Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3' and/or 5' end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells. CONCLUSIONS: Our study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis

Berry Flesh and Skin Ripening Features in Vitis vinifera as Assessed by Transcriptional Profiling

Background Ripening of fleshy fruit is a complex developmental process involving the differentiation of tissues with separate functions. During grapevine berry ripening important processes contributing to table and wine grape quality take place, some of them flesh- or skin-specific. In this study, transcriptional profiles throughout flesh and skin ripening were followed during two different seasons in a table grape cultivar ‘Muscat Hamburg’ to determine tissue-specific as well as common developmental programs. Methodology/Principal Findings Using an updated GrapeGen Affymetrix GeneChip® annotation based on grapevine 12×v1 gene predictions, 2188 differentially accumulated transcripts between flesh and skin and 2839 transcripts differentially accumulated throughout ripening in the same manner in both tissues were identified. Transcriptional profiles were dominated by changes at the beginning of veraison which affect both pericarp tissues, although frequently delayed or with lower intensity in the skin than in the flesh. Functional enrichment analysis identified the decay on biosynthetic processes, photosynthesis and transport as a major part of the program delayed in the skin. In addition, a higher number of functional categories, including several related to macromolecule transport and phenylpropanoid and lipid biosynthesis, were over-represented in transcripts accumulated to higher levels in the skin. Functional enrichment also indicated auxin, gibberellins and bHLH transcription factors to take part in the regulation of pre-veraison processes in the pericarp, whereas WRKY and C2H2 family transcription factors seems to more specifically participate in the regulation of skin and flesh ripening, respectively. Conclusions/Significance A transcriptomic analysis indicates that a large part of the ripening program is shared by both pericarp tissues despite some components are delayed in the skin. In addition, important tissue differences are present from early stages prior to the ripening onset including tissue-specific regulators. Altogether, these findings provide key elements to understand berry ripening and its differential regulation in flesh and skin.This study was financially supported by GrapeGen Project funded by Genoma España within a collaborative agreement with Genome Canada. The authors also thank The Ministerio de Ciencia e Innovacion for project BIO2008-03892 and a bilateral collaborative grant with Argentina (AR2009-0021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Cartilage-like tissue engineering using silk scaffolds and mesenchymal stem cells

Silk fibroin scaffolds were studied as a new biomaterial option for tissue-engineered cartilage-like tissue. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on silk, collagen, and crosslinked collagen scaffolds and cultured for 21 days in serum-free chondrogenic medium. Cells proliferated more rapidly on the silk fibroin scaffolds than on the collagen matrices. The total content of glycosaminoglycan deposition was three times higher on silk as compared to collagen scaffolds. Glycosaminoglycan deposition coincided with overexpression of collagen type II and aggrecan genes. Cartilage-like tissue was homogeneously distributed throughout the entire silk scaffolds, while on the collagen and crosslinked collagen systems tissue formation was restricted to the outer rim, leaving a doughnut appearance. Round or angular-shaped cells resided in deep lacunae in the silk systems and stained positively for collagen type II. The aggregate modulus of the tissue-engineered cartilage constructs was more than 2-fold higher than that of the unseeded silk scaffold controls. These results suggest that silk fibroin scaffolds are suitable biomaterial substrates for autologous cartilage tissue engineering in serum-free medium and enable mechanical improvements along with compositional features suitable for durable implants to generate or regenerate cartilage. © Mary Ann Liebert, Inc

Silk fibroin as an organic polymer for controlled drug delivery

The pharmaceutical utility of silk fibroin (SF) materials for drug delivery was investigated. SF films were prepared from aqueous solutions of the fibroin protein polymer and crystallinity was induced and controlled by methanol treatment. Dextrans of different molecular weights, as well as proteins, were physically entrapped into the drug delivery device during processing into films. Drug release kinetics were evaluated as a function of dextran molecular weight, and film crystallinity. Treatment with methanol resulted in an increase in beta-sheet structure, an increase in crystallinity and an increase in film surface hydrophobicity determined by FTIR, X-ray and contact angle techniques, respectively. The increase in crystallinity resulted in the sustained release of dextrans of molecular weights ranging from 4 to 40 kDa, whereas for less crystalline films sustained release was confined to the 40 kDa dextran. Protein release from the films was studied with horseradish peroxidase (HRP) and lysozyme (Lys) as model compounds. Enzyme release from the less crystalline films resulted in a biphasic release pattern, characterized by an initial release within the first 36 h, followed by a lag phase and continuous release between days 3 and 11. No initial burst was observed for films with higher crystallinity and subsequent release patterns followed linear kinetics for HRP, or no substantial release for Lys. In conclusion, SF is an interesting polymer for drug delivery of polysaccharides and bioactive proteins due to the controllable level of crystallinity and the ability to process the biomaterial in biocompatible fashion under ambient conditions to avoid damage to labile compounds to be delivered

Non-invasive time-lapsed monitoring and quantification of engineered bone-like tissue

The formation of bone-like tissue from human mesenchymal stem cells (hMSC) cultured in osteogenic medium on silk fibroin scaffolds was monitored and quantified over 44 days in culture using non-invasive time-lapsed micro-computed tomography (µCT). Each construct was imaged nine times in situ. From µCT imaging, detailed morphometrical data on bone volume density, surface-to-volume ratio, trabecular thickness, trabecular spacing, and the structure model index and tissue mineral density were obtained. µCT irradiation did not impact the osteogenic performance of hMSCs based on DNA content, alkaline phosphatase activity, and calcium deposition when compared to non-exposed control samples. Bone-like tissue formation initiated at day 10 of the culture with the deposition of small mineralized clusters. Tissue mineral density increased linearly over time. The surface-to-volume ratio of the bone-like tissues converged asymptotically to 26 mm-1. Although in vitro formation of bone-like tissue started from clusters, the overall bone volume was not predictable from the time, number, and size of initially formed bone-like clusters. Based on microstructural analysis, the morphometry of the tissue-engineered constructs was found to be in the range of human trabecular bone. In future studies, non-invasive, time-lapsed monitoring may enable researchers to culture tissues in vitro, right until the development of a desired morphology is accomplished. Our data demonstrate the feasibility of qualitatively and quantitatively detailing the spatial and temporal mineralization of bone-like tissue formation in tissue engineering. © 2007 Biomedical Engineering Society