Association of combined low physical activity and low dietary diversity with mild cognitive impairment among community-dwelling Japanese older adults.

Objective: This study aimed to investigate the potential association between the combination of low physical activity and low dietary diversity with mild cognitive impairment (MCI) in older Japanese adults. Methods: Data from 600 older adults (mean age 74.1 ± 6.4 years; 62.0% women) were analyzed. We evaluated dietary variety based on the Food Frequency Score (FFS) (maximum 30 points) by assessing the one-week consumption frequencies of ten foods. An FFS of ≤ 16 indicated low dietary diversity. We assessed MCI using the National Center for Geriatrics and Gerontology (NCGG) Functional Assessment Tool. Physical activity levels was determined based on participant responses to two questions: "Do you engage in moderate levels of physical exercise or sports aimed at health?" and "Do you engage in low levels of physical exercise aimed at health?". Participants who responded "No" to both questions were classified as having low physical activity levels. We classified the participants into robust, low-dietary diversity, low-physical activity, and coexistence groups. Results: The overall prevalence of MCI was 20.7%, with rates in the robust, low dietary diversity, low physical activity, and coexistence groups of 17.7%, 24.7%, 25.0%, and 41.9%, respectively. Multiple logistic regression analysis revealed that low dietary diversity and physical activity were associated with MCI in older adults (odds ratio [OR] 2.80, 95% confidence interval [CI] 1.22-6.28). Conclusions: The results of the present study demonstrated the association of the co-occurrence of low dietary diversity and low physical activity with MCI. Older adults with both risk factors may require early detection, as well as physical activity and dietary interventions.

Associations of eating out and dietary diversity with mild cognitive impairment among in community-dwelling older adults.

Background: Dementia is a critical later life health issue that occurs among members of aging societies. This study examined the relationships between eating out, dietary diversity, and mild cognitive impairment (MCI) among community-dwelling older adults. Methods: We analyzed data from 597 older adults (median age 73.0 years [interquartile range 69.0-78.0] and 62.6% females). We applied the Food Frequency Score (FFS) to evaluate diet variety and the weekly consumption frequencies of ten food items were determined. The Functional Assessment Tool from the National Center for Geriatrics and Gerontology was used to evaluate MCI. Finally, we asked the participants how often they ate out each month; those who replied 'none' were categorized into the "non-eating out" group. Results: The overall prevalence of MCI was 122 (20.4%), with a higher prevalence in the low dietary diversity group than in the high dietary diversity group (28.6% vs. 18.6%). After adjusting for covariates, the participants who self-described as not eating out were independently associated with low dietary diversity (odds ratio [OR]: 1.97, 95% confidence interval [CI]: 1.20-3.20), while low dietary diversity was associated with MCI (OR: 1.72, 95% CI: 1.02-2.87). Structural equation models revealed that not eating out had no direct effect on MCI but was associated with MCI via low dietary diversity (root mean square error of approximation = 0.030, goodness-of-fit index=0.999, and adjusted goodness-of-fit index = 0.984). Conclusions: Although non-eating out may not have a direct effect on MCI, an indirect relationship may exist between eating-out habits and MCI via dietary diversity status.

A Lipid Nanoparticle-Based Method for the Generation of Liver-Specific Knockout Mice.

Knockout mice are useful tools that can provide information about the normal function of genes, including their biochemical, developmental, and physiological roles. One problem associated with the generation of knockout mice is that the loss of some genes of interest produces a lethal phenotype. Therefore, the use of conditioned knockout mice, in which genes are disrupted in specific organs, is essential for the elucidation of disease pathogenesis and the verification of drug targets. In general, conditional knockout mice are produced using the Cre/loxP system; however, the production of the large numbers of Cre/flox knockout and control mice required for analysis requires substantial time and effort. Here, we describe the generation of liver-specific conditional knockout mice via the introduction of lipid nanoparticles encapsulating Cre mRNA into the liver of floxed mice. This technique does not require the production of offspring by mating floxed mice and is therefore more convenient than the conventional method. The results presented here demonstrate that the LNP-based method enables liver-specific gene knockout in a short period of time.

Efficient generation of epigenetic disease model mice by epigenome editing using the piggyBac transposon system.

BACKGROUND: Epigenome-edited animal models enable direct demonstration of disease causing epigenetic mutations. Transgenic (TG) mice stably expressing epigenome-editing factors exhibit dramatic and stable changes in target epigenome modifications. Successful germline transmission of a transgene from founder mice to offspring will yield a sufficient number of epigenome-edited mice for phenotypic analysis; however, if the epigenetic mutation has a detrimental phenotypic effect, it can become difficult to obtain the next generation of animals. In this case, the phenotype of founder mice must be analyzed directly. Unfortunately, current TG mouse production efficiency (TG founders per pups born) is relatively low, and improvements would increase the versatility of this technology. RESULTS: In the current study, we describe an approach to generate epigenome-edited TG mice using a combination of both the dCas9-SunTag and piggyBac (PB) transposon systems. Using this system, we successfully generated mice with demethylation of the differential methylated region of the H19 gene (H19-DMR), as a model for Silver-Russell syndrome (SRS). SRS is a disorder leading to growth retardation, resulting from low insulin-like growth factor 2 (IGF2) gene expression, often caused by epimutations at the H19-IGF2 locus. Under optimized conditions, the efficiency of TG mice production using the PB system was approximately threefold higher than that using the conventional method. TG mice generated by this system showed demethylation of the targeted DNA region and associated changes in gene expression. In addition, these mice exhibited some features of SRS, including intrauterine and postnatal growth retardation, due to demethylation of H19-DMR. CONCLUSIONS: The dCas9-SunTag and PB systems serve as a simple and reliable platform for conducting direct experiments using epigenome-edited founder mice.

The Association between Dietary Variety and Physical Frailty in Community-Dwelling Older Adults.

The aim of this cross-sectional study was to examine the association between diet variety and physical frailty in community-dwelling older adults. Data of 577 older adults (mean age: 74.0 ± 6.3 years, women: 62.5%) were analyzed. Diet variety was assessed using the Food Frequency Score (FFS) (maximum, 30 points). The FFS assessed the one-week consumption frequency of ten foods (meat, fish/shellfish, eggs, milk & dairy products, soybean products, green & yellow vegetables, potatoes, fruits, seafood, and fats & oil). Physical frailty was assessed using Fried's component (slowness, weakness, exhaustion, low physical activity, and weight loss). The participants were classified into frail, pre-frail, and non-frail groups. The prevalence of physical frailty was 6.6%. This study found significant associations between physical frailty and low FFS after adjusting for covariates (odds ratio (OR) 0.90, 95% confidence interval (CI) 0.84-0.97, p < 0.01). The optimal cutoff point of the FFS for physical frailty was ≤16 points. FFS lower than the cutoff point were significantly associated with physical frailty after adjusting for covariates (OR 3.46, 95% CI 1.60-7.50, p < 0.01). Diet variety assessed using the FFS cutoff value of ≤16 points was related to the physical frailty status in community-dwelling older adults.

Application of decarboxylation reactions for improvement of dielectric properties of a methacrylic polymer.

In this work, we present a method based on thermally induced decarboxylation to solve the incompatibility of dielectric properties and the developability in aqueous developer solution of dielectric materials for the application in photodefinable insulating materials. Herein, malonic acid 2-hydroxyethyl methacrylate monoester, a methacrylate monomer with ß-keto acid is synthesized via phosphotungstic acid catalyzed esterification of 2-hydroxyethyl methacrylate and malonic acid. Further polymerisation is conducted with the obtained monomer to prepare poly(2-hydroxyethyl methacrylate-co-malonic acid 2-hydroxyethyl acrylate monoester). The thermally induced decarboxylation behaviors of the prepared polymer are confirmed with TGA, pyrolysis-GCMS analysis, and IR analysis. D f/D k tests and dissolution tests are carried out to investigate the influences of decarboxylation on the dielectric properties and dissolubility in aqueous solution, respectively. The results demonstrate that the significant changes in dielectric properties and dissolubility are due to the thermally induced decarboxylation.

Positive Association between High Protein Food Intake Frequency and Physical Performance and Higher-Level Functional Capacity in Daily Life.

Nutritional factors, including low protein intake and poor dietary variety, affect age-associated impairment in physical performance resulting in physical frailty. This cross-sectional study investigated the association between intake frequency of major high protein foods and both physical performance and higher-level functional capacity using the food frequency score (FFS) and high protein food frequency score (PFFS) among community-dwelling older adults. The data of 1185 older adults categorized into quartiles based on FFS and PFFS were analyzed. After adjusting for covariates, FFS and PFFS were significantly associated with physical performance [FFS, usual gait speed (p for trend = 0.007); PFFS, usual gait speed (p for trend < 0.001), maximum gait speed (p for trend = 0.002), timed up and go (p for trend = 0.025)], and higher-level functional capacity [FFS (p for trend < 0.001); PFFS (p for trend < 0.001)]. After excluding PFFS data, the participants' scores were associated with only higher-level functional capacity. Multi-regression analysis with higher-level functional capacity as the covariate showed that FFS and PFFS were significantly correlated with physical performance. Hence, improving food intake frequency, particularly that of high protein foods, and dietary variety may help maintain higher-level functional capacity and physical performance in community-dwelling older adults.

Calcium-Free and Cytochalasin B Treatment Inhibits Blastomere Fusion in 2-Cell Stage Embryos for the Generation of Floxed Mice via Sequential Electroporation.

The generation of conditional knockout mice using the Cre-loxP system is advantageous for the functional analysis of genes. Flanked by two loxP sites (floxed) mice can be directly obtained from fertilized eggs by the CRISPR/Cas9 genome editing system. We previously reported that sequential knock-in (KI) of each loxP site by electroporation (EP) at the 1- and 2-cell embryonic stages increases the number of mice with floxed alleles compared with simultaneous KI. However, EP at the 2-cell stage frequently induced blastomere fusion. These fused embryos cannot develop to term because they are tetraploidized. In this study, we examined the following three conditions to inhibit blastomere fusion by EP at the 2-cell stage: (1) hypertonic treatment, (2) Calcium (Ca2+)-free treatment, and (3) actin polymerization inhibition. Hypertonic treatment of 2-cell stage embryos prevented blastomere fusion and facilitated blastocyst development; however, KI efficiency was decreased. Ca2+-free treatment and actin polymerization inhibition by cytochalasin B (CB) reduced fusion rate, and did not have negative effects on development and KI efficiency. These results suggest that Ca2+-free and CB treatment at the 2-cell stage is effective to generate floxed mice in combination with a sequential EP method.

Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome.

BACKGROUND: Epigenetic modifications, including DNA methylation, play an important role in gene silencing and genome stability. Consequently, epigenetic dysregulation can cause several diseases, such as cancer, obesity, diabetes, autism, and imprinting disorders. RESULTS: We validate three methods for the generation of epigenome-edited mice using the dCas9-SunTag and single-chain variable fragment-TET1 catalytic domain. We generate model mice for Silver-Russell syndrome (SRS), an imprinting disorder, by target-specific DNA demethylation in the H19 differentially methylated region. Like SRS patients, these mice show H19 upregulation and Igf2 downregulation, leading to severe intrauterine and postnatal growth retardation. CONCLUSION: This is the first report of an imprinting disease model animal generated by targeted demethylation of specific loci of the epigenome in fertilized eggs. Epigenome-edited animals are also useful for exploring the causative epimutations in epigenetic diseases.

Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform.

Overexpression of a gene of interest is a general approach used in both basic research and therapeutic applications. However, the conventional approach involving overexpression of exogenous genes has difficulty achieving complete genome coverage, and is also limited by the cloning capacity of viral vectors. Therefore, an alternative approach would be to drive the expression of an endogenous gene using an artificial transcriptional activator. Fusion proteins of dCas9 and a transcription activation domain, such as dCas9-VP64, are widely used for activation of endogenous genes. However, when using a single sgRNA, the activation range is low. Consequently, tiling of several sgRNAs is required for robust transcriptional activation. Here we describe the screening of factors that exhibit the best synergistic activation of gene expression with TET1 in the dCas9-SunTag format. All seven factors examined showed some synergy with TET1. Among them, VP64 gave the best results. Thus, simultaneous tethering of VP64 and TET1 to a target gene using an optimized dCas9-SunTag format synergistically activates gene expression using a single sgRNA.

DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.

Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.

[Mail-based intervention for improvement of dietary habits among community-dwelling older adults living in heavy snowfall regions].

Objectives　The aim of this study was to investigate the effect of mail-based intervention using the TAKE10! Program to improve dietary habits in cases where direct intervention is not possible.Methods　Subjects aged 70-91 years (77.6±5.0) were randomly assigned to two groups: 72 in the intervention group and 71 in the control group. The intervention group received monthly mail, which included self-check sheets (TAKE10! Check sheet and TAKE10! Calendar) and a letter with feedback and comments for 5 months. The outcome measures were changes in the intake frequency of 10 food groups, Dietary Variety Score (DVS), and Food Frequency Score (FFS).Results　Compared to baseline, the post-intervention intake frequencies for 9 of 10 food groups, DVS, and FFS significantly increased in the intervention group. No significant differences were observed between baseline and post-intervention in the control group. In the subgroup analysis of the intervention group, post-intervention DVS and FFS of both subjects who cooked their own food and those who did not showed significant increases compared to baseline.Conclusion　The mail-based TAKE10! Program resulted in improved dietary habits and could be shared with families in addition to direct interventions and could also be used in regions with inadequate transportation systems or frequent poor weather conditions.

Arid1b Haploinsufficiency Causes Abnormal Brain Gene Expression and Autism-Related Behaviors in Mice.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with core symptoms that include poor social communication, restricted interests, and repetitive behaviors. Several ASD mouse models exhibit impaired social interaction, anxiety-like behavior, and elevated perseveration. Large-scale whole exome sequencing studies identified many genes putatively associated with ASD. Like chromodomain helicase DNA binding protein 8 (CHD8), the most frequently mutated gene in individuals with ASD, the candidate gene AT-rich interaction domain 1B (ARID1B) encodes a chromatin remodeling factor. Arid1b heterozygous knockout (hKO) mice exhibited ASD-like traits related to social behavior, anxiety, and perseveration, in addition to associated features reported in some cases of ASD, such as reduced weight, impaired motor coordination, and hydrocephalus. Hydrocephalus was present in 5 of 91 hKO mice, while it was not observed in wild-type littermates (0 of 188). Genome-wide gene expression patterns in Arid1b hKO mice were similar to those in ASD patients and Chd8-haploinsufficient mice, an ASD model, and to developmental changes in gene expression in fast-spiking cells in the mouse brain. Our results suggest that Arid1b haploinsufficiency causes ASD-like phenotypes in mice.

Efficient generation of conditional knockout mice via sequential introduction of lox sites.

Conditional knockout using Cre/lox is essential for functional analysis of genes. CRISPR/Cas in combination with two sets of guide RNAs and a single-stranded oligonucleotide enables simultaneous insertion of two lox sequences. However, this method induces double-strand breaks at two sites on the same chromosome, which causes an undesirable chromosomal deletion and reduces the flanked lox (flox) rate. To solve this problem, we investigated a method that sequentially introduces each lox sequence at the 1-cell and 2-cell embryonic stages, respectively. The sequential method was applied to both microinjection and electroporation systems. Sequential electroporation improved the flox efficiency compared with ordinary simultaneous microinjection, leading to a high yield of offspring with floxed alleles. Finally, we directly produced Cre/lox mice containing both the Cre transgene and floxed allele via sequential electroporation using Cre zygotes, which accelerated the generation of conditional knockout mice compared with the ordinary method.

Evaluation of the comprehensive health program "Sumida TAKE10!" for community-dwelling older adults, which aims to prevent or delay the need for long-term nursing care.

Objective The purpose of this study was to evaluate the comprehensive health program "Sumida TAKE10!", which aims to improve dietary habits and promote physical activity among community-dwelling older adults including the pre-frail elderly. This study has been ongoing since 2005 in Sumida Ward, Tokyo with the ultimate aim of preventing or delaying the need for long-term nursing care. We used the term "pre-frail elderly" for older adults who are at risk of requiring long-term care.Methods "Sumida TAKE10!" consists of a general lecture in a public hall followed by 5 educational sessions biweekly at 4-6 community centers. From 2008 to 2013, 402 participants aged ≥65 years were enrolled and included as subjects of the study. The main outcome measures were changes in 10 food intake frequencies, food frequency score (FFS), dietary variety score (DVS), frequency of exercise (obtained via questionnaire) and physical fitness (5-meter maximal walking time, 5-meter walking time, handgrip strength, one-leg standing time with eyes opened (time to upright posture for standing on one leg with eyes open), and the timed up & go test). The secondary outcome measures were changes in the Tokyo Metropolitan Institute of Gerontology (TMIG) Index of Competence score, appetite, frequency of walking and sports, self-rated health, frequency of leaving the house, communication with neighbors, engagement in hobbies, participation in group activities and participation in volunteer activities (obtained via questionnaire).Results Compared to baseline, all outcomes showed significant improvement. "Sumida TAKE10!" can improve dietary habits and increase the physical activity of participants. Positive secondary effects were seen for life function, self-rated health, and social activities. Almost identical positive results were obtained from the pre-frail elderly group, while improvement was also seen in the dietary habits of the subjects who do not cook.Conclusion These results suggest that this program may be useful for population-based approach programs. In addition, comprehensive programs like TAKE10! may increase the health consciousness of community-dwelling older adults.

Targeted DNA demethylation in vivo using dCas9-peptide repeat and scFv-TET1 catalytic domain fusions.

Despite the importance of DNA methylation in health and disease, technologies to readily manipulate methylation of specific sequences for functional analysis and therapeutic purposes are lacking. Here we adapt the previously described dCas9-SunTag for efficient, targeted demethylation of specific DNA loci. The original SunTag consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, which induces demethylation, we changed the linker length to 22 amino acids. The system attains demethylation efficiencies >50% in seven out of nine loci tested. Four of these seven loci showed demethylation of >90%. We demonstrate targeted demethylation of CpGs in regulatory regions and demethylation-dependent 1.7- to 50-fold upregulation of associated genes both in cell culture (embryonic stem cells, cancer cell lines, primary neural precursor cells) and in vivo in mouse fetuses.

Structure and Engineering of Francisella novicida Cas9.

The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5'-NGG-3' PAM, and used the structural information to create a variant that can recognize the more relaxed 5'-YG-3' PAM. Furthermore, we demonstrated that the FnCas9-ribonucleoprotein complex can be microinjected into mouse zygotes to edit endogenous sites with the 5'-YG-3' PAM, thus expanding the target space of the CRISPR-Cas9 toolbox.

Gene expression profiling of white adipose tissue reveals paternal transmission of proneness to obesity.

Previously, we found that C57BL/6J (B6) mice are more prone to develop obesity than PWK mice. In addition, we analyzed reciprocal crosses between these mice and found that (PWK × B6) F1 mice, which have B6 fathers, are more likely to develop dietary obesity than (B6 × PWK) F1 mice, which have B6 mothers. These results suggested that diet-induced obesity is paternally transmitted. In this study, we performed transcriptome analysis of adipose tissues of B6, PWK, (PWK × B6) F1, and (B6 × PWK) F1 mice using next-generation sequencing. We found that paternal transmission of diet-induced obesity was correlated with genes involved in adipose tissue inflammation, metal ion transport, and cilia. Furthermore, we analyzed the imprinted genes expressed in white adipose tissue (WAT) and obesity. Expression of paternally expressed imprinted genes (PEGs) was negatively correlated with body weight, whereas expression of maternally expressed imprinted genes (MEGs) was positively correlated. In the obesity-prone B6 mice, expression of PEGs was down-regulated by a high-fat diet, suggesting that abnormally low expression of PEGs contributes to high-fat diet-induced obesity in B6 mice. In addition, using single-nucleotide polymorphisms that differ between B6 and PWK, we identified candidate imprinted genes in WAT.

p53 suppresses tetraploid development in mice.

Mammalian tetraploid embryos die in early development because of defects in the epiblast. Experiments with diploid/tetraploid chimeric mice, obtained via the aggregation of embryonic stem cells, clarified that while tetraploid cells are excluded from epiblast derivatives, diploid embryos with tetraploid extraembryonic tissues can develop to term. Today, this method, known as tetraploid complementation, is usually used for rescuing extraembryonic defects or for obtaining completely embryonic stem (ES) cell-derived pups. However, it is still unknown why defects occur in the epiblast during mammalian development. Here, we demonstrated that downregulation of p53, a tumour suppressor protein, rescued tetraploid development in the mammalian epiblast. Tetraploidy in differentiating epiblast cells triggered p53-dependent cell-cycle arrest and apoptosis, suggesting the activation of a tetraploidy checkpoint during early development. Finally, we found that p53 downregulation rescued tetraploid embryos later in gestation.

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering.

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.