To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

Objective To evaluate the effect of BML-111 on NF-κB pathway during acute lung injury induced by hemorrhagic shock and resuscitation in rats.Methods Thirty-two adult male Sprague-Dawley rats,weighing 200-250 g,were randomly divided into 4 groups (n =8 each) using a random number table:sham operation group (group S),hemorrhagic shock and resuscitation group (group HSR),BML-111 group,and BML-111 + BOC-2 (lipoxin A4 receptor antagonist) group (group BOC-2).The animals were anesthetized with intraperitoneal pentobarbital sodium.Hemorrhagic shock was induced by blood letting and maintained for 30 min.The animals were then resuscitated for 30 min by infusion of the shed blood and lactated Ringer's solution.In group BOC-2,BOC-2 (50 μg/kg) was injected intraperitoneally before blood letting.In BML-111 and BOC-2 groups,BML-111 (1 mg/kg) was injected intraperitoneally at the beginning of resuscitation.The rats were sacrificed at 2 h after the end of resuscitation and lungs were removed for determination of pathological changes,myeloperoxidase (MPO) activity,intercellular adhesion molecule-1 (ICAM-1) expression (by immunohistochemistry),tumor necrosis factor-alpha (TNF-α) content (by ELISA),and NF-κB p65 and IκB-α expression (by Western blot).Results Compared with group S,the MPO activity,ICAM-1 expression,and TNF-α content were significantly increased,NF-κB p65 expression was up-regulated,and IκB-α expression was down-regulated in group HSR.Compared with group.HSR,the MPO activity,ICAM-1 expression,and TNF-α content were significantly decreased,NF-κB p65 expression was down-regulated,IκB-α expression was up-regulated,and pathological changes of lung were attenuated in group BML-111.Compared with group BML-111,the MPO activity,ICAM-1 expression,and TNF-α content were significantly increased,NF-κB p65 expression was up-regulated,and lκ:B-α expression was down-regulated,and pathological changes of lung were aggravated in group BOC-2.Conclusion BML-1 11 inhibits activation of NF-κB pathway and inflammatory responses,thus mitigating acute lung injury induced by hemorrhagic shock and resuscitation in rats.

OBJECTIVETo investigate the effects of pro-inflammatory cytokine interleukin-1β (IL-1β) on mineralization potential of dental pulp stem cells (DPSC).

METHODSRat DPSC were cultured in vitro and randomly divided into three groups, IL-1β (10 µg/L), osteogenic inductive medium and non-osteogenic inductive medium. After 3, 7, and 12 days of treatment, the cultures were evaluated for cell proliferation and calcium deposit. Real-time polymerase chain reaction was used to detect the gene expression levels of osteocalcin (OC), bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) and dentin matrix protein 1 (DMP-1). In vivo test, after 3 day's treatment with IL-1β, the cell-scaffold complexes were implanted subcutaneously in mice for 8 weeks. Histological analysis was performed to evaluate hard tissue formation.

RESULTSIn vitro test, after 3-day's treatment, IL-1β improved cell proliferation to 137.22 DNA µg/L and cell viability becomes (97.12 ± 7.18)% of control. The gene expression levels of OC, BSP, DSPP and DMP-1 are (378.19 ± 16.22)%, (427.12 ± 18.22)%, (247.19 ± 10.11)% and (198.29 ± 10.23)% respectively. The results of IL-1β's group was notable increased compared with non-osteogenic induction medium and the statistical differences are significant. IL-1β induced the odontogenic differentiation of DPSC. However, these effects tended to continuously decrease with treatment time. Histological analysis demonstrated that in the group treated with IL-1β hard tissue was markedly formed in vivo.

CONCLUSIONSIL-1β may induce the mineralization of DPSC and play an important role in host defenses and tissue repair.

Animals ; Calcification, Physiologic ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dental Pulp ; cytology ; Extracellular Matrix Proteins ; metabolism ; Integrin-Binding Sialoprotein ; metabolism ; Interleukin-1beta ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Osteocalcin ; metabolism ; Phosphoproteins ; metabolism ; Rats ; Rats, Wistar ; Sialoglycoproteins ; metabolism

OBJECTIVETo study the antigenic properties of mutant hepatitis B virus surface antigen, to understand the sensitivity of the commercially available HBsAg assays to the variants and to reduce the undetectability of the variants.

METHODSRecombinant eukaryotic expression plasmids for HBsAg. The recombinant eukaryotic expression plasmids pSS1adr, pSS1adw2, pSS1adw2- 145Arg, pSS1adr-126 Asn and pSS1adr-126Ser were transfected into COS-7 cells. HBsAg in the supernatants of transfected cells was detected by using different commercial ELISA kits.

RESULTSThe absorbance value of pSS1adr-126 Asn and pSS1adr-126Ser plasmids were similar to that of the wild type HBsAg, the absorbance value of pSS1adw2-145Arg plasmids was lower than that of the wild type HBsAg.

CONCLUSIONIt is estimated that the antigenicity of HBsAg mainly depended on the amino acid sequence of "a" antigen determinant and its conformation, so 145 amino acid substitutions led to the change of conformation and the antigenicity of variant HBsAg was lower than that of the wild type.

Animals ; COS Cells ; Cercopithecus aethiops ; Culture Media, Conditioned ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; analysis ; genetics ; immunology ; Mutation ; Transfection ; Viral Envelope Proteins ; genetics

To analyze the constituents in supercritical fluid CO2 extraction (SFE-CO2) of Radix caulophylli, the Radix caulophylli was extracted with SFE-CO2, and analyzed by gas chromatography-mass spectrometry (GC-MS). The GC-MS analysis with a DB-5MS capillary column (30 mm x 0.32 mm ID, 0.25 microm film thickness) was used. The inlet temperature was maintained at 280 degrees C. The column oven was held at 80 degrees C for 2 min, then programmed from 80 to 280 degrees C at 5 degrees C x min(-1) and, finally, held for 4 min. Helium at a constant flow rate of 2.0 mL x min(-1) was used as the carrier gas. The mass spectrometry conditions were as follows: ionization energy, 70 eV; ion source temperature, 200 degrees C. The mass selective detector was operated in the TIC mode (m/z was from 40 - 500). For the first time 49 peaks were separated and identified, the compounds were quantitatively determined by normalization method, and the identified compounds represent 97.44% of total GC peak areas. Viz, n-hexadecanoic acid (31.4%), (E, E) -9, 12-octadecadienoic acid (26.54%), (Z)-7-tetradecenal (9.4%), hexadecenoic acid (3.23%), 10-undecenal (3.22%), octadecanoic acid (2.25%), and caulophylline (1.76%) etc. The results will provide important foundation for understanding the constituents and further exploitation of Radix caulophylli.
Carbon Dioxide ; Caulophyllum ; chemistry ; Chromatography, Supercritical Fluid ; Gas Chromatography-Mass Spectrometry ; Linoleic Acid ; analysis ; Palmitic Acid ; analysis ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry

OBJECTIVETo construct full-length hepatitis B core particles presenting preS1 aa 21-47 epitope and truncated core particles presenting preS1 aa 37-45 epitope on their surface and compare their antigenicity.

METHODSPreS1 aa21-47 epitope and aa 37-45 epitope were inserted respectively into full-length hepatitis B core (aa 1-183) and truncated HBcAg (aa 1-144), between the 78th (Asp) and 79th (Pro). The genes synthesized after the codon optimization were ligated to the pET43. 1a vector with the same cohesive terminal (NdeI and XhoI) and expressed in the E. coli expression system. The morphology of the proteins of interest were observed by electron microscope and characterized by ELISA and Western Blotting.

RESULTSThe morphology of the virus-like particles were confirmed by electron microscope. H2 were solid particles with a diameter of (31.61 +/- 1.27) nm, while H3 were hollow particles with a diameter of (28.46 +/- 1.16) nm. Statistical analysis showed that H2 is larger than H3 in the diameter (P < 0.01). The antigenicity of the inserted epitopes and carrier protein were identified by ELISA and Western Blotting.

CONCLUSIONChimeric hepatitis B core particles presenting the preS1 neutralizing epitopes on their surface have been expressed, purified and identified, which lays the foundation for its application in vaccine research.

Epitopes ; chemistry ; genetics ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B Core Antigens ; chemistry ; genetics ; immunology ; Hepatitis B Surface Antigens ; chemistry ; genetics ; immunology ; Hepatitis B virus ; chemistry ; genetics ; immunology ; Humans ; Neutralization Tests ; Protein Precursors ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology

OBJECTIVETo evaluate the vulnerability to floods in Guangdong province at district level.

METHODSData were collected from the sixth census, the 2010 Statistical Yearbook of Guangdong, the 2010 Health Statistics Yearbook of Guangdong and China Disease Prevention and Control information systems, etc. The weight of each indicator was determined based on subjective method and objective method respectively; and finally the results of the two methods were compared.

RESULTS13 indicators were selected for the assessment of vulnerability to floods, including 6 sensitivity indicators, 5 adaptability indicators and 2 exposure indicators. Indicators with large weight (subjective weight/objective weight) were the proportion of population older than 65 years old (0.31/0.30), the proportion of population older than 65 years old (0.16/0.23), infant mortality rate (0.18/0.20), the total Gross Domestic Product (GDP) per capita (0.33/0.21), the proportion of illiterate in the population older than 15 years old (0.19/0.28), history frequency of floods (0.75/0.75). The mean vulnerability index (VI) calculated by subjective method was 0.35 with the standard deviation of 0.10; the mean vulnerability index calculated by objective method was 0.31 with the standard deviation of 0.08. The two weighting methods showed consistent results of vulnerability index (ICC = 0.975, P < 0.01). VI of most districts dropped in the interval of 0.30 - 0.39. Districts with subjective VI > 0.50 or objective VI > 0.40 should pay more attention to floods, including parts of the coastal areas, Beijiang River Basin, the eastern tributary area of Dongjiang River and the northern part of Pearl River Delta. Dapu district of Meizhou (0.55/0.45), Dianbai district and Maogang district of Maoming (0.54/0.48) were most vulnerable. Districts of Heyuan, Dongguan, Zhaoqing and Huizhou were less vulnerable, Yuancheng district of Heyuan showed least vulnerable to floods (0.15/0.12) followed by Dongguan (0.18/0.16), Duanzhou district (0.18/0.16) and Guangning (0.17/0.15) district of Zhaoqing. The score of indicators differed among different level of vulnerability (P < 0.05).

CONCLUSIONDifferent regions of Guangdong province showed different vulnerability to floods, vulnerable areas should be priority in the prevention and control of floods.

China ; Climate ; Demography ; Disasters ; Floods ; Humans ; Risk Assessment ; Rivers

OBJECTIVETo determine the main genotype of hepatitis B virus (HBV) in Xinjiang.

METHODS200 HBsAg positive serum specimens were detected from more than 2000 serum of Xinjiang inhabitants, and HBV S gene was detected by using nPCR amplifying, and compared with the standard S region HBV nucleotide sequences of genotypes A-H retrieved from GenBank, then analyzed and drawn the polygenetic tree by MEGA3 software.

RESULTGene in 127 (63.5%) serum specimens was detected from 200 samples. Among 127 serum specimens, 10 (7.8%) was genotype B, 58 (45.7%) was genotype C, and 59 (46.5%) was genotype D.

CONCLUSIONGenotype B, C and D have been found in Xinjiang.

Adolescent ; Adult ; Aged ; Child ; China ; Female ; Genotype ; Hepatitis B ; ethnology ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Rural Health ; Young Adult

OBJECTIVETo evaluate the role of nutritional status on serum immunoglobulins, body weight and postoperative infectious-related complications in patients with Crohn's disease receiving perioperative parenteral nutrition (PN).

METHODS32 patients with Crohn's disease receiving perioperative parenteral nutrition in our department between 1984 and 1994 were enrolled in this survey. 16 patients with loss of body weight in the range of 15%-30% were assigned to the malnutrition group, the other 16 patients with normal weight or loss of body weight less than 15% to the control group. Serum IgM, IgG and IgA levels were measured before and after PN by enzyme-linked immunosorbent assays. Liver function, body weight changes and postoperative complications were also analyzed.

RESULTSIgM levels were elevated before PN in both groups [control group: (133 +/- 16) mg/dl, malnutrition group: (139 +/- 41) mg/dl; normal value: (110 +/- 35) mg/dl; P = 0.04], decreased to normal value [(105 +/- 29) mg/dl, P = 0.02] in the malnutrition group while having no obvious changes in the control group [(129 +/- 13) mg/dl, P = 0.34]. No significant changes in concentrations of IgG and IgA were found (P in the range of 0.20-0.57). The average weight gain was 1.862 kg in malnutrition group [before PN: (45.8 +/- 8.9) kg, after PN: (48.0 +/- 8.8) kg; P = 0.005] and no significant changes in the control group [before PN: (55.6 +/- 6.1) kg, after PN: (56.3 +/- 6.0) kg; P = 0.46]. There was an increase in infectious complications in the control group (control group: 4 cases, 25%, malnourished group: 2 cases, 12.5%; P = 0.13).

CONCLUSIONSPerioperative parenteral nutrition ameliorated the humoral immunity, increased the body weight in patients with obvious malnutrition, whereas it had little value for those without or with mild malnutrition.

Adult ; Aged ; Body Weight ; Crohn Disease ; immunology ; surgery ; therapy ; Female ; Humans ; Immunoglobulins ; blood ; Male ; Malnutrition ; etiology ; Middle Aged ; Nutritional Status ; Parenteral Nutrition ; Pneumonia ; etiology ; Postoperative Complications ; etiology

Objective To investigate the effect of Zheng's 4C suspension transumbilical laparoendoscopic single-site surgery(TU-LSSS)in the treatment of cervical cancer,and analyze its prognosis.Methods A total of 92 patients with cervical cancer admitted to our hospital were selected and randomly divided into the control group(traditional laparoscopic surgery)and the observation group(Zheng's 4C suspension TU-LSSS),with 46 cases in each group.The perioperative related indicators of patients between the two groups were compared.The patients were followed up for 3 years,the progression-free survival(PFS)and overall survival(OS)were recorded,and the influencing factors of prognosis were analyzed.Results Compared with the control group,patients in the observation group had longer surgical time(P<0.05),less intraoperative bleeding(P<0.05),shorter recovery time for postoperative bowel sounds and hospital stay(P<0.05),higher postoperative 3-year PFS rate and 3-year OS rate(P<0.05).During 3-year follow-up,there were 11 cases of disease progression or death.The univariate analysis results showed that there were statistically significant differences in tumor diameter,clinical stage,lymph node metastasis,vascular infiltration,and treatment methods of patients between the good prognosis group and the poor prognosis group(P<0.05).The binary Logistic regression results showed that tumor diameter(≥4 cm),clinical stage(≥stage ⅠB2),lymph node metastasis,and vascular infiltration were the risk factors for the prognosis of patietns with cervical cancer(P<0.05),while Zheng's 4C suspension TU-LSSS was the protective factors(P<0.05).Conclusion Zheng's 4C suspension TU-LSSS can effectively promote the recovery of patients with cervical cancer,and improve 3-year PFS rate and 3-year OS rate.The prognosis is related to many factors,and targeted treatment should be performed according to different risk factors.