Among the 12 P-type ATPases encoded by the genome of Mycobacterium tuberculosis(Mtb), CtpF responds to the greatest number of stress conditions, including oxidative stress, hypoxia, and infection. CtpF is the mycobacterial homolog of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) of higher eukaryotes. Its expression is regulated by the global regulator of latency, DosR. However, the role that CtpF plays in the mycobacterial plasma membrane remains unknown. In this study, different functional analyses showed that CtpF is associated with calcium pumping from mycobacterial cells. Specifically, Mtb CtpF expression in Mycobacterium smegmatis cells prevents Ca2+ accumulation compared with wild type (WT) cells. In addition, plasma membrane vesicles from recombinant membranes, in which the direction of ion transport is inverted, accumulate more Ca2+ compared with vesicles obtained from the WT strain. This findings support the hypothesis that CtpF contributes to calcium efflux from mycobacterial cells. Accordingly, Mtb cells defective in ctpF (MtbΔctpF) accumulate more Ca2+ compared with WT cells, while the Ca2+-dependent ATPase activity is significantly lower in the mutant cells. Interestingly, the deletion of ctpF in Mtb impairs the tolerance of the bacteria to oxidative and nitrosative stress. Overall, our results indicate that CtpF is associated with calcium pumping from mycobacterial cells and the response to oxidative stress.
A fast and simple method for the extraction and Flame Atomic Absorption Spectroscopy (FAAS) quantification of ZnO in different cosmetic matrices, including lipsticks, water-in-oil foundations, and oil-in-water creams, was developed and validated, according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and the United States Pharmacopeial Convention guidelines. The sample preparation consisted of an ultrasound-assisted ethanolic extraction of ZnO followed by digestion with 1 M nitric acid (HNO3). Samples were analyzed by Flame Atomic Absorption Spectroscopy (FAAS). Specificity, linearity, the limit of detection (LOD), the limit of quantification (LOQ), sensitivity, precision, and accuracy parameters were studied. The robustness of the method was evaluated with a five-variable Youden–Steiner model. The method was specific for ZnO, and the extraction procedure did not affect the stability of the signal compared to the background. The method was linear in the range 0.2–1.0 mg/L with LOD/LOQ values equal to 0.0156 (mg·L−1)/0.0473 (mg·L−1), 0.0098 (mg·L−1)/0.0297 (mg·L−1), 0.0113 (mg·L−1)/0.0341 (mg·L−1), and 0.0131 (mg·L−1)/0.0397 (mg·L−1), respectively, for raw material, lipstick, liquid foundation, and emulsion matrices. Regarding precision, the %RSD values were below 3.0% for repeatability and intermediate precision. Global reproducibility RSD was below 8.0% for all matrices. The percentage of recovery was not statistically different from 100% in all cases. The final concentration was found to be a critical variable for all matrices except for the raw material. The variables associated with the extraction step (ethanol volume, bath temperature, and extraction time) were critical in the extraction of liquid foundations and cream emulsions. The method reduces the number and concentration of mineral acids spent on the digestion of ZnO, and its application is extendable to raw materials. This development is an adequate tool for routine analysis and cosmetic quality control of chemically different products that contain ZnO as ultraviolet radiation (UV) filter, to guarantee regulatory compliance and ensure the safety and efficacy of products delivered to consumers.
A new method for extraction of titanium dioxide (TiO2) from cosmetic matrices using borate salts for its quantification by flame atomic absorption spectroscopy (FAAS) was developed and validated. Following International Commission for Harmonization (ICH) and the United States Pharmacopeial Convention (USP) guidelines, the parameters of the method considered in this study were specificity, linearity, sensitivity, precision, and accuracy. In addition, critical factors of the method were assessed using a Youden–Steiner model. The method was able to differentiate the titanium signal from matrix and background signals, for which it is considered specific. The method is also linear for all cosmetic matrices and the raw material in the range 20–80 ppm with LOD and LOQ around 2 ppm and 7 ppm, respectively. Repeatability and intermediate precision were below 5.0%RSD, and Global Reproducibility was below 8.0% RSD. A digestion step free of HF or strong oxidizers makes this method a safer and easily transferable alternative to classical methods for quality control. It is thus a convenient tool for routine analysis of cosmetic products that need to comply with current regulations to ensure the safety of consumers and to guarantee batch-to-batch quality.
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