Project description:Berry skin total protein from Cabernet Sauvignon, Merlot, Pinot Noir, Chardonnay and Semillon. Treatments were control (well-watered) versus restricted irrigation (water-deficit). Samples were taken from harvest-ripe whole berry clusters following a seasonal water deficit in treatment vines. A comparative analysis between the cultivars and treatments was performed. Associated dataset identifiers: GSE72421, PRJNA268857.
Project description:The vacuole occupies a large portion of plant cell volume, it is especially true to fruit tissues. Berry flesh cell vacuole serves as storage organelle for water, sugars, acids, secondary metabolites and others, which largely determining berry quality (Fontes et al., 2011a, b; Shiratake and Martinoia, 2007, Conde et al., 2006). However, the molecular basis of these compartmentation processes is still poorly understood. As in many species, the major bottle neck to study these aspects in grapevine is to obtain highly purified vacuoles with a good yield (Fontes et al., 2010). Up to date, several vacuole or tonoplast proteome researches were applied on a few plants mainly on Arabidopsis thaliana, vacuoles or tonoplast were derived from mesophyll cells (Carter et al., 2004, Endler et al., 2006, Schulze, et al., 2012) or cell culture (Jaquinod et al 2006, Shimaoka et al 2004), cauliflower buds (Schmidt et al., 2007) and sugar beet taproots (Jung et al., 2015). Though the grape berry protoplasts and intact vacuoles were successfully isolated from Cabernet Sauvignon berry suspension-cultured cells (Fontes et al., 2010), the vacuoles isolated from grape berry or different development and ripening stages of grape berry mesocarp tissues were not achieved.
Project description:Blanc Du bois grapes are gaining popularity in the South eastern US due to its distinctive flavor and disease tolerance characteristics. Berry composition at harvest is a major contributing factor of wine quality. Blanc Du bois grapes are harvested from EL-38 and EL-39 stages depending on the style of wine desired or harvested early to avoid rain nearing harvest. In the current study, gel-free proteome analysis was applied to investigate changes in enzymes, primary and secondary metabolism proteins during ripening and late ripe stage. Grape berries from EL-33, EL-34, EL-36, EL-38 and EL-39 were collected based on brix, acidity and density. Protein extracts from different berry stages were resolved by electrophoresis. Proteins were extracted from the gel as a single band, detained and subjected to proteolysis with sequencing grade trypsin. Trypsin digested peptides from different berry protein extracts were separated on a nano LC and the eluent was sprayed onto to a LTQ Orbitrap Velos mass spectrometer. The raw files were analyzed using Proteome Discoverer with Sequest and Mascot search nodes using Vitis species FASTA database (70,263 entries) and the data were further validated by Scaffold software. A total of 1091, 1131, 1078, 1042 and 1066 proteins were detected in EL-33, EL-34, EL-36, EL-38 and EL-39 of berries respectively. Statistical ANOVA analysis revealed 927 proteins present across the stages that are involved in various biochemical and metabolic pathways. Seventeen proteins including dihydroflavonol reductase, sucrose phosphate synthase, PR proteins increased more than three-fold between ripe and late ripe berry stages. Other proteins that increased during ripe and late ripe stage berries were alcohol dehydrogenase 1, anthocyanidin reductase, phospho-2-dehydro-3-deoxyheptonate aldolase, fatty acid hydroperoxide lyase, cinnamyl alcohol dehydrogenase, -isopiperitenol (-)-carveol and SAM-methyltransferases.
Project description:The experiment was designed to investigate transcriptional dynamics during definitive endoderm differentiation of human embryonic stem cells (hESCs) in early G1 phase of the cell cycle. For this, the FUCCI system was used to sort hESCs in Early G1 (EG1). Samples were taken at 0h (hESCs), 12h, 24h, 36h, 48h, 60h and 72h in triplicates. Total RNA was extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich) and residual genomic DNA was removed using the On-Column DNase I Digestion Set (Sigma-Aldrich) following manufacturer’s instructions. The libraries were generated at a library fragment size between 100 bp to 1 kb with Stranded RNAseq Standard with Oligo dT pulldown. All samples were amplified with a standard 10 PCR cycle before sequencing. The samples were multiplexed and analysed by Illumina Hiseq V4 with a paired end read-length PE75. RNA-seq data analysis was performed following recommendations described in Conesa et al. (DOI https://doi.org/10.1186/s13059-016-0881-8).
Project description:The E3 SUMO ligase PIAS2 is expressed at high levels in differentiated papillary thyroid carcinomas but at low levels in anaplastic thyroid carcinomas (ATC), an undifferentiated cancer with very high mortality. Double-stranded RNA–directed RNA interference (dsRNAi) targeting the PIAS2 isoform beta (PIAS2b) inhibits growth of ATC cell lines and patient primary cultures in vitro and orthotopic patient-derived xenografts (oPDX) in vivo, but not of thyroid cell lines or non-anaplastic primary thyroid cultures (differentiated carcinoma, benign lesions, or normal). PIAS2b-dsRNAi also has an anti-cancer effect on other anaplastic human cancers (pancreas, lung, and gastric). Mechanistically, PIAS2b is required for proper mitotic spindle and centrosome assembly, and it is a dosage-sensitive protein in ATC. Strikingly, PIAS2b-dsRNAi induces mitotic catastrophe at prophase. High-throughput proteomics revealed the proteasome (PSMC5) and spindle cytoskeleton as direct targets of PIAS2b SUMOylation at mitotic initiation. PIAS2b-dsRNAi is a promising therapy for ATC and other aggressive anaplastic cancers.
Project description:Single-stranded DNA (ssDNA) widely exists as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors out-compete RPA and access to ssDNA. Using the budding yeast, Saccharomyces cerevisiae, as a model system, we discovered that Dna2, a key nuclease in DNA replication and repair, employs a bimodal interface to act with RPA both in cis and in trans. The cis-action renders RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA actively delivers its bound ssDNA to Dna2. The trans-action mediated by an acidic patch from Dna2, on the other hand, enables Dna2 to operatie with a sub-optimal amount of RPA or to overcome DNA secondary structures. Genetically, this trans-action mode is not required for cell viability, but indispensable for successful DSB repair.
Project description:Sea cucumbers (Holothuroidea; Echinodermata), cycle annually between aestivation when water temperature is above about 25°C in the summer and active life when temperature is below about 18°C. We used RNA-Seq to determine gene expression profiles of intestine tissue of A.japonicus during non-aestivation (NA), deep-aestivation (DA), and arousal from aestivation (AA). At least 15 individuals per stage, intestines at NA, DA and AA stages were used for our experiments.
Project description:Sea cucumbers (Holothuroidea; Echinodermata) cycle annually between aestivation, when water temperature is above about 25°C in the summer, and active life, when temperature is below about 18°C. We used RNA-Seq to determine gene expression profiles of respiratory tree tissue of A. japonicus during non-aestivation (NA), deep-aestivation (DA), and arousal from aestivation (AA). At least 15 individuals per stage, respiratory tree at NA, DA and AA stages were used for our experiments.
Project description:In order to circumvent environmental changes throughout fruit development, young and ripening berries were sampled simultaneously on continuously flowering microvines acclimated to controlled circadian light and temperature changes. Gene expression profiles along fruit development were monitored during both day and night with whole genome microarray Nimbelgen® vitis 12x, yielding a total number of 9273 developmentally modulated probesets. All day-detected transcripts were modulated at night, whereas 1755 genes were night-specific. Very similar developmental patterns of gene expression were observed upon independent hierarchical clustering of day and night data, whereas functional categories of allocated transcripts varied according to time of the day. Many transcripts within pathways, known to be upregulated during ripening, in particular those linked to secondary metabolism exhibited a clearer developmental regulation at night than during the day. Functional enrichment analysis also indicated that diurnally modulated genes considerably varied during fruit development, with a shift from cellular organization and photosynthesis in green berries to secondary metabolism and stress-related genes in ripening ones. These results reveal critical changes in gene expression during night development that differ from day development which have not been observed in other transcriptomic studies on fruit development so far. A total of 24 samples were analyzed representing four berry developmental stages (two during green development, two during ripening). Sample were drawn simultaneously in triplicates at day and night on the microvine dwarf (DRCF) GAI mutant.
Project description:Transcriptional profiling of C. tropicalis a/alpha cells (CAY1511) in white state, opaque state, overexpressing Wor1, or wor1 mutants hybridized against a universal mixed reference sample from all 4 states. 4 condition experiment: white, opaque, tdh3-wor1, Δ/Δwor1; 4 biological replicates of each