Objective To sythesize 99Tcm labeled asparagine-glycine-arginine (NGR)- interferon (INF)-α2a and investigate its biodistribution by scintigraphy in tumor bearing mice. Methods NGR-INFα2a was labeled with 99Tcm by a two-step method. Ethylenedicysteine (EC) and MDP were used as bifunctional and transferring chelating agents. The bioactivities of 99Tcm-NGR-IFN-EC-NGR-IFN-α2a, EC-NGRIFN-α2a and NGR-IFN-α2a were compared using least significant difference t-test. The hepatoma bearing mice models were established by subcutaneous injection of MHCC97-H cells. The mice were randomly divided into eight groups and 7.4 MBq 99Tcm-NGR-IFN-α2a was injected via the tail vein. The tissue uptake of the radiolabeled compound was measured as % ID/g. The scintigraphy was performed at 0.5, 1, 2, 4,6, 8, 12 and 24 h after injection. ROI were drawn around tumor and non-tumor tissue and the radioactivity ratio of T/NT was calculated. Results Both the labeling efficiency and radiochemical purity of 99Tcm-EC-NGR-IFN-α2a were more than 90%. The radiochemical purity was 71% after 24 h in saline. The bioactivity showed no significant difference among three compounds (t = 0.416, 0. 120 and 1. 300, all P >0.05). The tracer was mainly excreted through alimentary and urinary tract within 24 h after injection. The peak values of % ID/g in kidney, liver, interstinal tract and tumor were 41.5 ± 8.0_ (at 8 h), 31.3 ± 5.0(at 6 h), 36.0 ± 7.8 (at 6 h), 43.0 ± 4.8 (at 4 h), respectively. The tracer was cleared quickly from the blood and the highest T/NT ratio was 16.5. The optimal imaging time ranged from 4 to 8 h after injection. Conclusions The sythesis of 99Tcm-NGR-IFN-α2a is applicable and it may be used as a potential tumor imaging agent.

A fusion gene CTB-PROIN, in which Proinsulin gene was fused to the 3' end of CTB gene by a hinge peptide 'GPGP', was constructed and cloned into pET-30a(+) to obtain a prokaryotic expression vector pETCPI. Subsequently the recombinant plasmid pETCPI was transformed into E. coli stain BL21 (DE3). After induced by IPTG, the expression product was analyzed by sodium dodecyl sulphate-polyacrylamide gel (15%) electrophoresis (SDS-PAGE), and its result indicated that the recombinant protein CTB-PROIN was expressed and accumulated as inclusion bodies. The recombinant CTB-PROIN protein accumulated to the level of 25% of total bacterial proteins. After inclusion bodies was denaturalized and refolded in vitro, significant assembly of monomers had occurred, and the recombinant protein represented assembled pentamers. The results of western blotting analysis also demonstrated that the fusion protein could be recognized by the anti-CT and anti-insulin antibody, respectively. In addition, the result of the CTB-PROIN-GM1 binding assay, that the protein could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. These results provided the possibility of developing a cheaper and more efficient oral vaccine for type I diabetes using such constructs.
Artificial Gene Fusion ; Cholera Toxin ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; G(M1) Ganglioside ; metabolism ; Proinsulin ; genetics ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVESTo detect the loss of heterozygosity (LOH) of circulating DNA in the plasma of patients with hepatocellular carcinoma (HCC), and to assess its potential as a clinical predictive marker.

METHODSThree high-polymorphic microsatellite markers D8S277, D8S298 and D8S1771 located at chromosome 8p were selected to detect LOH in plasma DNA of 62 HCC patients. The associations between LOH and its clinicopathological features, including HBsAg, liver cirrhosis, serum AFP level, tumor size, tumor cell differentiation, and intrahepatic metastasis were also examined.

RESULTSIn plasma DNA of the 62 HCC patients, LOH was found at one or several loci in 36 (58.1%), and heterozygosity at D8S277, D8S298, and D8S1771 loci was 74.2% (46/62), 75.8% (47/62), and 69.4% (43/62), respectively. LOH frequency at D8S277, D8S298 and D8S1771 was 32.6% (15/46), 44.7% (21/47), and 46.5% (20/43), respectively. LOH in plasma DNA was more frequently detected in the patients with intrahepatic cancer metastasis than those without metastasis (62.5 percent vs. 26.1 percent, P < 0.05); however, no statistically significant correlations were observed between LOH at these loci and other clinicopathological features analyzed in this study.

CONCLUSIONSLOH at D8S298 in plasma DNA may be a potential predictive marker of intrahepatic metastatic recurrence after surgical resection of the HCC.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; blood ; genetics ; Chromosomes, Human, Pair 8 ; DNA ; blood ; Female ; Humans ; Liver Neoplasms ; genetics ; Loss of Heterozygosity ; Male ; Middle Aged

BACKGROUNDCorneal dystrophy is a group of inherited blinding diseases of the cornea. This study was to identify the mutations of the keratoepithelin (KE) gene for proper diagnosis of corneal dystrophy.

METHODSThree families with corneal dystrophy were analysed. Thirteen individuals at risk for corneal dystrophy in family A, the proband and her son in family B, and the proband in family C were examined after their blood samples were obtained. Mutation screening of human transforming growth factor beta-induced gene (BIGH3 gene) was performed.

RESULTSFive individuals in family A were found by clinical evaluation to be affected with granular corneal dystrophy and carried the BIGH3 mutation W555R. However, both probands in families B and C, also diagnosed with granular corneal dystrophy, harboured the BIGH3 mutation R124H.

CONCLUSIONMolecular genetic analysis can improve accurate diagnosis of corneal dystrophy.

Adolescent ; Adult ; Child ; Child, Preschool ; Corneal Dystrophies, Hereditary ; genetics ; pathology ; Extracellular Matrix Proteins ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Transforming Growth Factor beta ; genetics

Vp1 gene of O type foot-and-mouth diseases virus and M. tuberculosis HSP70 were expressed in methylotrophic yeast Pichia pastoris expression system. The results of cellular immune responses and humoral immune response were examined after BALB/c mice were immunized with fusion protein expressed in methylotrophic yeast Pichia pastoris. The genes was cloned into the vector pPICZalpha-A by routine molecular technique. The plasmid fusion (pPICZalphaA-vp1-HSP70) was created that HSP70 located downstream of VP1 gene of O type foot-and-mouth disease virus. Vp1 was expressed by fusing to the amino terminus of M. tuberculosis hsp70 in yeast Pichia pastoris. The recombined fusion plasmid was transformed into methylotrophic yeast Pichia pastoris X-33 by electrophoration. The recombinant transformants were selected by Zeocin and induced by the addition of methanol every 24h. The expressived product analyzed by SDS-PAGE and Western blotting. The result indicated that the fusion protein(vp1-HSP70) has specific antigenicity. Mice were inoculated transcutaneous three times at a two-weeks interval with fusion protein, PBS and conventional inactivated vaccines. To evaluate the prophylaxtic efficacy of fusion protein, Titers of antibodies was detected by ELISA and proliferation of lymphocytes were determined by MTT. The results indicated that fusion protein could elicit specific humoral immune and cellular immune responses. Compared with conventional inactivated vaccines, fusion protein elicited slightly lower FMDV antibody level but stronger T cell proliferation.
Animals ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; Enzyme-Linked Immunosorbent Assay ; Foot-and-Mouth Disease Virus ; immunology ; HSP70 Heat-Shock Proteins ; genetics ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Pichia ; genetics ; Recombinant Fusion Proteins ; immunology ; Vaccines, Synthetic ; immunology ; Viral Vaccines ; immunology

This study was aimed to compare HHGV678 with imatinib (IM) in growth inhibition of Bcr-Abl wild type and IM-resistant cell lines, investigate the possibility of replacing IM with HHGV678 in treatment of chronic myeloid leukemia (CML) and IM-resistant CML patients. Viability of two Bcr-Abl wild type cell lines (K562 and 32Dp210) and 16 IM-resistant cell lines (K562R and 15 Bcr-Abl point mutant cell lines) treated with HHGV678 and IM was analyzed by MTT. The apoptosis of those cells was identified by flow cytometry with Annexin V staining and DNA ladder analysis. Western blot was applied for detecting the expression of Bcr-Abl and phosphotyrosine protein levels. The results indicated that HHGV678 significantly inhibited the growth of two Bcr-Abl wild types and IM-resistant cell lines in dose-dependent manner except cell line of T315I point mutant. IC(50) results showed that the growth inhibition of HHGV678 was 15.5 and 28-fold higher than that of IM in K562, 32Dp210 and 1.4 to 124.3-fold higher than that of IM in 15 IM-resistant cell lines respectively. Compared with IM, HHGV678 more significantly inhibited phosphotyrosine kinase protein of the cells mentioned above at different concentrations. With most importance, HHGV678 of 10.0 micromol/L induced cell apoptosis of 40.06% and 33.32% in K562R and 32Dp210(T315I) cell lines, which were much higher than that of IM (19.77% and 10.68%). It is concluded that HHGV678 is more effective than IM in the growth inhibition of Bcr-Abl wild type cell lines and IM-resistant cell lines, especially in strongest IM-resistant cell lines. Further studies are needed to show whether HHGV678 may be a novel targeting drug in treatment of CML and IM-resistant CML patients.
Aminopyridines ; Apoptosis ; drug effects ; Benzamides ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fusion Proteins, bcr-abl ; metabolism ; Humans ; Imatinib Mesylate ; Piperazines ; pharmacology ; Protein Kinase Inhibitors ; pharmacology ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; Pyrimidines ; pharmacology

OBJECTIVES: To investigate the clinical treatment outcomes and the changes of the outcomes over time in extremely preterm twins in Guangdong Province, China. METHODS: A retrospective analysis was performed for 269 pairs of extremely preterm twins with a gestational age of <28 weeks who were admitted to the department of neonatology in 26 grade A tertiary hospitals in Guangdong Province from January 2008 to December 2017. According to the admission time, they were divided into two groups: 2008-2012 and 2013-2017. Besides, each pair of twins was divided into the heavier infant and the lighter infant subgroups according to birth weight. The perinatal data of mothers and hospitalization data of neonates were collected. The survival rate of twins and the incidence rate of complications were compared between the 2008-2012 and 2013-2017 groups. RESULTS: Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of severe asphyxia and smaller head circumference at birth (P<0.05). The mortality rates of both of the twins, the heavier infant of the twins, and the lighter infant of the twins were lower in the 2013-2017 group compared with the 2008-2012 group (P<0.05). Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of pulmonary hemorrhage, patent ductus arteriosus (PDA), periventricular-intraventricular hemorrhage (P-IVH), and neonatal respiratory distress syndrome (NRDS) and a higher incidence rate of bronchopulmonary dysplasia (P<0.05). CONCLUSIONS There is a significant increase in the survival rate over time in extremely preterm twins with a gestational age of <28 weeks in the 26 grade A tertiary hospitals in Guangdong Province. The incidences of severe asphyxia, pulmonary hemorrhage, PDA, P-IVH, and NRDS decrease in both the heavier and lighter infants of the twins, but the incidence of bronchopulmonary dysplasia increases. With the improvement of diagnosis and treatment, the multidisciplinary collaboration between different fields of fetal medicine including prenatal diagnosis, obstetrics, and neonatology is needed in the future to jointly develop management strategies for twin pregnancy.
Bronchopulmonary Dysplasia/epidemiology* ; Female ; Gestational Age ; Humans ; Infant ; Infant, Extremely Premature ; Infant, Newborn ; Pregnancy ; Respiratory Distress Syndrome, Newborn/epidemiology* ; Retrospective Studies ; Treatment Outcome

Aortic Valve/surgery* ; Aortic Valve Stenosis/surgery* ; China/epidemiology* ; Heart Valve Prosthesis Implantation ; Humans ; Severity of Illness Index ; Treatment Outcome