Genetic manipulation of folate in rice

Folate malnutrition is a major problem in many countries around the world especially in Asia and Africa. Stable foods such as rice contain very low amounts of folate. White rice which is the most popular form for human consumption contains less than 80 µg folate per 100 g. Given that it forms a major part of the South East Asian diet, rice represents an important target for enhancing folate levels. The objectives of this thesis is study the mechanisms that regulate total folate levels in rice grains and attempt to enhance and stabilise folate in rice endosperm. Three main strategies were adopted. First, the natural variation of folate biosynthesis gene expression was probed using RT- qPCR. Second, functional genomic approaches were used to manipulate the activity of rice folylpolyglutamate synthetase (FPGS), the enzyme which adds glutamate residues to folate. Third, genetic engineering was used to express FPGS enzymes and mammalian folate binding proteins in rice endosperm. RT-qPCR revealed that the variation in folate biosynthesis transcript abundance was closely correlated with total folate levels among rice varieties. High transcript abundance of all folate biosynthesis genes was associated with high total folate levels in Moroberekan rice mature seed. Comparative genomic studies revealed that rice FPGS is encoded by two distinct genes, FPGS Os03g02030 and FPGS Os10g35940. Transcript abundance of FPGS Os03g02030 appeared higher than that of FPGS Os10g35940 in seed, whilst, transcript abundance of FPGS Os10g35940 was higher in leaf. To determine the function of the FPGS Os03g02030 gene in rice seed, a TDNA knock out line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed maturation and decreased mono- and polyglutamylated folate pools in mutant seed. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knock out plant, whereas the folate deglutamylating enzyme y-glutamyl hydrolase (GGH) mRNA level was reduced. A potential feedback mechanism to maintain folate abundance during rice development was uncovered through the alternative functional FPGS Os10g35940 activity and reduction of folate breakdown. Protein-bound folate forms are better protected from oxidative degradation resulting in greater folate stability (Suh et al. 2001). Two rice FPGS and mammalian folate binding proteins was successfully introduced into rice endosperm using Agrobacterium based transformation in an attempt to retain and stabilise folate pool within rice endosperm. Analysis in terms of folate abundance and bioavailability will form part of future studies

Genetic manipulation of folate in rice

Folate malnutrition is a major problem in many countries around the world especially in Asia and Africa. Stable foods such as rice contain very low amounts of folate. White rice which is the most popular form for human consumption contains less than 80 µg folate per 100 g. Given that it forms a major part of the South East Asian diet, rice represents an important target for enhancing folate levels. The objectives of this thesis is study the mechanisms that regulate total folate levels in rice grains and attempt to enhance and stabilise folate in rice endosperm. Three main strategies were adopted. First, the natural variation of folate biosynthesis gene expression was probed using RT- qPCR. Second, functional genomic approaches were used to manipulate the activity of rice folylpolyglutamate synthetase (FPGS), the enzyme which adds glutamate residues to folate. Third, genetic engineering was used to express FPGS enzymes and mammalian folate binding proteins in rice endosperm. RT-qPCR revealed that the variation in folate biosynthesis transcript abundance was closely correlated with total folate levels among rice varieties. High transcript abundance of all folate biosynthesis genes was associated with high total folate levels in Moroberekan rice mature seed. Comparative genomic studies revealed that rice FPGS is encoded by two distinct genes, FPGS Os03g02030 and FPGS Os10g35940. Transcript abundance of FPGS Os03g02030 appeared higher than that of FPGS Os10g35940 in seed, whilst, transcript abundance of FPGS Os10g35940 was higher in leaf. To determine the function of the FPGS Os03g02030 gene in rice seed, a TDNA knock out line was characterised. Disrupting Os03g02030 gene expression resulted in delayed seed maturation and decreased mono- and polyglutamylated folate pools in mutant seed. RT-qPCR detected an increase in the transcript abundance of folate biosynthesis genes in seed of the knock out plant, whereas the folate deglutamylating enzyme y-glutamyl hydrolase (GGH) mRNA level was reduced. A potential feedback mechanism to maintain folate abundance during rice development was uncovered through the alternative functional FPGS Os10g35940 activity and reduction of folate breakdown. Protein-bound folate forms are better protected from oxidative degradation resulting in greater folate stability (Suh et al. 2001). Two rice FPGS and mammalian folate binding proteins was successfully introduced into rice endosperm using Agrobacterium based transformation in an attempt to retain and stabilise folate pool within rice endosperm. Analysis in terms of folate abundance and bioavailability will form part of future studies

Ultrarapid and high-resolution HLA class I typing using transposase-based nanopore sequencing applied in pharmacogenetic testing

Nanopore sequencing has been examined as a method for rapid and high-resolution human leukocyte antigen (HLA) typing in recent years. We aimed to apply ultrarapid nanopore-based HLA typing for HLA class I alleles associated with drug hypersensitivity, including HLA-A*31:01, HLA-B*15:02, and HLA-C*08:01. Most studies have used the Oxford Nanopore Ligation Sequencing kit for HLA typing, which requires several enzymatic reactions and remains relatively expensive, even when the samples are multiplexed. Here, we used the Oxford Nanopore Rapid Barcoding kit, which is transposase-based, with library preparation taking less than 1 h of hands-on time and requiring minimal reagents. Twenty DNA samples were genotyped for HLA-A, -B, and -C; 11 samples were from individuals of different ethnicity and nine were from Thai individuals. Two primer sets, a commercial set and a published set, were used to amplify the HLA-A, -B, and -C genes. HLA-typing tools that used different algorithms were applied and compared. We found that without using several third-party reagents, the transposase-based method reduced the hands-on time from approximately 9 h to 4 h, making this a viable approach for obtaining same-day results from 2 to 24 samples. However, an imbalance in the PCR amplification of different haplotypes could affect the accuracy of typing results. This work demonstrates the ability of transposase-based sequencing to report 3-field HLA alleles and its potential for race- and population-independent testing at considerably decreased time and cost

Iron–Quercetin Complex Preconditioning of Human Peripheral Blood Mononuclear Cells Accelerates Angiogenic and Fibroblast Migration: Implications for Wound Healing

Cell-based therapy is a highly promising treatment paradigm in ischemic disease due to its ability to repair tissue when implanted into a damaged site. These therapeutic effects involve a strong paracrine component resulting from the high levels of bioactive molecules secreted in response to the local microenvironment. Therefore, the secreted therapeutic can be modulated by preconditioning the cells during in vitro culturing. Herein, we investigated the potential use of magnetic resonance imaging (MRI) probes, the “iron–quercetin complex” or IronQ, for preconditioning peripheral blood mononuclear cells (PBMCs) to expand proangiogenic cells and enhance their secreted therapeutic factors. PBMCs obtained from healthy donor blood were cultured in the presence of the iron–quercetin complex. Differentiated preconditioning PBMCs were characterized by immunostaining. An enzyme-linked immunosorbent assay was carried out to describe the secreted cytokines. In vitro migration and tubular formation using human umbilical vein endothelial cells (HUVECs) were completed to investigate the proangiogenic efficacy. IronQ significantly increased mononuclear progenitor cell proliferation and differentiation into spindle-shape-like cells, expressing both hematopoietic and stromal cell markers. The expansion increased the number of colony-forming units (CFU-Hill). The conditioned medium obtained from IronQ-treated PBMCs contained high levels of interleukin 8 (IL-8), IL-10, urokinase-type-plasminogen-activator (uPA), matrix metalloproteinases-9 (MMP-9), and tumor necrosis factor-alpha (TNF-α), as well as augmented migration and capillary network formation of HUVECs and fibroblast cells, in vitro. Our study demonstrated that the IronQ-preconditioning PBMC protocol could enhance the angiogenic and reparative potential of non-mobilized PBMCs. This protocol might be used as an adjunctive strategy to improve the efficacy of cell therapy when using PBMCs for ischemic diseases and chronic wounds. However, in vivo assessment is required for further validation