Project description:Purpose: Analyze changes in the transcriptome of Arabidopsis thaliana in response to chronic exposure to silver nitrate at 4 μg/mL concentration. Methods: mRNA was extracted from non-treated and silver nitrate-treated 14-day old Arabidopsis thaliana seedlings using the RNAeasy extraction kit (Qiagen). RNA-seq libraries (3 rep/treatment and 3 reps/control) constructed with the TruSeq Stranded mRNA Sample Preparation kit (Illumina) were paired-end sequenced (100-nt read length) on an Illumina Nova Seq6000 system. Reads were mapped to the A. thaliana TAIR10 reference genome sequence and transcript levels were analyzed using the softare CLC Genomics Workbench (version 20.0.4, Qiagen). Results: Chronic exposure of A. thaliana plants to silver nitrate caused a change in the abundance of transcripts: AT2G01520 and AT4G12550, but no measureable impact on the rest of the transcriptome. Conclusions: Exposure of A. thaliana to silver nitrate at 4 μg/mL has minor impact on the transcriptome.
Project description:In this project, we treated Arabidopsis wild type (Col-0) with low nitrate (0.2 mM KNO3) and high nitrate (5 mM KNO3) after nitrate starvation. Next we harvested root, and then we performed memebrane fraction and cytosolic fraction isolation for trypsin digestion and phosphopeptide enrichment.
Project description:Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out. In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A+ enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3. This nitrate-responsive miRNA/target module might be involved in controlling carbon flux to assimilate nitrate into amino acids, suggesting that microRNAs can have both developmental and metabolic functions in the nitrate response of Arabidopsis roots.
Project description:Nitrate and other nitrogen metabolites can act as signals that regulate global gene expression in plants. Adaptive changes in plant morphology and physiology triggered by changes in nitrate availability are partly explained by these changes in gene expression. Despite several genome-wide efforts to identify nitrate-regulated genes, no comprehensive study of the Arabidopsis root transcriptome under contrasting nitrate conditions has been carried out. In this work, we employed the Illumina high throughput sequencing technology to perform an integrated analysis of the poly-A+ enriched and the small RNA fractions of the Arabidopsis thaliana root transcriptome in response to nitrate treatments. Our sequencing strategy identified new nitrate-regulated genes including 40 genes not represented in the ATH1 Affymetrix GeneChip, a novel nitrate-responsive antisense transcript and a new nitrate responsive miRNA/TARGET module consisting of a novel microRNA, miR5640 and its target, AtPPC3. This nitrate-responsive miRNA/target module might be involved in controlling carbon flux to assimilate nitrate into amino acids, suggesting that microRNAs can have both developmental and metabolic functions in the nitrate response of Arabidopsis roots. Arabidopsis thaliana wild-type Col-0 plants were grown in hydroponic nitrate-free medium with 0.5 mM ammonium succinate as the only N-source for two weeks and were treated with 5 mM KNO3, or 5 mM KCl as control, for 2 hours. Total RNA from two independent sets of plants (biological replicates) was extracted from roots, and poly-A+ enriched and sRNA fractions were used to construct libraries for Illumina sequencing.
Project description:NLP1-9 are plant unique transcrition facotrs. To identify NLP1-9 target genes, we transiently expressed NLP1-9 in Arabidopsis mesophyll protoplasts and performed RNA-seq analysis. NLP1-9 individually induced specific gene expression and co-activated nitrate responsive genes expression. To study genome-wide transcriptional landscape modulated by NLP, we performed transcriptome analysis at 20 min after nitrate induction in wild type and nlp2,4,5,6,7,8,9. All nitrate responsive genes were significant reduced in nlp2,4,5,6,7,8,9 indicated that NLP transcription factors are central regulators in PNR.
Project description:Kilian2024 - Immune cell dynamics in Cue-Induced Extended Human Colitis Model
Single-cell technologies such as scRNA-seq and flow cytometry provide critical insights into immune cell behavior in inflammatory bowel disease (IBD). However, integrating these datasets into computational models for dynamic analysis remains challenging. Here, Kilian et al., (2024) developed a deterministic ODE-based model that incorporates these technologies to study immune cell population changes in murine colitis. The model parameters were optimized to fit experimental data, ensuring an accurate representation of immune cell behavior over time. It was then validated by comparing simulations with experimental data using Pearson’s correlation and further tested on independent datasets to confirm its robustness. Additionally, the model was applied to clinical bulk RNA-seq data from human IBD patients, providing valuable insights into immune system dynamics and potential therapeutic strategies.
Figure 4c, obtained from the simulation of human colitis model is highlighted here.
This model is described in the article:
Kilian, C., Ulrich, H., Zouboulis, V.A. et al. Longitudinal single-cell data informs deterministic modelling of inflammatory bowel disease. npj Syst Biol Appl 10, 69 (2024). https://doi.org/10.1038/s41540-024-00395-9
Abstract:
Single-cell-based methods such as flow cytometry or single-cell mRNA sequencing (scRNA-seq) allow deep molecular and cellular profiling of immunological processes. Despite their high throughput, however, these measurements represent only a snapshot in time. Here, we explore how longitudinal single-cell-based datasets can be used for deterministic ordinary differential equation (ODE)-based modelling to mechanistically describe immune dynamics. We derived longitudinal changes in cell numbers of colonic cell types during inflammatory bowel disease (IBD) from flow cytometry and scRNA-seq data of murine colitis using ODE-based models. Our mathematical model generalised well across different protocols and experimental techniques, and we hypothesised that the estimated model parameters reflect biological processes. We validated this prediction of cellular turnover rates with KI-67 staining and with gene expression information from the scRNA-seq data not used for model fitting. Finally, we tested the translational relevance of the mathematical model by deconvolution of longitudinal bulk mRNA-sequencing data from a cohort of human IBD patients treated with olamkicept. We found that neutrophil depletion may contribute to IBD patients entering remission. The predictive power of IBD deterministic modelling highlights its potential to advance our understanding of immune dynamics in health and disease.
This model was curated during the Hackathon hosted by BioMed X GmbH in 2024.
Project description:The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 um nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism. Keywords: Expression profilling by array
Project description:Nitrate regulates plant growth and development and acts as a potent signal to control gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis thaliana roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly and quickly after nitrate treatments in root organs in a tissue-specific manner. Phenotypic analysis of tga1/tga4 double mutant plants indicated that TGA1 and TGA4 are necessary for nitrate modulation of both primary and lateral root growth. Global gene expression analysis revealed that 97% of the genes with altered expression in tga1/tga4 double mutant plants are regulated by nitrate treatments indicating these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. Among the nitrate-responsive genes that depend on TGA1/TGA4 for normal regulation of gene expression, we found nitrate transporters NRT2.1, NRT2.2 and nitrite reductase (NIR) genes. Specific binding of TGA1 to its cognate DNA sequence on the target gene promoters was confirmed by chromatin immunoprecipitation assays. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots.
Project description:Nitrate regulates plant growth and development and acts as a potent signal to control gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis thaliana roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly and quickly after nitrate treatments in root organs in a tissue-specific manner. Phenotypic analysis of tga1/tga4 double mutant plants indicated that TGA1 and TGA4 are necessary for nitrate modulation of both primary and lateral root growth. Global gene expression analysis revealed that 97% of the genes with altered expression in tga1/tga4 double mutant plants are regulated by nitrate treatments indicating these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. Among the nitrate-responsive genes that depend on TGA1/TGA4 for normal regulation of gene expression, we found nitrate transporters NRT2.1, NRT2.2 and nitrite reductase (NIR) genes. Specific binding of TGA1 to its cognate DNA sequence on the target gene promoters was confirmed by chromatin immunoprecipitation assays. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots. Arabidopsis seedlings of the Col-0 and tga1/tga4 genotypes were grown on hydroponic medium containing 1X MS salts without Nitrogen, supplemented with 0.5 mM ammonium succinate as Nitrogen source and 3 mM sucrose on a Percival chamber under a photoperiod of 16 hours of light (100 ?E/m2/sec) and 8 hours of dark at 22°C for 14 days. The plants were treated at the onset of the light cycle with 5 mM KNO3 or 5 mM KCl as control for 2 hours. Whole roots were cut from seedlings and frozen on liquid Nitrogen. Total RNA was extracted using the TriZol reagent. 3 independent biological replicates were performed.
Project description:Plants modulate gene expression profile in response to nitrate through NIN-LIKE PROTEIN (NLP) transcription factors in order to promote nitrate uptake and utilization. Although there are 9 NLP proteins in Arabidopsis, NLP7 had attracted most of the attention. Here, we focused on NLP2, because the nlp2 and nlp7 knockout mutants displayed different phenotypes. To gain insight into genes whose expression was affected by the nlp2 mutation, we compared gene expression profiles in the wild-type Columiba and the nlp2 mutant that were treated with 10 mM KNO3 for 1 hour. Columbia and nlp2 that were treated with 10 mM KCl for 1 hour were used as controls. We found that nitrate induction of 6 genes was compromised only in the nlp2 mutant.