10 research outputs found
Data in support of in vivo studies of silk based gold nano-composite conduits for functional peripheral nerve regeneration
In the present data article we report the in vitro and in vivo biocompatibility of fabricated nerve conduits described in Das et al. [1]. Green synthesised gold nanoparticles (GNPs) were evaluated for their cytotoxicity in rat Schwann cells (SCTM41). We also describe herein the adhesion and proliferation of Schwann cells over the nanofibrous scaffolds. Methods describing surgical implantation of conduits in a rat sciatic nerve injury model, confirming its accurate implantation as well as the porosity and swelling tendency of the nerve conduits are illustrated in the various figures and graphs
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 ERĪ± in breast cancer is carried out using ERĪ±-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 ERĪ± 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 ERĪ± (<i>K</i>a = 1.55Ā±0.298Ć10<sup>8</sup> M<sup>-1</sup>; Ī<i>H</i> = 4.32Ć10<sup>4</sup>Ā±801.1 cal/mol; Ī<i>S</i> = -108 cal/mol/deg). Interestingly, the specificity measurements suggest that the ERaptD4 internalizes into ERĪ±-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 ERĪ± expression in breast cancer samples, we performed the aptamer-assisted histochemical analysis of ERĪ± in tissue samples from breast cancer patients. The results were validated by performing the immunohistochemistry on same samples with an ERĪ±-antibody. We found that the two methods agree strongly in assay output (kappa value = 0.930, p-value <0.05 for strong ERĪ± positive and the ERĪ± negative samples; kappa value = 0.823, p-value <0.05 for the weak/moderate ER+ve samples, n = 20). Further, the aptamer stain the ERĪ±-positive cells in breast tissues without cross-reacting to ERĪ±-deficient fibroblasts, adipocytes, or the inflammatory cells. Our results demonstrate a significant consistency in the aptamer-assisted detection of ERĪ± in strong ERĪ± positive, moderate ERĪ± positive and ERĪ± negative breast cancer tissues. We anticipate that the ERaptD4 aptamer targeting ERĪ± may potentially be used for an efficient grading of ERĪ± expression in cancer tissues.</p></div
Analysing the binding site of ERaptD4 on ERĪ±.
<p><b>(a)</b> Effect of increasing concentrations (100 nM, 250 nM, 500 nM, 1000 nM, 2000 nM, 3000 nM, 4000 nM and 5000 nM) of unlabelled EREs (ERE1: 5'- ttccagtgccaccggtg -3'; ERE2: 5'- ggtccagtgtcactggac- 3') on saturation binding of biotinylated ERaptD4 (100 nM). <b>(b)</b> Binding of ERaptD4 to the ligand binding domain and full length ERĪ± is probed using aptamer-ELISA. PR-DBD, PR-LBD and human serum are taken as sample controls. Non-enriched aptamer library is taken as ligand control.</p
List of the selected most probable ERĪ± aptamers and their copy number as obtained after high throughput sequencing.
<p>List of the selected most probable ERĪ± aptamers and their copy number as obtained after high throughput sequencing.</p
Analysis of the affinity and specificity of selected probable ERĪ±-aptamers.
<p>(<b>a</b>) Relative binding of selected DNA sequences is analyzed through an aptamer-assisted ELISA. ERĪ±-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 ERĪ± interactions with aptamer ERaptD4 is determined by titrating aptamer (10 Ī¼M; in the syringe) into ERĪ± (1Ī¼M, 1.4 ml in sample cell). The top panel represents the raw heats of binding obtained upon titration of aptamer to ERĪ± protein. The lower panel is the binding isotherm fitted to the raw data using one site model.</p
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 ERĪ±-positive MCF-7 and ERĪ±-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
Staining of ERĪ±-positive cells in breast cancer tissue samples.
<p>A comparative analysis of the aptahistochemistry and immunohistochemistry methods is made by analyzing the expression of ERĪ± in breast tissue sections with ERĪ±-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 ERĪ± aptamer screening using modified SELEX method. (<b>b</b>) Monitoring the enrichment of ERĪ± binding sequences through photocolorimetric approach. The relative binding of enriched sequences (equivalent amounts) obtained after 2, 4, 6, 8 and 9<sup>th</sup> 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