Neuro-Fuzzy Based Hybrid Model for Web Usage Mining

AbstractWeb Usage mining consists of three main steps: Pre-processing, Knowledge Discovery and Pattern Analysis. The information gained from the analysis can then be used by the website administrators for efficient administration and personalization of their websites and thus the specific needs of specific communities of users can be fulfilled and profit can be increased. Also, Web Usage Mining uncovers the hidden patterns underlying the Web Log Data. These patterns represent user browsing behaviours which can be employed in detecting deviations in user browsing behaviour in web based banking and other applications where data privacy and security is of utmost importance. Proposed work pre-process, discovers and analyses the Web Log Data of Dr. T.M.A.PAI polytechnic website. A neuro-fuzzy based hybrid model is employed for Knowledge Discovery from web logs

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4β

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC‐specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β‐nanovaccine to guide further optimization and in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol% 1,8‐bis(p‐carboxyphenoxy)‐3,6‐dioxaoctane and 80 mol% 1,6‐bis(p‐carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β‐specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β‐loaded nanoparticles generated MUC4β‐specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy

Development and characterization of carboxy-terminus specific monoclonal antibodies for understanding MUC16 cleavage in human ovarian cancer

<div><p>MUC16 is overexpressed in ovarian cancer and plays important roles in invasion and metastasis. Previously described monoclonal antibodies against cell surface expressed MUC16 recognize the N-terminal tandemly repeated epitopes present in cancer antigen 125 (CA125). MUC16 is cleaved at a specific location, thus, releasing CA125 into the extracellular space. Recent reports have indicated that the retained carboxy-terminal (CT) fragment of MUC16 might play an important role in tumorigenicity in diverse types of cancers. However, limited data is available on the fate and existence of CT fragment on the surface of the cancer cell. Herein, we characterize two monoclonal antibodies (mAbs) showing specificity to the retained juxtamembrane region of MUC16. For the first time, we demonstrate that MUC16 is cleaved in ovarian cancer cells (NIH:OVCAR-3 [OVCAR-3]) and that the cleaved MUC16 subunits remain associated with each other. Immunohistochemical analyses on different grades of ovarian tumor tissues indicated differential reactivity of CA125 and MUC16 CT mAbs. The CA125 (M11) mAb detected 32/40 (80%), while the CT mAb (5E6) detected 33/40 (82.5%) of total ovarian cancer cases. For serous and serous papillary cases, the CA125 (M11) mAb stained 27/31 cases (87%), while CT mAb (5E6) stained 29/31 cases (93.5%). The CT mAb(s) accurately predict expression of MUC16 since their epitopes are not tandemly repeated and their reactivity may not be dependent on O-linked glycosylation. These antibodies can serve as valuable reagents for understanding MUC16 cleavage and may also serve as potential therapeutic agents for treatment of ovarian cancer.</p></div

Chimeric antibody targeting unique epitope on onco-mucin16 reduces tumor burden in pancreatic and lung malignancies

Abstract Aberrantly expressed onco-mucin 16 (MUC16) and its post-cleavage generated surface tethered carboxy-terminal (MUC16-Cter) domain are strongly associated with poor prognosis and lethality of pancreatic (PC) and non-small cell lung cancer (NSCLC). To date, most anti-MUC16 antibodies are directed towards the extracellular domain of MUC16 (CA125), which is usually cleaved and shed in the circulation hence obscuring antibody accessibility to the cancer cells. Herein, we establish the utility of targeting a post-cleavage generated, surface-tethered oncogenic MUC16 carboxy-terminal (MUC16-Cter) domain by using a novel chimeric antibody in human IgG1 format, ch5E6, whose epitope expression directly correlates with disease severity in both cancers. ch5E6 binds and interferes with MUC16-associated oncogenesis, suppresses the downstream signaling pFAK(Y397)/p-p70S6K(T389)/N-cadherin axis and exert antiproliferative effects in cancer cells, 3D organoids, and tumor xenografts of both PC and NSCLC. The robust clinical correlations observed between MUC16 and N-cadherin in patient tumors and metastatic samples imply ch5E6 potential in targeting a complex and significantly occurring phenomenon of epithelial to mesenchymal transition (EMT) associated with disease aggressiveness. Our study supports evaluating ch5E6 with standard-of-care drugs, to potentially augment treatment outcomes in malignancies inflicted with MUC16-associated poor prognosis

Generation and characterization of monoclonal antibodies (mAbs) to MUC16 C-terminal (CT) domain.

<p>(<b>A</b>) Structure of MUC16 CT domain indicating the two membrane-proximal cleavage sites. The fragment used for generation of hybridomas is indicated by a line with double arrow heads. This fragment incorporates the last putative cleavage site. Other important domains are indicated: TM- Transmembrane domain; Cyt. Tail- Cytoplasmic tail. (<b>B</b>) Binding of selected anti-MUC16 CT monoclonal antibodies with purified MUC16 CT protein using an indirect ELISA. MUC16 CT protein was coated in ELISA wells and hybridoma supernatants of the indicated antibodies were added. Antibody binding was detected using a secondary antibody labeled with horseradish peroxidase and TMB substrate. (<b>C</b>) Flow cytometry analysis showing relative binding of anti-MUC16 CT mAbs (5E6 and 3H1) to MIA PaCa-2 cells transfected either with control vector or MUC16 CT FL 321 construct (last 321 amino acids of MUC16). Binding was also analyzed on OVCAR-3 (MUC16_HIGH) and OVCAR-5 (MUC16_LOW) cells. Anti- tandem repeat mAb M11 served as a positive control. Cells were stained with the indicated antibodies and the signal was detected using Alexa-Fluor 488 anti-mouse IgG secondary antibody. A mouse IgG1 antibody served as the irrelevant isotype control and is indicated by the gray shaded curve. (<b>D</b>) Flow cytometry analysis of OVCAR-3 cells using mAbs pre-incubated with either MUC16 peptide or irrelevant control peptide. (<b>E and F</b>) OVCAR-3 and OVCAR-5 cells were seeded on coverslips, fixed with 4% Paraformaldehyde in PBS and were either permeabilized with 0.1% Triton X-100 in PBS (E) or not permeabilized (F) and incubated with 10 μg/ml of indicated mAbs. Signal was detected using Alexa Fluor 488 conjugated secondary antibody. Coverslips were placed on glass slides containing a drop of anti-fade Vectashield mounting medium and observed under a ZEISS confocal laser scanning microscope (magnification, 630X).</p

MUC16 CT mAb 5E6 exhibits heterogeneous staining on human ovarian cancer tissues.

<p>Sections of ovarian cancer tissues indicating strong membranous and cytoplasmic staining in tumor cells (A), focal apical membranous staining on tumor cells (B), and strong intra-luminal and membranous staining (C) of MUC16 CT. In all cases, the surrounding stroma was negative for MUC16 CT expression. Original magnification 200х.</p

Narrowing down the epitope recognized by MUC16 CT mAbs.

<p>(<b>A</b>) The last 12 and 29 amino acids from TM domain of MUC16 were deleted in the F114HA construct, tagged with FLAG at N-terminus and HA at C-terminus, to generate 2 deletion constructs (Δ12 and Δ29). The Δ12 and Δ29 constructs were transfected into HEK293T cells and immunoblotted with the indicated antibodies. (<b>B</b>) Schematic representation of MUC16 C-terminal region indicating various domains, cleavage site and approximate location of the putative epitope recognized by mAbs 5E6and 3H1. <u>Key</u>: Purple—represents the last SEA domain. Red portion—indicates the start of the transmembrane domain. Boxed region—indicates the putative epitope. Underlined region—represents the predicted putative cleavage site.</p

Partial epitope mapping using MUC16 CT constructs transfected into HEK293T cells.

<p><b>(A</b>) Schematic representation of different lengths of MUC16 CT fragments with C-terminal HA-tag cloned into the p3X-FLAG-CMV9 vector (Empty vector) with a preprotrypsin leader peptide (LP). The predicted cleavage sites in the last (site #1, PLARRVDR) and penultimate (site #2, DSVLV) SEA domains and the transmembrane (TM) domain are indicated. (<b>B</b>) Partial epitope mapping using various constructs of MUC16 CT (given in (<b>A</b>)) transfected into HEK293T cells was performed. Lysates from transfected cells were immunoblotted with the indicated antibodies. (<b>C</b>) The Flag tagged F114HA MUC16 CT construct was domain swapped with the various domains of MUC4 as indicated. (<b>D</b>) The constructs described in (<b>C</b>) were transfected into HEK293T cells. Lysates from these cells were immunoblotted with the respective antibodies.</p

List of antibodies used in this study.

<p>List of antibodies used in this study.</p