Novel vitamin E analogue and 9-nitro-camptothecin administered as liposome aerosols decrease syngeneic mouse mammary tumor burden and inhibit metastasis

Purpose To test the anticancer properties of a nonhydrolyzable ether-linked acetic acid analogue of vitamin E, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (-TEA), and a derivative of camptothecin, 9-nitrocamptothecin (9-NC) singly and in combination against mouse mammary tumor cells (line 66 clone 4 stably transfected with green fluorescent protein; 66c1-4-GFP) cultured in vitro or transplanted subcutaneously into the inguinal region of female BALB/c mice to form established tumors.Methods Following in vitro treatment of 66cl-4-GFP cells with -TEA and suboptimal concentrations of 9-NC, singly or in combination, apoptosis was measured by morphological evaluation of nuclei stained with 4,6-diamidino-2-phenylindole (DAPI), and DNA synthesis arrest was measured by tritiated thymidine uptake. For in vivo analyses -TEA and 9-NC, both water-insoluble compounds, were formulated into liposomes using dilauroylphosphatidylcholine and administered by aerosol to deliver doses calculated to be 36 and 0.4 g/mouse per day, respectively, (singly or each separately for combined treatments) 7 days per week.Results Treatment of 66cl-4-GFP cells in culture for 3 days with a combination of -TEA (10 g/ml; singly produces 38% apoptosis), and suboptimal concentrations of 9-NC (15.6, 31.3, 62.5, or 125 ng/ml; singly produce 2–7% apoptosis), produced 47%, 58%, 64%, and 69% apoptosis. Likewise, combinations of -TEA + 9-NC inhibited DNA synthesis more than either agent administered singly. A significant reduction (P<0.001) in growth of subcutaneous transplanted tumors was observed with liposome-formulated and aerosolized delivery of -TEA + 9-NC to BALB/c mice. The incidence of macroscopic lung metastasis was 83% in control vs 8% in -TEA-, 9-NC-, or combination-treated mice. Fluorescence microscopic examination of lungs and axillary and brachial lymph nodes showed a statistically significant decrease in metastasis observed in -TEA-, 9-NC-, and combination- vs control-treated animals. Analyses of primary tumor tissue for proliferation and apoptosis showed treatment groups to have lower Ki-67 and higher terminal deoxynucleotidyl transferase-mediated nick end labeling, respectively. Treatments showed no measurable effects on two angiogenesis parameters, namely intratumoral blood volume as assessed by hemoglobin content and intratumoral blood vessel density as assessed with CD31 staining.Conclusions Combination treatments enhanced antiproliferative and proapoptotic activities in cell culture, and when formulated in liposomes and delivered via aerosolization to treat an aggressive and metastatic syngeneic murine mammary tumor, the combination treatment showed a significant reduction in tumor volume in comparison to either treatment alone. Mechanistically, it appears that neither enhanced apoptosis, reduced cell proliferation, nor reduced blood vessel density can fully account for the enhanced effects of the combination treatment.Abbreviations -TEA 2,5,7,8-Tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid - 9-NC 9-Nitro-camptothecin - 66cl-4-GFP BALB/c mouse mammary tumor line 66 clone 4 stably transfected with GFP - DAPI 4,6-Diamidino-2-phenylindole - DMSO Dimethylsulfoxide - GFP Green fluorescent protein - PECAM-1 Platelet-endothelial cell adhesion molecule - THF Tetrahydrofuran - TUNEL Terminal deoxynucleotidyl transferase-mediated nick end labeling     

Easy listening to sleep recordings: tools and examples

BACKGROUND AND PURPOSE: To specify a simple conversion of neurophysiological signals contained in sleep recordings into standard audio files and to illustrate how our cerebral audio processor can then detect specific signal characteristics. METHODS: A software package (freely accessible from the Internet) has been developed that converts signals from standard EDF (or EDF+) format to standard audio (WAV) format, a process usually called audification. The software has been applied to sleep EEG, EOG and ECG. The software is easy to apply. RESULTS: A wide range of audified signals is described, stressing the analogy with familiar sounds. Audio properties of EEG in different sleep stages, EOG, ECG and respiration signals are discussed. Auditive presentation of the signals invokes brain processes that differ essentially from the commonly applied visual interpretation, including physiological frequency analysis. CONCLUSIONS: Such auditive interpretation may complement the visual one. The widespread use of EDF+ and multimedia computers makes such audification simple and straightforward.     

Assessing the agreement between two quantitative assays with repeated measurements

We propose a statistical test in evaluating the equivalence (or agreement) between assay values x and y obtained using two methods in assay or instrument validation, where both x and y are measurements of an unobserved analyte z. Our method builds on the availability of repeated measurements from each sampled subject, which enables us to assess the agreement in terms of the conditional mean of the difference x - y given z as well as the conditional variance of x - y given z. With the help of repeated measurements and a large number of sampled subjects, our method does not require any distributional assumption on (x, y, z) or any model assumption on the conditional mean (or variance) of x - y. Furthermore, our test is designed so that when the null hypothesis is rejected, we can conclude that the two assay methods do not have practically meaningful difference with a high statistical assurance, which is an approach adopted in the assessment of bioequivalence between two drug products.