Objective:To investigate the essential determinants that are critical to initiating the assembly of outer membrane proteins by replacing the POTRA domains of the translocator protein FhaC and the insertase protein TtOmp85 of the Omp85 family. Methods:FhaC, TtOmp85 proteins and their recombinant chimeric proteins after replacing the POTRA domain were obtained by overexpression and purification in vitro. An in vitro reconstitution system was used to investigate the effects of the different domains on the transport efficiency of the substrate outer membrane protein FhaB and the membrane insertion efficiency of OmpA. Results:Replacing the POTRA domain of FhaC with that of TtOmp85 led to the loss of the transport function of FhaB. During the membrane insertion process of OmpA, the FhaC mutant containing the POTRA of TtOmp85 protein acquired the ability to assemble OmpA. Conclusion:The compositional differences in the POTRA domain of Omp85 family proteins determine their abilities to recognize their substrate proteins.

Objective:To investigate the essential determinants that are critical to initiating the assembly of outer membrane proteins by replacing the POTRA domains of the translocator protein FhaC and the insertase protein TtOmp85 of the Omp85 family. Methods:FhaC, TtOmp85 proteins and their recombinant chimeric proteins after replacing the POTRA domain were obtained by overexpression and purification in vitro. An in vitro reconstitution system was used to investigate the effects of the different domains on the transport efficiency of the substrate outer membrane protein FhaB and the membrane insertion efficiency of OmpA. Results:Replacing the POTRA domain of FhaC with that of TtOmp85 led to the loss of the transport function of FhaB. During the membrane insertion process of OmpA, the FhaC mutant containing the POTRA of TtOmp85 protein acquired the ability to assemble OmpA. Conclusion:The compositional differences in the POTRA domain of Omp85 family proteins determine their abilities to recognize their substrate proteins.

Objective:To screen active antibacterial components from licorice extract using BamA and BamD, the core components of Escherichia coli ( E. coli) β-barrel assembly machinery (BAM), as targets in order to combat the increasingly serious problem of antibiotic resistance. Methods:Affinity ultrafiltration combined with high performance liquid chromatography-mass spectrometry (HPLC-MS) was used to screen the potential components interacting with BamA and BamD from licorice extract. Changes in the expression of bamA and bamD genes of E. coli after treatment with the compounds were detected by fluorescence quantitative PCR, and the effects of the compounds on the function of the BAM complex to integrate outer membrane proteins into the bacterial outer membrane were analyzed using an in vitro recombination system. The influence of the compounds on the integrity of bacterial membranes was evaluated through analyzing the accumulation of SDS within the bacterial cells. Results:Bioaffinity ultrafiltration combined with HPLC-MS screening revealed that 18β-glycyrrhetinic acid could interact with BamD. After 18β-glycyrrhetinic acid treatment, the expression of bamA gene increased by 1.5 times, and the expression of bamD gene increased by 2 times. However, the inhibitory effect of 18β-glycyrrhetinic acid on the membrane insertion function of the BAM complex was not observed in the in vitro recombinant system assay, and the cell membrane integrity assay experiments did not reveal any disruption of the E. coli cell membrane by 18β-glycyrrhetinic acid. Conclusions:Using BamA and BamD proteins as targets, a natural product screening method using affinity ultrafiltration combined with HPLC-MS is established. The screening result shows that 18β-glycyrrhetinic acid can interact with BamD and affect the expression of outer membrane proteins in E. coli. Therefore, the screening and experimental procedures established in this study are of good reference value for the screening of novel antimicrobial drugs from other sources targeting outer membrane proteins, and this study also suggests that the selection of the relevant target sites is crucial for the successful screening of the corresponding natural products.

Humans ; China/epidemiology* ; SARS-CoV-2 ; COVID-19

Objective To observe the characteristics of brain activation of multiple sclerosis (MS) patients and healthy controls at functional magnetic resonance imaging (fMRI) with olfactory stimulation,determine the locations of activation in areas of olfactory center and explore the MS olfactory related network.Methods Eighteen MS patients from the Affiliated Hospital of North Sichuan Medical College from February 2017 to March 2018 were enrolled as MS group,and 20 matched healthy adults during the same period served as controls.The Visual Analogue Scale was used to evaluate olfactory function in all subjects,the rest structure MRI was performed first,and volatile gases of lavender and rose solution were used to alternately stimulate olfactory during fMRI scanning.The brain activation was obtained by using matlab2013a and SPM8 softwares.The distribution and quantity of demyelination lesions were counted on T2 weighted image,and Spearman correlation analysis was done with SPSS 17.0 software package.Results The activated brain areas in the healthy control group included bilateral middle frontal gyrus,bilateral insula,bilateral supramarginal gyrus,bilateral orbitofrontal gyrus,right thalamus,right central anterior gyrus,bilateral cingulated gyrus,bilateral hippocampus,bilateral amygdala and bilateral superior frontal gyrus (t =2.11,P＜0.05).The activated brain areas in the MS group included right cerebellum,left insula,left superior temporal gyrus,right inferior frontal gyrus (t=2.19,P＜0.05).Compared with the control group,the MS group showed statistically significant decrease in activated values in right insula,right amygdala,right inferior frontal gyrus,right frontal middle gyrus,and the left supramarginal gyrus (t=2.04,P＜0.05).The distribution and number of demyelination lesions and major activation of brain regions with olfactory in the MS group demonstrated no significant correlation (r=-0.524,P=0.054).Conclusions Multiple brain areas involved in the olfactory processing and olfactory-related brain network existed.The activation of olfactory center had dominance in the right brain.The activation of the brain area in the MS group was significantly reduced,and the activation voxel and activation intensity were weakened.The olfactory-related brain network changed in MS patients.The distribution and number of demyelination lesions had no significant effect on the major activation of brain regions with olfactory stimulation.

Objective To investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia. Methods Hyperthermic HepG2 cell models established by exposure of the cells to 42 ℃ for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed. Results ROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001). Conclusion The intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.

Objective To investigate the mechanism by which heat shock protein 90 (HSP90) regulates 26S proteasome in hyperthermia. Methods Hyperthermic HepG2 cell models established by exposure of the cells to 42 ℃ for 3, 6, 12, and 24 h were examined for production of reactive oxygen species (ROS) and cell proliferation, and the changes in Hsp90α and 26S proteasome were analyzed. Results ROS production in the cells increased significantly after hyperthermia (F=28.958, P<0.001), and the cell proliferation was suppressed progressively as the heat exposure time extended (F=621.704, P<0.001). Hyperthermia up-regulated Hsp90α but decreased the expression level (F=164.174, P<0.001) and activity (F=133.043, P<0.001) of 26S proteasome. The cells transfected with a small interfering RNA targeting Hsp90α also showed significantly decreased expression of 26S proteasome (F=180.231, P<0.001). Conclusion The intracellular ROS production increases as the hyperthermia time extends. Heat stress and ROS together cause protein denature, leading to increased HSP90 consumption and further to HSP90 deficiency for maintaining 26S proteasome assembly and stability. The accumulation of denatured protein causes unfolded protein reaction in the cells to eventually result in cell death.

Objective To discuss the clinical effects of using CT positioning keyhole approach to treat hy-pertensive intracerebral hemorrhage (HICH).Methods 85 cases of patients with hypertensive intracerebral hemor-rhage(HICH) were chosen and divided into two groups according to the operation methods:the observation group had 55 cases given CT positioning keyhole approach ,while the control group had 30 cases treated with traditional cranioto-my hematoma removal operation .All patients were supplemented by postoperative blood pressure control and nutrition -al support treatment .The average operation time ,hematoma disappearing time ,the length of hospital stay and re-bleed-ing rates and postoperative ability of daily life ( ADL) scores of the two groups were all carefully recorded and com-pared.Results The average operation time,hematoma disappearing time and hospital stay of the observation group were (66.5 ±12.8)min,(3.4 ±1.3)d,and (9.3 ±1.7)day which were all significantly lower than those of the con-trol group(193.5 ±23.7)min,(5.8 ±2.1)d and (15.2 ±3.8)d;T-test values of the two groups were 2.874,3.125 and 3.433 separately(P<0.05);there were 2 cases(3.6%) of postoperative hemorrhage in the observation group of while 6 cases(20.0%) in the control group,whose difference was statistically significant (χ2 =6.097,P<0.05);In the observation group 4 cases(7.3%) died after operation and also 4 cases(13.3%) died in the control group ,and the mortality of the two groups had no statistical significance (χ2 =0.836,P>0.05).6 months′follow-up after opera-tion,in the control group 2 cases were lost to follow-up while in the observation group 3 cases were lost to follow-up;Using ADL to evaluate the two groups of patients with survival and continuous follow-up,we found that the observation group′s postoperative quality of life was better than that of the control group′s(μ=3.325,P<0.05).Conclusion Using CT positioning keyhole approach has smaller trauma , shorter operation time and faster postoperative recovery and other characteristics,which is an effective method for the treatment of hypertensive intracerebral hemorrhage(HICH).

Objective To compare the clinical effects and safety of surgical clipping and intravascular interventional therapy in treatment of intracranial wide-necked aneurysm.Methods The clinical data of 158 patients with intracranial wide-necked aneurysm from February 2010 to February 2013 were retrospectively analyzed,all patients were divided into two groups:surgical clipping group with 92 cases and intravascular interventional therapy group with 66 cases,the postoperative curative effects,treatment time,hospital stay,hospital expenses and postoperative complications between two groups were compared.Followed up for 10-46 months,the recurrence rate were compared.Results The good prognosis and defective rates between surgical clipping group and intravasular interventional therapy group had no significant difference [90.2％(83/92) vs.90.9％(60/66),9.8％(9/92) vs.9.1％ (6/66)] (x2 =0.298,P ＞ 0.05).The preoperative Hunt-Hess classification and CT Fisher classification between two groups had no significant difference (P ＞ 0.05).Six months after discharge,mRS score was used to evaluate the curative effect,the defective rates in same level patients between two kinds of treatment methods had no significantdifference (P ＞ 0.05).The treatment time,hospital stay in surgical clipping group were significantly longer than those in intravascular interventional therapy group [(4.03 ± 1.01) h vs.(1.61 ± 0.98) h,(15.90 ± 2.03) dvs.(13.20 ± 1.95) d],hospital expenses was significantly lower than that in intravascular intervention therapy group [61 829.4 ±320.6) yuan vs.(99 876.2 ±371.5) yuan] (P ＜0.05).The postoperative complications rate between two groups had no significant difference (P ＞ 0.05).Followed up for 31.3 (10-46) months,the recurrence rate in surgical clipping group was significantly lower than that in intravascular intervention therapy group [1.1％ (1/94) vs.8.8％ (6/68)] (P ＜ 0.05).Conclusion Surgical clipping and intravascular interventional therapy in treatment of intracranial wide-necked aneurysm has their own different characteristics,so patients' treatment methods should be based on their preoperative status (especially preoperative Hunt-Hess and Fisher classification) and patients' economic conditions.

Objective To investigate the effect of quetiapine on the behavior and expression of pERK1/2 in chronic unpredictable stress(CUS) model rats.Methods 32 adult male Sprague Dawley rats were randomly divided into four groups (n =8 for each group):control group,CUS group,CUS + QUE (5 mg/kg,L) group and CUS + QUE(10 mg/kg,M)group.The rats in control group were left undisturbed in their home cage for 28 days and the other groups were exposed to 28 consecutive days of CUS,then the rats in control group and CUS group were treated with 1％ DMSO in saline (5 ml/kg,intraperitoneal injection),the rats in CUS + QUE (L)group and CUS + QUE(M) group respectively treated with quetiapine (5 mg/kg)or quetiapine(10 mg/kg) for consecutive 7 days.The weight data of each group were recorded,and the behavioral changes in these rats were analyzed by open field test and forced swimming test;and the expression of pERK1/2 was measured by Western blot.Results (1)Compared with control group,quetiapine (10 mg/kg) ameliorated the inhibition of body weight gain that induced by chronic unpredictable stress (P ＜ 0.05),but quetiapine (5 mg/kg) did not have this effect.(2)Open field and Forced swimming test showed significant difference (P ＜ 0.05) of horizontal motion distance (F =17.846),the number of central region entering(F=4.720) and the immobility time(F=26.090) in each group.And these tests showed that horizontal motion distance and the number of central region entering in CUS group ((6696.30 ±1061.19)mm,(19.63 ±9.15)times) were significantly lower than that of control group ((10824.61 ± 1399.37) mm,(37.75 ± 13.02) times) and CUS + QUE (M) group ((9637.51 ± 1630.16) mm,(32.38 ± 6.23)),while the immobility time (110.73 ± 15.98)s were significantly higher than that of control group((66.13 ± 5.18)s)and CUS + QUE (M) group((73.40 ± 11.99) s,P ＜ 0.05).But there was no significant difference between that of CUS group and CUS + QUE(L) group(P＞0.05).(3)The expression of pERK1/2 in CUS group showed significant decrease when compared with control group or CUS + QUE (M) group,but showed no significant difference with CUS + QUE(L) group(F=6.641,P＜ 0.01).Conclusion Quetiapine can ameliorate depressive-like behaviors induced by chronic unpredictable stress,and this effect may be carried out by up-regulation the expression of pERK1/2 in the hippocampus.