Quantitative real-time polymerase chain reaction (qPCR) is a sensitive gene quantification method that has been extensively used in biological and biomedical fields. The currently used methods for PCR data analysis, including the threshold cycle method and linear and nonlinear model-fitting methods, all require subtracting background fluorescence. However, the removal of background fluorescence can hardly be accurate and therefore can distort results. We propose a new method, the taking-difference linear regression method, to overcome this limitation. Briefly, for each two consecutive PCR cycles, we subtract the fluorescence in the former cycle from that in the latter cycle, transforming the n cycle raw data into n-1 cycle data. Then, linear regression is applied to the natural logarithm of the transformed data. Finally, PCR amplification efficiencies and the initial DNA molecular numbers are calculated for each reaction. This taking-difference method avoids the error in subtracting an unknown background, and thus it is more accurate and reliable. This method is easy to perform, and this strategy can be extended to all current methods for PCR data analysis.

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Therapeutics, BioNTx, and Polaris. L.L. serves on advisory boards for Servier. S.C.W. and J.P.A. are inventors of a patent application submitted by The University of Texas MD Anderson Cancer Center related to a genetic mouse model of immune checkpoint blockade induced immune-related adverse events. S.C.W. is currently an employee of Spotlight Therapeutics. E.M.W has ownership interest in Pathogenesis, LLC. W.C.M. was supported by funding from the Niels Stensen Fellowship and the Netherlands Heart Institute. D.B.J serves on advisory boards for Array Biopharma, BMS, Merck, Novartis; research funding from BMS and Incyte. J.E.S serves on advisory boards for BMS. J.E.S, D.B.J and J.J.M are inventors of a patent application submitted by The Assistance Publique-Hopitaux de Paris related to abatacept for the treatment of immune-related adverse events associated with immune checkpoint inhibitors. Research.

Although cell-based studies have shown that c-tocotrienol (cTE) exhibits stronger anticancer activities than other forms of vitamin E including c-tocopherol (cT), the molecular bases underlying cTE-exerted effects remains to be elucidated. Here we showed that cTE treatment promoted apoptosis, necrosis and autophagy in human prostate PC-3 and LNCaP cancer cells. In search of potential mechanisms of cTE-provoked effects, we found that cTE treatment led to marked increase of intracellular dihydroceramide and dihydrosphingosine, the sphingolipid intermediates in de novo sphingolipid synthesis pathway but had no effects on ceramide or sphingosine. The elevation of these sphingolipids by cTE preceded or coincided with biochemical and morphological signs of cell death and was much more pronounced than that induced by cT, which accompanied with much higher cellular uptake of cTE than cT. The importance of sphingolipid accumulation in cTE-caused fatality was underscored by the observation that dihydrosphingosine and dihydroceramide potently reduced the viability of both prostate cell lines and LNCaP cells, respectively. In addition, myriosin, a specific inhibitor of de novo sphingolipid synthesis, counteracted cTE-induced cell death. In agreement with these cell-based studies, cTE inhibited LNCaP xenograft growth by 53% (p < 0.05), compared to 33% (p 5 0.07) by cT, in nude mice. These findings provide a molecular basis of cTE-stimulated cancer cell death and support the notion that elevation of intracellular dihydroceramide and dihydrosphingosine is likely a novel anticancer mechanism.Vitamin E has eight lipophilic antioxidants, that is, a-, b-, cand d-tocopherol (aT, bT, cT and dT) and a-, b-, c-and dtocotrienol (aTE, bTE, cTE and dTE). Specific forms of vitamin E have been proposed to be promising anticancer agents.

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