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The IL-6 amplifier, which describes the simultaneous activation of STAT3 and NF-kB, in synovial fibroblasts causes the infiltration of immune cells into the joints of F759 mice. The result is a disease that resembles human rheumatoid arthritis. However, the kinetics and regulatory mechanisms of how augmented transcriptional activation by STAT3 and NF-kB leads to F759 arthritis is unknown. We here show that the STAT3-NF-κB complex is present in the cytoplasm and nucleus and accumulates around NFkB binding sites of the IL-6 promoter region and established a computer model that shows IL-6 and IL-17 signaling promotes the formation of the STAT3-NF-κB complex followed by its binding on promoter regions of NF-kB target genes to accelerate inflammatory responses, including the production of IL-6, epiregulin, and CCL2, phenotypes consistent with in vitro experiments. The binding also promoted cell growth in the synovium and the recruitment of Th17 cells and macrophages in the joints. Anti-IL-6 blocking antibody treatment inhibited inflammatory responses even at the late phase, but anti-IL-17 and anti-TNFα antibodies did not. However, anti-IL-17 antibody at the early phase showed inhibitory effects, suggesting that the IL-6 amplifier is dependent on IL-6 and IL-17 stimulation at the early phase, but only on IL-6 at the late phase. These findings demonstrate the molecular mechanism of F759 arthritis can be recapitulated in silico and identify a possible therapeutic strategy for IL-6 amplifier-dependent chronic inflammatory diseases.

We recently discovered a (to our knowledge) new neuroimmune interaction named the gateway reflex, in which the activation of specific neural circuits establishes immune cell gateways at specific vessel sites in organs, leading to the development of tissue-specific autoimmune diseases, including a multiple sclerosis (MS) mouse model, experimental autoimmune encephalomyelitis (EAE). We have reported that peripheral-derived myeloid cells, which are CD11b+MHC class II+ and accumulate in the fifth lumbar (L5) cord during the onset of a transfer model of EAE (tEAE), play a role in the pain-mediated relapse via the pain-gateway reflex. In this study, we investigated how these cells survive during the remission phase to cause the relapse. We show that peripheral-derived myeloid cells accumulated in the L5 cord after tEAE induction and survive more than other immune cells. These myeloid cells, which highly expressed GM-CSFRα with common β chain molecules, grew in number and expressed more Bcl-xL after GM-CSF treatment but decreased in number by blockade of the GM-CSF pathway, which suppressed pain-mediated relapse of neuroinflammation. Therefore, GM-CSF is a survival factor for these cells. Moreover, these cells were colocalized with blood endothelial cells (BECs) around the L5 cord, and BECs expressed a high level of GM-CSF. Thus, GM-CSF from BECs may have an important role in the pain-mediated tEAE relapse caused by peripheral-derived myeloid cells in the CNS. Finally, we found that blockade of the GM-CSF pathway after pain induction suppressed EAE development. Therefore, GM-CSF suppression is a possible therapeutic approach in inflammatory CNS diseases with relapse, such as MS.

The homeostatic regulation of sleep is characterized by rebound sleep after prolonged wakefulness, but the molecular and cellular mechanisms underlying this regulation are still unknown. We show here that CaMKII-dependent activity control of parvalbumin (PV)-expressing cortical neurons is involved in sleep homeostasis regulation. Prolonged wakefulness enhances cortical PV-neuron activity. Chemogenetic suppression or activation of cortical PV neurons inhibits or induces rebound sleep, implying that rebound sleep is dependent on increased activity of cortical PV neurons. Furthermore, we discovered that CaMKII kinase activity boosts the activity of cortical PV neurons, and that kinase activity is important for homeostatic sleep rebound. We propose that CaMKII-dependent PV-neuron activity represents negative feedback inhibition of cortical neural excitability, which serves as the distributive cortical circuits for sleep homeostatic regulation.

Using a zoobiquity concept, we directly connect animal phenotypes to a human disease mechanism: the reduction of local plasminogen levels caused by matrix metalloproteinase-9 (MMP9) activity is associated with the development of inflammation in the intestines of dogs and patients with inflammatory bowel disease. We first investigated inflammatory colorectal polyps (ICRPs), which are a canine gastrointestinal disease characterized by the presence of idiopathic chronic inflammation, in Miniature Dachshund (MD) and found 31 missense disease-associated SNPs by whole-exome sequencing. We sequenced them in 10 other dog breeds and found five, PLG, TCOF1, TG, COL9A2 and COL4A4, only in MD. We then investigated two rare and breed-specific missense SNPs (T/T SNPs), PLG: c.477G > T and c.478A>T, and found that ICRPs with the T/T SNP risk alleles showed less intact plasminogen and plasmin activity in the lesions compared to ICRPs without the risk alleles but no differences in serum. Moreover, we show that MMP9, which is an NF-κB target, caused the plasminogen reduction and that intestinal epithelial cells expressing plasminogen molecules were co-localized with epithelial cells expressing MMP9 in normal colons with the risk alleles. Importantly, MMP9 expression in patients with ulcerous colitis or Crohn’s disease also co-localized with epithelial cells showing enhanced NF-κB activation and less plasminogen expression. Overall, our zoobiquity experiments showed that MMP9 induces the plasminogen reduction in the intestine, contributing to the development of local inflammation and suggesting the local MMP9-plasminogen axis is a therapeutic target in both dogs and patients. Therefore, zoobiquity-type experiments could bring new perspectives for biomarkers and therapeutic targets.

Juzen-taiho-to, a traditional Chinese herbal medicine, is used for patients with anorexia and fatigue in human medicine. In our previous study, granulated Juzen-taiho-to improved vincristine-induced gastrointestinal adverse effects through increasing gastric motility in dogs. As the effect of Hozen-S, the sweet liquid form of Juzen-taiho-to, on dog gastric motility has not been investigated, we examined the effect of administration of Hozen-S on gastric motility. Furthermore, we assessed dog plasma ghrelin level to further elucidate the mechanism of the effect of Hozen-S on gastric contraction. Finally, we assessed the palatability of Hozen-S compared to granulated Juzen-taiho-to and its effect on body weight in dogs. Administration of Hozen-S significantly increased gastric motility, plasma ghrelin concentration, and body weight. A palatability evaluation revealed that the dogs preferred Hozen-S to granulated Juzen-taiho-to. In conclusion, Hozen-S administration to dogs promoted gastric motility by raising plasma ghrelin levels. Considering these functional and palatability data, Hozen-S may replace granulated type Juzen-taiho-to and become a prominent traditional Chinese veterinary medicament.

Three-dimensional (3D) organoid culture holds great promises in cancer precision medicine. However, Matrigel and stem cell-stimulating supplements are necessary for culturing 3D organoid cells. It costs a lot of money and consumes more time and effort compared with 2D cultured cells. Therefore, the establishment of cheaper and Matrigel-free organoid culture that can maintain the characteristics of a part of 3D organoids is demanded. In the previous study, we established a dog bladder cancer (BC) 3D organoid culture system by using their urine samples. Here, we successfully isolated cells named “2.5D organoid” from multiple strains of dog BC 3D organoids using 2.5 organoid media. The cell proliferation speed of 2.5D organoids was faster than parental 3D organoid cells. The expression pattern of stem cell markers was close to 3D organoids. Injection of 2.5D organoid cells into immunodeficient mice formed tumors and showed the histopathological characteristics of urothelial carcinoma similar to the injection of dog BC 3D organoids. The 2.5D organoids had a similar sensitivity profile for anti-cancer drug treatment to their parental 3D organoids. These data suggest that our established 2.5D organoid culture method might become a reasonable and useful tool instead of 3D organoids in dog BC research and therapy.

Objective The oxygen concentration within cancer tissue is known to be low, but is expected to increase rapidly when oxygen is supplied by angiogenesis and hematogenous metastasis, suggesting that rapid increases in oxygen levels might influence cancer cell physiology. Therefore, we investigated the effects of oxygen concentration fluctuations on the glucose metabolism of cancer cells. Methods The glucose metabolism of oral squamous cell carcinoma (HSC-2 and HSC-3) and normal epithelial (HaCaT) cells cultured under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions was measured using a pH-stat system under normoxic or hypoxic conditions. The acidic end-products and reactive oxygen species (ROS) generated by glucose metabolism were also measured. Results Under normoxic conditions, the metabolic activity of hypoxically cultured cancer cells was significantly increased, and the production of acids other than lactate was upregulated, while the normal cells did not respond to rapid increases in oxygen levels. ROS production was higher in normoxic conditions in all cells, especially the hypoxically cultured HSC-3 cells. Conclusions Rapid increases in oxygen levels might enhance the glucose metabolism of hypoxically cultured cancer cells by mainly activating the TCA cycle and electron transport system, which might activate cancer cells through the ATP and ROS generation.

In human and dogs, bladder cancer (BC) is the most common neoplasm affecting the urinary tract. Dog BC resembles human muscle‐invasive BC in histopathological characteristics and gene expression profiles, and could be an important research model for this disease. Cancer patient‐derived organoid culture can recapitulate organ structures and maintains the gene expression profiles of original tumor tissues. In a previous study, we generated dog prostate cancer organoids using urine samples, however dog BC organoids had never been produced. Therefore we aimed to generate dog BC organoids using urine samples and check their histopathological characteristics, drug sensitivity, and gene expression profiles. Organoids from individual BC dogs were successfully generated, expressed urothelial cell markers (CK7, CK20, and UPK3A) and exhibited tumorigenesis in vivo. In a cell viability assay, the response to combined treatment with a range of anticancer drugs (cisplatin, vinblastine, gemcitabine or piroxicam) was markedly different in each BC organoid. In RNA‐sequencing analysis, expression levels of basal cell markers (CK5 and DSG3) and several novel genes (MMP28,CTSE,CNN3,TFPI2,COL17A1, and AGPAT4) were upregulated in BC organoids compared with normal bladder tissues or two‐dimensional (2D) BC cell lines. These established dog BC organoids might be a useful tool, not only to determine suitable chemotherapy for BC diseased dogs but also to identify novel biomarkers in human muscle‐invasive BC. In the present study, for the 1st time, dog BC organoids were generated and several specifically upregulated organoid genes were identified. Our data suggest that dog BC organoids might become a new tool to provide fresh insights into both dog BC therapy and diagnostic biomarkers.

Vincristine, one of the anti-cancer drugs used in veterinary practice, has adverse hematological and gastrointestinal effects in dogs. Juzen-taiho-to is a traditional Chinese medicine used for patients with anorexia in human medicine. However, the protective effects of Juzen-taiho-to against anti-cancer drug-induced toxicity in dogs have not been investigated. We therefore examined whether the administration of Juzen-taiho-to to dogs affects gastric motility, and vincristine-induced gastrointestinal and hematological toxicity. The study was composed of three trials. In the first trial, Juzen-taiho-to (450 mg/kg/day) was orally administered to five dogs. In the second and third trials, vincristine (0.75 mg/m2) was intravenously administered to each dog in the absence or presence of Juzen-taiho-to (450 mg/kg/day). During these trials, gastric motility and blood parameters were assessed. Juzen-taiho-to increased gastric motility and improved vincristine-induced gastrointestinal, but not hematological, adverse effects in dogs. This study suggested that Juzen-taiho-to may be applicable for gastrointestinal care in dogs receiving chemotherapy.

Bladder cancer (BC), a main neoplasm of urinary tract, is usually inoperable and unresponsive to chemotherapy, indicating a necessity for more effective therapies. As a novel experimental model for muscle-invasive BC, we previously established a culture method of dog BC organoids. In the present study, the detailed in vitro and in vivo anti‐tumor effects of trametinib were investigated by using this model. In each BC organoid strain, epidermal growth factor receptor (EGFR)/ERK signaling was upregulated compared with normal bladder cells. Trametinib even at a low concentration inhibited the cell viability of BC organoids and the activation of ERK through decreasing expression of c-Myc, ELK1, SIK1, and PLA2G4A. Trametinib arrested cell cycle of BC with few apoptoses. Additionally, trametinib decreased expression of CD44, while YAP1 was unexpectedly upregulated. Dual treatment of BC organoids with trametinib and YAP inhibitor, verteporfin extremely inhibited the cell viability with apoptosis induction. Moreover, trametinib induced basal to luminal transformation of BC organoids by upregulating luminal markers (ERBB2 and GATA3) and downregulating basal ones (CK5 and DSG3). In vivo, trametinib decreased the tumor growth of BC organoids in mice and the xenograft-derived organoids from trametinib-administered mice showed enhanced sensitivity to carboplatin due to MSH2 upregulation. Our data suggested a new strategy of trametinib-YAP inhibitor or trametinib-carboplatin combination as a promising treatment of BC. Dog BC organoid model may hopefully become a promising research tool for human muscle-invasive BC in near future.

Plasmodium falciparum, the most virulent human malaria parasite, causes serious diseases among the infected patients in the world and is particularly important in African regions. Although artemisinin combination therapy is recommended by the WHO for treatment of P. falciparum-malaria, the emergence of artemisinin-resistant parasites has become a serious issue which underscores the importance of sustained efforts to obtain novel chemotherapeutic agents against malaria. As a part of such efforts, thirty-nine herbal extracts from traditional Chinese medicine (TCM) were assayed for their anti-malarial activity using 3D7 strain of P. falciparum. Three herbal supplements appeared to possess higher specific anti-malarial activity than the others. One of them (D3) was separated by two sequential fractionations with reverse-phase (the first step) and normal-phase (the second step) liquid chromatography, in which some fractions resulted in higher specific activities than those of D3 or the previous fractions. Cell toxicity assay was performed with the fractions of the first fractionation and demonstrated no obvious cell toxicity. These results suggest that structure determination of the major compound for the anti-malarial activity in D3 may help the development of more potent chemicals in the future.

Oral bacteria may contribute to postoperative infectious complications including postoperative pneumonia or surgical site infection. The aim of this study was to investigate the impact of preoperative dental care on postoperative outcomes among surgical patients under general anesthesia.Retrospective cohort study.We analyzed clinical records of major surgical patients at a university hospital between 2016 and 2018. Subjects were categorized into either the preoperative dental care group, those being referred to dentists by their surgeons based on an individual surgeon's judgment for dental care before surgery, or the control group.The primary outcome was postoperative infectious complications. Secondary outcomes were postoperative inflammation markers (C-reactive protein and fever), and economic outcomes (postoperative length of hospital stay and medical expenses). As the main analysis, the average treatment effects of the preoperative dental care were obtained from the augmented inverse-probability weighting (AIPW) method with consideration of demographics and perioperative risk factors to estimate causal effect of the intervention from the observational data. Then, stratified analyses by age and surgical sites were conducted with the inverse-probability weighting and linear regression methods, respectively.In the AIPW estimation, compared with the control group, the care group saw a significantly lower rate of postoperative infection (average treatment effect -3.02) and shorter fever duration (-2.79 days). The stratified analysis by age revealed significant positive impact of dental care in all age groups, including the highest treatment effects observed among patients younger than 60. Also, treatment effect was observed in wider surgical sites than previously known.This study indicates a significant impact of preoperative dental care on preventing postoperative infection and inflammation. Along with old age or certain types of surgeries in which advantages of dental referral have been already known, preoperative dental referral could be beneficial for broader types of patients.

Despite its adverse effects, chemotherapy is generally used for the treatment of colorectal cancer (CRC). Development of supplement preparations targeting cancer stem cells (CSCs) that cause distant metastasis and drug resistance is required. Although curcumin is known to have anti-tumor, hepatoprotective, and hypoglycemic-like actions, its low water solubility, oral absorption, and bioavailability impede its therapeutic uses. Patient-derived organoid cultures can recapitulate heterogeneity, epithelial structures, and molecular imprints of their parental tissues. In the present study, anti-carcinogenic properties of amorphous curcumin (AC), a compound with improved solubility and bioavailability, were evaluated against human CRC organoids. Treatment with AC inhibited the cell viability of CRC organoids in a concentration-dependent manner. AC arrested the cell cycle of CRC organoids and induced apoptosis. AC inhibited phosphorylation of ERK. Expression of downstream signals of ERK, namely c-MYC and cyclin-D1, were inhibited. Expressions of CSC markers, CD44, LGR5, and CD133, were declined in the AC-treated CRC organoids. The combinational treatment of CRC organoids with AC and anti-cancer drugs, oxaliplatin, 5-FU, or irinotecan showed a synergistic activity. In vivo, AC decreased the tumor growth of CRC organoids in mice with the induction of necrotic lesions. In conclusion, AC diminished the cell viability of CRC organoids through the inhibition of proliferation-related signals and CSC marker expression in addition to arresting the cell cycle. Collectively, these data suggest the value of AC as a promising supplement that could be used in combination with anti-cancer drugs to prevent the recurrence and metastasis of CRC.

Simple Summary In cases of cancer metastasis, some tumor cells circulate in blood (CTCs) and function as a seed for metastasis. These cells are important for detailed study of the molecular basis of metastasis. However, their few numbers and difficult isolation hinder its analysis. Apheresis, a process used to isolate a specific component of blood, can efficiently separate peripheral blood monocyte (PBMC), whose density is closely like that of CTCs. In the present study we used the dogs as a precious model of human’s cancers to check the safety and feasibility of apheresis and to separate PBMC and infused MCF7 cells using Spectra Optia apheresis machine (Terumo). The process was performed safely to capture PBMC and MCF7. The captured MCF7 cells were regrown up in vitro and characterized. In conclusion, Spectra Optia apheresis machine (Terumo) can be used safely to isolate CTCs from dogs blood with precautions to keep hemodynamic stability. Abstract In patients with solid tumors, circulating tumor cells (CTCs) spread in their blood and function as a seed for metastases. However, the study of CTCs has been limited by their rarity, low frequency, and heterogeneity. The efficient collection of CTCs will contribute to further research of metastatic cancers. Apheresis is a process in which the whole blood of an individual is passed through a machine that isolates a particular constituent and returns the remainder to the circulation. In the present study, we investigated the safety and feasibility of apheresis to separate peripheral blood monocytes (PBMCs), whose density is closely similar to that of CTCs, and to capture intravenously administered human breast cancer cells, MCF7s, from the dogs. No life-threatening events were observed in dogs during the apheresis process. The changes in the hemogram were transient and recovered gradually within a few days after apheresis. During apheresis, 50 mL of PBMCs could be collected from each dog. Notably, a thrombus was formed along the circuit wall during apheresis, which decreased the blood collection pressure. MCF7 cells were successfully captured by the apheresis machine. The captured cells were regrown in vitro and characterized compared with the original cells. In conclusion, apheresis could be safely performed in dogs to isolate CTCs with precautions to maintain hemodynamic stability.

This study aimed to establish a real-time monitoring system for evaluating the acid-producing activity of cells and the effects of microenvironmental pH on their metabolism. Oral squamous cell carcinoma (HSC-2, HSC-3) and normal (HaCaT) cells were used. Their acid-producing activity from glucose, glutamine, and glutamate was monitored at various pH values using a pH stat system. Their production of lactic acid and ammonia was also measured. The acid-producing activity was monitored successfully. Both the cancer and normal cells produced acids from glucose, glutamine, and glutamate. All of the cells decreased their acid-producing activity as the environmental pH fell, but in glucose-derived acid-producing activity the cancer cells were more acid-tolerant than HaCaT cells. In the cancer cells, the proportion of lactic acid among all acids produced from glucose at the acidic environment tended to be higher than that in HaCaT cells. All of the cells produced ammonia from glutamine, while only HaCaT cells produced ammonia from glutamate. We established a real-time monitoring system for evaluating the acid-producing activity of cells. Our results suggest that the cancer cells possess acid-tolerant glucose metabolism with a tendency of metabolic shift to lactic acid production at acidic pH and they metabolise glutamate without ammonia production.

Non-alcoholic steatohepatitis (NASH) is associated with liver fibrosis and cirrhosis, which eventually leads to hepatocellular carcinoma. Although several animal models were developed to understand the mechanisms of NASH pathogenesis and progression, it remains obscure. A 3D organoid culture system can recapitulate organ structures and maintain gene expression profiles of original tissues. We therefore tried to generate liver organoids from different degrees [defined as mild (NASH A), moderate (NASH B) and severe (NASH C)] of methionine- and choline-deficient diet-induced NASH model mice and analyzed the difference of their architecture, cell components, organoid-forming efficacy, and gene expression profiles. Organoids from each stage of NASH model mice were successfully generated. Interestingly, epithelial-mesenchymal transition was observed in NASH C organoids. Expression of Collagen I and an activated hepatic stellite cell marker, α-sma was upregulated in the liver organoids from NASH B and C mice. The analysis of RNA sequencing revealed that several novel genes were upregulated in all NASH liver organoids. These results suggest that our generated liver organoids from different stages of NASH diseased mice might become a useful tool for in vitro studies of the molecular mechanism of NASH development and also for identifying novel biomarkers for early diagnosis of NASH disease.

Raman spectroscopy was used for rapid in-situ measurement of alcohols in ethanol-methanol-water ternary systems. Mass fractions of the individual components were determined using calibration curves for binary systems of ethanol-water, methanol-water, and ethanol-methanol. Calibration curves were constructed by calculating the ratio of the Raman peak intensity of a component and that of an external standard (acetonitrile). Assuming additivity of the spectra, simultaneous equations were written, and mass fractions of ethanol, methanol, and water in the ternary solutions were determined by solving the system of equations through calculating an inverse matrix. The relative errors between the mass fractions obtained from the Raman spectra and those obtained from mass measurements were

Summary Temperature compensation is a striking feature of the circadian clock. Here we investigate biochemical mechanisms underlying temperature-compensated, CKIδ-dependent multi-site phosphorylation in mammals. We identify two mechanisms for temperature-insensitive phosphorylation at higher temperature: lower substrate affinity to CKIδ-ATP complex and higher product affinity to CKIδ-ADP complex. Inhibitor screening of ADP-dependent phosphatase activity of CKIδ identified aurintricarboxylic acid (ATA) as a temperature-sensitive kinase activator. Docking simulation of ATA and mutagenesis experiment revealed K224D/K224E mutations in CKIδ that impaired product binding and temperature-compensated primed phosphorylation. Importantly, K224D mutation shortens behavioral circadian rhythms and changes the temperature dependency of SCN's circadian period. Interestingly, temperature-compensated phosphorylation was evolutionary conserved in yeast. Molecular dynamics simulation and X-ray crystallography demonstrate that an evolutionally conserved CKI-specific domain around K224 can provide a structural basis for temperature-sensitive substrate and product binding. Surprisingly, this domain can confer temperature compensation on a temperature-sensitive TTBK1. These findings suggest the temperature-sensitive substrate- and product-binding mechanisms underlie temperature compensation.

Raman spectroscopy was employed for the precise quantitative analysis of a gaseous mixture. The band ratio between the Raman band of the target gas and that of the external standard was calculated and found to be proportional to the pressure of the target gas. The linearity of the calibration curves was very good (higher than 0.999). The mole fractions of atmospheric nitrogen and atmospheric oxygen were determined from their partial pressures. The average molar fraction of atmospheric nitrogen was calculated to be 0.790, similar to the literature value. Those of methane, hydrogen, and carbon dioxide, which are produced by fermentation, were also determined from the Raman spectrum. The values were identical to those obtained from the measured volume of each gas. The possibility of quantifying gas molecules by Raman spectroscopy is demonstrated.

Absolute values of protein expression levels in cells are crucial information for understanding cellular biological systems. Precise quantification of proteins can be achieved by liquid chromatography (LC)-mass spectrometry (MS) analysis of enzymatic digests of proteins in the presence of isotope-labeled internal standards. Thus, development of a simple and easy way for the preparation of internal standards is advantageous for the analyses of multiple target proteins, which will allow systems-level studies. Here we describe a method, termed MS-based Quantification By isotope-labeled Cell-free products (MS-QBiC), which provides the simple and high-throughput preparation of internal standards by using a reconstituted cell-free protein synthesis system, and thereby facilitates both multiplexed and sensitive quantification of absolute amounts of target proteins. This method was applied to a systems-level dynamic analysis of mammalian circadian clock proteins, which consist of transcription factors and protein kinases that govern central and peripheral circadian clocks in mammals. Sixteen proteins from 20 selected circadian clock proteins were successfully quantified from mouse liver over a 24-h time series, and 14 proteins had circadian variations. Quantified values were applied to detect internal body time using a previously developed molecular timetable method. The analyses showed that single time-point data from wild-type mice can predict the endogenous state of the circadian clock, whereas data from clock mutant mice are not applicable because of the disappearance of circadian variation.

We have developed novel push–pull-type 8-arylbutadienyl 2′-deoxyguanosine derivatives, AB G ( 1a ) and CB G ( 1b ). These nucleosides exhibit strong solvent polarity dependent fluorescence emission at long wavelength (ca. 490–550 nm). These environmentally sensitive fluorescent deoxyguanosines are powerful tools for structural studies of nucleic acids and also in molecular diagnostics.

We have synthesized various substituted 8-arylethynylated 2′-deoxyguanosine derivatives. Among them, acetyl substituted deoxyguanosine analogue 4c showed a remarkable solvent dependent fluorescence property, that is, an intense fluorescence in non-polar solvents but extremely weak fluorescence in polar solvents like methanol. By using solvatofluorochromic deoxyguanosine analogue 4c, we have developed highly thymine (T) selective fluorescent DNA probes that can sense T opposite 4c in a target DNA regardless of the flanking sequences. We were able to demonstrate that 4c can be used as a T specific base-discriminating fluorescent (BDF) nucleoside in homogeneous fluorescence assay.