Galectin-10 in serum extracellular vesicles reflects asthma pathophysiology.

BACKGROUND: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. OBJECTIVE: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs). METHODS: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps. RESULTS: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo. CONCLUSION: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches.

Changes in the fecal gut microbiome of home healthcare patients with disabilities through consumption of malted rice amazake.

The aim of the present study was to investigate changes in the gut microbiome both during and after consumption of malted rice amazake (MR-Amazake), a fermented food from Japan, in-home healthcare patients with disabilities, including patients with severe motor and intellectual disabilities. We monitored 12 patients who consumed MR-Amazake for 6 wk and investigated them before and after the intervention as well as 6 wk after the end of intake to compare their physical condition, diet, type of their medication, constipation assessment scale, and analysis of their comprehensive fecal microbiome using 16S rRNA sequencing. Their constipation symptoms were significantly alleviated, and principal coordinate analysis revealed that 30% of patients showed significant changes in the gut microbiome after MR-Amazake ingestion. Furthermore, Bifidobacterium was strongly associated with these changes. These changes were observed only during MR-Amazake intake; the original gut microbiome was restored when MR-Amazake intake was discontinued. These results suggest that 6 wk is a reasonable period of time for MR-Amazake to change the human gut microbiome and that continuous consumption of MR-Amazake is required to sustain such changes.NEW & NOTEWORTHY The consumption of malted rice amazake (MR-Amazake) showed significant changes in the gut microbiome according to principal coordinate analysis in some home healthcare patients with disabilities, including those with severe motor and intellectual disabilities. After discontinuation of intake, the gut microbiome returned to its original state. This is the first pilot study to examine both the changes in the gut microbiome and their sustainability after MR-Amazake intake.

Flavivirus recruits the valosin-containing protein-NPL4 complex to induce stress granule disassembly for efficient viral genome replication.

Flaviviruses are human pathogens that can cause severe diseases, such as dengue fever and Japanese encephalitis, which can lead to death. Valosin-containing protein (VCP)/p97, a cellular ATPase associated with diverse cellular activities (AAA-ATPase), is reported to have multiple roles in flavivirus replication. Nevertheless, the importance of each role still has not been addressed. In this study, the functions of 17 VCP mutants that are reportedly unable to interact with the VCP cofactors were validated using the short-interfering RNA rescue experiments. Our findings of this study suggested that VCP exerts its functions in replication of the Japanese encephalitis virus by interacting with the VCP cofactor nuclear protein localization 4 (NPL4). We show that the depletion of NPL4 impaired the early stage of viral genome replication. In addition, we demonstrate that the direct interaction between NPL4 and viral nonstructural protein (NS4B) is critical for the translocation of NS4B to the sites of viral replication. Finally, we found that Japanese encephalitis virus and dengue virus promoted stress granule formation only in VCP inhibitor-treated cells and the expression of NS4B or VCP attenuated stress granule formation mediated by protein kinase R, which is generally known to be activated by type I interferon and viral genome RNA. These results suggest that the NS4B-mediated recruitment of VCP to the virus replication site inhibits cellular stress responses and consequently facilitates viral protein synthesis in the flavivirus-infected cells.

TargetMine 2022: a new vision into drug target analysis.

SUMMARY: We introduce the newest version of TargetMine, which includes the addition of new visualization options; integration of previously disaggregated functionality; and the migration of the front-end to the newly available Bluegenes service. AVAILABILITY AND IMPLEMENTATION: TargeteMine is accessible online at https://targetmine.mizuguchilab.org/bluegenes. Users do not need to register to use the software. Source code for the different components listed in the article is available from TargetMine's organizational account at http://github.com/targetmine. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Prediction of the Contribution Ratio of a Target Metabolic Enzyme to Clearance from Chemical Structure Information.

The contribution ratio of metabolic enzymes such as cytochrome P450 to in vivo clearance (fraction metabolized: fm) is a pharmacokinetic index that is particularly important for the quantitative evaluation of drug-drug interactions. Since obtaining experimental in vivo fm values is challenging, those derived from in vitro experiments have often been used alternatively. This study aimed to explore the possibility of constructing machine learning models for predicting in vivo fm using chemical structure information alone. We collected in vivo fm values and chemical structures of 319 compounds from a public database with careful manual curation and constructed predictive models using several machine learning methods. The results showed that in vivo fm values can be obtained from structural information alone with a performance comparable to that based on in vitro experimental values and that the prediction accuracy for the compounds involved in CYP induction or inhibition is significantly higher than that by using in vitro values. Our new approach to predicting in vivo fm values in the early stages of drug discovery should help improve the efficiency of the drug optimization process.

Altered gut microbiota and its association with inflammation in patients with chronic thromboembolic pulmonary hypertension: a single-center observational study in Japan.

BACKGROUND: The pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) is considered to be associated with chronic inflammation; however, the underlying mechanism remains unclear. Recently, altered gut microbiota were found in patients with pulmonary arterial hypertension (PAH) and in experimental PAH models. The aim of this study was to characterize the gut microbiota in patients with CTEPH and assess the relationship between gut dysbiosis and inflammation in CTEPH. METHODS: In this observational study, fecal samples were collected from 11 patients with CTEPH and 22 healthy participants. The abundance of gut microbiota in these fecal samples was assessed using 16S ribosomal ribonucleic acid (rRNA) gene sequencing. Inflammatory cytokine and endotoxin levels were also assessed in patients with CTEPH and control participants. RESULTS: The levels of serum tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and macrophage inflammatory protein (MIP)-1α were elevated in patients with CTEPH. Plasma endotoxin levels were significantly increased in patients with CTEPH (P < 0.001), and were positively correlated with TNF-α, IL-6, IL-8, and MIP-1α levels. The 16S rRNA gene sequencing and the principal coordinate analysis revealed the distinction in the gut microbiota between patients with CTEPH (P < 0.01) and control participants as well as the decreased bacterial alpha-diversity in patients with CTEPH. A random forest analysis for predicting the distinction in gut microbiota revealed an accuracy of 80.3%. CONCLUSION: The composition of the gut microbiota in patients with CTEPH was distinct from that of healthy participants, which may be associated with the elevated inflammatory cytokines and endotoxins in CTEPH.

Comprehensive analysis of gut microbiota of a healthy population and covariates affecting microbial variation in two large Japanese cohorts.

BACKGROUND: Inter-individual variations in gut microbiota composition are observed even among healthy populations. The gut microbiota may exhibit a unique composition depending on the country of origin and race of individuals. To comprehensively understand the link between healthy gut microbiota and host state, it is beneficial to conduct large-scale cohort studies. The aim of the present study was to elucidate the integrated and non-redundant factors associated with gut microbiota composition within the Japanese population by 16S rRNA sequencing of fecal samples and questionnaire-based covariate analysis. RESULTS: A total of 1596 healthy Japanese individuals participated in this study via two independent cohorts, NIBIOHN cohort (n = 954) and MORINAGA cohort (n = 642). Gut microbiota composition was described and the interaction of these microorganisms with metadata parameters such as anthropometric measurements, bowel habits, medical history, and lifestyle were obtained. Thirteen genera, including Alistipes, Anaerostipes, Bacteroides, Bifidobacterium, Blautia, Eubacterium halli group, Faecalibacterium, Fusicatenibacter, Lachnoclostridium, Parabacteroides, Prevotella_9, Roseburia, and Subdoligranulum were predominant among the two cohorts. On the basis of univariate analysis for overall microbiome variation, 18 matching variables exhibited significant association in both cohorts. A stepwise redundancy analysis revealed that there were four common covariates, Bristol Stool Scale (BSS) scores, gender, age, and defecation frequency, displaying non-redundant association with gut microbial variance. CONCLUSIONS: We conducted a comprehensive analysis of gut microbiota in healthy Japanese individuals, based on two independent cohorts, and obtained reliable evidence that questionnaire-based covariates such as frequency of bowel movement and specific dietary habit affects the microbial composition of the gut. To our knowledge, this was the first study to investigate integrated and non-redundant factors associated with gut microbiota among Japanese populations.

Network modeling and inference of peroxisome proliferator-activated receptor pathway in high fat diet-linked obesity.

Systems biology aims to understand how holistic systems theory can be used to explain the observable living system characteristics, and mathematical modeling tools have been successful in understanding the intricate relationships underlying cellular functions. Lately, researchers have been interested in understanding molecular mechanisms underlying obesity, which is a major health concern worldwide and has been linked to several diseases. Various mechanisms such as peroxisome proliferator-activated receptors (PPARs) are known to modulate obesity-induced inflammation and its consequences. In this study, we have modeled the PPAR pathway using a Bayesian model and inferred the sub-pathways that are potentially responsible for the activation of the output processes that are associated with high fat diet (HFD)-induced obesity. We examined a previously published dataset from a study that compared gene expression profiles of 40 mice maintained on HFD against 40 mice fed with chow diet (CD). Our simulations have highlighted that GPCR and FATCD36 sub-pathways were aberrantly active in HFD mice and are therefore favorable targets for anti-obesity strategies. We further cross-validated our observations with experimental results from the literature. We believe that mathematical models such as those presented in the present study can help in inferring other pathways and deducing significant biological relationships.

Gut microbial composition in patients with atrial fibrillation: effects of diet and drugs.

Atrial fibrillation (AF) reduces the quality of life by triggering stroke and heart failure. The association between AF onset and gut metabolites suggests a causal relationship between AF and gut microbiota dysbiosis; however, the relationship remains poorly understood. We prospectively enrolled 34 hospitalized patients with AF and 66 age-, sex-, and comorbidity-matched control subjects without a history of AF. Gut microbial compositions were evaluated by amplicon sequencing targeting the 16S ribosomal RNA gene. We assessed differences in dietary habits by using a brief-type self-administered diet history questionnaire (BDHQ). Gut microbial richness was lower in AF patients, although the diversity of gut microbiota did not differ between the two groups. At the genus level, Enterobacter was depleted, while Parabacteroides, Lachnoclostridium, Streptococcus, and Alistipes were enriched in AF patients compared to control subjects. The BDHQ revealed that the intake of n-3 polyunsaturated fatty acids and eicosadienoic acid was higher in AF patients. Our results suggested that AF patients had altered gut microbial composition in connection with dietary habits.

Distinct Age-Specific miRegulome Profiling of Isolated Small and Large Intestinal Epithelial Cells in Mice.

The intestinal epithelium serves as a dynamic barrier to protect the host tissue from exposure to a myriad of inflammatory stimuli in the luminal environment. Intestinal epithelial cells (IECs) encompass differentiated and specialized cell types that are equipped with regulatory genes, which allow for sensing of the luminal environment. Potential inflammatory cues can instruct IECs to undergo a diverse set of phenotypic alterations. Aging is a primary risk factor for a variety of diseases; it is now well-documented that aging itself reduces the barrier function and turnover of the intestinal epithelium, resulting in pathogen translocation and immune priming with increased systemic inflammation. In this study, we aimed to provide an effective epigenetic and regulatory outlook that examines age-associated alterations in the intestines through the profiling of microRNAs (miRNAs) on isolated mouse IECs. Our microarray analysis revealed that with aging, there is dysregulation of distinct clusters of miRNAs that was present to a greater degree in small IECs (22 miRNAs) compared to large IECs (three miRNAs). Further, miRNA-mRNA interaction network and pathway analyses indicated that aging differentially regulates key pathways between small IECs (e.g., toll-like receptor-related cascades) and large IECs (e.g., cell cycle, Notch signaling and small ubiquitin-related modifier pathway). Taken together, current findings suggest novel gene regulation pathways by epithelial miRNAs in aging within the gastrointestinal tissues.

Relationship between Nutrient Intake and Human Gut Microbiota in Monozygotic Twins.

Background and Objectives: The gut microbiota is associated with human health and dietary nutrition. Various studies have been reported in this regard, but it is difficult to clearly analyze human gut microbiota as individual differences are significant. The causes of these individual differences in intestinal microflora are genetic and/or environmental. In this study, we focused on differences between identical twins in Japan to clarify the effects of nutrients consumed on the entire gut microbiome, while excluding genetic differences. Materials and Methods: We selected healthy Japanese monozygotic twins for the study and confirmed their zygosity by matching 15 short tandem repeat loci. Their fecal samples were subjected to 16S rRNA sequencing and bioinformatics analyses to identify and compare the fluctuations in intestinal bacteria. Results: We identified 12 genera sensitive to environmental factors, and found that Lactobacillus was relatively unaffected by environmental factors. Moreover, we identified protein, fat, and some nutrient intake that can affect 12 genera, which have been identified to be more sensitive to environmental factors. Among the 12 genera, Bacteroides had a positive correlation with retinol equivalent intake (rs = 0.38), Lachnospira had a significantly negative correlation with protein, sodium, iron, vitamin D, vitamin B6, and vitamin B12 intake (rs = -0.38, -0.41, -0.39, -0.63, -0.42, -0.49, respectively), Lachnospiraceae ND3007 group had a positive correlation with fat intake (rs = 0.39), and Lachnospiraceae UCG-008 group had a negative correlation with the saturated fatty acid intake (rs = -0.45). Conclusions: Our study is the first to focus on the relationship between human gut microbiota and nutrient intake using samples from Japanese twins to exclude the effects of genetic factors. These findings will broaden our understanding of the more intuitive relationship between nutrient intake and the gut microbiota and can be a useful basis for finding useful biomarkers that contribute to human health.

B cell-intrinsic MyD88 signaling controls IFN-γ-mediated early IgG2c class switching in mice in response to a particulate adjuvant.

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR-dependent and -independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR-independent MOAs of adjuvants is poorly understood. Here, using the T-dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class-switch recombination (CSR) in mice. Using cell-type-specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell-intrinsic MyD88 signaling. Notably, IFN-γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B-cell intrinsic MyD88 is required for IFN-γ production. Moreover, IFN-γ receptor (IFNγR) deficiency abolished sHZ-induced IgG2c production, while recombinant IFN-γ administration successfully rescued IgG2c CSR impairment in mice lacking B-cell intrinsic MyD88. Together, our results show that B cell-intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.

Update of the GRIP web service.

G protein-coupled receptors (GPCRs) can form homodimers, heterodimers, or higher-order molecular complexes (oligomers). The reports on the change of functions through the oligomerization have been accumulated. Inhibition of GPCR oligomerization without affecting the protomer's overall structure would clarify the oligomer-specific functions although inhibition experiments are costly and require accurate information about the interface location. Unfortunately, the number of experimentally determined interfaces is limited. The precise prediction of the oligomerization interfaces is, therefore, useful for inhibition experiments to examine the oligomer-specific functions, which would accelerate investigations of the GPCR signaling. However, interface prediction for GPCR oligomerization is difficult because different GPCR subtypes belonging to the same subfamily often use different structural regions as their interfaces. We previously developed a high-performance method to predict the interfaces for GPCR oligomerization, by identifying the conserved surfaces with the sequence and structure information. Then, the structural characteristic of a GPCR structure is regarded to be a thick-tube like conformation that is approximately perpendicular to the membrane plane. Our method had successfully predicted all of the interfaces available on that day. We had launched a web server for our interface prediction of GPCRs (GRIP). We have improved the previous version of GRIP server and enhanced its usability. First, we discarded the approximation of the GPCR structure as the thick-tube-like conformation. This improvement increased the number of structures for the prediction. Second, the FUGUE-based template recommendation service was introduced to facilitate the choice of an appropriate structure for the prediction. The new prediction server is available at http://grip.b.dendai.ac.jp/∼grip/.

Essential Role of CARD14 in Murine Experimental Psoriasis.

Caspase recruitment domain family member 14 (CARD14) was recently identified as a psoriasis-susceptibility gene, but its immunological role in the pathogenesis of psoriasis in vivo remains unclear. In this study, we examined the role of CARD14 in murine experimental models of psoriasis induced by either imiquimod (IMQ) cream or recombinant IL-23 injection. In all models tested, the psoriasiform skin inflammation was abrogated in Card14-/- mice. Comparison of the early gene signature of the skin between IMQ-cream-treated Card14-/- mice and Tlr7-/-Tlr9-/- mice revealed not only their similarity, but also distinct gene sets targeted by IL-23. Cell type-specific analysis of these mice identified skin Langerinhigh Langerhans cells as a potent producer of IL-23, which was dependent on both TLR7 and TLR9 but independent of CARD14, suggesting that CARD14 is acting downstream of IL-23, not TLR7 or TLR9. Instead, a bone marrow chimera study suggested that CARD14 in radio-sensitive hematopoietic cells was required for IMQ-induced psoriasiform skin inflammation, controlling the number of Vγ4+ T cells producing IL-17 or IL-22 infiltrating through the dermis to the inflamed epidermis. These data indicate that CARD14 is essential and a potential therapeutic target for psoriasis.

An Antigen-Free, Plasmacytoid Dendritic Cell-Targeting Immunotherapy To Bolster Memory CD8+ T Cells in Nonhuman Primates.

The priming, boosting, and restoration of memory cytotoxic CD8+ T lymphocytes by vaccination or immunotherapy in vivo is an area of active research. Particularly, nucleic acid-based compounds have attracted attention due to their ability to elicit strong Ag-specific CTL responses as a vaccine adjuvant. Nucleic acid-based compounds have been shown to act as anticancer monotherapeutic agents even without coadministration of cancer Ag(s); however, so far they have lacked efficacy in clinical trials. We recently developed a second-generation TLR9 agonist, a humanized CpG DNA (K3) complexed with schizophyllan (SPG), K3-SPG, a nonagonistic Dectin-1 ligand. K3-SPG was previously shown to act as a potent monoimmunotherapeutic agent against established tumors in mice in vivo. In this study we extend the monoimmunotherapeutic potential of K3-SPG to a nonhuman primate model. K3-SPG activated monkey plasmacytoid dendritic cells to produce both IFN-α and IL-12/23 p40 in vitro and in vivo. A single injection s.c. or i.v. with K3-SPG significantly increased the frequencies of activated memory CD8+ T cells in circulation, including Ag-specific memory CTLs, in cynomolgus macaques. This increase did not occur in macaques injected with free CpG K3 or polyinosinic-polycytidylic acid. Injection of 2 mg K3-SPG induced mild systemic inflammation, however, levels of proinflammatory serum cytokines and circulating neutrophil influx were lower than those induced by the same dose of polyinosinic-polycytidylic acid. Therefore, even in the absence of specific Ags, we show that K3-SPG has potent Ag-specific memory CTL response-boosting capabilities, highlighting its potential as a monoimmunotherapeutic agent for chronic infectious diseases and cancer.

Integrating sequence and gene expression information predicts genome-wide DNA-binding proteins and suggests a cooperative mechanism.

DNA-binding proteins (DBPs) perform diverse biological functions ranging from transcription to pathogen sensing. Machine learning methods can not only identify DBPs de novo but also provide insights into their DNA-recognition dynamics. However, it remains unclear whether available methods that can accurately predict DNA-binding sites in known DBPs can also identify novel DBPs. Moreover, sequence information is blind to the cellular- and disease-specific contexts of DBP activities, whereas the under-utilized knowledge from public gene expression data offers great promise. To address these issues, we have developed novel methods for predicting DBPs by integrating sequence and gene expression-derived features and applied them to explore human, mouse and Arabidopsis proteomes. While our sequence-based models outperformed the gene expression-based ones, some proteins with weaker DBP-like sequence features were correctly predicted by gene expression-based features, suggesting that these proteins acquire a tangible DBP functionality in a conducive gene expression environment. Analysis of motif enrichment among the co-expressed genes of top 100 candidates DBPs from hitherto unannotated genes provides further avenues to explore their functional associations.

Impact of quality trimming on the efficiency of reads joining and diversity analysis of Illumina paired-end reads in the context of QIIME1 and QIIME2 microbiome analysis frameworks.

BACKGROUND: To increase the accuracy of microbiome data analysis, solving the technical limitations of the existing sequencing machines is required. Quality trimming is suggested to reduce the effect of the progressive decrease in sequencing quality with the increased length of the sequenced library. In this study, we examined the effect of the trimming thresholds (0-20 for QIIME1 and 0-30 for QIIME2) on the number of reads that remained after the quality control and chimera removal (the good reads). We also examined the distance of the analysis results to the gold standard using simulated samples. RESULTS: Quality trimming increased the number of good reads and abundance measurement accuracy in Illumina paired-end reads of the V3-V4 hypervariable region. CONCLUSIONS: Our results suggest that the pre-analysis trimming step should be included before the application of QIIME1 or QIIME2.

CLINE: a web-tool for the comparison of biological dendrogram structures.

BACKGROUND: When visually comparing the results of hierarchical clustering, the differences in the arrangements of components are of special interest. However, in a biological setting, identifying such differences becomes less straightforward, as the changes in the dendrogram structure caused by permuting biological replicates, do not necessarily imply a different biological interpretation. Here, we introduce a visualization tool to help identify biologically similar topologies across different clustering results, even in the presence of replicates. RESULTS: Here we introduce CLINE, an open-access web application that allows users to visualize and compare multiple dendrogram structures, by visually displaying the links between areas of similarity across multiple structures. Through the use of a single page and a simple user interface, the user is able to load and remove structures form the visualization, change some aspects of their display and set the parameters used to match cluster topology across consecutive pairs of dendrograms. CONCLUSIONS: We have implemented a web-tool that allows the users to visualize different dendrogram structures, showing not only the structures themselves, but also linking areas of similarity across multiple structures. The software is freely available at http://mizuguchilab.org/tools/cline/ . Also, the source code, documentation and installation instructions are available on GitHub at https://github.com/RodolfoAllendes/cline/ .

High-Contrast Facile Imaging with Target-Directing Fluorescent Molecular Rotors, the N3-Modified Thioflavin T Derivatives.

Immunostaining methods have generally been used not only for biological studies but also for clinical diagnoses for decades. However, recently, for these methods, improved rapidity and simplicity have been required for relevant techniques in laboratory research and medical applications. To this end, we present here a novel approach for designing fluorescent molecular rotor probes, i.e., N3-modified thioflavin T (ThT) derivatives, which enabled specific detection of interesting protein targets with sensitive fluorescence turn-on. As an example, we synthesized N3-( d-desthiobiotinyl-PEGylated) thioflavin T (ThT-PD) and N3-(cortisolyl-PEGylated) thioflavin T (ThT-PC) that carried d-desthiobioin and cortisol, respectively, via PEG linkers. Compared to those of the probes without the targets, ThT-PD and ThT-PC exhibited around 27- and 8-fold fluorescence intensities, respectively, with the target streptavidin and anti-cortisol antibody in excess of saturation, enabling quantitative detection of the targets. Furthermore, we successfully demonstrated the feasibility of ligand-tethering N3-ThT derivatives by the rapid specific staining of glucocorticoid receptors in cells, which was completed within only several minutes using ThT-PC after cell fixation, whereas it took ∼24 h for immunostaining to capture the corresponding fluorescence images.

Development of Simplified in Vitro P-Glycoprotein Substrate Assay and in Silico Prediction Models To Evaluate Transport Potential of P-Glycoprotein.

For efficient drug discovery and screening, it is necessary to simplify P-glycoprotein (P-gp) substrate assays and to provide in silico models that predict the transport potential of P-gp. In this study, we developed a simplified in vitro screening method to evaluate P-gp substrates by unidirectional membrane transport in P-gp-overexpressing cells. The unidirectional flux ratio positively correlated with parameters of the conventional bidirectional P-gp substrate assay ( R2 = 0.941) and in vivo Kp,brain ratio (mdr1a/1b KO/WT) in mice ( R2 = 0.800). Our in vitro P-gp substrate assay had high reproducibility and required approximately half the labor of the conventional method. We also constructed regression models to predict the value of P-gp-mediated flux and three-class classification models to predict P-gp substrate potential (low-, medium-, and high-potential) using 2397 data entries with the largest data set collected under the same experimental conditions. Most compounds in the test set fell within two- and three-fold errors in the random forest regression model (71.3 and 88.5%, respectively). Furthermore, the random forest three-class classification model showed a high balanced accuracy of 0.821 and precision of 0.761 for the low-potential classes in the test set. We concluded that the simplified in vitro P-gp substrate assay was suitable for compound screening in the early stages of drug discovery and that the in silico regression model and three-class classification model using only chemical structure information could identify the transport potential of compounds including P-gp-mediated flux ratios. Our proposed method is expected to be a practical tool to optimize effective central nervous system (CNS) drugs, to avoid CNS side effects, and to improve intestinal absorption.