The Rice NRT2.4 Alters Metabolism and Nitrogen Uptake through Root Modulation

Purpose The expression patterns of the NRT2 genes have been well described, however, it is not well known the role of OsNRT2.4 in root growth. In this study we thus aimed at investigating the role of high-affinity NO3- transport OsNRT2.4 in NO3--regulated and the modulation of root growth.Methods Through the gene silencing technique amiRNA-mediated we successfully obtained osnrt2.4 knockdown lines to study the role of OsNRT2.4 on root growth under low nitrate conditions. We performed real time RT-PCR analysis to investigate the relative gene expression level in root and shoot, soluble metabolites, and measurement of root system.Results Knockdown of OsNRT2.4 did not affect rice growth. In comparison with wild-type (WT) plants showed that knockdown of OsNRT2.4 inhibited root formation under low NO3- supply. We demonstrated that the mutant lines had significantly increased NO3- uptake than WT plants when growth in different nitrate supply; osnrt2.4 knockdown lines showed an alteration in nitrogen metabolism, and this affected the root growth. The downregulation of OsNRT2.4 enhanced the expression of genes response of low external NO3- concentrations.Conclusion Herein we provide new insights in OsNRT2.4 functions. Our data demonstrated that OsNRT2.4 plays a role in root growth, nitrogen metabolic pathway and probably have functions in nitrate transport from root to shoot under low nitrate availability in rice.

The Spatial Distribution Characteristics of Typical Pathogens and Nitrogen and Phosphorus in the Sediments of Shahe Reservoir and their Relationships

Using samples collected in Shahe Reservoir in the upper North Canal in China, this research analyzes the structure of a microorganism group in sediment and the gene expression levels of two typical pathogenic bacteria (Escherichia coli and Enterococcus), and their relationship with environmental factors including total nitrogen (TN) and total phosphorus (TP). The study of samples collected from the surface (0–20 cm) and sediment cores shows that the absolute gene expression level of E. coli in in horizontal distribution in the sediment is higher than the relative gene expression level in the downstream of the reservoir and contaminated area. In vertical distribution, the absolute gene expression level of the two pathogenic bacteria in the sediment tends to decrease with increasing depth, although the relative gene expression level has its highest value at 10–30 cm depth. The relative gene expression level of the two pathogenic bacteria is much greater in the sediment of Shahe Reservoir with the structure of horizontal groups including Clortridium sensu stricto, unclassified Anaeroineaceae, and Povalibacter, while Anaeroineaceae is much more abundant in the group structure of the vertical distribution. Pearson correlation analysis suggests positive correlation in horizontal distribution for E. coli and TN and TP (P < 0.05) and for Enterococcus and TP (P < 0.05). The results clearly show that the amount of pathogenic bacteria in the sediment in Shahe Reservoir is most likely due to water eutrophication.

A152 INVESTIGATING THE ROLE OF NOVEL NUDT15 VARIANT IN AZATHIOPRINE-RELATED MYELOTOXICITY

Background Azathioprine (AZA), an immunosuppressant, has classically been used to treat patients with inflammatory bowel disease (IBD). AZA inhibits purine synthesis, and its metabolism occurs via a pathway involving thiopurine methyltransferase (TPMT). While standard TPMT genetic screening is conducted for IBD patients initiating AZA treatment to minimize adverse drug effects (ADE), a majority of patients experiencing ADE have wildtype TPMT. Another gene, NUDT15, has been found to be associated with AZA-related myelotoxicity Aims In this study we report two novel variants in NUDT15 and aim to evaluate the impact of NUDT15 variation on its gene expression. We hypothesize that the mutations found within novel NUDT15 variant are detrimental either to the gene’s expression levels or its translation process, resulting in a lower amount of NUDT15 product present and hence translating to AZA-related myelotoxicity observed clinically. Methods IBD patients experiencing AZA-related myelotoxicity were recruited for this study. Patients were then genotyped and the NUDT15 variants were replicated through site-directed mutagenesis. The NUDT15 variants were subsequently transformed into mammalian cell lines then E. coli cells. DNA products were isolated, and transcription levels were assessed through RT-PCR. Results Patient cohort consisted of 27 AZA-exposed IBD patients who developed myelotoxicity despite their TPMT wildtype genotype. Two novel NUDT15 variants were found. The mutation in one of the variants was placed in 3’ UTR, and hence further research was not pursued. Further analysis was conducted for the variant with mutation in coding region. RT-PCR was conducted to assess and compare gene transcription levels between wildtype and variant NUDT15. Wildtype NUDT15 had a relative gene expression level of 0.8x107, whereas variant NUDT15’s relative gene expression level was at 1.1x107. The two groups were not significantly different in terms of gene expression. Conclusions Contrary to our initial hypothesis, it appears that the mutation in the start codon for variant NUDT15 gene does not significantly impact its gene expression as compared to the wildtype gene. We are currently pursuing protein expression analysis studies to assess for translational deficits possibly present in the novel NUDT15 variant. Funding Agencies SRTP - Schulich School of Medicine

Association between the CEBPA and c-MYC genes expression levels and acute myeloid leukemia pathogenesis and development

CEBPA and c-MYC genes belong to TF and play an essential role in hematologic malignancies development. Furthermore, these genes also co-regulate with RUNX1 and lead to bone marrow differentiation and may contribute to the leukemic transformation. Understanding the function and full characteristics of selected genes in the group of patients with AML can be helpful in assessing prognosis, and their usefulness as prognostic factors can be revealed. The aim of the study was to evaluate CEBPA and c-MYC mRNA expression level and to seek their association with demographical and clinical features of AML patients such as: age, gender, FAB classification, mortality or leukemia cell karyotype. Obtained results were also correlated with the expression level of the RUNX gene family. To assess of relative gene expression level the qPCR method was used. The expression levels of CEBPA and c-MYC gene varied among patients. Neither CEBPA nor c-MYC expression levels differed significantly between women and men (p=0.8325 and p=0.1698, respectively). No statistically significant correlation between age at the time of diagnosis and expression of CEBPA (p=0.4314) or c-MYC (p=0.9524) was stated. There were no significant associations between relative CEBPA (p=0.4247) or c-MYC (p=0.4655) expression level and FAB subtype and mortality among the enrolled patients (p=0.5858 and p=0.8437, respectively). However, it was observed that c-MYC and RUNX1 expression levels were significantly positively correlated (rS=0.328, p=0.0411). Overall, AML pathogenesis involves a complex interaction among CEBPA, c-MYC and RUNX family genes.

Elevated Lipocalin-2 Can Indicate the Vascular Inflammation in Patients with Ischemic Stroke

Purpose: Elevated level of Lipocalin-2 (LCN2), a new acute phase adipokine, was described after ischemic stroke. A number of researchers feel as though that LCN2 originated from the infiltrating neutrophils and other cells in brain after stroke. Others measured elevated LCN2 expression in arteriosclerotic plaque. Therefore we have investigated LCN2 relative gene expression level of blood neutrophil granulocytes in patients with ischemic stroke to assess if elevated LCN2 is the cause or consequence of ischemic stroke. Methods: Laboratory and anamnestic data were collected, which could have a role in development of thrombo-embolic events in patients with ischemic stroke. RNA based method was used to evaluate the relative gene expression level of LCN2. We calculated Odds Ratio (OR) and Confidence Interval (CI) for the association between LCN2 and ischemic stroke. Results: 34 samples were available for evaluation. The LCN 2 relative gene expression level was decreased in 12 cases. In this group, 91% of patients have Atrial Fibrillation (AF) at the time of hospitalisation. The mean LCN2 relative gene expression value was 64.25% (ranges: 34%-115%) in patients with AF. It was significantly lower than in patients with normal sinus rhythm (409.2%; ranges: 127%-1127%; p=0.0003). The elevated LCN2 relative gene expression level significantly (p=0.012) increases the risk of stroke (OR: 12.6) independently from other factors. Conclusions: High LCN2 expression level seems to have strong positive predictive value on ischemic stroke, and may be useful in thrombotic risk stratification of plaque vulnerability in these patients.