Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.

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Background: The Y-box protein MSY3/Csda represses myogenin transcription in skeletal muscle by binding a highly conserved cis-acting DNA element located just upstream of the myogenin minimal promoter (myogHCE). It is not known how this MSY3 activity is controlled in skeletal muscle. In this study, we provide multiple lines of evidence showing that the post-translational phosphorylation of MSY3 by Akt kinase modulates the MSY3 repression of myogenin. Methods: Skeletal muscle and myogenic C2C12 cells were used to study the effects of MSY3 phosphorylation in vivo and in vitro on its sub-cellular localization and activity, by blocking the IGF1/PI3K/Akt pathway, by Akt depletion and over-expression, and by mutating potential MSY3 phosphorylation sites. Results: We observed that, as skeletal muscle progressed from perinatal to postnatal and adult developmental stages, MSY3 protein became gradually dephosphorylated and accumulated in the nucleus. This correlated well with the reduction of phosphorylated active Akt. In C2C12 myogenic cells, blocking the IGF1/PI3K/Akt pathway using LY294002 inhibitor reduced MSY3 phosphorylation levels resulting in its accumulation in the nuclei. Knocking down Akt expression increased the amount of dephosphorylated MSY3 and reduced myogenin expression and muscle differentiation. MSY3 phosphorylation by Akt in vitro impaired its binding at the MyogHCE element, while blocking Akt increased MSY3 binding activity. While Akt over-expression rescued myogenin expression in MSY3 overexpressing myogenic cells, ablation of the Akt substrate, (Ser126 located in the MSY3 cold shock domain) promoted MSY3 accumulation in the nucleus and abolished this rescue. Furthermore, forced expression of Akt in adult skeletal muscle induced MSY3 phosphorylation and myogenin derepression. Conclusions: These results support the hypothesis that MSY3 phosphorylation by Akt interferes with MSY3 repression of myogenin circuit activity during muscle development. This study highlights a previously undescribed Akt-mediated signaling pathway involved in the repression of myogenin expression in myogenic cells and in mature muscle. Given the significance of myogenin regulation in adult muscle, the Akt/MSY3/myogenin regulatory circuit is a potential therapeutic target to counteract muscle degenerative disease.

Diabetic kidney disease (DKD) is a major complication of diabetes and the leading cause of end-stage renal failure. Epigenetics has been associated with metabolic memory in which prior periods of hyperglycemia enhance the future risk of developing DKD despite subsequent glycemic control. To understand the mechanistic role of such epigenetic memory in human DKD and to identify new therapeutic targets, we profiled gene expression, DNA methylation, and chromatin accessibility in kidney proximal tubule epithelial cells (PTECs) derived from subjects with and without type 2 diabetes (T2D). T2D-PTECs displayed persistent gene expression and epigenetic changes with and without transforming growth factor-β1 treatment, even after culturing in vitro under similar conditions as nondiabetic PTECs, signified by deregulation of fibrotic and transport-associated genes (TAGs). Motif analysis of differential DNA methylation and chromatin accessibility regions associated with genes differentially regulated in T2D revealed enrichment for SMAD3, HNF4A, and CTCF transcription factor binding sites. Furthermore, the downregulation of several TAGs in T2D (including CLDN10, CLDN14, CLDN16, SLC16A2, and SLC16A5) was associated with promoter hypermethylation, decreased chromatin accessibility, and reduced enrichment of HNF4A, histone H3-lysine-27-acetylation, and CTCF. Together, these integrative analyses reveal epigenetic memory underlying the deregulation of key target genes in T2D-PTECs that may contribute to sustained renal dysfunction in DKD.

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The role of internal lubricants in the closure of large intergranular pores during dry-pressing was investigated. Alumina was spray-dried with and without an internal lubricant to yield granules with similar characteristics other than lubricant effects. Green and sintered microstructures were evaluated at different compaction pressures. The defects were quantified by evaluating the fracture surface of ϳ90% dense sintered compacts. The samples that contained an internal lubricant had higher green densities and fewer defects at comparable compaction pressures. The internal lubricant did not cause any significant reduction in green strength or increase in springback.

The effects of an externally applied plasticizer on compaction behavior and green microstructure quality of spray-dried powders was investigated. The plasticizer was applied to the external surfaces of already spray-dried powders by spraying it on tumbling spray-dried granules. The apparent yield point of the spray-dried powder was reduced when the plasticizer was added. Microstructures of compacts made from these granules (with and without the externally applied plasticizer) were compared at different compaction pressures. Better knitting across granule interfaces and fewer defects were obtained for the granules with the externally applied plasticizer.

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