Island-Model Genomic Selection for Long-Term Genetic Improvement of Autogamous Crops

Acceleration of genetic improvement of autogamous crops such as wheat and rice is necessary to increase cereal production in response to the global food crisis. Population and pedigree methods of breeding, which are based on inbred line selection, are used commonly in the genetic improvement of autogamous crops. These methods, however, produce a few novel combinations of genes in a breeding population. Recurrent selection promotes recombination among genes and produces novel combinations of genes in a breeding population, but it requires inaccurate single-plant evaluation for selection. Genomic selection (GS), which can predict genetic potential of individuals based on their marker genotype, might have high reliability of single-plant evaluation and might be effective in recurrent selection. To evaluate the efficiency of recurrent selection with GS, we conducted simulations using real marker genotype data of rice cultivars. Additionally, we introduced the concept of an “island model” inspired by evolutionary algorithms that might be useful to maintain genetic variation through the breeding process. We conducted GS simulations using real marker genotype data of rice cultivars to evaluate the efficiency of recurrent selection and the island model in an autogamous species. Results demonstrated the importance of producing novel combinations of genes through recurrent selection. An initial population derived from admixture of multiple bi-parental crosses showed larger genetic gains than a population derived from a single bi-parental cross in whole cycles, suggesting the importance of genetic variation in an initial population. The island-model GS better maintained genetic improvement in later generations than the other GS methods, suggesting that the island-model GS can utilize genetic variation in breeding and can retain alleles with small effects in the breeding population. The island-model GS will become a new breeding method that enhances the potential of genomic selection in autogamous crops, especially bringing long-term improvement.     

Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

We have previously isolated a lactic acid bacterium (LAB), Pediococcus pentosaceus LP28, from the longan fruit Euphoria longana. Since the plant-derived LAB strain produces an extracellular polysaccharide (EPS), in this study, we analyzed the chemical structure and the biosynthesizing genes for the EPS.The EPS, which was purified from the LP28 culture broth, was classified into acidic and neutral EPSs with a molecular mass of about 50 kDa and 40 kDa, respectively. The acidic EPS consisted of glucose, galactose, mannose, and N-acetylglucosamine moieties. Interestingly, since pyruvate residue was detected in the hydrolyzed acidic EPS, one of the four sugars may be modified with pyruvate. On the other hand, the neutral EPS consisted of glucose, mannose, and N-acetylglucosamine; pyruvate was scarcely detected in the polysaccharide molecule.As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs) with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.     

Synthesis and Anti-HCV Activity of 4-Methoxy-7H-Pyrrolo[2,3-d] Pyrimidine Carbocyclic Nucleosides

The present study includes the exploration of new possible nucleoside mimetics based on 4-methoxy-7H-pyrrolo[2,3-d]pyrimidine carbocyclic nucleosides (4a–g), which were synthesized by 10–15 synthetic steps and characterized adequately. We report the anti-HCV activities and cytotoxicities of 4a–g. Compound 4a was analyzed by single crystal X-ray diffraction which showed some puckering in the cyclopentene ring with a 2′-endo conformation and anti-base disposition (χ = ?125.7°).     

HTLV-1 bZIP Factor Impairs Anti-viral Immunity by Inducing Co-inhibitory Molecule,T Cell Immunoglobulin and ITIM Domain (TIGIT)

Human T-cell leukemia virus type 1 (HTLV-1) infects CD4⁺ T cells and induces proliferation of infected cells in vivo, which leads to the onset of adult T-cell leukemia (ATL) in some infected individuals. The HTLV-1 bZIP factor (HBZ) gene, which is encoded in the minus strand of HTLV-1, plays critical roles in pathogenesis. In this study, RNA-seq and ChIP-seq analyses using HBZ transduced T cells revealed that HBZ upregulates the expression and promoter acetylation levels of a co-inhibitory molecule, T cell immunoglobulin and ITIM domain (TIGIT), in addition to those of regulatory T cells related genes, Foxp3 and Ccr4. TIGIT was expressed on CD4⁺ T cells from HBZ-transgenic (HBZ-Tg) mice, and on ATL cells and HTLV-1 infected CD4⁺ T cells of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in vivo. Expression of Blimp1 and IL-10 was upregulated in TIGIT⁺CD4⁺ cells of HBZ-Tg mice compared with TIGIT^-CD4⁺ T cells, suggesting the correlation between TIGIT expression and IL-10 production. When CD4⁺ T cells from HBZ-Tg mice were stimulated with TIGIT’s ligand, CD155, their production of the inhibitory cytokine IL-10 was enhanced. Furthermore, dendritic cells from HBZ-Tg mice produced high levels of IL-10 after stimulation. These data suggest that HBZ alters immune system to suppressive state via TIGIT and IL-10. Importantly, TIGIT suppressed T-cell responses to another HTLV-1 virus protein, Tax, in vitro. Blocking of TIGIT and PD-1 slightly increased anti-Tax T-cell activity in some HAM/TSP patients. These results suggest that HBZ-induced TIGIT on HTLV-1 infected cells impairs T-cell responses to viral antigens. This study shows that HBZ-induced TIGIT plays a pivotal role in attenuating host immune responses and shaping a microenvironment favorable to HTLV-1.     

Nondestructive evaluation of photosynthesis by delayed luminescence in Arabidopsis in Petri dishes

Nondestructive evaluation of photosynthesis is a valuable tool in the field and laboratory. Delayed luminescence (DL) can reflect charge recombination through the backflow of electrons. However, DL detection has not yet been adapted for whole plants in Petri dishes. To compensate for differences in DL decay between sibling Arabidopsis plants grown under the same conditions, we developed a time-sequential double measurement method. Using this method, we examined the influence of photosynthetic electron flow inhibitors, and differences in the DL decay curves were categorized by considering the initial and late phases of the decay curves, as well as their intermediate slopes. The appearance of concavity and convexity in DL curves in Arabidopsis was different from unicellular algae, suggesting complexity in the photosynthetic machinery of higher plants. This detection method should be invaluable for evaluating photosynthetic defects in higher plants under sterile conditions without interrupting plant culture.     

Physicochemical and biological properties of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose (6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin)

A unique multibranched cyclomaltooligosaccharide (cyclodextrin, CD) of 6(1),6(3),6(5)-tri-O-alpha-maltosyl-cyclomaltoheptaose [6(1),6(3),6(5)-tri-O-alpha-maltosyl-beta-cyclodextrin, (G(2))(3)-betaCD] was prepared. The physicochemical and biological properties of (G(2))(3)-betaCD were determined together with those of monobranched CDs (6-O-alpha-D-glucopyranosyl-alpha-cyclodextrin (G(1)-alphaCD), 6-O-alpha-D-glucopyranosyl-beta-cyclodextrin (G(1)-betaCD), and 6-O-alpha-maltosyl-beta-cyclodextrin (G(2)-betaCD)). NMR spectra of (G(2))(3)-betaCD were measured using various 2D NMR techniques. The solubility of (G(2))(3)-betaCD in water and MeOH-water solutions was extremely high in comparison with nonbranched betaCD and was about the same as that of the other monobranched betaCDs. The formation of an inclusion complex of (G(2))(3)-betaCD with stereoisomers (estradiol, retinoic acid, quinine, citral, and glycyrrhetinic acid) depends on the cis-trans isomers of guest compounds. The cis isomers of estradiol, retinoic acid, and glycyrrhetinic acid were included more than their trans isomers, while the trans isomers of citral and quinine fit more tightly than their cis isomers. (G(2))(3)-betaCD was the most effective host compound in the cis-trans resolution of glycyrrhetinic acid. Among the branched betaCDs, (G(2))(3)-betaCD exhibited the weakest hemolytic activity in human erythrocytes and showed negligible cytotoxicity in Caco-2 cells up to 200 microM. These results indicate unique characteristics of (G(2))(3)-betaCD in some biological responses of cultured cells.     

Transcriptional suppression of nephrin in podocytes by macrophages: roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1beta and TNF-alpha markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms' tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.     

Rapid, transient induction of ER stress in the liver and kidney after acute exposure to heavy metal: evidence from transgenic sensor mice

Endoplasmic reticulum (ER) stress-responsive alkaline phosphatase (ES-TRAP) serves as a sensitive indicator for ER stress. In response to heavy metals including cadmium, nickel and cobalt, hepatocytes and renal tubular cells expressing ES-TRAP exhibited ER stress and decreased ES-TRAP activity. In ES-TRAP transgenic mice, acute exposure to cadmium showed rapid, transient decreases in the activity of serum ES-TRAP. It was inversely correlated with the induction of endogenous ER stress markers in the liver and kidney. Our result provides first evidence for the acute, reversible induction of ER stress in vivo after exposure to heavy metal.     

Fucosyl-GM1a, an endoglycoceramidase-resistant ganglioside of porcine brain

The use of bovine brain has been prohibited in many countries because of the world-wide prevalence of mad cow disease, and thus porcine brain is expected to be a new source for the preparation of gangliosides. Here, we report the presence of a ganglioside in porcine brain which is strongly resistant to hydrolysis by endoglycoceramidase, an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. Five major gangliosides (designated PBG-1, 2, 3, 4, 5) were extracted from porcine brain by Folch's partition, followed by mild alkaline hydrolysis and PBA column chromatography. We found that PBG-2, but not the others, was strongly resistant to hydrolysis by the enzyme. After the purification of PBG-2 with Q-Sepharose, Silica gel 60 and Prosep-PB chromatographies, the structure of PBG-2 was determined by GC, GC-MS, FAB-MS and NMR spectroscopy as Fucalpha1-2Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer (fucosyl-GM1a). The ceramide was mainly composed of C18:0 and C20:0 fatty acids and d18:1 and d20:1 sphingoid bases. The apparent kcat/Km for fucosyl-GM1a was found to be 30 times lower than that for GM1a, indicating that terminal fucosylation makes GM1a resistant to hydrolysis by the enzyme. This report indicates the usefulness of endoglycoceramidase to prepare fucosyl-GM1a from porcine brain.     

Limited suppression of the interferon-beta production by hepatitis C virus serine protease in cultured human hepatocytes

Toll-like receptors and RNA helicase family members [retinoic acid-inducible gene I (RIG-I) and melanoma differentiation associated gene-5 (MDA5)] play important roles in the induction of interferon-beta as a major event in innate immune responses after virus infection. TRIF (adaptor protein of Toll-like receptor 3)-mediated and Cardif (adaptor protein of RIG-I or MDA5)-mediated signaling pathways contribute rapid induction of interferon-beta through the activation of interferon regulatory factor-3 (IRF-3). Previously, it has been reported that the hepatitis C virus NS3-4A serine protease blocks virus-induced activation of IRF-3 in the human hepatoma cell line HuH-7, and that NS3-4A cleaves TRIF and Cardif molecules, resulting in the interruption of antiviral signaling pathways. On the other hand, it has recently been reported that non-neoplastic human hepatocyte PH5CH8 cells retain robust TRIF- and Cardif-mediated pathways, unlike HuH-7 cells, which lack a TRIF-mediated pathway. In the present study, we further investigated the effect of NS3-4A on antiviral signaling pathways. Although we confirmed that PH5CH8 cells were much more effective than HuH-7 cells for the induction of interferon-beta, we obtained the unexpected result that NS3-4A could not suppress the interferon-beta production induced by the TRIF-mediated pathway, although it suppressed the Cardif-mediated pathway by cleaving Cardif at the Cys508 residue. Using PH5CH8, HeLa, and HuH-7-derived cells, we further showed that NS3-4A could not cleave TRIF, in disagreement with a previous report describing the cleavage of TRIF by NS3-4A. Taken together, our findings suggest that the blocking of the interferon production by NS3-4A is not sufficient in HCV-infected hepatocyte cells.