RASSF1A promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis

<p>Abstract</p> <p>Background</p> <p>Epigenetic silencing of the RAS association domain family 1A (<it>RASSF1A</it>) tumor suppressor gene promoter has been demonstrated in renal cell carcinoma (RCC) as a result of promoter hypermethylation. Contradictory results have been reported for <it>RASSF1A </it>methylation in normal kidney, thus it is not clear whether a significant difference between <it>RASSF1A </it>methylation in normal and tumor cells of the kidney exists. Moreover, RASSF1A expression has not been characterized in tumors or normal tissue as yet.</p> <p>Results</p> <p>Using combined bisulfite restriction analysis (COBRA) we compared RASSF1A methylation in 90 paired tissue samples obtained from primary kidney tumors and corresponding normal tissue. Bisulfite sequence analysis was carried out using both pooled amplicons from the tumor and normal tissue groups and subclones obtained from a single tissue pair. Expression of RASSF1A was analyzed by the use of tissue arrays and immunohistochemistry. We found significantly increased methylation in tumor samples (mean methylation, 20%) compared to corresponding normal tissues (mean methylation, 11%; <it>P </it>< 0.001). Densely methylated sequences were found both in pooled and individual sequences of normal tissue. Immunohistochemical analysis revealed a significant reduced expression of RASSF1A in most of the tumor samples. Heterogeneous expression patterns of RASSF1A were detected in a subgroup of histologically normal tubular epithelia.</p> <p>Conclusion</p> <p>Our methylation and expression data support the hypothesis that <it>RASSF1A </it>is involved in early tumorigenesis of renal cell carcinoma.</p

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Vestibular effects of a 7 Tesla MRI examination compared to 1.5 T and 0 T in healthy volunteers.

Ultra-high-field MRI (7 Tesla (T) and above) elicits more temporary side-effects compared to 1.5 T and 3 T, e.g. dizziness or "postural instability" even after exiting the scanner. The current study aims to assess quantitatively vestibular performance before and after exposure to different MRI scenarios at 7 T, 1.5 T and 0 T. Sway path and body axis rotation (Unterberger's stepping test) were quantitatively recorded in a total of 46 volunteers before, 2 minutes after, and 15 minutes after different exposure scenarios: 7 T head MRI (n = 27), 7 T no RF (n = 22), 7 T only B0 (n = 20), 7 T in & out B0 (n = 20), 1.5 T no RF (n = 20), 0 T (n = 15). All exposure scenarios lasted 30 minutes except for brief one minute exposure in 7 T in & out B0. Both measures were documented utilizing a 3D ultrasound system. During sway path evaluation, the experiment was repeated with eyes both open and closed. Sway paths for all long-lasting 7 T scenarios (normal, no RF, only B0) with eyes closed were significantly prolonged 2 minutes after exiting the scanner, normalizing after 15 minutes. Brief exposure to 7 T B0 or 30 minutes exposure to 1.5 T or 0 T did not show significant changes. End positions after Unterberger's stepping test were significantly changed counter-clockwise after all 7 T scenarios, including the brief in & out B0 exposure. Shorter exposure resulted in a smaller alteration angle. In contrast to sway path, reversal of changes in body axis rotation was incomplete after 15 minutes. 1.5 T caused no rotational changes. The results show that exposure to the 7 Tesla static magnetic field causes only a temporary dysfunction or "over-compensation" of the vestibular system not measurable at 1.5 or 0 Tesla. Radiofrequency fields, gradient switching, and orthostatic dysregulation do not seem to play a role

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-1

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p>ylated (M) and unmethylated (U) band signals: L, length marker; C1, negative methylation control (plasmid pCU); C2, positive methylation control (plasmid pCM); CO, negative control; T1 – T3, tumoral and P1 – P3, normal paired tissue samples

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-8

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p>COBRA in 45 tumor and paired normal renal tissue samples as well as plasmid – DNA negative controls. Note that 44 (98%) tumors and 44 (98%) of 45 paired normal tissues demonstrated relative methylation greater than 2.75% determined as limit of analytical sensitivity. b. Quantitative methylation analysis of the promoter in 45 tumoral and normal tissue pairs using combined bisulfite restriction analysis (COBRA). Note that most of the tumors demonstrate a substantial relative increase in methylation when compared to their corresponding normal tissue (< 0.001

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-3

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p> 50 clones analyzed by the use of bisulfite sequencing. For each clone the methylation status of analyzed CpG sites is shown (solid circles: methylation, open circles: no methylation). Numbers on x-axis refer to base pair positions as indicated in Figure 1. Asterisks indicate amplicons derived from densely methylated promoters demonstrating at least 11 methylated CpG sites

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-6

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p>e sequence analysis. Numbers refer to the position of transcription start site while bracketed numbers indicate CpG sites. Solid triangles show TaqI restrictions sites used for COBRA. The solid and dashed lines indicate sequences amplified for COBRA and sequence analysis following bisulfite conversion of DNA. Inner primer positions were indicated by solid arrows

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-2

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p>COBRA in 45 tumor and paired normal renal tissue samples as well as plasmid – DNA negative controls. Note that 44 (98%) tumors and 44 (98%) of 45 paired normal tissues demonstrated relative methylation greater than 2.75% determined as limit of analytical sensitivity. b. Quantitative methylation analysis of the promoter in 45 tumoral and normal tissue pairs using combined bisulfite restriction analysis (COBRA). Note that most of the tumors demonstrate a substantial relative increase in methylation when compared to their corresponding normal tissue (< 0.001

Promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis-5

<p><b>Copyright information:</b></p><p>Taken from "promoter methylation and expression analysis in normal and neoplastic kidney indicates a role in early tumorigenesis"</p><p>http://www.molecular-cancer.com/content/6/1/49</p><p>Molecular Cancer 2007;6():49-49.</p><p>Published online 16 Jul 2007</p><p>PMCID:PMC1939711.</p><p></p>SSF1A protein using immunohistochemistry (counterstaining hematoxylin). a. Immunohistochemical positive control: CaSki cell line demonstrating absence of promoter methylation in COBRA (see box; U, unmethylated signals; M, methylation signals) and immunopositivity depending on the absence () or presence () of an antibody blocking peptide, respectively. b. Immunohistochemical negative control: HEK293 cell line showing strong methylation in COBRA (see box; U, unmethylated signals; M, methylation signals). Only residual unspecific nuclear immunopositivity can be observed, which is independent from the absence () and presence () of an antibody blocking peptide. c. Tumor cells of a clear cell RCC (magnification, 400×). d. Histopathologically normal tissue with immunopositive epithelial tubular cells (magnification, 400×). e. Heterogeneous staining patterns demonstrating immunopositive () and -negative () epithelia in renal tubules. f. Mosaic-like staining signals in a single tubular epithelium (see arrows, magnified from panel d as indicated)