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 discuss the topical action characteristics of the biological transmission of moxibustion heat via temperature collection and numerical modeling.Methods:Temperature of moxibustion was measured at multiple points at a distance of 3 cm to obtain the moxibustion temperature field nephograms by the high-accuracy temperature measure array.Finite element analysis was used to imitate the three-dimensional dynamic distribution of temperature in acupoint tissues.Results:Through numerical analysis,the one-dimensional,two-dimensional and three-dimensional distributions of temperature in human acupoint tissues at 5 min of moxibustion were established.The result showed that moxibustion heat mainly transmitted from the surface of the tissue to the internal,and the influence of moxibustion heat decreased with the depth of the tissue.The analysis of the nephograms of acupoint tissue temperature at 5,10,15 and 20 min of moxibustion showed that with the increase of the moxibustion time,the temperature in acupoint tissues constantly rose,and the transmission depth of moxibustion heat also further expanded inside acupoint.Conclusion:By establishing the three-dimensional dynamic model of heat transmission inside acupoint tissues with the biological parameters of human tissues and the temperature values obtained,this study used finite element analysis software ANSYS 14.0 and discovered the rules in the transmission of heat in body tissues during moxibustion,and the features in moxibustion heat transmission (from the proximal to the distant) and heat penetration (from the surface to the internal).This study provides theoretical and experimental support for the application of moxibustion in clinical practice.

OBJECTIVE: MicroRNAs (miRNAs) play a vital role in pathogenesis and progression of many cancers, including cervical cancer. However, importance of serum level of miR-101 in cervical cancer has rarely been studied. In the present study, clinical significance and prognostic value of serum miR-101 for cervical cancer was investigated. METHODS: Association between miR-101 level in cervical cancer tissues and prognosis of patients was analyzed by using data retrieved from The Cancer Genome Atlas (TCGA) database, which was followed with our clinical study in which miR-101 serum level comparison between cervical cancer patients and healthy controls was conducted by real-time quantitative polymerase chain reaction (PCR). RESULTS: TCGA database demonstrated that miR-101 was down-regulated in cervical cancer tissues compared with normal cervical tissues, and univariate Cox regression analysis indicated that decreased miR-101 expression was a highly significant negative risk factor. Similar trend was found in the serum miR-101. Serum level of miR-101 was associated with International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.003), lymph node metastasis (p=0.001), and serum squamous cell carcinoma antigen (SCC-Ag) level >4 (p=0.007). The overall survival time of cervical cancer patients with a higher level of serum miR-101 was significantly longer than that of patients with a lower level of serum miR-101. Moreover, multivariate Cox regression analysis indicated that the down-regulated serum level of miR-101 was an independent predictor for the unfavorable prognosis of cervical cancer. CONCLUSION: Serum level of miR-101 is closely associated with metastasis and prognosis of cervical cancer; and, hence could be a potential biomarker and prognostic predictor for cervical cancer.
Carcinoma, Squamous Cell ; Clinical Study ; Disease Progression ; Genome ; Gynecology ; Humans ; Lymph Nodes ; MicroRNAs ; Neoplasm Metastasis ; Obstetrics ; Polymerase Chain Reaction ; Prognosis* ; Risk Factors ; Uterine Cervical Neoplasms*

The essence of endogenous turbidity in Chinese medicine (CM) is different from cream, fat, phlegm, retention, damp, toxicity, and stasis. Along with the development of modern scientific technologies and biology, researches on the essence of endogenous turbidity should keep pace with the time. Its material bases should be defined and new connotation endowed at the microscopic level. The essence of turbidity lies in abnormal functions of zang-fu organs. Sugar, fat, protein, and other nutrient substances cannot be properly decomposed, but into semi-finished products or intermediate metabolites. They are inactive and cannot participate in normal material syntheses and decomposition. They cannot be transformed to energy metabolism, but also cannot be synthesized as executive functioning of active proteins. If they cannot be degraded by autophagy-lysosome or ubiquitin-prosome into glucose, fatty acids, amino acids, and other basic nutrients to be used again, they will accumulate inside the human body and become endogenous turbidity. Therefore, endogenous turbidity is different from final metabolites such as urea, carbon dioxide, etc., which can transform vital qi. How to improve the function of zang-fu organs, enhance its degradation by autophagy-lysosome or ubiquitin-prosome is of great significance in normal operating of zang-fu organs and preventing the emergence and progress of related diseases.
Autophagy ; Humans ; Medicine, Chinese Traditional ; Proteasome Endopeptidase Complex

OBJECTIVETo study the effect of budesonide aerosol inhalation on the expression of glucocorticoid receptor (GR) and nuclear factor (NF)-κB in asthmatic mice.

METHODSTwenty-four healthy male BALB/c mice aged 6 to 8 weeks were randomly divided into three groups (n=8 each): normal saline (control group), asthma model (asthma group) and budesonide-treated asthma (BUD group). Asthma was induced by intraperitoneal injection of ovalbumin (OVA) and aluminium hydroxide suspension and aerosol inhalation of OVA solution. Mice were sacrificed 24 hours after the last challenge. Eosinophil count in the bronchoalveolar lavage fluid (BALF) was determined. Pathological examination of the lung tissues was performed and the expression levels of GR and NF-κB were measured by immunohistochemical analysis.

RESULTSEosinophil count in the BALF was significantly higher in the asthma and BUD groups than in the control group (P<0.05). BUD treatment decreased eosinophil count in the BALF compared with the asthma group (P<0.05). The lung tissues in the BUD group showed a less severe infiltration of eosinophils and lymphocytes compared with the asthma group. The percentage of GR-positive cells in the asthma group decreased significantly compared with the control group (P<0.05), and the percentage of GR-positive cells in the BUD group increased significantly compared with the asthma group (P<0.05). Compared with the control group, the percentage of NF-κB-positive cells increased significantly in the asthma group (P<0.05), and the percentage of NF-κB positive cells in the BUD group was significantly reduced compared with the asthma group (P<0.05).

CONCLUSIONSThe action mechanism of budesonide in treating asthmatic mice may be related to the upregulation of GR expression and the inhibition of NF-κB activity.

Aerosols ; Animals ; Asthma ; drug therapy ; metabolism ; Budesonide ; administration & dosage ; Eosinophils ; Male ; Mice ; Mice, Inbred BALB C ; NF-kappa B ; analysis ; Receptors, Glucocorticoid ; analysis

Objective:To understand the molecular pat hophysiology of hepatocellular carcinoma and pancreatic cancer.Methods: We studied novel gene expression by cDNA microarray method. The PCR pro ducts of 4 096 genes and 12 800 gene were spotted onto a kind of chemical-mater ial-coated-glass slide in array. Both the mRNAs from 5 cases of hepatocellular carcinoma and 3 cases of pancreatic cancer were reversely transcribed to cDNAs with the incorporation of fluorescent-labeled dUTP to prepare the hybridization probes. After hybridization, BioDoor4096 and BioDoor12800 cDNA microarray were scanned for the fluorescent intensity. Tumor invasion-related gene expression w as screened through the analysis of difference in gene expression profile.Results:Among 4 096 and 12 800 target genes, there were 15 genes who se expression level differed from normal and carcinoma tissues. Therefore, they might be associated with metastasis.Conclusion:Further analysis of these differentially expressed metastasis-associated genes will be helpful for understanding the molecular mechanism of malignant carcinoma.

OBJECTIVETo investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak.

METHODBetween June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.

RESULTOut of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.

CONCLUSIONTwo major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Adolescent ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Viral ; Female ; Hemagglutinins, Viral ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; drug therapy ; epidemiology ; pathology ; virology ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Oseltamivir ; pharmacology ; Pandemics ; Viral Vaccines ; genetics ; immunology

We integrate web-based learning with team based learning,which is called WTBL method (Web-based and team-based Learning). WTBL method is constructed and applied to the teaching practice of core curriculum group courses of clinical medicine. We build some small private online courses. The students can preview online and do the case discussions by teamwork in class. The application of WTBL teaching method has realized the flipping of classroom, and helps to enhance students' self-learning ability and teamwork ability.

Peptide and protein biologics possess high specificity and high biological activity, but their poor stability and short plasma half-life have limited clinical application. One established strategy to increase half-life of therapeutic proteins is chemical conjugation of the biologic with PEG. Nevertheless, PEGylation technology has some drawbacks, so recombinant polypeptide mimetics of PEG have gradually developed in recent years. Pharmaceutically active protein can be fused with specific amino acid sequences using recombinant DNA technology, and then increase hydrodynamic volume or produce charge effect, which retards kidney filtration and eventually prolongs the half-life. This article mainly reviews kinds of polypeptides and the research progress in half-life extension of therapeutic proteins.

Objective To discuss the method,mid-long term clinical therapeutic effect and safety of coil embolization in treating patients with hepatic arterial pseudoaneurysm(HAPA).Methods Seven patients with repeatedly massive hemorrhage of gastrointestinal tract were undertaken DSA of celiac arteries and hepatic arteries and embolization of the feeding artery by coils or microcoils after correct diagnosis.All cases underwent follow-up from 6 to 60 months(mean 38).Results The blood loss before angiography was ranged from 1200 to 4000(mean 2385)ml.There were 3 cases with normal hepatic function and 4 with hepatic dysfunction including ALT increase in 2 and obstructive jaundice in another.Digital substraction angiography(DSA)clearly showed the location,shape and feeding arteries of HAPA.There were 2 types of HAPA namely intrahepatic (n=3)and extrahepatic(n=4),adding one case with arteriovenous fistula(AVF).Embolization was successful in all cases by coils(n=13)or microeoils(n=12).No recurrence and any definite clinical complication occurred during follow-up.Conclusion Coil embolization in treating HAPA is safe and effective with mid-long term positive clinical therapeutic efficiency without severe complications.(J Intervent Radiol, 2007,16:803-806)