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

BACKGROUND:For dislocation of acromioclavicular joint induced by coracoclavicular ligament fracture,single EndoButton Plate reconstruction and double EndoButton Plates reconstruction are common repair methods.Further study on the stress distribution and fracture risk of the two repair methods is of great significance. OBJECTIVE:To study the biomechanical properties of the coracoclavicular ligament,and compare the fixation effect,stress distribution and failure mode of single and double EndoButton Plates reconstruction. METHODS:(1)Finite element simulation analysis:Mimics,Wrap and SolidWorks were used to establish normal coracoclavicular ligament,single EndoButton Plate reconstruction and double EndoButton Plates reconstruction.Ansys software was used to analyze the stress and deformation of the scapula and clavicle of each model under vertical load.(2)Sample experiment:Fifteen intact scapular-clavicle specimens were randomly grouped into five groups,with three specimens in each group.In group A,the acromioclavicular ligament was severed and the coracoclavicular ligament remained intact.In group B,acromioclavicular ligaments and trapeoid ligaments were severed,leaving intact conical ligaments.In group C,acromioclavicular ligaments and conical ligaments were cut off,and the intact traprex ligaments were retained.In group D,acromioclavicular and coracoclavicular ligaments were severed,and coracoclavicular ligaments were repaired by single EndoButton Plate reconstruction.In group E,acromioclavicular and coracoclavicular ligaments were severed,and the coracoclavicular ligaments were repaired by double EndoButton Plates reconstruction.The mechanical experiment was carried out by a mechanical testing machine to analyze the biomechanical status,stress distribution and failure patterns of the scapular-clavicle and clavicle. RESULTS AND CONCLUSION:(1)Finite element simulation analysis:The average stress of coracoclavicular ligament attached specimens was the lowest,and the risk of coracoclavicular fracture was less than that of single and double EndoButton Plates reconstruction.The mean stress of the coracoid process was similar in single and double EndoButton Plates reconstruction,and the fracture risk was similar.(2)Sample experiment:In groups A,B,C,D and E,the stiffness of specimens was(26.4±3.5),(19.8±2.8),(21.3±3.2),(57.7±4.1),and(46.2±2.8)N/mm,respectively;the ultimate loads were(545.5±53.7),(360.1±42.1),(250.9±44.4),(643.5±39.1),and(511.9±31.7)N,respectively;global stiffness in groups D and E was higher than that in group A(P=0.000 06,0.000 3);ultimate load in group D was higher than that in group A(P<0.05);the ultimate load was not significantly different between the group E and group A(P>0.05).Ligament fracture was observed in groups A,B and C and coracoid process fracture was found in groups D and E.(3)These results suggest that from the biomechanical analysis,Single EndoButton Plate reconstruction and double EndoButton Plates reconstruction are effective treatment techniques for coracoclavicular ligament fracture in acromioclavicular joint dislocation,but increase the risk of fracture.The double EndoButton Plates reconstruction dispersed the stress of the steel plate and reduced the contact force between the steel plate and bone,but slightly reduced the ultimate bearing capacity.Single and double EndoButton Plates reconstruction should be selected according to the actual clinical situation.

BACKGROUND:The stability of the pelvis is mainly determined by the posterior pelvic ring and the sacroiliac joint.The posterior pelvic ring injury and the dislocation of the sacroiliac joint caused by high energy impacts such as car accidents increase year by year.Surgical treatment is the best method,and there are many kinds of endophytorepair methods in clinical practice,but which treatment method has the best biomechanical properties is still controversial. OBJECTIVE:To compare the biomechanical properties of three kinds of internal implants:anterior double plates,posterior bridging plate and tension nail in the repair of unilateral unstable pelvic posterior ring injury,to provide a reference for the clinical treatment and development of a new pelvic tension screw. METHODS:(1)Finite element simulation:Mimics,Wrap and SolidWorks were used to establish normal pelvic model,unilateral injured pelvis model,and three kinds of internal implant repaired models(anterior double plates,posterior bridging plate and tension nail).Ansys was used to analyze the stress and deformation of the models.(2)Biomechanical test:A total of 15 intact pelvic specimens were randomly grouped into five groups,normal pelvic model,unilateral injured pelvis model,anterior double plates,posterior bridging plate and tension nail groups.The mechanical test was performed using an Instron E10000 testing machine. RESULTS AND CONCLUSION:(1)Simulation:In the normal pelvic model,the average displacement of the sacrum was 0.174 mm,and the maximum stress of the sacral iliac bone was 10.51 MPa,and the stress distribution was uniform.The mean sacral displacement of the unilateral injured pelvis model was 0.267 mm,and the stress concentration of the model was obvious.The mean displacement of the sacrum in the three repaired models was close to that in the normal pelvic model,and the stress distribution of the sacral iliac bone in the tension nail repaired model was uniform.(2)Mechanical test:The stiffness of the normal pelvic model was(226.38±4.18)N/mm,and that of the unilateral unstable pelvic model was the smallest(130.02±2.19)N/mm.The deviation of the normal pelvic model stiffness and the three repaired models'stiffness were all within(±10%),and the repair effect was obvious.(3)The simulation results were in agreement with the experimental results.(4)The biomechanics of the tension nail repaired model was the most similar to that of the normal pelvis,and this method was the best.The repairing stiffness of the anterior double plate was too large,and the stress shielding effect was more significant.The posterior bridging plate repair could not solve the compensatory effect of the normal side soft tissue and had defects.This study provides an optimal basis for clinical surgery.(5)The new type of pelvic tension nail should be improved from the point of view of the tension nail to retain the good biomechanical properties of the tension nail,while adding other advantages,such as being used for the osteoporotic pelvis.

AIM To rapidly screen xanthine oxidase(XOD)inhibitors from Smilax glabra Roxb.by enzyme-immobilized magnetic microspheres and LC-MS/MS,and to confirm the anti-uric acid constituents from S.glabra Roxb.METHODS The immobilized xanthine oxidase was prepared by covalent coupling with carboxyl magnetic beads as a carrier.The xanthine oxidase inhibitors in S.glabra were screened by the specific adsorption of immobilized enzyme.LC-MS/MS and standard substances were used for analysis and comparison,and the inhibitory activity and inhibition type of the screened and identified components were investigated.RESULTS The successful synthesis of immobilized xanthine oxidase was characterized by scanning electron microscopy and infrared spectroscopy.The enzyme loading was 70.50 μg/mg and the relative activity was 79.44%.Thirteen active compounds were screened from the extract of S.glabra,and eleven compounds were identified.The enzyme activity test showed that the inhibitory activites of engeletin and isoengeletin were the strongest,which was close to the positive control allopurinol.The IC50 value and inhibition type were 32.25 μg/mL,mixed inhibition,35.12 μg/mL,competitive inhibition.CONCLUSION The method is simple,rapid,accurate and suitable for directly screened active ingredients which can inhibit XOD from complex extract of traditional Chinese medicines.

Objective To explore the postmortem diffusion rule of Aconitum alkaloids and their metabo-lites in poisoned rabbits,and to provide a reference for identifying the antemortem poisoning or post-mortem poisoning of Aconitum alkaloids.Methods Twenty-four rabbits were sacrificed by tracheal clamps.After 1 hour,the rabbits were administered with aconitine LD50 in decocting aconite root powder by intragastric administration.Then,they were placed supine and stored at 25℃.The biological samples from 3 randomly selected rabbits were collected including heart blood,peripheral blood,urine,heart,liver,spleen,lung and kidney tissues at 0 h,4 h,8 h,12 h,24 h,48 h,72 h and 96 h after intragastric administration,respectively.Aconitum alkaloids and their metabolites in the biological samples were ana-lyzed by high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS).Results At 4 h after intragastric administration,Aconitum alkaloids and their metabolites could be detected in heart blood,peripheral blood and major organs,and the contents of them changed dynamically with the preservation time.The contents of Aconitum alkaloids and their metabolites were higher in the spleen,liver and lung,especially in the spleen which was closer to the stomach.The average mass fraction of benzoylmesaconine metabolized in rabbit spleen was the highest at 48 h after intragastric administration.In contrast,the contents of Aconitum alkaloids and their metabolites in kidney were all lower.Aconi-tum alkaloids and their metabolites were not detected in urine.Conclusion Aconitum alkaloids and their metabolites have postmortem diffusion in poisoned rabbits,diffusing from high-content organs(stomach)to other major organs and tissues as well as the heart blood.The main mechanism is the dispersion along the concentration gradient,while urine is not affected by postmortem diffusion,which can be used as the basis for the identification of antemortem and postmortem Aconitum alkaloids poisoning.

Objective To investigate the biomechanical properties of a novel sacroiliac lag screw with a spiral blade.Methods Percutaneous sacroiliac lag screws were used as the controls.Polyurethane material was used to simulate the trabecular bone,and the pullout resistance performance was tested on an Instron mechanical testing machine.Subsequently,pelvic specimens were utilized to analyze the static stiffness and dynamic stability of the novel sacroiliac lag screw in repairing sacroiliac joint injuries under normal standing conditions,with normal pelvis,single-sided sacroiliac joint injury pelvis,percutaneous sacroiliac lag screw-single screw repair,and percutaneous sacroiliac lag screw-double screw repair as controls.Results The damage to the polyurethane material after screw extraction was smaller in the novel sacroiliac lag screw group.The average effective holding displacement of the novel sacroiliac lag screw was significantly greater than that of the percutaneous sacroiliac lag screw(P＜0.05).However,the maximum resistance to the pullout force for the percutaneous sacroiliac lag screw was significantly higher than that for the novel sacroiliac lag screw(P＜0.05).The stiffness after repair of sacroiliac joint injuries was significantly higher when using a single sacroiliac lag screw than when using two percutaneous sacroiliac lag screws(P＜0.05).The displacement amplitude was the highest in the sacroiliac joint injury group,followed by that in the normal group.The displacement amplitudes in the other groups were similar;however,the differences were statistically significant(P＜0.05).The dynamic stability of the sacroiliac lag screw repair group was the best,slightly better than that of the percutaneous sacroiliac lag screw-double screw repair group,and the dynamic stability of the sacroiliac joint injury group was the worst.The novel sacroiliac lag screw effectively repaired the sacroiliac joint injuries.Conclusions The novel sacroiliac lag screw can effectively hold the trabecular bone and has practical clinical utility.

Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.

In the electroencephalography (EEG) monitoring of patients with hypoxic-ischemic encephalopathy, generalized periodic discharges are often monitored abnormal waveforms. When there are some features of generalized periodic discharges (e.g., frequency≥1.5 Hz or plus), it indicates that the patient is at high risk for seizures or has a poor prognosis. Compared with conventional EEG, the time of continuous EEG monitoring is longer, so the detection rate of these waveforms is higher. At present, scholars at home and abroad have studied these waveforms, but there is controversy about the significance of these waveforms. In this paper, the definition and characteristics of these waveforms and their significance in determining prognosis and guiding treatment in patients with hypoxic-ischemic encephalopathy are reviewed.

Aristolochic acid (AA), extracted from Aristolochiaceae plants, plays an essential role in traditional herbal medicines and is used for different diseases. However, AA has been found to be nephrotoxic and is known to cause aristolochic acid nephropathy (AAN).AA-induced acute kidney injury (AKI) is a syndrome in AAN with a high morbidity that manifests mitochondrial damage as a key part of its pathological progression. Melatonin primarily serves as a mitochondria-targeted antioxidant. However, its mitochondrial protective role in AA-induced AKI is barely reported. In this study, mice were administrated 2.5 mg/kg AA to induce AKI. Melatonin reduced the increase in Upro and Scr and attenuated the necrosis and atrophy of renal proximal tubules in mice exposed to AA. Melatonin suppressed ROS generation, MDA levels and iNOS expression and increased SOD activities in vivo and in vitro. Intriguingly, the in vivo study revealed that melatonin decreased mitochondrial fragmentation in renal proximal tubular cells and increased ATP levels in kidney tissues in response to AA. In vitro, melatonin restored the mitochondrial membrane potential (MMP) in NRK-52E and HK-2 cells and led to an elevation in ATP levels. Confocal immunofluorescence data showed that puncta containing Mito-tracker and GFP-LC3A/B were reduced, thereby impeding the mitophagy of tubular epithelial cells. Furthermore, melatonin decreased LC3A/B-II expression and increased p62 expression. The apoptosis of tubular epithelial cells induced by AA was decreased. Therefore, our findings revealed that melatonin could prevent AA-induced AKI by attenuating mitochondrial damage, which may provide a potential therapeutic method for renal AA toxicity.

Objective: To identify the risk factors in mortality of pediatric acute respiratory distress syndrome (PARDS) in pediatric intensive care unit (PICU). Methods: Second analysis of the data collected in the "efficacy of pulmonary surfactant (PS) in the treatment of children with moderate to severe PARDS" program. Retrospective case summary of the risk factors of mortality of children with moderate to severe PARDS who admitted in 14 participating tertiary PICU between December 2016 to December 2021. Differences in general condition, underlying diseases, oxygenation index, and mechanical ventilation were compared after the group was divided by survival at PICU discharge. When comparing between groups, the Mann-Whitney U test was used for measurement data, and the chi-square test was used for counting data. Receiver Operating Characteristic (ROC) curves were used to assess the accuracy of oxygen index (OI) in predicting mortality. Multivariate Logistic regression analysis was used to identify the risk factors for mortality. Results: Among 101 children with moderate to severe PARDS, 63 (62.4%) were males, 38 (37.6%) were females, aged (12±8) months. There were 23 cases in the non-survival group and 78 cases in the survival group. The combined rates of underlying diseases (52.2% (12/23) vs. 29.5% (23/78), χ²=4.04, P=0.045) and immune deficiency (30.4% (7/23) vs. 11.5% (9/78), χ²=4.76, P=0.029) in non-survival patients were significantly higher than those in survival patients, while the use of pulmonary surfactant (PS) was significantly lower (8.7% (2/23) vs. 41.0% (32/78), χ²=8.31, P=0.004). No significant differences existed in age, sex, pediatric critical illness score, etiology of PARDS, mechanical ventilation mode and fluid balance within 72 h (all P>0.05). OI on the first day (11.9(8.3, 17.1) vs.15.5(11.7, 23.0)), the second day (10.1(7.6, 16.6) vs.14.8(9.3, 26.2)) and the third day (9.2(6.6, 16.6) vs. 16.7(11.2, 31.4)) after PARDS identified were all higher in non-survival group compared to survival group (Z=-2.70, -2.52, -3.79 respectively, all P<0.05), and the improvement of OI in non-survival group was worse (0.03(-0.32, 0.31) vs. 0.32(-0.02, 0.56), Z=-2.49, P=0.013). ROC curve analysis showed that the OI on the thind day was more appropriate in predicting in-hospital mortality (area under the curve= 0.76, standard error 0.05,95%CI 0.65-0.87,P<0.001). When OI was set at 11.1, the sensitivity was 78.3% (95%CI 58.1%-90.3%), and the specificity was 60.3% (95%CI 49.2%-70.4%). Multivariate Logistic regression analysis showed that after adjusting for age, sex, pediatric critical illness score and fluid load within 72 h, no use of PS (OR=11.26, 95%CI 2.19-57.95, P=0.004), OI value on the third day (OR=7.93, 95%CI 1.51-41.69, P=0.014), and companied with immunodeficiency (OR=4.72, 95%CI 1.17-19.02, P=0.029) were independent risk factors for mortality in children with PARDS. Conclusions: The mortality of patients with moderate to severe PARDS is high, and immunodeficiency, no use of PS and OI on the third day after PARDS identified are the independent risk factors related to mortality. The OI on the third day after PARDS identified could be used to predict mortality.
Female ; Male ; Humans ; Child, Preschool ; Infant ; Child ; Critical Illness ; Pulmonary Surfactants/therapeutic use* ; Retrospective Studies ; Risk Factors ; Respiratory Distress Syndrome/therapy*