Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.     

Embryo defective 14 encodes a plastid‐targeted cGTPase essential for embryogenesis in maize

The embryo defective (emb) mutants in maize genetically define a unique class of loci that is required for embryogenesis but not endosperm development, allowing dissection of two developmental processes of seed formation. Through characterization of the emb14 mutant, we report here that Emb14 gene encodes a circular permuted, YqeH class GTPase protein that likely functions in 30S ribosome formation in plastids. Loss of Emb14 function in the null mutant arrests embryogenesis at the early transition stage. Emb14 was cloned by transposon tagging and was confirmed by analysis of four alleles. Subcellular localization indicated that the EMB14 is targeted to chloroplasts. Recombinant EMB14 is shown to hydrolyze GTP in vitro (K_m = 2.42 ± 0.3 μm ). Emb14 was constitutively expressed in all tissues examined and high level of expression was found in transition stage embryos. Comparison of emb14 and WT indicated that loss of EMB14 function severely impairs accumulation of 16S rRNA and several plastid encoded ribosomal genes. We show that an EMB14 transgene complements the pale green, slow growth phenotype conditioned by mutations in AtNOA1, a closely related YqeH GTPase of Arabidopsis. Taken together, we propose that the EMB14/AtNOA1/YqeH class GTPases function in assembly of the 30S subunit of the chloroplast ribosome, and that this function is essential to embryogenesis in plants.     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Inhibition of Transforming Growth Factor β (TGF-β) Signaling can Substitute for Oct4 Protein in Reprogramming and Maintain Pluripotency

Mouse pluripotent stem cells (PSCs), such as ES cells and induced PSCs (iPSCs), are an excellent system to investigate the molecular and cellular mechanisms involved in early embryonic development. The signaling pathways orchestrated by leukemia inhibitor factor/STAT3, Wnt/β-catenin, and FGF/MEK/ERK play key roles in the generation of pluripotency. However, the function of TGF-β signaling in this process remains elusive. Here we show that inhibiting TGF-β signaling with its inhibitor SB431542 can substitute for Oct4 during reprogramming. Moreover, inhibiting TGF-β signaling can sustain the pluripotency of iPSCs and ES cells through modulating FGF/MEK/ERK signaling. Therefore, this study reveals a novel function of TGF-β signaling inhibition in the generation and maintenance of PSCs.