Discovery of a novel spotted fever group Rickettsia, "Candidatus Rickettsia kedanie," in unfed larval chigger mites, Leptotrombidium scutellare.

Spotted fever group (SFG) rickettsia, the causative agent of SFG rickettsiosis, is predominantly carried by ticks, whereas Orientia tsutusgamushi, the causative agent of scrub typhus, is primarily transmitted by chigger mites in Japan. In this study, we attempted to isolate intracellular eubacteria from Leptotrombidium scutellare, a major vector of O. tsutsugamushi; moreover, we isolated an SFG rickettsia using a mosquito-derived cell line. Draft genome sequences of this unique isolate, by applying criteria for species delimitation, classified this isolate as a novel strain, proposed as "Rickettsia kedanie." Further genetic analysis identified conserved virulence factors, and the isolate successfully propagated in mammalian cells, suggesting its ability to cause diseases in humans. The presence of SFG rickettsia in unfed larvae implies potential dual-pathogen carriage and reflects a symbiotic relationship similar to that between the mites and O. tsutsugamushi, indicating possibility of its transovarial transmission from female adults. Furthermore, conserved genomic similarity of the novel isolate to known SFG rickettsia suggests potential multiple hosts, including chiggers and ticks. In the natural environment, ticks, chigger mites, and wild animals may carry new isolates, complicating the infection cycle and increasing the transmission risks to humans. This discovery challenges the conventional association of SFG rickettsia with ticks, emphasizing its implications for research and disease control. However, this study was confined to a particular species of chigger mites and geographic area, underscoring the necessity for additional studies to comprehend the ecological dynamics, host interactions, and health implications linked to this newly identified SFG rickettsia.

[Dystrophia myotonica Type 1 associated with glioblastoma: a case report].

This case involved a 65-year-old woman, who had been suffered from weakness in both legs for 10 years. She had not been diagnosed of dystrophia myotonica type 1 (DM1) despite her son's diagnosis of DM and her distinct facial features and gait anomaly. During her son's recent clinical visit, she was finally suspected of having DM. She was sent to our institution, where a distinct muscle atrophy and grip myotonia were observed and a genetical examination was performed. The sequencing data confirmed her diagnosis of DM1 due to the distinct 230-900 CTG repeats found in the dystrophia myotonica protein kinase gene 3' untranslated region. A brain MRI revealed an abnormal lesion with irregular ring-enhancement at the right temporal lobe. Because of the steady growth of the lesion during one month observation, a surgical intervention was performed in our institution. The histopathological examination gave a diagnosis of glioblastoma multiforme (GBM). The clinical management of the patient required special cares during the perioperative periods due to the distinct pathological manifestation of DM. The risk of developing cancer in DM patients has been estimated about twice as much as general population. Since GBM developed in the DM patient is rarely reported, we present this rare case with a few insights: the difficulties of the clinical management of DM patients under the perioperative stress; the pathological contribution of DM to the malignant transformation of the glial cells.

Adolescent Health Promotion Interventions Using Well-Care Visits and a Smartphone Cognitive Behavioral Therapy App: Randomized Controlled Trial.

BACKGROUND: Adolescent health promotion is important in preventing risk behaviors and improving mental health. Health promotion during adolescence has been shown to contribute to the prevention of late onset of the mental health disease. However, scalable interventions have not been established yet. OBJECTIVE: This study was designed to test the efficacy of two adolescent health promotion interventions: a well-care visit (WCV) with a risk assessment interview and counseling and self-monitoring with a smartphone cognitive behavioral therapy (CBT) app. Our hypothesis was that participants who had received both WCV and the CBT app would have better outcomes than those who had received only WCV or those who had not received any intervention. We conducted a prospective multi-institutional randomized controlled trial. METHODS: Participants were 217 adolescents aged 13-18 years. They were randomly divided into two intervention groups (WCV group and WCV with CBT app group) and a nonintervention group. WCV comprised a standardized physical examination along with a structured interview and counseling for youth risk assessment, which was designed with reference to the Guideline for Health Supervision of Adolescents of Bright Futures. A smartphone-based CBT program was developed based on the CBT approach. The CBT app comprised a 1-week psychoeducation component and a 1-week self-monitoring component. During the CBT program, participants created several self-monitoring sheets based on the CBT model with five window panels: event, thoughts, feelings, body response, and actions. The primary outcome was the change in scores for depressive symptoms. Secondary outcomes included changes in scores for self-esteem, quality of life, self-monitoring, and an adolescent health promotion scale. These outcomes were evaluated at baseline and at 1, 2, and 4 months after baseline. The exploratory outcome was the presence of suicidal ideation during the observation period. Intervention effects were estimated using mixed effect models. RESULTS: In total, 94% (204/217) of the participants completed the 4-month evaluation. Both intervention groups showed a significant effect in the form of reduced scores for depressive symptoms at 1 month in high school students; however, these effects were not observed at 2 and 4 months. The intervention effect was significantly more predominant in those scoring above cutoff for depressive symptoms. There was significantly less suicidal ideation in the intervention groups. As for secondary outcomes, there was significant increase in health promotion scale scores at the 4-month follow-up among junior high school students in the WCV group. Moreover, the CBT app was significantly effective in terms of obtaining self-monitoring skills and reducing depressive symptoms. CONCLUSIONS: Although adolescent health promotion interventions may have short-term benefits, the frequency of WCV and further revision of the CBT app should be considered to evaluate long-term effectiveness. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry UMIN 000036343; https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000041246.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Inhibition of xanthine oxidase in the acute phase of myocardial infarction prevents skeletal muscle abnormalities and exercise intolerance.

AIMS: Exercise intolerance in patients with heart failure (HF) is partly attributed to skeletal muscle abnormalities. We have shown that reactive oxygen species (ROS) play a crucial role in skeletal muscle abnormalities, but the pathogenic mechanism remains unclear. Xanthine oxidase (XO) is reported to be an important mediator of ROS overproduction in ischaemic tissue. Here, we tested the hypothesis that skeletal muscle abnormalities in HF are initially caused by XO-derived ROS and are prevented by the inhibition of their production. METHODS AND RESULTS: Myocardial infarction (MI) was induced in male C57BL/6J mice, which eventually led to HF, and a sham operation was performed in control mice. The time course of XO-derived ROS production in mouse skeletal muscle post-MI was first analysed. XO-derived ROS production was significantly increased in MI mice from Days 1 to 3 post-surgery (acute phase), whereas it did not differ between the MI and sham groups from 7 to 28 days (chronic phase). Second, mice were divided into three groups: sham + vehicle (Sham + Veh), MI + vehicle (MI + Veh), and MI + febuxostat (an XO inhibitor, 5 mg/kg body weight/day; MI + Feb). Febuxostat or vehicle was administered at 1 and 24 h before surgery, and once-daily on Days 1-7 post-surgery. On Day 28 post-surgery, exercise capacity and mitochondrial respiration in skeletal muscle fibres were significantly decreased in MI + Veh compared with Sham + Veh mice. An increase in damaged mitochondria in MI + Veh compared with Sham + Veh mice was also observed. The wet weight and cross-sectional area of slow muscle fibres (higher XO-derived ROS) was reduced via the down-regulation of protein synthesis-associated mTOR-p70S6K signalling in MI + Veh compared with Sham + Veh mice. These impairments were ameliorated in MI + Feb mice, in association with a reduction of XO-derived ROS production, without affecting cardiac function. CONCLUSION: XO inhibition during the acute phase post-MI can prevent skeletal muscle abnormalities and exercise intolerance in mice with HF.

Type 2 diabetes is an independent predictor of lowered peak aerobic capacity in heart failure patients with non-reduced or reduced left ventricular ejection fraction.

BACKGROUND: Although type 2 diabetes mellitus (T2DM) is one of the most frequent comorbidities in patients with chronic heart failure (CHF), the effects of T2DM on the exercise capacity of CHF patients are fully unknown. Here, we tested the hypothesis that the coexistence of T2DM lowers CHF patients' peak aerobic capacity. METHODS: We retrospectively analyzed the cases of 275 Japanese CHF patients with non-reduced ejection fraction (left ventricular ejection fraction [LVEF] ≥ 40%) or reduced EF (LVEF < 40%) who underwent cardiopulmonary exercise testing. We divided them into diabetic and nondiabetic groups in each CHF cohort. RESULTS: The mean peak oxygen uptake (VO2) value was 16.87 mL/kg/min in the non-reduced LVEF cohort and 15.52 mL/kg/min in the reduced LVEF cohort. The peak VO2 was lower in the diabetics versus the nondiabetics in the non-reduced LVEF cohort with the mean difference (95% confidence interval [95% CI]) of - 0.93 (- 1.82 to - 0.04) mL/kg/min and in the reduced LVEF cohort with the mean difference of - 1.05 (- 1.96 to - 0.15) mL/kg/min, after adjustment for age-squared, gender, anemia, renal function, LVEF, and log B-type natriuretic peptide (BNP). The adjusted VO2 at anaerobic threshold (AT), a submaximal aerobic capacity, was also decreased in the diabetic patients with both non-reduced and reduced LVEFs. Intriguingly, the diabetic patients had a lower adjusted peak O2 pulse than the nondiabetic patients in the reduced LVEF cohort, but not in the non-reduced LVEF cohort. A multivariate analysis showed that the presence of T2DM was an independent predictor of lowered peak VO2 in CHF patients with non-reduced LVEF and those with reduced LVEF. CONCLUSIONS: T2DM was associated with lowered peak VO2 in CHF patients with non-reduced or reduced LVEF. The presence of T2DM has a negative impact on CHF patients' exercise capacity, and the degree of impact is partly dependent on their LV systolic function.

Empagliflozin restores lowered exercise endurance capacity via the activation of skeletal muscle fatty acid oxidation in a murine model of heart failure.

Decreased exercise capacity, which is an independent predictor of the poor prognosis of patients with heart failure (HF), is attributed to markedly impaired skeletal muscle mitochondrial function and fatty acid oxidation. Previous studies reported that the administration of an inhibitor of sodium-glucose cotransporter 2 (SGLT2) increases ketone body production and fat utilization in type 2 diabetic mice. In this study, we investigated the effects of SGLT2 inhibitor administration on exercise endurance and skeletal muscle mitochondrial function with fatty acid oxidation in a murine model of HF after the induction of myocardial infarction (MI). Two weeks post-MI, HF mice were divided into 2 groups, i.e., with or without treatment with the SGLT2 inhibitor empagliflozin (Empa, 300 mg/kg of food). Consistent with previous studies, urinary glucose and blood beta-hydroxybutyrate levels were increased in the HF+Empa mice compared with the sham and HF mice 4 weeks after the start of Empa administration. Exercise endurance capacity was limited in the HF mice but was ameliorated in the HF+Empa mice, without any effects on cardiac function, food intake, spontaneous physical activity, skeletal muscle strength, and skeletal muscle weight. Mitochondrial oxidative phosphorylation capacity with fatty acid substrates was reduced in the skeletal muscle of HF mice, and this decrease was ameliorated in the HF+Empa mice. Our results demonstrate that SGLT2 inhibitors may be novel therapeutics against reduced exercise endurance capacity in HF, by improving mitochondrial fatty acid oxidation in skeletal muscle.

Mitochondrial reactive oxygen species generation in blood cells is associated with disease severity and exercise intolerance in heart failure patients.

Systemic oxidative stress plays a key role in the development of chronic heart failure (CHF). We tested the hypothesis that mitochondrial reactive oxygen species (ROS) generation in circulating peripheral blood mononuclear cells (PBMCs) contributes to CHF progression. A total of 31 patients who had a history of hospital admission due to worsening HF were enrolled and grouped as having either mild CHF defined as New York Heart Association (NYHA) functional class I-II or moderate-to-severe CHF defined as NYHA functional class III. ROS levels in PBMC mitochondria were significantly increased in CHF patients with NYHA functional class III compared to those with NYHA functional class I-II, accompanied by impaired mitochondrial respiratory capacity in PBMCs. ROS generation in PBMC mitochondria was positively correlated with urinary 8-hydroxydeoxyguanosine, a systemic oxidative stress marker, in CHF patients. Importantly, mitochondrial ROS generation in PBMCs was directly correlated with plasma levels of B-type natriuretic peptide, a biomarker for severity of HF, and inversely correlated with peak oxygen uptake, a parameter of exercise capacity, in CHF patients. The study showed that ROS generation in PBMC mitochondria was higher in patients with advanced CHF, and it was associated with disease severity and exercise intolerance in CHF patients.

A mitochondrial delivery system using liposome-based nanocarriers that target myoblast cells.

Mitochondrial function is reduced in skeletal muscles of many patients with systemic diseases and it is difficult to deliver medicinal substances to mitochondria in such tissue. In this study, we report on attempts to develop liposome-based carriers for mitochondrial delivery using mouse myoblasts (C2C12) by varying the lipid composition of the carriers. We found that a liposome that contains an optimal lipid modified with the KALA peptide (a cellular uptake and mitochondrial targeting device) was the most effective nanocarrier for achieving mitochondrial delivery in C2C12 cells. We also report on successful mitochondrial transgene expression using the carriers encapsulating a mitochondrial DNA vector as we previously reported.

Impaired mitochondrial oxidative phosphorylation capacity in epicardial adipose tissue is associated with decreased concentration of adiponectin and severity of coronary atherosclerosis.

Epicardial adipose tissue (EAT), a source of adipokines, is metabolically active, but the role of EAT mitochondria in coronary artery disease (CAD) has not been established. We investigated the association between EAT mitochondrial respiratory capacity, adiponectin concentration in the EAT, and coronary atherosclerosis. EAT samples were obtained from 25 patients who underwent elective cardiac surgery. Based on the coronary angiographycal findings, the patients were divided into two groups; coronary artery disease (CAD; n = 14) and non-CAD (n = 11) groups. The mitochondrial respiratory capacities including oxidative phosphorylation (OXPHOS) capacity with non-fatty acid (complex I and complex I + II-linked) substrates and fatty acids in the EAT were significantly lowered in CAD patients. The EAT mitochondrial OXPHOS capacities had a close and inverse correlation with the severity of coronary artery stenosis evaluated by the Gensini score. Intriguingly, the protein level of adiponectin, an anti-atherogenic adipokine, in the EAT was significantly reduced in CAD patients, and it was positively correlated with the mitochondrial OXPHOS capacities in the EAT and inversely correlated with the Gensini score. Our study showed that impaired mitochondrial OXPHOS capacity in the EAT was closely linked to decreased concentration of adiponectin in the EAT and severity of coronary atherosclerosis.

Analytical Model and Experimental Evaluation of the Micro-Scale Thermal Property Sensor for Single-Sided Measurement.

We report a new analytical model of the MEMS-based thermal property sensor for samples which are difficult to handle and susceptible to damage by thermal stimulus, such as living cells. Many sensor designs had been reported for thermal property measurements, but only a few of them have considered the analytical model of the single-sided measurement in which a measurement sample is placed on the sensor substrate. Even in the few designs that have considered the analytical model, their applicable limits are restricted to more than 1 mm length in practical situations. Our new model considers both the sample and the sensor substrate thermal properties and is applicable to a sensor length less than 1 µm. In order to minimize the influence of the heat stimulus to the sample, the model formulates the required heat dissipating time for different sensor geometries. We propose fast and precise detection circuit architecture to realize our model, and we discuss the sensor performance for a number of different designs.

Impact of High Respiratory Exchange Ratio During Submaximal Exercise on Adverse Clinical Outcome in Heart Failure.

BACKGROUND: Oxygen uptake (V̇O2) at peak workload and anaerobic threshold (AT) workload are often used for grading heart failure (HF) severity and predicting all-cause mortality. The clinical relevance of respiratory exchange ratio (RER) during exercise, however, is unknown. Methods and Results: We retrospectively studied 295 HF patients (57±15 years, NYHA class I-III) who underwent cardiopulmonary exercise testing. RER was measured at rest; at AT workload; and at peak workload. Peak V̇O2 had an inverse correlation with RER at AT workload (r=-0.256), but not at rest (r=-0.084) or at peak workload (r=0.090). Using median RER at AT workload, we divided the patients into high RER (≥0.97) and low RER (<0.97) groups. Patients with high RER at AT workload were characterized by older age, lower body mass index, anemia, and advanced NYHA class. After propensity score matching, peak V̇O2 tended to be lower in the high-RER than in the low-RER group (14.9±4.5 vs. 16.1±5.0 mL/kg/min, P=0.06). On Kaplan-Meier analysis, HF patients with a high RER at AT workload had significantly worse clinical outcomes, including all-cause mortality and rate of readmission due to HF worsening over 3 years (29% vs. 15%, P=0.01). CONCLUSIONS: High RER during submaximal exercise, particularly at AT workload, is associated with poor clinical outcome in HF patients.

The Effect of Cerebellar Transcranial Direct Current Stimulation on A Throwing Task Depends on Individual Level of Task Performance.

The effect of cerebellar transcranial direct current stimulation (tDCS) on motor performance remains controversial. Some studies suggest that the effect of tDCS depends upon task-difficulty and individual level of task performance. Here, we investigated whether the effect of cerebellar tDCS on the motor performance depends upon the individual's level of performance. Twenty-four naïve participants practiced dart throwing while receiving a 2-mA cerebellar tDCS for 20 min under three stimulus conditions (anodal-, cathodal-, and sham-tDCS) on separate days with a double-blind, counter-balanced cross-over design. Task performance was assessed by measuring the distance between the center of the bull's eye and the dart's position. Although task performance tended to improve throughout the practice under all stimulus conditions, improvement within a given day was not significant as compared to the first no-stimulus block. In addition, improvement did not differ among stimulation conditions. However, the magnitude of improvement was associated with an individual's level of task performance only under cathodal tDCS condition (p < 0.05). This resulted in a significant performance improvement only for the sub-group of participants with lower performance levels as compared to that with sham-tDCS (p < 0.05). These findings suggest that the facilitation effect of cerebellar cathodal tDCS on motor skill learning of complex whole-body movements depends on the level of an individual's task performance. Thus, cerebellar tDCS would facilitate learning of a complex motor skill task only in a subset of individuals.

A Case of Dermatomyositis with Severe Myalgia and Muscle Weakness Testing Positive for Anti-melanoma Differentiation-associated Gene 5 Antibody.

We report a case of a woman with typical dermatomyositis (DM) with skin manifestations, severe myalgia and muscle weakness complicated by interstitial lung disease (ILD) and pneumomediastinum. Pneumomediastinum persisted despite treatment with immunosuppressive therapy (steroids and cyclosporine). After the test for anti-melanoma differentiation-associated gene 5 (MDA5) antibody came out positive, we doubled the cyclosporine dose and her condition improved. Despite typical clinical features of DM, in cases complicated by pneumomediastinum or steroid resistance, measurement of anti-MDA5 antibody may be useful for immunosuppressant dose titration.

A Case of Streptobacillus moniliformis Infection with Cutaneous Leukocytoclastic Vasculitis.

A 71-year-old man presented with a high fever, polyarthralgia, petechiae and palpable purpura accompanied by livedoid change on his legs and feet. Histopathological findings of the purpura revealed perivascular infiltration of neutrophils, mononuclear cells, and nuclear debris, and extravasation of red cells mainly in the upper dermis: all signs consistent with leukocytoclastic vasculitis. Small vessel thrombi, which are characteristic features of septic vasculopathy, were also observed. Direct immunofluorescence showed negative results. Blood culture revealed the growth of gram-negative bacilli. Subsequently, 16S rRNA sequencing of DNA confirmed the organism as Streptobacillus moniliformis, which is the causative pathogen of rat-bite fever. He had frequently encountered wild rats in his house although there was no evidence of rat bite on his body. Empiric therapy with intravenous administration of ceftriaxone in combination with azithromycin hydrate led to a prompt resolution of the symptoms. Precise history-taking related to contact with rats and detection of skin eruptions suggestive of leukocytoclastic vasculitis on the extremities, especially on the feet, can be clues to Streptobacillus moniliformis infection. Familiarity with its cutaneous features is important for early diagnosis; the evidence herein may also help in understanding its underlying pathogenesis.

Diffusion-weighted magnetic resonance neurography for parapharyngeal schwannomas: preoperative determination of the originating nerves.

OBJECTIVE: The aim of this study was to assess the feasibility of diffusion-weighted (DW) magnetic resonance neurography (MRN) for determining the originating nerve of parapharyngeal schwannomas preoperatively. METHODS: Six patients who underwent DW-MRN preoperatively for a parapharyngeal schwannoma were studied. Prediction of the originating nerve was performed. With the conventional method, a tumor showing "separation" between the internal jugular vein and carotid artery was determined to originate from the vagus nerve, with "no separation" from the sympathetic chain. With DW-MRN, the relationships between the vagus nerve and sympathetic chain to the tumor were characterized as "connected" or "dislocated." A nerve connected to the tumor was determined as the origin. RESULTS: Surgeries revealed that the origins included 1 vagus nerve and 5 sympathetic chains. Using a conventional method, all 6 cases were diagnosed correctly, whereas DW-MRN successfully predicted only 4 cases with a sympathetic chain origin. CONCLUSIONS: The DW-MRN is a feasible approach for determining an originating nerve.

One-minute mental status examination for category fluency is more useful than mini-mental state examination to evaluate the reliability of insulin self-injection in elderly diabetic patients.

AIMS/INTRODUCTION: We investigated the factors associated with the reliability of insulin self-injection in elderly diabetic patients receiving insulin therapy. MATERIALS AND METHODS: We enrolled diabetic patients aged ≥65 years and receiving insulin therapy, and assessed their cognitive function by the mini-mental state examination and 1-min mental status examination for category fluency. We also observed their technique of insulin self-injection, and evaluated whether or not patients were able to inject insulin by themselves according to nine defined details in terms of insulin self-injection. The predictive factors for the reliability of insulin self-injection were determined by univariate and multivariate logistic regression analysis. There were 278 participants (135 males, 143 females) enrolled in the present study. RESULTS: According to multivariate logistic regression analysis, only the 1-min mental status examination score was found to be a significant independent predictor of the reliability of insulin self-injection (odds ratio 0.75; 95% confidence interval 0.62-0.90; P = 0.002). CONCLUSIONS: The 1-min mental status examination for category fluency can be considered more useful than mini-mental state examination to evaluate the reliability of insulin self-injection in elderly diabetic patients receiving insulin therapy.

New approach for fish breeding by chemical mutagenesis: establishment of TILLING method in fugu (Takifugu rubripes) with ENU mutagenesis.

BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.