OBJECTIVE: To study the types and characteristics of TUBB1 mutation in children with congenital hypothyroidism (CH) and thyroid dysgenesis (TD) in Shandong, China. METHODS: Mutations of the whole coding region of the TUBB1 gene were analyzed for 289 children with CH and TD in Shandong. Whole-genome DNA was extracted from peripheral blood leukocytes. PCR multiplication was performed for the whole coding region of the TUBB1 gene. Sanger sequencing was performed for the PCR products, and a biological information analysis was performed. RESULTS: Among the 289 children with CH and TD, 4 (1.4%) were found to have a c.952C>T(p.R318W) heterozygous mutation in the TUBB1 gene, resulting in the change of tryptophan into arginine at codon 318 of TUBB1 protein. This mutation was evaluated as "potentially pathogenic" based on the classification criteria and guidelines for genetic variation by American College of Medical Genetics and Genomics. CONCLUSIONS A novel mutation is detected in the exon of the TUBB1 gene in children with CH and TD in Shandong, suggesting that the TUBB1 gene may be a candidate pathogenic gene for CH children with TD.
Child ; China ; Congenital Hypothyroidism ; genetics ; DNA Mutational Analysis ; Humans ; Mutation ; Thyroid Dysgenesis ; genetics ; Tubulin ; genetics

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

BACKGROUND: We investigated the species distribution and amphotericin B (AMB) susceptibility of Korean clinical Aspergillus isolates by using two Etests and the CLSI broth microdilution method. METHODS: A total of 136 Aspergillus isolates obtained from 11 university hospitals were identified by sequencing the internal transcribed spacer (ITS) and beta-tubulin genomic regions. Minimal inhibitory concentrations (MICs) of AMB were determined in Etests using Mueller-Hinton agar (Etest-MH) and RPMI agar (Etest-RPG), and categorical agreement with the CLSI method was assessed by using epidemiological cutoff values. RESULTS: ITS sequencing identified the following six Aspergillus species complexes: Aspergillus fumigatus (42.6% of the isolates), A. niger (23.5%), A. flavus (17.6%), A. terreus (11.0%), A. versicolor (4.4%), and A. ustus (0.7%). Cryptic species identifiable by beta-tubulin sequencing accounted for 25.7% (35/136) of the isolates. Of all 136 isolates, 36 (26.5%) had AMB MICs of > or =2 microg/mL by the CLSI method. The categorical agreement of Etest-RPG with the CLSI method was 98% for the A. fumigatus, A. niger, and A. versicolor complexes, 87% for the A. terreus complex, and 37.5% for the A. flavus complex. That of Etest-MH was < or =75% for the A. niger, A. flavus, A. terreus, and A. versicolor complexes but was higher for the A. fumigatus complex (98.3%). CONCLUSIONS: Aspergillus species other than A. fumigatus constitute about 60% of clinical Aspergillus isolates, and reduced AMB susceptibility is common among clinical isolates of Aspergillus in Korea. Molecular identification and AMB susceptibility testing by Etest-RPG may be useful for characterizing Aspergillus isolates of clinical relevance.
Amphotericin B/*pharmacology ; Antifungal Agents/*pharmacology ; Aspergillus/*drug effects/isolation & purification ; DNA, Fungal/chemistry/genetics/metabolism ; Hospitals ; Humans ; Microbial Sensitivity Tests ; Mycoses/diagnosis/microbiology ; Republic of Korea ; Sequence Analysis, DNA ; Tubulin/genetics

Geosmithia argillacea, an anamorph of Talaromyces eburneus, is a thermophilic filamentous fungus that has a phenotype similar to that of the Penicillium species, except for the creamy-white colonies and cylindrical conidia. Recently, a new genus called Rasamsonia has been proposed, which is to accommodate the Talaromyces and Geosmithia species. Here, we report the first Korean case of G. argillacea isolated from a patient with a fungal ball. The patient was a 44-yr-old Korean man with a history of pulmonary tuberculosis and aspergilloma. The newly developed fungal ball in his lung was removed and cultured to identify the fungus. The fungal colonies were white and slow-growing, and the filaments resembled those of Penicillium. Molecular identification was carried out by sequencing the internal transcribed spacer (ITS) region of the 28S rDNA and the beta-tubulin genes. A comparative sequence analysis using the GenBank (http://blast.ncbi.nlm.nih.gov/) database was performed with the basic local alignment search tool (BLAST) algorithm. The results revealed a 97-100% similarity with the G. argillacea ITS sequence. This case should increase awareness among physicians about the pathogenic potential of G. argillacea in humans and help them accurately identify this fungus, because it can be easily confused with Penicillium and Paecilomyces species owing to their similar phenotypic and microscopic characteristics. A molecular approach should be employed to enable accurate identification of G. argillacea.
Adult ; Databases, Genetic ; Eurotiales/classification/*isolation & purification ; Humans ; Lung/microbiology/radiography ; Male ; Phylogeny ; RNA, Ribosomal, 28S/chemistry/genetics ; Sequence Analysis, DNA ; Tomography, X-Ray Computed ; Tuberculosis/*diagnosis/microbiology/radiography ; Tubulin/chemistry/genetics

Twenty Penicillium isolates were recovered during the investigation of fungal community in the soil samples collected from Wando (Jeonnam Province, Korea). Among them, one species was identified and described as P. cainii based on phylogentic analysis of internal transcribed spacer and beta-tubulin (BT2) genes and morphological characteristics. This is a first report of P. cainii in Korea.
Korea ; Penicillium ; Sequence Analysis ; Soil ; Tubulin

BACKGROUND: The identification of molds in clinical laboratories is largely on the basis of phenotypic criteria, the classification of which can be subjective. Recently, molecular methods have been introduced for identification of pathogenic molds in clinical settings. Here, we employed comparative sequence analysis to identify molds. METHODS: A total of 47 clinical mold isolates were used in this study, including Aspergillus and Trichophyton. All isolates were identified by phenotypic properties, such as growth rate, colony morphology, and reproductive structures. PCR and direct sequencing, targeting the internal transcribed spacer (ITS) region, the D1/D2 region of the 28S subunit, and the beta-tubulin gene, were performed using primers described previously. Comparative sequence analysis by using the GenBank database was performed with the basic local alignment search tool (BLAST) algorithm. RESULTS: For Aspergillus, 56% and 67% of the isolates were identified to the species level by using ITS and beta-tubulin analysis, respectively. Only D1/D2 analysis was useful for Trichophyton identification, with 100% of isolates being identified to the species level. Performances of ITS and D1/D2 analyses were comparable for species-level identification of molds other than Aspergillus and Trichophyton. In contrast, the efficacy of beta-tubulin analysis was limited to genus identification because of the paucity of database information for this gene. CONCLUSIONS: The molecular methods employed in this study were valuable for mold identification, although the different loci used had variable usefulness, according to mold genus. Thus, a tailored approach is recommended when selecting amplification targets for molecular identification of molds.
Aspergillus/genetics/isolation & purification ; DNA, Fungal/analysis/isolation & purification ; Databases, Genetic ; Fungi/genetics/*isolation & purification ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 28S/*genetics ; Sequence Analysis, DNA ; Trichophyton/genetics/isolation & purification ; Tubulin/*genetics

OBJECTIVETo study the effects of miR-124-1 on neuronal differentiation of rat bone marrow mesenchymal stem cells (MSCs).

METHODSMSCs cells were assigned into three groups: control (uninfected and untransfected), miR-124-1+ (infected with miR-124-1), and miR-124-1- (transfected with Anti-rno-miR-124* Inhibitor). MSCs were induced by β-mercaptoethanol (β-ME) to differentiate into neurons. The fluorescence expressed by infected MSCs was observed under an inverted fluorescence microscope. MTT method was used to measure cell survival rate after transfection or infection. Immunocytochemistry, RT-PCR and Western blot methods were used to detect the expression of β3 tubulin, MAP-2 and GFAP 6 days after β-ME induction.

RESULTSThe expression of miR-124-1 in the miR-124-1+ group was significantly higher 2 days after infection of lentivirus vector compared with the control group (P<0.01). In the miR-124-1- group, the cell survival rate and the miR-124-1 expression level decreased significantly 24 hrs after transfection of anti-rno-miR-124* inhibitor (P<0.01). After 6 days of β-ME induction, the protein and mRNA expression levels of β3 tubulin and MAP-2 in the miR-124-1+ group were much higher than the other two groups (P<0.01); while the expression levels of β3 tubulin and MAP-2 in the miR-124-1-group were lower than the control group (P<0.01). The expression of GFAP in the three groups was weak (<1%).

CONCLUSIONSmiR-124 might promote neuronal differentiation of rat MSCs.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Male ; Mesenchymal Stromal Cells ; cytology ; MicroRNAs ; physiology ; Microtubule-Associated Proteins ; analysis ; Neurons ; cytology ; Rats ; Rats, Wistar ; Tubulin ; analysis

In the present study, a phylogenetic analysis was undertaken based on the internal transcribed spacer (ITS) rDNA and partial beta-tubulin gene sequence of the Ganoderma species. The size of the ITS rDNA regions from different Ganoderma species varied from 625 to 673 bp, and those of the partial beta-tubulin gene sequence were 419 bp. Based on the results, a phylogenetic tree was prepared which revealed that Korean Ganoderma lucidum strains belong in a single group along with a G. lucidum strain from Bangladesh.
Bangladesh ; DNA, Ribosomal ; Ganoderma ; Korea ; Reishi ; Sequence Analysis ; Sprains and Strains ; Tubulin

OBJECTIVEThe purpose of this study was to culture and identify neural stem cells from mouse embryos in vitro using a modified method and provide a basis for further study of the biology of neural stem cells under hypoxia.

METHODSThe cells were isolated mechanically from the front cortex of fetal Institute of Cancer Research (ICR) mice on embryonic day 14. They were passaged by mechanical dissociation and enzymatic digestion. The neurospheres were identified by immunofluorescent staining of nestin. Cell differentiation was induced by 1% fetal bovine serum and then the cells were identified by immunohistochemistry of β-tubulin III and GFAP.

RESULTSThe cells obtained from the front cortex of fetal ICR mice had the capacity of forming neurospheres which showed nestin immunoreactive positivity. After being induced by 1% fetal bovine serum, the cells were differentiated into β-tubulin III-positive cells and GFAP-positive cells.

CONCLUSIONSUsing mechanical dissociation of primary cells and mechanical dissociation with enzymatic digestion of primary cells, the NSCs from the front cortex of mouse embryos can be obtained.

Animals ; Cell Differentiation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Female ; Glial Fibrillary Acidic Protein ; analysis ; Intermediate Filament Proteins ; analysis ; Mice ; Mice, Inbred ICR ; Nerve Tissue Proteins ; analysis ; Nestin ; Neural Stem Cells ; chemistry ; cytology ; Tubulin ; analysis