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Insulin regulates various cellular metabolic processes by activating specific isoforms of the Akt family of kinases. Here, we elucidated metabolic pathways that are regulated in an Akt2-dependent manner. We constructed a transomics network by quantifying phosphorylated Akt substrates, metabolites, and transcripts in C2C12 skeletal muscle cells with acute, optogenetically induced activation of Akt2. We found that Akt2-specific activation predominantly affected Akt substrate phosphorylation and metabolite regulation rather than transcript regulation. The transomics network revealed that Akt2 regulated the lower glycolysis pathway and nucleotide metabolism and cooperated with Akt2-independent signaling to promote the rate-limiting steps in these processes, such as the first step of glycolysis, glucose uptake, and the activation of the pyrimidine metabolic enzyme CAD. Together, our findings reveal the mechanism of Akt2-dependent metabolic pathway regulation, paving the way for Akt2-targeting therapeutics in diabetes and metabolic disorders.

Long non-coding RNAs (lncRNAs) play vital roles in the pathogenesis of infectious diseases, but the role of lncRNAs in herpes simplex virus 1 (HSV-1) infection remains unknown. Using RNA sequencing analysis, we explored lncRNAs that were highly expressed in murine retinal photoreceptor cell-derived 661W cells infected with HSV-1. U90926 RNA (522 nucleotides) was the most upregulated lncRNA detected post HSV-1 infection. The level of U90926 RNA was continuously increased post HSV-1 infection, reaching a 100-fold increase at 24 h. Cellular fractionation showed that U90926 RNA was located in the nucleus post HSV-1 infection. Downregulation of U90926 expression by RNA interference markedly suppressed HSV-1 DNA replication (80% reduction at 12 h post infection) and HSV-1 proliferation (93% reduction at 12 h post infection) in 661W cells. The survival rates of U90926-knockdown cells were significantly increased compared to those of control cells (81% and 21%, respectively; p U90926 is crucial for HSV-1 proliferation in retinal photoreceptor cells and consequently leads to host cell death by promoting HSV-1 proliferation.

Salmonella enterica serovar Typhimurium (Salmonella), a pathogenic bacterium, is a major cause of foodborne diseases worldwide. Salmonella injects multiple virulence factors, called effectors, into cells and causes multiple rearrangements of cellular biological reactions that are important for Salmonella proliferation and virulence. Previously, we reported that Salmonella infection causes loss of MTR4 and RRP6, which are nuclear RNA degradation factors, resulting in the stabilization and accumulation of unstable nuclear RNAs. This accumulation is important for the cellular defense for Salmonella infection. In this study, we examined a series of Salmonella mutant strains, most of which are strains with genes related to effectors translocated by T3SSs encoded on Salmonella pathogenic islands, SPI-1 and SPI-2, that have been depleted. Among 42 Salmonella mutants, 6 mutants' infections canceled loss of MTR4 and RRP6. Proliferation assay of Salmonella in the cell revealed that six mutants showed poor proliferation in the host cell, demonstrating that poor proliferation contributed to cancellation of MTR4 and RRP6 loss. This result indicates that certain events associated with Salmonella proliferation in host cells cause loss of MTR4 and RRP6.

Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of “Dyrec-seq,” which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes’ biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.

As intracellular RNA levels are determined by the balance between RNA synthesis and decay, RNA stability is a critical factor in the quantity of intracellular RNA. Recent advances in epitranscriptomic measuring techniques have shown that RNA modifications, such as methylation and acetylation, affect RNA stability, including those of messenger RNA (mRNA) and noncoding RNA (ncRNA) such as transfer RNA (tRNA) and ribosomal RNA (rRNA). In addition, some viruses infected into cells avoid innate immunogenicity through the RIG-I signaling pathway by modifying their genomic RNAs. In this chapter, we focus on epitranscriptomic regulation for RNA stability. These epitranscriptomic regulations of RNA stability have also been utilized in commercial applications, such as development of gene therapy agents. This chapter also addresses biotechnological applications of RNA stabilization that use modified nucleosides and measurement technologies that evaluate RNA stability.

Summary: Skeletal muscle adaptation is mediated by cooperative regulation of metabolism, signal transduction, and gene expression. However, the global regulatory mechanism remains unclear. To address this issue, we performed electrical pulse stimulation (EPS) in differentiated C2C12 myotubes at low and high frequency, carried out metabolome and transcriptome analyses, and investigated phosphorylation status of signaling molecules. EPS triggered extensive and specific changes in metabolites, signaling phosphorylation, and gene expression during and after EPS in a frequency-dependent manner. We constructed trans-omic network by integrating these data and found selective activation of the pentose phosphate pathway including metabolites, upstream signaling molecules, and gene expression of metabolic enzymes after high-frequency EPS. We experimentally validated that activation of these molecules after high-frequency EPS was dependent on reactive oxygen species (ROS). Thus, the trans-omic analysis revealed ROS-dependent activation in signal transduction, metabolome, and transcriptome after high-frequency EPS in C2C12 myotubes, shedding light on possible mechanisms of muscle adaptation.

Most viruses inhibit the innate immune system and/or the RNA degradation processes of host cells to construct an advantageous intracellular environment for their survival. Characteristic RNA sequences within RNA virus genomes or RNAs transcribed from DNA virus genomes contribute toward this inhibition. In this study, we developed a method called “Fate-seq” to comprehensively identify the RNA sequences derived from RNA and DNA viruses, contributing RNA stability in the cells. We examined the stabilization activity of 5,924 RNA fragments derived from 26 different viruses (16 RNA viruses and 10 DNA viruses) using next-generation sequencing of these RNAs fused 3′ downstream of GFP reporter RNA. With the Fate-seq approach, we detected multiple virus-derived RNA sequences that stabilized GFP reporter RNA, including sequences derived from severe acute respiratory syndrome-related coronavirus (SARS-CoV). Comparative genomic analysis revealed that these RNA sequences and their predicted secondary structures are highly conserved between SARS-CoV and the novel coronavirus, SARS-CoV-2, which is responsible for the global outbreak of the coronavirus-associated disease that emerged in December 2019 (COVID-19). These sequences have the potential to enhance the stability of viral RNA genomes, thereby augmenting viral replication efficiency and virulence., Highlights • RNA stability is an important regulator for viral life-cycle. • Fate-seq enables measurement of stabilization activity of RNAs derived from viruses. • Conserved sequences among coronaviruses including SARS-CoV-2 have stabilizing activity.

SummaryGene expression is one of the factors determining cellular conditions, and its level is determined by the balance between transcription and RNA degradation. However, it has been not well elucidated how transcription and degradation are related to each other. To elucidate the relationship of them, methods enabling simultaneous measurement of transcription and degradation are required. Here, we report the development of “Dyrec-seq” to evaluate transcription and RNA degradation rates simultaneously, by using 4-thiouridine and 5-bromouridine. Using Dyrec-seq, we quantified the transcription and degradation rates of 4,702 genes in HeLa cells. Functional enrichment analysis showed that the transcription and degradation rates of genes are actually determined by the genes’ biological functions. Comparison of theoretical and experimental analysis results revealed that the amount of RNA was determined by the ratio of transcription to degradation rates, while the rapidity of responses to external stimuli was determined only by the degradation rate. This study emphasizes that degradation as well as transcription is important in determining the behavior of RNA.

Cellular signaling regulates various cellular functions via protein phosphorylation. Phosphoproteomic data potentially include information for a global regulatory network from signaling to cellular functions, but a procedure to reconstruct this network using such data has yet to be established. In this paper, we provide a procedure to reconstruct a global regulatory network from signaling to cellular functions from phosphoproteomic data by integrating prior knowledge of cellular functions and inference of the kinase-substrate relationships (KSRs). We used phosphoproteomic data from insulin-stimulated Fao hepatoma cells and identified protein phosphorylation regulated by insulin specifically over-represented in cellular functions in the KEGG database. We inferred kinases for protein phosphorylation by KSRs, and connected the kinases in the insulin signaling layer to the phosphorylated proteins in the cellular functions, revealing that the insulin signal is selectively transmitted via the Pi3k-Akt and Erk signaling pathways to cellular adhesions and RNA maturation, respectively. Thus, we provide a method to reconstruct global regulatory network from signaling to cellular functions based on phosphoproteomic data.

Summary The concentrations of insulin selectively regulate multiple cellular functions. To understand how insulin concentrations are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells using transcriptomic data, western blotting analysis of signaling proteins, and metabolomic data. By integrating sensitivity into the trans-omic network, we identified the selective trans-omic networks stimulated by high and low doses of insulin, denoted as induced and basal insulin signals, respectively. The induced insulin signal was selectively transmitted through the pathway involving Erk to an increase in the expression of immediate-early and upregulated genes, whereas the basal insulin signal was selectively transmitted through a pathway involving Akt and an increase of Foxo phosphorylation and a reduction of downregulated gene expression. We validated the selective trans-omic network in vivo by analysis of the insulin-clamped rat liver. This integrated analysis enabled molecular insight into how liver cells interpret physiological insulin signals to regulate cellular functions., Graphical Abstract, Highlights • We constructed a trans-omic network of insulin action using multi-omic data • The trans-omic network integrates phosphorylation, transcription, and metabolism • We divided induced- and basal-insulin-dependent trans-omic network by sensitivity • Induced and basal insulin triggered selective trans-omic network in the liver, Systems Biology; Omics; Metabolomics; Transcriptomics

SUMMARYThe concentration and temporal pattern of insulin selectively regulate multiple cellular functions. To understand how insulin dynamics are interpreted by cells, we constructed a trans-omic network of insulin action in FAO hepatoma cells from three networks—a phosphorylation-dependent cellular functions regulatory network using phosphoproteomic data, a transcriptional regulatory network using phosphoproteomic and transcriptomic data, and a metabolism regulatory network using phosphoproteomic and metabolomic data. With the trans-omic regulatory network, we identified selective regulatory networks that mediate differential responses to insulin. Akt and Erk, hub molecules of insulin signaling, encode information of a wide dynamic range of dose and time of insulin. Down-regulated genes and metabolites in glycolysis had high sensitivity to insulin (fasting insulin signal); up-regulated genes and dicarboxylic acids in the TCA cycle had low sensitivity (fed insulin signal). This integrated analysis enables molecular insight into how cells interpret physiologically fed and fasting insulin signals.HighlightsWe constructed a trans-omic network of insulin action using multi-omic data.The trans-omic network integrates phosphorylation, transcription, and metabolism.We classified signaling, transcriptome, and metabolome by sensitivity to insulin.We identified fed and fasting insulin signal flow across the trans-omic network.

UTokyo Research掲載「細胞内のビッグデータから大規模ネットワークの再構築に成功」URI: http://www.u-tokyo.ac.jp/ja/utokyo-research/research-news/reconstruction-of-molecular-network-from-cellular-big-data/, UTokyo Research "Reconstruction of molecular network from cellular big data" URI: http://www.u-tokyo.ac.jp/en/utokyo-research/research-news/reconstruction-of-molecular-network-from-cellular-big-data/

Secretion of insulin transiently increases after eating, resulting in a high circulating concentration. Fasting limits insulin secretion, resulting in a low concentration of insulin in the circulation. We analyzed transcriptional responses to different temporal patterns and doses of insulin in the hepatoma FAO cells and identified 13 up-regulated and 16 down-regulated insulin-responsive genes (IRGs). The up-regulated IRGs responded more rapidly than did the down-regulated IRGs to transient stepwise or pulsatile increases in insulin concentration, whereas the down-regulated IRGs were repressed at lower concentrations of insulin than those required to stimulate the up-regulated IRGs. Mathematical modeling of the insulin response as two stages-(i) insulin signaling to transcription and (ii)transcription and mRNA stability-indicated that the first stage was the more rapid stage for the down-regulated IRGs, whereas the second stage of transcription was the more rapid stage for the up-regulated IRGs. A subset of the IRGs that were up-regulated or down-regulated in the FAO cells was similarly regulated in the livers of rats injected with a single dose of insulin. Thus, not only can cells respond to insulin but they can also interpret the intensity and pattern of signal to produce distinct transcriptional responses. These results provide insight that may be useful in treating obesity and type 2 diabetes associated with aberrant insulin production or tissue responsiveness.

It was reported that one-dimensionally elongated pores in implants promote the production of new bone tissue possessing both high bone density and the preferential alignment of biological apatite (BAp) c-axis/collagen as a bone quality parameter. This finding indicates that the anisotropic orientation and/or migration of osteoblasts guided by the grooved-pore surface affected the establishment of the anisotropic microstructure of bone tissue. In this study, a grooved polytetrafluoroethylene (Teflon) implant, which may have a role in regulating osteoblast arrangement, was prepared to investigate the relationship between cell behavior and bone microstructure. A cylindrical Teflon implant with 8 grooves on its side was prepared. The width and depth of the groove cross-section were 0.5 and 0.75 mm, respectively. Each implant was inserted in a drill-hole defect created on a rabbit femur such that the groove direction was parallel or perpendicular to the long bone axis in which the BAp c-axis aligns one-dimensionally. The Young’s modulus of Teflon is approximately 0.5 GPa, much lower than that of bone; therefore, the effects of applied stress can be eliminated in this model. The oriented new bone was preferentially produced along the grooved surface. The alignment direction of the BAp c-axis was almost parallel to the grooved surface even near the surface vertically aligned to the long bone axis. The geometry of the implant surface can control the organization of BAp alignment through the arrangement of osteoblasts to orient and subsequently to migrate along the surface direction; hence, implant geometry, particularly the groove, is considered an important factor controlling the BAp orientation of regenerated bone tissues.

Basic information on the molecular weights and structures of poly(p-phenylene) PPP, poly(m-phenylene) PMP, poly(thiophene-2,5-diyl) PTh, and related copolymers has been obtained. PPPs prepared by a Ni-catalyzed dehalogenative polycondensation of p-C6H4Br2 with Mg (PPP(Ni)) and an oxidative polymerization of benzene using CuCl2 (PPP(Cu)) became soluble in DMF after nitration. The nitration proceeded essentially without crosslinking, and GPC analysis gave Mn values of 8300 and 6100 for nitrated PPP(Ni) and PPP(Cu), respectively. Light scattering analysis indicated that nitrated PPP(Ni) and PPP(Cu) assumed a rather stiff structure in DMF with ρv (degree of depolarization) in the range of 0.12−0.13. PMP and PTh also became soluble in DMF after nitration, and GPC analysis of nitrated PMP and PTh gave Mn values of 5500 and 8500, respectively. Light scattering analysis of nitrated PTh gave a ρv of 0.33, which indicated that the polymer had a stiff structure in DMF. Copolymers of p-phenylene (PP) and m-phenylene ...

Preparation of Polyphenylenes with Higher Molecular Weights via Dehalogenative Polycondensation Using Magnesium and Aggregating Property of the Polymer in Solutions

Reference LPI-CONF-2002-004View record in Web of Science Record created on 2006-02-21, modified on 2017-05-12

Reagents containing carbon–tin–halogen inter-element linkages were effective for palladium-catalyzed carbostannylation. In situ generated allyltin trichlorides add to carbon–carbon double bonds of bicyclo[2.2.1]hept-2-ene (norbornene) and bicyclo[2.2.1]hepta-2,5-diene (norbornadiene), stereoselectively, under the catalysis of palladium(0) species in good yields. The regioselectivity of unsymmetrically substituted allylic reagents dramatically alters with the choice of the tin(II) salt. Aryltin trichlorides undergo palladium-catalyzed arylstannylation of norbornene, giving a mixture of products composed of that taking one norbornene and that taking two norbornenes. The product ratio is dependent on the choice of the solvent and electronic nature of the aromatic substituent.

The sections in this article are Introduction Electrolyte Properties of Ionic Liquids Electrochemical Stability Dye-sensitized Solar Cells Keywords: ionic liquids; electrolytes; electrochemical; solar cell; green chemistry

[reaction: see text]. In contrast to the Pd(0)-catalyzed mechanism by Uemura, Mizoroki-Heck type reaction of boronic acids is found to proceed under a Pd(II)-mediated pathway using a catalytic amount of Pd(OAc)2 in the presence of Cu(OAc)2 as an oxidant. Treatment of a variety of alkenes with boronic acids, boronates, and sodium tetraphenylborate furnishes beta-arylated and alkenylated products in good to excellent yields. The reactions with norbornene, norbornadiene, and diphenylacetylene are also performed to give 1:2 or 2:1 coupling products.

The three-dimensional (3D) visualisation of the photovoltaic active layer in a dye-sensitised solar cell (DSSC) was newly achieved by employing an optical interference measurement system. The technique was used to measure with high precision the active layer thickness of a complete cell to evaluate the diffusion resistance of the electrode layer made of titania and the electrolyte layer containing tetracyanoborate ionic liquid. Additionally, the visualised image was compared with the local photovoltaic characteristics in a region with diameter 100 µm studied using an AC impedance technique as well as by transient photocurrent and photovoltage decay measurements to elucidate the charge transport properties of the ionic-liquid-based electrolyte.

The first photo-regulation of non-enzymatic RNA hydrolysis has been achieved. A positively-charged and bulky regulator has been linked to a catalytically active Zn(II) complex, via a photo-responsive linker involving an azobenzene. The rate of RNA hydrolysis has been reversibly modulated by photo-irradiation.

We present a durability test of polymer electrolyte membrane fuel cell (PEMFC) in open circuit condition. Such a condition enhances the deterioration of the membrane electrode assembly. ac impedance spectroscopy measurements and scanning electron microscopy observation suggest that the degradation occurred at the cathode. Direct gas mass spectroscopy of the cathode outlet gas indicated the formation of HF, H 2 O 2 , CO 2 , SO, SO 2 , H 2 SO 2 , and H 2 SO 3 . A kinetic model is presented assuming that the H 2 gas cross leakage from the anode caused the cathode degradation. The model determines the rate of degradation using the permeability across the electrolyte membrane measured for crossover H 2 gas.