Biology subjects

Ito, C.

Publications and source records attributed to Ito, C..

9 recordsLinked to original sources

bioRxiv2026

In vivo brain temperature measurement using quantum dot imaging temperature sensors

Temperature regulation in the brain is essential for maintaining neuronal function and preventing thermally induced damage. Here, we report the development and in vivo application of quantum dots (QDs) to high-resolution thermometry in the mouse brain using two-photon excitation microscopy. These QDs, via the red-to-green photoluminescence (PL) intensity ratios, enabled stable temperature measurements in both normal and chronically hypo-perfused cerebral tissue. Our findings show that localized neuronal activity leads to transient heat generation, which is rapidly dissipated by cerebrovascular responses. In a chronic hypoperfusion model, impaired vascular function resulted in exaggerated and prolonged brain temperature elevations. This thermometry system provides unprecedented insight into the mechanisms of cerebral thermoregulation and highlights the importance of vascular cooling in protecting the brain from heat-induced stress, particularly in pathological conditions such as stroke.

bioengineering↗

bioRxiv2025

Structural basis of stereochemical promiscuity by an umami taste receptor ortholog, Tas1r1/Tas1r3 from pufferfish

Taste receptor type 1 (TAS1R), which consists of sweet and umami receptors in humans, senses nutrients (such as sugars and amino acids) with substrate specificity that is often broad and varies among animals and subtypes. However, the structural basis for achieving diverse specificities remains largely elusive. Here, we present the crystal structure of the ligand-binding domain (LBD) of Tas1r1/Tas1r3 heterodimer from pufferfish, an ortholog of the human umami taste receptor. The overall structure of Tas1r1/Tas1r3LBD resembles previously reported TAS1R structures, indicating a conserved core architecture within the family. Nevertheless, pufferfish Tas1r1/Tas1r3 was found to bind and respond to both L- and D-amino acids, even though TAS1Rs are considered to respond to either enantiomer. Structural and mutational analyses revealed that this non-rigorous stereochemical recognition is attributed to inter- subdomain interactions that latch the cleft containing the amino acid-binding site. These interactions prevent the cleft from fully opening, thereby stabilizing the active conformation, even if the ligand shares a different chirality. These results suggest that different substrate specificities in TAS1Rs can be acquired not only by the gain or loss of direct interactions with the substrate but also by the gain of intramolecular interactions, which alter the conformational equilibrium of the receptor.

biochemistry↗

bioRxiv2025

In vivo deep-brain microscopy at submicrometer resolution with refractive index-matched prism interfaces

The mammalian brain is a thick and densely layered structure comprising a huge number of neurons that work together to process information and regulate brain functions. Although various optical methods have been developed to investigate deep brain dynamics, they are limited by technical constraints, invasiveness, suboptimal spatial resolution, and/or a restricted field of view. To overcome these limitations, we developed an implantable, optically optimized microprism interface with a refractive index matched to that of brain tissue and water, enabling minimally-invasive, wide-field two-photon imaging method with enhanced brightness and sub-micron resolution in deep prefrontal areas.

neuroscience↗

bioRxiv2024

Low-invasive, wide-field, and cellular resolution two-photon imaging of neural population activity in brainstem and nucleus tractus solitarii

Brain-viscera communication plays a crucial role in regulating mental health, with the vagus nerve being a key structure mediating this interaction. Clinically, artificial vagus nerve stimulation (VNS) is used to treat various neuropsychiatric disorders, highlighting the importance of vagal afferent fibers in regulating emotion. The nucleus tractus solitarii (NTS) is a brainstem structure proposed to receive signals from vagal afferents and relay them to brain networks for emotion regulation. However, due to the anatomical complexity and difficulty in accessing the deep-brain NTS region in living animals, the mechanisms remain unclear. Here, we developed a wide-field and deep-brain two-photon imaging method using a double-prism based optical interface. This approach enables the identification of cellular-resolution neural activities in the NTS while preserving the cerebellum, which covers the NTS and is important for emotion regulation, intact. We systematically evaluated how NTS neurons respond to VNS and a gastrointestinal hormone, suggesting the usefulness of this method for investigating the role of the vagus-NTS pathway in vivo.

neuroscience↗

bioRxiv2023

Translation in a Box: Orthogonal Evolution in the Saccharomyces cerevisiae Mitochondrion

The ability to re-engineer and creatively evolve the translation system (TS) would allow invention of new coded polymers by altering the amino acid sidechain inventory and by shifting the polypeptide backbone into new chemical spaces. Unfortunately, the TS is difficult to manipulate and is more constrained over evolution than any other biological system. An orthogonal TS, running in parallel to the primary TS within a given host cell, would release constraints and allow TS manipulation. A fully orthogonal TS requires dedicated rRNAs, rProteins, aminoacyl-tRNA synthetases, and initiation and termination factors, none of which interact with the primary TS. The S. cerevisiae mitochondrial TS is fully orthogonal to the cytosolic TS. Mito-rRNAs, mito- rProteins, mito-tRNAs, mito-aminoacyl tRNA synthetases, and mito-translation factors are distinct from, physically separated from, and functionally independent of their cytosolic counterparts. Here, the S. cerevisiae mitochondrial translation system was subjected to various stresses including antibiotics, mutagenesis and truncation of mito-rProteins, or wholesale replacement of mito-rProteins. Directed evolution of these stressed systems was facilitated by controlled transitions between fermentation and respiration, by changing the carbon source in the growth medium; the dependence of S. cerevisiae survival on mitochondrial translation can be toggled on and off. Specific recreation of the resulting mutations recapitulate the evolved phenotypes. The method developed here appears to be a general approach for discovering functional dependencies. Suppressor mutations reveal functional dependencies within the S. cerevisiae mitochondrial TS. For example proteins Rrg9 or Mrx1 interact with the mito-TS and have critical role in its function. The combined results indicate that the S. cerevisiae mitochondrial TS can be engineered and evolved in isolation of the cytosolic TS. SignificanceThe Central Dogma of Molecular Biology rules life on Earth. Information flows from DNA to mRNA to protein. In the last step of the Central Dogma, the translation system decodes mRNA and produces coded proteins by linking amino acids into polymers. Engineering and evolving the translation system could permits full technical control over this process and could lead to the generation of novel polymers. Here, we use the mitochondrial translation system in the budding yeast Saccharomyces cerevisiae for directed evolution of translation. We modify and evolve the translation system both directly and indirectly using antibiotics and gene editing tools and then measure resulting functionality. Our results show this secondary translation system inside S. cerevisiae mitochondria can be used as an approach for translation engineering.

synthetic biology↗

bioRxiv2022

Chloride ion evokes taste sensation by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

Salt taste sensation is multifaceted: NaCl at low or high concentrations is preferably or aversively perceived through distinct pathways. Cl- is thought to participate in taste sensation through an unknown mechanism. Here we describe Cl- ion binding and the response of taste receptor type 1 (T1r), a receptor family composing sweet/umami receptors. The T1r2a/T1r3 heterodimer from the medaka fish, currently the sole T1r amenable to structural analyses, exhibited a specific Cl- binding in the vicinity of the amino-acid-binding site in the ligand-binding domain (LBD) of T1r3, which is likely conserved across species, including human T1r3. The Cl- binding induced a conformational change in T1r2a/T1r3LBD at sub-to low-mM concentrations similar to canonical taste substances. Furthermore, oral Cl- application to mice increased impulse frequencies of taste nerves connected to T1r-expressing taste cells and promoted their behavioral preferences attenuated by a T1r-specific blocker or T1r3 knock-out. These results suggest that the Cl- evokes taste sensations by binding to T1r, thereby serving as another preferred salt taste pathway at a low concentration.

biophysics↗

bioRxiv2020

Human Ribosomal G-Quadruplexes Regulate Heme Bioavailability

The in vitro formation of stable G-quadruplexes (G4s) in human ribosomal RNA (rRNA) was recently reported. However, their formation in cells and their cellular roles have not been resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest ribosomes are central hubs of heme metabolism.

biochemistry↗

bioRxiv2019

Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

Taste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested L-amino acids, but no D-amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

biochemistry↗

bioRxiv2018

Dynamic G-quadruplexes on the surface of the human ribosome

Profound similarities and critical differences mark ribosomes across phylogeny. The ribosomal core, approximated by the prokaryotic ribosome, is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. Differences in size are due in part to rRNA expansion segments. Here we show rRNA tentacles of Expansion Segment 7 (ES7) of Homo sapiens can form G-quadruplexes in vitro. G-quadruplex-forming regions are located on the most surface-exposed regions of the ribosome, near the termini of rRNA tentacles. We characterized rRNA of the large ribosomal subunit by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. We investigated ES7 and oligomers derived from ES7, intact 28S rRNA, and 80S ribosomes and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. Proteins that associate with rRNA G-quadruplexes include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins (hnRNPs). And finally, by multiple sequence alignments, we observed that G-quadruplex-forming sequences appear to be a general feature LSU rRNA of the phylum Chordata but not in other phyla. It is known that G-quadruplexes form in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported previously in ribosomes.

biochemistry↗