A facile gold nanoparticle-based ELISA system for detection of osteopontin in saliva: Towards oral cancer diagnostics.

In the current study, we emphasize that osteopontin is overexpressed in oral squamous cell carcinoma. Overexpression of osteopontin levels was confirmed by mRNA quantification studies and immunohistochemistry analysis. Based on this, a gold nanoparticle-based ELISA system was developed for non-invasive osteopontin detection. The incorporation of AuNRs (Gold nanorods) or AuNSs (Gold nanospheres) in the conventional ELISA improved the sensitivity of analyses. A considerably lowered detection limit in case of AuNR (detection limit: 0.02ngmL-1) and AuNS (detection limit: 0.03ngmL-1) modified assay was obtained as compared to commercially available OPN ELISA kit (detection limit: 0.14ngmL-1). The modified ELISA had a wide linear detection range (0.31-20ngmL-1), good reproducibility, and specificity against the tested interferents in the saliva. Finally, the nanoELISA was validated with osteopontin spiked in artificial and normal saliva samples and observed to show good recovery (95.4-97.85%), which indicates the application potential of the developed kit for real sample analysis.

p53 Expression Helps Identify High Risk Oral Tongue Pre- malignant Lesions and Correlates with Patterns of Invasive Tumour Front and Tumour Depth in Oral Tongue Squamous Cell Carcinoma Cases.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cancer subtype with a maximum propensity for regional spread. Our objective was to study if p53 expression might have any correlation with aggressive patterns of invasion within oral tongue cancers as well as with the histologically identified degree of oral tongue dysplasia. p53 immunoexpression was studied using immunohistochemistry in early staged OTSCCs (n=155), oral tongue dysplasias, (n=29) and oral tongue normal specimens (n=10) and evaluated for correlations with histological and clinicopathological parameters. Our study (n=194) showed a pattern of p53 expression increasing with different grades of tongue dysplasia to different grades of invasive OTSCC (p=0.000). Among the OTSCC tumours, positive p53 expression was seen in 43.2% (67/155) and a higher p53 labelling index was significantly associated with increased Bryne's grade of the tumour invasive front (p=0.039) and increased tumour depth (p=0.018). Among the OTSCC patients with tobacco habits, (n=91), a higher p53 labelling index was significantly associated with increased risk of local recurrence (p=0.025) and with lymphovascular space involvement (p=0.014). Evaluation of p53 through varying degrees of dysplasia to oral tongue cancer indicates that p53 expression is linked to aggressive features of oral tongue cancers and tongue precancers entailing a closer monitoring in positive cases. Among the OTSCCs, p53 expression is associated with tumour aggressiveness correlating with increased grading of invasive tumour front and tumour depth.

Use of Fast Transfer Analysis Cartridges for Cervical Sampling and Real Time PCR Based High Risk HPV Testing in Cervical Cancer Prevention - a Feasibility Study from South India.

BACKGROUND: Molecular testing for human papillomavirus (HPV) is the most objective and reproducible of all cervical cancer screening tests and also less demanding in terms of training and quality assurance. However, there is an impending need for cost effective molecular HPV testing methods with sampling ease, easy storage measures and minimum turn around times suitable for a low resource setting. OBJECTIVE: Our aim was to evaluate the feasibility of using a fast transfer analysis (FTA) mini elute cartridge for cervical sampling to identify high risk HPV by real time PCR and to compare molecular HPV testing and Pap cytology testing to predict histologically confirmed cervical precancer (CIN 2+ lesions) in a cervical cancer prevention program. MATERIALS AND METHODS: This was conducted as a pilot study (n=200) on women sampled using FTA mini elute cartridges, genotyped by two different real time PCR assays, detecting 13 high risk HPV (HR HPV) species, including HPV16 along with its physical DNA status. Results obtained from each of the tests were compared and analysed using suitable statistical tests. RESULTS: With FTA mini elute cartridge samples HR HPV positivity was seen in 48/200 (24%). Of these, presence of HPV 16 DNA was observed in 28/48 (58.3%) women. High risk HPV was positive in 20% (37/185) of women with benign cytology and 73.3% (11/15) of women with abnormal cytology findings. A very significant correlation (χ2 = 22.090 ; p=0.000) was observed between cytology and HR HPV findings showing an increasing trend of HR HPV prevalence in 50% (1/2) of LSIL, 75% (3/4) of HSIL and 100% (3/3) of SCC. Of the CIN 2+ lesions identified by histopathology, 88.9% (8/9) had HR HPV. A significant association (χ2=11.223 ; p=0.001) of HR HPV and histopathologically confirmed CIN 2+ lesions was found. Sensitivity of the two tests were comparable but specificity of Pap testing was better (90.7% vs 70.4%) to predict histopathologically diagnosed cervical precancers. CONCLUSIONS: The current study explored the feasibility of using a FTA mini elute cartridge for cervical sampling for the first time in India as a part of a community based cervical cancer prevention program. We suggest that FTA based sampling is suitable and feasible for real time based HPV testing. Molecular HR HPV testing can be more sensitive and useful to identify high risk women requiring Pap testing which is more specific to detect histologically confirmed cervical precancer.

A unique phosphorylation-dependent eIF4E assembly on 40S ribosomes co-ordinated by hepatitis C virus protein NS5A that activates internal ribosome entry site translation.

We previously reported that the HCV (hepatitis C virus) protein NS5A up-regulated mRNA cap binding eIF4F (eukaryotic initiation factor 4F) complex assembly through mTOR (mechanistic target of rapamycin)-4EBP1 (eIF4E-binding protein 1) pathway and that NS5A (non-structural protein 5A) physically interacted with translation apparatus. In the present study, we demonstrate that NS5A co-ordinates a unique assembly of the cap binding protein eIF4E and 40S ribosome to form a complex that we call ENR (eIF4E-NS5A-ribosome). Recruitment of NS5A and eIF4E to 40S ribosome was confirmed by polysome fractionation, subcellular fractionation and high-salt-wash immunoprecipitation. These observations were also confirmed in HCV-infected cells, validating its biological significance. eIF4E phosphorylation was critical for ENR assembly. 80S ribosome dissociation and RNase integrity assays revealed that, once associated, the ENR complex is stable and RNA interaction is dispensable. Both the N- and C-terminal regions of NS5A domain 1 were indispensable for this assembly and for the NS5A-induced HCV IRES (internal ribosome entry site) activation. The present study demonstrates that NS5A initially associates with phosphorylated eIF4E of eIF4F complex and subsequently recruits it to 40S ribosomes. This is the first time the interaction of viral protein with both eIF4E and ribosomes has been reported. We propose that this assembly would determine the outcome of HCV infection and pathogenesis through regulation of viral and host translation.

Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition.

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.