Although transplantation of c-kit+ cardiac stem cells (CSCs) alleviates post-myocardial infarction left ventricular dysfunction, there are no reliable methods that enable measurement of the absolute number of CSCs that persist in the recipient heart. To overcome this limitation, we developed a highly sensitive and accurate method to quantify the absolute number of murine CSCs after transplantation. This method has two unique features: i) real-time PCR-based detection of a novel male-specific, multiple-copy gene, Rbmy, which significantly increases the sensitivity of detection of male donor cells in a female recipient, and ii) an internal standard, which permits quantification of the absolute number of CSCs as well as the total number of cells in the recipient organ. Female C57BL/6 mice underwent coronary occlusion and reperfusion; 2 days later, 105 male mouse CSCs were injected intramyocardially. Tissues were analyzed by real-time PCR at serial time points. In the risk region, >75% of CSCs present at 5 min were lost in the ensuing 24 h; only 7.6±2.1% of the CSCs present at 5 min could still be found at 7 days after transplantation and only 2.8±0.5% (i.e., 1,224±230 cells/heart) at 35 days. Thus, even after direct intramyocardial injection, the total number of CSCs that remain in the murine heart is minimal (at 24 h, ~10% of the cells injected; at 35 days, ~1%). This new quantitative method of stem cell detection, which enables measurement of absolute cell number, should be useful to optimize cell-based therapies, not only for CSCs but also for other stem cells and other organs.

Increased oxidative stress is implicated in the etiology of cancer. Exposure to cigarette smoke is well documented to increase oxidative stress in smoker tissues and probably plays a role in the incidence of cervical cancer. The cervical pre-cancerous lesions, initiated by HPV infection, generally regress in the absence of the known risk factors such as smoking. The oxidation of deoxyguanosine results in a highly mutagenic and extensively studied oxidative DNA lesion, 8-oxodeoxyguanosine (8-oxodG). The present study was performed to determine i) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in HPV-transfected cervical cell lines (ECT1/E6 E7); ii) the removal of the oxidative damage from HPV-positive (CaSki), HPV-negative (C33A) and ECT1/E6 E7 cells; and iii) the cell-cycle progression and apoptosis in the CSC-treated ECT1/E6 E7. We found that CSC induced 8-oxodG lesion in a dose- and time-dependent manner in the transfected cervical cell line. A 2.4-fold higher damage was observed in the HPV-positive tumor cell line compared to the HPV-negative cell line. Flow-cytometric and ImageStream (Amnis) analysis indicated that CaSki cells most efficiently removed the oxidative lesion in 48 h (93%). On the contrary, C33A cells did not reveal any significant removal of 8-oxodG up to 48 h. This difference could be explained by the presence of residual p53 activity in CaSki cells while the p53 is mutated in the C33A cell line. ECT1/E6 E7 cell line showed only a modest (22%) removal after 48 h. Interestingly, 8-oxodG was almost completely removed 72 h post-exposure in all the cervical cell lines. Furthermore, a dose-dependent increase in apoptosis was observed in ECT1/E6 E7 cells with an arrest at G0/G1 phase of cell cycle. We conclude that cigarette smoke induces oxidative stress with higher burden in HPV-positive cells; however, HPV exposure may not play a role in the kinetics of 8-oxodG removal (Supported from Agnes Brown Duggan Endowment and, in part, USPHS grant ES-011564). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4367.

Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

Purpose This study assessed the impact of flipped classrooms on physician assistant (PA) students' performance and opinions. Methods Students completed quizzes and an opinion survey in Genetics, Human Pathophysiology (HPP), Clinical Medicine (CM) (n = 105) and Physical Exam (PE) (n = 98) courses. Results In Genetics and PE, the quiz scores were significantly higher for flipped classrooms (Genetics 95.00 ± 6.56; PE 83.09 ± 11.47) compared to the traditional lectures (Genetics 90.00 ± 10.53; PE 55.43 ± 16.66). In HPP, students performed better with traditional lectures (86.54 ± 8.82) compared to the flipped classrooms (75.12 ± 8.54). In CM, students' gain score was significantly higher for flipped classrooms (37.85 ± 16.73) than for traditional lectures (20.97 ± 15.55). The opinion surveys showed that the students surveyed preferred traditional lectures over flipped classrooms in Genetics (4.58 ± 0.46 vs. 2.29 ± 0.71) and HPP (4.14 ± 0.35 vs. 2.09 ± 0.53). Conclusion Flipped classrooms improved the quality of learning in courses that deliver a hands-on skill or use case-based scenarios. They may not be an ideal choice for courses that require explanation of intricate scientific concepts.

Exposure to cigarette smoke is well documented to increase oxidative stress and could account for higher risk of cervical cancer in smokers. Cervical pre-cancerous lesions that are initiated by human papillomavirus (HPV) infection generally regress in the absence of known risk factors such as smoking. 8-oxodeoxyguanosine (8-oxodG) is a highly mutagenic oxidative DNA lesion that is formed by the oxidation of deoxyguanosine. In the present study, we examined: a) the effect of cigarette smoke condensate (CSC) on 8-oxodG formation in and its removal from HPV-transfected (ECT1/E6 E7), HPV-positive (CaSki) and HPV-negative (C33A) human cervical cancer cells, and b) the cell cycle progression and apoptosis in CSC-treated ECT1/E6 E7 cells. CSC induced 8-oxodG in a dose-(p=0.03) and time (p=0.002)-dependent fashion in ECT1/E6 E7 cells as determined by flow cytometry. A 2.4-fold higher level of 8-oxodG was observed in HPV-positive compared with HPV-negative cells. However, 8-oxodG lesions were almost completely removed 72 h post-exposure in all cell lines as determined by ImageStream analysis. This observation correlates with the 2- and 5-fold increase in the p53 levels in ECT1/E6 E7 and CaSki cells with no significant change in C33A cells. We conclude that: a) cigarette smoke constituents induce oxidative stress with higher burden in HPV-positive cervical cancer cells and b) the significant increase observed in p53 levels in wild-type cervical cells (ECT1/E6 E7 and CaSki) may be attributed to the p53-dependent DNA repair pathway while a p53-independent pathway in C33A cells cannot be ruled out.