Bone Marrow Cells ; Cell Line ; Cell Proliferation ; Humans ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes ; cytology ; T-Lymphocytes, Regulatory ; cytology

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

Cochlea ; metabolism ; Deafness ; genetics ; Humans ; Mutation

ObjectiveTo explore the risk factors of obstructive hydrocephalus secondary to intracranial hemorrhage in premature infants.MethodsA total of 304 premature infants were selected who were diagnosed as sever intracranial hemor-rhage (grade III and IV) by cranial bedside ultrasound admitted to our hospital from Jun. 2013 to Sep. 2014. According to wheth-er the obstructive hydrocephalus was followed, all infants were divided into hydrocephalus group (n=59) and non-hydrocephalus group (n=185). The risk factors of obstructive hydrocephalus secondary to intracranial hemorrhage were analyzed and the lateral ventricle size was measured dynamically.ResultsThe univariate analysis showed the factors related with obstructive hydro-cephalus were as follows: gestational age≤32 weeks, birth weight< 1500g, severe asphyxia, cesarean section, RDS, neonatal infection, heart failure, PDA, acidosis, thrombocytopenia, coagulation abnormalities, and intracranial hemorrhage (gradeⅢ orⅣ) (allP<0.05). Multivariate logistic regression analysis showed that acidosis, thrombocytopenia, coagulation abnormalities, gesta-tional age≤ 32 weeks, severe asphyxia, intracranial hemorrhage (gradeⅢ orⅣ) were independent risk factors for obstructive hydrocephalus (OR: 1.76~20.46, allP<0.05). At each time point after birth, the ratio of posterior horn of lateral ventricle was signiifcantly higher in hydrocephalus group than that in non-hydrocephalus group (P<0.05). There were signiifcant differences in the changes of the posterior horn ratio of left or right lateral ventricle with time in hydrocephalus group (P=0.000), increasing at 14 days and reaching the peak at 28 days after birth.ConclusionsThe risk factors for obstructive hydrocephalus secondary to intracranial hemorrhage in neonates are important. Regular and dynamical monitoring of ventricle size by cranial ultrasound is needed in infants with sever intracranial hemorrhage.

OBJECTIVE:To study the stability and to predicate the expiration date of betamethasone cream.METHODS:The HPLC method was used to determination the content of betamethasone,the stability test of cream was done by initial mean velocity method and strong light exposure experiment,respectively.RESULTS:The linear concentration range for betametha?sone cream was0.25～8?g/ml(r=0.9991),the average recovery rate was98.56%,RSD=0.78%;The content was highly correlated to temperature,whereas on which the light exposure did little effect.CONCLUSIONS:Betamethasone cream should be stored under room temperature and in a cool place,it is predicted that the expiration date of betamethasone cream is1year.

Objective: To investigate the effect of urinastatin on the expression levels of NF-?B and TNF-? liver transplantation in patients. Methods: Thirty-six patients of late stage hepatic cirrhosis were randomly divided into two eaquel groups: the experiment group was administrated by urinastatin 300 000 U and saline 10 mL via venous injection during liver transplantation, the control group was administrated 10 mL saline by venous injection at the same time. The blood samples were harvestd at 1, 2, 4 and 6 h after the blood recovery of donated liver. The nuclear factor-?B (NF-?B) P65 relative level of blood was detected by Western blot analysis and the TNF-? level of blood was detected by double antibody Sandwich enzyme linked immunosorbent assay. Serum ALT and AST were also measured. Results: The expression level of serum NF-?B p65, TNF-? in the experiment group were lower than those in the control group, and the difference was significant(P

Objective To report the clinical and magnetic resonance imaging(MRI)features of 5 eases with idiopathic orbital myositis.Methods Four females and one male,aged 27 to 57 years,presented department of neurology in the First Hospital of Peking University in October 2008 to September 2009.The duration of disease Was between 3 months and 4 years.Recurrent course appeared in 3 of them.0rbital MRI Was performed in all of them.After diagnosis they underwent long.term corticosteroid treatment.Results All patients presented ocular pain,asymmetrical and incomplete ophthalmoplegia and mild proptosis.EMG revealed no significant decline in repetitive stimulation.Muscle biopsies of limb muscle were unremarkable.Creatine kinase and thyroid function test were in normal limits.MRI revealed unilateral.focal or difluse enlargement and enhancement of extraocular muscles,involving 1 extraocular muscle in 2 cases,2extraocular muscles in 2 cases,more extraocular muscles in 1 case.No evidence indicated bone destruction or cavernous sinus abnormalities.Five Cases showed improvement and remission after long-term administration of steroids.Conclusion Persistent and asymmetrical ophthalmoplegia is connnon in orbital myositis.Extraocular muscle swelling characterized the MRI changes.

Objective To investigate the possible existence of HBoV in children with acute respiratory infections in Nanjing area and explore its relationship with clinical characteristics.Methods A total of 397 nasopharyngeal secretion samples were collected from children with acute respiratory infection,admitted from July 2009 to June 2010 in Nanjing Children'S Hospital affiliated to Nanjing Medical University,and 50 cases of children without symptoms of respiratory infection were recruited as control group,whose nasopharyngeal secretion samples were also collected.HBoV was determined by real-time fluorescence quantitative PCR.MP and CT were detected by real-time fluorescence quantitative PCR in those HBoV-positive samples.RSV,ADV,IVA,IVB,PIV-1,PIV-2,PIV-3 and hMPV were detected by direct antigen-specific immunofluorescence assays.HBoV NP-1 fragments were amplified and sequenced in 5 HBoV positive samples randomly selected.The results were compared with the known GenBank sequence,and thereby the phylogenetic tree was established.The epidemiological characteristics,clinical presentation and the final clinical diagnosis of HBoV were analyzed according to the clinical data of the HBoV-positive patients.Results Thirty-three HBoV-positive cases were detected by real-time fluorescence quantitative PCR method with a positivity rate of 8. 3% ( 33/397 ). Among the 33 HBoV-positive cases, 19 cases (57.6%) were multiple infections with HBoV and other pathogens, the top three of which were MP (27.3% ,9/33 ),RSV (24.2% , 8/33 ) and PIV-3 ( 12. 1% ,4/33 ). Affected children aged from 7 to 36 months old accounted for 75.8% of the total ( 25/33 ). The measured HBoV NP-1 gene sequences of 5 specimens were consistent,indicating a high homology (99% to 100% ) with the stl, st2 and WHL-1. Conclusions HBoV is one of the pathogens of children's acute respiratory infections in Nanjing. HBoV NP-1 gene is highly conserved,with little variation in different seasons and in different regions and therefore can be used as a marker for real-time fluorescence quantitative PCR and other methods.

Objective To elucidate the expression of Toll-like receptor 3 (TLR3) on dendritic cells(DCs) in patients with chronic hepatitis B (CHB), and to explore the correlation between hepatitis B virus (HBV) persistent infection and TLR3 expression. Methods Sixty CHB patients (CHB group) and 20 healthy controls (control group) were enrolled. The peripheral blood mononuclear cells (PBMCs) were isolated and CD14~+ monocytes were sorted by immunomagnetic beads. Immature DCs (imDC) were induced and proliferated in vitro and mature DCs (mDC) were obtained after the poly I:C stimulation. The expression of intracellular TLR3 mRNA was detected by real-time polymerase chain reaction (PCR), and surface markers [CD80 and human leucocyte antigen (HLA)-DR] were determined by flow cytometry after 48 h of stimulation. The comparison of quantitative data was done using t test. The qualitative data were compared using chi-square test.Results The mean fluorescence intensities (MFI) of intracellular TLR3 of imDC before poly I:C stimulation in CHB group and control group were 1212.05 ± 250.80 and 1192.95 ± 301.40,respectively, which were not significantly different (t = 0. 280, P>0. 05). While after stimulation,those were 1352.98± 313.67 and 1593. 00± 349. 65, respectively, the latter was significantly higher than the former (t = 2. 880, P<0. 05). The levels of TLR3 mRNA inside mDCs in both groups were increased after poly I:C stimulation, which were 0. 1204 ±0.0267 and 0. 1780 ± 0.0664, respectively in CHB group and control group, and that in control group was significantly higher (t = 3. 909, P<0.05). Furtherly, patients in CHB group were divided into HBeAg(+ ) and HBeAg( -) subgroups.After stimulation, the MFI and mRNA of TLR3 inside mDC were greatly elevated in both subgroups,but there were no difference between these two subgroups (t = 0. 366, P>0. 05). Conclusions The intracellular expressions of TLR3 in mDC in CHB group and control group are obviously increased after the poly I:C stimulation, but the increased level in CHB group is lower than that in control group. The results suggest that the insufficiency of TLR3 synthesis may be related to the HBVpersistent infection.

Objective To detect the expression of type Ⅰ interferon in monocyte-derived dendritic cells(MoDCs)after Toll like receptor(TLR)3 triggered in patients with chronic hepatitis B(CHB),and to evaluate immune responses of CHB patients and its roles in the mechanisms of persistent infection of hepatitis B virus(HBV)and chronicity of hepatitis.Methods Peripheral blood mononuclear cells(PBMCs)were isolated and purified using magnetic beads(plasma was saved simultaneously)from 26 CHB patients and 18 healthy volunteers(HV).Dendritic cells(DCs)were induced and proliferated in a culture medium with recombinant human granulocyte macrophage colony stimulating factor(rhGM-CSF)and recombinant human interleukin(rhIL-4).EX3s were stimulated with Poly Ⅰ:C and the supernatants were collected at 0 h and 24 h after stimulation.Type Ⅰ interferon(IFN-α and IFN-β)in plasma and supernatants were examined by enzyme linked immunosorbent assay (ELISA).Results The levels of type Ⅰ interferon in plasma were not significantly different in groups of HV and CH B.IFN-α and IFN-β expressions in supernatants before Poly Ⅰ:C stimulation were(80.00±16.15)ng/L,(36.39±13.90)ng/L in CHB group and(76.76±15.90)ng/L,(37.14±13.68)ng/L in HV group,respectively.And there were no statistical differences between two groups(t=1.651,t=0.178;both P＞0.05).IFN-α expressions in supernatants at 24 h after stimulation in two groups were both higher than those before stimulation(at 0 h),but there were no statistical differences(t=1.534,t=1.243;both P＞0.05).IFN-β expressions in supernatants at 24 h after stimulation in HV group was(54.57±16.80)ng/L,which was significantly higher than that at 0 h(37.14±13.68)ng/L(t=4.061,P＜0.05).However,there was no significant difference at 24 h than tht at 0 h in CHB group(t=1.796,P＞0.05).At 24 h after stimulation.IFN-β level was(54.57±16.80)ng/L in HV group,which was significantly higher than that[(41.64±12.57)ng/L]in CHB group(t=2.921,P＜0.05).Conclusions Functions of MoDCs from CHB patients are impaired and MoDCs could not express type Ⅰ interferon normally.Expression of type Ⅰ interferon after TLR3 triggered in CHB patients is mainly IFN-β.