Calcineurin activation contributes to Hcy-induced dephosphorylation and mitochondrial translocation of Bad.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic +mitochondrial fractions) localized to the mitochondrial fraction was determined by densitometric analysis of the data from B. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Hcy induces apoptosis and suppresses 14-3-3ε expression in cultured hippocampal neurons.

<p>A, Cultures were exposed for 24 h to either saline (<i>Con</i>) or 500 µM Hcy (<i>Hcy</i>) and were subsequently stained with the fluorescent DNA-binding dye Hoechst 33342 (<i>top</i>) or photographed under phase-contrast optics (<i>bottom</i>). Note the nuclear DNA condensation and fragmentation in neurons exposed to Hcy (arrow). Scale bar: 100 µm. B, Cultures were exposed to the indicated concentrations of Hcy for 24 h, and the percentages of neurons with apoptotic nuclei were quantified. Values are the mean ±S.E.M. of counts made in four to six cultures. C, Cells were treated as in B, and the levels of cleaved PARP were determined by western blotting. Top, representative western blots. Bottom, densitometric analysis of cleaved PARP normalized by β-actin (<i>n</i> = 3). D, 14-3-3ε mRNA and protein was measured by real-time quantitative PCR (<i>top</i>) and western blotting (<i>mid and bottom</i>), respectively. *<i>P</i><0.05 vs. control.</p

Calcineurin activation and 14-3-3ε suppression together contribute to Hcy-induced apoptosis.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were then pretreated with 1 µM CsA for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. B, Western blot analysis of Bad in mitochondrial (Mit) and cytoplasmic (Cyto) fractions from hippocampal neurons. C, Percentage of total Bad (cytoplasmic+mitochondrial fractions) localized in the mitochondrial fraction as determined by densitometric analysis of the data from B. D, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. E, Cleaved PARP was measured using western blotting. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group. ^Δ<i>P</i><0.05 vs. CsA group.</p

Inhibition of Hcy-induced apoptosis by a calcineurin inhibitor or intracellular Ca²⁺ chelator.

<p>Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before treatment with 500 µM Hcy (<i>Hcy</i>) for 24 h. A, The level of apoptosis was determined by counting the number of neurons with apoptotic nuclei. Four randomly selected fields of Hoechst-stained cells were counted and the average percentage of apoptotic cells per total number of cells was determined. B, Cleaved PARP was studied by western blotting. Results are expressed as the mean ± S.E.M. of three experiments. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

The effect of lentiviral 14-3-3ε transduction on apoptosis in hippocampal neurons.

<p>Hippocampal neurons transduced with lentiviral 14-3-3ε for 48 h were treated with Hcy for 24 h. A, 14-3-3ε mRNA and proteins were analyzed by real-time quantitative PCR or western blotting, respectively. B, The percentages of neurons with apoptotic nuclei (Hoechst staining) were quantified. C, Cleaved PARP was determined by western blotting. D, Total Bad and phospho-Bad levels were assessed by western blotting and densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Activation of calcineurin by Hcy and inhibition by specific blockers.

<p>A, Cultures were exposed to the indicated concentrations of Hcy (<i>Hcy</i>) for 24 h, and cellular calcineurin activity was then determined. B, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>). Cellular calcineurin activity was measured at the indicated time periods after the addition of Hcy to the cells. Values represents the mean ± S.E.M. of four or more assays. C, Cells were pretreated with 1 µM CsA or 10 µM BAPTA-AM for 30 min before exposed to 500 µM Hcy (<i>Hcy</i>) for 24h. Calcineurin A (Calcineurin) protein level was determined by western blotting. Top, representative western blots. Bottom, densitometric analysis. *<i>P</i><0.05 vs. control. ^#<i>P</i><0.05 vs. Hcy treatment group.</p

Pyrosequencing of Plaque Microflora In Twin Children with Discordant Caries Phenotypes

<div><p>Despite recent successes in the control of dental caries, the mechanism of caries development remains unclear. To investigate the causes of dental decay, especially in early childhood caries, the supragingival microflora composition of 20 twins with discordant caries phenotypes were analyzed using high-throughput pyrosequencing. In addition, the parents completed a lifestyle questionnaire. A total of 228,789 sequencing reads revealed 10 phyla, 84 genera, and 155 species of microflora, the relative abundances of these strains varied dramatically among the children, Comparative analysis between groups revealed that <i>Veillonella</i>, <i>Corynebacterium</i> and <i>Actinomyces</i> were presumed to be caries-related genera, <i>Fusobacterium</i>, <i>Kingella</i> and <i>Leptotrichia</i> were presumed to be healthy-related genus, yet this six genera were not statistically significant (P>0.05). Moreover, a cluster analysis revealed that the microbial composition of samples in the same group was often dissimilar but that the microbial composition observed in twins was usually similar. Although the genetic and environmental factors that strongly influence the microbial composition of dental caries remains unknown, we speculate that genetic factors primarily influence the individual's susceptibility to dental caries and that environmental factors primarily regulate the microbial composition of the dental plaque and the progression to caries. By using improved twins models and increased sample sizes, our study can be extended to analyze the specific genetic and environmental factors that affect the development of caries.</p></div

Weighted Unifrac clustering results of the samples.

<p>Samples having similar plaque microbiota compositions are usually clustered in a sub-branch. The different colors denote different kindergartens.</p

Genera and species with significant differences in representation (P<0.05).

<p>A: Comparisons between different kindergartens. B: Comparisons between different samples in the same group.</p

The barplot graph of samples microorganisms and the predominant bacteria of three groups.

<p>(A, B, C) Abundance and prevalence of bacteria at the phylum, genus, and species level in the 30 plaque samples. (a, b, c) Mean levels of the predominant bacteria in groups H1, H2 and C2 at the phylum, genus, and species level.</p