Objective To construct the recombinant plasmid pYES6-S and express and purify spike protein of severe acute respiratory syndrome(SANS)coronavirus in Saccharomyces cerevisiae. Methods DNA fragments of SANS coronavirus were obtained by reverse transeription.Four over- lapped fragments of spike protein genes were amplified by polymerase chain reaction(PCR)and ligated into an integral spike protein gene by restriction enzyme digestion.The spike protein gene recombined with pYES6 and cloned into E.coll.The recombinant plasmid pYES6-S was induced and expressed in Saccharomyces cerevisiae(INVScl)by galactose.Results The recombinant plasmid pYES6-S was confirmed that inserted fragment was right in length,direction and base matching by restriction enzyme digestion and sequencing.The purified protein encoded by the whole spike protein gene was about Mr 110?10~3 identified by electrophoresis.Conclusion The whole spike protein gene of SARS coronavirus is cloned into E.coli and the protein is expressed in Saccharomyces cerevisiae successful ly.which can be helpful in SARS vaccine research.

The rate of corneal graft rejection is still high for high-risk keratoplasty although immune suppression drug is routinely used.The role of traditional Chinese medicine in corneal transplantation is concerned gradually.Heijingpaichitang on the prevention and treatment of rats with high-risk corneal allograft rejection needs further study.Objective This study was to investigate the inhibitory effect of heijingpaichitang on high-risk corneal transplantation immune rejection in rats.Methods Sixteen female SD rats were used as the donors and 32female Wistar rats were served as recipients.The high-risk corneal trasplantation models were established by corneal suture in 32 Wistar rats,and then homogeneity variant SD-Wistar corneal transplantation was performed.The recipients were randomized into model control group,cyclosporinc A (CsA)group,heijingpaichitang group and CsA +heijingpaichitang group.CsA,heijingpaichitang and CsA + heijingpaichitang was orally administered 4 days after operation once per day for 15 days,and normal saline solution was used at the same way in the model control group.Ocular anterior segment reaction was examined under the slit lamp and corneal opacification,edema and neovasculation were scored based on Larkin' s criteria.Rejection index of the corneal graft was recorded and the graft survival time was calculated.The pathological examination of the corneal graft was carried out in all rats,and the inflammatory cells in the corneas and CD4+ cells in the periphery blood were assayed using flow cytometry.The use of the animals complied with ARVO Statement.Results Corneal graft rejection occurred in 10 days after operation in the model control group,12-13 days in the CsA group and heijingpaichitang group and 22 days in the CsA +heijingpaichitang group.Compared with model control group,the scores of the corneal opacification,corneal edema and neovascularization were significantly lower in the CsA group,heijingpaichitang group and CsA+heijingpaichitang group (P＜0.05),and all the scores were declined in the CsA+ heijingpaichitang group compared with CsA group and heijingpaichitang group(P＜0.01),but no significant differences were seen in the scores between the CsA group and heijingpaichitang group(P＞0.05).The mean survival time of grafts was (10.38 ±1.69)days in the model control group,(22.50 ± 3.07) days in the CsA + heijingpaichitang group,with the significant difference (t =-9.790,P =0.000).The pathological examination of graft showed that the lymphocytes and new blood vessels were less in the CsA+heijingpaichitang group compared with CsA group and heijingpaichitang group 15 days after operation.Flow cytometry verified that the number of lymphocytes in graft,CD4+cells and CD4+/CD8+ in periphery blood were significantly lower in the heijingpaichitang group,CsA group and CsA+heijingpaichitang group compared with model control group (P＜0.05).Conclusions Heijingpaichitang can inhibit immune rejection to certain extent in high-risk corneal transplantation rat and has a similar effect to 0.1％ CsA.Heijingpaichitang and 0.1％ CsA have a synergistic effect.

Background Age-related cataract is one of the common causes of blindness.Although the pathophysiology of age-related cataract is far from clearly understood,it is well accepted that DNA damage plays an important role in the disease pathogenesis.Objective The purpose of this study was to quantitatively evaluate the DNA damage in peripheral lymphocytes of age-related cataract.Methods A cross-sectional study was carried out.This study complied Declaration of Helsinki and approved by Ethic Committee of Affiliated Hospital of Nantong University.Written informed consent was obtained from each subject.Two hundred and eleven patients with agerelated cataract and 147 normal subjects were enrolled from a “ Jiangsu Eye Study:Funing 2011 Eye Disease Epidemic Survey”.All the subjects aged from 50 through 80 years with matched age and gender between the two groups.The percentage of tail DNA and Olive tail moment (OTM) were detected by comet assay to assess the extent of DNA damage in peripheral lymphocytes.Statistical analyses were performed with SPSS 17.0 software,and the differences of the percentage of tail DNA and OTM were compared between the age-related cataract group and normal control group by independent sample t test as well as among the 50-59 years group,60-69 years group and ≥70 years group by one-way analysis of variance.Results Comet assay showed a round lymph cell with the clear border in the normal group;while in the age-related cataract group,the cell was bigger with a comet-like tail.The percentage of tail DNA and OTM in peripheral lymphocytes were (21.75 ± 3.51) ％ and 6.54 ± 1.65 in the age-related cataract group,and those in the normal control group were (9.31 ±3.60)％ and 2.18 ± 1.10,respectively,with significant differences between them (t =32.67,P =0.00 ; t =28.02,P =O.00).In the 50-59 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.04±2.86) ％ and 5.92± 1.14,and in the 60-69 years subgroup of the age-related cataract group,the percentage of tail DNA and OTM in peripheral lymphocytes were (20.77 ±2.93) ％ and 6.13 ± 1.14,which were significantly reduced in comparison with (22.79 ± 3.67)％ and 6.95±1.91 of the ≥70years subgroup(TailDNA％:q=2.75,P=0.00; q=2.02,P=0.00;OTM:q=1.03,P =0.02 ; q =0.82,P =0.00).Conclusions The pathogenesis and development of age-related cataract probably is associated with DNA damage.

OBJECTIVE: To investigate the polymorphism and forensic efficiency values of STR loci D12S391/D18S865 METHODS: Population studies on D12S391 and D18S865 were carried out in a sample of 454 unrelated Wenzhou Han individuals using PCR followed by PAGE and silver stain detection. RESULTS: 11/7 alleles and 50/21 genotypes of D12S391/D18S865 were respectively observed among the 454 unrelated individuals. The genotype frequency matched the Hardy-Weinberg equilibrium, The heterozygotes (H) in D12S391/D18S865 were 0.7974/0.7379, Power of Exclusion were 0.9566/0.9077, polymorphism information content (PIC) were 0.8260/0.7279 respectively. CONCLUSION D12S391 and D18S865 were high polymorphic loci and the frequencies were in accordance with Hardy-Weinberg equilibrium (P>0.5), so the two loci can be used in forensic medicine and in other genetic research areas.
Alleles ; China/ethnology* ; DNA/isolation & purification* ; Electrophoresis, Polyacrylamide Gel ; Forensic Medicine ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Tandem Repeat Sequences

OBJECTIVE: To study genetic polymorphism of D7S2846, D19S400 and D18S535 in Han population in Wenzhou. METHODS: DNA was extracted with chelex-100 method from EDTA-blood samples of 194 unrelated individuals in Wenzhou and amplified with PCR technique. The PCR products were analyzed by PAG vertical electrophoresis and silver-stain. RESULTS: 6 alleles and 15 genotypes of D7S2846, 10 alleles and 36 genotypes of D19S400, 8 alleles and 26 genotypes of D18S535 was observed. The heterozygosities of the three loci are 0.644, 0.724 and 0.772; The power of discriminating are 0.854, 0.940 and 0.938. CONCLUSION The heterozygosities of the three loci are high and the frequencies was in accordance with Hardy-Weinberg equilibrium (P>0.05), so the three loci can be used in forensic medicine and in other genetic researches.
Alleles ; China/ethnology* ; DNA/isolation & purification* ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Tandem Repeat Sequences

OBJECTIVEThis study investigated the pathogen distribution and resistance patterns in childhood urinary tract infection in order to provide references for optimal use of antibiotics in the treatment of this disorder.

METHODSThe clinical data of 152 children with community acquired urinary tract infection (urinary culture positive) between December 2001 and December 2004 were studied retrospectively. The bacterial pathogens of urinary tract infection and antimicrobial resistance were analyzed.

RESULTSGram-negative bacilli was predominant pathogenic bacteria, accounting for 79.0% of the cases, and Escherichia coli (E. coli) was most commonly found (56.2%). Gram-positive cocci accounted for 18.4%, including 15.1% of Enterococcus faecalis. Fungi was rarely seen, accounting for only 2.6%. E. coli had a resistance rate of more than 50% to ampicillin, amoxicillin/clavulate, co-trimoxazole, cefradine, and fosomycin, but a very low resistance rate (< 4%) to 3rd generation cefalosporin, nitrofurantoi, azactom and amikacin. Enterococcus faecalis had a low resistance rate (< 20%) to ampicillin, vancomycin, penicillin, and nitrofurantoin.

CONCLUSIONSE. coli is the major pathogen in community acquired pediatric urinary tract infection, and Enterococcus has been become another important pathogen. Selection of antibiotics for the treatment of this disorder should base on drug-sensitive test results.

Adolescent ; Bacteria ; drug effects ; isolation & purification ; Child ; Child, Preschool ; Community-Acquired Infections ; drug therapy ; microbiology ; Drug Resistance, Bacterial ; Female ; Humans ; Infant ; Male ; Urinary Tract Infections ; drug therapy ; microbiology

OBJECTIVETo develop a procedure by which Schwann cells and myelin in the peripheral nerve could be removed while the basal lamina tubes remained intact, and to obtain a thick and long acellular nerve allograft in humans.

METHODSFour ulnar nerves 10.0 cm long and 4.0 - 5.0 mm in diameter were excised from a donated male body and cleaned from external debris. The nerves were treated with a solution of Triton X-100 and a solution of sodium deoxycholate at room temperature. After a final wash in water, the nerves were stored in phosphate-buffered saline (PBS, pH 7.2) at 4 degrees C. HE, luxol fast blue and fibrin staining were performed to visualize cells, myelin and basal membranes respectively and immunohistochemical staining was performed to visualize the presence of laminin, a Schwann cell lamina component, both in fresh and acellular nerve segments. To reveal overall structure better, methylene blue-fuchsin staining was performed in semithin section. The ultrastructure of acellular and fresh nerves were observed and photographed in a transmission electron microscope.

RESULTSThe acellular human ulnar nerve was white long cylinder with well elasticity and ductility. HE, myelin and fibrin staining revealed that cells, axons and myelin sheath were removed and basal membrane was preserved after extraction procedure. Staining for the presence of laminin showed that the Schwann cell basal lamina component were present in the nerves after chemical treatment. Methylene blue-fuchsin staining and transmission electron microscopy showed that the myelin sheaths were absent in the extracted nerve segments and empty basal lamina tubes remained in the endoneurium.

CONCLUSIONSWe developed an extracted procedure with the detergents of Triton X-100 and deoxycholate, by which cells, axons and myelin sheaths could be removed from a human ulnar nerve while the basal lamina tubes remain intact and a thick long acellular nerve allograft is obtained. The laminin, a Schwann cell basal lamina component, can be preserved in the acellular nerve.

Adult ; Axons ; drug effects ; Cell Separation ; methods ; Deoxycholic Acid ; pharmacology ; Humans ; Male ; Myelin Sheath ; drug effects ; Octoxynol ; pharmacology ; Transplantation, Homologous ; Ulnar Nerve ; cytology ; transplantation ; ultrastructure

It is a challenging and important project to prolong the in vivo half life of protein and peptide drugs by physicochemical methods without new molecular entities generation. Protein crystallization provides a new strategy for improving the stability and in vivo delivery of these drugs. We show here that recombinant human interferon-alpha (rhIFN) can form spherical crystals. The physical and chemical features of the crystals were characterized, and drug dissolution was determined in vitro. The pharmacokinetics of crystallized interferon after sc injection in rabbit at 1.5 x 10(7) U x kg(-1) was compared to that of soluble form. The crystals were characterized as mono-dispersed spheres, with yield of > 80%, mean diameter size of about 16 microm and crystallinity of 23.2%. The in vitro dissolution behavior of crystallized rhIFN was featured as low initial burst release (21% within the first 2 h) and prolonged cumulative dissolution time up to 72 h without biological potency lost. After sc administration of soluble and crystallized interferon in rabbits, the peak time (T(max)) and half life (t1/2) were prolonged from (1.80 +/- 0.45) h and (1.35 +/- 0.35) h to (13.20 +/- 2.68) h and (10.68 +/- 1.97) h, respectively. The corresponding peak concentration decreased from (1 411.10 +/- 575.28) U x mL(-1) to (721.37 +/- 206.55) U x mL(-1). PK/PD analysis indicated that (96.87 +/- 20.30) % of relative bioavailability was obtained. The research results of this work will provide important academic value and application prospect for improving clinical therapeutic effect and development of biomacromolecules delivery system for protein and peptide drugs.
Animals ; Antiviral Agents ; administration & dosage ; chemistry ; pharmacokinetics ; Biological Availability ; Crystallization ; Delayed-Action Preparations ; Drug Delivery Systems ; Half-Life ; Humans ; Injections, Subcutaneous ; Interferon-alpha ; administration & dosage ; chemistry ; pharmacokinetics ; Male ; Rabbits ; Recombinant Proteins ; administration & dosage ; chemistry ; pharmacokinetics ; Solubility ; Surface Properties

Objective To explore the diagnostic value of urine neutrophil gelatinase-associated lipocalin (NGAL) in children with acute pyelonephritis. Methods A total of 104 children with urinary tract infection admitted to Tianjin Children's Hospital from December 2016 to May 2017 were selected in this study, including 61 cases with acute pyelonephritis (group APN) and 43 with lower urinary tract infection (group non-APN). The serum levels of beta 2-Microglobulin (β2-MG), cystatin C (CysC), C-reactive protein (CRP), procalcitonin (PCT) and urine levels of NGAL were compared between two groups.Receiver operating characteristic(ROC)curves were drawn to evaluate the diagnostic values of serum β2-MG,CysC,CRP,PCT and urine NGAL.Results The serum levels of CRP,PCT,β2-MG and urinary NGAL were significantly higher in APN group than those in non-APN group (P < 0.05). There was no significant difference in serum CysC level between two groups(P>0.05).The areas under the ROC curve(AUC)for serum CRP,PCT,and urinary NGAL were 0.838,0.898 and 0.963.The optimal cutoff value of serum CRP was 22.6 mg/L,the sensitivity was 75.4% and the specificity was 83.7%. The optimal cutoff value of serum PCT was 0.285 μg/L, the sensitivity was 77.0% and the specificity was 93.0%.The optimal cutoff value of urine NGAL was 473 μg/L,the sensitivity was 82.0% and the specificity was 97.7%.Conclusion Urinary NGAL has high diagnostic value for APN in children,and which is helpful for the early identification of APN.

Objective To determine the urinary iodine level of people in Yunyang and Bishan County of Chongqing and explore into its influencing factors. Methods Using multistage cluster stratified simple random sample method, Yunyang and Bishan County were chosen as research spots, then thirty children aged 8-10 in each 3 primary school of the 2 counties were selected using stratified randomization sampling method to inspected their urine and household salt for iodine and the iodine content in drinking water. Results Five hundred and seventy-one urine samples were inspected and the urinary iodine median was 261.47 μg/L. 5.78% (33/571) and 37.48%(214/571) of samples had an urinary iodine median less than 100 μg/L and more than 300 μg/L. The urinary iodine median of Yunyang County was higher than that of Bishan (H = 7.42, P < 0.01). The iodine salt coverage rate, the qualified rate and edible qualified iodine salt rate respectively were 99.64%(554/556), 94.22% (522/554) and 93.88% (522/556) in 556 samples of family table salt. Eighty-seven samples of drinking water were inspected, resulting an averaged iodine content of 8.81 and 2.97 μg/L, respectively in the 2 counties. Conclusions The 2 counties are all the area of iodine deficiency. The urinary iodine level, although meeting the demand of eliminating iodine deficiency diseases, is a little bit higher given that iodized salt of present doage has been taken for a long time. The content of iodized salt should be adjusted accordingly.