Chemoproteomic Identification of Blue-Light-Damaged Proteins.

Visible light, particularly in the blue region of the spectrum, can cause cell dysfunction through the generation of singlet oxygen, contributing to cellular aging and age-related pathologies. Although photooxidation of nucleic acids, lipids, and amino acids has been extensively studied, the magnitude and span of blue-light-induced protein damages within proteome remain largely unknown. Herein we present a chemoproteomic approach to mapping blue-light-damaged proteins in live mammalian cells by exploiting a nucleophilic alkyne chemical probe. A gene ontology enrichment analysis revealed that cell surface proteins are more readily oxidized than other susceptible sets of proteins, including mitochondrial proteins. In particular, the integrin family of cell surface receptors (ITGs) was highly ranked in the mammalian cells tested, including human corneal endothelial cells. The blue-light-oxidized ITGB1 protein was functionally inactive in promoting cell adhesion and proliferation, suggesting that the photodamage of integrins contributes to the blue-light-induced cell dysfunction. Further application of our method to various cells and tissues should lead to a comprehensive analysis of light-sensitive proteins.

Development and multi-institutional validation of an artificial intelligence-based diagnostic system for gastric biopsy.

To overcome the increasing burden on pathologists in diagnosing gastric biopsies, we developed an artificial intelligence-based system for the pathological diagnosis of gastric biopsies (AI-G), which is expected to work well in daily clinical practice in multiple institutes. The multistage semantic segmentation for pathology (MSP) method utilizes the distribution of feature values extracted from patches of whole-slide images (WSI) like pathologists' "low-power view" information of microscopy. The training dataset included WSIs of 4511 gastric biopsy tissues from 984 patients. In tissue-level validation, MSP AI-G showed better accuracy (91.0%) than that of conventional patch-based AI-G (PB AI-G) (89.8%). Importantly, MSP AI-G unanimously achieved higher accuracy rates (0.946 ± 0.023) than PB AI-G (0.861 ± 0.078) in tissue-level analysis, when applied to the cohorts of 10 different institutes (3450 samples of 1772 patients in all institutes, 198-555 samples of 143-206 patients in each institute). MSP AI-G had high diagnostic accuracy and robustness in multi-institutions. When pathologists selectively review specimens in which pathologist's diagnosis and AI prediction are discordant, the requirement of a secondary review process is significantly less compared with reviewing all specimens by another pathologist.

Small molecule-based detection of non-canonical RNA G-quadruplex structures that modulate protein translation.

Tandem repeats of guanine-rich sequences in RNA often form thermodynamically stable four-stranded RNA structures. Such RNA G-quadruplexes have long been considered to be linked to essential biological processes, yet their physiological significance in cells remains unclear. Here, we report a approach that permits the detection of RNA G-quadruplex structures that modulate protein translation in mammalian cells. The approach combines antibody arrays and RGB-1, a small molecule that selectively stabilizes RNA G-quadruplex structures. Analysis of the protein and mRNA products of 84 cancer-related human genes identified Nectin-4 and CapG as G-quadruplex-controlled genes whose mRNAs harbor non-canonical G-quadruplex structures on their 5'UTR region. Further investigations revealed that the RNA G-quadruplex of CapG exhibits a structural polymorphism, suggesting a possible mechanism that ensures the translation repression in a KCl concentration range of 25-100 mM. The approach described in the present study sets the stage for further discoveries of RNA G-quadruplexes.

Discovery of a phase-separating small molecule that selectively sequesters tubulin in cells.

Phase-separated membraneless organelles or biomolecular condensates play diverse functions in cells, however recapturing their characteristics using small organic molecules has been a challenge. In the present study, cell-lysate-based screening of 843 self-assembling small molecules led to the discovery of a simple organic molecule, named huezole, that forms liquid droplets to selectively sequester tubulin. Remarkably, this small molecule enters cultured human cells and prevents cell mitosis by forming tubulin-concentrating condensates in cells. The present study demonstrates the feasibility of producing a synthetic condensate out of non-peptidic small molecules for exogenous control of cellular processes. The modular structure of huezole provides a framework for designing a class of organelle-emulating small molecules.

Stasis and diversity in living fossils: Species delimitation and evolution of lingulid brachiopods.

The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision.

Glucose as a Protein-Condensing Cellular Solute.

The present study reports a surprising protein-condensing effect of glucose, prompted by our accidental observation during chemical library screening under a high-glucose condition. We noticed "glucosing-out" of certain compounds, in which physiological concentrations of glucose induced compound aggregation. Adapting the "glucosing-out" concept to proteins, our proteomic analysis identified three cellular proteins (calmodulin, rho guanine nucleotide exchange factor 40, and polyubiquitin-C) that displayed robust glucose-dependent precipitation. One of these proteins, calmodulin, formed glucose-dependent condensates that control cellular glycogenolysis in hepatic cells. Our findings suggest that glucose is a heretofore underappreciated driver of protein phase separation that may have profound effects on cellular homeostasis.

Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe.

Covalent inhibitors of enzymes are increasingly appreciated as pharmaceutical seeds, yet discovering non-cysteine-targeting inhibitors remains challenging. Herein, we report an intriguing experience during our activity-based proteomic screening of 1601 reactive small molecules, in which we monitored the ability of library molecules to compete with a cysteine-reactive iodoacetamide probe. One epoxide molecule, F8, exhibited unexpected enhancement of the probe reactivity for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-limiting glycolysis enzyme. In-depth mechanistic analysis suggests that F8 forms a covalent adduct with an aspartic acid in the active site to displace NAD+, a cofactor of the enzyme, with concomitant enhancement of the probe reaction with the catalytic cysteine. The mechanistic underpinning permitted the identification of an optimized aspartate-reactive GAPDH inhibitor. Our findings exemplify that activity-based proteomic screening with a cysteine-reactive probe can be used for discovering covalent inhibitors that react with non-cysteine residues.

RNA-based cooperative protein labeling that permits direct monitoring of the intracellular concentration change of an endogenous protein.

Imaging the dynamics of proteins in living cells is a powerful means for understanding cellular functions at a deeper level. Here, we report a versatile method for spatiotemporal imaging of specific endogenous proteins in living mammalian cells. The method employs a bifunctional aptamer capable of selective protein recognition and fluorescent probe-binding, which is induced only when the aptamer specifically binds to its target protein. An aptamer for ß-actin protein preferentially recognizes its monomer forms over filamentous forms, resulting in selective G-actin staining in both fixed and living cells. Through actin-drug treatment, the method permitted direct monitoring of the intracellular concentration change of endogenous G-actin. This protein-labeling method, which is highly selective and non-covalent, provides rich insights into the study of spatiotemporal protein dynamics in living cells.

Sensorineural hearing dysfunction after discharge from critical care in adults: A retrospective observational study.

BACKGROUND: Patients undergoing intensive care are exposed to risk factors for hearing impairment. This study assessed the worse changes in pure tone average (PTA) thresholds after intensive care and identified the factors associated with worse hearing function. METHODS: We conducted a single-centre retrospective study, and included adult patients admitted to the intensive care unit (ICU) of Kurashiki Central Hospital between January 2014 and September 2019, who had regular pure tone audiometry performed before and after ICU admission. Correlations between changes in PTA threshold and patient characteristics, were evaluated. The included ears were classified as those with worse hearing (>10 dB increase in the PTA threshold) and those without worse hearing, and the baseline characteristics were compared. RESULTS: During the study period, 125 ears of 71 patients (male:female ratio, 35:36; mean age, 72.5 ± 12.3 years) met the eligibility criteria. Age, sex, and the use of furosemide were not correlated with changes in PTA threshold. Univariate analysis showed that ears with worse hearing were associated with a lower serum platelet count than ears without worse hearing (153 ± 85 × 109/L vs. 206 ± 85 × 109/L, respectively; P = 0.010), and the rate of planned ICU admission (elective surgery) was higher in the worse hearing group (57.1% vs. 28.8%, respectively; p = 0.011). CONCLUSIONS: Age, sex, and the use of furosemide did not have adversely affect hearing function. Low serum platelet count and planned admission appear to be risk factors for worse hearing.

Longer DNA exhibits greater potential for cell-free gene expression.

Cell-free gene expression systems have been valuable tools for understanding how transcription/translation can be regulated in living cells. Many studies have investigated the determining factors that affect gene expression. Here we report the effect of the length of linearized reporter DNAs encoding the firefly luciferase gene so as to exclude the influence of supercoiling. It is found that longer DNA molecules exhibit significantly greater potency in gene expression; for example, the expression level for DNA with 25.7 kbp is 1000-times higher than that for DNA of 1.7 kbp. AFM observation of the DNA conformation indicates that longer DNA takes shrunken conformation with a higher segment density in the reaction mixture for gene expression, in contrast to the stiff conformation of shorter DNA. We propose an underlying mechanism for the favorable effect of longer DNA on gene expression in terms of the enhancement of access of RNA polymerase to the shrunken conformation. It is expected that the enhancement of gene expression efficiency with a shrunken DNA conformation would also be a rather general mechanism in living cellular environments.

Non-genetic cell-surface modification with a self-assembling molecular glue.

This report describes the development of a non-genetic cell-surface modification method, in which a self-assembling small molecule is combined with Halo-tag proteins. Cell-surface functionalization with cancer-linked extracellular proteins led to enhanced cell motility, angiogenesis, and immune shielding of the cells, paving the way for translational opportunities for cell therapy.

Discovery of a Small-Molecule-Dependent Photolytic Peptide.

We accidentally found that YM-53601, a known small-molecule inhibitor of squalene synthase (SQS), selectively depletes SQS from mammalian cells upon UV irradiation. Further analyses indicated that the photodepletion of SQS requires its short peptide segment located at the COOH terminus. Remarkably, when the 27 amino acid peptide was fused to green fluorescent protein or unrelated proteins at either the NH2 or COOH terminus, such fusion proteins were selectively depleted when the cells were treated with both YM-53601 and UV exposure. Product analysis and electron spin resonance experiments suggested that the UV irradiation promotes homolytic C-O bond cleavage of the aryl ether group in YM-53601. It is likely that the radical species generated from UV-activated YM-53601 abstract hydrogen atoms from the SQS peptide, leading to the photolysis of the entire protein. The pair of the SQS peptide and YM-53601 discovered in the present study paves the way for the design of a new small-molecule-controlled optogenetic tool.

Nutrient-Based Chemical Library as a Source of Energy Metabolism Modulators.

Covalent conjugates of multiple nutrients often exhibit greater biological activities than each individual nutrient and more predictable safety profiles than completely unnatural chemical entities. Here, we report the construction and application of a focused chemical library of 308 covalent conjugates of a variety of small-molecule nutrients. Screening of the library with a reporter gene of sterol regulatory element-binding protein (SREBP), a master regulator of mammalian lipogenesis, led to the discovery of a conjugate of docosahexaenoic acid (DHA), glucosamine, and amino acids as an inhibitor of SREBP (molecule 1, DHG). Mechanistic analyses indicate that molecule 1 impairs the SREBP activity by inhibiting glucose transporters and thereby activating AMP-activated protein kinase (AMPK). Oral administration of molecule 1 suppressed the intestinal absorption of glucose in mice. These results suggest that such synthetic libraries of nutrient conjugates serve as a source of novel chemical tools and pharmaceutical seeds that modulate energy metabolism.

Multifunctionalization of Cells with a Self-Assembling Molecule to Enhance Cell Engraftment.

Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.

Approaches of validation of a 2-week combined repeated oral dose toxicity study with plasma micro sampling toxicokinetics (PMS-TK) in common marmosets.

We investigated the viability of a combined repeated dose toxicity study, including toxicokinetics (TK), in common marmosets according to the ICH-S4, ICH-S3A and ICH-S7A Guidelines using valsartan as test article whose non-clinical repeated dose toxicity studies had been conducted using this species for regulatory purpose. Valsartan was administered orally to 3 animals/sex at 200 mg/kg/day for 2 weeks. In addition to the routine parameters in repeated dose toxicity studies, safety pharmacology parameters (examinations of the central nervous, respiratory and cardiovascular systems) were also evaluated. The Plasma Micro Sampling Toxicokinetics (PMS-TK) method required ultrasensitive quantitation, was employed to evaluate the relationship between toxic changes and plasma concentrations as well as the effects of frequent blood sampling in individual animals. In valsartan, toxic findings (a deteriorated physical condition; moribundity of one male and one female on Day 14; sporadic vomitus; decreases in body weights and food consumption; decreases in erythrocytic parameters; and renal changes such as an increase in urea nitrogen, dilation of the tubules and hypertrophy of the tubular epithelium) were similar and plasma concentrations comparable to the results in the approval information. Furthermore, no side effects caused by frequent blood sampling were confirmed in the negative control group. Consequently, a combined repeated dose toxicity study including TK analysis using the PMS-TK method is viable in common marmosets and contributes to animal welfare.

Generalization tests using different dosing routes from those of drug discrimination training in rats.

The purpose of this study was to investigate the discriminative stimulus properties of morphine and codeine using different administration routes to that used at drug discrimination training. Rats were trained to discriminate morphine at 3 mg/kg from saline by the intraperitoneal route in a standard two-lever drug discrimination paradigm. Generalization of morphine by the subcutaneous and the oral routes, and codeine by the intraperitoneal and the oral routes to the discriminative stimulus properties of the morphine training dose were investigated. Morphine at 3 mg/kg by the subcutaneous route generalized to the morphine training dose and 10 of 12 rats showed 80% or more morphine-lever responses. In the administration of morphine by the oral route, morphine at 30 mg/kg generalized to the morphine training dose and all rats showed 80% or more morphine-lever responses within the range of 3 to 30 mg/kg. In the administration of codeine by the intraperitoneal route, codeine at 20 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 3 to 20 mg/kg. In the administration of codeine by the oral route, codeine at 60 mg/kg generalized to the morphine training dose and 14 of the 15 animals showed 80% or more morphine-lever responses within the range of 10 to 60 mg/kg. Thus, the discriminative stimulus properties of morphine and codeine were comparable when using different administration routes to those at discrimination training.

Live-cell imaging of multiple endogenous mRNAs permits the direct observation of RNA granule dynamics.

Here, we developed two pairs of high-contrast chemical probes and their RNA aptamers with distinct readout channels that permitted simultaneous live-cell imaging of endogenous ß-actin and cortactin mRNAs. Application of this technology allowed the direct observation of the formation process of stress granules, protein-RNA assemblies essential for cellular response to the environment.

Stapes surgery preserving the superstructure of stapes (Takagi's stapedotomy) in otosclerosis: A retrospective study of 24 consecutive cases.

OBJECTIVE: The aim of this study was to evaluate the hearing outcomes and complications of stapedotomy in which the stapes superstructure was preserved (Takagi's stapedotomy). In this surgical approach, the lenticular process of the incus rather is removed, than the superstructure of the stapes. METHODS: A single-center retrospective observational study was performed. We included all patients having Takagi's stapedotomy for otosclerosis between January 2005 and April 2016. Both primary and revision stapes surgery were included. We evaluated audiometric outcomes and surgical complications. RESULTS: Twenty-four patients who underwent stapedotomy preserving superstructure were included in this study. The postoperative air-bone gap at 1year postoperatively was ≤10dB in 66.7% of patients and ≤20dB in all cases. In longer follow-up period, elevation of the air-bone gap was not observed over the 5 postoperative years in available cases. The postoperative air-bone gap was ≤10dB in 72.2% at 3years and 81.8% at 5years postoperatively. CONCLUSION: Takagi's stapedotomy restore ossicular conduction without the removal of superstructure of stapes. The air-bone gap did not get worse in long-term follow-up, although audiometric results would be unsatisfactory. Further larger studies are needed to evaluate the efficacy and safety of Takagi's stapedotomy.

Chemical decontamination of iPS cell-derived neural cell mixtures.

This report describes the design and evaluation of phosphorylated 7-ethyl-10-hydroxycamptothecin (SN38-P), which selectively eliminates tumor-forming proliferative stem cells, including human induced pluripotent stem cells (hiPSCs) and neural stem cells, from iPSC-derived neural cell mixtures. Results of the present study demonstrate that simple phosphorylation of an anticancer drug can provide a safe, cost-effective, and chemically-defined tool for decontaminating hiPSC-derived neuron.