HWTX-I is a peptide neurotoxin purified from the crude venom of the Chinese bird Spider Selenocosmia Huwena, which has analyesic activity. rHWTX-I expressed by P. pastoris and secreted to culture supernatant was first precipitated by (NH4)2SO4, then it was isolated and desalted by ultrofiltration following by ion exchange chromatography of CM column, after reverse phase HPLC of C18 column and vacuum drying, the pure HWTX-I protein was obtained which was proved to be recombinant HWTX-I by Tricine SDS-PAGE, MALDI-TOF mass spectrometry, amino acid composition analysis, the N-terminal amino acid sequence and its biological activity. The final yield of the purified HWTX-I was about 80 mg/L accounting for 23.6% of its total secretory proteins.
Amino Acid Sequence ; Animals ; Chromatography, High Pressure Liquid ; methods ; Chromatography, Ion Exchange ; methods ; Culture Media ; Gene Expression ; Hydrogen-Ion Concentration ; Male ; Mice ; Molecular Sequence Data ; Neuromuscular Junction ; drug effects ; Neurotoxins ; genetics ; isolation & purification ; pharmacology ; Pichia ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; pharmacology ; Reptilian Proteins ; Seminiferous Tubules ; drug effects ; Sequence Analysis, Protein ; Spider Venoms ; genetics ; isolation & purification ; pharmacology ; Spiders ; Synaptic Transmission ; drug effects ; Time Factors

To seek the reason of heterogeneity of recombinant HWTX-I (rHWTX-I) expressed in Pichia pastoris. We expressed HWTX-I gene of interest in Pichia pastoris GS115/HWTX-I. The heterogenous product expressed was separated, purified and identified by using Ion exchange HPLC, reverse HPLC, Tricine SDS-PAGE and MALDI-TOF Mass Spectrometry and then sequenced in both N-terminus and C-terminus. These results show that the heterogeneity of rHWTX-I results from the incomplete processing of signal peptide of N-terminus and the internal degradation of C-terminus. Biological activity assay shows that the activity of the heterogenous rHWTX-I only showed 30% activity compared with the native HWTX-I. The Solutions to how to avoid the heterogeneity are also discussed.
Animals ; Neurotoxins ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Reptilian Proteins ; biosynthesis ; genetics ; Spider Venoms ; biosynthesis ; genetics

OBJECTIVETo investigate the chemical constituents of the leaves of Aquilaria sinensis, and provide a certain of basis for the comprehensive uses of the plant of A. sinensis.

METHODThe chemical constituents were isolated by various column chromatographic method. The structures were identified by spectral analyses of NMR, MS, et al.

RESULTThirteen compounds were isolated and identified as 7-hyroxy-5, 4'-dimethoxy flovone (1), 5-hydroxy-7, 4'-dimethoxy flavone (2), luteolin-7-3',4'-trimethyl (3), isocorydine (4), 4-hydroxybenzoic acid (5), triacontenoic (6), hentriacontane (7), alpha-stigmasterol (8), epifriedelanol (9), friedelan (10), friedelin (11), genkwanin (12), 5, 4'-dihyroxy-7, 3'-dimethoxy flovone (13).

CONCLUSIONCompound 4 was obtained from this genus for the first time, compounds 1, 6-11, 13 were obtained from this species for the first time.

Magnetic Resonance Spectroscopy ; Organic Chemicals ; analysis ; chemistry ; isolation & purification ; Plant Leaves ; chemistry ; Thymelaeaceae ; chemistry

BACKGROUND:Delayed graft function (DGF) occurs frequently in kidney transplants from donation after cardiac death if creatinine level is high in kidney recipients. OBJECTIVE:To analyze the clinical effects of renal transplantation with kidneys from donors dying of cardiac death in organophosphate poisoning. METHODS:Data were col ected from kidney transplants from two donors dying of cardiac death in organophosphate poisoning. After some donor maintenance, donor organ were obtained and perfused with impulse type machine. Recipients were treated with intervention of immunity induction, anti-rejection drugs and infection prevention drugs during and after renal transplantation. Pathological data of donor kidney zero needle biopsy, DGF after kidney transplantation, complication rate (such as acute rejection), renal al ograft recovery situation, the survival rate of recipients and kidney transplants were col ected and analyzed. RESULTS AND CONCLUSION:Needle biopsy results from four donor kidneys showed that glomerular morphology was normal, but there were edema and degeneration in kidney tubules in some degree. Donor DGF rate was 75%(3/4), acute rejection rate was 0%(0/4), perioperative period donor kidney and recipient survival rate were 100%(4/4). Al recipients showed a good result of transplanted kidney, their creatinine and urea nitrogen were at low level, and had no proteinuria. One recipient died of severe pulmonary infection 4 months after surgery. For some organophosphate poisoning donors dying of cardiac death, donor kidney quality can be improved by suitable donor maintenance and high-quality donor kidney preservation using machine perfusion. Kidney transplants from donors dying of cardiac death in organophosphate poisoning who receive the maintenance of organ function may be a promising candidate for renal transplantation due to a severe lack of kidney donor sources.

Embryonic stem cells are pluripotent and their differentiation in vitro can serve as an experimental model to explore the molecular mechanisms of early embryonic development. To investigate the effect of stromal cell conditioned medium combined with cytokines (sccm + cys) on the differentiation from human embryonic stem cells to hematopoietic cells and endothelial cells, the mouse fibroblast feeder cells to make human embryonic stem cells grown into embryonic bodies (EBs) were initially deleted. After culture for 3 days, EB cells were trypsinized into single cells and induced for 8 days by sccm + cys. Then, the differentiated cells were cultured in the semisolid medium containing 0.9% methylcellulose and cytokines to study the colony forming and self-renewal ability of cells. Immunocytochemical staining was used to check the surface markers of the colony cells. During the induction, mRNA expression of flk-1, BMI-1, scl, and Zeta-globin genes was tested by RT-PCR. Surface markers, such as flk-1, CD34 were tested by the flow cytometry. The results demonstrated that: (1) cell clusters containing 20-30 cells were formed after culture for 8 - 14 days in the semisolid medium, replanting these cells resulted in similar cell cluster forming. In addition, CD45 positive in big cell colonies were also found in the semisolid medium; (2) attached cell colonies appeared after culture for 8 days in the semisolid medium and VIII factor, UEA and KDR could be detected as negative by immunocytochemical staining; (3) on the 4(th) day of induction, mRNAs of flk-1, BMI-1, scl and Zeta-globin were all expressed. On the 8(th) day of induction, all of the above genes except Zeta-globin were expressed, while ES cell and EB cells which served as controls did not express scl and Zeta-globin genes; (4) on the 8(th) day of induction, the proportions of flk-1(+) cells and CD34(+) cells among all the inducing population were 9.8% and 16.8%, respectively, while the corresponding positive populations were 0.36% and 1.16% in spontaneously differentiated 11(th) day's EB, and 0.04% and 0.16%, respectively, in ES cells. If is concluded that embryonic stem cells can differentiate into hematopoietic cells and endothelial cells in combinant culture system of this study.
Cell Differentiation ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; Endothelial Cells ; cytology ; Flow Cytometry ; Globins ; genetics ; metabolism ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunohistochemistry ; Nuclear Proteins ; genetics ; metabolism ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

Animals ; Calcium ; metabolism ; Diaphragm ; physiology ; ultrastructure ; Male ; Muscle Contraction ; Rats ; Rats, Sprague-Dawley ; Respiration, Artificial ; Sarcoplasmic Reticulum ; metabolism

The quality control process throughout the Ebola virus nucleic acid detection in Sierra Leone-China Friendship Biological Safety Laboratory (SLE-CHN Biosafety Lab) was described in detail, in order to comprehensively display the scientific, rigorous, accurate and efficient practice in detection of Ebola virus of first batch detection team in SLE-CHN Biosafety Lab. Firstly, the key points of laboratory quality control system was described, including the managements and organizing, quality control documents and information management, instrument, reagents and supplies, assessment, facilities design and space allocation, laboratory maintenance and biosecurity. Secondly, the application of quality control methods in the whole process of the Ebola virus detection, including before the test, during the test and after the test, was analyzed. The excellent and professional laboratory staffs, the implementation of humanized management are the cornerstone of the success; High-level biological safety protection is the premise for effective quality control and completion of Ebola virus detection tasks. And professional logistics is prerequisite for launching the laboratory diagnosis of Ebola virus. The establishment and running of SLE-CHN Biosafety Lab has landmark significance for the friendship between Sierra Leone and China, and the lab becomes the most important base for Ebola virus laboratory testing in Sierra Leone.
China ; Ebolavirus ; classification ; genetics ; isolation & purification ; Hemorrhagic Fever, Ebola ; diagnosis ; virology ; Humans ; Laboratories ; manpower ; standards ; Laboratory Infection ; Quality Control ; RNA, Viral ; genetics ; Sierra Leone

The correct thoughts and principles of diagnosis and treatment of chronic refractory wounds need to be formulated. Through the relevant domestic and international consensus and based on clinical experience, the Thoughts and principles of diagnosis and treatment of chronic refractory wounds in China is proposed. It is considered that in the diagnosis and treatment of chronic refractory wounds, in the case of fully understanding the patient′s medical history, the following thoughts and principles should be complied in order. (1) Pay attention to the cleanliness of the wound after being cleaned. (2) Reasonably perform debridement to avoid being " excessive" or " not thorough". (3) Reasonably perform examination, diagnosis, and differential diagnosis of pathogenic factors. (4) Treat according to etiology. (5) Find comorbidities and prevent adverse outcomes. (6) Select the correct wound treatment method reasonably and timely. When the conservative wound care treatment is considered, pay attention to embodying the concept of etiological treatment, treat the wound according to the principles of safety, phase, selectivity, and effectiveness, and make a reasonable choice of continuing conservative treatment or surgical treatment in time after completing the preparation of the wound bed. When surgical treatment is considered, pay attention to the selection of reasonable surgical method and donor site, pay attention to the healing rate of surgical wound site and the outcome of donor site, and give reasonable protection to the wound site after surgery. (7) Carry out rehabilitation treatment after wound healing and related health education.

ObjectiveTo evaluate the effect of ejaculatory duct dilation combined with seminal vesicle clysis in the treatment of refractory hematospermia.

METHODSUsing ureteroscopy, we treated 32 patients with refractory hematospermia by transurethral dilation of the ejaculatory duct combined with clysis of the seminal vesicle with diluent gentamicin.

RESULTSThe operation was successfully accomplished in 31 cases, with the mean operation time of 32 (26－47) minutes. The patients were followed up for 6－39 (mean 23.6) months. No complications, such as urinary incontinence and retrograde ejaculation, were found after operation. Hematospermia completely disappeared in 27 cases, was relieved in 1, and recurred in 3 after 3 months postoperatively. Those with erectile dysfunction or mental anxiety symptoms showed significantly decreased scores of IIEF-Erectile Function (IIEF-EF) and Self-Rating Anxiety Scale (SAS).

CONCLUSIONSEjaculatory duct dilation combined with seminal vesicle clysis under the ureteroscope, with its the advantages of high effectiveness and safety, minimal invasiveness, few complications, and easy operation, deserves general clinical application in the treatment of refractory hematospermia.

Dilatation ; Ejaculatory Ducts ; surgery ; Genital Diseases, Male ; Hemospermia ; surgery ; Humans ; Male ; Postoperative Period ; Recurrence ; Seminal Vesicles ; surgery ; Ureteroscopy

Objective: To study the variation patterns of rhizospheric soil nutrient and the content of nutrient elements for Fritillaria taipaiensis,in order to provide the theoretical base for the soil improvement and balanced fertilization during the artificial cultivation. Method: Totally 14 samples of rhizospheric soil of Fritillaria taipaiensis from different origins and years were taken as the research objects. Total N,available N,total P,available P,total K,available K,organic matter,pH and 7 nutrient element contents (Ca,Mg,Na,Mn,Zn,Cu and Ni) were analyzed by the soil agrochemical analysis method combined with the atomic absorption spectrophotometry. SPSS 22.0 software was applied for data multiple comparison and correlation analysis. Result: The all results showed significant differences (P<0.05) and certain relative correlations between samples of different origins or years, and the rhizospheric soil nutrient or the content of nutrient elements of F. taipaiensis. The content of total N,available N,total P,available P and organic matter of rhizospheric soil collected from cultivated varieties decreased with the increase of years,and the content of total K,available K and pH decreased first and then increased. However,the soil physical and chemical properties of wild varieties had no obvious change with the increase of years. The content of Ca,Mg,Na and Cu of soil from cultivated varieties decreased with the increase of years,while the content of Mn decreased first and then increased. And Zn and Ni showed no significant change with the increase of years. Compared with cultivated varieties,the content of Ca,Mg,Na,Mn and Cu increased first and then decreased. The content of Zn and Ni showed no obvious change. In general,the rhizospheric soil nutrient and the content of nutrient elements for wild F. taipaiensis were superior to those of cultivated varieties. Conclusion: The third year is the turning point of F. taipaiensis growth. The rhizospheric soil nutrient and the content of nutrient elements decreased obviously after three years. Attention shall be given to the balanced fertilization,the improvement of soil quality and the prevention of the cropping during cultivation of F. taipaiensis.