OBJECTIVETo identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene.

METHODSGenomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations.

RESULTSA c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing.

CONCLUSIONThe Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

DNA Mutational Analysis ; methods ; Female ; Humans ; Male ; Mutation ; genetics ; Polymerase Chain Reaction ; methods ; Receptor, Fibroblast Growth Factor, Type 3 ; genetics ; Transition Temperature

OBJECTIVES: To determine the optimal timing for post space preparation of root canals sealed with epoxy resin-based AH Plus sealer in terms of its polymerization and influence on apical leakage. MATERIALS AND METHODS: The epoxy polymerization of AH Plus (Dentsply DeTrey) as a function of time after mixing (8, 24, and 72 hours, and 1 week) was evaluated using Fourier transform infrared (FTIR) spectroscopy and microhardness measurements. The change in the glass transition temperature (Tg ) of the material with time was also investigated using differential scanning calorimetry (DSC). Fifty extracted human single-rooted premolars were filled with gutta-percha and AH Plus, and randomly separated into five groups (n = 10) based on post space preparation timing (immediately after root canal obturation and 8, 24, and 72 hours, and 1 week after root canal obturation). The extent of apical leakage (mm) of the five groups was compared using a dye leakage test. Each dataset was statistically analyzed by one-way analysis of variance and Tukey's post hoc test (α = 0.05). RESULTS: Continuous epoxy polymerization of the material with time was observed. Although the T(g) values of the material gradually increased with time, the specimens presented no clear T(g) value at 1 week after mixing. When the post space was prepared 1 week after root canal obturation, the leakage was significantly higher than in the other groups (p < 0.05), among which there was no significant difference in leakage. CONCLUSIONS: Poor apical seal was detected when post space preparation was delayed until 1 week after root canal obturation.
Bicuspid ; Calorimetry, Differential Scanning ; Dataset ; Dental Pulp Cavity* ; Fourier Analysis ; Glass ; Gutta-Percha ; Humans ; Polymerization ; Polymers ; Post and Core Technique ; Root Canal Obturation ; Root Canal Preparation ; Spectrum Analysis ; Transition Temperature

OBJECTIVETo establish a rapid, accurate, noninvasive and low cost method for screening MT3243A>G mutation in mitochondrial diabetes.

METHODSBlood, saliva, and urine sediment samples were collected from 6 patients with confirmed mitochondrial diabetes and 50 healthy controls from Shanghai Children's Hospital and Shanghai Sixth People's Hospital. The heterozygosity levels of MT3243A>G mutation in above samples were detected with pyrosequencing, and the data were compared. MT3243A>G mutations were rapidly screened with high resolution melting curve analysis (HRM) in the urine sediment samples of 1070 diabetic patients from 4 communities in Shanghai. Furthermore, pyrosequencing was used to validate the suspected positive samples, and the heterozygosity levels were also quantified.

RESULTSComparative experiments found that heterozygosity of MT3243A>G mutation was 2 to 7 times higher in urine sediment than in saliva and blood samples from the 6 patients with confirmed mitochondrial diabetes. However, the heterozygosity was slightly higher in saliva than blood samples. MT3243A>G mutation was not detected in the 50 healthy controls. Two samples with suspected MT3243A>G mutation were identified in the 1070 urine sediment samples of diabetes patients with HRM screening, which were validated by pyrosequencing. The heterozygosity of MT3243A>G mutation were 33.32% and 14.67% in the urine sediment samples, respectively.

CONCLUSIONUrine sediment samples can be used for rapid screening of MT3243A>G mutation for its ease to collect, noninvasiveness and higher level of heterozygosity. HRM is suitable for rapid screening for mitochondrian mutations for its low cost, while such mutations could be detected with sensitivity and accuracy by pyrosequencing.

DNA, Mitochondrial ; genetics ; Diabetes Mellitus ; genetics ; Heterozygote ; Humans ; Mutation ; Sequence Analysis, DNA ; methods ; Transition Temperature

In this study, porous scaffolds were produced by a thermal crosslinking of polycaprolactone diacrylate in the presence of hydroxyapatite (HA) and particulate leaching technique with sodium chloride as the water soluble porogen for bone tissue engineering applications. The prepared scaffolds were characterized using techniques such as Field Emission Scanning Electron Microscopy, Differential Scanning Calorimetry, and Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. Moreover, dynamic mechanical properties were investigated using Dynamic Mechanical Thermal Analysis. The obtained scaffolds present a porous structure with interconnected pores and porosity around 73%. It was found that the incorporation of HA particles to polycaprolactone (PCL) matrix resulted in an increased crystallinity. Moreover, both the storage modulus (E′) and glass transition temperature (T(g)) increased, while the loss factor (tan δ) decreased due to the hindrance of the HA particles to the mobility of polymer segments. Cytocompatability of the scaffolds was assessed by MTT assay and cell attachment studies. Osteoconductivity of the scaffolds was investigated with cells alkaline phosphatase extraction. The levels of alkaline phosphatase activity were found to be higher for PCL/HA network scaffold than for PCL network scaffold. In addition, cytocompatibility of the PCL/HA network scaffold indicated no toxicity, and cells were attached and spread to the scaffold walls.
Alkaline Phosphatase ; Bone and Bones* ; Calorimetry, Differential Scanning ; Crystallins ; Durapatite* ; Glass ; Microscopy, Electron, Scanning ; Polymers ; Porosity ; Sodium Chloride ; Spectrum Analysis ; Transition Temperature ; Water

Hyoscyami Semen, the mature dried seed of Hyoscyamus niger L., has long been used as a traditional Chinese medicine to treat human diseases. Hyoscyami Semen is found in local markets in China. In markets, sellers and buyers commonly inadvertently mix the seeds of H. niger with the seeds of related species such as Hygrophila salicifolia (Vahl) Nees, Astragalus complanatus R. Br., Cuscuta australis R. Br., Cuscuta chinensis Lam., and Impatiens balsamina L. because of their similar morphologies or similar names. Thus, developing a reliable method for discriminating H. niger seeds from its adulterants is necessary to reduce confusion and ensure the safe use of Hyoscyami Semen. The present study was designed to evaluate the efficiency of high-resolution melting analysis combined with DNA barcoding (Bar-HRM) with internal transcribed spacer 2 to discriminate H. niger. Our results show that Bar-HRM successfully identified the adulterants and detected the proportion of H. niger DNA extract within an admixture. In particular, HRM detected H. niger DNA extract in A. complanatus DNA extract at concentrations as low as 1%. In conclusion, the Bar-HRM method developed in the present study for authenticating H. niger is rapid and cost-effective. It can be used in the future to guarantee the purity of Hyoscyami Semen for the clinical use.
China ; DNA Barcoding, Taxonomic ; methods ; DNA, Intergenic ; chemistry ; genetics ; DNA, Plant ; chemistry ; genetics ; Discriminant Analysis ; Drug Contamination ; Drugs, Chinese Herbal ; chemistry ; Hyoscyamus ; genetics ; growth & development ; Seeds ; genetics ; growth & development ; Transition Temperature

High resolution melting (HRM), an important technology for genotyping and mutation scanning, has broad prospects in the authentification of traditional Chinese medicine. This paper selected universal trnH-psbA primers and used HRM to establish a new methods for identification of Akebia herbs. PCR was conduct at the annealing temperature of 58 degrees C and 35 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further analyzed. The results showed the Tm values of Caulis Akebiae was (81.84 ± 0.16), (85.28 ± 0.16) degrees C and Caulis Clematidis Armandii was (83.22 ± 0.19) degrees C and Caulis Aristolochiae manshuriensis was (81.67 ± 0.14) degrees C, (84.24 ± 0.10) degrees C with 5-125 mg - L-' DNA template, 0.4 μmol x L(-1) primer, 2.0 mmol x L(-1) Mg2+. This method can achieve the authentification of Akebia herbs and is simple, fast, high-throughput, visual.
Chemistry Techniques, Analytical ; methods ; DNA, Plant ; chemistry ; genetics ; Genotype ; Magnoliopsida ; chemistry ; classification ; genetics ; Phylogeny ; Transition Temperature

In order to improve the interfacial bonding strength of hydroxyapatite/polyurethane implanted material and dispersion of hydroxyapatite in the polyurethane matrix, we in the present study synthesized nano-hydroxyapatite/polyurethane composites by in situ polymerization. We then characterized and analyzed the fracture morphology, thermal stability, glass transition temperature and mechanical properties. We seeded MG63 cells on composites to evaluate the cytocompatibility of the composites. In situ polymerization could improve the interfacial bonding strength, ameliorate dispersion of hydroxyapatite in the properties of the composites. After adding 20 wt% hydroxyapatite into the polyurethane, the thermal stability was improved and the glass transition temperatures were increased. The tensile strength and maximum elongation were 6.83 MPa and 861.17%, respectively. Compared with those of pure polyurethane the tensile strength and maximum elongation increased by 236.45% and 143.30%, respectively. The composites were helpful for cell adhesion and proliferation in cultivation.
Biocompatible Materials ; chemistry ; Cell Adhesion ; Cell Line ; Durapatite ; chemistry ; Humans ; Polymerization ; Polyurethanes ; Tensile Strength ; Transition Temperature

High resolution melting (HRM) , an important technology for genotyping and mutation scanning, has broad prospects in the authenticity of traditional Chinese medicine. This paper selected universal CO I primers and used HRM to establish a new method for authenticity of Hairy Antler. PCR was conducted at the annealing temperature of 60 °C and 45 cycles. The range of the DNA template concentration, the primer concentration and the Mg2+ ion concentration were further optimized. The results showed that the Tm values of Cervus nippon were (81.96 ± 0.07), (84.51 ± 0.03) °C and Cervus elaphus was(82.58 ± 0.13), (85.95 ± 0.05) °C with 10-100 mg · L(-1) DNA template, 0.2 µLmol · L(-1) primer, 2.0 mmol · L(-1) Mg2+. This method can authenticate of hairy antler and is simple, fast, high-throughput, visualization.
Animals ; Antlers ; chemistry ; DNA ; chemistry ; genetics ; DNA Primers ; genetics ; Deer ; classification ; genetics ; Genotype ; Medicine, Chinese Traditional ; standards ; Polymerase Chain Reaction ; Transition Temperature

Crystal structures of chemical drugs has been being investigated widely. But few attention has been paid to polymorphs-phenomena of active ingredients from Traditional Chinese Medicine(TCM). Taking anhydrous dehydroandrographolide and hydrousprim-O-glucosylcimifugin as example, differences between TCM reference substances (RSs) with different crystal structures were discussed by using microscopy, melting point determination, differential thermal analysis (DTA) and infrared (IR) methods. The results showed that different crystal structures could lead to change of melting points, thermal behaviors and IR spectrum. It's indicated that polymorphs may be considered if different physicochemical properties were obtained when applying TCM RS. Differences of chemical properties of active ingredients from TCM with different crystal structures need further investigation.
Crystallization ; Differential Thermal Analysis ; methods ; standards ; Drugs, Chinese Herbal ; chemistry ; standards ; Medicine, Chinese Traditional ; Molecular Structure ; Reference Standards ; Spectrophotometry, Infrared ; methods ; standards ; Transition Temperature

OBJECTIVETo explore the effects and anti-depression mechanisms of Kaixin Jieyu Decoction (, KJD).

METHODSThe rat vascular depression (VD) model was established by ligation of bilateral common carotid arteries (LBCCA) combined with chronic unpredictable mild stress (CUMS). Forty Wistar rats were randomly divided into sham, VD model, VD + high-dose KJD [15.4 g/(kg·d) of crude drug], VD + medium-dose KJD [7.7 g/(kg·d) of crude drug], and VD + fluoxetine [2.4 mg/(kg·d)] groups (n=8 in each group), and the treatments lasted for 21 days. Changes of behavior and hippocampus pathology were observed. The level of glial fibrillary acidic protein (GFAP) protein and mRNA in hippocampus was detected respectively by immunohistochemistry and real-time polymerase chain reaction.

RESULTSCompared with the sham group, rats in model group showed a variety of behavioral obstacles, including a significant reduction in sucrose consumption percentage, horizontal and vertical activity scores in open-field tests (P<0.05 or P<0.01), pathological damage like neuronal degeneration, necrosis, and a significant decrease of GFAP protein and mRNA in hippocampus (P<0.01); compared with the model group, rats in the high-dose KJD group, medium-dose KJD group and fluoxetine group obtained notable higher behavioral scores, and pathological injury lessened in hippocampus with a increased expression of GFAP protein and mRNA P<0.05 or P<0.01); compared with the medium-dose KJD group and fluoxetine group, GFAP mRNA in high-dose KJD group expressed higer (P<0.05).

CONCLUSIONLBCCA combined with CUMS may cause depression-like behavioral changes resulting in the VD model of rats whose depression state can be ameliorated by KJD, and the mechanism of cerebral protection is related possibly with promoting expression of GFAP in hippocampus.

Animals ; Behavior, Animal ; Depression ; drug therapy ; genetics ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Electrophoresis, Agar Gel ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; pathology ; Immunohistochemistry ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats, Wistar ; Transition Temperature