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

Objective:To analyze the levels of serum soluble intercellular adhesion molecule-1(sICAM-1),soluble vascular cell adhesion molecule-1(sVCAM-1),and superoxide dismutase(SOD)activity in the patients with maintenance hemodialysis(MHD),and to discuss their relationships with coronary artery calcification(CAC).Methods:The clinical materials from 102 MHD patients(MHD group)were retrospectively analyzed.Additionally,74 volunteers underwent routine health examination at the same time(health examination group)were selected.The CAC scores(CACs)of the patients in MHD group were detected by multi-slice computed tomography(MSCT),and the patients were categorized into non-calcification group,mild calcification group,moderate calcification group,and severe calcification group.The general data and serum levels of sICAM-1,sVCAM-1,and SOD activities of the subjects in two groups were compared.The levels of calcium(Ca),phosphorus(P),parathyroid hormone(PTH),sICAM-1,sVCAM-1,and SOD activities in serum of the patients with different degrees of calcification were analyzed.Pearson's correlation analysis was used to analyze the correlations between the levels of sICAM-1,sVCAM-1,and SOD activity in serum of the MHD patients and CACs.Results:Compared with health examination group,the levels of sICAM-1 and sVCAM-1 in serum of the patients in MHD group were significantly increased(P<0.01),and the SOD activity was significantly decreased(P<0.01).Compared with non-calcification group,the levels of PTH,sICAM-1,and sVCAM-1 in serum of the MHD patients in mild,moderate,and severe calcification groups were significantly increased(P<0.05),and the SOD activities were significantly decreased(P<0.05);the levels of P in serum of the MHD patients in moderate and severe calcification groups were significantly increased(P<0.05).Compared with mild calcification group,the levels of P,PTH,sICAM-1,and sVCAM-1 in serum of the MHD patients in moderate and severe calcification groups were significantly increased(P<0.05),and the SOD activities significantly decreased(P<0.05).Compared with moderate calcification group,the levels of sICAM-1,sVCAM-1,and P in serum of the MHD patients in severe calcification group were significantly increased(P<0.05),and the SOD activity was significantly decreased(P<0.05).The SOD activity in serum of the MHD patients was negatively correlated with CACs(r=-0.484,P<0.01),while the levels of sICAM-1 and sVCAM-1 were positively correlated with CACs(r=0.441,P<0.01;r=0.561,P<0.01).Conclusion:The levels of sICAM-1 and sVCAM-1 and activity of SOD in serum of the MHD patients are abnormal.With the decreasing of the SOD activity and increasing of the levels of sICAM-1 and sVCAM-1,the degree of CAC in the MHD patients is aggravated.

Objective:To clarify the transitional components in the blood of compound Banlangen Granules; To explore the mechanism of drugs in the treatment of epidemic encephalitis B, hepatitis and parotitis.Methods:The transitional components in blood of compound Banlangen Granules were analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). The regulatory targets and pathways of compound Banlangen Granules in the treatment of epidemic encephalitis B, hepatitis and parotitis were analyzed based on UPLC-MS/MS and network pharmacology.Results:A total of 9 blood components were identified, of which 8 were prototype components, including sucrose, o-aminobenzoic acid, uridine, adenosine, guanosine, indole-3-acetonitrile-2 murine-S-β-D-glucopyranoside and salicylic acid. Through network pharmacological analysis, it was concluded that compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.Conclusion:The 9 blood components of compound Banlangen Granules may treat epidemic encephalitis B, hepatitis and parotitis by regulating lipid and atherosclerosis, insulin resistance, IL-17 and other signal pathways.

Objective:To investigate the imaging quality of CBCT under different exposure parameters and the relationship between subjective and objective evaluation.Methods:CBCT scanners of 6 different suppliers[3D Accuitomo(Morita),i-mCAT(Kavo),5G(NewTom),Smart3D(Beijing Langshi),DCT Pro(Vatech),Vgi(NewTom)]were used to scan the spatial resolution models and head simulators under typical exposure conditions(different device with different tube voltage and current for exposure).The visibility of SedentexCT IQ phantom and anatomical structures generated by different CBCT scanners were compared.For objective evaluation,7 professional evaluators scored CBCT imaging qualities.For subjective evaluation,the resolution(LP/mm)of the images were com-pared among the devices.Results:No significant difference was found by 7 evaluators either intra or inter of the teams.By objective e-valuation,the score of NewTom 5G was 2,that of i-CAT was 5 and that of the others was 4.By subjective evaluation,the LP/mm of i-CAT was 1.8,that of Smart3D was 2.0 and that of the others was 1.0-1.7.The subjective evaluation of image quality obtained un-der different tube voltages and tube currents were significantly different.Conclusion:The subjective and objective evaluations of image quality are approximately consistent.The difference of the subjective image quality may be produced by the different tube voltage and current for exposure.

Objective:To investigate the changes of left atrial structure and function in patients with apical hypertrophic cardiomyopathy (ApHCM) by three-dimensional (3D) echocardiography.Methods:From September 2020 to December 2022, 112 patients with ApHCM(ApHCM group) diagnosed at Tongji Hospital Tongji Medical College, Huazhong University of Science and Technology and 41 age- and sex-matched normal controls(control group) were finally enrolled. In 'pure’ ApHCM patients, cardiac hypertrophy was confined to the apical segment below the papillary muscle. The wall thickness of apical and intermediate segments in 'mixed’ ApHCM patients increased, but the wall thickness of apical segment was the largest. Two-dimensional(2D) and 3D volume and strain parameters of left atrium were compared between control group and ApHCM group, 'pure’ and 'mixed’ ApHCM patients.The correlations between 2D and 3D volume and strain parameters of left atrium and intraclass correlation coefficient (ICC) of those parameters were analyzed. The ROC curve was performed to determine the cutoff values of 3D left atrial volume abnormalities in all subjects. Logistics regression analysis was performed to analyze the impact factors of the left atrial enlargement in patients with ApHCM.Results:Compared with the control group, 2D left atrial maximum volume index (2D LAVimax), 2D left atrial minimum volume index (2D LAVimin), 3D left atrial maximum volume index (3D LAVimax), 3D left atrial minimum volume index (3D LAVimin), and 3D left atrial presystolic volume index (3D LAVipreA) significantly increased in ApHCM group( Z=-6.54, -6.38, -6.98, -7.40, -6.96; all P<0.001). However, 2D left atrial ejection fraction (2D LAEF) ( Z=-3.75, P<0.001), 2D left atrial expansion index (2D LAEI) ( t=-4.15, P<0.001), 3D left atrial ejection fraction (3D LAEF) ( Z=-5.09, P<0.001), 3D left atrial expansion index (3D LAEI) ( t=-5.49, P<0.001), 2D left atrial reservoir strain (2D LASr) ( t=-12.03, P<0.001), 2D left atrial conduit strain (2D LAScd) ( t=7.91, P<0.001), 2D left atrial contractile strain (2D LASct) ( t=6.06, P<0.001), 3D left atrial reservoir strain (3D LASr) ( t=-9.23, P<0.001), 3D left atrial conduit strain (3D LAScd) ( t=7.12, P<0.001) and 3D left atrial contractile strain (3D LASct) ( t=4.78, P<0.001) significantly decreased in ApHCM group. Compared with the 'pure’ ApHCM group, 2D LAVimax, 3D LAVimax, 2D LAVimin, 3D LAVimin, 3D LAVipreA in patients with mixed ApHCM increased more significantly, while 2D LAEF, 2D LAEI and 2D LASr decreased more significantly. The measurements of left atrial volume and strain by 3D echocardiography were significantly correlated with 2D measurements ( P<0.05). The correlations between 2D LAVimax and 3D LAVimax, 2D LAVimin and 3D LAVimin, 2D LAEF and 3D LAEF, 2D LASr and 3D LASr, 2D LAEI and 3D LAEI ( r=0.91, 0.93, 0.72, 0.76, 0.57; all P<0.05) were more than moderate. The repeatability of 3D left atrial strain was lower than 2D results, while the repeatability of 3D left atrial volume was higher than 2D results. ROC curve analysis showed that 3D echocardiography parameters could identify left atrial volume abnormality in all subjects. The cutoff values of 3D LAVimax, 3D LAVimin, 3D LAVipreA in all subjects were 36 ml/m 2, 18 ml/m 2 and 27 ml/m 2, respectively. Multivariate binary logistic regression analyses showed that ratio of LV systolic obliteration to cavity was independent factor affecting left atrial enlargement in ApHCM patients( OR=1.20, P<0.001). Conclusions:Three-dimensional echocardiography is significant for the accurate evaluation of left atrial structural changes in patients with ApHCM. Ratio of left ventricular systolic obliteration to cavity was an independent impact factor of left atrial enlargement in ApHCM patients.

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)-induced cytokine storms constitute the primary cause of coronavirus disease 19(COVID-19)progression,severity,criticality,and death.Gluco-corticoid and anti-cytokine therapies are frequently administered to treat COVID-19,but have limited clinical efficacy in severe and critical cases.Nevertheless,the weaknesses of these treatment modalities have prompted the development of anti-inflammatory therapy against this infection.We found that the broad-spectrum anti-inflammatory agent inosine downregulated proinflammatory interleukin(IL)-6,upregulated anti-inflammatory IL-10,and ameliorated acute inflammatory lung injury caused by mul-tiple infectious agents.Inosine significantly improved survival in mice infected with SARS-CoV-2.It indirectly impeded TANK-binding kinase 1(TBK1)phosphorylation by binding stimulator of interferon genes(STING)and glycogen synthase kinase-3β(GSK3β),inhibited the activation and nuclear trans-location of the downstream transcription factors interferon regulatory factor(IRF3)and nuclear factor kappa B(NF-κB),and downregulated IL-6 in the sera and lung tissues of mice infected with lipopoly-saccharide(LPS),H1N1,or SARS-CoV-2.Thus,inosine administration is feasible for clinical anti-inflammatory therapy against severe and critical COVID-19.Moreover,targeting TBK1 is a promising strategy for inhibiting cytokine storms and mitigating acute inflammatory lung injury induced by SARS-CoV-2 and other infectious agents.

Depressive disorder ranks as a major bur-den of disease worldwide,yet the current antidepressant medications are limited by frequent non-responsiveness and significant side effects.The lateral septum(LS)is thought to control of depression,however,the cellular and circuit substrates are largely unknown.Here,we identified a subpopulation of LS GABAergic adenosine A2A receptors(A2AR)-positive neurons mediating depres-sive symptoms via direct projects to the lateral habenula(LHb)and the dorsomedial hypothalamus(DMH).Activa-tion of A2AR in the LS augmented the spiking frequency of A2AR-positive neurons leading to a decreased activation of surrounding neurons and the bi-directional manipula-tion of LS-A2AR activity demonstrated that LS-A2ARs are necessary and sufficient to trigger depressive pheno-types.Thus,the optogenetic modulation(stimulation or inhibition)of LS-A2AR-positive neuronal activity or LS-A2AR-positive neurons projection terminals to the LHb or DMH,phenocopied depressive behaviors.Moreover,A2AR are upregulated in the LS in two male mouse mod-els of repeated stress-induced depression.This identifica-tion that aberrantly increased A2AR signaling in the LS is a critical upstream regulator of repeated stress-induced depressive-like behaviors provides a neurophysiological and circuit-based justification of the antidepressant poten-tial of A2AR antagonists,prompting their clinical transla-tion.

Medication-related osteonecrosis of the jaw (MRONJ) is primarily associated with administering antiresorptive or antiangiogenic drugs. Despite significant research on MRONJ, its pathogenesis and effective treatments are still not fully understood. Animal models can be used to simulate the pathophysiological features of MRONJ, serving as standardized in vivo experimental platforms to explore the pathogenesis and therapies of MRONJ. Rodent models exhibit excellent effectiveness and high reproducibility in mimicking human MRONJ, but classical methods cannot achieve a complete replica of the pathogenesis of MRONJ. Modified rodent models have been reported with improvements for better mimicking of MRONJ onset in clinic. This review summarizes representative classical and modified rodent models of MRONJ created through various combinations of systemic drug induction and local stimulation and discusses their effectiveness and efficiency. Currently, there is a lack of a unified assessment system for MRONJ models, which hinders a standard definition of MRONJ-like lesions in rodents. Therefore, this review comprehensively summarizes assessment systems based on published peer-review articles, including new approaches in gross observation, histological assessments, radiographic assessments, and serological assessments. This review can serve as a reference for model establishment and evaluation in future preclinical studies on MRONJ.
Animals ; Bisphosphonate-Associated Osteonecrosis of the Jaw/drug therapy* ; Bone Density Conservation Agents/adverse effects* ; Diphosphonates/therapeutic use* ; Humans ; Reproducibility of Results ; Rodentia

Adventitia ; Atherosclerosis/genetics* ; Cell Communication ; Humans ; Hyperhomocysteinemia/genetics* ; Sequence Analysis, RNA

Objective:To investigate the cardiac presentations and the possible influencing factors of severe and critical coronavirus disease 2019 (COVID-19).Methods:A retrospective study was conducted. Patients with severe and critical COVID-19 admitted to the Eighth People's Hospital of Guangzhou from January 21st to February 24th 2020 were enrolled. According to the clinical classification, the patients were divided into severe group and critical group. The myocardial injury markers, such as lactate dehydrogenase (LDH), aspartate aminotransferase (AST), creatine kinase (CK), cardiac troponin I (cTnI), myoglobin (MYO), MB isoenzyme of creatine kinase (CK-MB), B-type natriuretic peptide (BNP) and electrocardiogram (ECG) changes were compared between the two groups.Results:A total of 55 COVID-19 patients were selected, including 15 critical cases and 40 severe cases. The patients with severe and critical COVID-19 were male-dominated (61.8%), the average age was (61.2±13.0) years old, 83.6% (46 cases) of them had contact history of Hubei, 38.2% (21 cases) of them were complicated with hypertension. There was no significant difference in baseline data between the critical group and the severe group. Myocardial injury markers of critical and severe COVID-19 patients were increased in different proportion, LDH increased in most patients (20 severe cases and 7 critical cases), followed by AST (16 severe cases and 5 critical cases). There was significant difference in the number of patients with elevated CK between severe group and critical group (cases: 1 vs. 4, P = 0.027). Abnormal ECG was found in 39 of 42 patients with ECG examination. Nonspecific change of T wave was the most common. Before and after treatment, 9 of 15 patients with changes of ECG and myocardial injury markers had oxygenation index less than 100 mmHg (1 mmHg = 0.133 kPa), and the prominent changes of ECG were heart rate increasing and ST-T change. Conclusions:The increase of myocardial injury markers and abnormal ECG were not specific to the myocardial injury of severe and critical COVID-19 patients. At the same time, the dynamic changes of myocardial injury markers and ECG could reflect the situation of myocardial damage.