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Aim: The cancer stem cells (CSCs) are known to be responsible for drug resistance and cancer relapse in the treatment of cancer. Identification and isolation of CSCs and study of their properties will play a crucial role in developing an effective drug against these targets. The aim of the study was to isolate CSCs from primary cancer by the tumorspheres enrichment method, to confirm by indirect immunofluorescence and gene expression of stem cell markers by using real-time polymerase chain reaction (RT-PCR) technique. Materials and Methods: In this in vitro study, we enriched oral CSCs through tumorsphere formation assay from seven primary cultures of OSCC patients with defined serum media. The expression and localization of the cell surface markers of CD133 and CD44 were tested by indirect immunofluorescence. Gene expression of stem cell markers such as CD44, CD133, Oct4, Sox2, and Nanog were quantified by RT-PCR technique. One-way analysis of variance was applied to analyze gene expression. Results: Tumorsphere formation has been used to isolate the CSCs from the OSCC tissue culture. Both CD133 and CD44 antibody confirmed the presence of CSCs through indirect immunofluorescence. In comparison to parental cell lines, the expression levels of CD133, CD44, Oct4, Sox2, and Nanog stem cell were significantly higher in CSC-enriched subpopulations. Conclusions: The cost-effective spheroid enrichment and the indirect immunofluorescence methods are useful for the isolation of CSCs from the primary tumor.

Background and Aims:Porphyromonas gingivalis ( P. gingivalis) is considered as an important pathogen responsible for periodontal disease which is characterized by inflammation of gingiva and destruction of periodontal ligament and alveolar bone leading to loss of tooth. Along with clinical investigations, suitable microbiological analysis needs to be performed which could provide more insight into the disease severity. We aim to quantify P. gingivalis by real-time PCR (RT-PCR) and analyze its association with demographic data including clinical parameters.Materials and Methods:The study consisted of chronic periodontitis patients (CP group) and healthy subjects (H group) with 120 samples in each group. RT-PCR was carried out by the SYBR Green assay to target 16S ribosomal ribonucleic acid species-specific region of P. gingivalis. Standard strain of P. gingivalis ATCC 33277 was used as a control.Results:In the CP group, 79.16% samples were found positive for P. gingivalis, whereas 29.17% samples were positive in the H group. A significant difference was found when the prevalence was compared within males and females ( P < .001 for both). In the older age groups, we found a higher rate of detection of P. gingivalis. As analyzed by Spearman’s correlation test, the number of cells of P. gingivalis was significantly associated with probing depth ( P = .02) and clinical attachment level ( P = .01) in the CP group. The mean cell number of P. gingivalis was found to be increasing with increasing levels of probing depth and clinical attachment level ( P < .001 and P = .01, respectively).Conclusion:The present study reaffirms that the P. gingivalis microbe is significantly associated with the chronic periodontitis and that its level varies with the severity of the disease. Colonization of the bacterium is significantly associated with severe forms of the disease.

Aims and Objectives: The oral microbial flora is highly complex and diverse with obligate anaerobic bacteria as the predominant component. Most of these are not yet cultivated/difficult to cultivate due to technical limitations. In this study, we aim to detect two novel oral bacterial species Dialister invisus and Olsenella uli by simplified and economical procedure of polymerase chain reaction (PCR) and study their association with primary and persistent endodontic infections. Material and Methods: The study involved 60 patients that included 30 patients of primary endodontic infections and 30 with persistent endodontic infections. The sample collection from the root canal was performed by universally accepted protocol by using sterile paper points. The deoxyribonucleic acid (DNA) extraction was done, followed by PCR with species specific primers. We made several changes to the protocol mentioned by original authors. We adopted a one-step protocol for amplification of bacterial DNA, omitting the 16SrDNA amplification step with universal primers. Results: It was seen that 7 (23.3 %) samples in primary endodontic infection group and 24 (80 %) samples in persistent endodontic infection group were positive for D. invisus. On the other hand, 11 (36.6 %) patients of primary endodontic infection showed positivity for O. uli in comparison to 9 (30 %) of persistent endodontic infection. Conclusion: The results from the present study showed efficient amplification of both O. uli and D. invisus in a single-step PCR. Hence, we conclude that the modified protocol used here with taq polymerase enzyme offers a faster and cheaper alternative to nested PCR without compromising the quality of amplification process.