Purpose To investigate the positive rate and concordance rate of BRAF mutation in papillary thyroid carcinoma detected by real-time PCR method and Sanger sequencing. Methods 312 papillary thyroid carcinomas patients were enrolled in this study. Real-time PCR method and Sanger sequencing were performed to detect BRAF gene mutations. The frequency of BRAF mutation and the concordance of two methods were analyzed. Results BRAF mutation was detected in 65. 4% (204/312) and 63. 8% (199/312) of 312 papillary thyroid carcinoma samples by using real-time PCR method and Sanger sequencing, respectively. There was no significant correlation between BRAF gene mutations and patients’ gender. There was significant correlation between BRAF gene mutations and patients’ age. The overall concordance between real-time PCR method and Sanger sequencing for BRAF mutation detection was 98. 4%. Conclusion Real-time PCR method provides an effective method in BRAF gene mutation detection.

OBJECTIVETo evaluate the time course of granulocyte-colony-stimulating-factor (G-CSF), estrogen and various doses of atorvastatin on endothelial progenitor cells (EPCs) mobilization.

METHODA total of 48 male New Zealand White rabbits were treated with placebo, estrogen (0.25 mg.k(-1).d(-1)), Atorvastatin (2.5, 5, or 10 mg) and G-CSF (50 microg/rabbit/d), respectively. Peripheral EPCs number was surveyed weekly for 4 weeks by FACS analysis (double-positive for PE-CD34/FITC-CD133) and under fluorescent microscope (double-positive for FITC-UEA-1/Dil-acLDL). Serum nitric oxide (NO) and lipids were also measured at the third week.

RESULTSPeripheral EPCs was significantly increased in G-CSF treated animals and remained constant for 4 weeks compared to placebo treated animals. Atorvastatin increased peripheral EPCs dose-dependently from 2.5 to 5 mg and peaked at the third week while peripheral EPCs number was not affected by 10 mg.k(-1).d(-1) atorvastatin during the first 3 weeks and was significantly higher only in the fourth week compared to placebo group. Estrogen also significantly increased peripheral EPCs at the third and fourth week compared to placebo group. At the third week, serum NO was similar in G-CSF group, significantly higher in atorvastatin 5 mg.k(-1).d(-1) and estrogen groups while significantly lower in atorvastatin 10 mg.k(-1).d(-1) group compared to placebo group. Serum lipids were similar among various groups.

CONCLUSIONAtorvastatin, estrogen and G-CSF could mobilize EPCs. The mobilization efficacy is as follows: G-CSF > atorvastatin 5 mg.k(-1).d(-1) > estrogen > atorvastatin 2.5 mg.k(-1).d(-1) > atorvastatin 10 mg.k(-1).d(-1). NO might partly contribute to the mobilizing effect of estrogen and atorvastatin.

Animals ; Atorvastatin Calcium ; Endothelial Cells ; cytology ; drug effects ; Estrogens ; pharmacology ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Heptanoic Acids ; pharmacology ; Hypolipidemic Agents ; pharmacology ; Lipids ; blood ; Male ; Nitric Oxide ; blood ; Pyrroles ; pharmacology ; Rabbits ; Recombinant Proteins ; Stem Cells ; drug effects

OBJECTIVETo investigate the effects of retroviral vector containing shRNA targeting rat angiotensin II type 1 receptor (AT1R) gene (Ad5-AT1R-shRNA) on blood pressure and AT1R mRNA expression in spontaneously hypertensive rats (SHR).

METHODSRetroviral vector containing shRNA targeting rat AT1R gene was constructed and propagated further in 293 cells. SHR rats were randomly divided into SHR + Ad5-AT1R-shRNA (1.7 x 10(9) TCID(50)/ml) group and SHR (Ad5-EGFP, 7.9 x 10(9) TCID(50)/ml, n = 11 each) and 11 male Wistar-Kyoto rats (WKY) serve as normal controls (Ad5-EGFP, 7.9 x 10(9) TCID(50)/ml). Systolic blood pressure was measured before and after single intravenous injection of Ad5-AT1R-shRNA or Ad5-EGFP. Heart, liver, kidney, aorta and adrenal gland were removed after blood pressure measurement. Tissue Ad5-AT1R-shRNA expression was detected with fluorescence microscope and AT1R mRNA in liver, kidney and aorta was measured by fluorescence quantitative PCR.

RESULTSAd5-AT1R-shRNA significantly reduced blood pressure compared with controls (-29 mm Hg, 1 mm Hg = 0.133 kPa, P < 0.05) 24 hours after single injection and this antihypertensive effect could last for 5 to 7 days. Ad5-AT1R-shRNA expression detected with fluorescence microscope was significantly increased in heart, liver, kidney, aorta and adrenal gland post Ad5-AT1R-shRNA injection. AT1R mRNA in kidney and aorta (0.086 +/- 0.014, 0.051 +/- 0.023) were significantly decreased in Ad5-AT1R-shRNA treated rats compared with SHR control rats (0.362 +/- 0.042, 0.463 +/- 0.045, P < 0.01).

CONCLUSIONThe results indicate that Ad5-AT1R-shRNA could inhibit the tissue AT1R mRNA expression and produce prolonged antihypertensive effects in SHR rats.

Adenoviridae ; Animals ; Blood Pressure ; Genetic Vectors ; Heart Rate ; Hypertension ; genetics ; metabolism ; physiopathology ; Male ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism

OBJECTIVE: To study genetic polymorphism of 7 Y-specific short tandem repeats (STR) and assess their usefulness in forensic casework. METHODS: 7 Y-STR have been amplified in two multiplex reactions, (Multiplex I:DYS391, GATA-A4, GATA-A10 and GATA-H4. Multiplex II:DYS439,DYS437 and DYS434). PCR products were separated by polyacrylamide gels electrophoresis followed by silver stain. RESULTS: When 372 unrelated individuals from the Han population in Guangdong were detected by those system, DYS391, GATA-A4, GATA-A10, GATA-H4,DYS439,DYS437 and DYS434 showed 5,7,6,5,6,4,4 alleles, respectively. A total of 254 different haplotypes were identified, of which 201 (79.13%) were found in single individuals. The overall haplotypes diversity reached 0.9960. CONCLUSION The 7 Y-STR loci are highly genetic polymorphism and they will be very powerful for establishing Y-STR database, understanding human origin, paternity testing and personal identification.
Alleles ; Asian People ; Chromosomes, Human, Y/genetics* ; Forensic Medicine ; Gene Frequency ; Genetic Markers ; Genetics, Population ; Genotype ; Haplotypes ; Humans ; Male ; Microsatellite Repeats/genetics* ; Paternity ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Sequence Analysis, DNA

OBJECTIVE: To establish a multiplexing Y-STR system and study haplotype frequencies of 4 Y-specific loci in China Han population. METHODS: DYS439, DYS390, GATA-A7.2 and DYS393 loci were amplified simultaneously and were analyzed by polyacrylamide gel electrophoresis and silver staining. RESULTS: When 558 unrelated male individuals from the Han population in China were tested by the multiplex system, DYS439, DYS390, GATA-A7.2 and DYS393 show 7,7,7,6 alleles, respectively. 180 different haplotypes were detected. The power of discrimination of this system was 0.9853. CONCLUSION The multiplex amplified system of these 4 Y-specific loci and their database are useful for human origin exploration and forensic practice.
Alleles ; China ; Chromosomes, Human, Y ; DNA/isolation & purification* ; Electrophoresis, Polyacrylamide Gel ; Ethnicity/genetics* ; Female ; Forensic Medicine ; Gene Frequency ; Haplotypes ; Humans ; Male ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Silver Staining ; Tandem Repeat Sequences/genetics*

OBJECTIVETo search for the best program for treatment of peripheral nerve incomplete injury.

METHODSNinety cases were randomly divided into a treatment group, a control group I and a control group II, 30 cases in each group. The treatment group were treated with electroacupuncture at Jianyu (LI 15), Hegu (LI 4), Quchi (LI 11), etc. plus functional training, and the control group I with electroacupuncture and the control group H with functional training. After treatment for 3 months, basic function, practical function, EMG, nerve conduction velocity were compared among the 3 groups.

RESULTSThe good rate of basic function of 50.0%, the curemarkedly effective rate of practical function of 50.0% and the total effective rate of neurophysiology of 64.3% in the treatment group were better than 20.7%, 17.2%, 41.4% in the control group I (P < 0.05) and 23.3%, 20.0% and 36.O7% in the control group II (P 0.05).

CONCLUSIONElectroacupuncture combined with functional training can accelerate nervous repair, promote functional recovery of the denervated muscles, so as to shorten the restoring time of nerve-muscle and increase life quality of the patient.

Adult ; Electroacupuncture ; methods ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Peripheral Nerve Injuries ; Peripheral Nerves ; physiopathology ; Physical Therapy Modalities ; Upper Extremity ; innervation

OBJECTIVETo investigate the feasibility of real-time PCR-optimized oligonucleotide probe method for detection of KRAS mutations in lung and colorectal carcinomas, as compared with Sanger sequencing method.

METHODSGenomic DNA was extracted from formalin fixed paraffin embedded samples of 221 lung carcinomas and 131 colorectal carcinomas after tumor cell content assessment and macrodissection. Real-time PCR-optimized oligonucleotide probe method and Sanger sequencing were performed to detect KRAS gene mutations. The frequency of KRAS mutation, mutation types, and their concordance were analyzed.

RESULTSKRAS mutation was detected in 6.3% (14/221) and 4.5% (10/221) of 221 lung cancer samples by using real-time PCR-optimized oligonucleotide probe method and Sanger sequencing, respectively, while in 41.2% (54/131) and 40.5% (53/131) of 131 colorectal cancer samples, respectively. There was no significant correlation between KRAS gene mutations and patients' gender and age (P > 0.05). The positive rate of KRAS codon 12 mutation was significantly higher than that of KRAS codon 13 mutation (P < 0.05). The overall concordance between real-time PCR-optimized oligonucleotide probe method and Sanger sequencing for KRAS mutation detection was 97.4%.

CONCLUSIONReal-time PCR-optimized oligonucleotide probe method provides an alternative method with high consistency and sensitivity as compared to Sanger sequencing in gene mutation detection.

Codon ; Colorectal Neoplasms ; genetics ; DNA Mutational Analysis ; methods ; Female ; Humans ; Lung Neoplasms ; genetics ; Male ; Middle Aged ; Mutation ; Oligonucleotide Probes ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins p21(ras) ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; ras Proteins ; genetics

OBJECTIVETo investigate the mutation frequencies of KRAS, BRAF, PIK3CA and EGFR genes that were effective on the targeted therapies in colorectal carcinoma.

METHODSThe tissue specimens from 331 colorectal cancer patients were collected and subject to KRAS, BRAF, PIK3CA and EGFR mutation analysis. Paraffin-embedded tissue samples were obtained and macrodissection was performed to enrich the tumor cells for DNA extraction when necessary. PCR-based direct DNA sequencing was used to investigate the codons 12 and 13 in exon 2 of KRAS gene, exons 11 and 15 of BRAF gene, exons 9 and 20 of PIK3CA gene and exons 18-21 of EGFR gene.

RESULTSActivating mutations were detected in KRAS (44.1%, 137/311), BRAF (5.8%, 9/156), PIK3CA (2.6%, 4/156) and EGFR (1.3%, 2/156) in the study cohort of colorectal carcinoma cases. Among KRAS gene mutations, 81.0% (111/137) occurred in codon 12, with p.G12D as the most common variant (45.3%, 62/137); 19.0% (26/137) occurred in codon 13, with 38G > A (G13D) as the most common variant (17.5%, 24/137).

CONCLUSIONSThe KRAS mutation frequency is the highest among the four genes (KRAS, BRAF, PIK3CA and EGFT) tested in colorectal carcinoma. The presence of these gene mutations may provide therapeutic information for targeted therapy. Mutation analyses of BRAF and PIK3CA in addition to KRAS should be a part of the standard diagnostic algorithm for colorectal carcinoma patients.

Class I Phosphatidylinositol 3-Kinases ; Codon ; Colorectal Neoplasms ; genetics ; pathology ; DNA Mutational Analysis ; Exons ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins B-raf ; genetics ; Proto-Oncogene Proteins p21(ras) ; Receptor, Epidermal Growth Factor ; genetics ; ras Proteins ; genetics

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.
Animals ; Cellular Reprogramming ; Humans ; Induced Pluripotent Stem Cells ; physiology ; Transduction, Genetic ; Transfection ; Transgenes

AIMTo investigate the effects of shivering on airway rewarming.

METHODSThe hypothermic dog model without shivering was established by immersing an anesthetized dog in cold water and administering atracurium to inhibit the dog shivering. The model dog respired warm fully humidified (40-45 degrees C, RH 99.9%) air and room temperature air(19 +/- 1 degrees C, RH 30% - 75%) to rewarm each for 2 hours, the priority of different temperature air respired was arranged randomly. After rewarming for 4 hours, the relaxed dog breathed warm humidified air by positive pressure ventilation in order to restore its spontaneous respiratory. Then the dog continued to respire warm humidified air spontaneously until the esophageal (Te) and rectal temperature (Tr) of the dog achieved the same degrees as the dog was immersed in the water. The metabolic heat production was detected by indirect calorimetry during the experiment.

RESULTS(1) When the shivering was inhibited, inhaling warm humidified air for 2 hours made the Tr and Te of the dogs increase 0.26-0.39 degrees C and 0.44-1.11 degrees C per hour respectively, inhaling air at room temperature for 2 hours made Tr and Te of the dogs decrease 0.24-0.51 degrees C and 0.58-0.67 degrees C per hour, respectively. And the changes in Tr and Te of the dogs were unrelated to the priority of inhaling air at different temperature. (2) When the dog with shivering respired spontaneously warm humidified air, the rewarming rates of Tr and Te were 2.26-2.33 degrees C/h and 1.96-2.38 degrees C/h respectively, quicker than those of the dogs whose shivering was inhibited. (3) Compared with metabolic heat production of the unshivering dog respiring warm humidified air by positive pressure ventilation, that of the shivering dog respiring warm humidified air spontaneously increased outstandingly, shivering thermogenesis made the rewarming rates increased obviously.

CONCLUSIONAirway rewarming is a method conducive to rewarming of hypothermia. When the body is shivering, the metabolic heat production increases obviously, that makes the rewarming rate increase markedly. So the shivering must be inhibited in order to eliminate the interference of shivering thermogenesis when the effects of airway rewarming are detected.

Animals ; Body Temperature Regulation ; Cold Temperature ; Dogs ; Hypothermia ; physiopathology ; therapy ; Hypothermia, Induced ; Male ; Respiratory Physiological Phenomena ; Shivering