ABSTRACT
Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.
Subject(s)
Disease Resistance , Plant Diseases , Plant Leaves , Solanum lycopersicum , Solanum lycopersicum/microbiology , Solanum lycopersicum/genetics , Plant Diseases/microbiology , Plant Diseases/genetics , Plant Diseases/immunology , Disease Resistance/genetics , Plant Leaves/microbiology , Plant Leaves/genetics , Genetic Loci , Alleles , Basidiomycota/physiology , Mutation/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Plant Proteins/genetics , Plant Proteins/metabolismSubject(s)
Computer Simulation , Drug Development , Drug Development/methods , Drug Development/trends , HumansABSTRACT
Drug-induced cholestasis results in drug discontinuation and market withdrawal, and the prediction of cholestasis risk is critical in the early stages of drug development. Animal tests and membrane vesicle assay are currently being conducted to assess the risk of cholestasis in the preclinical stage. However, these methods have drawbacks, such as species differences with humans and difficulties in evaluating the effects of drug metabolism and other transporters, implying the need for a cholestasis risk assessment system using human hepatocytes. However, human hepatocytes hardly form functional, extended bile canaliculi, a requirement for cholestasis risk assessment. We previously established a culture protocol for functional, extended bile canaliculi formation in human iPSC-derived hepatocytes. In this study, we modified this culture protocol to support the formation of functional, extended bile canaliculi in human cryopreserved hepatocytes (cryoheps). The production of bile acids, which induces bile canaliculi extension, increased time-dependently during bile canaliculi formation using this protocol, suggesting that increased bile acid production may be involved in the extended bile canaliculi formation. We have also shown that our culture protocol can be applied to cryoheps from multiple donors and that bile canaliculi can be formed stably among different culture batches. Furthermore, this protocol enables long-term maintenance of bile canaliculi and scaling down to culture in 96-well plates. We expect our culture protocol to be a breakthrough for in vitro cholestasis risk assessment.
Subject(s)
Bile Acids and Salts , Bile Canaliculi , Cholestasis , Cryopreservation , Culture Media , Hepatocytes , Humans , Cryopreservation/methods , Hepatocytes/metabolism , Hepatocytes/drug effects , Bile Acids and Salts/metabolism , Bile Canaliculi/metabolism , Cholestasis/metabolism , Cholestasis/chemically induced , Cell Culture Techniques/methods , Cells, Cultured , Induced Pluripotent Stem Cells/metabolismABSTRACT
Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.
Subject(s)
Coloring Agents , Cytochrome P-450 CYP3A , Humans , Fluorescence , Mercaptoethanol , RhodaminesABSTRACT
Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.
Subject(s)
Aging , Osteoporosis , Phosphoric Diester Hydrolases , Pyrophosphatases , Animals , Humans , Mice , Aging/genetics , Cartilage/metabolism , Luciferases , Mice, Knockout , Phosphoric Diester Hydrolases/metabolism , Pyrophosphatases/genetics , Pyrophosphatases/metabolismABSTRACT
Online hemodiafiltration (OL-HDF) is a treatment modality using diffusion and ultrafiltration. There are two types of dilution methods in OL-HDF: pre-dilution, which is commonly provided in Japan, and post-dilution, which is commonly provided in Europe. The optimal OL-HDF method for individual patients is not well studied. In this study, we compared the clinical symptoms, laboratory data, spent dialysate, and adverse events of pre- and post-dilution OL-HDF. We conducted a prospective study of 20 patients who underwent OL-HDF between January 1, 2019 and October 30, 2019. Their clinical symptoms and dialysis efficacy were evaluated. All patients underwent OL-HDF every 3 months in the following sequence: first pre-dilution, post-dilution, and second pre-dilution. We evaluated 18 patients for the clinical study and 6 for the spent dialysate study. No significant differences in spent dialysates regarding small and large solutes, blood pressure, recovery time, and clinical symptoms were observed between the pre- and post-dilution methods. However, the serum α1-microglobulin level in post-dilution OL-HDF was lower than that in pre-dilution OL-HDF (first pre-dilution: 124.8 ± 14.3 mg/L; post-dilution: 116.6 ± 13.9 mg/L; second pre-dilution: 125.8 ± 13.0 mg/L; first pre-dilution vs. post-dilution, post-dilution vs. second pre-dilution, and first pre-dilution vs. second pre-dilution: p = 0.001, p < 0.001, and p = 1.000, respectively). The most common adverse event was an increase in transmembrane pressure in the post-dilution period. Compared to pre-dilution, the post-dilution method decreased the α1-microglobulin level; however, there were no significant differences in clinical symptoms or laboratory data.
Subject(s)
Hemodiafiltration , Humans , Hemodiafiltration/methods , Prospective Studies , Renal Dialysis/methods , Blood Pressure , Dialysis SolutionsABSTRACT
Osteosarcoma is rare but is the most common bone tumor. Diagnostic tools such as magnetic resonance imaging development of chemotherapeutic agents have increased the survival rate in osteosarcoma patients, although 5-year survival has plateaued at 70%. Thus, development of new treatment approaches is needed. Here, we report that IL-17, a proinflammatory cytokine, increases osteosarcoma mortality in a mouse model with AX osteosarcoma cells. AX cell transplantation into wild-type mice resulted in 100% mortality due to ectopic ossification and multi-organ metastasis. However, AX cell transplantation into IL-17-deficient mice significantly prolonged survival relative to controls. CD4-positive cells adjacent to osteosarcoma cells express IL-17, while osteosarcoma cells express the IL-17 receptor IL-17RA. Although AX cells can undergo osteoblast differentiation, as can patient osteosarcoma cells, IL-17 significantly inhibited that differentiation, indicating that IL-17 maintains AX cells in the undifferentiated state seen in malignant tumors. By contrast, IL-17RA-deficient mice transplanted with AX cells showed survival comparable to wild-type mice transplanted with AX cells. Biopsy specimens collected from osteosarcoma patients showed higher expression of IL-17RA compared to IL-17. These findings suggest that IL-17 is essential to maintain osteosarcoma cells in an undifferentiated state and could be a therapeutic target for suppressing tumorigenesis.
Subject(s)
Bone Neoplasms , Osteosarcoma , Humans , Mice , Animals , Receptors, Interleukin-17/metabolism , Interleukin-17/genetics , Interleukin-17/metabolism , Osteosarcoma/pathology , Cell Differentiation , Bone Neoplasms/pathologyABSTRACT
When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.
Subject(s)
Anterior Cruciate Ligament Injuries , Anterior Cruciate Ligament Reconstruction , Humans , Adult , Rats , Animals , Mice , Anterior Cruciate Ligament/surgery , Tendons/surgery , Anterior Cruciate Ligament Injuries/surgery , Knee Joint/surgeryABSTRACT
Lumbar spinal stenosis (LSS) is a degenerative disease characterized by intermittent claudication and numbness in the lower extremities. These symptoms are caused by the compression of nerve tissue in the lumbar spinal canal. Ligamentum flavum (LF) hypertrophy and spinal epidural lipomatosis in the spinal canal are known to contribute to stenosis of the spinal canal: however, detailed mechanisms underlying LSS are still not fully understood. Here, we show that surgically harvested LFs from LSS patients exhibited significantly increased thickness when transthyretin (TTR), the protein responsible for amyloidosis, was deposited in LFs, compared to those without TTR deposition. Multiple regression analysis, which considered age and BMI, revealed a significant association between LF hypertrophy and TTR deposition in LFs. Moreover, TTR deposition in LF was also significantly correlated with epidural fat (EF) thickness based on multiple regression analyses. Mesenchymal cell differentiation into adipocytes was significantly stimulated by TTR in vitro. These results suggest that TTR deposition in LFs is significantly associated with increased LF hypertrophy and EF thickness, and that TTR promotes adipogenesis of mesenchymal cells. Therapeutic agents to prevent TTR deposition in tissues are currently available or under development, and targeting TTR could be a potential therapeutic approach to inhibit LSS development and progression.
Subject(s)
Ligamentum Flavum , Spinal Stenosis , Humans , Spinal Stenosis/complications , Ligamentum Flavum/metabolism , Prealbumin/metabolism , Spinal Canal/metabolism , Hypertrophy/metabolism , Lumbar Vertebrae/metabolismABSTRACT
The entomopathogenic fungus Beauveria bassiana is used commercially as a microbial insecticides against a wide range of agricultural insect pests. Some strains of B. bassiana protect the plants from pathogens, but the underlying mechanisms are largely unknown. Here, we found that prophylactic sprays of commercial bioinsecticide Botanigard on cucumber, tomato, and strawberry plants suppressed the severity of economically damaging powdery mildews. On leaf surfaces, hyphal elongation and spore germination of cucumber powdery mildew, Podosphaera xanthii, were inhibited, but B. bassiana strain GHA, the active ingredient isolated from Botanigard, only inhibited hyphal elongation but had no effect on spore germination of P. xanthii. In addition, strain GHA suppressed powdery mildew symptoms locally, not systemically. Treatment with Botanigard and strain GHA induced a hypersensitive response (HR)-like cell death in epidermal cells of the cucumber leaves in a concentration-dependent manner and inhibited penetration by P. xanthii. Transcriptome analysis and mass spectrometry revealed that GHA induced expression of salicylic acid (SA)-related genes, and treatment with Botanigard and GHA increased the SA level in the cucumber leaves. In NahG-transgenic tomato plants, which do not accumulate SA, the biocontrol effect of tomato powdery mildew by GHA was significantly reduced. These results suggested that B. bassiana GHA induces SA accumulation, leading to the induction of HR-like cell death against powdery mildew and subsequent suppression of fungal penetration. Thus, Botanigard has the potential to control both insect pests and plant diseases.
ABSTRACT
Severe diarrhea is a common side effect of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We aimed to evaluate the risk of EGFR-TKI-induced diarrhea using spheroids of human and monkey crypt-derived intestinal stem cells. Intestinal spheroids exhibited higher toxic susceptibility to EGFR-TKIs than Caco-2 cells. As concentration of EGFR-TKIs increased, cellular ATP first decreased relative to the control condition, followed by an increase in LDH release, in contrast with their simultaneous changes with traditional cytotoxic anticancer drugs. The toxic sensitivity of spheroids to various EGFR-TKIs corresponded to clinical diarrhea incidence. Afatinib, a second-generation EGFR-TKI, exhibited higher toxic sensitivity compared with the first-generation ones, corresponding to the clinical evidence that afatinib-induced diarrhea is almost inevitable and severe. By contrast, the third-generation osimertinib, which reduces the risk of diarrhea, showed mitigated cytotoxicity compared with afatinib. For irreversible EGFR-TKIs, the decreased ATP level persisted or its recovery was delayed even after drug removal compared with reversible ones. Furthermore, the highest drug accumulation in spheroids (TKIspheroids) and inhibition potency against EGFR (TKIspheroids/Ki) were observed for afatinib. This system would be useful for predicting the risk of EGFR-TKI-induced diarrhea; moreover, on-target cytotoxicity against intestinal stem cells might contribute to clinically observed diarrhea.
Subject(s)
Antineoplastic Agents , Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Humans , Animals , Afatinib/toxicity , Afatinib/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/toxicity , Haplorhini/metabolism , Caco-2 Cells , ErbB Receptors/metabolism , Mutation , Antineoplastic Agents/pharmacology , Diarrhea/chemically induced , Adenosine TriphosphateABSTRACT
Hip fractures are fragility fractures frequently seen in persons over 80-years-old. Although various factors, including decreased bone mineral density and a history of falls, are reported as hip fracture risks, few large-scale studies have confirmed their relevance to individuals older than 80, and tools to assess contributions of various risks to fracture development and the degree of risk are lacking. We recruited 1395 fresh hip fracture patients and 1075 controls without hip fractures and comprehensively evaluated various reported risk factors and their association with hip fracture development. We initially constructed a predictive model using Extreme Gradient Boosting (XGBoost), a machine learning algorithm, incorporating all 40 variables and evaluated the model's performance using the area under the receiver operating characteristic curve (AUC), yielding a value of 0.87. We also employed SHapley Additive exPlanation (SHAP) values to evaluate each feature importance and ranked the top 20. We then used a stepwise selection method to determine key factors sequentially until the AUC reached a plateau nearly equal to that of all variables and identified the top 10 sufficient to evaluate hip fracture risk. For each, we determined the cutoff value for hip fracture occurrence and calculated scores of each variable based on the respective feature importance. Individual scores were: serum 25(OH)D levels (<10 ng/ml, score 7), femoral neck T-score (<-3, score 5), Barthel index score (<100, score 3), maximal handgrip strength (<18 kg, score 3), GLFS-25 score (≥24, score 2), number of falls in previous 12 months (≥3, score 2), serum IGF-1 levels (<50 ng/ml, score 2), cups of tea/day (≥5, score -2), use of anti-osteoporosis drugs (yes, score -2), and BMI (<18.5 kg/m2, score 1). Using these scores, we performed receiver operating characteristic (ROC) analysis and the resultant optimal cutoff value was 7, with a specificity of 0.78, sensitivity of 0.75, and AUC of 0.85. These ten factors and the scoring system may represent tools useful to predict hip fracture.
Subject(s)
Hip Fractures , Osteoporosis , Humans , Aged , Aged, 80 and over , Bone Density , Hand Strength , Risk Assessment/methods , Hip Fractures/etiology , Osteoporosis/complications , Risk FactorsABSTRACT
Focal nodular hyperplasia (FNH) or FNH-like lesions of the liver are benign lesions that can be mostly diagnosed by hepatobiliary phase gadoxetic acid-enhanced magnetic resonance imaging (MRI). Accurate imaging diagnosis is based on the fact that most FNHs or FNH-like lesions show characteristic hyper- or isointensity on hepatobiliary phase images. We report a case of an FNH-like lesion in a 73-year-old woman that mimicked a malignant tumor. Dynamic contrast-enhanced computed tomography (CT) and MRI using gadoxetic-acid revealed an ill-defined nodule showing early enhancement in the arterial phase and gradual and prolonged enhancement in the portal and equilibrium/transitional phases. Hepatobiliary phase imaging revealed inhomogeneous hypointensity, accompanied by a slightly isointense area compared to the background liver. Angiography-assisted CT showed a portal perfusion defect of the nodule, inhomogeneous arterial blood supply in the early phase, and less internal enhancement in the late phase, accompanied by irregularly shaped peritumoral enhancement. No central stellate scar was identified in any of the images. Imaging findings could not exclude the possibility of hepatocellular carcinoma, but the nodule was pathologically diagnosed as an FNH-like lesion by partial hepatectomy. In the present case, an unusual inhomogeneous hypointensity on hepatobiliary phase imaging made it difficult to diagnose the FNH-like lesions.
ABSTRACT
In this study, the ethnic ratios (ERs) of oral clearance between Japanese and Western populations were subjected to model-based meta-analysis (MBMA) for 81 drugs evaluated in 673 clinical studies. The drugs were classified into eight groups according to the clearance mechanism, and the ER for each group was inferred together with interindividual variability (IIV), interstudy variability (ISV), and inter-drug variability within a group (IDV) using the Markov chain Monte Carlo (MCMC) method. The ER, IIV, ISV, and IDV were dependent on the clearance mechanism, and, except for particular groups such as drugs metabolized by polymorphic enzymes or their clearance mechanism is not confirmative, the ethnic difference was found to be generally small. The IIV was well-matched across ethnicities, and the ISV was approximately half of the IIV as the coefficient of variation. To adequately assess ethnic differences in oral clearance without false detections, phase I studies should be designed with full consideration of the mechanism of clearance. This study suggests that the methodology of classifying drugs based on the mechanism that causes ethnic differences and performing MBMA with statistical techniques such as MCMC analysis is helpful for a rational understanding of ethnic differences and for strategic drug development.
Subject(s)
Computer Simulation , Humans , Markov ChainsABSTRACT
Prediction of intestinal absorption of drugs in humans is one of the critical elements in the development process for oral drugs. However, it remains challenging, because intestinal absorption of drugs is influenced by multiple factors, including the function of various metabolic enzymes and transporters, and large species differences in drug bioavailability hinder the prediction of human bioavailability directly from in vivo animal experiments. For the screening of intestinal absorption properties of drugs, a transcellular transport assay with Caco-2 cells is still routinely used by pharmaceutical companies because of its convenience, but the predictability of the fraction of the oral dose that goes to the portal vein of metabolic enzyme/transporter substrate drugs was not always good because the cellular expression of metabolic enzymes and transporters is different from that in the human intestine. Recently, various novel in vitro experimental systems have been proposed such as the use of human-derived intestinal samples, transcellular transport assay with induced pluripotent stem-derived enterocyte-like cells, or differentiated intestinal epithelial cells derived from intestinal stem cells at crypts. Crypt-derived differentiated epithelial cells have an excellent potential to characterize species differences and regional differences in intestinal absorption of drugs because a unified protocol can be used for the proliferation of intestinal stem cells and their differentiation into intestinal absorptive epithelial cells regardless of the animal species and the gene expression pattern of differentiated cells is maintained at the site of original crypts. The advantages and disadvantages of novel in vitro experimental systems for characterizing intestinal absorption of drugs are also discussed. SIGNIFICANCE STATEMENT: Among novel in vitro tools for the prediction of human intestinal absorption of drugs, crypt-derived differentiated epithelial cells have many advantages. Cultured intestinal stem cells are rapidly proliferated and easily differentiated into intestinal absorptive epithelial cells simply by changing the culture media. A unified protocol can be used for the establishment of intestinal stem cell culture from preclinical species and humans. Region-specific gene expression at the collection site of crypts can be reproduced in differentiated cells.
Subject(s)
Epithelial Cells , Intestines , Animals , Humans , Caco-2 Cells , Epithelial Cells/metabolism , Stem Cells , Membrane Transport Proteins/metabolism , Cell Differentiation , Intestinal Absorption , Intestinal Mucosa/metabolismABSTRACT
The Japan Association of Radiological Technologists (JART) and the Japan Medical Imaging and Radiological Systems Industries Association jointly conducted a nationwide survey to reveal the current situation of diagnostic displays in Japan using a questionnaire on the performance and quality control (QC) of diagnostic displays for mammography and common use. The questionnaire for radiological technologists (RTs) was distributed via email to 4519 medical facilities throughout Japan, where RTs affiliated with JART were employed; 613 (13.6%) facilities responded. Diagnostic displays with suitable maximal luminance (500 cd/m2 or higher for mammography and 350 cd/m2 or higher for common use) and resolution (5 megapixels for mammography) have been widely used. However, while 99% of the facilities recognized the necessity of QC, only approximately 60% implemented it. This situation arose due to several barriers to QC implementation, such as insufficient devices, time, staff, knowledge, and the recognition of QC as a duty. The implementation of QC can lead to the avoidance of incidents or accidents caused by a decrease in luminance, variation in luminance response, and the influence of ambient light. Moreover, the barriers discouraging the implementation of QC are mainly related to a lack of human resources and budgets. Therefore, to popularize the QC of diagnostic displays in all facilities, it is crucial to identify countermeasures to eliminate these barriers and to continue positive actions for popularization.
Subject(s)
Mammography , Radiology , Humans , Japan , Quality Control , Radiology/methods , Surveys and QuestionnairesABSTRACT
Cholestatic toxicity causes the failure of pharmaceutical agents during drug development and, thus, should be identified at an early stage of drug discovery and development. The formation of functional bile canaliculi in human hepatocytes is required for in vitro cholestasis toxicity tests conducted during the early stage of drug development. In this study, we investigated the culture conditions required for the formation of bile canaliculi using human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps). When hiPSC-Heps were sandwich-cultured under the condition we established, extended bile canaliculi were formed on the whole well surfaces. Biliary efflux transporters were localized in the formed bile canaliculi structures which had junctional complexes. After the model substrates of the biliary efflux transporters were taken up into cells, their subsequent excretion into the bile canaliculi was observed and was found to be impeded by each inhibitor of the biliary efflux transporter. These findings suggest that bile canaliculi have transporter-specific bile excretion abilities. We will continue to study the application of this culture protocol to cell-based cholestasis assay system. As a result, the culture protocol could lead to a highly predictable, robust cell-based cholestasis assay system because it forms functional bile canaliculi reproducibly and efficiently.
Subject(s)
Cholestasis , Induced Pluripotent Stem Cells , Bile , Bile Canaliculi , Cells, Cultured , Hepatocytes , Humans , Membrane Transport ProteinsABSTRACT
Pharmacokinetic research plays an important role in the development of new drugs. Accurate predictions of human pharmacokinetic parameters are essential for the success of clinical trials. Clearance (CL) and volume of distribution (Vd) are important factors for evaluating pharmacokinetic properties, and many previous studies have attempted to use computational methods to extrapolate these values from nonclinical laboratory animal models to human subjects. However, it is difficult to obtain sufficient, comprehensive experimental data from these animal models, and many studies are missing critical values. This means that studies using nonclinical data as explanatory variables can only apply a small number of compounds to their model training. In this study, we perform missing-value imputation and feature selection on nonclinical data to increase the number of training compounds and nonclinical datasets available for these kinds of studies. We could obtain novel models for total body clearance (CLtot) and steady-state Vd (Vdss) (CLtot: geometric mean fold error [GMFE], 1.92; percentage within 2-fold error, 66.5%; Vdss: GMFE, 1.64; percentage within 2-fold error, 71.1%). These accuracies were comparable to the conventional animal scale-up models. Then, this method differs from animal scale-up methods because it does not require animal experiments, which continue to become more strictly regulated as time passes.
Subject(s)
Drug Elimination Routes , Machine Learning , Animals , Humans , Models, Biological , Pharmaceutical PreparationsABSTRACT
AIM: Previous reports suggest that the null genotype (*0/*0) of glutathione S-transferase (GST) M1 and/or GSTT1 could be risk factors for drug-induced liver injury (DILI). However, multi-institutional pharmacogenetic research with various suspected drugs has rarely been performed in Japan. Therefore, the aim of this study was to investigate the role of GSTM1 and GSTT1 null genotype in the occurrence of DILI in Japanese patients. METHODS: Blood samples of 270 DILI patients from 23 hospitals throughout Japan collected between 2010 and 2018 were subjected to genotyping of null genotypes of GSTM1 and GSTT1 using the SmartAmp-2 method. We also collected information on DILI types, time to onset of DILI, pharmacological classification of suspected drugs and Digestive Disease Week-Japan score, as well as genotypes of GSTM1 and GSTT1 in each patient with DILI. RESULTS: The distribution of a combination of null genotypes of GSTM1 and GSTT1 in Japanese patients with DILI was significantly different from that reported in the general Japanese population. Notably, the incidence of the GSTM1 null genotype in patients with DILI was significantly higher than that of the control population. A significant relationship between the frequency of GSTM1 and GSTT1 null genotypes and pharmacological classification of suspected drugs, clinical laboratory data for liver function, time to onset of DILI, and Digestive Disease Week-Japan scores was not observed. CONCLUSIONS: The GSTM1 null genotype was associated with an increased incidence of DILI in Japanese patients.
ABSTRACT
The fungus Cladosporium fulvum causes the leaf mould in tomatoes. During the colonization of the host, it secretes plenty of effector proteins into the plant apoplast to suppress the plant's immune system. Here, we characterized and functionally analyzed the Ecp20-2 gene of C. fulvum using combined omics approaches. RNA-sequencing of susceptible tomato plants inoculated with C. fulvum race 0WU showed strongly induced expression of the Ecp20-2 gene. Strong upregulation of expression of the Ecp20-2 gene was confirmed by qPCR, and levels were comparable to those of other known effectors of C. fulvum. The Ecp20-2 gene encodes a small secreted protein of 149 amino acids with a predicted signal peptide of 17 amino acids. Mass spectrometry of apoplastic fluids from infected tomato leaves revealed the presence of several peptides originating from the Ecp20-2 protein, indicating that the protein is secreted and likely functions in the apoplast. In the genome of C. fulvum, Ecp20-2 is surrounded by various repetitive elements, but no allelic variation was detected in the coding region of Ecp20-2 among 120 C. fulvum isolates collected in Japan. Δecp20-2 deletion mutants of strain 0WU of C. fulvum showed decreased virulence, supporting that Ecp20-2 is an effector required for full virulence of the fungus. Virulence assays confirmed a significant reduction of fungal biomass in plants inoculated with Δecp20-2 mutants compared to those inoculated with wild-type, Δecp20-2-complemented mutants, and ectopic transformants. Sequence similarity analysis showed the presence of Ecp20-2 homologs in the genomes of several Dothideomycete fungi. The Ecp20-2 protein shows the best 3D homology with the PevD1 effector of Verticillium dahliae, which interacts with and inhibits the activity of the pathogenesis-related protein PR5, which is involved in the immunity of several host plants.