A versatile Plasmodium falciparum reporter line expressing NanoLuc enables highly sensitive multi-stage drug assays.

Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study.

Exploring natural microbial resources for the discovery of anti-malarial compounds.

Microorganisms in nature are highly diverse biological resources, which can be explored for drug discovery. Some countries including Brazil, Columbia, Indonesia, China, and Mexico, which are blessed with geographical uniqueness with diverse climates and display remarkable megabiodiversity, potentially provide microorganismal resources for such exploitation. In this review, as an example of drug discovery campaigns against tropical parasitic diseases utilizing microorganisms from such a megabiodiversity country, we summarize our past and on-going activities toward discovery of new antimalarials. The program was held in a bilateral collaboration between multiple Indonesian and Japanese research groups. In order to develop a new platform of drug discovery utilizing Indonesian bioresources under an international collaborative scheme, we aimed at: 1) establishment of an Indonesian microbial depository, 2) development of robust enzyme-based and cell-based screening systems, and 3) technology transfer necessary for screening, purification, and identification of antimalarial compounds from microbial culture broths. We collected, characterized, and deposited Indonesian microbes. We morphologically and genetically characterized fungi and actinomycetes strains isolated from 5 different locations representing 3 Indonesian geographical areas, and validated genetic diversity of microbes. Enzyme-based screening was developed against two validated mitochondrial enzymes from Plasmodium falciparum, dihydroorotate dehydrogenase and malate:quinone oxidoreductase, while cell-based proliferation assay was developed using the erythrocytic stage parasite of 3D7 strain. More than 17 thousands microbial culture extracts were subjected to the enzyme- and cell-based screening. Representative anti-malarial compounds discovered in this campaign are discussed, including a few isolated compounds that have been identified for the first time as anti-malarial compounds. Our antimalarial discovery campaign validated the Indonesian microbial library as a powerful resource for drug discovery. We also discuss critical needs for selection criteria for hits at each stage of screening and hit deconvolution such as preliminary extraction test for the initial profiling of the active compounds and dereplication techniques to minimize repetitive discovery of known compounds.

Administration of FK506 from Late Stage of Disease Prolongs Survival of Human Prion-Inoculated Mice.

Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.

Generation of Novel Plasmodium falciparum NF135 and NF54 Lines Expressing Fluorescent Reporter Proteins Under the Control of Strong and Constitutive Promoters.

Transgenic reporter lines of malaria parasites that express fluorescent or luminescent proteins are valuable tools for drug and vaccine screening assays as well as to interrogate parasite gene function. Different Plasmodium falciparum (Pf ) reporter lines exist, however nearly all have been created in the African NF54/3D7 laboratory strain. Here we describe the generation of novel reporter lines, using CRISPR/Cas9 gene modification, both in the standard Pf NF54 background and in a recently described Cambodian P. falciparum NF135.C10 line. Sporozoites of this line show more effective hepatocyte invasion and enhanced liver merozoite development compared to Pf NF54. We first generated Pf NF54 reporter parasites to analyze two novel promoters for constitutive and high expression of mCherry-luciferase and GFP in blood and mosquito stages. The promoter sequences were selected based on available transcriptome data and are derived from two housekeeping genes, i.e., translation initiation factor SUI1, putative (sui1, PF3D7_1243600) and 40S ribosomal protein S30 (40s, PF3D7_0219200). We then generated and characterized reporter lines in the Pf NF135.C10 line which express GFP driven by the sui1 and 40s promoters as well as by the previously used ef1α promoter (GFP@ef1α, GFP@sui1, GFP@40s). The GFP@40s reporter line showed strongest GFP expression in liver stages as compared to the other two lines. The strength of reporter expression by the 40s promoter throughout the complete life cycle, including liver stages, makes transgenic lines expressing reporters by the 40s promoter valuable novel tools for analyses of P. falciparum.

Generation of a Genetically Modified Chimeric Plasmodium falciparum Parasite Expressing Plasmodium vivax Circumsporozoite Protein for Malaria Vaccine Development.

Chimeric rodent malaria parasites with the endogenous circumsporozoite protein (csp) gene replaced with csp from the human parasites Plasmodium falciparum (Pf) and P. vivax (Pv) are used in preclinical evaluation of CSP vaccines. Chimeric rodent parasites expressing PfCSP have also been assessed as whole sporozoite (WSP) vaccines. Comparable chimeric P. falciparum parasites expressing CSP of P. vivax could be used both for clinical evaluation of vaccines targeting PvCSP in controlled human P. falciparum infections and in WSP vaccines targeting P. vivax and P. falciparum. We generated chimeric P. falciparum parasites expressing both PfCSP and PvCSP. These Pf-PvCSP parasites produced sporozoite comparable to wild type P. falciparum parasites and expressed PfCSP and PvCSP on the sporozoite surface. Pf-PvCSP sporozoites infected human hepatocytes and induced antibodies to the repeats of both PfCSP and PvCSP after immunization of mice. These results support the use of Pf-PvCSP sporozoites in studies optimizing vaccines targeting PvCSP.

A proper reference metabolic equivalent value to assess physical activity intensity in Japanese female nurses.

BACKGROUND: Various questionnaires have been developed to assess physical activity, but only a few simple questionnaires are suitable for self-administration in large groups of midlife working women. This study examined the usefulness of the Japan Nurses' Health Study (JNHS) questionnaire for self-administered physical activity surveys. METHODS: The JNHS physical activity questionnaire consisted of items covering seven degrees of intensity. The metabolic equivalents (METs) for the physical activity intensity of the questionnaire were estimated from energy expenditure as measured by a uniaxial accelerometer with the Markov Chain Monte Carlo (MCMC) simulation. The estimated METs were then assigned to the JNHS baseline survey data, and the total energy expenditure (TEE) and the time spent performing ≥3 METs hour of physical activity, called moderate to vigorous intensity physical activity (MVPA), were calculated. RESULTS: For working situations, application of the MCMC simulation resulted in estimated reference values of 1.2 METs for "sitting work", 1.6 METs for "standing work", 1.8 METs for "walking work", and 4.5 METs for "heavy work". For non-working situations, the estimated values were 1.1 METs for sedentary time, 2.4 METs for "moderate physical activity", 4.4 METs for "vigorous physical activity", and 9.4 METs for "very vigorous physical activity". When these estimated METs were used, the mean TEE/day was 1808 kcal. This corresponded to - 3.0% of the TEE/day generated by the accelerometer. These estimated MET values showed similar results as a previous study measuring activity using the doubly-labeled water method. The number of hours per week of MVPA significantly decreased with age, which is also consistent with previous findings. CONCLUSIONS: Estimated reference MET values in this study were similar to those in previous studies of Japanese women. The JNHS questionnaire is therefore useful for epidemiological surveys of midlife working women because it assigns estimated MET values as physical activity intensities.

Identification of Plasmodium falciparum Mitochondrial Malate: Quinone Oxidoreductase Inhibitors from the Pathogen Box.

Malaria is one of the three major global health threats. Drug development for malaria, especially for its most dangerous form caused by Plasmodium falciparum, remains an urgent task due to the emerging drug-resistant parasites. Exploration of novel antimalarial drug targets identified a trifunctional enzyme, malate quinone oxidoreductase (MQO), located in the mitochondrial inner membrane of P. falciparum (PfMQO). PfMQO is involved in the pathways of mitochondrial electron transport chain, tricarboxylic acid cycle, and fumarate cycle. Recent studies have shown that MQO is essential for P. falciparum survival in asexual stage and for the development of experiment cerebral malaria in the murine parasite P. berghei, providing genetic validation of MQO as a drug target. However, chemical validation of MQO, as a target, remains unexplored. In this study, we used active recombinant protein rPfMQO overexpressed in bacterial membrane fractions to screen a total of 400 compounds from the Pathogen Box, released by Medicines for Malaria Venture. The screening identified seven hit compounds targeting rPfMQO with an IC50 of under 5 µM. We tested the activity of hit compounds against the growth of 3D7 wildtype strain of P. falciparum, among which four compounds showed an IC50 from low to sub-micromolar concentrations, suggesting that PfMQO is indeed a potential antimalarial drug target.

Identification of Alprenolol Hydrochloride as an Anti-prion Compound Using Surface Plasmon Resonance Imaging.

Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.

Genetic characterization of Lassa virus strains isolated from 2012 to 2016 in southeastern Nigeria.

Lassa virus (LASV) is endemic in parts of West Africa where it causes Lassa fever (LF), a viral hemorrhagic fever with frequent fatal outcomes. The diverse LASV strains are grouped into six major lineages based on the geographical location of the isolated strains. In this study, we have focused on the lineage II strains from southern Nigeria. We determined the viral sequences from positive cases of LF reported at tertiary hospitals in Ebonyi and Enugu between 2012 and 2016. Reverse transcription-polymerase chain reaction (RT-PCR) showed that 29 out of 123 suspected cases were positive for the virus among which 11 viral gene sequences were determined. Phylogenetic analysis of the complete coding sequences of the four viral proteins revealed that lineage II strains are broadly divided into two genetic clades that diverged from a common ancestor 195 years ago. One clade, consisting of strains from Ebonyi and Enugu, was more conserved than the other from Irrua, although the four viral proteins were evolving at similar rates in both clades. These results suggested that the viruses of these clades have been distinctively evolving in geographically separate parts of southern Nigeria. Furthermore, the epidemiological data of the 2014 outbreak highlighted the role of human-to-human transmission in this outbreak, which was supported by phylogenetic analysis showing that 13 of the 16 sequences clustered together. These results provide new insights into the evolution of LASV in southern Nigeria and have important implications for vaccine development, diagnostic assay design, and LF outbreak management.

Selective Cytotoxicity of Dihydroorotate Dehydrogenase Inhibitors to Human Cancer Cells Under Hypoxia and Nutrient-Deprived Conditions.

Human dihydroorotate dehydrogenase (HsDHODH) is a key enzyme of pyrimidine de novo biosynthesis pathway. It is located on the mitochondrial inner membrane and contributes to the respiratory chain by shuttling electrons to the ubiquinone pool. We have discovered ascofuranone (1), a natural compound produced by Acremonium sclerotigenum, and its derivatives are a potent class of HsDHODH inhibitors. We conducted a structure-activity relationship study and have identified functional groups of 1 that are essential for the inhibition of HsDHODH enzymatic activity. Furthermore, the binding mode of 1 and its derivatives to HsDHODH was demonstrated by co-crystallographic analysis and we show that these inhibitors bind at the ubiquinone binding site. In addition, the cytotoxicities of 1 and its potent derivatives 7, 8, and 9 were studied using human cultured cancer cells. Interestingly, they showed selective and strong cytotoxicity to cancer cells cultured under microenvironment (hypoxia and nutrient-deprived) conditions. The selectivity ratio of 8 under this microenvironment show the most potent inhibition which was over 1000-fold higher compared to that under normal culture condition. Our studies suggest that under microenvironment conditions, cancer cells heavily depend on the pyrimidine de novo biosynthesis pathway. We also provide the first evidence that 1 and its derivatives are potential lead candidates for drug development which target the HsDHODH of cancer cells living under a tumor microenvironment.

Biochemical studies of membrane bound Plasmodium falciparum mitochondrial L-malate:quinone oxidoreductase, a potential drug target.

Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc1 complex inhibitor.

Smoking cessation in pregnancy: psychosocial interventions and patient-focused perspectives.

BACKGROUND: Smoking during pregnancy causes obstetric and fetal complications, and smoking cessation may have great benefits for the mother and the child. However, some pregnant women continue smoking even in pregnancy. OBJECTIVE: To review the literature addressing the prevalence of smoking during pregnancy, explore psychosocial factors associated with smoking, and review the evidence of psychosocial interventions for smoking cessation during pregnancy in recent years. LITERATURE REVIEW: Computerized Internet search results in PubMed for the years spanning from 2004 to 2014, as well as references cited in articles, were reviewed. A search for the keywords "smoking cessation pregnancy" and "intervention" and "clinical trials" yielded 52 citations. Thirty-five citations were identified as useful to this review for the evidence of psychosocial interventions for smoking cessation during pregnancy. RESULTS: The prevalence of smoking during pregnancy differs by country, reflecting the countries' social, cultural, and ethnic backgrounds. Women who had socioeconomic disadvantages, problems in their interpersonal relationships, higher stress, depression, less social support, and who engaged in health-risk behaviors were more prone to smoking during pregnancy. Psychosocial interventions, such as counseling, are effective methods for increasing smoking cessation. CONCLUSION: Smokers may have various psychosocial problems in addition to health problems. It is important to understand each individual's social situation or psychosocial characteristics, and a psychosocial intervention focused on the characteristics of the individual is required.

[Comparison of the Pressure on the Larynx and Tongue Using McGRATH® MAC Video Laryngoscope--Direct Vision versus Indirect Vision].

BACKGROUND: Studies show that McGRATH® MAC (McG) is useful during direct laryngoscopy. However, no study has examined whether McG re- duces pressure on the upper airway tract We compared direct vision with indirect vision concerning pressure on the larynx and tongue. METHODS: Twenty two anesthesiologists and 16 junior residents attempted direct laryngoscopy of airway management simulator using McG with direct vision and indirect vision. Pressure was measured using pressure measurement film. RESULTS: In anesthesiologists group, pressure on larynx was 14.8 ± 2.7 kgf · cm(-2) with direct vision and 12.7 ± 2.7 kgf · cm(-2) with indirect vision (P < 0.05). Pressure on the tongue was 8.8 ± 3.2 kgf cm(-2) with direct vision and 7.6 ± 2.8 kgf · cm(-2) with indirect vision (P = 0.18). In junior residents group, pressure on larynx was 19.0 ± 1.3 kgf · cm(-2) with direct vision and 14.1 ± 3.1 kgf · cm(-2) with indirect vision (P < 0.05). Pressure on the tongue was 15.4 ± 3.6 kgf · cm(-2) with direct vision and 11.2 ± 4.7 kgf · cm(-2) with indirect vision (P < 0.05). CONCLUSIONS: McG with indirect vision can reduce pressure on the upper airway tract.

Analysis of Assembly and Budding of Lujo Virus.

The recently identified arenavirus Lujo virus (LUJV) causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein Z and the cell surface glycoprotein precursor GPC. The L domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor PF-429242 reduced LUJV production.

Contamination of rice by etofenprox, diethyl phthalate and alkylphenols: effects on first delivery and sperm count in mice.

We observed that first delivery was delayed when a group of paired mice fed with non-organic common rice compared to a group fed with organic rice. This led us to hypothesize that pesticides or other soil contaminants may be responsible for the effect on mice reproduction. We then found that the non-organic rice was contaminated with a pesticide etofenprox and nonylphenol, butylphenol and diethyl phthalate which are used as agricultural detergents or plasticizers of agricultural film, that are all suspected to be estrogenic. Therefore, the chemicals were administered to mice at the levels detected in rice, and we subsequently observed that first delivery and sperm count of the animals were significantly impaired. This study is the first to show that rice contaminated with agricultural chemicals affects reproduction in the mammal.

[Frequency and prevention of childhood domestic injury according to age].

OBJECTIVE: This study aimed to: 1) obtain data about occurrence of childhood domestic injuries in Gunma prefecture according to children's age; 2) ascertain parental awareness of injury prevention; and 3) develop ideas for creating concrete strategies of childhood injury prevention. METHODS: The participants were 551 parents of children living in 14 cities/towns in Gunma prefecture that showed interest in cooperating with this survey. A self-reported questionnaire was handed to parents when they took their children to health check-ups provided by the cities/towns either during the child's first year, at 18 months, or at 3 years. Parents completed the questionnaire asking whether their child had been injured at home during the past year, and if so, they were asked about the type of injury, the cause of injury, and what action they took. We also asked whether the parents took specific injury prevention measures at home. Data were analyzed statistically. RESULTS: The injury experienced most frequently during the first year of life was "fall" (30.8%), followed by "ingestion of a foreign body" (22.7%), and then "choking" (11.5%). For children around the age of 18 months, the most frequently experienced injury was "fall" (41.0%), followed by "burn" (20.3%), and "ingestion of a foreign body" (19.3%). At 3 years, "burn" was reported most frequently (32.3%), followed by "fall" (31.0%), and "choking" (14.5%). Chi2-test revealed significant correlations among the three age groups concerning the rate of burn injury, foreign body ingestion, and drowning. The rate of burn injury was higher at 3 years than at 18 months, and also higher at 18 months as compared to under the age of one. In contrast, the rate of foreign body ingestion was higher under the age of one than at 18 months, and also higher at 18 months as compared to the age of 3 years. Drowning was more common at 18 months and 3 years than under the age of one. As for prevention of domestic injury, investigation of preventive means taken according to type of injury revealed that parents taking any of the measures to prevent an injury were significantly more likely to also take other means to prevent that injury. CONCLUSION: Characteristics of injury differed according to children's age. Furthermore, it was clarified that parents' performance of injury prevention depends on their awareness of preventive measures.

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues.

We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named "HS-ovalbumin" in the present study, was found to have a T(m) value of 83 degrees C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.

Dietary habits and selenium intake of residents in mountain and coastal communities in Japan.

We used a Simple Food Frequency Questionnaire (SFFQ) in combination with other dietary approaches to estimate the selenium intake from different food groups based on the average long-term diet, in two rural communities in Japan, one in a mountain area and the other in a coastal area. The intake frequencies of rice and wheat products were significantly different in the two districts. The intake frequencies of fish, meat, and eggs, which are rich in selenium, were not significantly different. The mean dietary selenium intake, estimated from the SFFQ and the 24-h recall method, was 82.7 microg/d (n=234) (range 19.2-180.1 microg/d) in the mountain community. The mean dietary selenium intake estimated from the SFFQ and average value of the normal portion size was 118.0 microg/d (n=123) (range 22.6-255.3 microg/d) in the coastal community. These estimated mean values exceeded the Japanese RDA, although the range of daily selenium intake was large. In the mountain community, fish made the largest contribution to dietary selenium intake (48.2% of daily total), followed by eggs (24.3%), and meat (17.0%). In the coastal community, fish accounted for 57.7% of daily total selenium intake, followed by meat (17.5%), and eggs (16.1%). In both districts, the total contribution of rice and wheat products was around 10%. It was found that the contribution of fish to dietary selenium intake was high and the contribution of cereals was low among Japanese.