A Generalized Method for Determining Free Soluble Phenolic Acid Composition and Antioxidant Capacity of Cereals and Legumes.

Phenolic acids are a class of organic compounds that bear both a phenolic group, and a carboxylic group. They are found in grains and concentrate in the bran of cereals or seed coat of legumes. They possess antioxidant properties that have generated much research interest in recent years, about their potential antioxidant protective health functions. This work presents a generalized method for the extraction of free soluble phenolic acids from whole grains and analysis of their antioxidant capacity. Five whole grain samples comprising two cereals (wheat and yellow corn) and three legumes (cowpea bean, kidney bean, and soybean), were used. The grains were milled into flour and their free soluble phenolic acids extracted using aqueous methanol. The compounds were then identified using a high-pressure liquid chromatograph (HPLC). The Folin-Ciocalteu method was used to determine their total phenolic content while their antioxidant capacities were determined using the DPPH radical scavenging capacity, Trolox equivalent antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC) assays. The phenolic acids identified included vanillic, caffeic, p-coumaric and ferulic acids. Vanillic acid was identified only in cowpea while caffeic acid was identified only in kidney bean. p-Coumaric acid was identified in yellow corn, cowpea, and soybean, while ferulic acid was identified in all the samples. Ferulic acid was the predominant phenolic acid identified. The total concentration of phenolic acids in the samples decreased in the following order: soybean > cowpea bean > yellow corn = kidney bean > wheat. The total antioxidant capacity (sum of values of DPPH, TEAC and ORAC assays) decreased as follows: soybean > kidney bean > yellow corn = cowpea bean > wheat. This study concluded that HPLC analysis as well as DPPH, TEAC, and ORAC assays provide useful information about the phenolic acid composition and antioxidant properties of whole grains.

Phenolic Composition and Antioxidant Properties of Cooked Rice Dyed with Sorghum-Leaf Bio-Colorants.

White rice is an important staple food globally. It is a rich source of energy but is low in dietary phenolic antioxidants. This current research aimed at providing scientific evidence for an alternative rice dish that has increased phenolic-antioxidant health-promoting potential by combining white rice with red cowpea beans and cooking with dye sorghum leaves hydrothermal extract, as a source of natural colorant. Boiled white rice and the rice-cowpea-sorghum extract dish were freeze-dried, and the free and bound phenolic compounds of raw and cooked samples were extracted. Phenolic composition, total phenolic content (TPC), and antioxidant activities (measured by 2,2-diphenyl-1-picrylhydrazyl radical scavenging capacity, Trolox equivalent antioxidant capacity, and oxygen radical absorbance capacity methods) of the raw and cooked samples were determined. Combining white rice with cowpea seeds and sorghum leaves extract significantly (p < 0.0001) increased the TPC and antioxidant activities of the rice due to the higher TPC and antioxidant activities of cowpea and sorghum leaves. Although boiling caused substantial losses of flavonoids and anthocyanins in the rice-cowpea-sorghum extract composite meal, the resulting dish had higher TPC and antioxidant activities than boiled white rice. Compositing white rice with phenolic-rich pulses can be an innovative approach to providing alternative healthy rice dishes to consumers.

An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity.

Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the Drosophila midgut, we identified adenosine receptor (AdoR) as a top candidate gene required for ISC proliferation. We demonstrate that Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and that Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of adenosine deaminase-related growth factor A (Adgf-A) from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As Adgf-A expression and its enzymatic activity decrease following tissue damage, our study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation.

A cytosine deaminase for programmable single-base RNA editing.

Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive ß-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.

RNA editing with CRISPR-Cas13.

Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided ribonuclease Cas13. We profiled type VI systems in order to engineer a Cas13 ortholog capable of robust knockdown and demonstrated RNA editing by using catalytically inactive Cas13 (dCas13) to direct adenosine-to-inosine deaminase activity by ADAR2 (adenosine deaminase acting on RNA type 2) to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineered this system to create a high-specificity variant and minimized the system to facilitate viral delivery. REPAIR presents a promising RNA-editing platform with broad applicability for research, therapeutics, and biotechnology.