Objective To make sciatic nerve traction injury models of rabbit , in order to prospectively evaluate possibility and accuracy of diffusion tensor tracking (DTT) in sciatic nerve injures. Methods The right sciatic nerves of 32 New Zealand white rabbits were selected for traction injury , and the left sciatic nerves were the sham operation side. DTI scan was performed before and after the operation on 1st day , 3rd day, and 1 week, 2, 3, 4, 6 and 8th week respectively, and DTT was reconstructed. Then the length of reconstructed fiber tracts and fiber density index were calculated. After the MRI scan , the sciatic nerve was removed to perform pathologic examination at different time points. Results The difference of the length of reconstructed fiber tracts between nerve traction injury and sham operation side was significant at 1 day~2weeks after operation (P<0.05), while the difference was not significant at 3~8 weeks.The fiber density index of nerve traction injury and sham operation side was significantly different at 1 day~8 weeks(P<0.05). 1day after operation, myelin sheath of traction portion was obviously twisting. 3 days after operation , a large amount of myelin sheath broke down. 2 weeks after operation , axon, myelin sheath degeneration and regeneration coexisted at the same time. 8 weeks after operation , nerve fibers regenerated and restored to normal structure. Conclusion The length time curve and density index-time curve of nerve traction injury are consistent with the changes of pathology , which can be used as a supplementary method to evaluate the degeneration and regeneration in nerve injury.

BACKGROUND:Magnetic resonance diffusion tensor imaging can display the dispersion changes of peripheral nerve injury and be used to conduct quantitative research, so it has good application prospects in displaying the nerve injury and regeneration. OBJECTIVE:To investigate the possibility of magnetic resonance diffusion tensor imaging of rabbit acute sciatic nerve traction injury, and to figure out the value of diffusion tensor parameters in the diagnosis of peripheral nerve injuries and to reveal the pathologic basis. METHODS:The right hind limb sciatic nerves of 32 New Zealand white rabbits were selected to make the regeneration and repair models, the left hind limb nerves as the sham-operation side. Diffusion tensor imaging examination of sciatic nerves were performed at 1 and 3 days, 1, 2, 3, 4, 6 and 8 weeks after operation with 1.5 T MRI. Fractional anisotropy and apparent diffusion coefficient were measured through diffusion tensor tracing
reconstruction, and then the pathological examination was performed. RESULTS AND CONCLUSION:Diffusion tensor imaging revealed only the proximal nerve, injured nerve as wel as the middle of the distal nerve at 1 day after traction injury. At 1 week, the nerve of distal portion appeared thinner and shorter fiber bundle. At 2-6 weeks after operation, the fiber bundle was increased and thickened. At 8 weeks after operation, the distal nerve fibers had nearly restored to the level before injury. There was significant difference in the fractional anisotropy value of traction portion and distal portions between traction injury and sham-operation group at 1 day-8 weeks after operation (P<0.05). While there was significant difference in the fractional anisotropy value of proximal traction portion between traction injury and sham-operation group 1 day-1 week after operation (P<0.05). There were no significant differences in the apparent diffusion coefficient values between traction injury and sham-operation group at 1 day-8 weeks after operation. Fal of fractional anisotropy value in the early stage of nerve traction injury was the result of myelin sheath broke down and axonal disintegrated;recovery of fractional anisotropy value resulted from myelin sheath proliferated and myelin sheath grew slowly to mature. Diffusion tensor tracing can show the abnormal change of the sciatic nerve with traction injury in rabbit clearly and early, and the measurement of fractional anisotropy value can be used as the sensitive method to monitor the degeneration and regeneration after nerve traction injury.

Objective To investigate the correlation between eigenvalues(λ∥ andλ⊥)of sciatic nerve and limb function in a rabbit model of acute nerve traction inj ury for finding the role ofλ∥ andλ⊥ on diagnosis of nerve inj uries.Methods The right sciatic nerves of 32 Newland rabbits were selected to be performed as traction injury,and then left sciatic nerves were treated as control for the sham operation side.MRI DTI scan was performed before and after the operation on 1 day ,3 day,and 1 week,2,3,4,6 and 8 weeks respectively.Theλ∥ andλ⊥ of inj ured sciatic nerves and sham operated nerves were measured,meanwhile functional exami-nations were evaluated,and then analyzing the correlation betweenλ⊥ of inj ured sciatic nerves and the score of inj ured limb function in the periods of 3 days to 8 weeks;finally different portions of sciatic nerve were removed for histological examinations.Results Theλ⊥ of inj ured sciatic nerves in the proximal portion was slightly increased at 3 days and recovered to the normal at 2 weeks.Theλ⊥ of inj ured sciatic nerves in the traction portion and distal portion were slightly raised at 1 day,reached to its maximum value at 3 days,and then decreased gradually after 1 week and dropped to the pre-operation level after 4 weeks.Theλ⊥ of inj ured sciatic nerves in the proximal portions was significantly higher than that of the sham-operated nerves from 1 day to 1 week(F=7.275,P=0.000). Theλ⊥ of inj ured sciatic nerves in the traction portion and distal portion were significantly higher than that of the sham-operated nerves from 1 day to 3 weeks(F=5.851,F=3.794,P=0.001,P=0.000).However,there was no significantly difference between theλ∥ of inj ured nerves in each portion and sham-operated nerves .The negative correlation between theλ⊥ of inj ured sciatic nerves in the distal portion and the score of the nerve function in the injured limb was found (r=-0.938,r=-0.897,P<0.01).Conclu-sion Theλ⊥ of inj ured nerves in the traction portion and distal portion could monitor the process of degeneration and regeneration of sciatic nerve in traction inj ury,while theλ∥ plays no obvious role in diagnosis of nerve inj uries.

Objective: To investigate the feasibility of in vitro perfusion culture of human adipose tissue and its induction into muscle tissue. Methods: Human abdominal adipose tissue were cultured in vitro by perfusion culture. After 1, 3, 5 or 7 weeks, FAD/PI staining was used to detect the tissue vitality. Histological staining was used to observe the changes of its histomorphology. Protein expressions of myogenic molecules Myf-5 and myoD1 as well as muscle specific protein Desmin were measured by immunohistochemistry and Western blot assay. Results: The adipose tissues cultured in myogenic induction media were still in the appearance of adipose tissue at 7 weeks. While in the basal medium without inducing, vascular pedicles shed after 7 weeks and could not continue to be cultured. FAD/PI staining showed that the tissue cultured in the induction media remained viable at 7 weeks, while the viability of the tissue in the basal culture medium decreased significantly at 5 weeks. Histologically, Myf-5, myoD1 and Desmin were all positively expressed in muscle tissues, while in adipose tissues, some mesenchymal and vascular endothelial cells expressed Myf-5 but not myoD1, and only separate vascular smooth muscle cells expressed Desmin. Interestingly, in adipose tissues cultured in myogenic induction medium, partial muscle-like tissue formed, evidenced as positive expression of Myf-5, myoD1 and Desmin. There was no muscle-like tissue formation in adipose tissue cultured in basal medium and the expression patterns were similar to that of the control group. Western blot results showed that the expression levels of Myf-5 and Desmin in muscle tissue were significantly higher than that of the other groups (P<0.05). The expression level of Myf-5 was slightly higher in inducing group than that in non-inducing group at the same time points, but the difference was not statistically significant. Similarly, the expression of Desmin was higher in inducing group than that in non-inducing group, yet there was statistically significant difference only in the first week. The protein expression of myoD1 was not detected in any group using Western blot. Conclusions The adipose tissue cultured in the perfusion bioreactor can survive for up to 7 weeks, and it can be partially induced into muscle-like tissue in the myogenic induction medium.

Rhesus macaque (Macaca mulatta) is the best model to study of human immunodeficiency virus (HIV) infection and to develop acquired immunodeficiency syndrome (AIDS) vaccine. The crystal structure of its major histocompatibility antigen complex (MHC) is helpful to understand the mechanism of HIV immune evasion. In this study, we cloned the light chain (beta2m) of MHC class I allele of rhesus macaques, Mamu-A*02, and inserted it into pET21a(+) vector. We transfected the recombinant plasmid pET21a(+)-Mamu-beta2m and pET21a(+)-Mamu-alpha into BL21(DE3). Mamu-A*02 and beta2m were expressed in the form of inclusion bodies in BL21 (DE3). We co-refolded the inclusion bodies of Mamu-alpha and Mamu-beta2m with SIV nonapeptide YY9 and obtained the correct refolded protein complex. Then we purified the protein complex by the gel filtration and anion-exchange column. With hanging-drop method, we screened and optimized for the protein crystal. We managed to collect a X-ray diffraction with the resolution to 2.8 angstroms in the condition of 0.1 mol/L BIS-TRIS (pH5.5), 2.0 mol/L(NH4)2SO4. This crystal belong to perpendicular space group P2(1)2(1)2(1), with unit-cell parameters a = 128.99 angstroms, b = 129.01 angstroms, c = 129.03 angstroms. This data is available for the structure determination.
Animals ; Crystallography, X-Ray ; Epitopes ; immunology ; Escherichia coli ; genetics ; metabolism ; Histocompatibility Antigens Class I ; biosynthesis ; genetics ; Macaca mulatta ; Oligopeptides ; biosynthesis ; genetics ; Simian Immunodeficiency Virus ; immunology

Objective : To learn the passive smoking exposure and hazard awareness among the residents aged 18 years and over in Zhengzhou,so as to provide evidence for tobacco control. Methods: By multi-stage stratified and clustered sampling method,the residents aged 18 years and over in Zhengzhou were selected. From June to October in 2018,a questionnaire for risk factors of non-communicable diseases,designed by Chinese Center for Disease Control and Prevention,was used to collect their passive smoking exposure and hazard awareness and then analyzed. Results : A total of 6 793 questionnaires were qualified in 6 809 questionnaires and the effective rate was 99.77%. Among 5 387 non-smokers,2 131 people were exposed to passive smoking,and the crude and standardized rate was 39.56% and 40.14%. The standardized rate of passive smoking exposure was higher in men than in women（42.44% vs. 38.67%,P<0.05）,in rural residents than in urban residents（43.90% vs. 36.62%,P<0.05）,and it decreased with the age increase（P<0.05）. The standardized rate of passive smoking exposure in family,indoor workplace and indoor public place was 30.99%,36.99% and 68.02%,respectively. The standardized awareness rate of “passive smoking exposure may lead to heart disease,lung disease in children and lung cancer in adults” was 56.63%,which was higher in urban rural residents than in rural residents（75.69% vs. 36.33%,P<0.05）. Conclusions The passive smoking exposure rate was high in Zhengzhou,especially in indoor public places. The awareness of the hazards of passive smoking exposure was scarce, especially in rural residents.

There is a great need for new vaccine development against influenza A viruses due to the drawbacks of traditional vaccines that are mainly prepared using embryonated eggs. The main component of the current split influenza A virus vaccine is viral hemagglutinin (HA) which induces a strong antibody-mediated immune response. To develop a modern vaccine against influenza A viruses, the current research has been focused on the universal vaccines targeting viral M2, NP and HA proteins. Crystallographic studies have shown that HA forms a trimer embedded on the viral envelope surface, and each monomer consists of a globular head (HA1) and a "rod-like" stalk region (HA2), the latter being more conserved among different HA subtypes and being the primary target for universal vaccines. In this study, we rationally designed the HA head based on the crystal structure of the 2009-pandemic influenza A (H1N1) virus HA as a model, tested its immunogenicity in mice, solved its crystal structure and further examined its immunological characteristics. The results show that the HA globular head can be easily prepared by in vitro refolding in an E. coli expression system, which maintains its intact structure and allows for the stimulation of a strong immune response. Together with recent reports on some similar HA globular head preparations we conclude that structure-based rational design of the HA globular head can be used for subtype-specific vaccines against influenza viruses.
Animals ; Antibodies, Viral ; immunology ; Crystallography, X-Ray ; Drug Design ; Female ; Freund's Adjuvant ; administration & dosage ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; immunology ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza Vaccines ; administration & dosage ; biosynthesis ; Influenza, Human ; immunology ; prevention & control ; virology ; Mice ; Mice, Inbred BALB C ; Models, Molecular ; Protein Folding ; Recombinant Proteins ; genetics ; immunology ; Structure-Activity Relationship ; Vaccination ; Vaccines, Subunit ; administration & dosage ; biosynthesis

PD-L1 is a member of the B7 protein family, most of whose members so far were identified as dimers in a solution and crystalline state, either complexed or uncomplexed with their ligand(s). The binding of PD-L1 with its receptor PD-1 (CD279) delivers an inhibitory signal regulating the T cell function. Simultaneously with the Garboczi group, we successfully solved another structure of human PD-L1 (hPD-L1). Our protein crystallized in the space group of C222(1) with two hPD-L1 molecules per asymmetric unit. After comparison of reported B7 structures, we have found some intrinsic factors involved in the interaction of these two molecules. Based on these results, we tend to believe this uncomplexed hPD-L1 structure demonstrated its potential dimeric state in solution, although it could just be an evolutionary relic, too weak to be detected under present technology, or still a functional unit deserved our attentions.
Antigens, CD ; chemistry ; immunology ; B7-H1 Antigen ; Crystallography, X-Ray ; Evolution, Molecular ; Humans ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Secondary ; T-Lymphocytes ; chemistry ; immunology

Avian influenza A virus continues to pose a global threat with occasional H5N1 human infections, which is emphasized by a recent severe human infection caused by avian-origin H7N9 in China. Luckily these viruses do not transmit efficiently in human populations. With a few amino acid substitutions of the hemagglutinin H5 protein in the laboratory, two H5 mutants have been shown to obtain an air-borne transmission in a mammalian ferret model. Here in this study one of the mutant H5 proteins developed by Kawaoka's group (VN1203mut) was expressed in a baculovirus system and its receptor-binding properties were assessed. We herein show that the VN1203mut had a dramatically reduced binding affinity for the avian α2,3-linkage receptor compared to wild type but showed no detectable increase in affinity for the human α2,6-linkage receptor, using Surface Plasmon Resonance techonology. Further, the crystal structures of the VN1203mut and its complexes with either human or avian receptors demonstrate that the VN1203mut binds the human receptor in the same binding manner (cis conformation) as seen for the HAs of previously reported 1957 and 1968 pandemic influenza viruses. Our receptor binding and crystallographic data shown here further confirm that the ability to bind the avian receptor has to decrease for a higher human receptor binding affinity. As the Q226L substitution is shown important for obtaining human receptor binding, we suspect that the newly emerged H7N9 binds human receptor as H7 has a Q226L substitution.
Air Microbiology ; Crystallography, X-Ray ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; metabolism ; Humans ; Influenza A Virus, H5N1 Subtype ; chemistry ; metabolism ; Influenza A Virus, H7N9 Subtype ; chemistry ; Models, Molecular ; Mutant Proteins ; chemistry ; genetics ; metabolism ; Protein Binding ; Protein Stability ; Receptors, Cell Surface ; genetics ; metabolism ; Solubility ; Surface Plasmon Resonance ; Temperature

Enolase is a conserved cytoplasmic metalloenzyme existing universally in both eukaryotic and prokaryotic cells. The enzyme can also locate on the cell surface and bind to plasminogen, via which contributing to the mucosal surface localization of the bacterial pathogens and assisting the invasion into the host cells. The functions of the eukaryotic enzymes on the cell surface expression (including T cells, B cells, neutrophils, monocytoes, neuronal cells and epithelial cells) are not known. Streptococcus suis serotype 2 (S. suis 2, SS2) is an important zoonotic pathogen which has recently caused two large-scale outbreaks in southern China with severe streptococcal toxic shock syndrome (STSS) never seen before in human sufferers. We recently identified the SS2 enolase as an important protective antigen which could protect mice from fatal S.suis 2 infection. In this study, a 2.4-angstrom structure of the SS2 enolase is solved, revealing an octameric arrangement in the crystal. We further demonstrated that the enzyme exists exclusively as an octamer in solution via a sedimentation assay. These results indicate that the octamer is the biological unit of SS2 enolase at least in vitro and most likely in vivo as well. This is, to our knowledge, the first comprehensive characterization of the SS2 enolase octamer both structurally and biophysically, and the second octamer enolase structure in addition to that of Streptococcus pneumoniae. We also investigated the plasminogen binding property of the SS2 enzyme.
Amino Acid Sequence ; Binding Sites ; Crystallography, X-Ray ; Humans ; Models, Molecular ; Molecular Sequence Data ; Phosphopyruvate Hydratase ; chemistry ; metabolism ; Plasminogen ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Solutions ; Species Specificity ; Streptococcus suis ; enzymology