Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening

Building The Sugarcane Genome For Biotechnology And Identifying Evolutionary Trends

Background: Sugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.Results: Three hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.Conclusion: This release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery. © 2014 de Setta et al.; licensee BioMed Central Ltd.151European Commission: Agriculture and Rural Development: Sugar http://ec.europa.eu/agriculture/sugar/index_en.htmKellogg, E.A., Evolutionary history of the grasses (2001) Plant Physiol, 125, pp. 1198-1205Grivet, L., Arruda, P., Sugarcane genomics: depicting the complex genome of an important tropical crop (2001) Curr Opin Plant Biol, 5, pp. 122-127Piperidis, G., Piperidis, N., D'Hont, A., Molecular cytogenetic investigation of chromosome composition and transmission in sugarcane (2010) Mol Genet Genomics, 284, pp. 65-73D'Hont, A., 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A Molecular Linkage Map For Drosophila Mediopunctata Confirms Synteny With Drosophila Melanogaster And Suggests A Region That Controls The Variation In The Number Of Abdominal Spots

The classic approach to gene discovery relies on the construction of linkage maps. We report the first molecular-based linkage map for Drosophila mediopunctata, a neotropical species of the tripunctata group. Eight hundred F2 individuals were genotyped at 49 microsatellite loci, resulting in a map that is ≈450 centimorgans long. Five linkage groups were detected, and the species' chromosomes were identified through cross-references to BLASTn searches and Müller elements. Strong synteny was observed when compared with the Drosophila melanogaster chromosome arms, but little conservation in the gene order was seen. 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Characterization Of New Polymorphic Functional Markers For Sugarcane

Expressed sequence tags (ESTs) offer the opportunity to exploit single, low-copy, conserved sequence motifs for the development of simple sequence repeats (SSRs). The authors have examined the Sugarcane Expressed Sequence Tag database for the presence of SSRs. To test the utility of EST-derived SSR markers, a total of 342 EST-SSRs, which represent a subset of over 2005 SSR-containing sequences that were located in the sugarcane EST database, could be designed from the nonredundant SSR-positive ESTs for possible use as potential genic markers. These EST-SSR markers were used to screen 18 sugarcane (Saccharum spp.) varieties. A high proportion (65.5%) of the above EST-SSRs, which gave amplified fragments of foreseen size, detected polymorphism. The number of alleles ranged from 2 to 24 with an average of 7.55 alleles per locus, while polymorphism information content values ranged from 0.16 to 0.94, with an average of 0.73. The ability of each set of EST-SSR markers to discriminate between varieties was generally higher than the polymorphism information content analysis. When tested for functionality, 82.1% of these 224 EST-SSRs were found to be functional, showing homology to known genes. As the EST-SSRs are within the expressed portion of the genome, they are likely to be associated to a particular gene of interest, improving their utility for genetic mapping; identification of quantitative trait loci, and comparative genomics studies of sugarcane. The development of new EST-SSR markers will have important implications for the genetic analysis and exploitation of the genetic resources of sugarcane and related species and will provide a more direct estimate of functional diversity.522191209Aggarwal, R.K., Hendre, P.S., Varshney, R.K., Bhat, P.R., Krishna-kumar, V., Singh, L., Identification, characterization and utilization of EST-derived genic microsatellite markers for genome analyses of coffee and related species (2007) Theor. Appl. Genet, 114, pp. 359-372. , PMID:17115127, doi:10.1007/s00122- 006-0440-xhttp://cran.r-project.org/web/packages/kinship/index.html, Atkinson, B, and Therneau, T. 2007. Kinship: mixed-effects Cox models, sparse matrices, and modeling data from large pedigrees, R package version 1.1.0-17. 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Marker-trait Association And Epistasis For Brown Rust Resistance In Sugarcane

Sugarcane brown rust, caused by Puccinia melanocephala, is one of the major sugarcane diseases. The detection of molecular markers associated to brown rust resistance and also of its epistatic interactions were investigated in a mapping population obtained by crossing the brown rust susceptible clone IACSP953018 and the resistant cultivar IACSP933046. Resistance was evaluated in a field trial in plant cane and first ratoon under natural infection and scored using a diagrammatic scale from 1 (most resistance) to 9 (most susceptible). A total of 488 single dose markers (amplified fragment length polymorphism AFLP, genomic microsatellite gSSR and expressed sequence tag derived microsatellites EST-SSRs) were evaluated through a single marker trait association approach for brown rust resistance. Sixty one putative quantitative trait alleles (QTA) for brown rust (30 in plant cane, 31 in ratoon cane; 10 of them was common for both crop years) were detected of which several were related to resistance. Twenty one (34 %) of the markers associated to QTA derived from ESTs. Some of them have similarity to genes/proteins related to disease response pathways. The estimates of the proportion of the total phenotypic variation ((Formula presented.)) explained by each significant main QTA effect ranged from 1.84 to 7.22 %, while the total explained variance estimates were 37.25 % (plant cane) and 43.26 % (ratoon cane) considering all main significant QTA effects. Fifty significant digenic epistatic interactions were suggested with the majority (68 %) contributing to increase brown rust resistance. Together, these probable epistatic effects explain 16.26 % (plant cane) and 17.22 % (ratoon cane) of the total phenotypic variance. Although evidence of epistasis was observed, linkage cannot be ruled out as the majority of the markers involved in the digenic interaction could not be addressed to any linkage group. The results suggest that epistasis may have an important contribution on sugarcane resistance to brown rust