The Antifungal Eugenol Perturbs Dual Aromatic and Branched-Chain Amino Acid Permeases in the Cytoplasmic Membrane of Yeast

Eugenol is an aromatic component of clove oil that has therapeutic potential as an antifungal drug, although its mode of action and precise cellular target(s) remain ambiguous. To address this knowledge gap, a chemical-genetic profile analysis of eugenol was done using ∼4700 haploid Saccharomyces cerevisiae gene deletion mutants to reveal 21 deletion mutants with the greatest degree of susceptibility. Cellular roles of deleted genes in the most susceptible mutants indicate that the main targets for eugenol include pathways involved in biosynthesis and transport of aromatic and branched-chain amino acids. Follow-up analyses showed inhibitory effects of eugenol on amino acid permeases in the yeast cytoplasmic membrane. Furthermore, phenotypic suppression analysis revealed that eugenol interferes with two permeases, Tat1p and Gap1p, which are both involved in dual transport of aromatic and branched-chain amino acids through the yeast cytoplasmic membrane. Perturbation of cytoplasmic permeases represents a novel antifungal target and may explain previous observations that exposure to eugenol results in leakage of cell contents. Eugenol exposure may also contribute to amino acid starvation and thus holds promise as an anticancer therapeutic drug. Finally, this study provides further evidence of the usefulness of the yeast Gene Deletion Array approach in uncovering the mode of action of natural health products.     

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.     

The association between fat mass and obesity-associated (FTO) genotype and serum vitamin D level in breast cancer patients

The preventive effect of vitamin D against breast cancer can be influenced by gene polymorphisms. This study aimed to investigate the association between serum level of 25(OH) vitamin D and FTO genotype in breast cancer patients. A cross-sectional study was carried out on 180 newly diagnosed patients with breast cancer in Tehran, Iran. The blood samples were collected from the participants in order to assess the FTO gene rs9939609 polymorphism by the tetra-primer amplification refractory mutation system (Tetra-ARMS) PCR method. The serum level of 25(OH) vitamin D was measured using the direct competitive enzyme-linked immunosorbent assay (ELISA) method. The association between vitamin D and the FTO genotype in patients with breast cancer was assessed after adjustment for cofounders. The frequency of TT, AT and AA genotypes in the breast cancer patients were 43% (n = 77), 49% (n = 89) and 8% (n = 14), respectively. All patients with higher than 40 ng/dl of serum 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele (p = 0.019). No linear association was found between the number of FTO risk allele and the level of serum vitamin D. All patients with high serum level of 25(OH) vitamin D had one or two copies of FTO rs9939609 risk allele. FTO gene polymorphisms may counteract the beneficial effects of vitamin D in breast cancer prevention. Further studies can help to better understand the genetic factors predisposing to breast cancer and their effect on the association between vitamin D and breast cancer.     

The role of Hippo signaling pathway and mechanotransduction in tuning embryoid body formation and differentiation

Embryoid bodies (EBs) are the three-dimensional aggregates of pluripotent stem cells that are used as a model system for the in vitro differentiation. EBs mimic the early stages of embryogenesis and are considered as a potential biomimetic body in tuning the stem cell fate. Although EBs have a spheroid shape, they are not formed accidentally by the agglomeration of cells; they are formed by the deliberate and programmed aggregation of stem cells in a complex topological and biophysical microstructure instead. EBs could be programmed to promisingly differentiate into the desired germ layers with specific cell lineages, in response to intra- and extra-biochemical and biomechanical signals. Hippo signaling and mechanotransduction are the key pathways in controlling the formation and differentiation of EBs. The activity of the Hippo pathway strongly relies on cell–cell junctions, cell polarity, cellular architecture, cellular metabolism, and mechanical cues in the surrounding microenvironment. Although the Hippo pathway was initially thought to limit the size of the organ by inhibiting the proliferation and the promotion of apoptosis, the evidence suggests that this pathway even regulates stem cell self-renewal and differentiation. Considering the abovementioned explanations, the present study investigated the interplay of the Hippo signaling pathway, mechanotransduction, differentiation, and proliferation pathways to draw the molecular network involved in the control of EBs fate. In addition, this study highlighted several neglected critical parameters regarding EB formation, in the interplay with the Hippo core component involved in the promising differentiation.     

Determination of floral initiation in <Emphasis Type="Italic">Malus domestica</Emphasis>: a novel morphogenetic approach

Floral initiation in apple (Malus domestica Borkh) was studied by a novel morphogenetic approach. Developmental stages of apices were evaluated based on the morphology of shoot apical meristem (SAM) from various collection dates. Besides, the frequency of each stage was calculated within apices populations after full blooming (DAFB). Prior to doming of apex, three marked phases were found based on SAM morphology: 1) narrow appearance (vegetative phase), 2) broadened form (transition phase), and 3) prominent shape (commitment phase). A furrow region was formed at the base of leaf primordium during the bract initiation, while significant broadening of SAM was observed. Cell division patterns manifested in modification of anisotropic clusters from isotropic cellular packets, as a result of which profound morphological changes of apices occurred. Based on these findings, we propose that the structural alterations prior to doming may be taken into account for determination of the initial development and reproduction signs in apple trees.