The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.
Animals ; Animals, Genetically Modified ; genetics ; Base Sequence ; Caseins ; genetics ; Cloning, Organism ; DNA, Complementary ; genetics ; Gene Knock-In Techniques ; Genetic Engineering ; methods ; Genetic Vectors ; chemical synthesis ; Goats ; genetics ; Humans ; Mammary Glands, Animal ; cytology ; metabolism ; Molecular Sequence Data ; Mutant Proteins ; genetics ; Tissue Plasminogen Activator ; genetics

The present study was to explore the functional and morphological changes in cochleas of guinea pig models of early endolymphatic hydrops. Thirty albino guinea pigs were randomly divided into three groups: control, 4-week model and 8-week model groups. For each group, n = 10. Model groups were operated on the right ears to result in endolymphatic hydrops with the method of slight destruction of endolymphatic sac and duct from extradural posterior cranial fossa approach, and the animals in control group were sham operated. Electrocochleogram recorded by trans-tympanic approach and auditory brainstem response (ABR) were tested in preoperative model groups, control group, 4-week model group and 8-week model group to assess the hearing changes. Histologic morphometry was used to quantify hydrops by testing scala media area (SMA) ratio. Scanning electron microscope was used to assess the changes of cochlea hair cells. The results showed that the summating potential/compound action potential (SP/AP) ratio of electrocochleogram in 4-week model group (0.33 ± 0.14) and 8-week model group (0.43 ± 0.14) increased significantly, compared with that in control group (0.07 ± 0.06). The maximum SMA ratio in 4-week model group (2.64 ± 0.10) and 8-week model group (3.54 ± 0.13) increased significantly, compared with that in control group (1.06 ± 0.08). The results of maximum SMA ratio correlated with SP/AP ratio of electrocochleogram (r = 0.86). The results of hearing threshold of ABR revealed that the operated ears of model groups were higher than the preoperative results at frequencies of 2 kHz and 4 kHz. And the damage of cochlea hair cells in operated ears occurred in apical and subapical turns. These results suggest the increased SP/AP ratio of electrocochleogram can indicate early endolymphatic hydrops. There is low-tone hearing loss in guinea pig models of early endolymphatic hydrops, and it may be associated with the abnormalities of the stereocilia among the outer hair cells in operated ears which occurs in apical and subapical turns.
Animals ; Cochlea ; pathology ; physiopathology ; Endolymphatic Hydrops ; complications ; physiopathology ; Guinea Pigs ; Hair Cells, Auditory, Outer ; pathology ; Hearing Loss, Sensorineural ; etiology ; pathology ; physiopathology ; Male

OBJECTIVETo study the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) and detect JAK2 mutation in BMSCs from myeloproliferative neoplasms (MPN) patients.

METHODSJAK2 V617F mutation and exon 12 mutation in 70 MPN patients' blood or bone marrow samples were detected. Isolated BMSCs were then characterized their phenotype, mesenchymal differentiation capacity and existence of JAK2 mutation.

RESULTSBMSCs derived from the patients were similar with healthy donors in terms of morphology, surface antigen and differentiation ability. Of them, 38 patients' blood or bone marrow samples harbored JAK2 V617F, and identified that 3 V617F-negative-patients' samples existed JAK2 exon 12. No patients' BMSC harbored JAK2 mutation though their blood or bone marrow samples carried JAK2 mutation.

CONCLUSIONBMSCs from MPN patients had similar biological characteristics with healthy donors. BMSCs from MPN patients known to bear JAK2 mutation in blood or bone marrow cells didn't carry the mutation.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow Cells ; cytology ; Bone Marrow Neoplasms ; genetics ; Case-Control Studies ; Child ; DNA Mutational Analysis ; Female ; Humans ; Janus Kinase 2 ; genetics ; Male ; Mesenchymal Stromal Cells ; cytology ; Middle Aged ; Mutation ; Myeloproliferative Disorders ; genetics ; Young Adult

OBJECTIVETo study a new implant material (carbonated hydroxyapatite, CHA) united pedicle screw to cure spine fracture.

METHODSThirty-two cases of spine compressed fracture were used with pedicle screw fixator and vertebroplasty. Before operation, patients' vertebral body were compressed (46 + 21)% (20% approximately 70%) on average. In operation, broken vertebral body was reposition through pedicle screw technique, then used self-made syringe to inject CHA into anterior and central column of broken vertebral body through pedicle. And all of patients were not given any bone-graft.

RESULTSIn 6 - 26 months followed-up, no immunologic rejection was found about hydroxyapatite, and no any broken of the screws and shafts was found, no loosing and other complications either. All the patients could move in 3 - 5 days after operation. The height of the broken vertebral body were reduced 97% compared with pre-operation. And CHA in vertebral body was degraded gradually, and at the same time it was replace by new bone in vertebral body. After operation, VAS score was 61 +/- 32, and there was significant difference compared with pre-operation.

CONCLUSIONSThe pedicle screw fixation united vertebroplasty is an efficient way to prevent the failure of the treatment of spine fracture.

Adult ; Bone Screws ; Bone Substitutes ; therapeutic use ; Durapatite ; therapeutic use ; Female ; Follow-Up Studies ; Fracture Fixation, Internal ; instrumentation ; methods ; Fractures, Compression ; surgery ; Humans ; Male ; Middle Aged ; Spinal Fractures ; surgery ; Vertebroplasty ; methods

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD (OmpW) was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced (such as AnsB, AspA, FrdAB, and KatG) or repressed (such as AceEF, OmpX, SodA, and SucAB) during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.
Bacterial Proteins ; analysis ; Computational Biology ; Electrophoresis, Gel, Two-Dimensional ; Gene Expression Profiling ; methods ; Kinetics ; Peptide Mapping ; Proteome ; analysis ; Proteomics ; methods ; Shigella flexneri ; growth & development ; metabolism ; pathogenicity ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Temperature ; Trypsin ; pharmacology ; Virulence

OBJECTIVETo explore the concentration of polybrominated diphenyl ethers (PBDEs) in umbilical cord serum and analyze the influence of exposure to PBDEs during fetal stage on newborn birth outcomes in Shanghai.

METHODSFifty delivery women in a Shanghai hospital were surveyed by questionnaire, and the umbilical cord serum were collected from September 2006 to April 2007. All the delivery women were singleton pregnancies, excluding high blood pressure, diabetes, HIV infection and adverse medical history. Seven congeners (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154 and BDE-183) were measured by gas chromatography-negative chemical ionization-mass spectrometry and the influencing factors were analyzed.

RESULTSNewborns' length, weight, chest circumference, head circumference and body mass index (BMI) were (50.15 ± 0.75) cm, (3.49 ± 0.42) kg, (34.76 ± 1.51) cm, (35.03 ± 1.40) cm, (13.76 ± 1.36) kg/m(2), respectively. The median of Σ(7PBDEs) concentration in umbilical cord serum was 14.06 (1.03 - 379.73) ng/g lipid weight (lw). The detection rate of BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154 and BDE-183 were 22% (11/50), 22% (11/50), 98% (49/50), 72% (36/50), 76% (38/50), 90% (45/50) and 14% (7/50), respectively. The median (range) of PBDEs (BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183) congeners were < LOD (< LOD-137.20 ng/g lw), < LOD (< LOD-33.17 ng/g lw), 7.54 ng/g lw (< LOD-94.01 ng/g lw), 1.57 ng/g lw (< LOD-46.95 ng/g lw), 0.63 ng/g lw (< LOD-79.08 ng/g lw), 0.63 ng/g lw (< LOD-22.30 ng/g lw) and < LOD (< LOD-21.63 ng/g lw), respectively. The newborns' BMI showed a negative correlation with BDE-99 (r = -0.347, P < 0.05) and BDE-154 (r = -0.292, P < 0.05). BDE-99 in low-length group (≤ 50 cm, 10.59 ng/g lw) was significantly higher (t = 2.32, P = 0.03) than that in high-length group (> 50 cm, 3.60 ng/g lw).

CONCLUSIONPBDEs were commonly detected in newborns' umbilical cord serum in this study. Our findings indicated that exposure to PBDEs adversely affected the development of the newborns.

China ; Female ; Fetal Blood ; chemistry ; Humans ; Infant, Newborn ; Maternal Exposure ; Polybrominated Biphenyls ; blood ; Pregnancy ; Prenatal Exposure Delayed Effects ; Surveys and Questionnaires

This study is to investigate the protective effect of quercetin against adriamycin-induced cardiotoxicity and its mechanism. The cardiotoxicity was induced by intraperitoneal injection of adriamycin (ADR) at a single dose of 20 mg x kg(-1). Mice were randomly divided into 5 groups (n=20): normal control group, ADR 20 mg x kg(-1) group, quercetin (50, 100, and 200 mg x kg(-1) groups, intragastric administration, once a day, for 7 days before ADR administration). The health conditions, electrocardiogram, activity of iNOS, SOD and LDH, levels of NO and MDA in serum or tissue homogenate, the ultrastructure and the expression of p53 protein in cardiac tissue of mice were observed. Compared with the normal control group, ADR decreased the amplitude of ECG's R wave (P < 0.001), increased the incidence of arrhythmia (to 60%), injured myocardial ultrastructure, increased the activity of LDH and iNOS, and levels of NO and MDA, decreased the activity of SOD, and increased the expression of p53 (P < 0.001). Compared with ADR 20 mg x kg(-1) group, the quercetin decreased the levels of LDH, iNOS, NO and MDA, increased the activity of SOD, restored the amplitude of R wave, decreased the incidence of arrhythmia and p53 expression (P < 0.001 , P < 0.01 or P < 0.05), and markedly reduced the myocardial ultrastructure injury. Quercetin had protective effect against adriamycin-induced cardiotoxicity. The mechanism may be related to its enhancing myocardial SOD activity, decreasing iNOS activity and inhibiting myocardial apoptosis.
Animals ; Apoptosis ; drug effects ; Arrhythmias, Cardiac ; blood ; chemically induced ; metabolism ; pathology ; Doxorubicin ; Female ; L-Lactate Dehydrogenase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Mice ; Myocardium ; metabolism ; ultrastructure ; Myocytes, Cardiac ; metabolism ; ultrastructure ; Nitric Oxide ; blood ; Nitric Oxide Synthase Type II ; blood ; Protective Agents ; pharmacology ; Quercetin ; pharmacology ; Random Allocation ; Superoxide Dismutase ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo explore the risk factors of acute lymphoblastic leukemia (ALL) recurrence in adult patients and establish a prognosis index (PI) calculation model in order to improve the prevention strategy of ALL in adults.

METHODS104 adult ALL patients from Blood Diseases Hospital & Chinese Academy of Medical Sciences between August 2008 and November 2011 were enrolled. COX proportional hazards regression stratified by Dummy variable was used to set up the prediction model; Kaplan-Meier method and Log-rank test were used to estimate and compare the survival. After calculated individual PI value, patients' expected survival should be estimated by groups.

RESULTSThe overall median survival of adult ALL patients was 22.00 months (95% CI 17.00-27.00). COX regression analysis showed that chemotherapy group patients had a higher risk of recurrence than of ASCT group while setting treatment as the dummy variable (RR=2.052, 95%CI 0.877-4.799, P=0.007). Stratified Analysis showed that the risk factors of B-ALL recurrence in adult patients included HGB <100 g/L (RR=0.186, 95% CI 0.068-0.512, P=0.001), CNSL (RR=7.767,95% CI 2.951- 20.433, P=0.001), number of consolidation chemotherapy<3 (RR=0.445, 95% CI 0.211-0.940, P=0.034) and Ph chromosome positive (RR=2.771, 95% CI 1.353-5.674, P=0.005). Grouped by the PI value, the expected survival of each individual patient could be estimated as PI=0.58 base.

CONCLUSIONHGB, CNSL, number of consolidation chemotherapy and Ph chromosome were independent risk factors of B-ALL recurrence in adult patients. PI value could predict the survival of adult ALL patients and provide reference for individual therapy and prognostic evaluation.

Adolescent ; Adult ; Female ; Humans ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; epidemiology ; pathology ; Prognosis ; Recurrence ; Risk Assessment ; Risk Factors ; Young Adult

Objective To explore the efficacy and safety of sodium valproate in a higher dose in the evening for treating nocturnal and early-morning seizures.Methods A total of 69 newly diagnosed pediatric patients with nocturnal or early-morning seizures were enrolled.They were randomly distributedinto experimental group (35 cases) and control group (34 cases) and treated with sodium valproate tablets.The initial dose was little.It was gradually increased to the effective maintenance dose.With sodium valproate given twice a day,the post meridiem(PM) dose was twice the ante meridiem(AM) dose in the experimental group,while the PM/AM dose was equal in the control group.All patients were at least been followed up for 6 months.Results In the experimental group,28 cases were seizure free (80.0％),and the total effective rate was 85.7％.In the control group,20 cases were seizure free (58.8％),and the total effective rate was 64.7％.The difference in the total effective rate between the 2 groups was significant (P ＜ 0.05).No severe adverse effect was found among all patients.The incidence of daytime sleepiness (0/35 cases) in the experimental group was lower than that in the control group (2/34 cases).Conclusions A higher dose of sodium valproate in the evening for nocturnal and early-morning seizures led to better seizure control,better nocturnal sleep,less daytime somnolence,and the side effects are slight.

This study was aimed to explore whether multiple common gene mutations of leukemia synergistically involved in acute promyelocytic leukemia (APL) pathogenesis, and to investigate their relevance to clinical features, cytogenetics and molecular risk stratification. 84 specimens of admitted de novo APL patients from February 2005 to October 2010 were collected, the gene mutations of bone marrow mononuclear cells and clinical features of mutation-positive patients were analyzed by genomic DNA-PCR. The results indicated that the prevalence of mutations was 60.7% (51/84), in which the mutations with the highest incidence were found as FLT3-ITD, reaching 27.4% (23/84). Next, there were 12 cases WT1 mutation, 9 for FLT3-TKD, 7 for TET2, 5 for N-RAS, 4 for ASXL1, 2 for EZH2 mutation and 1 positive case in MLL-PTD, IDH1 and CBL mutation respectively. No mutation was found in other JAK1, DNMT3, c-Kit, NPM1, IDH2, RUNX1 and JAK2 (V617F) common leukemia-related genes. Combined analysis with clinical data demonstrated that the patients with FLT3-ITD mutation displayed higher white blood cell counts, while the patients with N-RAS mutation showed lower platelet counts. Overall survival of these patients was obviously shorten as compared with patients with wild-type. This difference between mutant and wild-type of all above mentioned cases was statistically significant (P < 0.05). The difference between APL with simple t (15;17) and additional abnormal karyotype was not statistically significant. It is concluded that the FLT3-ITD mutation is recurrent genetic change in APL, and together with N-RAS mutation indicates poor prognosis. Additional abnormal karyotype does not associate with prognosis of APL.
Adolescent ; Adult ; Aged ; Child ; DNA Mutational Analysis ; DNA-Binding Proteins ; genetics ; Enhancer of Zeste Homolog 2 Protein ; Female ; Genes, ras ; Humans ; Leukemia, Promyelocytic, Acute ; genetics ; Male ; Middle Aged ; Mutation ; Nuclear Proteins ; genetics ; Polycomb Repressive Complex 2 ; genetics ; Prognosis ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Repressor Proteins ; genetics ; Tandem Repeat Sequences ; Young Adult ; fms-Like Tyrosine Kinase 3 ; genetics