with primary site in the cardia has a lower risk of death from gastric cancer compared to those with other primary sites.