Southern blot analysis of the wild-type, mutant, and complemented strains.

<p>Schematic representation of the genomic <i>yapA</i> locus of <i>T</i>. <i>marneffei</i>, <i>Nco</i>I cleavage sites, and the position of the probe hybridization are indicated (A). The chromosomal DNA of wild type (lane 1), mutant (lane 2), and complemented strain (lane 3) was digested with <i>Nco</i>I. The 2.5 kb containing the 5’ UTR of <i>yapA</i> gene and <i>pyrG</i> marker was used as a probe. The bands characteristic of wild type, mutant, and complemented strain displayed the expected size differences (B).</p

The <i>yapA</i> Encodes bZIP Transcription Factor Involved in Stress Tolerance in Pathogenic Fungus <i>Talaromyces marneffei</i> - Fig 4

<p>The microscopic examinations of mold phase using a slide culture technique (A). Microscopic examinations of the yeast phase from the 1% peptone culture broth (B). Both mold and yeast phase were stained with calcofluor white to visualize chitin deposition in cell walls and septa.</p

Growth studies of the <i>ΔyapA</i> deletion mutant.

<p>The growth curves (radial diameter) of <i>T</i>. <i>marneffei</i> wild type, <i>ΔyapA</i> mutant, and complemented strain grown in ANM medium for 1 to 12 d at 25°C (****, p value < 0.05) (A). The germination rates of wild type, <i>ΔyapA</i> mutant, and complemented strain which grown in SDB medium for 24 h at 25°C (****, p value < 0.001) (B) at 37°C (****, p value < 0.001) (C). The conidia number of wild type, <i>ΔyapA</i> mutant, and complemented strain cultured on ANM medium (****, p value < 0.05) (D).</p

The <i>T. marneffei ΔyapA</i> mutant is more sensitive to oxidative and nitrosative stress.

<p>The wild type, mutant, and complemented strain were grown for 7 day in ANM agar at 25°C (A), ANM plus 0.1 mM Menadione (B), ANM plus 1 mM H₂O₂ (C), ANM plus 12 mM NaNO₂ (D), BHA agar at 37°C (E), BHA plus 0.05 mM Menadione (F), BHA plus 0.5 mM H₂O₂ (G), and BHA plus 6 mM NaNO₂ (H).</p

The macroscopic examinations of <i>T</i>. <i>marneffei</i> wild type G809, Δ<i>yapA</i> mutant, and complemented strain.

<p>The mold and yeast form were examined by optical visualization, the mold phase was represented on the top and yeast on the bottom, respectively (A). The stereomicroscope examination of the mold colonies at day 7 and 12, and yeast at day 12 (B).</p

Protein sequence alignment of the bZIP transcription factor AP-1/Yap1 homologs from <i>S</i>. <i>cerevisiae</i> (gi|4798|emb|CAA41536.1), <i>C</i>. <i>glabrata</i> (gi|49526305|emb|CAG59929.1), <i>S</i>. <i>pombe</i> (gi|5001|emb|CAA40363.1), <i>M</i>. <i>oryzae</i> (gi|145608602|ref|XP_001408783.1), <i>T</i>. <i>marneffei</i> (gi|212531151|ref|XP_002145732.1), and <i>A</i>. <i>fumigatus</i> (gi|70992066|ref|XM_745789.1).

<p>The domains important for function are indicated as follows: the bZIP_Yap region is shaded black; cysteine amino acids are shaded grey in the N-terminal cysteine rich domain and the C-terminal cysteine rich domain.</p

Role of genomic DNA methylation in detection of cytologic and histologic abnormalities in high risk HPV-infected women.

Cervical cancer is the fourth most common malignancy affecting women worldwide. The development of disease is related to high-risk human papillomavirus (hrHPV) infection. Cytology has been the most recommended triage for primary cervical (pre)cancer screening despite relatively low sensitivity. Recently, genomic DNA methylation has been proposed as an additional marker to increase sensitivity for detecting cervical precancerous lesion. This study aimed to evaluate the performance of methylation status of three tumor suppressor genes (CADM1, FAM19A4, and MAL) and HPV genotyping in detection of cytologic and histologic abnormalities in cervical cancer screening. Two hundred and sixty samples with available frozen cell pellets including 70 randomly selected cases of negative for intraepithelial lesion or malignancy (NILM)&HPV-negative, 70 randomly selected cases of NILM&HPV-positive, and 120 cytologic abnormalities & HPV-positive from a population-based cervical cancer screening program (n = 7,604) were investigated for the DNA methylation pattern of CADM1, FAM19A4, and MAL. Of 120 cytologic abnormalities & HPV-positive cases, there were 115 available histologic results. HPV52 and HPV58 were most commonly found in histologic HSIL+. The methylation levels of CADM1, FAM19A4, and MAL were elevated with the severity of cytologic abnormality which significantly increased by 3.37, 6.65 and 2 folds, respectively, in cytologic HSIL comparing with NILM. A significant increase in methylation levels of these three genes was also observed in histologic HSIL+ compared with negative histology but only CADM1 showed a significant higher methylation level than histologic LSIL. Using the ROC curve analysis, DNA methylation levels of FAM19A4 performed best in differentiating high-grade cytology (ASC-H+ from NILM/ASC-US/LSIL), followed by CADM1 and MAL. Whilst the CADM1 methylation performed best in distinguishing histologic HSIL+ from negative/LSIL with an area under the ROC curve of 0.684, followed by MAL (0.663) and FAM19A4 (0.642). Interestingly, after combining high DNA methylation levels to HPV16/18 genotypes, rates of histologic HSIL+ detection were substantially increased from 25% to 79.55% for CADM1, 77.27% for FAM19A4, and 72.73% for MAL, respectively. The rate further increased up to 95.45% when at least one of three genes had a high methylation level. This suggests a possible role of genomic DNA methylation, especially CADM1, in detecting histologic HSIL+ lesions in combination with hrHPV testing