pPIC9 was used as a template and ?-factor gene of Saccharomyces cerevisiae was amplified by PCR.The gene was cloned in the intracellular expression vector pYES2/CT for Saccharomyces cerevisiae and the secreting expression vector named pYES2/CT/?-factor(pYC?)was constructed.The gene of mannase(man)from recombinant vector of pKLAC1-man(pKLman) was cut by restriction enzymes and linked with pYC?.This recombinant vector pYC?-man was used to determine the secretory ability and stability of pYC?.The excellent secretory ability of pYC? was proved by two experiments.One showed that INVSc1/pYC?-man clones formed the clear rings around the clones on the medium contained trypan blue,while INVSc1/pYC? clones had no rings.Further analysis of mannase activity of extracellular supernatant and intracellular extracts showed that both extracellular and intracellular mannase activities of INVSc1/pYC? were not detected,while INVSc1/pYC?-man had evident extracellular mannase activities and no intracellular mannase activities.The stability of pYC? was also very good proved by continuous cultivation for about 150h.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADMSCs) can improve the liver function of rats with liver failure, which illustrates the important research value in the field of tissue engineering and cell transplantation.OBJECTIVE: To evaluate the therapeutic potential of human ADMSCs in heart failure rats and to discuss the possible biological mechanisms involved.METHODS: Heart failure rats were randomized into model and ADMSCs groups, which were given normal saline or DAPI-labeled human ADMSCs (3.0×106) via the tail vein. At 1, 3, 7 days after transplantation, we detected the biochemical indexes for liver function in rats. At 3 days after transplantation, the serum levels of cytokines, such as tumor necrosis factor α and interleukin-10, were detected, the histomorphological changes in the liver were observed by hematoxylin-eosin staining, and the protein expression of proliferating cell nuclear antigen was detected by western blot. RESULTS AND CONCLUSION: We found that human ADMSCs could migrate to the liver and lung tissues in rats after the transplantation via the tail vein. At 1 and 3 days after transplantation, the levels of serum alanine aminotransferase and aspartate aminotransferase were significantly reduced in the ADMSCs group as compared with the model group (P< 0.05); furthermore, the secretion of tumor necrosis factor α and interleukin-10 was significantly suppressed at 3 days after cell transplantation (P < 0.05). The results of hematoxylin-eosin staining indicated a significant improvement in liver degeneration and necrosis. The expression of proliferating cell nuclear antigen protein in the ADMSCs group was significantly up-regulated compared with the model group. To conclude, human ADMSCs can inhibit the inflammatory reaction and up-regulate the expression of proliferating cell nuclear antigen, to promote the regeneration of liver cells and he recovery of liver function.

Firstly, We used error-prone PCR to induce mutations on Armillariella tabescens MAN47 beta-mannanase gene, Secondly, we cloned the mutated fragments into secreted expression vector pYCalpha, Then the recombinant plasmids were transformed into Saccharomyces cerevisiae BJ5465 after amplified and extracted in DH5alpha cells. Through three cycles of error-prone PCR we built a mutant database, Then we screened one optimum (named M262) from about 104 mutants. The evoluted MAN47 beta-mannanase displayed both higher thermal stability and activity than wide type. The evoluted enzyme M262 retained high activity after treatment at 80 degrees C for 30 min, whereas, the wild type nearly lost activity under this condition. Meanwhile, the activity of M262 can reach to 25 U/mL, which is 4.3 times as wide type under optimum temperature. In addition, pH stability and pH range of evoluted enzyme M262 were both improved compared with wild-type enzyme. The optimum pH was estimated to be similar to that of wild-type enzyme. The sequence comparison illustrated that there were three nucleotide substitutions (T343A/C827T/T1139C) which carried corresponding amino acid changes (Ser115Thr/Thr276Met/Val380Ala). According to homologous modeling by SWISS-MODEL Repository, three mutated amino acids located at the sixth amino acid of the fourth beta-sheet, the first amino acid of the sixth alpha-helix, the turn between the tenth and eleventh beta-sheet, respectively.
Armillaria ; classification ; enzymology ; genetics ; Directed Molecular Evolution ; Enzyme Stability ; Escherichia coli ; enzymology ; genetics ; Hot Temperature ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering ; Recombinant Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; genetics ; beta-Mannosidase ; chemistry ; genetics ; metabolism

The experiment was conducted by directed evolution strategy (error-prone PCR) to improve the activity of aflatoxin detoxifzyme with the high-throughput horse radish peroxidas and recessive brilliant green (HRP-RBG) screening system. We built up a mutant library to the order of 10(4). Two rounds of EP-PCR and HRP-RBG screening were used to obtain three optimum mutant strains A1773, A1476 and A2863. We found that mutant A1773 had upper temperature tolerance of 70 degrees C and that its enzyme activity was 6.5 times higher than that of the parent strain. Mutant strains A1476 worked well at pH 4.0 and its enzyme activity was 21 times higher than that of the parent strain. Mutant A2863 worked well at pH 4.0 and pH 7.5, and its enzyme activity was 12.6 times higher than that of the parent strain. With DNA sequencing we found that mutant A1773 revealed two amino acid substitutions, Glu127Lys and Gln613Arg. Mutant A1476 revealed four amino acid substitutions: Ser46Pro, Lys221Gln, Ile307Leu and Asn471lle. Mutant A2863 revealed four amino acid substitutions: Gly73Ser, Ile307Leu, Va1596Ala and Gln613Arg. The results provided a useful illustration for the deep understanding of the relationship between the function and structure of aflatoxin detoxifzyme.
Aflatoxin B1 ; antagonists & inhibitors ; chemistry ; Amino Acid Substitution ; Directed Molecular Evolution ; Enzyme Activation ; Enzyme Stability ; Multienzyme Complexes ; genetics ; metabolism ; Mutant Proteins ; genetics ; metabolism ; Point Mutation ; Polymerase Chain Reaction ; methods ; Protein Engineering

Objective To compare the effect of microsurgery and conventional surgery in the repair of tendon rupture and the effect of postoperative adhesion prevention. Methods From February 2011 to December 2016,seventy?two patients with finger tendon rupture treated in Dongfeng Hospital Affiliated to Hubei Medical College were selected as the research objectand then randomly divided into the observation group and the control group,36 cases in each group. The observation group was treated with microsurgery treatment,the control group was treated with the traditional surgical treatment to record postoperative prognosis and adhesion of the two groups. Results At 12 weeks after operation,the recovery rate of tendon in the observation group and the control group were 94. 4％ (34/36)and 75％(27/36),respectively,the data in the observation group was significantly higher than that in the control group (χ2=5. 552, P<0. 05 ) . At the same time, Carroll score of upper limb function in the observation group at 4 weeks and 12 weeks after the surgery were (85. 55±8. 29) and (102. 49 ±10.82),significantly higher than those in the control group ( (74.25±8.53)％,(91.44±12.49) ),the differences were statistically significant (t=6. 948,5. 323,P<0. 05). At 12 weeks after operation,the adhesion rate of tendon in the observation group was 2. 8％ ( 1/36 ) , significantly lower than that of the control group ( 16. 7％ ( 6/36) ) , ( P<0. 05 ) . Conclusion Compares with the conventional surgery, microsurgery in the treatment of tendon rupture repair can promote the repair of tendon tissue,improve the upper limb function,and reduce the occurrence of postoperative tendon adhesion.

Objective To evaluate the correlation between 3D morphological parameters of aneurysms based on the computer-assisted semi-automated measurement and the risk of aneurysm rupture.Methods From October 2019 to October 2022,patients with ruptured multiple aneurysms admitted to the Department of Neurosurgery of Xuanwu Hospital,Capital Medical University were retrospectively included.Aneurysmal morphological parameters(including aneurysmal diameter,maximum diameter,width,neck width,volume,flow angle,parental artery diameter,surface area,wave index and non-spherical index)were measured by computer-assisted semi-automated measurement methods.The length-to-width ratio,wide-to-neck ratio,aspect ratio and size ratio were calculated,and the aneurysm location information was recorded.The ruptured aneurysms in multiple aneurysms were included in the ruptured group,and the remaining aneurysms were included in the unruptured group.Uni variable analysis and binary Logistic analysis were used to evaluate the differences in morphological parameters and location information between the ruptured and unruptured groups.Results All 56 patients with multiple ruptured aneurysms and a total of 126aneurysms were included in the group for analysis.Concerning morphology,including diameter＞5 mm(51.8％[29/56]vs.15.7％[11/70],P＜0.01),maximum diameter＞6mm(57.1％[32/56]vs.25.7％[18/70],P＜0.01),flow angle＞107°(57.1％[32/56]vs.35.7％[25/70],P=0.016),wide-to-neck ratio＞1.1(50.0％[28/56]vs.30.0％[21/70],P=0.022),aspect ratio＞1.1(46.4％[26/56]vs.25.7％[18/70],P=0.015)and size ratio＞1.9(57.1％[32/56]vs.10.0％[7/70],P＜0.01),there was significant difference between the ruptured and unruptured group;Concerning locations,aneurysms are mainly located in the posterior communicating segment of the internal carotid artery(39.3％[22/56])and the middle cerebral artery(23.2％[13/56])in ruptured group,while in the middle cerebral artery(28.6％[20/70])and the non-posterior communicating segment of internal carotid artery(27.1％[19/70])in unruptured group,and there was significant difference in distribution of aneurysm locations(P=0.003).Multivariate Logistic regression analysis showed that size ratio＞1.9 was an independent risk factor for aneurysm rupture(OR,11.62,95％CI 2.40-56.15;P=0.002).Concerning locations,posterior communicating artery aneurysms had a significantly higher risk of rupture compared with the non-posterior communicating segment of internal carotid artery(OR,19.25,95％CI 2.19-169.51;P=0.008).Conclusion For multiple intracranial aneurysms,the size ratio of the three-dimensional morphological parameters of aneurysms＞1.9 is an independent risk factor for aneurysm rupture,and the rupture risk of posterior communicating artery aneurysms is significantly higher than that of non-posterior communicating segment of internal carotid artery.

Objective To investigate the clinical application and effect of pick-stab skill in eyebrow embroidery.Methods From July 2015 to January 2016,36 cases were selected to receive eyebrow embroidery with pick-stab skill in our department,including 1 male and 35 females.The cosmetic outcomes,complications and satisfaction rate were observed in these cases.Results In these 36 cases,14 cases were colored in one time,19 cases were colored twice,and 3 cases were more than 3 times.All patients were followed up for 6 months.The color of the eyebrow was natural,and the blue color was not existed.Conclusions The pick-stab skill in eyebrow embroidery is worthy in clinic with better cosmetic effect,higher success rate and safety.

OBJECTIVE:To determine quality indexes of Hejiang Dendrobium nobile at different harvesting time and months,and to provide scientific reference for reasonable harvesting period of Hejiang D.nobile.METHODS:The stem of annual,biennial and triennial D.nobile were collected.The drying rate,the contents of polysaccharides (colorimetry),dendrobine (GC) and total alkaloids (colorimetry),effective component total rate (dendrobine×drying rate) were measured.Biennial D.nobile were collected in autumn and winter (Oct.of the second year-Mar.of the third year,15th day a month) to determine drying rate,the contents of polysaccharides and dendrobine.RESULTS:The drying rate,the contents of polysaccharides and effective component total rate were higher than annual and triennial D.nobile.The content of total alkaloids and dendrobine were in descending order:annual D.nobile (0.52％,0.48％) ＞biennial D.nobile(0.48％,0.44％) ＞triennial D.nobile (0.32％,0.22％).From Oct.of the second year to Mar.of the third year,the drying rate of biennial D.nobile was increasing month by month;the contents of polysaccharides and dendrobine increased firstly and then decreased;the content of polysaccharide was the highest in Feb.(17.32％),and the content of dendrobine reached the highest level in Dec.(0.51％).CONCLUSIONS:The optimal harvesting period is biennial Hejiang D.nobile in Dec.and Jan.(third years before flowering),considering flowering characteristics,drying rate,the contents of polysaccharide and dendrobine.

PURPOSE: Hrd1 has recently emerged as a critical regulator of B-cells in autoimmune diseases. However, its role in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unexplored. This study aimed to examine Hrd1 expression and B-cell accumulation and their possible roles in CRSwNP. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry, enzyme-linked immunosorbent assay and Western blotting were used to assess gene and protein expression in nasal tissue extracts. Cells isolated from nasal tissues and peripheral blood mononuclear cells were characterized by flow cytometry. Local antibody production was measured in tissue extracts with a Bio-Plex assay. Additionally, changes in Hrd1 expression in response to specific inflammatory stimuli were measured in cultured dispersed polyp cells. RESULTS: Nasal polyps (NPs) from patients with eosinophilic CRSwNP (ECRS) had increased levels of Hrd1, B-cells and plasma cells compared with NPs from patients with non-eosinophilic CRSwNP (non-ECRS) or other control subjects (P < 0.05). The average Hrd1 levels in B-cells in NPs from ECRS patients were significantly higher than those from non-ECRS patients and control subjects (P < 0.05). NPs also contained significantly increased levels of several antibody isotypes compared with normal controls (P < 0.05). Interestingly, Hrd1 expression in cultured polyp cells from ECRS patients, but not non-ECRS patients, was significantly increased by interleukin-1β, lipopolysaccharide and Poly(I:C) stimulation, and inhibited by dexamethasone treatment (P < 0.05). CONCLUSIONS: Differential Hrd1 expression and B-cell accumulation between the ECRS and non-ECRS subsets suggests that they can exhibit distinct pathogenic mechanisms and play important roles in NP.
Antibody Formation ; Autoimmune Diseases ; B-Lymphocytes* ; Blotting, Western ; Dexamethasone ; Enzyme-Linked Immunosorbent Assay ; Eosinophils* ; Flow Cytometry ; Humans ; Immunity, Innate ; Immunohistochemistry ; Nasal Polyps* ; Plasma Cells ; Polyps ; Real-Time Polymerase Chain Reaction ; Tissue Extracts

Objective:To investigate the effects of microwave radiation on associative learning and memory function and hippocampal structure of mice.Methods:C57BL/6N mice were ramdomly divided into sham-radiated group ( n=27) and radiation group ( n=2). The radiation group was exposed to microwave at 2.856 GHz, 8 mW/cm 2 for 15 min, then their spatial and associative learning and memory function were examined with the morris water maze and shuttle box behavior experiment. The pathological changes of hippocampal tissue were observed by HE staining and light microscope, the ultrastructural changes of hippocampal tissue were observed by transmission electron microscope. Results:After microwave radiation, the times of mice crossing the platform for the reverse space exploration decreased from(3.60±0.79) times to (2.55±0.47) times( t=2.21, P=0.046), the average active escape rate decreased significantly ( t = 2.70, P<0.05), and the average active latency and the total shock time was significantly prolonged ( t = -3.09, -3.02, P < 0.05). At 8 d after microwave radiation, the nuclei of some neurons in the CA3 and DG regions of the hippocampus were pyknosis. The neurons were apoptotic, the synaptic spaces blurred, the glial cells swollen, and the perivascular spaces widened in the CA3 region of the hippocampus. Conclusions:Microwave radiation can decline the spatial reference memory ability and associative learning and memory ability of mice. The morphological and pathological changes of hippocampus are the structural basis of this dysfunction.