Trait stacking simultaneously enhances provitamin A carotenoid and mineral bioaccessibility in biofortified Sorghum bicolor.

Vitamin A, iron, and zinc deficiencies are major nutritional inadequacies in sub-Saharan Africa and disproportionately affect women and children. Biotechnology strategies have been tested to individually improve provitamin A carotenoid or mineral content and/or bioaccessibility in staple crops including sorghum (Sorghum bicolor). However, concurrent carotenoid and mineral enhancement has not been thoroughly assessed and antagonism between these chemical classes has been reported. This work evaluated two genetically engineered constructs containing a suite of heterologous genes to increase carotenoid stability and pathway flux, as well as phytase to catabolize phytate and increase mineral bioaccessibility. Model porridges made from transgenic events were evaluated for carotenoid and mineral content as well as bioaccessibility. Transgenic events produced markedly higher amounts of carotenoids (26.4 µg g-1 DW) compared to null segregants (4.2 µg g-1 DW) and wild-type control (Tx430; 3.7 µg g-1 DW). Phytase activation by pre-steeping flour resulted in significant phytate reduction (9.4 to 4.2 mg g-1 DW), altered the profile of inositol phosphate catabolites, and reduced molar ratios of phytate to iron (16.0 to 4.1), and zinc (19.0 to 4.9) in engineered material, suggesting improved mineral bioaccessibility. Improved phytate : mineral ratios did not significantly affect micellarization and bioaccessible provitamin A carotenoids were over 23 times greater in transgenic events compared to corresponding null segregants and wild-type controls. A 200 g serving of porridge made with these transgenic events provide an estimated 53.7% of a 4-8-year-old child's vitamin A estimated average requirement. These data suggest that combinatorial approaches to enhance micronutrient content and bioaccessibility are feasible and warrant further assessment in human studies.

Installing the neurospora carotenoid pathway in plants enables cytosolic formation of provitamin A and its sequestration in lipid droplets.

Vitamin A deficiency remains a severe global health issue, which creates a need to biofortify crops with provitamin A carotenoids (PACs). Expanding plant cell capacity for synthesis and storing of PACs outside the plastids is a promising biofortification strategy that has been little explored. Here, we engineered PAC formation and sequestration in the cytosol of Nicotiana benthamiana leaves, Arabidopsis seeds, and citrus callus cells, using a fungal (Neurospora crassa) carotenoid pathway that consists of only three enzymes converting C5 isopentenyl building blocks formed from mevalonic acid into PACs, including ß-carotene. This strategy led to the accumulation of significant amounts of phytoene and γ- and ß-carotene, in addition to fungal, health-promoting carotenes with 13 conjugated double bonds, such as the PAC torulene, in the cytosol. Increasing the isopentenyl diphosphate pool by adding a truncated Arabidopsis hydroxymethylglutaryl-coenzyme A reductase substantially increased cytosolic carotene production. Engineered carotenes accumulate in cytosolic lipid droplets (CLDs), which represent a novel sequestering sink for storing these pigments in plant cytosol. Importantly, ß-carotene accumulated in the cytosol of citrus callus cells was more light stable compared to compared with plastidial ß-carotene. Moreover, engineering cytosolic carotene formation increased the number of large-sized CLDs and the levels of ß-apocarotenoids, including retinal, the aldehyde corresponding to vitamin A. Collectively, our study opens up the possibility of exploiting the high-flux mevalonic acid pathway for PAC biosynthesis and enhancing carotenoid sink capacity in green and non-green plant tissues, especially in lipid-storing seeds, and thus paves the way for further optimization of carotenoid biofortification in crops.

Provitamin A, lysine and tryptophan enrichment in shrunken2-based sweet corn genotypes through genomics-assisted breeding for crtRB1 and opaque2 genes.

BACKGROUND: Malnutrition affects large section of population worldwide. Vitamin A and protein deficiencies have emerged as the major global health-issue. Traditional shrunken2 (sh2)-based sweet corn is deficient in provitamin A (proA), lysine and tryptophan. Natural variant of ß-carotene hydroxylase1 (crtRB1) and opaque2 (o2) enhances proA, lysine and tryptophan in maize. So far, no sweet corn hybrid rich in these nutrients has been released elsewhere. Development of biofortified sweet corn hybrids would help in providing the balanced nutrition. METHODS AND RESULTS: We targeted three sh2-based sweet corn inbreds (SWT-19, SWT-20 and SWT-21) for introgression of mutant crtRB1 and o2 genes using molecular breeding. The gene-based 3'TE-InDel and simple sequence repeat (SSR) (umc1066) markers specific to crtRB1 and o2, respectively were utilized in foreground selection in BC1F1, BC2F1 and BC2F2. Segregation distortion was observed for crtRB1 and o2 genes in majority of populations. Background selection using 91-100 SSRs revealed recovery of recurrent parent genome (RPG) up to 96%. The introgressed progenies possessed significantly higher proA (13.56 µg/g) as compared to the original versions (proA: 2.70 µg/g). Further, the introgressed progenies had accumulated moderately higher level of lysine (0.336%) and tryptophan (0.082%) over original versions (lysine: 0.154% and tryptophan: 0.038%). Kernel sweetness among introgressed progenies (17.3%) was comparable to original sweet corn (17.4%). The introgressed inbreds exhibited higher resemblance with their recurrent parents for yield and morphological characters. CONCLUSION: These newly developed biofortified sweet corn genotypes hold immense promise to alleviate malnutrition.

Rapid Gene Target Tracking for Enhancing ß-Carotene Production Using Flow Cytometry-Based High-Throughput Screening in Yarrowia lipolytica.

ß-Carotene is a provitamin A precursor and an important antioxidant that is used widely in the aquaculture, food, cosmetic, and pharmaceutical industries. Oleaginous Yarrowia lipolytica has been demonstrated as a competitive producer microorganism for the production of hydrophobic ß-carotene through rational engineering strategies. However, the limited understanding of the complexity of the metabolic network between carotenoid biosynthesis and other cellular processes has hampered further advancement. Genome-scale mutagenesis and high-throughput screening of mutagenesis libraries have been extensively employed in gene mining or in the identification of key targets associated with particular phenotypes. In this study, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-ß-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased ß-carotene significantly. By engineering these targets in a high-ß-carotene production, a strain that produced 9.4 g/L ß-carotene was constructed. Here, we used a flow cytometry approach to improve screening efficiency and eliminate the interference of intermediate metabolites. The targets obtained in this study can be used in studies focusing on metabolic engineering in the future for improving carotenoid production. IMPORTANCE ß-Carotene is a high-value-added product that is widely used in the aquaculture, food, cosmetic, and pharmaceutical industries. In our previous study, Yarrowia lipolytica has been engineered extensively to produce ß-carotene. To further improve its production, high-throughput screening and the identification of new beneficial gene targets are required. Herein, we developed a fluorescence-activated cell-sorting approach for the effective high-throughput screening of high-ß-carotene-producing strains. Using this approach, millions of mutants were screened rapidly, and new gene targets involved in lipid metabolism, sterol metabolism, signal transduction, and stress response were identified. The disruption of the genes affecting fatty acid oxidation, lipid composition, and sterol transcriptional regulation (4CL-8, GCS, and YIsterTF) increased ß-carotene significantly. By engineering these targets in a high-ß-carotene production, a strain that produced 9.4 g/L ß-carotene was constructed.

Golden Rice-Lessons learned for inspiring future metabolic engineering strategies and synthetic biology solutions.

Golden Rice is a genetically modified rice variety that accumulates carotenoids with provitamin A activity in its endosperm. This biofortified crop was developed to fight provitamin A deficiency prevailing in countries with a diet based on rice as the major staple. Since its first version in 2000, Golden Rice was continuously developed to increase endosperm carotenoids, to achieve regulatory compliance, and to introgress the trait into regional rice varieties. Golden Rice was recently approved for planting in the Philippines. In this review, we recapitulate major steps leading to the current version of Golden Rice. We examine the knowledge regarding the carotenoid pathway and the technical capabilities to generate transgenic plants at each development stage. Moreover, we discuss whether the scientific approaches taken at the time would be any different today with the increased knowledge and analytical capacities available. We conclude that most decisions for each Golden Rice version would be very similar which is due to the peculiarities and the complexity of the carotenoid biosynthetic pathway. However, each step taken in the development of Golden Rice yielded much insight about regulation of the carotenoid pathway in plants as well as into specific requirements in rice endosperm. The knowledge gained and the recent advances in the field can provide strategies and molecular tools, including synthetic biology approaches, to help develop future versions of Golden Rice with improved provitamin A levels and carotenoid storage capabilities of the rice endosperm.

Status of carotenoids in elite and landrace maize genotypes: Implications for provitamin A biofortification in Tanzania.

Maize is among the crops containing carotenoids that are easily converted to vitamin A and have an enormous influence on consumers' health. Principally maize has high calories and proteins but has less number of other micronutrients such as vitamin A. Societies that use maize as their main and sole staple food are likely to be affected by vitamin A deficiency. Thus, development and production of maize varieties rich in micronutrients and vitamin A are important for improved health. This study characterized 5 carotenoid components in maize genotypes grown in Tanzania as a strategy for improving vitamin A content in maize. The study involved maize landraces, commercial or elite varieties, and inbred lines in determining their potential for provitamin A breeding programs for nutrition improvement. The study found that mean concentration of important carotenoid components, i.e., alpha carotene (AC), beta-carotene (BC), beta-cryptoxanthin (BCX), lutein (LU), zeaxanthin (ZX), provitamin A (ProVA), non-provitamin A (Non-ProVA), and total carotenoids (TC) varied significantly (P < 0.001) among maize genotypes. The 3 maize groups studied (landraces, commercial varieties, and breeding materials (BMs) varied significantly. For maize landraces, the concentration (µg/g) of studied carotenoids were AC (0.13-2.67), BC (0.60-3.72), BCX (0.36-1.01), ProVA (0.89-5.29), Retinol (0.25-0.87), LU (2.37-16.97). ZX (0.16-4.41), Non-ProVA (2.4-19.01), and TC (3.68-25.27); in commercial or elite maize varieties were (in µg/g): AC (0.31-3.84), BC (0.56-6.5), BCX (0.46-2.58), ProVA (0.92-11.80), Retinol (0.15-1.82), LU (3.28-22.39). ZX (0.05-11.31), Non-ProVA (2.56-28.81), and TC (4.23-37.84); and for maize BMs AC (0.53-6.64), BC (1.92-13.87), BCX (0.65-6.51), ProVA (2.69-18.62), Retinol (0.5-3.1), LU (4.86-34.99), ZX (0.06-18.58), Non-ProVA (4.8-53.57), and TC (9.86-76.94). Furthermore, the study found that the concentration of studied carotenoids was higher in pigmented (yellow or red) maize genotypes than in white maize genotypes. The current study found an appreciable amount of ProVA in studied materials, including maize landraces, commercial yellow varieties, and CIMMYT lines. The concentration of ProVA and retinol determined in studied maize genotypes were below 15 µg/g a daily vitamin A requirement, thus based on the current ProVA and retinol status it is difficult to meet Vitamin A requirement. Therefore, these maize genotypes with promising levels of carotenoid components are potential breeding materials that can be used in maize provitamin A biofortification program for improved food nutrition and livelihoods in Tanzania.

Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation.

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

Mutant combinations of lycopene ɛ-cyclase and ß-carotene hydroxylase 2 homoeologs increased ß-carotene accumulation in endosperm of tetraploid wheat (Triticum turgidum L.) grains.

Grains of tetraploid wheat (Triticum turgidum L.) mainly accumulate the non-provitamin A carotenoid lutein-with low natural variation in provitamin A ß-carotene in wheat accessions necessitating alternative strategies for provitamin A biofortification. Lycopene ɛ-cyclase (LCYe) and ß-carotene hydroxylase (HYD) function in diverting carbons from ß-carotene to lutein biosynthesis and catalyzing the turnover of ß-carotene to xanthophylls, respectively. However, the contribution of LCYe and HYD gene homoeologs to carotenoid metabolism and how they can be manipulated to increase ß-carotene in tetraploid wheat endosperm (flour) is currently unclear. We isolated loss-of-function Targeting Induced Local Lesions in Genomes (TILLING) mutants of LCYe and HYD2 homoeologs and generated higher order mutant combinations of lcye-A, lcye-B, hyd-A2, and hyd-B2. Hyd-A2 hyd-B2, lcye-A hyd-A2 hyd-B2, lcye-B hyd-A2 hyd-B2, and lcye-A lcye-B hyd-A2 hyd-B2 achieved significantly increased ß-carotene in endosperm, with lcye-A hyd-A2 hyd-B2 exhibiting comparable photosynthetic performance and light response to control plants. Comparative analysis of carotenoid profiles suggests that eliminating HYD2 homoeologs is sufficient to prevent ß-carotene conversion to xanthophylls in the endosperm without compromising xanthophyll production in leaves, and that ß-carotene and its derived xanthophylls are likely subject to differential catalysis mechanisms in vegetative tissues and grains. Carotenoid and gene expression analyses also suggest that the very low LCYe-B expression in endosperm is adequate for lutein production in the absence of LCYe-A. These results demonstrate the success of provitamin A biofortification using TILLING mutants while also providing a roadmap for guiding a gene editing-based approach in hexaploid wheat.

Marker based enrichment of provitamin A content in two tropical maize synthetics.

Most of the maize (Zea mays L.) varieties in developing countries have low content of micronutrients including vitamin A. As a result, people who are largely dependent on cereal-based diets suffer from health challenges due to micronutrient deficiencies. Marker assisted recurrent selection (MARS), which increases the frequency of favorable alleles with advances in selection cycle, could be used to enhance the provitamin A (PVA) content of maize. This study was carried out to determine changes in levels of PVA carotenoids and genetic diversity in two maize synthetics that were subjected to two cycles of MARS. The two populations, known as HGA and HGB, and their advanced selection cycles (C1 and C2) were evaluated at Ibadan in Nigeria. Selection increased the concentrations of ß-carotene, PVA and total carotenoids across cycles in HGA, while in HGB only α-carotene increased with advances in selection cycle. ß-cryptoxanthine increased at C1 but decreased at C2 in HGB. The levels of ß-carotene, PVA, and total carotenoids increased by 40%, 30% and 36% respectively, in HGA after two cycles of selection. α-carotene and ß-cryptoxanthine content improved by 20% and 5%, respectively after two cycles of selection in HGB. MARS caused changes in genetic diversity over selection cycles. Number of effective alleles and observed heterozygosity decreased with selection cycles, while expected heterozygosity increased at C1 and decreased at C2 in HGA. In HGB, number of effective alleles, observed and expected heterozygosity increased at C1 and decreased at C2. In both populations, fixation index increased after two cycle of selections. The greatest part of the genetic variability resides within the population accounting for 86% of the total genetic variance. In general, MARS effectively improved PVA carotenoid content. However, genetic diversity in the two synthetics declined after two cycles of selection.

Using black soldier fly larvae reared on fruits and vegetables waste as a sustainable dietary source of provitamin a carotenoids.

We showed that black soldier fly larvae reared on fruits and vegetables rich in provitamin A carotenoids can accumulate significant amounts of these vitamin A precursors. Using a simulated gastro-intestinal digestion model, we demonstrated that α- and ß-carotene from the larvae are as bioaccessible as from the fruits and vegetables they were reared on. We calculated that provitamin A carotenoid-rich larvae have the capacity to provide more vitamin A than fruits and vegetables rich in these molecules. Remarkably, the incorporation of usual quantities of these larvae in feed could cover the needs of several production animals for this vitamin. Thus, our findings suggest that rearing black soldier fly larvae on by-products or waste rich in provitamin A carotenoids could be a sustainable strategy to recycle a fraction of vitamin A back into the food chain and could represent a new approach to fight against vitamin A deficiency.

Low expression of carotenoids cleavage dioxygenase 1 (ccd1) gene improves the retention of provitamin-A in maize grains during storage.

Provitamin-A (proA) is essentially required for vision in humans but its deficiency affects children and pregnant women especially in the developing world. Biofortified maize rich in proA provides new opportunity for sustainable and cost-effective solution to alleviate malnutrition, however, significant loss of carotenoids during storage reduces its efficacy. Here, we studied the role of carotenoid cleavage dioxygenase 1 (ccd1) gene on degradation of carotenoids in maize. A set of 24 maize inbreds was analyzed for retention of proA during storage. At harvest, crtRB1-based maize inbreds possessed significantly high proA (ß-carotene: 12.30 µg/g, ß-cryptoxanthin: 4.36 µg/g) than the traditional inbreds (ß-carotene: 1.74 µg/g, ß-cryptoxanthin: 1.28 µg/g). However, crtRB1-based inbreds experienced significant degradation of proA carotenoids (ß-carotene: 20%, ß-cryptoxanthin: 32% retention) following 5 months. Among the crtRB1-based genotypes, V335PV had the lowest retention of proA (ß-carotene: 1.63 µg/g, ß-cryptoxanthin: 0.82 µg/g), while HKI161PV had the highest retention of proA (ß-carotene: 4.17 µg/g, ß-cryptoxanthin: 2.32 µg/g). Periodical analysis revealed that ~ 60-70% of proA degraded during the first three months. Expression analysis revealed that high expression of ccd1 led to low retention of proA carotenoids in V335PV, whereas proA retention in HKI161PV was higher due to lower expression. Highest expression of ccd1 was observed during first 3 months of storage. Copy number of ccd1 gene varied among yellow maize (1-6 copies) and white maize (7-35 copies) while wild relatives contained 1-4 copies of ccd1 gene per genome. However, copy number of ccd1 gene did not exhibit any correlation with proA carotenoids. We concluded that lower expression of ccd1 gene increased the retention of proA during storage in maize. Favourable allele of ccd1 can be introgressed into elite maize inbreds for higher retention of proA during storage.

Integration of Nitrogen and Biofertilizer on the Agronomic Efficiency and Quality of Shallot (Allium ascalonicum L.) Plant in the Rainy Season.

BACKGROUND AND OBJECTIVE: In the rainy season farmers don't interest to cultivate shallot because in addition to providing a high dosage of fertilizer they are also sensitive to pathogenic attacks so they are afraid of crop failure and cause low shallot production. This study aimed to knew effect of agronomic component and quality of shallot under different concentrations of biofertilizer and Ammonium Sulphate (AS) fertilizer dose in the rainy season. MATERIALS AND METHODS: The study was conducted in Cangkring, Srandakan, Bantul, Special Region of Yogyakarta Indonesia from August to October 2019. The study was arranged in RCBD factorial with three replications. The first factor was a various dose of ammonium sulphate (100, 200 and 300 kg ha-1). The second factor was various concentrations of biofertilizer (2, 3 and 4%), and control. The observed variables were the analysis of growth yield and quality component of shallot plant. The analyzed using analysis of variance at 5% of significance then continued by DMRT at 5% of significance. RESULTS: There was the interaction between the application of AS dosage and biofertilizer concentration on all of variable observations. There was a significant difference between treatment with control on all of the observation variables. CONCLUSION: The combination of AS fertilizer 200 kg ha-1 dose and 3% biofertilizer concentration increased agronomic efficiency, growth, bulbs yields, and quality of bulbs include provitamin A, oleoresin compounds.

Building the Synthetic Biology Toolbox with Enzyme Variants to Expand Opportunities for Biofortification of Provitamin A and Other Health-Promoting Carotenoids.

Carotenoids are a large class of structures that are important in human health and include both provitamin A and nonprovitamin A compounds. Vitamin A deficiency is a global health problem that can be alleviated by enriching provitamin A carotenoids in a range of food crops. Suitable plants for biofortification are those with high levels of the provitamin A biosynthetic precursor, lycopene, which is enzymatically converted by lycopene ß-cyclase (LCYB) to ß-carotene, a provitamin A carotenoid. Crops, such as citrus, naturally accumulate high levels of provitamin A and other health-promoting carotenoids. Such plants may have useful genes to expand the synthetic biology toolbox for producing a range of phenotypes, including both high provitamin A crops and crops with unique compositions of health-promoting carotenoids. To examine enzyme variants having different activity levels, we introduced two citrus LCYB alleles into tomato, a plant with fruit rich in lycopene. Overexpression in tomato of the stronger allele of the citrus chromoplast-specific lycopene ß-cyclase (CsLCYb2a) produced "golden" transgenic tomato fruits with 9.3-fold increased levels of ß-carotene at up to 1.5 mg/g dry weight. The use of the weaker allele, CsLCYb2b, also led to enhanced levels of ß-carotene but in the context of a more heterogeneous composition of carotenoids. From a synthetic biology standpoint, these allelic differences have value for producing cultivars with unique carotenoid profiles. Overexpression of the citrus LCYB genes was accompanied by increased expression of other genes encoding carotenoid biosynthetic enzymes and increased size and number of chromoplasts needed to sequester the elevated levels of carotenoids in the transgenic tomato fruits. The overexpression of the citrus LCYB genes also led to a pleiotropic effect on profiles of phytohormones and primary metabolites. Our findings show that enzyme variants are essential synthetic biology parts needed to create a wider range of metabolic engineering products. In this case, strong and weak variants of LCYB proved useful in creating dietary sources to alleviate vitamin A deficiency or, alternatively, to create crops with a heterogeneous composition including provitamin A and healthful, nonprovitamin A carotenoids.

Safety and Metabolism of Long-term Administration of NIAGEN (Nicotinamide Riboside Chloride) in a Randomized, Double-Blind, Placebo-controlled Clinical Trial of Healthy Overweight Adults.

Nicotinamide riboside (NR) is a newly discovered nicotinamide adenine dinucleotide (NAD+) precursor vitamin. A crystal form of NR chloride termed NIAGEN is generally recognized as safe (GRAS) for use in foods and the subject of two New Dietary Ingredient Notifications for use in dietary supplements. To evaluate the kinetics and dose-dependency of NR oral availability and safety in overweight, but otherwise healthy men and women, an 8-week randomized, double-blind, placebo-controlled clinical trial was conducted. Consumption of 100, 300 and 1000 mg NR dose-dependently and significantly increased whole blood NAD+ (i.e., 22%, 51% and 142%) and other NAD+ metabolites within 2 weeks. The increases were maintained throughout the remainder of the study. There were no reports of flushing and no significant differences in adverse events between the NR and placebo-treated groups or between groups at different NR doses. NR also did not elevate low density lipoprotein cholesterol or dysregulate 1-carbon metabolism. Together these data support the development of a tolerable upper intake limit for NR based on human data.

Compositional Analysis of Genetically Engineered GR2E "Golden Rice" in Comparison to That of Conventional Rice.

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.

Voriconazole inhibits cross-kingdom interactions between Candida albicans and Actinomyces viscosus through the ergosterol pathway.

Candida albicans and Actinomyces viscosus are prominent microbes associated with dental root caries. The aim of this study was to investigate the effect of C. albicans on A. viscosus biofilms and to identify the mechanisms associated with this interaction. A. viscosus and C. albicans strains (wide-type and mutants) were used to form biofilms in vitro and in vivo, which were subsequently analysed by crystal violet assay and scanning electron microscopy (SEM) to investigate the effect of C. albicans on A. viscosus growth. A viable plate count and survival curve for C. albicans mutants and A. viscosus combinations were used to identify which C. albicans pathway was crucial for cross-kingdom interactions. Voriconazole was used to block their interactions both in vitro and in vivo. SEM, fluorescence in situ hybridisation (FISH), quantitative PCR and survival curve analyses were performed to evaluate the activity of voriconazole on C. albicans and A. viscosus interactions. The biomass and virulence of mixed-species biofilms were significantly enhanced compared with the A. viscosus biofilm alone. However, this was not observed in the mixed-species biofilms with the C. albicans mutant erg11Δ/Δ in vitro and in vivo, indicating that azoles may work on the mixed-species biofilms. As expected, voriconazole can effectively reduce the biomass of mixed-species biofilms. A high concentration of voriconazole (1 µg/mL) reduced the abundance of C. albicans, whilst a low voriconazole concentration (0.25 µg/mL) blocked their interactions similar to the effect of the erg11Δ/Δ mutant. Voriconazole may be a candidate strategy to combat root caries pathogens.

The development and release of maize fortified with provitamin A carotenoids in developing countries.

Micronutrient deficiencies have been identified as major public health problems affecting a large part of the world's population. Biofortification of staple crops like maize has been proposed as one of the most cost effective and feasible approaches to combat micronutrient deficiencies. Studies have shown that provitamin A from biofortified crops is highly bioavailable and has the capacity to improve vitamin A status of vulnerable groups. Most people in sub-Saharan Africa subsist on maize and many people may benefit from consumption of provitamin A carotenoid biofortified maize, especially women and children. With the exception of transgenic golden rice, biofortified crops have received considerable acceptance by most communities. Negative perceptions associated with yellow maize do not affect orange maize, which is, for example, well-liked in rural Zambia. With proper policy frameworks and full commercialization, provitamin A maize can address the problem of vitamin A deficiencies among poor nations with maize-based diets.

Effects of Dietary ß-Carotene on the Melanization Response and Growth Rate of Trichoplusia ni (Lepidoptera: Noctuidae).

Animals rely on carotenoids as fundamental precursors for hormones and antioxidants, and animals must acquire carotenoids from their diet. Previous research has shown that insects often absorb carotenoids in amounts proportional to those in their diet, and that carotenoids play key roles in multitrophic interactions. The consumption of diets that provide high levels of antioxidant compounds is associated with high levels of immune responses; however, it is unknown whether individual carotenoids directly influence immune response. Here, the objective of this study was to examine the effect of the carotenoid ß-carotene on melanization, a measure of immune response, and growth rate of Trichoplusia ni Hübner (Lepidoptera: Noctuidae). To fulfill the objective, a low, medium, and high concentration of ß-carotene, representing the range found in typical host plants, were mixed in an artificial diet, and immune response and growth rate were assessed in fifth instar larvae. Immune response was induced by injection of chromatography beads in to the abdomen of the larvae, and percent melanization was measured after injection. Melanization was greatest when larvae were reared on high ß-carotene diets. Mass was measured at 5 and 10 d to assess growth rate. Larvae reared on high ß-carotene diets initially gained little mass, but after 10 d larvae reared on no and high ß-carotene diets were larger than those reared on other diets. This research has shown that ß-carotene has the potential to influence the immune response and growth rate of T. ni.

Non-Provitamin A and Provitamin A Carotenoids as Immunomodulators: Recommended Dietary Allowance, Therapeutic Index, or Personalized Nutrition?

Vegetables and fruits contain non-provitamin A (lycopene, lutein, and zeaxanthin) and provitamin A (ß-carotene, ß-cryptoxanthin, and α-carotene) carotenoids. Within these compounds, ß-carotene has been extensively studied for its health benefits, but its supplementation at doses higher than recommended intakes induces adverse effects. ß-Carotene is converted to retinoic acid (RA), a well-known immunomodulatory molecule. Human interventions suggest that ß-carotene and lycopene at pharmacological doses affect immune functions after a depletion period of low carotenoid diet. However, these effects appear unrelated to carotenoids and retinol levels in plasma. Local production of RA in the gut-associated lymphoid tissue, as well as the dependency of RA-induced effects on local inflammation, suggests that personalized nutrition/supplementation should be considered in the future. On the other hand, the differential effect of RA and lycopene on transforming growth factor-beta suggests that lycopene supplementation could improve immune functions without increasing risk for cancers. However, such preclinical evidence must be confirmed in human interventions before any recommendations can be made.