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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Domain swapping of complementarity-determining region in nanobodies produced by Pichia pastoris.

Easy preparation of chimeric nanobodies with various scaffolds is important for customizing abilities of nanobodies toward practical utilization. To accomplish high-throughput production of various nanobodies, utilization of microbes is an attractive option. In the present study, various chimeric nanobodies were prepared using the methylotrophic yeast Pichia pastoris. We designed chimeric nanobodies with complementarity-determining regions (CDRs) against green fluorescent protein (GFP) or cluster of differentiation 4 (CD4) based on the scaffold of GFP-nanobody. FLAG-tagged chimeric nanobodies were prepared by one-step cloning and produced using P. pastoris. Secreted chimeric nanobodies were purified from the culture media of P. pastoris transformants. Relative binding abilities of purified chimeric nanobodies to GFP and CD4 was tested using a BIACORE T-200. P. pastoris successfully produced a high yield of FLAG-tagged chimeric nanobodies. FLAG-tagged GFP- and CD4-nanobodies were shown to specifically bind to GFP and CD4, respectively. Chimeric nanobodies, in which the CDR2 or 3 of GFP-nanobody was replaced with CDRs of CD4-nanobody, acquired the ability to bind to CD4 without binding to GFP. These results demonstrate successful production of functional chimeric nanobodies using P. pastoris. These results also suggest that swapping of CDRs, especially CDRs 2 or 3, potentially enables a novel method of creating nanobodies.

High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry.

Proteome is extremely complex as many proteins with a large dynamic range are involved. Nano-liquid chromatography/mass spectrometry-based proteomics has made it possible to separate and identify thousands of proteins in one shot. Although the number of identified proteins in proteomics has significantly improved, it is necessary to increase detection sensitivity to clearly identify low-abundant proteins. In this study, we developed meter-long monolithic columns with a small inner diameter and applied them to selected reaction monitoring-based proteomics for improving proteomic detection sensitivity. Bovine serum albumin tryptic digests were analyzed with optimized selected reaction monitoring methods, and separation efficiency and detection sensitivity in each monolithic column were evaluated. As a result, peak capacity increased by about 1.8-fold and peak area of peptide levels increased by about 2.3-fold. Although flow rate was reduced during analysis with columns of a smaller inner diameter, the peak area reproducibility was maintained. These data displayed the value of meter-long monolithic columns with small inner diameter for selected reaction monitoring-based proteomics.

Peptide barcoding for establishment of new types of genotype-phenotype linkages.

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype-phenotype linkages.

Ruegeria sp. Strains Isolated from the Reef-Building Coral Galaxea fascicularis Inhibit Growth of the Temperature-Dependent Pathogen Vibrio coralliilyticus.

The coral microbiome has attracted increased attention because of its potential roles in host protection against deadly diseases. However, little is known about the role of coral-associated bacteria against the temperature-dependent opportunistic pathogen Vibrio coralliilyticus. In this study, we tested whether bacteria associated with the reef-building coral Galaxea fascicularis could inhibit the growth of V. coralliilyticus. Twenty-nine cultivable bacteria were successfully isolated from a healthy colony of G. fascicularis kept in an aquarium. Among the bacterial isolates, three Ruegeria sp. strains inhibited the growth of V. coralliilyticus P1 as a reference strain and Vibrio sp. isolated in this study. Ruegeria sp. strains were also detected from other G. fascicularis colonies in the aquarium and in previous field studies by 16S rRNA amplicon sequencing, suggesting that Ruegeria sp. strains are common among G. fascicularis colonies. These results illuminate the potential role of Ruegeria sp. in protecting corals against pathogenic Vibrio species.

Effect of cold and hot compress on neutrophilic migration to the site of doxorubicin extravasation.

In the present study, we investigated a part of the mechanism responsible for the effects of hot and cold compresses for extravasation of doxorubicin. We injected 20 µl of doxorubicin (DOX) (1 µg/µl) subcutaneously into the dorsal area in mice and observed the resulting skin lesions macroscopically and histologically from day 1 to day 14 thereafter in groups treated with a cold pack (18-20°C) and a hot pack (38-40°C) or left untreated (control). Immunofluorescence and RT-PCR for C5a receptor (CD88), interleukin-8 receptor (IL-8RA), and transient receptor potential cation channel subfamily V member 1 (TRPV1) were also performed. Macroscopic observation showed that the area of the skin lesion was significantly smaller in the cold group than in the control group, but was significantly larger in the hot group. The neutrophil count in the lesion was significantly higher in the hot group than in the cold (3 hrs) and control groups. The numbers of inflammatory cells expressing CD88 and IL-8RA were significantly lower in the cold group than that in the other groups at almost time points and in the hot groups at later time points, respectively. The number of nerve fascicles expressing TRPV1 was higher in the hot group than in the cold group on days 1, 3 and 14. mRNA for CD88, IL-8RA and TRPV1 was detectable by reverse transcription-polymerase chain reaction in both the cold and hot pack groups. Consequently, these results suggested that the cold pack for the extravasation of DOX might reduce inflammation.

Evaluation of Unconventional Protein Secretion by Saccharomyces cerevisiae and other Fungi.

Development of proteome analysis of extracellular proteins has revealed that a wide variety of proteins, including fungal allergens are present outside the cell. These secreted allergens often do not contain known secretion signal sequences. Recent research progress shows that some fungal allergens are secreted by unconventional secretion pathways, including autophagy- and extracellular-vesicle-dependent pathways. However, secretion pathways remain unknown for the majority of extracellular proteins. This review summarizes recent data on unconventional protein secretion in Saccharomyces cerevisiae and other fungi. Particularly, methods for evaluating unconventional protein secretion are proposed for fungal species, including S. cerevisiae, a popular model organism for investigating protein secretion pathways.

Three-dimensional alginate hydrogels for radiobiological and metabolic studies of cancer cells.

The purpose of this study is to demonstrate calcium alginate hydrogels as a system for in vitro radiobiological and metabolic studies of cancer cells. Previous studies have established calcium alginate as a versatile three-dimensional (3D) culturing system capable of generating areas of oxygen heterogeneity and modeling metabolic changes in vitro. Here, through dosimetry, clonogenic and viability assays, and pimonidazole staining, we demonstrate that alginate can model radiobiological responses that monolayer cultures do not simulate. Notably, alginate hydrogels with radii greater than 500 µm demonstrate hypoxic cores, while smaller hydrogels do not. The size of this hypoxic region correlates with hydrogel size and improved cell survival following radiation therapy. Hydrogels can also be utilized in hyperpolarized magnetic resonance spectroscopy and extracellular flux analysis. Alginate therefore offers a reproducible, consistent, and low-cost means for 3D culture of cancer cells for radiobiological studies that simulates important in vivo parameters such as regional hypoxia and enables long-term culturing and in vitro metabolic studies.

Protection of Coral Larvae from Thermally Induced Oxidative Stress by Redox Nanoparticles.

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNPO) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNPO treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNPO treatment, whereas these proteins were absent in RNPO-treated larvae, which suggested that RNPO effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNPO treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.