Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer

Supplementary Methods, Supplementary table, Supplementary Figure Legends. (DOCX 41 kb

A novel cancer testis antigen, A-kinase anchor protein 4 (AKAP4) is a potential biomarker for breast cancer.

BACKGROUND: Breast cancer is the second leading cause of cancer related deaths in women worldwide. Reports about the early diagnosis of breast cancer are suggestive of an improved clinical outcome and overall survival rate in cancer patients. Therefore, cancer screening biomarker for early detection and diagnosis is urgently required for timely treatment and better cancer management. In this context, we investigated an association of cancer testis antigen, A-Kinase anchor protein 4 (AKAP4) with breast carcinoma. METHODOLOGY/FINDINGS: We first compared the AKAP4 gene and protein expression in four breast cancer cells (MCF7, MDA-MB-231, SK-BR3 and BT474) and normal human mammary epithelial cells. In addition, 91 clinical specimens of breast cancer patients of various histotypes including ductal carcinoma in situ, infiltrating ductal carcinoma and infiltrating lobular carcinoma and 83 available matched adjacent non-cancerous tissues were examined for AKAP4 gene and protein expression by employing in situ RNA hybridization and immunohistochemistry respectively. Humoral response against AKAP4 was also investigated in breast cancer patients employing ELISA. Our in vitro studies in all breast cancer cells revealed AKAP4 gene and protein expression whereas, normal human mammary epithelial cells failed to show any expression. Using in situ RNA hybridization and immunohistochemistry, 85% (77/91) tissue specimens irrespective of histotypes, stages and grades of breast cancer clinical specimens revealed AKAP4 gene and protein expression. However, matched adjacent non-cancerous tissues failed to display any AKAP4 gene and protein expression. Furthermore, humoral response was observed in 79% (72/91) of total breast cancer patients. Interestingly, we observed that 94% (72/77) of breast cancer patients found positive for AKAP4 protein expression generated humoral response against AKAP4 protein. CONCLUSIONS: Collectively, our data suggests that AKAP4 may be used as serum based diagnostic test for an early detection and diagnosis of breast cancer and may be a potential target for immunotherapeutic use

Utility of inter-frequency amplitude ratio of vestibular-evoked myogenic potentials in identifying meniere's disease : a systematic review and meta-analysis

Objectives: A recently devised parameter of vestibular-evoked myogenic potential (VEMP) based on the principles of frequency tuning is the inter-frequency amplitude ratio (IFAR). It refers to the ratio of the amplitude of 1000 Hz tone burst evoked VEMP to 500 Hz evoked tone burst. A pathology like Meniere's disease changes the frequency response and alters the frequency tuning of the otolith organs. Because IFAR is based on the principle of frequency tuning of VEMP, it is likely to help identify Meniere's disease. Few studies in the last decade have investigated the utility of IFAR in identifying Meniere's disease. However, a systematic review and a meta-analysis on IFAR in Meniere's disease are lacking. The present study investigates whether the IFAR of VEMP helps identify Meniere's disease and differentiates it from healthy ears and other vestibular pathologies. Design: The present study is a systematic review and a meta-analysis. The studies investigating the IFAR of cervical and ocular VEMPs in Meniere's disease, healthy controls, and other vestibular pathologies were searched across research databases such as PubMed, Science Direct, and Scopus. The search strategy was developed using the PICO (population, intervention, comparison, and outcomes) format, and Medical Subject Headings (MeSH) terms and Boolean operators were employed. The systematic review was performed using the Rayyan software, whereas the Review Manager software was used to carry out the meta-analysis. A total of 16,605 articles were retrieved from the databases. After the duplicate removal, 2472 articles remained. These were eliminated using title screening, abstract screening, and full-length inspections. A total of nine articles were found eligible for quality assessment and meta-analysis, and the New Castle-Ottawa Scale was used for quality assessment. After the data extraction, 24 six articles were found to have the desired data format for the meta-analysis. Results: The results showed significantly higher IFAR in the affected ears of individuals in the Meniere's disease group than in the control group's unaffected ears. There was no significant difference between the unaffected ears of individuals in the Meniere's disease group and the ears of the control group. The only study on Meniere's disease and benign paroxysmal positional vertigo found significantly larger ocular VEMP IFAR in ears with Meniere's disease than in benign paroxysmal positional vertigo. Conclusions: This systematic review and meta-analysis found IFAR efficient in differentiating Meniere's disease from healthy controls. We also found an enhanced IFAR as a potential marker for Meniere's disease. However, more investigations are needed to confirm the utility of an enhanced IFAR value in the exclusive identification of Meniere's disease

Crystallization and preliminary X-ray characterization of phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv

The phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv was crystallized and diffraction data were collected to 2.8 Å resolution

Immunohistochemical analysis of AKAP4 protein expression in different stages of IDC tissue specimens.

<p>Panels A–D, H & E staining showing the histological cytostructure of representative stage I, stage II, stage III and stage IV tissue specimens respectively. Panels E–H, AKAP4 immunoreactivity was observed in the cytoplasm in all stages (brown color). Overall, 100% stage I (1/1), 86% stage II (44/51), 82% stage III (31/38) and 100% stage IV (100%) clinical specimens were found positive for AKAP4 protein expression. Panels I–L, no immunoreactivity was detected with control IgG probed specimens. Panels M- P, AKAP4 protein was not detected in any of the matched ANCT specimens probed with anti-AKAP4 antibodies. (Original magnification 400; Objective - X40).</p

Humoral response against AKAP4 in breast cancer patients.

<p>A, To detect circulating anti-AKAP4 antibodies in cancer patient’s sera, purified recombinant AKAP4 protein was coated in 96-well plates and subjected to ELISA. Line X denotes the mean +2SD value of healthy female’s sera (absorbance value = 0.308) above which the patient’s sera was designated positive and below which the patient’s sera was designated as negative for anti-AKAP4 antibodies. All healthy males were found to be negative for anti-AKAP4 antibodies. B, Western blotting experiments were carried out by resolving purified AKAP4 recombinant protein on SDS-PAGE. Two representative cancer patient’s sera from each histotypes (DCIS, IDC and ILC) are shown depicting AKAP4 immunoreactivity. C, two representative samples showing AKAP4 immunoreactivity in different grades (grade 1, grade 2 and grade 3). D, two representative early stages and late stages sera samples showing reactivity against AKAP4. However, normal healthy sera failed to show AKAP4 immunoreactivity. E, Band 1, Coomassie stained recombinant purified AKAP4 protein; Band 2, recombinant AKAP4 protein probed with polyclonal anti-AKAP4 antibody (positive control); Band 3, recombinant AKAP4 protein probed with neutralized polyclonal anti-AKAP4 antibody (1∶10 diluted polyclonal anti-AKAP4 antibody pre-incubated with 15 µg/ml recombinant AKAP4 protein) resulted in loss of reactivity; Band 4, <i>E.coli</i> BL 21 (DE3) whole cell lysate probed with polyclonal anti-AKAP4 antibody (non-specific <i>E.coli</i> BL21 background) showed no immune-reactive band; Band 5 and 6, Representative neutralized sera samples of two cancer patients (positive for anti-AKAP4 antibodies) showed complete loss of reactivity. F. To demonstrate the specificity of anti-AKAP4 antibody in tissue specimens, serial sections were probed with anti-AKAP4 antibody and neutralized anti-AKAP4 antibody. Representative IDC specimen showing H&E staining (left), anti-AKAP4 antibody (Pre-neutralization; middle)) and neutralized anti-AKAP4 antibody (Post-neutralization; right).</p

AKAP4 expression and localization in breast cancer cell lines.

<p>A, Reverse-transcriptase PCR analysis showing <i>AKAP4</i> gene expression in testis, all breast cancer cell lines, MCF7, MDA-MB 231, SK-BR3 and BT474 but not in normal human mammary epithelial cells. B, AKAP4 protein expression was detected by employing Western blotting. C, Cells were fixed, permeabilized and processed for indirect immunofluorescence studies which revealed cytoplasmic localization (green color) of AKAP4 protein. Nuclei were stained blue using DAPI. D, Flow cytometric analysis of live cells demonstrating surface expression of AKAP4 protein in all breast cancer cell lines, MCF7, MDA­MB-231, SK-BR3 and BT474. The surface expression of AKAP4 protein (blue histogram) is depicted by the shift of fluorescence on X-axis with respect to unstained cells (green histogram) and control IgG stained cells (red histogram). Analysis was done using BD cell quest software.</p