Enhanced Production of EPA-Derived Anti-Inflammatory Metabolites after Oral Administration of a Novel Self-Emulsifying Highly Purified EPA Ethyl Ester Formulation (MND-2119).

AIMS: MND-2119 is a novel once-daily dose self-emulsifying formulation of highly purified eicosapentaenoic acid ethyl ester (EPA-E) and is approved as an antihyperlipidemia agent in Japan. It has improved absorption and achieves higher plasma EPA concentrations at Cmax than conventional EPA-E. In the JELIS trial, concomitant use of EPA-E with statin therapy significantly reduced atherosclerotic cardiovascular disease (ASCVD) risks. As a potential mechanism of action of EPA, endogenous formation of EPA-derived anti-inflammatory metabolites is receiving greater attention. This study aims to investigate the endogenous formation of EPA-derived anti-inflammatory metabolites following single and multiple administrations of MND-2119. METHODS: Healthy adult male subjects were randomly assigned to a nonintervention (control) group, MND-2119 2-g/day group, MND-2119 4-g/day group, or EPA-E 1.8-g/day group for 7 days (N=8 per group). Plasma fatty acids and EPA-derived metabolites were evaluated. Peripheral blood neutrophils were isolated, and the production of EPA-derived metabolites from in vitro stimulated neutrophils was evaluated. RESULTS: After single and multiple administrations of MND-2119 2 g/day, there were significant increases in plasma EPA concentration, 18-hydroxyeicosapentaenoic acid (18-HEPE), and 17,18-epoxyeicosatetraenoic acid compared with those of EPA-E 1.8 g/day. They were further increased with MND-2119 4 g/day administration. In neutrophils, the EPA concentration in the MND-2119 2-g/day group was significantly higher compared with that in the EPA-E 1.8-g/day group after multiple administration, and 18-HEPE production was positively correlated with EPA concentration. No safety issues were noted. CONCLUSIONS: These results demonstrate that MND-2119 increases the plasma and cellular concentrations of EPA and EPA-derived metabolites to a greater extent than conventional EPA-E formulations.

Combination of violet light irradiation and collagenase treatments in a rabbit model of keratoconus.

Purpose: We evaluated the use of collagenase treatment to generate a rabbit model of keratoconus and the impact of violet light (VL) irradiation on the disease model in six Japanese White rabbits. Methods: After epithelial debridement, the collagenase group was treated with a collagenase type II solution for 30 min; the control group was treated with a solution without collagenase. Three rabbits also underwent VL irradiation (375 nm, irradiance 310 µW/cm2) for 3 h daily for 7 days after topical collagenase application. Slit-lamp microscopy results, steep keratometry (Ks), corneal astigmatism, central corneal thickness, and axial length were examined before and after the procedure. The corneas were obtained on day 7 for biomechanical evaluation. Results: A significant increase in Ks and corneal astigmatism was observed in the collagenase and VL irradiation groups compared with the control group on day 7. No significant difference was found in the change in corneal thickness between the groups. The elastic modulus at 3, 5, and 10% strain was significantly lower in the collagenase group than in the control group. There was no significant difference in the elastic modulus at any level of strain between the collagenase and VL irradiation groups. The average axial length at day 7 was significantly longer in the collagenase and VL irradiation groups than in the control group. Collagenase treatment induced a model of keratoconus by steepening the keratometric and astigmatic values. There was no significant difference in the observed elastic behavior of normal and ectatic corneas under physiologically relevant stress levels. Conclusion: VL irradiation did not cause regression of corneal steepening in a collagenase-induced model during short-term observation.

Models of Aviation Noise Impact in the Context of Operation Decrease at Tan Son Nhat Airport.

Air traffic bans in response to the spread of the coronavirus have changed the sound situation of urban areas around airports. This study aimed to investigate the effect of this unprecedented event on the community response to noise before and after the international flight operation at Tan Son Nhat Airport (TSN) in March 2020. The "before" survey was conducted in August 2019, and the two "after" surveys were conducted in June and September 2020. Structural equation models (SEMs) for noise annoyance and insomnia were developed by linking the questionnaire items of the social surveys. The first effort aimed to achieve a common model of noise annoyance and insomnia, corresponding to the situation before and after the change, respectively. Approximately, 1200 responses were obtained from surveys conducted in 12 residential areas around TSN in 2019 and 2020. The average daily flight numbers observed in August 2019 during the two surveys conducted in 2020 were 728, 413, and 299, respectively. The sound pressure levels of the 12 sites around TSN decreased from 45-81 dB (mean = 64, SD = 9.8) in 2019 to 41-76 dB (mean = 60, SD = 9.8) and 41-73 dB (mean = 59, SD = 9.3) in June and September 2020, respectively. The SEM indicated that the residents' health was related to increased annoyance and insomnia.

The Effect of an Alternative Definition of "Percent Highly Annoyed" on the Exposure-Response Relationship: Comparison of Noise Annoyance Responses Measured by ICBEN 5-Point Verbal and 11-Point Numerical Scales.

Since the development of the 5-point verbal and 11-point numerical scales for measuring noise annoyance by the ICBEN Team 6, these scales have been widely used in socio-acoustic surveys worldwide, and annoyance responses have been easily compared internationally. However, both the top two categories of the 5-point verbal scale and the top three ones of the 11-point numerical scale are correspond to high annoyance, so it is difficult to precisely compare annoyance responses. Therefore, we calculated differences in day-evening-night-weighted sound pressure levels (Lden) by comparing values corresponding to 10% highly annoyed (HA) on Lden_%HA curves obtained from measurements in 40 datasets regarding surveys conducted in Japan and Vietnam. The results showed that the Lden value corresponding to 10% HA using the 5-point verbal scale was approximately 5 dB lower than that of the 11-point numerical scale. Thus, some correction is required to compare annoyance responses measured by the 5-point verbal and the 11-point numerical scales. The results of this study were also compared with those of a survey in Switzerland.

Combination of violet light irradiation and collagenase treatments in a rabbit model.

PURPOSE: To investigate the use of collagenase type II for generating a rabbit model of keratoconus and to evaluate the impact of violet light (VL) irradiation on the disease model. METHODS: Six Japanese White rabbits were used. After epithelial debridement, the collagenase group was treated with a collagenase type II solution for 30 min; the control group was treated with a solution without collagenase. Three rabbits also underwent VL irradiation (375 nm, irradiance 310 µW/cm2) for 3 h daily for 7 days after topical collagenase application. Slit-lamp microscopy, steep keratometry (Ks), corneal astigmatism, central corneal thickness, and axial length were examined before and after the procedure. The corneas were obtained on day 7 for biomechanical evaluation. RESULTS: A significant increase in Ks and corneal astigmatism was observed in the collagenase and VL irradiation groups compared with the control group at day 7. No significant difference was found in the change in corneal thickness between the groups. The elastic modulus at 10% strain but not at 3% and 5% strain in the collagenase group was significantly lower than that in the control group. There was no significant difference in the elastic modulus at each level of strain between the collagenase and VL irradiation groups. The average axial length at day 7 in the collagenase group was significantly longer than that in the control group. CONCLUSIONS: Collagenase type II treatment can mimic keratoconus with increased corneal keratometry and astigmatism. There was no significant difference in the observed elastic behaviour of normal and ectatic corneas under physiologically relevant stress levels. VL irradiation did not cause regression of corneal steepening in this model with short-term observation.

Short-Chain Guide RNA for Site-Directed A-to-I RNA Editing.

Site-directed RNA editing is a promising genetic modification technology for therapeutic and pharmaceutical applications. We previously constructed adenosine deaminases acting on RNA (ADAR)-guiding RNAs (AD-gRNAs) that direct A-to-I RNA editing activity of native human ADAR2 into a programmable target site. In this study, we developed the short-chain AD-gRNA (shAD-gRNA) as a potential basic framework for practical RNA-editing oligonucleotides. Based on knowledge of previous AD-gRNA, shAD-gRNAs were designed to have the shortest possible sequence for the induction of editing activity. In vitro, compared to the original AD-gRNA, the shAD-gRNAs showed similar or superior editing induction activity, depending on the target RNA sequence, and had lower off-target editing activity around the target site, which is predicted to be a hotspot for off-target editing. Moreover, shAD-gRNAs achieved target RNA editing with both exogenous and endogenous human ADARs in cultured cells. Our results present shAD-gRNA as a short basic framework that would be applicable to further development for practical RNA-editing oligonucleotides.

Effects of Changes in Acoustic and Non-Acoustic Factors on Public Health and Reactions: Follow-Up Surveys in the Vicinity of the Hanoi Noi Bai International Airport.

Herein, the effects of changes in acoustic and non-acoustic factors on public health and reactions were assessed using two follow-up investigations; this was achieved after three surveys were conducted on the impact of the step change in noise caused by the increased number of flights at the Noi Bai International Airport in Hanoi (Vietnam) after the new terminal building was opened to the public. Exposure-response relationships established in the follow-up studies were less in number than those established in 2015 after the step change had occurred, and were almost similar to the relationship established in the survey conducted before the step change; however, these relationships were significantly greater than those established in the European Union position paper. Comparisons between respondents with high blood pressure and insomnia ratios at different noise level ranges showed that there is no significant association between ratios of high blood pressure and day-evening-night noise levels; however, an exposure-response relationship was discovered between insomnia and night-time noise levels. Non-acoustic factors such as noise sensitivity, sound insulation capacity of houses, and length of residence were found to curb the respondents' annoyance, insomnia, and high blood pressure. Thus, an improvement in residence quality and a restriction on nighttime flight operation is necessitated.

Metabolomic Analysis of Diet-Induced Obese Mice Supplemented with Eicosapentaenoic Acid.

Eicosapentaenoic acid (EPA) is an omega-3 fatty acid with anti-inflammatory effects. To determine the effects of EPA on metabolic pathways in obese adipose tissues and liver, mice were fed normal chow diet (NCD), high-fat diet (HFD), or 3% EPA-containing high fat diet (HFD+EPA) for 8 weeks. Metabolomic analysis was performed using epididymal adipose tissues (epi WAT) and liver. Metabolites that were specifically elevated in HFD+EPA, were assessed for their anti-inflammatory properties using RAW264.7 macrophage cells. Body and adipose tissue weights were significantly higher in HFD than NCD, and lower in HFD+EPA than HFD. Plasma insulin levels were significantly higher in HFD than NCD, and lower in HFD+EPA compared with HFD. Plasma monocyte chemotactic protein-1 (MCP-1) levels were higher in HFD than NCD, and tended to be lower in HFD+EPA than HFD. The levels of intermediate metabolites in the glycolytic pathways were lower in HFD compared with NCD and HFD+EPA in both epi WAT and liver, while intermediate metabolites of the TCA cycles were elevated in HFD and HFD+EPA compared with NCD in epi WAT. Among the metabolites in epi WAT, the levels of thiaproline, phenaceturic acid, and pipecolic acid were specifically elevated in HFD+EPA, but not in HFD or NCD. Treatment of RAW264.7 cells with thiaproline significantly ameliorated LPS-induced iNOS expression, while pipecolic acid inhibited LPS-induced IL-1ß expression. These results suggest that EPA normalizes glycolytic pathway intermediates in both epi WAT and liver, and induces metabolites with anti-inflammatory properties.

Effects of Railway Elevation, Operation of a New Station, and Earthquakes on Railway Noise Annoyance in Kumamoto, Japan.

This study investigated the effects of railway elevation, operation of a new station, and earthquakes on railway noise annoyance in two areas along a conventional railway line (CRL) adjacent to the Kyushu Shinkansen line: the north area with the CRL elevation and the south area with the operation of the new station, both of which occurred in March 2016. In April 2016, Kumamoto region was struck by a series of large earthquakes, prompting their inclusion in this study, as frequent aftershocks with loud ground rumbling might make people more sensitive to railway noise and vibration. Socioacoustic surveys were performed in both areas before and after the earthquakes. Because very few respondents in the north area reported that they were “highly annoyed,” further analysis was conducted on data from the south area. The exposure⁻annoyance relationship was found to be significantly higher in 2017 than in 2011 despite lower noise exposure. Multiple logistic regression analysis showed that Lden, noise sensitivity, and serious damage by the earthquakes in addition to the operation of the new station significantly affected the annoyance in both detached and apartment houses. However, when the earthquakes caused minimal damage, they did not significantly affect annoyance.

Community response to a step change in the aircraft noise exposure around Hanoi Noi Bai International Airport.

In December 2014, a new terminal building was opened at the Hanoi Noi Bai International Airport (HNBIA) with two runways, causing a 20%-30% increase in the number of flights. Three socio-acoustic surveys were conducted in August-September 2014, February-March 2015, and August-September 2015, to contribute not only to the environmental impact assessments and aircraft noise policies in Vietnam but also to more global intervention studies. Because of the change of runway use, in addition to the increased number of flights, noise exposure at each site changed considerably among the surveys. Changes in the noise exposure from the first to the second or third survey (ΔLden and ΔLnight) were used as a measure of exposure change. Multiple logistic regression analysis showed that ΔLden has a significant positive effect on annoyance regardless of ΔLden ranges, but the effect of ΔLnight on insomnia was significant only for ΔLnight > 0. Annoyance increase in the overall ΔLden range may be caused by the respondents' recognition of increase in emission in addition to practical increase in exposure. More severe attitudes to airplanes around HNBIA might increase annoyance even if noise exposure decreases. Thus, the change effect clearly occurs in annoyance but partially in insomnia.

Combined Effects of High-Speed Railway Noise and Ground Vibrations on Annoyance.

The Shinkansen super-express railway system in Japan has greatly increased its capacity and has expanded nationwide. However, many inhabitants in areas along the railways have been disturbed by noise and ground vibration from the trains. Additionally, the Shinkansen railway emits a higher level of ground vibration than conventional railways at the same noise level. These findings imply that building vibrations affect living environments as significantly as the associated noise. Therefore, it is imperative to quantify the effects of noise and vibration exposures on each annoyance under simultaneous exposure. We performed a secondary analysis using individual datasets of exposure and community response associated with Shinkansen railway noise and vibration. The data consisted of six socio-acoustic surveys, which were conducted separately over the last 20 years in Japan. Applying a logistic regression analysis to the datasets, we confirmed the combined effects of vibration/noise exposure on noise/vibration annoyance. Moreover, we proposed a representative relationship between noise and vibration exposures, and the prevalence of each annoyance associated with the Shinkansen railway.

Association Between Ocular Dominance and Anisometropic Hyperopia.

INTRODUCTION AND PURPOSE: Anisometropia, a relative difference in the refractive state of the two eyes, is common in hyperopic patients. We investigated the association between ocular dominance (sighting dominance) and refractive asymmetry in patients with hyperopia. METHODS: This retrospective study included 223 hyperopic patients with a mean age of 10.1 ± 3.6 years (range 3 to 21 years). Refractive error was measured with cycloplegic refraction, and axial length was measured with IOLMaster® (Carl Zeiss Meditec, Dublin, CA). Ocular dominance was assessed with the hole-in-the-card test. The amount of hyperopic anisometropia was subdivided into four groups: less than 0.50 D, 0.50-0.99 D, 1.00-1.99 D, and 2.00 D or greater. RESULTS: Ocular dominance of the right and left eye was seen in 66% and 34% of the patients, respectively. The nondominant eye had higher hyperopia, astigmatism, and shorter axial length than the dominant eye (P < 0.001). In the group with spherical equivalent anisometropia of ≥0.50 D in particular, the nondominant eye was significantly more hyperopic and had shorter axial length than the dominant eye (both P < 0.001). CONCLUSIONS: The current study revealed that the nondominant eye had a greater hyperopic refractive error and shorter axial length than the dominant eye, in patients who had a high degree of anisometropia in particular.

Exposure-response relationship of wind turbine noise with self-reported symptoms of sleep and health problems: A nationwide socioacoustic survey in Japan.

The association of wind turbine noise (WTN) with sleep and physical/mental health has not been fully investigated. To investigate the relationship of WTN with the prevalence of self-reported symptoms of sleep and health problems, a socioacoustic survey of 1079 adult residents was conducted throughout Japan (2010-2012): 747 in 34 areas surrounding wind turbine plants and 332 in 16 control areas. During face-to-face interviews, the respondents were not informed of the purpose of the survey. Questions on symptoms such as sleeplessness and physical/mental complaints were asked without specifying reasons. Insomnia was defined as having one or any combination of the following that occurs three or more times a week and bothers a respondent: Difficulty initiating sleep, difficulty maintaining sleep, premature morning awakening, and feeling of light overnight sleep. Poor health was defined as having high scores for health complaints, as determined using the Total Health Index, exceeding the criteria proposed by the authors of the index. The noise descriptor for WTN was LAeq,n outdoor, estimated from the results of actual measurement at some locations in each site. Multiple logistic analysis was applied to the LAeq,n and insomnia or poor health. The odds ratio (OR) of insomnia was significantly higher when the noise exposure level exceeded 40 dB, whereas the self-reported sensitivity to noise and visual annoyance with wind turbines were also independently associated with insomnia. OR of poor health was not significant for noise exposure, but significant for noise sensitivity and visual annoyance. The above two moderators appear to indicate the features of respondents who are sensitive to stimuli or changes in their homeostasis.

Efficient gene-targeting in rat embryonic stem cells by CRISPR/Cas and generation of human kynurenine aminotransferase II (KAT II) knock-in rat.

The relative proportion of kynurenine aminotransferase (KAT) I-IV activities in the brain is similar between humans and rats. Moreover, KAT II is considered to be the main enzyme for kynurenic acid production in the brain. Taken together, human KAT II knock-in (hKAT II KI) rats will become a valuable tool for the evaluation of KAT II targeted drugs as a human mimetic model. Although we initially tried the approach by conventional gene-targeting via embryonic stem cells (ESCs) to generate them, we had to give up the production because of no recombinant ESCs. Accordingly, we developed a method to improve the efficiency of homologous recombination (HR) in ESCs by the combination with the CRISPR/Cas system. Co-electroporation of Cas9 plasmid, single guide RNA plasmid and hKAT II KI vector increased the number of drug-resistant colonies and greatly enhanced the HR efficiency from 0 to 36 %. All the clones which we obtained showed the same sequence as designed. These recombinant clones resulted in chimeras that transmitted the hKAT II KI allele to their offspring. hKAT II KI rats showed no reduction of KATs mRNA expression and the amount of kynurenic acid was similar between the hKAT II KI rats and the wild type in their brains. These results indicate that the methodology presented in this report can overcome the problem encountered in conventional gene-targeting that prevented production of humanized rats.

Community response to noise in Vietnam: exposure-response relationships based on the community tolerance level.

Social surveys on noise annoyance have been conducted in five different cities in Vietnam. The surveys included both aircraft noise (three airports) and road traffic noise (five cities). The main objective for these studies was to establish dose-response functions that were representative for Vietnam. The results have been compared with results from similar surveys from other regions. Dose-response functions for aircraft noise in Vietnam showing the percentage of highly annoyed people versus the noise level are nearly identical to those presented in the European Noise Directive [European Commission (2002). http://ec.europa.eu/environment/noise/directive.htm]. For road traffic noise, however, the results indicate that people in Vietnam are more tolerant. The noise levels can be increased by 5-10 dB in order to have a response similar to the curve recommended by the European Commission.

Early-onset cognitive deficits and axonal transport dysfunction in P301S mutant tau transgenic mice.

Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD) are neurodegenerative "tauopathies" characterized by hyperphosphorylated tau accumulation and neurofibrillary tangles. The P301S mutation of tau, a causal mutation of a familial type of FTLD, is believed to be involved in neurodegenerative progression. We developed a transgenic mouse, named TPR50, harboring human P301S tau. Tau phosphorylation in the hippocampus of TPR50 mice increased with age, particularly at S202/T205. Insolubilization and intracellular accumulation of tau were detected in the hippocampus by 9 months of age. Expression of calbindin was significantly reduced in 6- and 9-month-old TPR50 mice but not in 3-month-old mice. TPR50 mice demonstrated cognitive dysfunction at 5 months. At this age or earlier, although no intracellular tau accumulation was observed in the hippocampus, abnormally increased microtubule (MT)-related proteins and MT hyperdynamics in the hippocampus, and impaired axonal transport in the septo-hippocampal pathway were already observed. Therefore, cognitive dysfunction in TPR50 mice may result from early MT dysfunction and impaired axonal transport rather than accumulation of insoluble tau and neurodegeneration. TPR50 mice are a valuable new model to study progression of tauopathies at both the behavioral and neurocellular levels and may also prove useful for testing new therapies for neurodegenerative diseases.

Generation of gene-targeted mice using embryonic stem cells derived from a transgenic mouse model of Alzheimer's disease.

Gene-targeting technology using mouse embryonic stem (ES) cells has become the "gold standard" for analyzing gene functions and producing disease models. Recently, genetically modified mice with multiple mutations have increasingly been produced to study the interaction between proteins and polygenic diseases. However, introduction of an additional mutation into mice already harboring several mutations by conventional natural crossbreeding is an extremely time- and labor-intensive process. Moreover, to do so in mice with a complex genetic background, several years may be required if the genetic background is to be retained. Establishing ES cells from multiple-mutant mice, or disease-model mice with a complex genetic background, would offer a possible solution. Here, we report the establishment and characterization of novel ES cell lines from a mouse model of Alzheimer's disease (3xTg-AD mouse, Oddo et al. in Neuron 39:409-421, 2003) harboring 3 mutated genes (APPswe, TauP301L, and PS1M146V) and a complex genetic background. Thirty blastocysts were cultured and 15 stable ES cell lines (male: 11; female: 4) obtained. By injecting these ES cells into diploid or tetraploid blastocysts, we generated germline-competent chimeras. Subsequently, we confirmed that F1 mice derived from these animals showed similar biochemical and behavioral characteristics to the original 3xTg-AD mice. Furthermore, we introduced a gene-targeting vector into the ES cells and successfully obtained gene-targeted ES cells, which were then used to generate knockout mice for the targeted gene. These results suggest that the present methodology is effective for introducing an additional mutation into mice already harboring multiple mutated genes and/or a complex genetic background.

Community response to noise.

Activities from 2008 to 2011 by ICBEN community response to noise team were summarized. That is, individual community-based indexes such as community tolerance Level, Zuricher Fluglarm Index (ZFI) and Frankfurter Fluglarm Index (FFI/FNI) were newly proposed, differences in railway bonus between Europe and Asia were discussed by a Swedish survey, socio-acoustic surveys were reported from developing countries, and annoyance equivalents and dominant source models were proposed as the adequate combined noise model. Furthermore, not only negative, but also positive aspects of sound were discussed as soundscape studies. Finally, seven items were listed as future team activities.

Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.

One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.