Large-scale preparation of yeast strains expressing condensates derived from a glycolytic enzyme via controlled dissolved oxygen levels under hypoxia.

Under hypoxia, Saccharomyces cerevisiae forms cytoplasmic condensates composed of proteins, including glycolytic enzymes, that are thought to regulate cellular metabolism. However, the hypoxic conditions required for condensate formation remain unclear. In this study, we developed a 300-mL-scale culture method to produce condensate-forming cells by precisely controlling the dissolved oxygen (DO) level in the media. Using enolase as a model, a foci formation rate of more than 50% was achieved at ∼0.1% DO, and the results showed that the DO level affected the foci formation rate. The foci formation rates of the previously reported foci-deficient strains and strains with single amino acid substitutions in the endogenous enolase were examined, and the effect of these amino acid substitutions on glucose consumption and ethanol and glycerol production under hypoxia was evaluated. The results of this study contribute to the investigation of the mechanisms that regulate biomacromolecular condensates under hypoxia.

Synthesis of [1-13 C-5-12 C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

Isocitrate dehydrogenase 1 (IDH1) mutations that generate the oncometabolite 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG) have been identified in many types of tumors and are an important prognostic factor in gliomas. 2-HG production can be determined by hyperpolarized carbon-13 magnetic resonance spectroscopy (HP-13 C-MRS) using [1-13 C]-α-KG as a probe, but peak contamination from naturally occurring [5-13 C]-α-KG overlaps with the [1-13 C]-2-HG peak. Via a newly developed oxidative-Stetter reaction, [1-13 C-5-12 C]-α-KG was synthesized. α-KG metabolism was measured via HP-13 C-MRS using [1-13 C-5-12 C]-α-KG as a probe. [1-13 C-5-12 C]-α-KG was synthesized in high yields, and successfully eliminated the signal from C5 of α-KG in the HP-13 C-MRS spectra. In HCT116 IDH1 R132H cells, [1-13 C-5-12 C]-α-KG allowed for unimpeded detection of [1-13 C]-2-HG. 12 C-enrichment represents a novel method to circumvent spectral overlap, and [1-13 C-5-12 C]-α-KG shows promise as a probe to study IDH1 mutant tumors and α-KG metabolism.

Small-scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae.

At normal oxygen concentration, glycolytic enzymes are scattered in the cytoplasm of Saccharomyces cerevisiae. Under hypoxia, however, most of these enzymes, including enolase, pyruvate kinase, and phosphoglycerate mutase, spatially reorganize to form cytoplasmic foci. We tested various small-scale hypoxic culture systems and showed that enolase foci formation occurs in all the systems tested, including in liquid and on solid media. Notably, a small-scale hypoxic culture in a bench-top multi-gas incubator enabled the regulation of oxygen concentration in the media and faster foci formation. Here, we demonstrate that the foci formation of enolase starts within few hours after changing the oxygen concentration to 1% in a small-scale cultivation system. The order of foci formation by each enzyme is tightly regulated, and of the three enzymes, enolase was the fastest to respond to hypoxia. We further tested the use of the small-scale cultivation method to screen reagents that can control the spatial reorganization of enzymes under hypoxia. An AMPK inhibitor, dorsomorphin, was found to delay formation of the foci in all three glycolytic enzymes tested. These methods and results provide efficient ways to investigate the spatial reorganization of proteins under hypoxia to form a multienzyme assembly, the META body, thereby contributing to understanding and utilizing natural systems to control cellular metabolism via the spatial reorganization of enzymes.

Design of novel primer sets for easy detection of Ruegeria species from seawater.

Some coral-associated bacteria show protective roles for corals against pathogens. However, the distribution of coral-protecting bacteria in seawater is not well known. In addition, compared with the methods for investigating coral pathogens, few methods have been developed to detect coral-protecting bacteria. Here we prepared a simple method for detecting Ruegeria spp., some strains of which inhibit growth of the coral pathogen Vibrio coralliilyticus. We successfully obtained two Ruegeria-targeting primer sets through in silico and in vitro screening. The primer sets r38F-r30R and r445F-r446R, in addition to the newly designed universal primer set U357'F-U515'R, were evaluated in vitro using environmental DNA extracted from seawater collected in Osaka. These methods and primers should contribute to revealing the distribution of Ruegeria spp. in marine environments.

Single-cell heterogeneity in suppression of PC12 differentiation by direct microinjection of a differentiation inhibitor, U0126.

Phenotypic and genomic heterogeneity among single cells in a cell population leads to inaccuracy and obscuration in research about mammalian cell differentiation. In order to address the problems regarding bulk analysis on heterogeneous cell populations, it is necessary to accurately regulate and analyze changes in differentiating cells at the single-cell level. To investigate the single-cell changes in PC12 neuronal differentiation that occur when inhibited by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), we directly injected the chemical into individual target cells and analyzed the outcomes (neurite outgrowth) at the single-cell level. As a result, we could accurately regulate the quantity of U0126 being introduced into each target cell, which was previously not possible using the common method of simply adding the inhibitor to the culture medium. It was possible to analyze the inhibitive effect of U0126 even when the injected quantity was lower than the lower limit for inhibition when added to culture medium (0.1 µM, identical to 1.2 × 10(8) molecules per cell on dish). In particular, injection of 1.5 × 10(7) molecules into each cell resulted in a 59% decrease of the mean total neurite length. Time-course analysis of neurite outgrowth at the single-cell level using fluorescence staining method showed that the changes in neurite length of differentiating PC12 cells were not homogeneous, but were largely variable across individual target cells.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

Spatial reorganization of Saccharomyces cerevisiae enolase to alter carbon metabolism under hypoxia.

Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-(13)C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.

Real-Time Monitoring of Hydrolysis Reactions of Pyrophosphates with Dissolution Dynamic Nuclear Polarization.

Dissolution dynamic nuclear polarization (d-DNP) has enabled applications such as the real-time monitoring of chemical reactions. Such applications are mainly for 13C and 15N spins with long spin-lattice relaxation times in the molecules of interest. However, the only applications for phosphorus using d-DNP are pH imaging and nucleation during crystallization due to the short relaxation times. Here we show that it is possible to observe enzyme reactions using d-DNP with phosphorus. Hyperpolarized 31P spins in pyrophosphate were obtained using bullet-DNP, which requires less dilution of highly polarized solid samples. Real-time monitoring of the hydrolysis reaction of pyrophosphate by inorganic pyrophosphatase from baker's yeast at physiological pH and was successfully achieved and the reaction rate was determined. This is an important reaction for a wide range of applications related to medicine, agriculture, and quantum life science.

Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering.

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods.

Tracing putative trafficking of the glycolytic enzyme enolase via SNARE-driven unconventional secretion.

Glycolytic enzymes are cytosolic proteins, but they also play important extracellular roles in cell-cell communication and infection. We used Saccharomyces cerevisiae to analyze the secretory pathway of some of these enzymes, including enolase, phosphoglucose isomerase, triose phosphate isomerase, and fructose 1,6-bisphosphate aldolase. Enolase, phosphoglucose isomerase, and an N-terminal 28-amino-acid-long fragment of enolase were secreted in a sec23-independent manner. The enhanced green fluorescent protein (EGFP)-conjugated enolase fragment formed cellular foci, some of which were found at the cell periphery. Therefore, we speculated that an overview of the secretory pathway could be gained by investigating the colocalization of the enolase fragment with intracellular proteins. The DsRed-conjugated enolase fragment colocalized with membrane proteins at the cis-Golgi complex, nucleus, endosome, and plasma membrane, but not the mitochondria. In addition, the secretion of full-length enolase was inhibited in a knockout mutant of the intracellular SNARE protein-coding gene TLG2. Our results suggest that enolase is secreted via a SNARE-dependent secretory pathway in S. cerevisiae.

Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress.

Scleractinian corals form symbiotic relationships with a variety of microorganisms, including endosymbiotic dinoflagellates of the family Symbiodiniaceae, and with bacteria, which are collectively termed coral holobionts. Interactions between hosts and their symbionts are critical to the physiological status of corals. Coral-microorganism interactions have been studied extensively, but dinoflagellate-bacterial interactions remain largely unexplored. Here, we developed a microbiome manipulation method employing KAS-antibiotic treatment (kanamycin, ampicillin, and streptomycin) to favor pigmented bacteria residing on cultured Cladocopium and Durusdinium, major endosymbionts of corals, and isolated several carotenoid-producing bacteria from cell surfaces of the microalgae. Following KAS-antibiotic treatment of Cladocopium sp. strain NIES-4077, pigmented bacteria increased 8-fold based on colony-forming assays from the parental strain, and 100% of bacterial sequences retrieved through 16S rRNA amplicon sequencing were affiliated with the genus Maribacter. Microbiome manipulation enabled host microalgae to maintain higher maximum quantum yield of photosystem II (variable fluorescence divided by maximum fluorescence [Fv/Fm]) under light-stress conditions, compared to the parental strain. Furthermore, by combining culture-dependent and -independent techniques, we demonstrated that species of the family Symbiodiniaceae and pigmented bacteria form strong interactions. Dinoflagellates protected bacteria from antibiotics, while pigmented bacteria protected microalgal cells from light stress via carotenoid production. Here, we describe for the first time a symbiotic relationship in which dinoflagellates and bacteria mutually reduce environmental stress. Investigations of microalgal-bacterial interactions further document bacterial contributions to coral holobionts and may facilitate development of novel techniques for microbiome-mediated coral reef conservation. IMPORTANCE Coral reefs cover less than 0.1% of the ocean floor, but about 25% of all marine species depend on coral reefs at some point in their life cycles. However, rising ocean temperatures associated with global climate change are a serious threat to coral reefs, causing dysfunction of the photosynthetic apparatus of endosymbiotic microalgae of corals, and overproducing reactive oxygen species harmful to corals. We manipulated the microbiome using an antibiotic treatment to favor pigmented bacteria, enabling their symbiotic microalgal partners to maintain higher photosynthetic function under insolation stress. Furthermore, we investigated mechanisms underlying microalgal-bacterial interactions, describing for the first time a symbiotic relationship in which the two symbionts mutually reduce environmental stress. Our findings extend current insights about microalgal-bacterial interactions, enabling better understanding of bacterial contributions to coral holobionts under stressful conditions and offering hope of reducing the adverse impacts of global warming on coral reefs.

Foci-forming regions of pyruvate kinase and enolase at the molecular surface incorporate proteins into yeast cytoplasmic metabolic enzymes transiently assembling (META) bodies.

Spatial reorganization of metabolic enzymes to form the "metabolic enzymes transiently assembling (META) body" is increasingly recognized as a mechanism contributing to regulation of cellular metabolism in response to environmental changes. A number of META body-forming enzymes, including enolase (Eno2p) and phosphofructokinase, have been shown to contain condensate-forming regions. However, whether all META body-forming enzymes have condensate-forming regions or whether enzymes have multiple condensate-forming regions remains unknown. The condensate-forming regions of META body-forming enzymes have potential utility in the creation of artificial intracellular enzyme assemblies. In the present study, the whole sequence of yeast pyruvate kinase (Cdc19p) was searched for condensate-forming regions. Four peptide fragments comprising 27-42 amino acids were found to form condensates. Together with the fragment previously identified from Eno2p, these peptide regions were collectively termed "META body-forming sequences (METAfos)." METAfos-tagged yeast alcohol dehydrogenase (Adh1p) was found to co-localize with META bodies formed by endogenous Cdc19p under hypoxic conditions. The effect of Adh1p co-localization with META bodies on cell metabolism was further evaluated. Expression of Adh1p fused with a METAfos-tag increased production of ethanol compared to acetic acid, indicating that spatial reorganization of metabolic enzymes affects cell metabolism. These results contribute to understanding of the mechanisms and biological roles of META body formation.

Condensate Formation by Metabolic Enzymes in Saccharomyces cerevisiae.

Condensate formation by a group of metabolic enzymes in the cell is an efficient way of regulating cell metabolism through the formation of "membrane-less organelles." Because of the use of green fluorescent protein (GFP) for investigating protein localization, various enzymes were found to form condensates or filaments in living Saccharomyces cerevisiae, mammalian cells, and in other organisms, thereby regulating cell metabolism in the certain status of the cells. Among different environmental stresses, hypoxia triggers the spatial reorganization of many proteins, including more than 20 metabolic enzymes, to form numerous condensates, including "Glycolytic body (G-body)" and "Purinosome." These individual condensates are collectively named "Metabolic Enzymes Transiently Assembling (META) body". This review overviews condensate or filament formation by metabolic enzymes in S. cerevisiae, focusing on the META body, and recent reports in elucidating regulatory machinery of META body formation.

Nonbacterial thrombotic endocarditis associated with cancer of unknown origin complicated with thrombus in the left auricular appendage: case report.

A 63-year-old man was admitted to our hospital with a complaint of right lateroabdominal pain. He was diagnosed with metastatic colon cancer, and then developed multiple brain embolic infarctions 7 days after admission. Transesophageal echocardiography showed that mobile, echo-dense masses were attached to the anterior and posterior mitral valve leaflet. Furthermore, there was a thrombus in the left auricular appendage despite sinus rhythm. These findings led to a diagnosis of suspected infectious endocarditis with subsequent multiple brain infarctions. The patient's general condition worsened and he died 13 days after admission. An autopsy was performed, and, while poorly differentiated cancer was observed in multiple organs, no primary tumor could be identified. Histological analysis showed that the masses of the mitral valve consisted mainly of fibrin without bacteria or oncocytes. This patient was therefore diagnosed with nonbacterial thrombotic endocarditis associated with cancer of unknown origin complicated with thrombus in the left auricular appendage.

Role of imaging studies in the diagnosis and evaluation of giant cell arteritis in Japanese: report of eight cases.

The objective of this study is to clarify the characteristics and imaging results of Japanese patients with giant cell arteritis (GCA). Eight patients with biopsy-proven GCA were enrolled. Their clinical data and imaging results were retrospectively examined from their medical records. All the patients met the criteria for the classification of GCA by the American College of Rheumatology. Although the clinical manifestations are similar to those previously reported, none of the eight patients presented ocular symptoms, and half of them presented jaw claudication. Ultrasonography (US) of temporal artery showed the halo sign in all the patients. Fluorodeoxyglucose positron emission tomography (FDG-PET) was performed in four patients and indicated the presence of aortitis of the patients. US is a quick and noninvasive test to detect inflammation of temporal artery, and FDG-PET is very helpful for early diagnosis of aortitis in GCA. Awareness of the disease and appropriate imaging tests will result in diagnosis of GCA.

Epidural spinal tumor and periaortitis as rare complications of Wegener's granulomatosis.

This case report describes findings in a 61-year-old woman who manifested scleritis, small pulmonary nodules, otitis media, periaortitis, and progressive epidural spinal tumor, associated with elevated serum myeloperoxidase anti-neutrophil cytoplasmic antibody (MPO-ANCA) levels. She was clinically diagnosed with Wegener's granulomatosis, although vasculitis was not diagnosed due to the lack of typical histological findings. We discuss the differential diagnosis in this patient, and the association of MPO-ANCA with periaortitis or epidural spinal tumor.

Peptide barcoding for one-pot evaluation of sequence-function relationships of nanobodies.

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.

Specific Detection of Coral-Associated Ruegeria, a Potential Probiotic Bacterium, in Corals and Subtropical Seawater.

Coral microbial flora has been attracting attention because of their potential to protect corals from environmental stresses or pathogens. Although coral-associated bacteria are considered to be acquired from seawater, little is known about the relationships between microbial composition in corals and its surrounding seawater. Here, we tested several methods to identify coral-associated bacteria in coral and its surrounding seawater to detect specific types of Ruegeria species, some of which exhibit growth inhibition activities against the coral pathogen Vibrio coralliilyticus. We first isolated coral-associated bacteria from the reef-building coral Galaxea fascicularis collected at Sesoko Island, Okinawa, Japan, via random colony picking, which showed the existence of varieties of bacteria including Ruegeria species. Using newly constructed primers for colony PCR, several Ruegeria species were successfully isolated from G. fascicularis and seawater. We further investigated the seawater microbiome in association with the distance from coral reefs. By seasonal sampling, it was suggested that the seawater microbiome is more affected by seasonality than the distance from coral reefs. These methods and results may contribute to investigating and understanding the relationships between the presence of corals and microbial diversity in seawater, in addition to the efficient isolation of specific bacterial species from coral or its surrounding seawater.

Simple Esterification of [1-13C]-Alpha-Ketoglutarate Enhances Membrane Permeability and Allows for Noninvasive Tracing of Glutamate and Glutamine Production.

Alpha-ketoglutarate (α-KG) is a key metabolite and signaling molecule in cancer cells, but the low permeability of α-KG limits the study of α-KG mediated effects in vivo. Recently, cell-permeable monoester and diester α-KG derivatives have been synthesized for use in vivo, but many of these derivatives are not compatible for use in hyperpolarized carbon-13 nuclear magnetic resonance spectroscopy (HP-13C-MRS). HP-13C-MRS is a powerful technique that has been used to noninvasively trace labeled metabolites in real time. Here, we show that using diethyl-[1-13C]-α-KG as a probe in HP-13C-MRS allows for noninvasive tracing of α-KG metabolism in vivo.

A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress.

Reef-building corals form a complex consortium with photosynthetic algae in the family Symbiodiniaceae and bacteria, collectively termed the coral holobiont. These bacteria are hypothesized to be involved in the stress resistance of the coral holobiont, but their functional roles remain largely elusive. Here, we show that cultured Symbiodiniaceae algae isolated from the reef-building coral Galaxea fascicularis are associated with novel bacteria affiliated with the family Flavobacteriaceae Antibiotic treatment eliminated the bacteria from cultured Symbiodiniaceae, resulting in a decreased maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and an increased production of reactive oxygen species (ROS) under thermal and light stresses. We then isolated this bacterial strain, named GF1. GF1 inoculation in the antibiotic-treated Symbiodiniaceae cultures restored the Fv/Fm and reduced the ROS production. Furthermore, we found that GF1 produces the carotenoid zeaxanthin, which possesses potent antioxidant activity. Zeaxanthin supplementation to cultured Symbiodiniaceae ameliorated the Fv/Fm and ROS production, suggesting that GF1 mitigates thermal and light stresses in cultured Symbiodiniaceae via zeaxanthin production. These findings could advance our understanding of the roles of bacteria in Symbiodiniaceae and the coral holobiont, thereby contributing to the development of novel approaches toward coral protection through the use of symbiotic bacteria and their metabolites.IMPORTANCE Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family Symbiodiniaceae, the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.