Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.
Project description:Background: Schistosoma japonicum is a pathogen of the phylum Platyhelminths that causes zoonotic schistosomiasis endemic in China and Southeast Asian countries. Control of the disease has been hampered by the lack of efficient measures. The development of tools for diagnosis of acute and chronic infection, and novel antiparasite reagents all relies on the understanding of the biological mechanism of the parasite. Results: In this study, the polyadenylated transcripts from the male and female S. japonicum were sequenced by a high-through-put RNA-seq technique. Bioinformatic and experimental analyses were focused on the posttranscriptional RNA processing, which at the first time revealed the extensive alternative splicing events occurred in the adult stage of the parasite. The number of protein-coding sequences identified in the transcriptomes of the female and male S. japonicum was 15,939 and 19,501 respectively, which is more than that predicted from the annotated genome sequence. Further, we identified four types of posttranscriptional processing, also called alternative splicing, in both female and male worms of S. japonicum, including exon skipping, intron retention, alternative donor site, and alternative acceptor site. Unlike mammalian organisms, alternative donor site and alternative acceptor site were found to be more common than the other two types of posttranscriptional processing in S. japonicum. In total, 13,438 and 16,507 alternative splicing events were respectively predicted in the transcriptomes of female and male S. japonicum. Conclusions: By using RNA-seq technology, we obtained the global transcriptomes of male and female S. japonicum. These results further provide a comprehensive view of the global transcriptome of S. japonicum. The findings of a substantial level of alternative splicing events dynamically occurring in the parasitation of the S. japonicum in the mammalian hosts suggested a complicate transcriptional and post-transcriptional regulation mechanisms employed by the parasite. The data will not only significantly improve the re-annotation of the genome sequences, but will also provide new information in the biology of the parasite. Two samples are analyzed.
Project description:In a previous study, we found that H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and by regulating H2S metabolism and the oxidative stress response. However, little is known about the molecular mechanisms behind H2S-regulated salt-stress tolerance in cucumber. Here, an integrated transcriptomic and proteomic analysis based on RNA-seq and 2-DE was used to investigate the global mechanism underlying H2S-regulated salt-stress tolerance. In total, 11 761 differentially expressed genes (DEGs) and 61 differentially expressed proteins (DEPs) were identified. Analysis of the pathways associated with the DEGs showed that salt stress enriched expression of genes in primary and energy metabolism, such as photosynthesis, carbon metabolism and biosynthesis of amino acids. Application of H2S significantly decreased these DEGs but enriched DEGs related to plant-pathogen interaction, sulfur-containing metabolism, cell defense and signal transduction pathways. Notably, changes related to sulfur-containing metabolism and cell defense were also observed through proteome analysis, such as Cysteine synthase 1, Glutathione S-transferase U25-like, Protein disulfide-isomerase and Peroxidase 2. We present the first global analysis of the mechanism underlying H2S regulation of salt-stress tolerance in cucumber through tracking changes in the expression of specific proteins and genes.
Project description:FBXO31 is a substrate adapter for the SCF-type ubiquitin ligase complex SCF/FBXO31 which ubiquitylates C-terminal amide-bearing proteins (CTAPs), thereby triggering their proteasomal degradation in human cells. Immunoprecipitation followed by MS (IP-MS) was performed on HA-tagged cDNA-expressed variants of FBXO31 from HEK293T cells to identify putative clients.
Project description:NIH 3T3 fibroblasts that express little detectable levels of the MIF binding receptor CD74 but do express the signalling component CD44 were used for the identification of interacting proteins that are shared by MIF and D-DT. Endogenously expressed MIF and D-DT were tagged at the C-terminus using a short sequence that is recognized and biotinylated by the bacterial biotin ligase BirA (Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice. Proc. Natl. Acad. Sci. U. S. A. 100, 7480–5) as described in detail for MIF Filip, A. M. et al. (2009). Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function. J. Biol. Chem. 284, 7977–7985). After pulling down biotinylated proteins from stable NIH 3T3 clones that express tagged MIF or D-DT and BirA ligase with streptavidin agarose, bound proteins were separated by SDS-PAGE and identified after elution and tryptic digestion by mass spectrometry in order to characterize the MIF and D-DT interactomes.
Project description:Fibroblasts are the most commonly used model in probing the complex process of somatic reprogramming. Although hematopoietic cells represents a more convenient and accesible/ or “an excellent alternative” starting cell type, the molecular mechanisms in hematopoietic reprogramming are poorly defined. In the present study, we showed that hematopoietic stem and progenitor cells (HSPCs) with long term repopulating potential, among several HSPC populations, are more amenable for reprogramming, and exhibit an efficient induction of transcriptional program involved in the promation of cell prolifieration and inhibition of apoptosis. In sharp comparison with fibroblasts, HSPCs possess distinct requirements for the activation of Tgf-b and wnt pathways in the initiation phase of reprograming, which can be attributable, at lease partially, to differentiall expression of key signaling genes in these two cell types. Our data demonstrate that lineage and developmental/differentiation stage-specific context define key early events in the reprogramming of hematopoietic cells to pluripotency. Examine the differential global gene expression among different hematopietic cells in reprogramming. LT-HSC, ST-HSC, MP and fibroblasts were used in this study. Doxcycline addition could induce expression of oct4, sox2, klf4 and c-myc, consequently reprogramming these cells into iPS cells. Hematopietic cells cultured with doxcycline for 0, 2, 4 days were sampled, and samples at the same timepoint without doxcycline were also taken to exclude vast gene expression change in hematopoietic cell in-vitro culture. Fibroblast samples induced by doxcycline for 0, 2, 4 days were also introduced to provide comparism between lineages.
Project description:Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression (DGE) profiles using Illumina’s high-throughput sequencing technology and the newly-assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in determinacy of axillary meristems. The RA3 gene encodes a trehalose-6-phosphate-phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of DGE libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20-21nt reads with frequencies spanning four orders of magnitude. Unique sequence tags were anchored to 3´-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of non-redundant signature tags to the maize genome, which associated with 37,117 working gene models and un-annotated regions of expression. 66% of maize genes were detected at ? nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis and rice in functional analyses of differentially expressed maize genes. Results from this study provide a basis for analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene. 3' tag-based DGE libraries were generated using Illumina 1G technology. Libraries were prepared from 2mm ear samples and represent: 3 biological replicates of wild-type B73, 3 biological replicates of ramosa3 (ra3) mutants, and 1 technical replicate of a ra3 sample. Each sample was sequenced in a single lane of a flow cell. Each lane includes library preparations from both DpnII and NlaIII digests for that sample. Sequences from DpnII digests start with GATC and those from NlaIII digests start with ATG.
Project description:MicroRNAs (miRNAs) represent a conserved class of small non-coding RNAs that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs are 20-25 nucleotides (nt) in length and have important regulatory functions in biological processes such as embryonic development, cell differentiation, hormone secretion or metabolism. Furthermore, miRNAs have been implicated in the pathology of various diseases including cancer. MiRNA expression profiles not only classify different types of cancer but also may even help to characterize distinct tumor stages, therefore constituting a valuable tool for prognosis. Here we report the miRNA profile of Epstein-Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) tissue samples characterized by cloning and sequencing. We find that all EBV miRNAs from the BART region are expressed in NPC tissues whereas ebv-miRNAs from the BHRF1 region are not found. Moreover, we identify two novel EBV miRNA genes originating from the BART region that have not been found in other tissues or cell lines before. We also identify three new human miRNAs, which might be specific for nasopharyngeal tissues. We further show that a number of different cellular miRNAs are upor down-regulated in NPC tissues compared to control tissue including miR-15a and miR-16. We find that the tumor suppressor BRCA-1 is a target of miR-15a as well as miR-16 suggesting a miRNA role in NPC pathogenesis. 2 pairs of NPC and control tissues from 2 patients (4 samples in total) were examined.
Project description:The maize smut fungus, Sporisorium reilianum f. sp. zeae, which is an important biotrophic pathogen responsible for extensive crop losses, infects maize by invading the root during the early seedling stage. In order to investigate disease-resistance mechanisms at this early seedling stage, digital gene expression (DGE) analysis, which applies a dual-enzyme approach (DpnII and NlaIII), was used to identify the transcriptional changes in roots of Huangzao4 (susceptible) and Mo17 (resistant) after inoculation with teliospores of S. reilianum. Before and after inoculation, pathogenesis-related genes were differentially regulated and enzymes involved in controlling reactive oxygen species (ROS) levels showed different activity between Huangzao4 and Mo17, which can potentially lead to changes in the growth of S. reilianum and ROS production in maize. Moreover, lignin depositions of roots were also changed differentially during root colonization of hyphae between Huangzao4 and Mo17. These results suggest that the interplays between S. reilianum and maize during the early infection stage involve many interesting transcriptional and physiological changes, which offer several novel insights for understanding the mechanisms of resistance to the fungal infection. Examination of control stage (ck), post-inoculation stage1 (P1) and post-inoculation stage2 (P2) in Huangzao4 (susceptible) and Mo17 (resistant)
Project description:The mRNA repertoire is specific to a given tissue type. This specificity, whether it is in the levels of gene expression or the splicing pattern of the transcripts, is important to study to increase our general knowledge of transcription and alternative splicing, as well as to identify novel coding sequence that can be important for elucidating the mechanisms of disease. We have performed RNA-Seq on three normal human retinal samples and have found a profound level of novel alternative splicing including novel exons, exon skipping, and 3M-bM-^@M-^Y and 5M-bM-^@M-^Y alternate splice sites. Additionally, we have identified hundreds of novel genes. With so many novel splicing events identified in the retinal transcriptome, we set out to determine if these novel features were indeed real. We developed a high throughput capture set to target 14,696 of the novel features. We found that 99% of the novel features do validate, including those initially detected at a depth of coverage as low as one. These results confirm that the transcripts expressed in the retina are much more diverse than previously identified. The complete human retinal transcriptome described here will be useful for investigators studying multiple aspects of retinal biology and disease. Characterization of alternative splicing in the normal human retinal transcriptome.