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.

Neuropathic pain is a chronic pain condition caused by damage or dysfunction of the nervous sys-tem.Symptoms of neuropathic pain include spontaneous pain,hyperalgesia,and sensory abnormalities.AMP-activated protein kinase(AMPK)is a key energy regulator that maintains cellular homeostasis by modulating carbohydrate and lipid metabolism.Recent studies have highlighted its role in synaptic plasticity and glial cell function,suggesting that AMPK signaling pathways contribute to the onset and progression of neurological disorders.Investigations using animal models of neuropathic pain as well as clinical research have illustrated aberrations in AMPK expression and activity at various sites along sensory conduction pathways.Treatment with AMPK agonists has been shown to alleviate pain symptoms,suggesting that dysregulation of AMPK expression/function contributes to the development and persistence of neuropathic pain.There-fore,the development of analgesics targeting AMPK has garnered considerable attention worldwide.This article provides a comprehensive review of the mechanisms by which AMPK participates in neuropathic pain,with the aim of guiding the de-velopment of related analgesic drugs.

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 explore the clinical efficacy and adverse reactions of albumin-bound paclitaxel (Nab-P) and conventional paclitaxel combined with cisplatin and concurrent radiotherapy for the treatment of patients with locally advanced esophageal squamous cell carcinoma.Methods:Forty-nine patients with locally advanced esophageal squamous cell carcinoma admitted to the First People's Hospital of Suqian from November 2016 to May 2020 were included. Of the 49 patients, 23 cases were treated with Nab-P combined with cisplatin and concurrent radiotherapy (NP group), 26 cases were treated with conventional paclitaxel combined with cisplatin and concurrent radiotherapy (TP group). All patients received 2 cycles of chemotherapy. The curative efficacy was evaluated one month after the end of radiotherapy, and the curative effect and adverse reactions of the two treatment regimens were compared.Results:The objective remission rate in NP group was 78.3% (18/23), and the disease control rate was 100.0% (23/23). The objective response rate in TP group was 61.5% (16/26), and the disease control rate was 92.3% (24/26). The objective response rate and disease control rate in NP group were higher than those in TP group, but the differences were not statistically significant (both P > 0.05). The common adverse reactions were mainly hair loss, loss of appetite, bone marrow suppression, radiation esophagitis, radiation pneumonia, malaise and myalgia. The incidence rate of grade 3-4 acute bone marrow suppression in NP group (8.7%, 2/23) was lower than that in TP group (38.5%, 10/26), and the difference was statistically significant ( χ2 = 4.35, P = 0.037). The incidence rate of myalgia in NP group (26.1%, 6/23) was lower than that in TP group (61.5%, 16/26), and the difference was statistically significant ( χ2 = 4.85, P = 0.028). Conclusions:Nab-P combined with cisplatin and concurrent radiotherapy has good efficacy in the treatment of patients with locally advanced esophageal squamous cell carcinoma, and the incidence rate of adverse reactions is lower than that of conventional paclitaxel combined with cisplatin and concurrent radiotherapy, so that the regimen is safe.

Despite the advancement of treatments, adults with relapsed/refractory (R/R) B-lineage acute lymphoblastic leukemia (B-ALL) have poor prognosis, with an expected five-year overall survival (OS) rate of 10%‒20% (Nguyen et al., 2008; Oriol et al., 2010). Extramedullary relapse of B-ALL is regarded as a high-risk factor generally associated with poor survival, occurring in about 15% to 20% of all relapsed patients (Ding et al., 2017; Sun et al., 2018). The central nervous system (CNS) and the testes are the most common sites of extramedullary relapse of B-ALL. In addition, extramedullary leukemia can appear in the skin, eyes, breasts, bones, muscles, and abdominal organs. The prognosis of relapsed extramedullary B-ALL after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is extremely poor (Spyridonidis et al., 2012; Dahlberg et al., 2019). Conventional chemotherapy or radiation is often ineffective in such patients. At present, there are no optimal treatment strategies for treating extramedullary leukemia after allo-HSCT.
Adult ; Antigens, CD19 ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunotherapy, Adoptive/adverse effects* ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy* ; Receptors, Chimeric Antigen ; Recurrence

Objective:To analyze the epidemiological and clinical characteristics of imported COVID-19 cases after SARS-CoV-2 vaccination and to provide evidence for the prevention and control of COVID-19.Methods:The imported COVID-19 cases in Chengdu as of April 15, 2021 were divided into the vaccinated group and unvaccinated group according to the history of SARS-CoV-2 vaccination. The epidemiological and clinical data of the cases were collected retrospectively, and the differences in epidemiological and clinical characteristics of the two groups were compared. Laboratory tests consisted of nucleic acid test, clinical index test, serum antibody test and lymphocyte test. Software WPS2019 was used for data management and software R 4.0.3 was used for statistical analysis.Results:A total of 75 COVID-19 cases were included in the analysis, in which 20 had received SARS-CoV-2 vaccination and only 4 with clinical symptoms, 55 patients did not receive SARS-CoV-2 vaccination, and 16 had clinical symptoms. In vaccinated group, the first injection time of vaccination ranged from July to November 2020, and 10 cases received two doses of vaccine simultaneously and 10 cases received two doses of vaccine at intervals of 14-57 days. The intervals between the completion of vaccination and the onset ranged from 87 days to 224 days. The differences in classification and clinical type between the two groups were significant. Significant differences were observed in case classification and clinical type between vaccinated group and unvaccinated group ( P<0.05). The vaccinated group had a relatively high proportion of asymptomatic infections (40.00%, 8/20), while mild infections were mainly observed in the unvaccinated group(76.36%,42/55). The differences in Ct values (ORF1ab gene and N gene) at the diagnosis were not significant between vaccinated group and unvaccinated group ( P>0.05), similar results were also observed in lymphocyte subtypes, procalcitonin and C-reactive protein level comparisons. Serum amyloid A level was higher in unvaccinated group than in vaccinated group ( P<0.05). However, the SARS-CoV-2 related serum antibody of IgM, IgG and total antibody levels were significantly higher in vaccinated group ( P<0.05). Conclusions:Risk of infection still exists with SARS-CoV-2 after vaccination, which can facilitate the production of specific serum antibody of IgM and IgG when people are exposed to the virus. It has a certain protective effect on SARS-CoV-2 infected persons. Vaccination can reduce the clinical symptoms and mitigate disease severity.

OBJECTIVE: To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (Na1.2 IQ). METHODS: The cDNA of Na1.2 IQ was constructed by PCR technique, CaM mutants CaM, CaM and CaM were constructed with Quickchange site-directed mutagenesis kit (QIAGEN). The binding of Na1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay. RESULTS: Na1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and Na1.2 IQ at [Ca]-free was greater than that at 100 nmol/L [Ca] ( < 0.05). In the absence of calcium, the binding amount of CaM wild-type to Na1.2 IQ was greater than that of its mutant, and the binding affinity of CaM to Na1.2 IQ was the weakest among the three mutants ( < 0.05). CONCLUSIONS The binding ability of CaM and CaM mutants to Na1.2 IQ is Ca-dependent. This study has revealed a new mechanism of Na1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.
Calcium ; metabolism ; Calmodulin ; genetics ; metabolism ; Mutation ; NAV1.2 Voltage-Gated Sodium Channel ; metabolism ; Protein Binding ; genetics

Objective To evaluate the clinical value of holographic image navigation in urological laparoscopic and robotic surgery.Methods The data of patients were reviewed retrospectively for whom accepted holographic image navigation laparoscopic and robotic surgery from Jan.2019 to Dec.2019 in Beijing United Family Hospital and other 18 medical centers,including 78 cases of renal tumor,2 cases of bladder cancer,2 cases of adrenal gland tumor,1 cases of renal cyst,1 case of prostate cancer,1 case of sweat gland carcinoma with lymph node metastasis,1 case of pelvic metastasis after radical cystectomy.All the patients underwent operations.In the laparoscopic surgery group,there were 27 cases of partial nephrectomy,1 case of radical prostatectomy,2 cases of radical cystectomy and 2 cases of adrenalectomy.In the da Vinci robotic surgery group of 54 cases,there were 51 cases of partial nephrectomy,1 case of retroperitoneal lymph node dissection,1 case of retroperitoneal bilateral renal cyst deroofing and 1 case of resection of pelvic metastasis.There were 41 partial nephrectomy patients with available clinical data for statistic,with a median age of 53.5 years (range 24-76),including 26 males and 15 females.The median R.E.N.A.L score was 7.8 (range 4-11).Before the operation,the engineers established the holographic image based on the contrast CT images and reports.The surgeon applied the holographic image for preoperative planning.During the operation,the navigation was achieved by real time fusing holographic images with the laparoscopic surgery images in the screen.Results All the procedures had been complete uneventfully.The holographic images helped surgeon in understanding the visual three-dimension structure and relation of vessels supplying tumor or resection tissue,lymph nodes and nerves.By manipulating the holographic images extracorporeally,the fused image guide surgeons about location vessel,lymph node and other important structure and then facilitate the delicate dissection.For the 41 cases with available clinical data including 23 cases of robotic-assisted partial nephrectomy and 18 cases of laparoscopic nephrectomy,the median operation time was 140 (range 50-225) min,the median warm ischemia time was 23 (range 14-60) min,the median blood loss was 80(range 5-1 200) ml.In the robotic surgery group,the median operation time was 140 (range 50-215)min,the median warm i schemia time was 21 (range 17-40)min,the median blood loss was 150(range 30-1 200)ml.In the laparoscopic surgery group,the median operation time was 160(range 80-225)min,the median warm ischemia time was 25 (range 14-60)min,the median blood loss was 50 (range 5-1 200) ml.All the patients had no adjacent organ injury during operation.There were 2 cases with Clavien Ⅱ complications.One required transfusion and the other one suffered hematoma post-operation.However,the tumors were located in the renal hilus for these 2 cases and the R.E.N.A.L scores were both 11.Conclusions Holographic image navigation can help location and recognize important anatomic structures during the surgical procedures..This technique will reduce the tissue injury,decrease the complications and improve the success rate of surgery.

OBJECTIVE: To establish a method of determination of difluorochloromethane in workplace air by direct sampling gas chromatography. METHODS: Difluorochloromethane in workplace air was collected with aluminum plastic composite film airbag and then directly injected. Difluorochloromethane was separated by DB-1 capillary column and detected with hydrogen flame ionization detector. RESULTS: Good linearity was obtained in the range of 1.50-15 042.90 mg/m~3 with the correlation coefficient of 0.999 96. The limit of detection was 1.26 mg/m~3 and the limit of quantitation was 3.78 mg/m~3. The minimum detection concentration was 1.26 mg/m~3 and the minimum quantification concentration was 3.78 mg/m~3. The standard recovery was 98.34%-99.19%. The within-run relative standard deviation(RSD) and the between-run RSD were 1.82%-3.72% and 2.17%-4.17%, respectively. The samples can be stored at room temperature for at least 7 days. CONCLUSION: This method is suitable for difluorochloromethane detection in workplace air.

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.