Satoko Matsukura

Increased arterial stiffness has emerged as a strong predictor of future cardiovascular events and all-cause mortality. The aim of this study was to elucidate influences of endothelin (ET)-related genetic polymorphisms and regular physical activity on age-related arterial stiffening through a 10-yr longitudinal study. A decadal change in brachial-ankle pulse wave velocity (baPWV), an index of arterial stiffness, was evaluated retrospectively among 92 volunteers (63 ± 14 yr, 51 men). The targeted single-nucleotide polymorphisms were ET-A receptor SNP rs5333 (ET-A) and ET-B receptor SNP rs5351 (ET-B). Subjects with either ET-A TC or CC genotypes exhibited significantly greater increases in baPWV (+15.3 ± 11.7 and +16.6 ± 15.7%/dec, respectively) than ET-A TT genotype holders (+9.2 ± 9.0%/dec), whereas subjects with the ET-B GG genotype showed a significantly greater increase in baPWV (+17.7 ± 14.1%/dec) than other ET-B genotype holders (AA: +9.5 ± 10.0%/dec; AG: +11.2 ± 9.6%/dec). The combination of these ET-related genetic risks was associated with a 2.4 times greater decadal increase in baPWV compared with no genetic risk (+8.1 ± 8.4 vs. 19.5 ± 16.0%/dec). In contrast, individuals engaging in >15 METs·h/wk of aerobic exercise showed substantially smaller increases in baPWV (+5.0 ± 9.7%/dec) compared with their physically inactive peers (approximately +13%/dec). These differences remained significant after adjusting for confounding factors, including baseline baPWV and ET-related genotype risk. Our current longitudinal study found that ET-related gene polymorphisms contribute to diverse age-related changes in arterial stiffness, and that regular sufficient aerobic exercise attenuates the age-related arterial stiffening independently of ET-related gene polymorphisms. NEW & NOTEWORTHY This 10-yr longitudinal study suggests that endothelin-related gene polymorphisms contribute to divergent increases in arterial stiffness with advancing age, whereas regular sufficient aerobic exercise attenuates age-related arterial stiffening independently of ET-related gene polymorphisms. This notion partly supports prevailing evidence that regular aerobic exercise contributes to a lower incidence of cardiovascular disease.

High-throughput sequencing of 16S rRNA gene amplicons (16S-seq) has become a widely deployed method for profiling complex microbial communities but technical pitfalls related to data reliability and quantification remain to be fully addressed. In this work, we have developed and implemented a set of synthetic 16S rRNA genes to serve as universal spike-in standards for 16S-seq experiments. The spike-ins represent full-length 16S rRNA genes containing artificial variable regions with negligible identity to known nucleotide sequences, permitting unambiguous identification of spike-in sequences in 16S-seq read data from any microbiome sample. Using defined mock communities and environmental microbiota, we characterized the performance of the spike-in standards and demonstrated their utility for evaluating data quality on a per-sample basis. Further, we showed that staggered spike-in mixtures added at the point of DNA extraction enable concurrent estimation of absolute microbial abundances suitable for comparative analysis. Results also underscored that template-specific Illumina sequencing artifacts may lead to biases in the perceived abundance of certain taxa. Taken together, the spike-in standards represent a novel bioanalytical tool that can substantially improve 16S-seq-based microbiome studies by enabling comprehensive quality control along with absolute quantification.

The purposes of this study were to identify signaling pathways that were specifically activated in ATDC5 cells cultured on poly (2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) gel in insulin-free maintenance medium and to evaluate the significance of the determined signaling pathways in the chondrogenic differentiation induced by this gel. In this study, ATDC5 cells cultured on PAMPS gel using the maintenance medium without insulin (PAMPS Culture) were compared with cells cultured on polystyrene using the differentiation medium containing insulin (PS-I Culture). The microarray analysis, Western blot analysis, and real-time PCR analysis demonstrated that the TGF-β/BMP signaling pathway was significantly enhanced at Days 1, 2, and 3 in the PAMPS Culture when compared with the PS-I Culture. Inhibition of the BMP type-I receptor reduced the phosphorylation level of Smad1/5 and expression of type-2 collagen and aggrecan mRNA in the cells accompanied by a reduction in cell aggregation at Day 13 in the PAMPS Culture. The inhibition of the TGF-β/BMP signaling pathway significantly inhibited the chondrogenic differentiation induced by the PAMPS gel. The present study demonstrated that synthetic PAMPS gel activates the TGF-β/BMP/Smad signaling pathway in the ATDC5 cells in the absence of insulin, and that this activation plays a significant role in the chondrogenic differentiation induced by PAMPS gel. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 734–746, 2016.

In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.

The mechanisms for plant growth restriction during stress conditions remains unclear. Here, we demonstrate that a phytochrome-interacting factor-like protein, OsPIL1/OsPIL13, acts as a key regulator of reduced internode elongation in rice under drought conditions. The level of OsPIL1 mRNA in rice seedlings grown under nonstressed conditions with light/dark cycles oscillated in a circadian manner with peaks in the middle of the light period. Under drought stress conditions, OsPIL1 expression was inhibited during the light period. We found that OsPIL1 was highly expressed in the node portions of the stem using promoter-glucuronidase analysis. Overexpression of OsPIL1 in transgenic rice plants promoted internode elongation. In contrast, transgenic rice plants with a chimeric repressor resulted in short internode sections. Alteration of internode cell size was observed in OsPIL1 transgenic plants, indicating that differences in cell size cause the change in internode length. Oligoarray analysis revealed OsPIL1 downstream genes, which were enriched for cell wall-related genes responsible for cell elongation. These data suggest that OsPIL1 functions as a key regulatory factor of reduced plant height via cell wall-related genes in response to drought stress. This regulatory system may be important for morphological stress adaptation in rice under drought conditions.

The genomes of three plants, Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and soybean (Glycine max), have been sequenced, and their many genes and promoters have been predicted. In Arabidopsis, cis-acting promoter elements involved in cold- and dehydration-responsive gene expression have been extensively analysed; however, the characteristics of such cis-acting promoter sequences in cold- and dehydration-inducible genes of rice and soybean remain to be clarified. In this study, we performed microarray analyses using the three species, and compared characteristics of identified cold- and dehydration-inducible genes. Transcription profiles of the cold- and dehydration-responsive genes were similar among these three species, showing representative upregulated (dehydrin/LEA) and downregulated (photosynthesis-related) genes. All (4⁶ = 4096) hexamer sequences in the promoters of the three species were investigated, revealing the frequency of conserved sequences in cold- and dehydration-inducible promoters. A core sequence of the abscisic acid-responsive element (ABRE) was the most conserved in dehydration-inducible promoters of all three species, suggesting that transcriptional regulation for dehydration-inducible genes is similar among these three species, with the ABRE-dependent transcriptional pathway. In contrast, for cold-inducible promoters, the conserved hexamer sequences were diversified among these three species, suggesting the existence of diverse transcriptional regulatory pathways for cold-inducible genes among the species.

DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.

DREB1A/CBF3 and DREB2A are transcription factors that specifically interact with a cis-acting dehydration-responsive element (DRE), which is involved in cold- and dehydration-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Overexpression of DREB1A improves stress tolerance to both freezing and dehydration in transgenic plants. In contrast, overexpression of an active form of DREB2A results in significant stress tolerance to dehydration but only slight tolerance to freezing in transgenic plants. The downstream gene products for DREB1A and DREB2A are reported to have similar putative functions, but downstream genes encoding enzymes for carbohydrate metabolism are very different between DREB1A and DREB2A. We demonstrate that under cold and dehydration conditions, the expression of many genes encoding starch-degrading enzymes, sucrose metabolism enzymes, and sugar alcohol synthases changes dynamically; consequently, many kinds of monosaccharides, disaccharides, trisaccharides, and sugar alcohols accumulate in Arabidopsis. We also show that DREB1A overexpression can cause almost the same changes in these metabolic processes and that these changes seem to improve freezing and dehydration stress tolerance in transgenic plants. In contrast, DREB2A overexpression did not increase the level of any of these metabolites in transgenic plants. Strong freezing stress tolerance of the transgenic plants overexpressing DREB1A may depend on accumulation of these metabolites.