Based on our assays, the increased level of AMPK phosphorylation may be the probable mechanism of activation of VPS34 tetramer induced by SD during M.tb infection.
By 24 h, several spherical clumps were visible at 14 mm, which, based on our assays in the control tube, was at ∼12 µM cyanide, similar to the concentration at which clumps formed in the Petri dish assay.
The results of our assay on the tested extracts are shown in Table 1.
As mitochondria are known to function quite well in vitro, most notably for metabolic and import reactions, we based the conditions of our assay on established buffering and energizing conditions [ 18, 19].
Additional exploratory analyses of our assay on the effect of frozen storage, which found concordant results in all samples, indicated the possibility to store freshly collected samples for a longer period and to process them later.
The subsequent reduction with NaBH4 and quantification of the formed 5,6-dihydro-M1dG showed no difference among the three tested buffers, suggesting no effect of Tris-HCl on the sensitivity of our assay.
One of the most interesting methodological advantages of our assay relies on the suitability of the inexpensive HotSHOT protocol for a rapid and feasible DNA extraction from bloodmeals.
We relied on microfiltration for our assay and had to centrifuge the food matrices to avoid clogging and did not monitor any losses of toxin or complex in that process nor adherence to the filters themselves.
This in turn infers, that when placed in the IGL locus of DT40 cells, the 3'E alone, just like the iE/MAR, works largely as a transcriptional enhancer with no detectable (based on the limit of our assays) targeting activity.
The extent to which the factors that are important for biofilm formation on abiotic surfaces (our assay) are also applicable for biofilm formation on biotic surfaces (e.g. in IBCs) needs to be further studied.
