Inheritance of song and stridulatory peg number divergence between Chorthippus brunneus and C. jacobsi, two naturally hybridizing grasshopper species (Orthoptera: Acrididae)

Knowledge of the genetic basis of divergence in mating signal characters that contribute to reproductive isolation is critical to understanding speciation. Here, we describe a semi-automated system for characterizing grasshopper acoustic signals. We used this system to study the genetic basis of divergence in three male calling song components [echeme (EL), syllable (SL) and phrase (PL) lengths] between Chorthippus brunneus and C. jacobsi, two species of grasshoppers that hybridize in northern Spain. We also studied the number of pegs in the stridulatory file. For all characters, additive effects accounted for most of the genetic differentiation between species. However, the three song components also showed small but significant epistatic effects. No sex linkage was detected. Wright-Castle-Lande estimates of the minimum numbers of genetic factors underlying song and peg number divergence were low: peg number (n(e)=5.87+/-5.84), SL (n(e)=2.37+/-4.79) and PL (n(e)=0.87+/-0.86). On the other hand, EL appeared to be controlled by many genes. These results suggest that divergence in SL and PL might be driven by sexual selection whereas EL might not be under selection. This is consistent with experimental results on female song preference in related species. However, the fact that few factors appear to underlie the differences in peg number is surprising. Peg number is not closely related to song characteristics. It often varies between closely related grasshopper species and it has been assumed to be a neutral character. The biometrical approaches used here tend to underestimate the number of factors influencing a trait but provide valuable background for subsequent quantitative trait loci analyses.     

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.     

Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.     

Molecular and morphological evidence of hybridization between native Ruditapes philippinarum and the introduced Ruditapes form in Japan

Marine aquaculture and stock enhancement are major causes of the introduction of alien species. A good example of such an introduction is the Japanese shortneck clam Ruditapes philippinarum, one of the most important fishery resources in the world. To meet the domestic shortage of R. philippinarum caused by depleted catches, clams were imported to Japan from China and the Korean peninsula. The imported clam is an alien species that has a very similar morphology, and was misidentified as R. philippinarum (hereafter, Ruditapes form). We genotyped 1,186 clams of R. philippinarum and R. form at four microsatellite loci, sequenced mitochondrial DNA (COI gene fragment) of 485 clams, 34 of which were R. variegatus, and measured morphometric and meristic characters of 754 clams from 12 populations in Japan and China, including the Ariake Sea and Tokyo Bay, where large numbers of R. form were released. Our analyses confirmed that R. form was from the genus Ruditapes, and the genetic differentiation between R. philippinarum and R. form was distinct, but small, compared with five bivalve outgroups. However, R. form had distinct shell morphology, especially larger numbers of radial ribs on the shell surface, suggesting that R. form might be a new Ruditapes species or a variation of R. philippinarum that originated from southern China. A genetic affinity of the sample from the Ariake Sea to R. form was found with the intermediate shell morphology and number of radial ribs, and the hybrid proportion was estimated at 51.3 ± 4.6 % in the Ariake Sea.     

Population genetics without intraspecific data

A central goal of computational biology is the prediction of phenotype from DNA and protein sequence data. Recent models of sequence change use in silico prediction systems to incorporate the effects of phenotype on evolutionary rates. These models have been designed for analyzing sequence data from different species and have been accompanied by statistical techniques for estimating model parameters when the incorporation of phenotype induces dependent change among sequence positions. A difficulty with these efforts to link phenotype and interspecific evolution is that evolution occurs within populations, and parameters of interspecific models should have population genetic interpretations. We show, with two examples, how population genetic interpretations can be assigned to evolutionary models. The first example considers the impact of RNA secondary structure on sequence change, and the second reflects the tendency for protein tertiary structure to influence nonsynonymous substitution rates. We argue that statistical fit to data should not be the sole criterion for assessing models of sequence change. A good interspecific model should also yield a clear and biologically plausible population genetic interpretation.     

Renormalized basal metabolic rate describes the human aging process and longevity

The question of why we age and finally die has been a central subject in the life, medical, and health sciences. Many aging theories have proposed biomarkers that are related to aging. However, they do not have sufficient power to predict the aging process and longevity. We here propose a new biomarker of human aging based on the mass‐specific basal metabolic rate (msBMR). It is well known by the Harris–Benedict equation that the msBMR declines with age but varies among individual persons. We tried to renormalize the msBMR by primarily incorporating the body mass index into this equation. The renormalized msBMR (RmsBMR) which was derived in one cohort of American men (n = 25,425) was identified as one of the best biomarkers of aging, because it could well reproduce the observed respective American, Italian, and Japanese data on the mortality rate and survival curve. A recently observed plateau of the mortality rate in centenarians corresponded to the lowest value (threshold) of the RmsBMR, which stands for the final stage of human life. A universal decline of the RmsBMR with age was associated with the mitochondrial number decay, which was caused by a slight fluctuation of the dynamic fusion/fission system. This decay form was observed by the measurement in mice. Finally, the present approach explained the reason why the BMR in mammals is regulated by the empirical algometric scaling law.     

Generation and Characterization of Functional Cardiomyocytes Derived from Human T Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have been proposed as novel cell sources for genetic disease models and revolutionary clinical therapies. Accordingly, human iPSC-derived cardiomyocytes are potential cell sources for cardiomyocyte transplantation therapy. We previously developed a novel generation method for human peripheral T cell-derived iPSCs (TiPSCs) that uses a minimally invasive approach to obtain patient cells. However, it remained unknown whether TiPSCs with genomic rearrangements in the T cell receptor (TCR) gene could differentiate into functional cardiomyocyte in vitro. To address this issue, we investigated the morphology, gene expression pattern, and electrophysiological properties of TiPSC-derived cardiomyocytes differentiated by floating culture. RT-PCR analysis and immunohistochemistry showed that the TiPSC-derived cardiomyocytes properly express cardiomyocyte markers and ion channels, and show the typical cardiomyocyte morphology. Multiple electrode arrays with application of ion channel inhibitors also revealed normal electrophysiological responses in the TiPSC-derived cardiomyocytes in terms of beating rate and the field potential waveform. In this report, we showed that TiPSCs successfully differentiated into cardiomyocytes with morphology, gene expression patterns, and electrophysiological features typical of native cardiomyocytes. TiPSCs-derived cardiomyocytes obtained from patients by a minimally invasive technique could therefore become disease models for understanding the mechanisms of cardiac disease and cell sources for revolutionary cardiomyocyte therapies.     

Bombyx small RNAs: genomic defense system against transposons in the silkworm, Bombyx mori

Selfish genetic elements called transposons can insert themselves at new locations in host genomes to modify gene structure and alter gene expression. Expansion of transposons can occur when novel transposition events are transmitted to subsequent generations after germline hopping. Therefore, organisms seem likely to have evolved defense mechanisms to silence transposons in the germline. Recently, small RNAs interacting with Piwi proteins (piwi-interacting RNAs: piRNAs) have been demonstrated to be involved in genomic defense mechanism against transposons. Here, we show that piRNA-like small RNAs are present abundantly in the Bombyx ovary. We cloned 38,493 kinds of Bombyx small RNA from the ovary and performed functional characterization. Bombyx small RNAs showed a unimodal length distribution with a peak at 28nt and a strong bias for U at the 5' end. We found that 12,869 kinds of Bombyx small RNAs were associated with transposons or repetitive sequences. We classified them as repeat-associated small interfering RNAs (rasiRNAs), a subclass of piRNAs. Notably, antisense rasiRNAs have a strong bias toward U at 5' ends; in contrast, sense rasiRNAs have a strong bias toward A at nucleotide position 10, indicating that the piRNA amplification loop proposed in Drosophila is evolutionarily conserved in Bombyx. These results suggest that Bombyx small RNAs regulate transposon activity.     

Population structure of chum salmon and selection on the markers collected for stock identification

Genetic stock identification (GSI) is a major management tool of Pacific salmon (Oncorhynchus Spp.) that has provided rich genetic baseline data of allozymes, microsatellites, and single‐nucleotide polymorphisms (SNPs) across the Pacific Rim. Here, we analyzed published data sets for adult chum salmon (Oncorhynchus keta), namely 10 microsatellites, 53 SNPs, and a mitochondrial DNA locus (mtDNA3, control region, and NADH‐3 combined) in samples from 495 locations in the same distribution range (n = 61,813). TreeMix analysis of the microsatellite loci identified the greatest convergence toward Japanese/Korean populations and suggested two admixture events from Japan/Korea to Russia and the Alaskan Peninsula. The SNPs had been purposively collected from rapidly evolving genes to increase the power of GSI. The largest expected heterozygosity was observed in Japanese/Korean populations for microsatellites, whereas it was largest in Western Alaskan populations for SNPs, reflecting the SNP discovery process. A regression of SNP population structures on those of microsatellites indicated the selection of the SNP loci according to deviations from the predicted structures. Specifically, we matched the sampling locations of the SNPs with those of the microsatellites and performed regression analyses of SNP allele frequencies on a 2‐dimensional scaling (MDS) of matched locations obtained from microsatellite pairwise F _ST values. The MDS first axis indicated a latitudinal cline in American and Russian populations, whereas the second axis showed differentiation of Japanese/Korean populations. The top five outlier SNPs included mtDNA3, U502241 (unknown), GnRH373, ras1362, and TCP178, which were identified by principal component analysis. We summarized the functions of 53 nuclear genes surrounding SNPs and the mtDNA3 locus by referring to a gene database system and propose how they may influence the fitness of chum salmon.     

Fluorescence demonstration of cathepsin B activity in fractionated alveolar macrophages.

Histochemical localization of cathepsin B in alveolar macrophages (AM) that separated into four different density fractions (I, II, III and IV) by discontinuous Percoll gradient centrifugation was demonstrated in fluorescence microscope using CBZ-Arg-Arg-4-methoxy-2- naphthylamide as a substrate and 5-nitrosalicylaldehyde as a coupling reagent. The least dense AM (fraction I) was found numerous bright yellow fluorescing particles with high intensity in small granules distributed throughout the cytoplasm when compared to the most dense cells (fraction IV). The different localization of cathepsin B activity in the fractionated cells suggested differentiation of lysosomal system and existence of maturational (or aging) sequence in rat AM.