Relationship of betel nut, tobacco, smoking, and alcohol and status of hr-HPV in Oral cavity patients only.

<p>* Statistically Significant OR = odds ratio; CI = confidence interval;</p><p>^† Reference group for OR calculation</p><p>Relationship of betel nut, tobacco, smoking, and alcohol and status of hr-HPV in Oral cavity patients only.</p

Demographic profiles and association with hr- HPV Positivity-.

<p>* Statistically Significant; OR = odds ratio; CI = confidence interval;</p><p>^† Reference group for OR calculation. Since only one variable is significant that will be significant in multivariate analysis also</p><p>Demographic profiles and association with hr- HPV Positivity-.</p

Relation of HPV -16 and HPV-18 with clinico-pathological characteristics.

<p>* Statistically Significant, OR = odds ratio; CI = confidence interval;</p><p>^† Reference group for OR calculation</p><p>Relation of HPV -16 and HPV-18 with clinico-pathological characteristics.</p

Relationship between clinico-pathological characteristics and hr- HPV Positivity.

<p>* Statistically Significant OR = odds ratio; CI = confidence interval;</p><p>^† Reference group for OR calculation</p><p>Relationship between clinico-pathological characteristics and hr- HPV Positivity.</p

Aptamer-Assisted Detection of the Altered Expression of Estrogen Receptor Alpha in Human Breast Cancer

<div><p>An increase in the expression of estrogen receptors (ER) and the expanded population of ER-positive cells are two common phenotypes of breast cancer. Detection of the aberrantly expressed ERα in breast cancer is carried out using ERα-antibodies and radiolabelled ligands to make decisions about cancer treatment and targeted therapy. Capitalizing on the beneficial advantages of aptamer over the conventional antibody or radiolabelled ligand, we have identified a DNA aptamer that selectively binds and facilitates the detection of ERα in human breast cancer tissue sections. The aptamer is identified using the high throughput sequencing assisted SELEX screening. Biophysical characterization confirms the binding and formation of a thermodynamically stable complex between the identified DNA aptamer (ERaptD4) and ERα (<i>K</i>a = 1.55±0.298×10⁸ M^-1; Δ<i>H</i> = 4.32×10⁴±801.1 cal/mol; Δ<i>S</i> = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERα-positive breast cancer cells in a target-selective manner and localizes specifically in the nuclear region. To harness these characteristics of ERaptD4 for detection of ERα expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERα in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERα-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERα positive and the ERα negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERα-positive cells in breast tissues without cross-reacting to ERα-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERα in strong ERα positive, moderate ERα positive and ERα negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERα may potentially be used for an efficient grading of ERα expression in cancer tissues.</p></div

Specificity analysis of ERaptD4 using flow cytometry, fluorescent microscopy and cytochemistry.

<p>(<b>a1-a2</b>) Treatment of MCF-7 cells with fluorescent-tagged ERaptD4 produced a shift of 84% relative to unstained MCF-7 cells. Figure in inset provides the fluorescent microscopy image of the FAM-ERaptD4 treated cells. (<b>b1-b2</b>) A shift of 20% is observed in MDA-MB-231 cell upon treatment with fluorescent-labelled ERaptD4. (<b>c1-c2</b>) The treatment of MCF-7 cells with a random sequence (FAM-labelled non-enriched aptamer library) produced no shift, indicating the lack of binding by the random sequence. FAM-labelling of the non-enriched aptamer library is carried out using PCR amplification with FAM-forward primer and biotin-reverse primer. Strand separation is performed on streptavidin magnetic beads. (<b>d-e</b>) Aptacytochemistry of ERα-positive MCF-7 and ERα-deficient MDA-MB-231 cells. Size-bar on images a-c is 100 pixels (imaging at 63X). Size-bar on images d-e is 200 pixels (imaging at 20X).</p

Analysis of the affinity and specificity of selected probable ERα-aptamers.

<p>(<b>a</b>) Relative binding of selected DNA sequences is analyzed through an aptamer-assisted ELISA. ERα-antibody is used as a positive control to normalize the binding. Non-enriched aptamer library is taken as negative control. (<b>b</b>) Target selectivity of selected sequences is analyzed in a similar manner using cellular extracts of MCF-7 and MDA-MB-231 cells. (<b>c</b>) ITC isotherms of ERα interactions with aptamer ERaptD4 is determined by titrating aptamer (10 μM; in the syringe) into ERα (1μM, 1.4 ml in sample cell). The top panel represents the raw heats of binding obtained upon titration of aptamer to ERα protein. The lower panel is the binding isotherm fitted to the raw data using one site model.</p

List of the selected most probable ERα aptamers and their copy number as obtained after high throughput sequencing.

<p>List of the selected most probable ERα aptamers and their copy number as obtained after high throughput sequencing.</p

Staining of ERα-positive cells in breast cancer tissue samples.

<p>A comparative analysis of the aptahistochemistry and immunohistochemistry methods is made by analyzing the expression of ERα in breast tissue sections with ERα-antibody (sc-543) and ERaptD4 aptamer. The 10 X and 40 X represents the two magnifications of same images stained using antibody or aptamer.</p

Monitoring the aptamer enrichment.

<p>(<b>a</b>) Schematic representation of ERα aptamer screening using modified SELEX method. (<b>b</b>) Monitoring the enrichment of ERα binding sequences through photocolorimetric approach. The relative binding of enriched sequences (equivalent amounts) obtained after 2, 4, 6, 8 and 9^th round of SELEX screening is analyzed through photocolorimetric method. The initial non-enriched library is used as a control sequence. (<b>c</b>) Copy number analysis (cut-off = 5) in the enriched DNA sequences obtained through Illumina sequencing.</p