Putative Mechanisms Underlying the Beneficial Effects of Polyphenols in Murine Models of Metabolic Disorders in Relation to Gut Microbiota.

The beneficial effects of polyphenols on metabolic disorders have been extensively reported. The interaction of these compounds with the gut microbiota has been the focus of recent studies. In this review, we explored the fundamental mechanisms underlying the beneficial effects of polyphenols in relation to the gut microbiota in murine models of metabolic disorders. We analyzed the effects of polyphenols on three murine models of metabolic disorders, namely, models of a high-fat diet (HFD)-induced metabolic disorder, dextran sulfate sodium (DSS)-induced colitis, and a metabolic disorder not associated with HFD or DSS. Regardless of the model, polyphenols ameliorated the effects of metabolic disorders by alleviating intestinal oxidative stress, improving inflammatory status, and improving intestinal barrier function, as well as by modulating gut microbiota, for example, by increasing the abundance of short-chain fatty acid-producing bacteria. Consequently, polyphenols reduce circulating lipopolysaccharide levels, thereby improving inflammatory status and alleviating oxidative imbalance at the lesion sites. In conclusion, polyphenols likely act by regulating intestinal functions, including the gut microbiota, and may be a safe and suitable therapeutic agent for various metabolic disorders.

Anti-Osteoporotic Mechanisms of Polyphenols Elucidated Based on In Vivo Studies Using Ovariectomized Animals.

Polyphenols are widely known for their antioxidant activity, i.e., they have the ability to suppress oxidative stress, and this behavior is mediated by the autoxidation of their phenolic hydroxyl groups. Postmenopausal osteoporosis is a common health problem that is associated with estrogen deficiency. Since oxidative stress is thought to play a key role in the onset and progression of osteoporosis, it is expected that polyphenols can serve as a safe and suitable treatment in this regard. Therefore, in this review, we aimed to elucidate the anti-osteoporotic mechanisms of polyphenols reported by in vivo studies involving the use of ovariectomized animals. We categorized the polyphenols as resveratrol, purified polyphenols other than resveratrol, or polyphenol-rich substances or extracts. Literature data indicated that resveratrol activates sirtuin 1, and thereafter, suppresses osteoclastogenic pathways, such as the receptor activator of the nuclear factor kappa B (RANK) ligand (RANKL) pathway, and promotes osteoblastogenic pathways, such as the wingless-related MMTV integration site pathway. Further, we noted that purified polyphenols and polyphenol-rich substances or extracts exert anti-inflammatory and/or antioxidative effects, which inhibit RANKL/RANK binding via the NF-κB pathway, resulting in the suppression of osteoclastogenesis. In conclusion, antioxidative and anti-inflammatory polyphenols, including resveratrol, can be safe and effective for the treatment of postmenopausal osteoporosis based on their ability to regulate the imbalance between bone formation and resorption.

Metabolic Fate of Orally Ingested Proanthocyanidins through the Digestive Tract.

Proanthocyanidins (PACs), which are oligomers or polymers of flavan-3ols with potent antioxidative activity, are well known to exert a variety of beneficial health effects. Nonetheless, their bioaccessibility and bioavailability have been poorly assessed. In this review, we focused on the metabolic fate of PACs through the digestive tract. When oligomeric and polymeric PACs are orally ingested, a large portion of the PACs reach the colon, where a small portion is subjected to microbial degradation to phenolic acids and valerolactones, despite the possibility that slight depolymerization of PACs occurs in the stomach and small intestine. Valerolactones, as microbiota-generated catabolites of PACs, may contribute to some of the health benefits of orally ingested PACs. The remaining portion interacts with gut microbiota, resulting in improved microbial diversity and, thereby, contributing to improved health. For instance, an increased amount of beneficial gut bacteria (e.g., Akkermansia muciniphila and butyrate-producing bacteria) could ameliorate host metabolic functions, and a lowered ratio of Firmicutes/Bacteroidetes at the phylum level could mitigate obesity-related metabolic disorders.

Epigenetic regulation affects gene amplification in Drosophila development.

In Drosophila melanogaster, in response to developmental transcription factors, and by repeated initiation of DNA replication of four chorion genes, ovarian follicle cells, form an onion skin-type structure at the replication origins. The DNA replication machinery is conserved from yeast to humans. Subunits of the origin recognition complex (ORC) is comprised of Orc1, Orc2, and Cdc6 genes. While mutations of Orc1 and Orc2 and not Cdc6can be lethal, overexpression of these genes lead to female sterility. Ecdysone, is a steroidal prohormone of the major insect molting hormone 20-hydroxyecdysone that in Drosophila, triggers molting, metamorphosis, and oogenesis. To this end, we identified several ecdysone receptor (EcR) binding sites around gene amplification loci. We also found that H3K4 was trimethylated at chorion gene amplification origins, but not at the act1 locus. Female mutants overexpressing Lsd1 (a dimethyl histone H3K4 demethylase) or Lid (a trimethyl histone H3K4 demethylase), but not a Lid mutant, were sterile. The data suggest that ecdysone signaling determines which origin initiates DNA replication and contributes to the development. Screening strategies using Drosophila offer the opportunity for development of drugs that reduce gene amplification and alter histone modification associated with epigenetic effects.

The function of replication and SCF complex during Drosophila wing development.

Chromosomal DNA replication machinery functions in the growing cells and organs in multicellular organisms. We previously demonstrated that its knockdown in several tissues of Drosophila led to a rough eye phenotype, the loss of bristles in the eye and female sterile. In this paper, I investigated in detail the wing phenotype using RNAi flies, and observed that the knockdown not only of Mcm10 but also of some other prereplicative complex components including Cdt1, Polα-primase, RPA, Psf2 (partner of SLD five 2; a subunit of GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese) and Rfc3 (replication factor C 3; a subunit of RFC complex) demonstrated wing phenotypes, using Gal4-driver flies. Surprisingly, some SCF complex components, which control cell cycle progression via protein degradation, also showed the wing phenotype. These results showed that the DNA replication machinery contributes to wing development independently of growth, probably through defects in DNA replication and protein degradation at specific places and times.

DNA replication machinery is required for development in Drosophila.

In Drosophila, some factors involved in chromosome replication seem to be involved in gene amplification and endoreplication, which are actively utilized in particular tissue development, but direct evidence has not been shown. Therefore, we examined the effect of depletion of replication factors on these processes. First, we confirmed RNAi knockdown can be used for the depletion of replication factors by comparing the phenotypes of RNAi knockdown and deletion or point mutants of the components of DNA licensing factor, MCM2, MCM4 and Cdt1. Next, we found that tissue-specific RNAi knockdown of replication factors caused tissue-specific defects, probably due to defects in DNA replication. In particular, we found that depletion inhibited gene amplification of the chorion gene in follicle cells and endoreplication in salivary glands, showing that chromosomal DNA replication factors are required for these processes. Finally, using RNAi, we screened the genes for chromosomal DNA replication that affected tissue development. Interestingly, wing specific knockdown of Mcm10 induced wing formation defects. These results suggest that some components of chromosomal replication machinery are directly involved in tissue development.

DNA replication machinery contributes to development of eye in Drosophila.

Chromosomal DNA replication machinery is essential for growth of all cells in all organs in multicellular organisms. Its knockdown in several tissues of Drosophila led to rough eye phenotype and the loss of bristles in the eye. These results show that the DNA replication machinery contributes to the body development independent of growth.

Chromosome and genetic testing using ChIP assay.

Chromatin immunoprecipitation (ChIP) assay can be used to easily visualize information about proteins, DNA, and RNA on chromosomes and is widely used for analysis of genomes, epigenomes, mRNAs, and non-coding RNAs. The ChIP assay can detect, not only DNA-binding proteins of various organisms, but also the temporal and spatial regulating mechanisms of RNA-binding proteins. Because of these features, demand for ChIP assay is expected to grow. Here, by using yeast and Drosophila as examples, we describe the superiority of the improved ChIP assay that we have developed.

A proposal for clinical genetics (genetics in medicine) education for medical technologists and other health professionals in Japan.

Since the completion of the Human Genome Project, technology has developed markedly in fields such as medical genetics and genetic counseling in the medical arena. In particular, this technology has advanced the discovery of and ways of understanding various genes responsible for genetic diseases, and genetic polymorphisms thought to be associated with disease. Some have been implicated as factors in common lifestyle diseases and have increased the significance of genetic testing. In Japan, doctors and other health professionals, such as nurse and medical technologists have been engaged in genetic testing and genetic disease treatment. Chromosomal and gene aberrations were detected mainly by medical technologists. However, due to the nature of medical technologists who have to provide various clinical tests, such as blood test, pre-medical technology students are required to cover tremendous knowledge of different academic fields to pass the national exam. Therefore, the time allowed for such students to study chromosomal and gene analysis is quite limited. Moreover, they are forced to enter the medical setting without receiving sufficient training. Among them, only few medical technologists specialize in chromosomal and gene analysis. However, with the advancement of clinical genetics and development of chromosomal and gene analysis, conducting clinical practice is becoming more and more difficult for medical technologists who just passed the national exam. Also, doctors and other health professionals have not been able to keep up with service demands either. This paper attempts to address knowledge and skills gaps (especially clinical genetics, English, and ICT literacy) of medical technologists and we propose educational methods to prepare medical genetics professionals in Japan to meet these gaps.

Problems and their solutions in genetic counseling education in Japan.

With the expansion of novel chromosome testing, a career as a certified genetic counselor has been gathering a lot of attention. However, few people certified as a genetic counselor after completing postgraduate courses are able to find employment as a genetic counselor, and their salaries are quite low. It is also questionable whether or not such newly graduated genetic counselors, who have limited life experience and knowledge, can fully understand family issues and properly perform counseling sessions. To address these issues, a wide range of education and training may be necessary. In this study, we examined current problems in genetic counseling education in Japan, and proposed effective measures to address these problems. Toward creating a new society, we are currently establishing a national qualification system and cultivating qualified professionals capable of providing patients with accurate information on chromosome and genetic testing. In addition, these professionals could encourage younger generations to have an interest in genetic counseling. I also hope that these professionals will work not only in Japan but all over the world.

Faster and easier chromatin immunoprecipitation assay with high sensitivity.

Chromatin immunoprecipitation (ChIP) assays are widely used to investigate where chromatin-binding proteins bind to the genome. The standard assay is very time consuming. We have developed a rapid ChIP assay in which the immunoprecipitates serve directly as PCR templates. This assay eliminates the step to reverse the crosslinking, shortening the assay by 1 day. It also requires a less immunoprecipitating antibody, permits many samples to be tested simultaneously, and is more sensitive than the standard ChIP assay.

Transcription factor Runx1 recruits the polyomavirus replication origin to replication factories.

Eukaryotic DNA replication takes place in the replication factories, where replication proteins are properly assembled to form replication forks. Thus, recruitment of DNA replication origins to the replication factories must be the key step for the regulation of DNA replication. The transcription factor Runx1 associates with the nuclear matrix, the putative substructure of DNA replication factories. An earlier report from our laboratory showed that Runx1 activates polyomavirus DNA replication, and that this requires its nuclear matrix-binding activity. Here, we show that Runx1 activates polyomavirus DNA replication by stimulating the binding of the viral-encoded replication initiator/helicase, large T antigen, to its replication origin. We found that newly replicated polyomavirus DNA is associated with the nuclear matrix and that large T antigen is targeted to replication factories, suggesting that polyomavirus is replicated in replication factories on the nuclear matrix. Although Runx1 did not co-localize with large T antigen-containing foci by itself, it co-localized with large T antigen-containing replication factories during Runx1-dependent polyomavirus DNA replication. These observations together suggest that Runx1 recruits the polyomavirus replication origin to the replication factory on the nuclear matrix, and that this requires the nuclear matrix-binding activity of Runx1.

Transcription factors and DNA replication origin selection.

The chromosomes of eukaryotic cells possess many potential DNA replication origins, of which a subset is selected in response to the cellular environment, such as the developmental stage, to act as active replication start sites. The mechanism of origin selection is not yet fully understood. In this review, we summarize recent observations regarding replication origins and initiator proteins in various organisms. These studies suggest that the DNA-binding specificities of the initiator proteins that bind to the replication origins and promote DNA replication are primarily responsible for origin selection. We particularly focus on the importance of transcription factors in the origin selection process. We propose that transcription factors are general regulators of the formation of functional complexes on the chromosome, including the replication initiation complex. We discuss the possible mechanisms by which transcription factors influence the selection of particular origins.