The Histone Deacetylases Hst1 and Rpd3 Integrate De Novo NAD+ Metabolism with Phosphate Sensing in Saccharomyces cerevisiae.

Nicotinamide adenine dinucleotide (NAD+) is a critical cofactor essential for various cellular processes. Abnormalities in NAD+ metabolism have also been associated with a number of metabolic disorders. The regulation and interconnection of NAD+ metabolic pathways are not yet completely understood. By employing an NAD+ intermediate-specific genetic system established in the model organism S. cerevisiae, we show that histone deacetylases (HDACs) Hst1 and Rpd3 link the regulation of the de novo NAD+ metabolism-mediating BNA genes with certain aspects of the phosphate (Pi)-sensing PHO pathway. Our genetic and gene expression studies suggest that the Bas1-Pho2 and Pho2-Pho4 transcription activator complexes play a role in this co-regulation. Our results suggest a model in which competition for Pho2 usage between the BNA-activating Bas1-Pho2 complex and the PHO-activating Pho2-Pho4 complex helps balance de novo activity with PHO activity in response to NAD+ or phosphate depletion. Interestingly, both the Bas1-Pho2 and Pho2-Pho4 complexes appear to also regulate the expression of the salvage-mediating PNC1 gene negatively. These results suggest a mechanism for the inverse regulation between the NAD+ salvage pathways and the de novo pathway observed in our genetic models. Our findings help provide a molecular basis for the complex interplay of two different aspects of cellular metabolism.

Plan-view transmission electron microscopy specimen preparation for atomic layer materials using a focused ion beam approach.

Using the focused ion beam (FIB) to prepare plan-view transmission electron microscopy (TEM) specimens is beneficial for obtaining structural information of two-dimensional atomic layer materials, such as graphene and molybdenum disulfide (MoS2) nanosheets supported on substrates. The scanning electron microscopy (SEM) image in a dual-beam FIB-SEM can accurately locate an area of interest for specimen preparation. Besides, FIB specimen preparation avoids damages and hydrocarbon contamination that are usually produced in other preparation methods, in which chemical etching and polymer adhesion layers are used. In order to reduce harmful ion-beam bombardment and re-deposition on the thin atomic layers during FIB specimen preparation, we develop a method to protect the atomic layers by making a "microcapsule" to insulate the sample surface. The method is applied respectively to prepare plan-view TEM specimens of a graphene sheet with multiple adlayers and MoS2 atomic layers. Useful electron diffraction results can be obtained from these specimens for understanding the interlayer orientation relationships in the two materials. Auger electron spectroscopy analysis further confirms that the sample surface is free from contamination under the sufficient protection given by the proposed method.