Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Huntington's disease (HD) is a deadly neurodegenerative disease with abnormal expansion of CAG repeats in the huntingtin gene. Mutant Huntingtin protein (mHTT) forms abnormal aggregates and intranuclear inclusions in specific neurons, resulting in cell death. Here, we tested the ability of a natural heat-shock protein 90 inhibitor, Gedunin, to degrade transfected mHTT in Neuro-2a cells and endogenous mHTT aggregates and intranuclear inclusions in both fibroblasts from HD patients and neurons derived from induced pluripotent stem cells from patients. Our data showed that Gedunin treatment degraded transfected mHTT in Neuro-2a cells, endogenous mHTT aggregates and intranuclear inclusions in fibroblasts from HD patients, and in neurons derived from induced pluripotent stem cells from patients in a dose- and time-dependent manner, and its activity depended on the proteasomal pathway rather than the autophagy route. These findings also showed that although Gedunin degraded abnormal mHTT aggregates and intranuclear inclusions in cells from HD patient, it did not affect normal cells, thus providing a new perspective for using Gedunin to treat HD.

Objective To establish a BALB/c mouse model of total hysterectomy and to investigate and analyze its characteristics and formation mechanism. Methods The control group was fed conventionally without any treatment. The mice in the model group were anesthetized and performed hysterectomy. The animals in the sham-operated group had abdominal cavity opened and only exposing the uterus. The changes of body weight were observed dynamically,and at the end of the fourth weeks. The concentration of serum estradiol was determined and histological structure of the ovaries was examined. Results The general status of all the mice after operation was good without obvious inflammation, and the incision healed completely. There was no significant difference in the body weight before operation among the mice, whereas the weight of model group mice was higher than the control group and sham-operation group at the end of the second,third and forth weeks(P< 0.05). The estradiol contents of the model group was significantly lower than that in control group and sham operation group(P< 0.01). The histological structure of ovaries in the control group and sham-operation group was normal,however the model group showed differences,such as irregular shape of the ovaries,changed number of mature follicles, loosely distributed granulosa cells, and apoptosis. Conclusions The mouse model of total hysterectomy established in our study can well simulate the clinical manifestations of ovarian dysfunction after operation, and is an ideal model for the study of complications after ovariectomy and screening of drugs.

Kallmann syndrome ( KS) is a rare disease and characteristic of an absence of puberty, infertility, and a defective sensation of smell (anosmia or hyposmia). Here, we analyze the features of a case of KS diagnosed clinically. In addition, the etiology, genetic features, clinical manifestations, diagnosis, and treatment of KS were reviewed.

Klinefelter syndrome(KS) is the most common sex chromosome disorder in males,which is caused by the presence of the extra X chromosome that maybe inherited from mother or father. Approximately 80% karyotype of the cases is 47,XXY. KS is characterized by small firm testes, hypergonadotropic hypogonadism, infertility,gynaecomastia, increased height. However, cognitive disabilities and psychiatric disorders are rarely diagnosed in KS because they lack screening in related aspects. At the present, the pathogenesis of cognitive disabilities and increased risk of psychiatric diseases in KS have not been delineated. In this article,we report two cases of KS,and review their clinical manifestations,diagnosis,and treatments.

Objective To discuss the effects of mutual management model on the recovery of multi-burn family members. Methods The multi-burn family members who were hospitalized the 180th Hospital of PLA from February 2010 to October 2014 were chosen as study objects, and 68 patients who were discharged to the hospital from February 2010 to March 2012 were chosen as the control group, while 70 patients who were discharged to the hospital from April 2012 to October 2014 were chosen as the observation group. The two groups of patients were hospitalized for the same treatment, and the patients in control group received general guidance for rehabilitation, while the patients in observation group were administered with the application of mutual management model being discharged, and the members of the family monitored, helped and encouraged each other; they received follow-up survey at 1 month, 6 months, 1 year and 3 years after being discharged, and the effects of mutual management on improving the quality of life and returning to the job (work and study) were analyzed. Results The quality of life after 6 months discharged in two groups had statistical significance (F=424.679,P<0.01), the quality of life in observation group were higher than that of control group in body, psychology and social function, and status of material life (t=5.115、4.088、4.314、4.760;P<0.01). The length of returning to job or school was shorter than that of control group (χ2=8.101,P<0.05). Conclusions The mutual management model could effectively utilize the resources, and the patients managed with each other, took care each other and encourage each other, so as to improve the quality of life.

Objective: To explore the clinical application of cone beam CT in the diagnosis of traumatic anterior teeth before orthodontic treatment in order to reduce the risk of orthodontic treatment failure due to adhesion. Methods : 48 fixed orthodontic treatment cases with the history of anterior teeth trama were selected. The anterior teeth were examined by CBCT to exclude root and alveolar bone adhesions. Results : 3 cases of root-alveolar bone adhesion were found, and the upper anterioa teeth of the remaining 45 cases were successfully moved by orthodontic treatment. Conclusion CBCT examination is recommended to check the periodontal ligament in patients of fixed orthodontic treatment with the history of anterior teeth trauma in order to exclude the root-alveolar bone adhesion cases.

Objective To understand tobacco use rate and explore the factors associated with tobacco use in middle school students in China.Methods A self-administered questionnaire survey was conducted among 155 117 eligible middle students selected through multi-stage stratified cluster sampling from 31 provinces of China.The questionnaire consists of the following topics:tobacco use and cessation,passive smoking,access to tobacco products,tobacco control and tobacco advertisement,and knowledge about and attitudes to tobacco.Software SAS 9.3 was used for sample weighting and data analysis.Results Current tobacco use rate was 6.9％ in middle school students in China.The rate was higher in boys (11.2％) than in girls (2.2％),and in rural area (7.8％) than in urban area (4.8％).Students in western area had the highest tobacco use rate (10.3％),followed by the students in middle area (6.4％),and the rate in students in eastern area was lowest (4.5％).Parent smoking (especially mother smoking),friend smoking,teacher smoking,pocket money for a week ＞ 20 yuan,tobacco advertisement or promotion,passive smoking,misconception of tobacco addiction,active attitude to smoking behavior were the risk factors for tobacco use.Conclusions Smoking rate in boys in China was higher than the average global level.A comprehensive intervention strategy by Framework Convention on Tobacco Control should be used to reduce tobacco use in adolescents,including health education,increasing cigarette price,banning tobacco advertisement and promotion,and smoke-free legislation.

[Summary] Kisspeptin is vital for the neuroendocrine regulation of GnRH secretion. Kisspeptin neurons are now recognized as a central pathway responsible for conveying key homeostatic information to GnRH neurons. A number of metabolic modulators have been proposed as regulators of kisspeptin including leptin, adiponectin, insulin, ghrelin, POMC, and neuropeptide Y. Recent data indicate that kisspeptin may have a direct role in regulating energy balance by its regulation of food intake and glucose homeostasis. Thus, kisspeptin may serve as a new connection between reproductive function and energy metabolism.

Objective To observe the changes of behavior,blood cortisol level,glucocorticoid receptors (Grs) and mineralocorticoid receptors (MRs) in hippocampus area after four weeks of unpredictable chronic mild stress,and to investigate the probable role of hypothalamus-pituitary-adrenal (HPA) axis in the pathogenesis of depression in aged people.Methods Aged male Wister rats were randomly assigned to control group and model group.The model group received unpredictable mild stress,including food and water deprivation,restrain,tail clipping,forced swimming,white noise,cage titling and cage rotating for 4 weeks,while the control group was undisturbed unless routine feeding and cage changing.After 4 weeks of procedure,the behavior changes were assessed by sucrose intake test,open-field test and state evaluation,serum cortisol level was measured by chemiluminescent assay,the qualitation and quantitation of GRs and MRs in hippocampus area were evaluated by immunohistochemistry and Western blotting respectively.All data were analyzed by using t-test.Results Body weight,the grooming score,activities in openfield test,food intake and sucrose intake were decreased in model group as compared with control group after 2 weeks of chronic mild stress (all P＜0.01),suggesting the stress induced depressive-like behavior effects on aged rats.Serum cortisol level was elevated in model group as compared with control group after 4 weeks of chronic mild stress (P＜0.01).A decrease of the neurons was found in CA3 of hippocampus,but not in DG area.In CA3 area,GR positive neurons were decreased,but no significant decrease was found in MR positive neurons.Conclusions The chronic mild stress leading to depressive-like behavior effects in aged rats induces overall HPA axis dysfunction,elevation of serum cortisol level,impairment of hippocampus neurons and decrease of GR positive neurons.The HPA axis dysfunction induced by chronic mild stress may play an important role in the pathogenesis of depression.