Project description:Plant-based human vaccines have been actively developed in recent years, and rice (Oryza sativa L.) is one of the best candidate crops for their production and delivery. By expressing a modified cholera toxin B (CTB) subunit, we previously developed MucoRice-CTB, a rice-based vaccine against cholera, which is caused by infection of the intestine with the bacteria Vibrio cholerae. MucoRice-CTB lines have been extensively characterized by whole-genome sequencing and proteome analyses to evaluate the mutation profiles and proteome status, respectively. Here, we report non-targeted metabolomic profiling of the MucoRice-CTB transgenic rice line 51A (MR-CTB51A), MucoRice-RNAi (MR-RNAi), and their non-transgenic parent line by using gas chromatography-time-of-flight mass spectrometry. The levels of several amino acids, organic acids, carbohydrates, lipids, and secondary metabolites were significantly increased in MR-CTB51A compared with the non-transgenic parent line. These metabolomics results complement essential information obtained by genome sequencing and proteomics approaches, thereby contributing to comprehensive understanding of the properties of MucoRice-CTB as a plant-based vaccine.
Project description:Plants are considered an important food and nutrition source for humans. Despite advances in plant seed metabolomics, knowledge about the genetic and molecular bases of rice seed metabolomes at different developmental stages is still limited. Here, using Zhenshan 97 (ZS97) and Minghui 63 (MH63), we performed a widely targeted metabolic profiling in seeds during grain filling, mature seeds and germinating seeds. The diversity between MH63 and ZS97 was characterized in terms of the content of metabolites and the metabolic shifting across developmental stages. Taking advantage of the ultra-high-density genetic map of a population of 210 recombinant inbred lines (RILs) derived from a cross between ZS97 and MH63, we identified 4681 putative metabolic quantitative trait loci (mQTLs) in seeds across the three stages. Further analysis of the mQTLs for the codetected metabolites across the three stages revealed that the genetic regulation of metabolite accumulation was closely related to developmental stage. Using in silico analyses, we characterized 35 candidate genes responsible for 30 structurally identified or annotated compounds, among which LOC_Os07g04970 and LOC_Os06g03990 were identified to be responsible for feruloylserotonin and l-asparagine content variation across populations, respectively. Metabolite-agronomic trait association and colocation between mQTLs and phenotypic quantitative trait loci (pQTLs) revealed the complexity of the metabolite-agronomic trait relationship and the corresponding genetic basis.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. Recently, disorders of metabolism are thought to be the center of many diseases such as OPLL. Advanced glycation end product (AGE) are accumulated in many extracellular matrixes such as ligament fibers, and it can functions as cellular signal through its receptor (RAGE), contributing to various events such as atherosclerosis or oxidative stress. However, its role in OPLL formation is not yet known. Therefore, we performed high-through-put RNA sequencing on primary posterior longitudinal ligament cells treated with different doses of AGEs (1µM, 5µM and negative control), with or without BMP2 (1µM). mRNA profiles of Primary human posterior longitudinal ligament cells stimulated with various stimuli (Control, 1µM AGE-BSA, 5µM AGE-BSA, 1µM AGE-BSA with BMP2, 5µM AGE-BSA with BMP2) were generated by deep sequencing on Ion Proton
Project description:Plant metabolites are crucial for both plant life and human nutrition. Despite recent advance in metabolomics, the genetic control of plant metabolome remains largely unknown. Here, we performed a genetic analysis of the rice metabolome that provided over 2,800 highly resolved metabolic quantitative trait loci for 900 metabolites. Distinct and overlapping accumulation patterns of metabolites were observed and complex genetic regulation of metabolism was revealed in two different tissues. We associated 24 candidate genes to various metabolic quantitative trait loci by data mining, including ones regulating important morphological traits and biological processes. The corresponding pathways were reconstructed by updating in vivo functions of previously identified and newly assigned genes. This study demonstrated a powerful tool and provided a vast amount of high-quality data for understanding the plasticity of plant metabolome, which may help bridge the gap between the genome and phenome.
Project description:We report for the first time movement of Correia Repeat Enclosed Elements, through inversion of the element at its chromosomal location. Analysis of Ion Torrent generated genome sequence data from Neisseria gonorrhoeae strain NCCP11945 passaged for 8 weeks in the laboratory under standard conditions and stress conditions revealed a total of 37 inversions: 24 were exclusively seen in the stressed sample; 7 in the control sample; and the remaining 3 were seen in both samples. These inversions have the capability to alter gene expression in N. gonorrhoeae through the previously determined activities of the sequence features of these elements. In addition, the locations of predicted non-coding RNAs were investigated to identify potential associations with CREE. Associations varied between strains, as did the number of each element identified. The analysis indicates a role for CREE in disrupting ancestral regulatory networks, including non-coding RNAs. RNA-Seq was used to examine expression changes related to Correia repeats in the strain
Project description:In order to determine whether dis-regulation of a genetic pathway could explain the increased apoptosis of parp-2-/- double positive thymocytes, the gene expression profiles in double positive thymocytes derived from wild-type and parp-2-/- mice were analysed using Affymetrix oligonucleotide chips (mouse genome 430 2.0).
Project description:This dataset was generated with the goal of comparative study of gene expression in three brain regions and two non-neural tissues of humans, chimpanzees, macaque monkeys and mice. Using this dataset, we performed studies of gene expression and gene splicing evolution across species and search of tissue-specific gene expression and splicing patterns. We also used the gene expression information of genes encoding metabolic enzymes in this dataset to support a larger comparative study of metabolome evolution in the same set of tissues and species. 120 tissue samples of prefrontal cortex (PFC), primary visual cortex (VC), cerebellar cortex (CBC), kidney and skeletal muscle of humans, chimpanzees, macaques and mice. The data accompanies a large set of metabolite measurements of the same tissue samples. Enzyme expression was used to validate metabolite measurement variation among species.
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a NeoR-based retrotransposition cassette. Cells were selected by G418 and used to prepare ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of G418-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016).
Project description:We used ATLAS-seq-neo to map the sites of integration of an engineered LINE-1 (L1) retrotransposon into the genome of HeLa S3 cells. In brief, we transfected cells with a plasmid-borne L1.3 element carrying a neomycin-resistance-based retrotransposition cassette, as well as a hygromycin-resistance cassette on the plasmid backbone. For this set of experiments, cells were only selected for transfection (hygromycin) but not for retrotransposition (neomycin). Then we prepared ATLAS-seq-neo libraries. Each sample corresponds to an independent transfection and pool of hygromycin-resistant cells. ATLAS-seq-neo relies on the random mechanical fragmentation of the genomic DNA to ensure high-coverage, ligation of adapter sequences, suppression PCR-amplification of the 3' end L1 junction with its flanking genomic sequence, and Ion Torrent sequencing using single-end 400 bp read chemistry. The primer used for suppression PCR specifically targets the engineered element and not endogenous copies as in the original ATLAS-seq protocol (Philippe et al. eLife 2016). For some libraries, the linker-ligated genomic DNA was digested with BamHI, which cuts downstream of L1 polyA site in the plasmid backbone, to limit amplification from the plasmid and enrich for retrotransposition-mediated insertion events into the genomic DNA.