OBJECTIVES: Uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy is a progressive neurodegenerative disease associated with homozygous or compound heterozygous missense mutations in the GNE gene. This study aims to explore the impact of the homozygous p.V543M mutation in on cell phenotype and to gain preliminary insights into the underlying mechanisms. METHODS: Human embryonic kidney 293T (HEK 293T) cells were used to construct wild-type (WT-GNE) and mutant (MUT-GNE) GNE overexpression models. Western blotting and immunofluorescence were used to assess GNE protein expression levels and subcellular localization. Cell adhesion, proliferation, apoptosis, and mitochondrial membrane potential were evaluated using the cell counting kit-8 (CCK-8) assay, crystal violet staining, flow cytometry, Hoechst 33342/propidium iodide (PI) staining, and tetramethylrhodamine ethyl ester (TMRE) staining. Sialic acid synthesis levels and GNE enzymatic activity were measured, and the mRNA expression of sialic acid biosynthesis-related enzymes was quantified by real-time PCR. RESULTS: Western blotting confirmed successful establishment of GNE overexpression models. Immunofluorescence showed significantly reduced co-localization of GNE protein with Golgin-97 in the MUT-GNE group compared to WT-GNE (Pearson's correlation coefficient: 0.65±0.08 vs 0.83±0.06, P<0.05). Compared with WT-GNE, cells in the MUT-GNE group exhibited increased adhesion, decreased proliferation, and reduced mitochondrial membrane potential (P<0.05). No significant differences in apoptosis were observed between groups. The MUT-GNE group showed reduced sialic acid production, significantly decreased kinase activity, and downregulated transcription of sialic acid biosynthesis-related enzymes compared to WT-GNE (P<0.001). CONCLUSIONS The p.V543M mutation in the GNE gene alters cellular phenotype by reducing GNE enzymatic activity and the transcription of sialic acid biosynthesis enzymes, ultimately impairing sialic acid production.
Humans ; Mutation, Missense ; HEK293 Cells ; Apoptosis/genetics* ; Phenotype ; Multienzyme Complexes/metabolism* ; Cell Proliferation ; Homozygote ; Cell Adhesion/genetics* ; Distal Myopathies/genetics*

Background Schizophrenia and the use of antipsychotic medications are identified to be the likely contributors to the development of metabolic syndrome(MS)and cardiovascular disease,and jeopardize the prognosis of schizophrenia.Therefore,effectively preventing or reducing the risk of developing MS in patients with schizophrenia is critical.Objective To explore the efficacy of aripiprazole combined with olanzapine for male schizophrenia patients and its effect on MS,so as to provide a certain reference for the selection of antipsychotic drugs for schizophrenia patients.Methods Male patients(n=80)who were hospitalized in The Third People's Hospital of Meizhou from February to June 2023 and fulfilling the International Classification of Diseases,tenth edition(ICD-10)diagnostic criteria for the schizophrenia were enrolled,and grouped using random number table method,each with 40 cases.Study group was treated with aripiprazole combined with olanzapine,while control group was given aripiprazole monotherapy.The treatment lasted for 6 continuous weeks in both groups.At the baseline,Positive and Negative Symptom Scale(PANSS)score,MS-related indices[fasting plasma glucose(FPG),hemoglobin A1c(HbA1c),body mass index(BMI),waist-to-hip ratio(WHR),lipid profile],S100 calcium-binding protein B(S100B)and high sensitivity C-reactive protein(hs-CRP)were recorded.Then the PANSS scores at the end of the 2nd,4th and 6th week of treatment,the Clinical Global Impression(CGI)scores at the end of the 2nd and 6th week of treatment,as well as the MS-related indices,S100B,hs-CRP,Treatment Emergent Symptom Scale(TESS)score and Rating Scale for Extrapyramidal Side Effects(RSESE)score at the end of the 6th week of treatment were recorded in all participants.Results Analysis on PANSS score revealed a significant group effect,time effect and group×time interaction effect(F=18.092,634.780,2.917,P<0.05 or 0.01).Analysis on CGI score revealed a significant group effect and time effect(F=20.492,99.190,P<0.01).At the end of the 6th week of treatment,study group detected lower serum concentrations of HbA1c and triglyceride(TG)compared with control group(t=-3.495,-3.293,P<0.05).The post-treatment hs-CRP level was lower in study group than that in control group(t=-3.916,P<0.05).Study group scored lower on TESS compared with control group(t=-4.684,P<0.01).Conclusion Aripiprazole combined with olanzapine can effectively alleviate psychotic states in male schizophrenia patients,and the combination therapy yields less impact on MS-related indices than olanzapine monotherapy.

Objective:To evaluate the role of Sirtuin 1/nuclear factor κB (SIRT1/NF-κB) signaling pathway in mild hypothermia-induced promotion of microglial polarization during oxygen-glucose deprivation and restoration (OGD/R).Methods:The well-grown BV2 microglia were divided into 4 groups ( n=36 each) using the random number table method: control group (group C), OGD/R group (group O), mild hypothermia group (group M), and mild hypothermia+ SIRT1 specific inhibitor EX527 group (group ME). Cells in group C were commonly cultured without any treatment. Cells in group O were subjected to 3 h of OGD followed by 21 h of restoration of O 2-glucose supply at 37 ℃. Cells in group M were subjected to 3 h of OGD followed by 21 h of restoration of O 2-glucose supply at 33 ℃. Cells in group ME were co-cultured with inhibitor EX527 (final concentration 5 nmol/L) for 12 h in the medium before OGD/R, and the other procedures were conducted as previously described in group M. The cell survival rate was detected by CCK-8 assay. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-10 (IL-10) in supernatant were detected by enzyme-linked immunosorbent assay. The expression of CD206, CD32, inducible nitric oxide synthase (iNOS) and arginine synthase 1 (Arg-1) mRNA was detected by quantitative real-time polymerase chain reaction. The expression of CD206 and CD32 was detected by immunofluorescent staining. The expression of iNOS, Arg-1, SIRT1, NF-κB p65 (p65) and acetylated NF-κB (Ac-p65) was detected by Western blot. Results:Compared with group C, the cell survival rate was significantly decreased, the concentrations of TNF-α, IL-6 and IL-10 in the supernatant were increased, the expression of CD206, Arg-1, CD32 and iNOS was up-regulated, the expression of SIRT1 was down-regulated, and the Ac-p65/p65 ratio was increased in group O ( P<0.05). Compared with group O, the cell survival rate was significantly increased, the concentrations of TNF-α and IL-6 in the supernatant were decreased, the concentration of IL-10 was increased, the expression of CD206, Arg-1 and SIRT1 was up-regulated, the expression of CD32 and iNOS was down-regulated, and the Ac-p65/p65 ratio was decreased in group M ( P<0.05). Compared with group M, the cell survival rate was significantly decreased, the concentrations of TNF-α and IL-6 in the supernatant were increased, the concentration of IL-10 was decreased, the expression of CD206, Arg-1 and SIRT1 was down-regulated, the expression of CD32 and iNOS was up-regulated, and the Ac-p65/p65 ratio was increased in group ME ( P<0.05). Conclusions:SIRT1/NF-κB signaling pathway is involved in mild hypothermia-induced promotion of microglial polarization during OGD/R.

Objective:To evaluate the effects of GSK484 on ventilator-induced lung injury (VILI) and neutrophil extracelluar traps (NETs) in mice.Methods:Forty-eight SPF healthy male C57BL/6 mice, aged 5-6 weeks, weighing 15-20 g, were divided into 4 groups ( n=12 each) by a random number table method: spontaneous breathing group (group S), spontaneous breathing+ GSK484 intervention group (group SG), VILI group (group V), and VILI + GSK484 intervention group (group VG). The animals kept spontaneous breathing for 4 h after tracheal intubation in S and SG groups. The animals were mechanically ventilated for 4 h (tidal volume 30 ml/kg, respiratory rate 75 breaths/min, inspiratory/expiratory ratio 1∶2, positive end-expiratory pressure 0 mmHg, fraction of inspired oxygen 21%) in V and VG groups. At 3 days before developing the VILI model, GSK484 4 mg/kg was intraperitoneally injected once a day in SG and VG groups, while the equal volume of normal saline was given instead in S and V groups. Blood samples were collected from the abdominal aorta for blood gas analysis at 4 h of spontaneous breathing or mechanical ventilation, and PaO 2 was recorded. The mice were then sacrificed and bronchoalveolar lavage fluid (BALF) was collected and lung tissues were obtained for microscopic examination of the pathological changes (with a light microscope after HE staining) which were scored and for determination of wet to dry weight ratio (W/D ratio), concentrations of interleukin-1beta (IL-1β), IL-6, tumor necrosis factor-alpha (TNF-α) and myeloperoxidase (MPO) in BALF (by enzyme-linked immunosorbent assay), expression of peptidylarginine deiminase 4 (PAD4), neutrophil elastase (NE), high mobility group box 1 (HMGB1) and citrullinated-histone 3 (Cit-H3) in lung tissues (by Western blot). Results:Compared with S and SG groups, the lung injury score and W/D ratio were significantly increased, PaO 2 was decreased, concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were increased, and the expression of PAD4, NE, HMGB1 and Cit-H3 in lung tissues was up-regulated in V and VG groups ( P<0.05). Compared with group V, the lung injury score and W/D ratio were significantly decreased, PaO 2 was increased, the concentrations of IL-1β, IL-6, TNF-α and MPO in BALF were decreased, and the expression of PAD4, NE, HMGB1 and Cit-H3 was down-regulated in group VG ( P<0.05). Conclusions:GSK484 can alleviate VILI in mice, and the mechanism is associated with inhibition of PAD4, reduction of the production of NETs and attenuation of inflammatory responses in lung tissues.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:To summarize the clinical characteristics and risk factors of combined pulmonary fibrosis and emphysema syndrome(CPFE)in the elderly.Methods:42 patients with CPFE and 83 patients with idiopathic pulmonary fibrosis(IPF)admitted to Beijing Hospital from January 2015 to January 2020 were included for this retrospective study, and their clinical data, laboratory test results, imaging, pulmonary function and treatment outcomes were compared.The correlations of diffusing capacity of the lungs for carbon monoxide(DLCO)with lung volume and other ventilation-related parameters were analyzed.Multivariate Logistic analysis was used to screen for risk factors.Results:Compared with the IPF group, the proportions of patients who were male(78.6% or 33/42 vs.50.6% or 42/83), were smokers(78.6% or 33/42 vs.45.8% or 38/83)and had allergies(23.8% or 10/42 vs.4.8% or 4/83)were significantly higher in the CPFE group( χ2=9.090, 12.219, 8.293, P=0.003, 0.000, 0.004, respectively). Eosinophil counts were significantly higher in the CPFE group than in the other group[0.17×10 9/L with a(0.12-0.25)×10 9/L range vs.0.10×10 9/L with a(0.03-0.21)×10 9/L range]. In terms of pulmonary function tests, patients with CPFE had higher levels of vital capacity(VC)[2.60 L, range: (2.18-3.08)L vs.1.99 L, range: (1.48-2.63)L], total lung capacity(TLC)[4.54 L, range: (3.89-5.45)L vs.3.74 L, range: (2.92-4.70)L], forced vital capacity(FVC)[2.46 L, range: (2.12-3.08)L vs.1.95 L, range: (1.43-2.58)L], and forced expiratory volume in 1 second(FEV1)/FVC%[84.18%, range: (75.59-88.83)% vs.80.94%, range: (69.07-83.08)%], with significant differences from patients in the IPF group( Z=2.032, 2.248, 2.357, 2.421, 3.102, P=0.042, 0.025, 0.018, 0.015, 0.002). Positive correlations of DLCO were found with residual volume of the predicted(RV%pred), vital capacity of the predicted(VC%pred), alveolar ventilation of the predicted(VA%pred), total lung capacity of the predicted(TLC%pred), forced vital capacity of the predicted(FVC%pred), and percentage of forced expiratory volume in first second as predicted(FEV1%pred)in the IPF group( r=0.422, 0.370, 0.473, 0.520, 0.356, 0.267, P=0.000, 0.002, 0.000, 0.000, 0.003, 0.029), but not in the CPFE group.According to multivariate Logistic regression analysis, smoking( OR=5.421, 95% CI: 1.458-20.154, P=0.012)and allergies( OR=7.458, 95% CI: 1.795-30.979, P=0.006)were independent predictors of CPFE. Conclusions:The eosinophil count and lung volume in elderly CPFE patients are higher than those in IPF patients, and the significant feature is the decrease of DLCO, which is not correlated with changes in lung ventilation parameters and lung volume.Smoking and allergies are risk factors for CPFE in the elderly.

Scaffold-free tissue engineered cell sheet is an emerging technology in biomedical field. It can avoid the adverse effects of scaffold materials, and can be further assembled to form more complex three-dimensional functional tissues. The construction of cell sheet is mainly based on the culture substrate composed of sensitive materials. By changing the stimulation factors such as temperature, enzyme, light, ion, redox, pH and sugar, the adhesion behavior of the substrate to the cells could be changed to make the cells detach naturally, thus generating the cell sheet. Recent years have seen the development of various simple and efficient construction technologies of cell sheet due to the development of a variety of novel sensitive culture substrates. The resulted cell sheets with excellent performance have greatly expanded their applications. This review summarized the construction methods of tissue engineered cell sheet and discussed the challenges and future perspectives in this field.
Temperature ; Tissue Engineering ; Tissue Scaffolds

Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.

Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.

Objective To investigate radiation-induced alternations of phospholipids in epithelial cells,and to provide experimental evidence for understanding the mechanism of radiation-induced intestinal injury.Methods The intestinal epithelial cells(IEC-6)in rats were divided into three groups:normal control group,8 Gy X-ray irradiation group and 12 Gy X-ray irradiation group.Phospholipids were extracted at 6 h or 24 h after radiation and then measured by high-performance liquid chromatography and mass spectrometry(HPLC-MS).Results At 6 h after radiation,the phospholipids in 8 Gy irradiation group didn't vary significantly,while those in 12 Gy irradiation group changed.The PG,PI and Lyso PC were significantly up-regulated(F=5.37,9.60,9.88,P＜0.05).However,at 24 h after radiation,many PE and PG species in both irradiation groups declined(F=5.15-99.77,P＜0.05)and SM species increased in 12 Gy irradiation group(F=4.35-7.92,P＜0.05).Conclusions The ionizing radiation could disorder phospholipid metabolism in IEC-6 cells with a dose-dependent manner.