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Water sampling and filtration of environmental DNA (eDNA) analysis have been performed by several different methods, and each method may yield a different species composition or eDNA concentration. Here, we investigated the eDNA of seawater samples directly collected by SCUBA to compare two widely used filtration methods: open filtration with a glass filter (GF/F) and enclosed filtration (Sterivex). We referred to biomass based on visual observation data collected simultaneously to clarify the difference between organism groups. Water samples were collected at two points in the Sea of Japan in May, September and December 2018. The respective samples were filtered through GF/F and Sterivex for eDNA extraction. We quantified the eDNA concentration of five fish and two cnidarian species by quantitative polymerase chain reaction (qPCR) using species-specific primers/probe sets. A strong correlation of eDNA concentration was obtained between GF/F and Sterivex; the intercepts and slopes of the linear regression lines were slightly different in fish and jellyfish. The amount of eDNA detected using the GF/F filtration method was higher than that detected using Sterivex when the eDNA concentration was high; the opposite trend was observed when the eDNA concentration was relatively low. The concentration of eDNA correlated with visually estimated biomass; eDNA concentration per biomass in jellyfish was approximately 700 times greater than that in fish. We conclude that GF/F provides an advantage in collecting a large amount of eDNA, whereas Sterivex offers superior eDNA sensitivity. Both filtration methods are effective in estimating the spatiotemporal biomass size of target marine species.

Preventing mosquito-borne infectious diseases requires that vector mosquitoes are monitored and controlled. Targeting immature mosquitoes (eggs, larvae, and pupae), which have less mobility than adults, is an effective management approach. However, conducting these surveys is often difficult due to the limitations of morphological classification and survey costs. The application of environmental DNA (eDNA) analysis can solve these issues because it allows easy estimation of species distribution and morphology-independent species identification. Although a few previous studies have reported mosquito eDNA detection, there is a gap in knowledge regarding the dynamics related to the persistence of immature mosquito eDNA. We used Culex pipiens pallens , a vector of West Nile fever, as a model species. First, we developed a species-specific detection assay and confirmed its specificity using in silico and in vitro tests. Next, we conducted laboratory experiments using breeding tanks. Water samples were collected at each developmental stage. In addition, water samples were collected daily until the seventh day after emergence from the pupae. We quantified eDNA using real-time PCR with the developed assay to investigate the dynamics of mosquito eDNA. The specificity of the developed assay was confirmed by in silico and in vitro tests. Mosquito eDNA was detected at all developmental stages and detected up to seven days after emergence of pupae. In particular, high concentrations of eDNA were detected immediately after hatching from eggs and after emergence from pupae. Highly frequent positive eDNA signals were continuously detected between egg hatching and pupa hatching. Mosquito eDNA was detected immediately after the eggs were introduced, and eDNA-positive detections continued until pupae emergence, suggesting that eDNA analysis is useful for monitoring mosquito larvae. In the future, monitoring immature mosquitoes using eDNA analysis will contribute to prevent mosquito-borne infectious diseases.

Aqueous environmental DNA (eDNA) analysis has been applied to the monitoring of various ecosystems and taxa, and the characteristics of aqueous eDNA have been previously studied. In contrast, although sedimentary eDNA has been used to restore past information, the characteristics of sedimentary eDNA are not well understood. In this study, we compared the properties of sedimentary and aqueous eDNA of macro‐organisms. First, to clarify the preservation ability of sediments, we compared the difference in decay rates between aqueous and sedimentary eDNA using samples collected from a biotope (an artificial pond prepared with concrete). Next, to clarify the biological information retained in sedimentary eDNA both qualitatively and quantitatively, we compared eDNA concentrations between sediment and water samples collected simultaneously from a lake, and the fish species detected by eDNA metabarcoding were also compared. The results demonstrated the following: (a) the decay rate (decreased eDNA copy number divided by the initial eDNA copy number per unit time) of sedimentary eDNA (0.00033 ± 0.000049 [mean ± SE]/hr) was lower than that of aqueous eDNA (0.01863 ± 0.0011/hr); (b) sedimentary eDNA concentration of the mitochondrial marker of three fish species was higher than aqueous eDNA concentration for the same sample weight (12.5–1,456.9 times); and (c) the species composition obtained by metabarcoding was not significantly different between sediment and water; however, considering the lower decay rate of sedimentary eDNA, using both sample types may provide more comprehensive information of species distribution. Thus, sedimentary eDNA analysis will expand future biomonitoring and ecological studies by providing a difference in timescale. We found that the degradation rate of sedimentary eDNA was lower than that of aqueous eDNA. Additionally, sedimentary eDNA concentration was higher than aqueous eDNA concentration for the same sample weight. Further, different fish species were detected in sediment and water samples by eDNA metabarcoding.

Summary We estimated the number of hip fracture patients in 2012 in Japan and investigated the trends in incidence during a 25-year period from 1987 to 2012. Despite the increasing number of patients, the incidence of hip fracture in both men and women aged 70–79 years showed the possibility of decline. Introduction The objectives of this study were to estimate the number of hip fracture patients in 2012, to investigate the trends in incidence during a 25-year period from 1987 to 2012, and to determine the regional differences in Japan. Methods Data were collected through a nationwide survey based on hospitals by a mail-in survey. Hip fracture incidences by sex and age and standardized incidence ratios by region were calculated. Results The estimated numbers of new hip fracture patients in 2012 were 175,700 in total (95 % CI 170,300–181,100), 37,600 (36,600–38,600) for men and 138,100 (134,300–141,900) for women. The incidence rates in both men and women aged 70–79 years were the lowest in the 20-year period from 1992 to 2012. The incidence was higher in western areas of Japan than that in eastern areas in both men and women; however, the difference in the incidence of hip fracture between western and eastern areas is becoming smaller. Conclusions Despite the increasing number of new patients, the incidence of hip fracture in both men and women aged 70–79 years showed the possibility of decline. The exact reasons for this are unknown, but various drugs for improving bone mineral density or preventing hip fracture might have influenced the results. A decrease in the differences in nutrient intake levels might explain some of the change in regional differences in Japan.

Mycetoma is a tropical disease caused by several fungi and bacteria present in the soil. Fungal mycetoma and eumycetoma are especially challenging to treat; therefore, prevention, early diagnosis, and early treatment are important, but it is also necessary to understand the geographic distribution of these pathogenic fungi. In this study, we used DNA metabarcoding methodology to identify fungal species from soil samples. Soil sampling was implemented at seven villages in an endemic area of Sennar State in Sudan in 2019, and ten sampling sites were selected in each village according to land-use conditions. In total, 70 soil samples were collected from ground surfaces, and DNA in the soil was extracted with a combined method of alkaline DNA extraction and a commercial soil DNA extraction kit. The region for universal primers was selected to be the ribosomal internal transcribed spacer one region for metabarcoding. After the second PCR for DNA library preparation, the amplicon-based DNA analysis was performed using next-generation sequencing with two sets of universal primers. A total of twelve mycetoma-causative fungal species were identified, including the prime agent, Madurella mycetomatis , and additional pathogens, Falciformispora senegalensis and Falciformispora tompkinsii , in 53 soil samples. This study demonstrated that soil DNA metabarcoding can elucidate the presence of multiple mycetoma-causative fungi, which may contribute to accurate diagnosis for patient treatment and geographical mapping.

Environmental DNA (eDNA) consists of DNA fragments shed from organisms into the environment, and can be used to identify species presence and abundance. This study aimed to reveal the dispersion and degradation processes of eDNA in the sea. Caged fish were set off the end of a pier in Maizuru Bay, the Sea of Japan, and their eDNA was traced at sampling stations located at the cage and 10, 30, 100, 300, 600 and 1000 m distances from the cage along two transect lines. Sea surface water was collected at each station at 0, 2, 4, 8, 24 and 48 h after setting the cage, and again after removing the cage. Quantitative PCR analyses using a species-specific primer and probe set revealed that the target DNA was detectable while the cage was present and for up to 1 h after removing the cage, but not at 2 h or later. Among the 57 amplified samples, 45 (79%) were collected within 30 m from the cage. These results suggest that eDNA can provide a snapshot of organisms present in a coastal marine environment.

Objectives To clarify the prevalence, frequent causes and distinct features of hypertrophic pachymeningitis (HP) according to background conditions in a nationwide survey in Japan. Methods The study began with a preliminary survey to determine the approximate number of HP patients diagnosed from 1 January 2005 to 31 December 2009, and was followed by a questionnaire survey for clinical and laboratory findings. HP was defined as a condition with thickening of the cranial or spinal dura mater with inflammation, evidenced by MRI or histology. Results Crude HP prevalence was 0.949/100 000 population. The mean age at onset was 58.3±15.8 years. Among 159 cases for whom detailed data were collated, antineutrophil cytoplasmic antibody (ANCA)-related HP was found in 54 cases (34.0%) and IgG4/multifocal fibrosclerosis (MFS)-related HP in 14 cases (8.8%). Seventy cases (44.0%) were classified as ‘idiopathic’ and 21 (13.2%) as ‘others’. ANCA-related HP cases showed a female preponderance, a higher age of onset, and higher frequencies of otological symptoms and elevated systemic inflammatory biomarkers, but lower frequencies of diplopia compared with idiopathic HP. IgG4/MFS-related HP cases showed a marked male predominance; all had cranial HP while none had isolated spinal HP or decreased sensation. Conclusions HP is not extremely rare. ANCA-related HP is the most frequent form, followed by IgG4/MFS-related HP. Both forms have unique features, which may help to differentiate background causes.

Invasive species are one of the most significant factors affecting biodiversity. American mink ( Neovison vison ) was introduced to Hokkaido, Japan, and is known to compete with other medium-sized mammals and prey on freshwater fish. Therefore, it is important to understand their distribution and the types of environments they prefer. We developed an N. vison -specific environmental DNA (eDNA) detection assay to estimate their distribution. Applying this assay to water samples from 48 rivers in the Shiretoko Peninsula, the World Natural Heritage site in Hokkaido, N. vison -specific DNA was identified in 10 rivers. Including seven rivers from a previous study on N. vision distribution in the peninsula, the environmental characteristics of the 17 rivers with the potential establishment of N. vison populations were investigated using a generalized linear model. The evaluated environmental factors included eDNA concentrations of two salmonid species ( Salvelinus curilus and Oncorhynchus masou, potential food resources for N. vison ), the presence of salmon hatchery and release programs, land uses around the rivers, and river structures. While the estimated N. vison distribution did not show a clear association with the eDNA concentrations of the two salmonid species, it showed positive and significant associations with the salmon release programs ( p  = 0.031) and with the proportion of farmland ( p  = 0.034). These findings imply that human activities have the potential not only to cause the introduction of invasive species but also unintentionally to contribute to the establishment of such species in new environments.

Opisthorchiasis, caused by the liver fluke Opisthorchis viverrini , is endemic to Southeast Asian countries and constitutes a major health problem as it increases the risk of cholangiocarcinoma. However, owing to the complicated life cycle of O. viverrini , there is no rapid method for monitoring the risk of infection in the environment. The inability to identify water sources at risk of infection presents a challenge in implementing preventive measures in areas where fish are a significant component of the local diet. Using the environmental DNA (eDNA) approach to detect parasitic DNA in water samples, it is possible to understand the distribution of O. viverrini and the dynamics of infection with high accuracy. However, the sensitivity of environmental samples remains a bottleneck. In this study, we developed a highly sensitive method for detecting O. viverrini eDNA by designing a multi-marker assay targeting two mitochondrial and two nuclear genes. We compared its performance with single-marker assays using tissue-derived DNA. The multi-marker assay exhibited the highest detection sensitivity. We also collected data from 56 sites in Sakon Nakhon, Thailand, in October 2022, and tested them for eDNA detection of O. viverrini . Eleven sites were positive in the multi-marker assay, whereas five sites were positive in the single-marker assay. This multi-marker assay can be used in the field to detect small organisms and rare species, other than parasites, that are expected to have low levels of eDNA.

To investigate the effects of continuous intervention with branched chain amino acids-enriched nutritional supplements from the acute phase to convalescent rehabilitation wards in inpatients with gait impairments. Open-label, randomized, parallel-group comparison study (UMIN Clinical Trials Registry ID: UMIN000018640). Acute care and convalescent rehabilitation wards We studied 80 patients undergoing stand/gait training. Participants in the intervention group (RJ group) received nutritional supplements (jelly foods comprising 2500 mg BCAA and 20 IU vitamin D) twice a day until hospital discharge. The primary outcome was the motor components of the Functional Independence Measure (FIM-m), and the secondary outcome was skeletal muscle mass index. Analyses were conducted on 55 patients who were able to perform stand/gait training continuously from the acute until the recovery phases. FIM-m was significantly elevated in the RJ group and the control group, but no difference was noted between the two groups. Only the RJ group showed a significant increase in skeletal muscle mass index, and the amount of variation was significantly different between the two groups (the control group decreased an average of 2.2% and the RJ group increased an average of 4.3%; P = 0.014). A significant decrease in body weight was found only in the control group (P = 0.084). Nutritional interventions using branched chain amino acids (BCAA)-enriched nutritional supplements demonstrated no significant difference in activities of daily living; however, an increase in skeletal muscle mass was noted. Skeletal muscle mass and body weight differed significantly between the two groups, and BCAA-enriched nutritional supplements intake in acute and convalescent rehabilitation wards may be effective for the prevention of malnutrition and sarcopenia.