Objective To explore the molecular mechanism of N6-methyladenosine(m6A)methylation mediated by methyltransferase 3(METTL3)in regulating lipopolysaccharide(LPS)-induced endothelial cell permeability changes.Methods Human umbilical vein endothelial cells(HUVECs)were cultured in vitro.HUVECs were treated with LPS 50,125,250,500,1 000,2 000 ng/ml for 24 h.METTL3 mRNA expression was detected by Real-time PCR.After HUVECs were intervened with 500 ng/ml for 24 h,the methylation level of m6A was detec-ted,and cell permeability was measured by cell permeability test.Real-time PCR and Western blot were used to detect mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-caherin).METTL3 overexpressed stable cell lines were constructed to measure the changes of m6A methylation level and per-meability of endothelial cells during METTL3 overexpression.Results Compared to the control group,LPS inhibi-ted the expression of HUVECs METTL3 mRNA,decreased the methylation of m6A,increased the cell permeabili-ty,and decreased the mRNA and protein expression of intercellular junction proteins(Claudin-5,Occludin and VE-Caherin).When METTL3 was overexpressed,the m6A methylation levels of endothelial cells were enhanced,and the increase of endothelial cell permeability induced by LPS was reversed.Conclusion METTL3-mediated m6A methylation can improve the permeability of endothelial cells induced by sepsis.

Objective To investigate the results of ambulatory electrocardiographic(ECG)monitoring in a community-based homebound elderly population and to explore the applicability of wearable long-range ambulatory ECG monitor for them.Methods Elderly volunteers were recruited in Shuangbei Community,Shapingba District,Chongqing,from November 2021 to June 2023.A single-lead wearable ambulatory ECG recorder was applied to them to obtain ECG for 7 consecutive days.The adverse reactions,acceptability,monitoring duration,and arrhythmia detection rate during the wearing were described and recorded.Serious arrhythmic events included frequent atrial premature,atrial flutter,atrial fibrillation(AF),frequent ventricular premature,and RR intervals ≥5 s.Results There were 416 individuals enrolled,with a mean age of 71.2±6.6 years,and a male percentage of 36.1％(150 men).Finally,384(92.3％)participants completed the wearing of the ECG monitor for 7 d,with an average time of 159.2±29.4 h.There were 179 participants(48.5％)reporting no discomfort during wearing,and 175 ones(47.4％)feeling itchy at the wearing site.The monitoring results showed that the common arrhythmias were atrial premature contractions(97.1％),premature ventricular contractions(93.3％),atrial tachycardia(84.6％),bradycardia(46.6％),frequent atrial premature contractions(15.1％),ventricular tachycardia(13.2％),and long RR interval(11.8％).Among them,29.1％of the participants experienced serious arrhythmic events,and the detection rate of certain serious arrhythmic events was comparatively higher in the individuals≥70 years of age and those with history of previous cardiac disease.Conclusion The detection rate of common arrhythmias is quite high in the community-based homebound elderly population.A 7-day long-range ambulatory ECG monitoring may be appropriate.

The human Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutations in a highly conserved ribosome assembly factor SBDS. The functional role of SBDS is to cooperate with another assembly factor, elongation factor 1-like (Efl1), to promote the release of eukaryotic initiation factor 6 (eIF6) from the late-stage cytoplasmic 60S precursors. In the present work, we characterized, both biochemically and structurally, the interaction between the 60S subunit and SBDS protein (Sdo1p) from yeast. Our data show that Sdo1p interacts tightly with the mature 60S subunit in vitro through its domain I and II, and is capable of bridging two 60S subunits to form a stable 2:2 dimer. Structural analysis indicates that Sdo1p bind to the ribosomal P-site, in the proximity of uL16 and uL5, and with direct contact to H69 and H38. The dynamic nature of Sdo1p on the 60S subunit, together with its strategic binding position, suggests a surveillance role of Sdo1p in monitoring the conformational maturation of the ribosomal P-site. Altogether, our data support a conformational signal-relay cascade during late-stage 60S maturation, involving uL16, Sdo1p, and Efl1p, which interrogates the functional P-site to control the departure of the anti-association factor eIF6.
Crystallography, X-Ray ; GTP Phosphohydrolases ; chemistry ; metabolism ; Humans ; Protein Domains ; Ribosome Subunits, Large, Eukaryotic ; chemistry ; metabolism ; Saccharomyces cerevisiae ; chemistry ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; metabolism

Objective : To investigate the molecular mechanism of Toll⁃like receptor 4 ( TLR4)/Ras homologue A (RhoA) signaling pathway involved in regulating the effect of septic serum on vascular endothelial cell permeability before and after continuous hemofiltration. Methods : The serum of 5 patients with sepsis before and after continuous hemofiltration treatment was collected , and the levels of inflammatory cytokines in serum before and after hemofiltration were detected. Human umbilical vein endothelial cells (HUVEC) were treated with serum before and after continuous hemofiltration for 24 hours. The expression of VE⁃cadherin , F ⁃actin , TLR4 and RhoA in vascular endothelial cells were detected by Western blot. A TLR4 low expression cell line was constructed to detect the effect of TLR4 low expression on the expression of VE⁃cadherin , F ⁃actin and RhoA and the permeability of endothelial cells. Results : After continuous blood treatment , the serum levels of TLR4 , RhoA , interleukin⁃1 (IL⁃1) , interleukin⁃6 (IL⁃6) and tumor necrosis factor⁃α (TNF⁃α ) significantly decreased. The expression levels of VE⁃cadherin , F ⁃actin , TLR4 and RhoA in the serum intervention group after continuous hemofiltration treatment significantly decreased , and the cell permeability significantly decreased. Low expression of TLR4 significantly promoted the expression of VE⁃cadherin and F ⁃actin , and inhibited the expression of RhoA protein. Conclusion TLR4/RhoA signaling pathway is involved in the regulation of changes in vascular endothelial cell permeability induced by septic serum after continuous hemofiltration treatment.

Objective To analyze and construct systolic blood pressure(SBP)fluctuation trajectory in a community population with hypertension and to analyze the factors influencing different trajectories.Methods This is a community-based retrospective cohort study.A latent class trajectory model was used to identify and construct longitudinal trajectories of blood pressure change.Multinomial logistic regression analysis was performed to identify the associated factors of blood pressure trajectories by adjusting for different confounders.Potential confounding factors were identified using a directed acyclic graph based on a priori knowledge.Results A total of 793 patients with hypertension were enrolled in the analysis.They were divided into 3 groups by LCTM-fitted systolic blood pressure trajectories,namely stable low-level group(n=561,70.74％),declining group(n=170,21.44％)and rising group(n=62,7.82％).Significant differences were observed among the 3 trajectories groups in terms of age,frequency of exercise,ways of follow-up,salt intake,compliance behavior,and referral(P＜0.05).Compared to the stable low-level group and adjusting for corresponding confounding factors,the male patients and the patients with"outpatient follow-up"were more likely to be classified into"declining group",with OR and 95％CI of 1.436(1.016～2.030)and 1.702(1.202～2.410),respectively.The participants aged ≥ 65 years,who did not exercise or occasionally exercised,and had moderate and severe salt intake,were more likely to be classified into the"rising group"(OR=1.949,2.284,2.433,4.540,95％CI:1.145～3.317,1.305～3.998,1.272～4.654,1.291～15.963).Conclusion SBP trajectories in community-dwelling hypertensive population can be divided into stable low-level,declining and rising groups.Gender,age,salt intake,exercise frequency,and follow-up methods may be influencing factors for SBP blood pressure trajectory.

Objective: To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism. Methods: Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test. Results: After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01). Conclusions HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.