Metabolome analysis of metabolic burden in Escherichia coli caused by overexpression of green fluorescent protein and delta-rhodopsin.

Overexpression of proteins by introducing a DNA vector is among the most important tools for the metabolic engineering of microorganisms such as Escherichia coli. Protein overexpression imposes a burden on metabolism because metabolic pathways must supply building blocks for protein and DNA synthesis. Different E. coli strains have distinct metabolic capacities. In this study, two proteins were overexpressed in four E. coli strains (MG1655(DE3), W3110(DE3), BL21star(DE3), and Rosetta(DE3)), and their effects on metabolic burden were investigated. Metabolomic analysis showed that E. coli strains overexpressing green fluorescent protein had decreased levels of several metabolites, with a positive correlation between the number of reduced metabolites and green fluorescent protein expression levels. Moreover, nucleic acid-related metabolites decreased, indicating a metabolic burden in the E. coli strains, and the growth rate and protein expression levels were improved by supplementation with the five nucleosides. In contrast, two strains overexpressing delta rhodopsin, a microbial membrane rhodopsin from Haloterrigena turkmenica, led to a metabolic burden and decrease in the amino acids Ala, Val, Leu, Ile, Thr, Phe, Asp, and Trp, which are the most frequent amino acids in the delta rhodopsin protein sequence. The metabolic burden caused by protein overexpression was influenced by the metabolic capacity of the host strains and the sequences of the overexpressed proteins. Detailed characterization of the effects of protein expression on the metabolic state of engineered cells using metabolomics will provide insights into improving the production of target compounds.

Metabolic pathway design for growth-associated phenylalanine production using synthetically designed mutualism.

Combination of growth-associated pathway engineering based on flux balance analysis (FBA) and adaptive laboratory evolution (ALE) is a powerful approach to enhance the production of useful compounds. However, the feasibility of such growth-associated pathway designs depends on the type of target compound. In the present study, FBA predicted a set of gene deletions (pykA, pykF, ppc, zwf, and adhE) that leads to growth-associated phenylalanine production in Escherichia coli. The knockout strain is theoretically enforced to produce phenylalanine only at high growth yields, and could not be applied to the ALE experiment because of a severe growth defect. To overcome this challenge, we propose a novel approach for ALE based on mutualistic co-culture for coupling growth and production, regardless of the growth rate. We designed a synthetic mutualism of a phenylalanine-producing leucine-auxotrophic strain (KF strain) and a leucine-producing phenylalanine-auxotrophic strain (KL strain) and performed an ALE experiment for approximately 160 generations. The evolved KF strain (KF-E strain) grew in a synthetic medium (with glucose as the main carbon source) supplemented with leucine, while severe growth defects were observed in the parental KF strain. The phenylalanine yield of the KF-E strain was 2.3 times higher than that of the KF strain.

Optogenetic reprogramming of carbon metabolism using light-powering microbial proton pump systems.

In microbial fermentative production, ATP regeneration, while crucial for cellular processes, conflicts with efficient target chemical production because ATP regeneration exhausts essential carbon sources also required for target chemical biosynthesis. To wrestle with this dilemma, we harnessed the power of microbial rhodopsins with light-driven proton pumping activity to supplement with ATP, thereby facilitating the bioproduction of various chemicals. We first demonstrated a photo-driven ATP supply and redistribution of metabolic carbon flows to target chemical synthesis by installing already-known delta rhodopsin (dR) in Escherichia coli. In addition, we identified novel rhodopsins with higher proton pumping activities than dR, and created an engineered cell for in vivo self-supply of the rhodopsin-activator, all-trans-retinal. Our concept exploiting the light-powering ATP supplier offers a potential increase in carbon use efficiency for microbial productions through metabolic reprogramming.

Bond durability and surface states of titanium, Ti-6Al-4V alloy, and zirconia for implant materials.

PURPOSE: Screw-retained implant crowns used as dental implants comprise a zirconia coping and titanium base bonded using resin cement. These devices are prone to debonding failures. This study investigated the bond characteristics of implant materials based on shear bond strength (SBS) and surface characteristics. METHODS: Chemically pure (CP) titanium grade-4 (Ti), Ti-6Al-4V alloy (Ti-6Al-4V), and tetragonal polycrystalline zirconia (zirconia) were evaluated as adherent materials. Plates of each material were polished, primed for the respective resin cements, and cemented using either methyl methacrylate-based resin cement (Super-Bond) or composite-based resin cement (Panavia). The cemented samples were subjected to 10,000 thermocycles alternating between 5 and 55 °C, and the SBS were obtained before and after thermocycling. The sample surfaces were characterized based on surface observations, roughness, and free energy (SFE). RESULTS: The SBSs of all materials bonded using Panavia were significantly compromised during thermocycling and reached zero. Although the SBSs of Ti and Ti-6Al-4V bonded using Super-Bond were not significantly affected by thermocycling, those of zirconia decreased significantly. The bond durability between zirconia and Super-Bond was improved via alumina air-abrasion, which caused no significant loss of SBS after thermocycling. Surface analyses of the air-abraded zirconia validated these results and confirmed that its surface roughness and SFE were significantly increased. CONCLUSION: The bond durability between resin cement and zirconia was lower than that between Ti and Ti-6Al-4V. The alumina air-abrasion pretreatment of zirconia improved the SFE and surface roughness, thereby enhancing bond durability.

Treatment of Retinoblastoma 1-Intact Hepatocellular Carcinoma With Cyclin-Dependent Kinase 4/6 Inhibitor Combination Therapy.

BACKGROUND AND AIMS: Synthetic cyclin-dependent kinase (CDK) 4/6 inhibitors exert antitumor effects by forcing RB1 in unphosphorylated status, causing not only cell cycle arrest but also cellular senescence, apoptosis, and increased immunogenicity. These agents currently have an indication in advanced breast cancers and are in clinical trials for many other solid tumors. HCC is one of promising targets of CDK4/6 inhibitors. RB family dysfunction is often associated with the initiation of HCC; however, this is revivable, as RB family members are not frequently mutated or deleted in this malignancy. APPROACH AND RESULTS: Loss of all Rb family members in transformation related protein 53 (Trp53)-/- mouse liver resulted in liver tumor reminiscent of human HCC, and re-expression of RB1 sensitized these tumors to a CDK4/6 inhibitor, palbociclib. Introduction of an unphosphorylatable form of RB1 (RB7LP) into multiple liver tumor cell lines induced effects similar to palbociclib. By screening for compounds that enhance the efficacy of RB7LP, we identified an I kappa B kinase (IKK)ß inhibitor Bay 11-7082. Consistently, RB7LP expression and treatment with palbociclib enhanced IKKα/ß phosphorylation and NF-κB activation. Combination therapy using palbociclib with Bay 11-7082 was significantly more effective in hepatoblastoma and HCC treatment than single administration. Moreover, blockade of IKK-NF-κB or AKT pathway enhanced effects of palbociclib on RB1-intact KRAS Kirsten rat sarcoma viral oncogene homolog mutated lung and colon cancers. CONCLUSIONS: In conclusion, CDK4/6 inhibitors have a potential to treat a wide variety of RB1-intact cancers including HCC when combined with an appropriate kinase inhibitor.

Effects of alumina airborne-particle abrasion on the surface properties of CAD/CAM composites and bond strength to resin cement.

The purpose of this study is to clarify physical and chemical changes in surfaces of CAD/CAM composites caused by alumina airborne-particle abrasion and its effect on adhesive bonding. Our study involved three dispersed filler (DF)-based and a polymer-infiltrated ceramic network (PICN)-based CAD/CAM composites. Changes in the surface morphologies of the composites were examined, and surface free energy (SFE) analysis was performed based on Owens-Wendt theory. The influence of the abrasion on the bond strengths of CAD/CAM composites to the resin cement was characterized by shear bond strength (SBS) test. The abrasion increased the roughness of the composites. The SFE analysis showed that the abrasion significantly increased the dispersive component but decreased the polar component of the SFE associated with the DF-based composites, while no change occurred for those of the PICN-based composite. The abrasion slightly improved the SBSs for the DF-based composites but not that of the PICN-based composite.

Adhesive bonding of alumina air-abraded Ag-Pd-Cu-Au alloy with 10-methacryloyloxydecyl dihydrogen phosphate.

The aim of this paper is to study changes in the Ag-Pd-Cu-Au alloy surfaces by alumina air-abrasion process and effect of those changes on the adhesive bonding characteristic. Surface roughness, surface composition and chemical state of the alumina air-abraded alloys were analyzed by a confocal laser scanning microscope, an energy dispersive X-ray spectroscopy and an X-ray photoelectron spectroscopy. The results showed that the alumina air-abrasion changed the alloy surface by mechanical roughening, alumina remain and copper oxidation. Effect of the changes in the alloy surface on the adhesive bonding characteristic was examined by using a methyl methacrylate/tri-n-butylborane derivative (MMA/TBB) resin cement with the 10-methacryloyloxydecyl dihydrogen phosphate (MDP) contained primer. The shear bond strength test results indicated that the surface oxidation by the abrasion is the main contributor that improved the adhesive bonding rather than other effects such as mechanical roughening or alumina remain.

Chemical alteration of Ag-Pd-Cu-Au alloy surface by alumina air-abrasion and its effect on bonding to resin cement.

The goal of this study was to investigate the chemical alteration of a dental alloy surface by alumina air-abrasion and its effect on bonding to resin cement. Alumina air-abrasion was carried out on an Ag-Pd-Cu-Au alloy. The surface morphology and chemical state of the abraded alloy were characterized. The effect of the air-abrasion on the shear bond strength between the alloy and a methyl methacrylate/tri-n-butyl borane (MMA/TBB) resin cement with some primers was evaluated. The surface characterization revealed that the alumina air-abrasion mechanically roughened and chemically altered the surface. The chemical alterations had two effects: (1) abraded alumina particles remained on the alloy surface and (2) copper ions were oxidized in the alloy surface. As the result, the shear bond strength test indicated that 10-methacryloyloxydecyl dihydrogen phosphate (MDP) contained primer worked with the abraded alloy surface, whereas it did not work with the non-abraded alloy surface.

Magnesium starvation improves production of malonyl-CoA-derived metabolites in Escherichia coli.

Starvation of essential nutrients, such as nitrogen, sulfur, magnesium, and phosphorus, leads cells into stationary phase and potentially enhances target metabolite production because cells do not consume carbon for the biomass synthesis. The overall metabolic behavior changes depend on the type of nutrient starvation in Escherichia coli. In the present study, we determined the optimum nutrient starvation type for producing malonyl-CoA-derived metabolites such as 3-hydroxypropionic acid (3HP) and naringenin in E. coli. For 3HP production, high production titer (2.3 or 2.0 mM) and high specific production rate (0.14 or 0.28 mmol gCDW-1 h-1) was observed under sulfur or magnesium starvation, whereas almost no 3HP production was detected under nitrogen or phosphorus starvation. Metabolic profiling analysis revealed that the intracellular malonyl-CoA concentration was significantly increased under the 3HP producing conditions. This accumulation should contribute to the 3HP production because malonyl-CoA is a precursor of 3HP. Strong positive correlation (r = 0.95) between intracellular concentrations of ATP and malonyl-CoA indicates that the ATP level is important for malonyl-CoA synthesis due to the ATP requirement by acetyl-CoA carboxylase. For naringenin production, magnesium starvation led to the highest production titer (144 ±â€¯15 µM) and specific productivity (127 ±â€¯21 µmol gCDW-1). These results demonstrated that magnesium starvation is a useful approach to improve the metabolic state of strains engineered for the production of malonyl-CoA derivatives.

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-L-methionine production.

BACKGROUND: Saccharomyces cerevisiae is a host for the industrial production of S-adenosyl-L-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol. RESULTS: The comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose. CONCLUSIONS: These results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.

Expression of Saccharomyces cerevisiae cDNAs to enhance the growth of non-ethanol-producing S. cerevisiae strains lacking pyruvate decarboxylases.

Metabolic engineering of Saccharomyces cerevisiae often requires a restriction on the ethanol biosynthesis pathway. The non-ethanol-producing strains, however, are slow growers. In this study, a cDNA library constructed from S. cerevisiae was used to improve the slow growth of non-ethanol-producing S. cerevisiae strains lacking all pyruvate decarboxylase enzymes (Pdc-, YSM021). Among the obtained 120 constructs expressing cDNAs, 34 transformants showed a stable phenotype with quicker growth. Sequence analysis showed that the open reading frames of PDC1, DUG1 (Cys-Gly metallo-di-peptidase in the glutathione degradation pathway), and TEF1 (translational elongation factor EF-1 alpha) genes were inserted into the plasmids of 32, 1, and 1 engineered strains, respectively. DUG1 function was confirmed by the construction of YSM021 pGK416-DUG1 strain because the specific growth rate of YSM021 pGK416-DUG1 (0.032 ± 0.0005 h-1) was significantly higher than that of the control strains (0.029 ± 0.0008 h-1). This suggested that cysteine supplied from glutathione was probably used for cell growth and for construction of Fe-S clusters. The results showed that the overexpression of cDNAs is a promising approach to engineer S. cerevisiae metabolism.

Intravenous IgG Reduces Pathogenic Autoantibodies, Serum IL-6 Levels, and Disease Severity in Experimental Bullous Pemphigoid Models.

Bullous pemphigoid (BP) is an autoimmune blistering disease characterized by autoantibodies to COL17. Currently, systemic corticosteroids are used as first-line treatments for BP; alternatively, intravenous administration of high-dose IgG (IVIG) has been shown to be effective for patients with steroid-resistant BP in clinical practice. However, the effect of IVIG on BP has not fully been investigated. To examine the effects and mechanisms of action of IVIG against BP, we performed IVIG experiments using two experimental BP mouse models. One is a passive-transfer BP model that reproduces subepidermal separation in neonatal mice by the passive transfer of IgGs against COL17, such as polyclonal or monoclonal mouse IgG or IgG from BP patients. The other is an active BP model that continuously develops a disease phenotype in adult mice. IVIG decreased pathogenic IgG and the disease scores in both models. Injected IVIG distributed throughout the dermis and the intercellular space of the lower epidermis. Notably, IVIG inhibited the increase of IL-6 in both models, possibly by suppressing the production of IL-6 by keratinocytes. These results suggest that the inhibitory effects of IVIG on BP are associated with the reduction of pathogenic IgG and the modulation of cytokine production.

(13)C-metabolic flux analysis in S-adenosyl-L-methionine production by Saccharomyces cerevisiae.

S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

Synergistic effect between Juzen-taiho-to, a Japanese traditional herbal medicine, and gemcitabine single-agent chemotherapy for advanced biliary tract cancer.

This is the first report of the successful treatment of advanced biliary tract cancer with gemcitabine single-agent chemotherapy in combination with Juzen-taiho-to (JTT), a Japanese traditional herbal medicine. An 84-year-old woman was referred to our hospital with general fatigue and appetite loss; she was diagnosed with advanced biliary tract cancer and accompanying colonic invasion and hepatic metastasis. The patient's response to combination chemotherapy was extremely good, and her tumors disappeared. Recent studies have confirmed the occurrence of spontaneous and induced antitumor immune responses, carried out by tumor-infiltrating lymphocytes in the tumor microenvironment. The availability, antigen presentation, and proliferation of these immune cells are increased by cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-2. Recently, JTT has gained recognition as a biological response modifier that has stimulatory effects on systemic immune responses such as enhancement of cytokine expression (GM-CSF, IL-2, etc.). In addition, some chemotherapy agents, such as anthracyclines and gemcitabine, are effective boosters of the immune response through tumor-specific antigen overexpression after apoptotic tumor cell destruction. These findings suggest that JTT enhances the antitumor effects of gemcitabine, in particular its tumor-specific effects on immune response, and these drugs are a good combination for advanced biliary tract cancer therapy.

Effect of embedded metal reinforcements and their location on the fracture resistance of acrylic resin complete dentures.

PURPOSE: This study evaluated the effect of metal reinforcement and its location on the flexural load at the proportional limit (FL-PL) and the flexural deflection of maxillary acrylic resin complete dentures. MATERIALS AND METHODS: Maxillary acrylic resin complete dentures reinforced with Remanium and without reinforcement were tested. The reinforcing material was embedded in the denture base resin in the doughy state and placed (1) under the ridge lap region; (2) in the anterior region; (3) in the middle region; and (4) in the anterior and posterior regions. The FL-PL (N) and the flexural deflection (mm) at 100 N of the reinforced maxillary denture specimens were tested using a load testing machine at a 5.0 mm/min crosshead speed. The data were analyzed statistically using one-way ANOVA; Tukey's post hoc comparisons test was applied when appropriate (95% confidence level). RESULTS: The FL-PL of the dentures without reinforcement (909 ± 195 N) and the dentures reinforced at the ridge lap (1094 ± 176 N) and in the middle (977 ± 215 N) regions were not significantly different (p > 0.05). The dentures reinforced in the anterior (1348 ± 205 N) and the anterior and posterior (1190 ± 191 N) regions had a higher FL-PL than the dentures without reinforcement (p < 0.05) and were not significantly different from each other (p > 0.05). The efficiency (times) of the reinforcing material on the dentures without reinforcement was 1.08 to 1.48. The flexural deflection of the dentures without reinforcement (0.133 ± 0.014 mm), the dentures reinforced at the ridge lap (0.125 ± 0.014 mm), in the anterior (0.122 ± 0.009 mm), and in the middle (0.132 ± 0.015 mm) regions were not significantly different (p > 0.05), and the dentures reinforced in the anterior and posterior (0.117 ± 0.011 mm) regions had significantly lower deflection than the dentures without reinforcement (p < 0.05). CONCLUSION: The location of the metal reinforcement affected the fracture resistance of the maxillary acrylic resin complete dentures.

Effect of surface preparation on the bond strength of heat-polymerized denture base resin to commercially pure titanium and cobalt-chromium alloy.

The aim of this study was to investigate the bond durability of heat-polymerized denture base resin to cast CP Ti and Co-Cr alloy. The alloy specimens were divided into five groups: 1) airborne-particle abraded with 50 µm alumina (SAND), 2) Rocatec tribochemical silica coating system (RO), 3) air-abraded followed by application of Epricord Opaque Primer (EP), 4) air-abraded followed by application of Super Bond C&B liquid (SB), 5) air-abraded followed by application of Alloy Primer (AL). Heat-polymerized denture resin was applied to the bonding area and polymerized according to the manufacturer's instructions. The halves of all specimens were thermocycled up to 10,000 cycles. Before thermocycling SB and AL showed significantly higher shear bond strengths than SAND, RO, EP for both metals. The shear bond strength of AL group after thermocycling was significantly higher than that of the other groups.

Transcriptome analysis of parallel-evolved Escherichia coli strains under ethanol stress.

BACKGROUND: Understanding ethanol tolerance in microorganisms is important for the improvement of bioethanol production. Hence, we performed parallel-evolution experiments using Escherichia coli cells under ethanol stress to determine the phenotypic changes necessary for ethanol tolerance. RESULTS: After cultivation of 1,000 generations under 5% ethanol stress, we obtained 6 ethanol-tolerant strains that showed an approximately 2-fold increase in their specific growth rate in comparison with their ancestor. Expression analysis using microarrays revealed that common expression changes occurred during the adaptive evolution to the ethanol stress environment. Biosynthetic pathways of amino acids, including tryptophan, histidine, and branched-chain amino acids, were commonly up-regulated in the tolerant strains, suggesting that activating these pathways is involved in the development of ethanol tolerance. In support of this hypothesis, supplementation of isoleucine, tryptophan, and histidine to the culture medium increased the specific growth rate under ethanol stress. Furthermore, genes related to iron ion metabolism were commonly up-regulated in the tolerant strains, which suggests the change in intracellular redox state during adaptive evolution. CONCLUSIONS: The common phenotypic changes in the ethanol-tolerant strains we identified could provide a fundamental basis for designing ethanol-tolerant strains for industrial purposes.

Glucocorticoids increase NPY gene expression in the arcuate nucleus by inhibiting mTOR signaling in rat hypothalamic organotypic cultures.

The mammalian target of rapamycin (mTOR) has been implicated in the regulation of physiological functions such as cell growth and proliferation, and glucocorticoids reportedly inhibit mTOR signaling in peripheral tissues. Recent studies suggest that the mTOR signaling in the hypothalamus plays a critical role in maintaining energy homeostasis. In this study, we examined whether the mTOR signaling in the hypothalamus is involved in the regulation of neuropeptide Y (Npy) gene expression in the arcuate nucleus by glucocorticoids. In the hypothalamic organotypic cultures, the incubation with rapamycin significantly inhibited the mTOR signaling which was shown by decreases in the levels of phosphorylated p70S6K1 and S6. Similar to the action of the mTOR inhibitor rapamycin, dexamethasone (DEX), a synthetic glucocorticoid, also inhibited the mTOR signaling in the hypothalamic explants. Analyses of the explants with in situ hybridization demonstrated that the DEX or rapamycin alone significantly increased Npy gene expression in the arcuate nucleus, but that there were no additive effects of DEX and rapamycin on the expression. These data suggest that glucocorticoids upregulate the Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling, at least in part.