ABSTRACT
Stress granules (SGs) are cytoplasmic messenger ribonucleoprotein granules transiently formed in stressed mammalian cells. Although SG components have been well characterized, detailed insights into the molecular behavior inside SGs remain unresolved. We investigated nanoscale dynamics and localization of endogenous mRNAs in SGs combining single mRNA tracking and super-resolution localization microscopy. First, we developed a methodology for tracking single mRNAs within SGs, revealing that although mRNAs in SGs are mainly stationary (â¼40%), they also move in a confined (â¼25%) or freely diffusing (â¼35%) manner. Second, the super-resolution localization microscopy showed that the mRNAs in SGs are heterogeneously distributed and partially form high-density clusters. Third, we simultaneously performed single mRNA tracking and super-resolution microscopy in SGs, demonstrating that single mRNA trajectories are mainly found around high-density clusters. Finally, a quantitative analysis of mRNA localization and dynamics during stress removal was conducted using live super-resolution imaging and single-molecule tracking. These results suggest that SGs have a highly organized structure that enables dynamic regulation of the mRNAs at the nanoscale, which is responsible for the ordered formation and the wide variety of functions of SGs.
ABSTRACT
The chemical derivatization of target analytes can enhance the sensitivity and selectivity of separation-based methods for metabolite analysis using microfluidic devices. However, the development of chromatography-based microfluidic devices with integrated derivatization units is challenging. In this study, a novel derivatization unit with a pillar array (PA)-based mixing channel was developed for postcolumn derivatization during on-chip liquid chromatography (LC). The PA mixer enhanced mixing between the derivatization reagents and analytes in the transverse direction, while preventing analyte dispersion in the flow direction. After the concept was confirmed using computational fluid dynamics analysis, microfluidic devices with a LC column and PA mixer were fabricated on a 20 × 20 mm silicon plate. Fluid experiments were performed using a PA mixer with a pillar size of 5 or 10 µm or a hollow-channel mixer, which revealed that the PA mixer enhanced transverse mixing without increasing the width of the analyte peak. Moreover, the developed device enabled the analysis of three amino acids within 40 s by separation via hydrophilic interaction chromatography followed by postcolumn fluorogenic derivatization with naphthalene-2,3-dicarboxaldehyde and fluorescence detection. Our results demonstrate the potential of integrated derivatization units for the development of micrototal analysis systems for use in bioanalysis.
ABSTRACT
Quantification of cytokine secretion has facilitated advances in the field of immunology, yet the dynamic and varied secretion profiles of individual cells, particularly those obtained from limited human samples, remain obscure. Herein, we introduce a technology for quantitative live-cell imaging of secretion activity (qLCI-S) that enables high-throughput and dual-color monitoring of secretion activity at the single-cell level over several days, followed by transcriptome analysis of individual cells based on their phenotype. The efficacy of qLCI-S was demonstrated by visualizing the characteristic temporal pattern of cytokine secretion of group 2 innate lymphoid cells, which constitute less than 0.01% of human peripheral blood mononuclear cells, and by revealing minor subpopulations with enhanced cytokine production. The underlying mechanism of this feature was linked to the gene expression of stimuli receptors. This technology paves the way for exploring gene expression signatures linked to the spatiotemporal dynamic nature of various secretory functions.
ABSTRACT
MicroRNA (miRNA) plays physiologically and pathologically important roles in post-transcriptional regulation. Although miRNA has been suggested to dynamically interact with cellular organelles, the dynamicity of intracellular miRNA behavior has remained unclear. Here, by introducing fluorescently labeled pre-miRNA into living cells, we improved the miRNA visualization method using exogenous miRNA precursors. Through the combination of our miRNA visualization method and single-molecule sensitive fluorescence microscopy, we quantitatively analyzed the process of miRNA maturation. Furthermore, single-particle tracking of fluorescent miRNA in cells revealed the directed movements of miRNA on cytoskeletal components (i.e., microtubules and actin filaments). Our results also suggest that cytoskeleton-dependent miRNA trafficking is associated with the interaction of miRNAs with the nucleus and the endoplasmic reticulum/Golgi apparatus. Our method should facilitate the elucidation of the mechanism and physiological significance of the subcellular localization and organelle interaction of miRNA.
ABSTRACT
Environmental microorganisms possess enzymes that can digest macromolecules such as agarose into smaller molecules that can be utilized for growth. These enzymes could be valuable for the effective utilization of global resources. However, since most of the microorganisms on Earth remain uncultured, there is significant untapped enzymatic potential in nature. Therefore, it is necessary to develop innovative tools and strategies for exploring these enzymatic resources. To address this, we developed a method for screening microbial cells that secrete hydrogel-degrading enzymes using deformability-based microfluidic microdroplet sorting. In this method, microbial cells are encapsulated as single cells in water-in-oil (W/O) microdroplets with a hydrogel whose shape becomes deformable as the hydrogel is progressively degraded into smaller molecules. Screening is achieved using a microfluidic device that passively sorts the deformed W/O microdroplets. Using this method, we successfully sorted agarose-containing microdroplets, encapsulating single bacterial cells that hydrolyzed agarose. This method can be used to screen various hydrogel-degrading microbial cells.
Subject(s)
Hydrogels , Microfluidics , Microfluidics/methods , Sepharose , Bacteria , WaterABSTRACT
The decision of whether cells are activated or not is controlled through dynamic intracellular molecular networks. However, the low population of cells during the transition state of activation renders the analysis of the transcriptome of this state technically challenging. To address this issue, we have developed the Time-Dependent Cell-State Selection (TDCSS) technique, which employs live-cell imaging of secretion activity to detect an index of the transition state, followed by the simultaneous recovery of indexed cells for subsequent transcriptome analysis. In this study, we used the TDCSS technique to investigate the transition state of group 2 innate lymphoid cells (ILC2s) activation, which is indexed by the onset of interleukin (IL)-13 secretion. The TDCSS approach allowed us to identify time-dependent genes, including transiently induced genes (TIGs). Our findings of IL4 and MIR155HG as TIGs have shown a regulatory function in ILC2s activation.
Subject(s)
Immunity, Innate , Lymphocytes , Immunity, Innate/genetics , Gene Expression Profiling , TranscriptomeABSTRACT
We report the complete genomic sequence of the agarolytic bacterium Pseudoalteromonas sp. strain MM1, recovered from deep seawater. The genome has two circular chromosomes with sizes of 3,686,652 bp and 802,570 bp and GC contents of 40.8 and 40.0%, and it carries 3,967 protein-coding sequences, 24 rRNA genes, and 103 tRNA genes.
ABSTRACT
We report the complete genomic sequences of two agarolytic Vibrio species strains, STUT-A11 and STUT-A16, isolated from the red algae Gracilaria. Genomic annotations revealed that both strains harbor four ß-agarases, α-neoagarooligosaccharide hydrolase, and agarolytic ß-galactosidase, which support efficient agarose catabolism.
ABSTRACT
Nod-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic innate immune receptor that senses organelle dysfunction induced by various stimuli, such as infectious, environmental, metabolic and drug stresses. Upon activation, NLRP3 forms an inflammasome with its adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1, to trigger the release of inflammatory cytokines. The development of effective anti-inflammatory drugs targeting the NLRP3 inflammasome is in high demand as its aberrant activation often causes inflammatory diseases. Here, we found that nanaomycin A (NNM-A), a quinone-based antibiotic isolated from Streptomyces, effectively inhibited NLRP3 inflammasome-mediated inflammatory responses induced by imidazoquinolines, including imiquimod. Interestingly, its epoxy derivative nanaomycin E (NNM-E) showed a comparable inhibitory effect against the NLRP3 inflammasome-induced release of interleukin (IL)-1ß and IL-18 from macrophages, with a much lower toxicity than NNM-A. NNM-E inhibited ASC oligomerization and caspase-1 cleavage, both of which are hallmarks of NLRP3 inflammasome activation. NNM-E reduced mitochondrial damage and the production of reactive oxygen species, thereby preventing the activation of the NLRP3 inflammasome. NNM-E treatment markedly alleviated psoriasis-like skin inflammation induced by imiquimod. Collectively, NNM-E inhibits NLRP3 inflammasome activation by preventing mitochondrial dysfunction with little toxicity and showed an anti-inflammatory effect in vivo. Thus, NNM-E could be a potential lead compound for developing effective and safe anti-inflammatory agents for the treatment of NLRP3 inflammasome-mediated inflammatory diseases.
Subject(s)
Inflammasomes , NLR Family, Pyrin Domain-Containing 3 Protein , Caspase 1/metabolism , Imiquimod/metabolism , Imiquimod/pharmacology , Interleukin-1beta/metabolism , Mitochondria/metabolism , NaphthoquinonesABSTRACT
Focused ion beamed (FIB) SEM has a higher spatial resolution than other volume-imaging methods owing to the use of ion beams. However, in this method, it is challenging to analyse entire biological structures buried deep in the resin block. We developed a novel volume-imaging method by combining array tomography and FIB-SEM tomography and investigated the chondrocyte ultrastructure. Our method imparts certainty in determining the analysis area such that cracks or areas with poor staining within the block are avoided. The chondrocyte surface showed fine dendritic processes that were thinner than ultrathin sections. Upon combination with immunostaining, this method holds promise for analysing mesoscopic architectures.
Subject(s)
Deceleration , Tomography , Imaging, Three-Dimensional/methods , Microscopy, Electron, Scanning , Tomography, X-Ray ComputedABSTRACT
Biothiols, such as cysteine and glutathione, play important roles in various intracellular reactions represented by the redox equilibrium against oxidative stress. In this study, a method for intracellular thiol quantification using HPLC-fluorescence detection was developed. Thiols were derivatized with a thiol-specific fluorescence derivatization reagent, viz. ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), followed by reversed-phase separation on an InertSustain AQ-C18 column. Six different SBD-thiols (homocysteine, cysteine, cysteinylglycine, γ-glutamylcysteine, glutathione, and N-acetylcysteine as an internal standard) were separated within 30 min using a citric buffer (pH 3.0)/MeOH mobile phase. The calibration curves of all the SBD-thiols had strong linearity (R2 > 0.999). Using this developed method, the thiol concentrations of human chronic myelogenous leukemia K562 cell samples were found to be 5.5-153 pmol/1 × 106 cells. The time-dependent effect of a thiol scavenger, viz. N-ethyl maleimide, on intracellular thiol concentrations was also quantified. This method is useful for elucidating the role of intracellular sulfur metabolism.
Subject(s)
Sulfhydryl Compounds/analysis , Chromatography, High Pressure Liquid , Fluorescence , Glutathione/metabolism , Humans , K562 Cells , Oxidative Stress/physiologyABSTRACT
Branched-chain α-keto acids (BCKAs, namely, α-ketoisovaleric acid (KIV), α-ketoisocaproic acid (KIC), and α-keto-ß-methylvaleric acid (KMV)) are related to many diseases such as myeloid leukemia, liver cancer, and diabetes mellitus. A rapid quantitative analytical method for BCKAs using pillar array columns was developed. α-Keto acids were labeled with 1,2-diamino-4,5-methylenedioxybenzene (DMB), followed by their separation on octadecylsilane-treated pillar array columns with MeOH/H2O as the mobile phase. Five DMB-labelled α-keto acids including the internal standard were separated in 160 s. The lower limits of quantification for DMB-α-keto acids were 2-5 µM. The intra- and interday precisions were 2.9-6.6 % and 5.2-10.7 %, respectively. The developed method was applied to BCKA quantification in human plasma samples; KIV, KIC, and KMV concentrations were determined to be 13.8, 24.2, and 15.2 µM, respectively. The method realized rapid, sensitive, and precise analysis of BCKAs and can be applied for clinical diagnosis.
Subject(s)
Keto Acids , HumansABSTRACT
Deep-sea Bathymodiolus mussels are generally thought to harbour chemosynthetic symbiotic bacteria in gill epithelial cells called bacteriocytes. However, previously observed openings at the apical surface of bacteriocytes have not been conclusively explained and investigated as to whether the Bathymodiolus symbiosis is intracellular or extracellular. In this study, we show that almost all the membranous chambers encompassing symbionts in a single bacteriocyte of Bathymodiolus septemdierum are interconnected and have pathways connecting to the external environment. Furthermore, the symbiont population colonising a single bacteriocyte is mostly clonal. This study hypothesises on a novel model of cellular localization at the interface between extra- and intracellular symbiosis, and the cellular-level process of symbiont acquisition in Bathymodiolus mussels.
ABSTRACT
Eukaryotic transcription is epigenetically regulated by chromatin structure and post-translational modifications (PTMs). For example, lysine acetylation in histone H4 is correlated with activation of RNA polymerase I-, II- and III-driven transcription from chromatin templates, which requires prior chromatin remodeling. However, quantitative understanding of the contribution of particular PTM states to the sequential steps of eukaryotic transcription has been hampered partially because reconstitution of a chromatin template with designed PTMs is difficult. In this study, we reconstituted a di-nucleosome with site-specifically acetylated or unmodified histone H4, which contained two copies of the Xenopus somatic 5S rRNA gene with addition of a unique sequence detectable by hybridization-assisted fluorescence correlation spectroscopy. Using a Xenopus oocyte nuclear extract, we analyzed the time course of accumulation of nascent 5S rRNA-derived transcripts generated on chromatin templates in vitro. Our mathematically described kinetic model and fitting analysis revealed that tetra-acetylation of histone H4 at K5/K8/K12/K16 increases the rate of transcriptionally competent chromatin formation â¼3-fold in comparison with the absence of acetylation. We provide a kinetic model for quantitative evaluation of the contribution of epigenetic modifications to chromatin transcription.
Subject(s)
Chromatin/genetics , Epigenesis, Genetic , Protein Processing, Post-Translational/genetics , Transcription, Genetic , Acetylation , Animals , Histones/genetics , Lysine/genetics , Nucleosomes/genetics , RNA, Ribosomal, 5S/genetics , Xenopus laevis/geneticsABSTRACT
Branched-chain keto acids and branched-chain amino acids are metabolites of branched-chain amino acid aminotransferases (BCATs), which catalyzes reversible transamination between them. We found that BCAT1 plays an important role in the progression of myeloid leukaemia, and a method for the analysis of intracellular α-keto acids including branched-chain keto acids was necessary to further investigate their role. In this study, we developed a method to analyze six α-keto acids (α-ketoglutaric acid (KG), pyruvic acid, α-ketobutyric acid, α-ketoisovaleric acid, α-ketoisocaproic acid, and α-keto-ß-methylvaleric acid) in K562 cells by HPLC with fluorescence detection, using 1,2-diamino-4,5-methylenedioxybenzene (DMB) as a derivatization reagent. Because split peaks of DMB-KG were observed when injection samples were too acidic, the derivatization solution was diluted with NaOH solution to obtain a single peak. Limits of detection and limits of quantification were 1.3-5.4 nM and 4.2-18 nM, respectively. Intracellular concentrations of α-keto acids were 1.55-316 pmol/1 × 106 K562 cells. The developed method realized reproducible and sensitive analysis of intracellular α-keto acids. Thus, the method could be used to elucidate the role of BCAT in myeloid leukaemia.
Subject(s)
Amino Acids, Branched-Chain , Keto Acids , Chromatography, High Pressure Liquid , Ketoglutaric Acids , Pyruvic AcidABSTRACT
The presence of approximately 200-bp cell-free DNA (cfDNA) in the urine has attracted attention as a biomarker for liquid biopsy. However, it is currently useful only for diagnoses of cancers in which a large amount of cfDNA is excreted in the urine. Therefore, the development of an efficient method for extracting cfDNA existing in small amounts in the urine is essential for diagnosing many other diseases. We examined the effect of particle size, small pore size (surface area), and surface modification of porous silica particles on the efficiency of DNA extraction. Our observations suggested that cfDNA could be captured by tertiary amine-modified particles and then removed from the particles by repeatedly washing with sodium bicarbonate (pH 11). Using this method with 30 mg of triamine-modified particles, we succeeded in extracting a few hundred nanograms of cfDNA from 15 mL urine. Furthermore, we could detect ~ 67 fg/mL caries DNA (211 bp) in 15 mL urine sample, suggesting that this method may be suitable for the extraction of genetic biomarkers for cfDNA-based liquid biopsy.
Subject(s)
Amines/chemistry , Biomarkers, Tumor/urine , Cell-Free Nucleic Acids/urine , Liquid Biopsy/methods , Silicon Dioxide/chemistry , HumansABSTRACT
A photo-responsive nanovesicle is fabricated by polyion complex (PIC) formation between poly(ethylene glycol) (PEG)-block-polypeptides and photo-reactive oligodeoxynucleotides (PROs)/anti-sense oligonucleotides (ASOs). The ultraviolet (UV) light triggers reversible crosslinking between PROs and ASOs in the vesicular membrane, providing the nanovesicle with switchable stability under physiological conditions. The resulting nanovesicle allows efficient cellular internalization, leading to significant UV-triggered gene knockdown in cultured cells.
Subject(s)
Gene Knockdown Techniques/methods , Nanostructures/chemistry , Oligodeoxyribonucleotides/chemistry , Ultraviolet Rays , A549 Cells , DNA Damage/drug effects , DNA Damage/radiation effects , Fluorescent Dyes/chemistry , Humans , Microscopy, Confocal , Nanostructures/toxicity , Peptides/chemistry , Polyethylene Glycols/chemistryABSTRACT
Mitochondria play a central role in the function of brown adipocytes (BAs). Although mitochondrial biogenesis, which is indispensable for thermogenesis, is regulated by coordination between nuclear DNA transcription and mitochondrial DNA transcription, the molecular mechanisms of mitochondrial development during BA differentiation are largely unknown. Here, we show the importance of the ER-resident sensor PKR-like ER kinase (PERK) in the mitochondrial thermogenesis of brown adipose tissue. During BA differentiation, PERK is physiologically phosphorylated independently of the ER stress. This PERK phosphorylation induces transcriptional activation by GA-binding protein transcription factor α subunit (GABPα), which is required for mitochondrial inner membrane protein biogenesis, and this novel role of PERK is involved in maintaining the body temperatures of mice during cold exposure. Our findings demonstrate that mitochondrial development regulated by the PERK-GABPα axis is indispensable for thermogenesis in brown adipose tissue.
Subject(s)
Adipose Tissue, Brown/metabolism , Endoplasmic Reticulum/metabolism , eIF-2 Kinase/metabolism , Adipocytes, Brown/metabolism , Animals , Cell Differentiation/genetics , DNA, Mitochondrial/metabolism , Female , Male , Mice , Mice, Inbred ICR , Mitochondria/metabolism , Organelle Biogenesis , Phosphorylation , Signal Transduction/genetics , Thermogenesis/physiology , Transcription, Genetic/geneticsABSTRACT
SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57-98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57-98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.
Subject(s)
Amino Acid Sequence/genetics , Escherichia coli Proteins/genetics , Protein Biosynthesis , Ribosomes/genetics , Transcription Factors/genetics , DNA Mutational Analysis , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Gene Expression Regulation, Bacterial/genetics , Mutation/genetics , Protein Transport/genetics , Ribosomal Proteins/genetics , Transcription Factors/chemistryABSTRACT
In this study, we investigated suitable conditions for the reduction of disulfides in mouse serum samples by tris(2-carboxyethyl) phosphine (TCEP) for fast analysis of total biothiols. Disulfides were reduced with TCEP, and then, thiols were derivatized with the fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Interference peaks on chromatograms of mouse serum samples disappeared when the TCEP reaction was conducted on ice instead of at room temperature, which is used classically. Low-molecular-weight disulfides, such as cystine and glutathione disulfide, were nearly completely reduced by TCEP on ice. Six SBD-biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, γ-glutamylcysteine, and N-acetylcysteine) were separated within 7.5 min on a sulfoalkylbetain-type column (ZIC-HILIC: 150 × 2.1 mm i.d., 3.5 µm), without interference peaks. The developed method showed good linearity and reproducibility, with inter- and intra-day precisions of less than 3%.