Project description:Besides the established selection criteria based on embryo morphology and blastomere number, new parameters for embryo viability are needed to improve the clinical outcome of in vitro fertilization (IVF) and more particular of elective single embryo transfer (eSET). The aim of the study was to analyse genome-wide whether the embryo viability was reflected by the expression of genes in the oocyte surrounding cumulus cells. Early cleavage (EC) was chosen as a parameter for embryo viability. Experiment Overall Design: Consenting patients visiting the IVF clinic underwent an IVF or ICSI treatment. Immediately following ultrasound-guided cumulus-oocyte-complex (COC) retrieval, a proportion of the cumulus cells surrounding the oocyte were removed. Gene expression in cumulus cells from eight oocytes resulting in an early cleavage embryo (EC-CC; n=8) and from eight oocytes resulting in a non-EC embryo (NEC-CC; n=8) derived from six patients were analysed using microarrays (n=16). To exclude a differential gene expression due to differences in patient characteristics, samples were paired. From four patients both an EC-CC and a NEC-CC sample were used. From two additional patients two EC-CC as well as two NEC-CC samples were used.
Project description:To understand the molecular foundation of the SE induction and development in avocado, we compared by proteomics approach, the embryogenic (EC) and non-embryogenic (NEC) cultures of two avocado varieties. Although Criollo and Hass EC exhibits particularities in the proteome and metabolome profile, in general, we observed a more active phenylpropanoid pathway in EC than NEC. Our proteomic pipeline consisted of a peptide labeled with TMT6plex and synchronous precursor selection (SPS) MS3
Project description:In somatic embryogenesis, embryos are formed from a single or small group of somatic cells in response to stress and hormonal stimuli. In this study, a high-throughput phosphoproteomic analysis was used to identify and quantify phosphoproteins related to the acquisition of embryogenic competence in sugarcane. Embryogenic (EC) and nonembryogenic (NEC) calli were compared at the multiplication phase, resulting in the identification of 1279 phosphoproteins. After the differential accumulation analysis, 163 phosphoproteins were identified as unique to EC, and nine were unique to NEC, while 51 were upaccumulated, and 40 were downaccumulated in EC compared to NEC. These phosphopeptides were mainly single phosphorylated peptides, with serine residues representing the major site of phosphorylation. The motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx] and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. Gene ontology enrichment revealed an overrepresentation of biological processes involved in embryonic development, showing that EC-related phosphoproteins (unique to EC and upaccumulated) were associated with stress responses, regulation of gene expression and epigenetic modifications. The NEC-related phosphoproteins (unique to NEC and downaccumulated) were associated with the translation process. In this sense, EC-related phosphoproteins described as potential regulators of abiotic stress tolerance in response to ABA-induced signals such as OSK3 (a catalytic subunit of SnRK1), ABF1, LEAs, REM4.1 and RD29Bs were identified. On the other hand, the NEC-related phosphoproteins EDR1 and PP2Ac-2 were negative regulators of ABA, suggesting a role of ABA in the acquisition of embryogenic competence linked to stress tolerance. Moreover, EC-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TPL, as well as those involved in embryo development, including ASIL1, M3KE1, MCM2, and GRV2, were identified as putative potential regulators of embryogenic competence. Our results provide novel data that may help elucidate the phosphorylation mechanisms controlling the activity, affinity and localization of specific proteins during somatic embryogenesis and suggest a role for these proteins in the hormonal signalling cascade leading to stress adaptation by reprogramming genetic expression.
Project description:Purpose: Maize somatic embryogenesis is usually required to achieve genetic transformation and represents an important alternative in plant development. Although many embryogenesis-related genes have been studied in this model, the molecular mechanisms underlying cell dedifferentiation and further plant regeneration are not completely understood. Methods: Immature embryos smRNA profiles of 15-day-after-pollination (IE) and Embryogenic Callus from one (C1), four (C4), and ten months (C10) were generated by deep sequencing, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed with two methods: Bowtie 1.1.2 and ShortStack 3.4. qRT–PCR validation for selected miRNAs was performed using SYBR Green assays. Results: We used high throughput sequencing to explore the sRNA populations during maize embryogenic callus induction and established subcultures from the Mexican cultivar VS-535, Tuxpeño landrace. We detected readjustments in 24 nt and 21-22 nt sRNA populations during the embryogenic callus establishment and maintenance. miRNAs related to stress response substantially increased upon callus proliferation establishment, correlating with a reduction in some of their target levels. On the other hand, while 24 nt-long hc-siRNAs derived from transposable retroelements transiently decreased in abundance during the embryogenic callus establishment, a population of 22 nt- hc-siRNAs increased. This was accompanied by reduction in transposon expression in the established callus subcultures. Conclusions: Stress- and development-related miRNAs are highly expressed upon maize EC callus induction and during maintenance subcultures, while miRNAs involved in hormone response only transiently increase during induction. The establishment of proliferative maize embryogenic callus is accompanied by important readjustments in the length of hc-siRNAs mapping to LTR retrotransposons, and their expression regulation.
Project description:Necrotizing enterocolitis (NEC) is an acute gut inflammatory disorder that mainly affects preterm neonates. A large proportion of NEC survivors develop neuronal deficits later in life, however the effect of early stages of NEC on the developing brain is unknown. Using preterm pigs as a NEC-sensitive animal model, we profiled the hippocampal gene expression in response to severe NEC lesions. The NEC-induced differentially expressed genes (DEGs) in the hippocampus segregated the piglets suffering from small intestinal NEC (Si-NEC) from those showing NEC lesions only in the colon (Co-NEC). Only when NEC lesions were observed in the small intestine, did piglets show reduced physical activity together with up-regulation of hippocampal genes related to inflammation and hypoxia, which was further verified by qPCR. Cluster analyses revealed key hippocampal NEC–related DEGs for Si-NEC (23 genes, including S100A8, PDK4, EDN1, IER3, Opalin, TXNIP) and Co-NEC (3 genes: GSTM3, TF, and S100A1). Both NEC phenotypes showed only two down-regulated DEGs (TMEM 167, HBB) and were devoid of any histological signs of microglia activation. Cerebrospinal fluid (CSF) from NEC-positive pigs contained elevated levels of several inflammatory proteins and in vitro exposure of immature hippocampal neurons to NEC-related CSF promoted neuritogenesis. Further in vitro experiments with neurite outgrowth indicated that VEGF, CINC-3, S100A9 and S100A8/S100A9 may play a role in NEC effects on hippocampal development. In conclusion, NEC lesions, especially when involving the small intestine, alter hippocampal gene expression with potential neuroinflammation and effects on neural circuit formation. Our results demonstrated that gut lesions affect the immature brain at early stages of disease progression. Thus, supportive care is important for preterm infants experienced with NEC to lessen possible later neurological dysfunctions.
Project description:Necrotizing Enterocolitis (NEC) is an inflammation causing injury to the bowel in newborns. This project uses a rodent model that mimics the intestinal pathological changes seen in NEC to study the effect of formula feeding and hypoxia on NEC development
Project description:To support our research of epigenomics in rice genome, we conducted massively parallel pyrosequencing of mRNAs (RNA-seq) using rice seedling and callus tissues. We obtained a total of 40.1 M reads from seedling and 29.6 M reads from callus.The RNA-seq data derived from the replicates showed high correlations (Spearman’s rank correlation coefficient, SC, 0.96 for seedling, 0.88 for callus). Our RNAseq data derived from the seedling also showed a high correlation with the recently published rice RNA-seq data that was also from two-week old seeding tissue (Lu et al., 2010, PMID: 20627892) (SC=0.87). Two biological replicates of each tissue was applied in RNA-seq. Each biological replicate was sequenced twice in separate lane.
Project description:Adult endothelial cells (ECs) are known to possess organ-specific gene expression, morphology and function, but whether organ-specific EC gene expression is present during human development is not known. Here, we used bulk RNA-sequencing (RNA-seq) to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC-gene signatures. FACS was used to isolate EC (CD31+CD144+, n=13) and non-EC (CD31-CD144-, n=16) populations from these three organs, profiling at least 4 biological replicates for each organ system. The biological specimens profiled were between 11-20 gestational weeks. We also sequenced cultured human umbilical vein endothelial cells (HUVECs) via bulk RNAseq. Computational approaches were used to identify organ-specific EC-enriched gene signatures across human fetal lung, intestine, and kidney ECs.
Project description:Necrotizing enterocolitis (NEC) is the most frequent life-threatening gastrointestinal disease experienced by premature infant occuring in neonatal intensive care units. NEC is associated with severe intestinal inflammation, intestinal perforation leading to mortality. The challenge for neonatologists is to detect early clinical manifestations of NEC. Therefore, one of the strategies to prevent or treat NEC would be to develop an early diagnostic tool allowing identification of preterm infants either at risk of developing NEC or at the onset of the disease. Illumina’s deep sequencing technology (RNA-seq) was used to establish the gene expression profile between resected ileal healthy preterm (control, n=5) and NEC diagnosed preterm infant (NEC, n=9) and analyzed by IPA Core analysis system. IPA analysis indicated that the most significant functional pathways overrepresented in NEC neonates were associated with innate immune functions, such as altered T and B cell signaling, B cell development, and the role of pattern recognition receptors in recognition of bacteria and viruses. Among genes that were strongly modulated in NEC neonates, we observed a high degree of similarity with those linked to the development of IBD. By comparing gene expression patterns between NEC and Crohn’s disease, we identified several new potential protein targets for helping to predict and/or diagnose NEC in preterm infant. Gene expression profile revealed an uncontrolled innate immune response in the intestine of NEC neonates. Moreover, comparative analysis between NEC and Crohn’s disease evidenced high degree of similarity between these two inflammatory diseases and allowed us to identify several new potential NEC biomarkers.
Project description:Background: Autophagy plays an essential role in the occurrence and progression of Necrotizing enterocolitis (NEC). We purposed to carry out the identification and validation of the probable autophagy-related genes of NEC via bioinformatics methods and experiment trials. Methods: The autophagy-related differentially expressed genes (arDEGs) of NEC were identified by analyzing the RNA sequencing data of experiment neonatal mouse model and dataset GSE46619. Protein-protein interactions (PPI), gene-ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used for the arDEGs. Then, co-expressed autophagy-related genes in two datasets were identified by Venn analysis and verified by qRT-PCR in experimental NEC. Results: Autophagy increased in experimental NEC and 47 arDEGs were identified in experimental NEC by RNA-sequencing. The PPI results proclaimed those genes interplayed with each other. The GO and KEGG enrichment results of arDEGs reported some certain enriched pathways related to autophagy and macroautophagy. Furthermore, 22 arDEGs were identified in human NEC from dataset GSE46619. The GO and KEGG enrichment analysis of these genes showed similar enriched terms with the results in experimental NEC. Finally, HIF-1a, VEGFA, ITGA3, ITGA6, ITGB4 and NAMPT were identified as co-expressed autophagy-related genes by Venn analysis in human NEC from dataset GSE46619 and experimental NEC. The result of qRT-PCR revealed the expression levels of HIF-1a and ITGA3 upregulated, VEGFA and ITGB4 downregulated in experimental NEC. Conclusion: We identified 47 arDEGs in experimental NEC and 22 arDEGs in human NEC via bioinformatics analysis. HIF-1a, ITGA3, VEGFA and ITGB4 may have effects on the progression of NEC through modulating autophagy.