Gene-Nutrient Interactions in Obesity: COBLL1 Genetic Variants Interact with Dietary Fat Intake to Modulate the Incidence of Obesity.

The COBLL1 gene is associated with leptin, a hormone important for appetite and weight maintenance. Dietary fat is a significant factor in obesity. This study aimed to determine the association between COBLL1 gene, dietary fat, and incidence of obesity. Data from the Korean Genome and Epidemiology Study were used, and 3055 Korean adults aged ≥ 40 years were included. Obesity was defined as a body mass index ≥ 25 kg/m2. Patients with obesity at baseline were excluded. The effects of the COBLL1 rs6717858 genotypes and dietary fat on incidence of obesity were evaluated using multivariable Cox proportional hazard models. During an average follow-up period of 9.2 years, 627 obesity cases were documented. In men, the hazard ratio (HR) for obesity was higher in CT, CC carriers (minor allele carriers) in the highest tertile of dietary fat intake than for men with TT carriers in the lowest tertile of dietary fat intake (Model 1: HR: 1.66, 95% confidence interval [CI]: 1.07-2.58; Model 2: HR: 1.63, 95% CI: 1.04-2.56). In women, the HR for obesity was higher in TT carriers in the highest tertile of dietary fat intake than for women with TT carriers in the lowest tertile of dietary fat intake (Model 1: HR: 1.49, 95% CI: 1.08-2.06; Model 2: HR: 1.53, 95% CI: 1.10-2.13). COBLL1 genetic variants and dietary fat intake had different sex-dependent effects in obesity. These results imply that a low-fat diet may protect against the effects of COBLL1 genetic variants on future obesity risk.

An Exercise-Induced Metabolic Shield in Distant Organs Blocks Cancer Progression and Metastatic Dissemination.

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor. SIGNIFICANCE: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124.

Genomic Approaches for Improvement of Tropical Fruits: Fruit Quality, Shelf Life and Nutrient Content.

The breeding of tropical fruit trees for improving fruit traits is complicated, due to the long juvenile phase, generation cycle, parthenocarpy, polyploidy, polyembryony, heterozygosity and biotic and abiotic factors, as well as a lack of good genomic resources. Many molecular techniques have recently evolved to assist and hasten conventional breeding efforts. Molecular markers linked to fruit development and fruit quality traits such as fruit shape, size, texture, aroma, peel and pulp colour were identified in tropical fruit crops, facilitating Marker-assisted breeding (MAB). An increase in the availability of genome sequences of tropical fruits further aided in the discovery of SNP variants/Indels, QTLs and genes that can ascertain the genetic determinants of fruit characters. Through multi-omics approaches such as genomics, transcriptomics, metabolomics and proteomics, the identification and quantification of transcripts, including non-coding RNAs, involved in sugar metabolism, fruit development and ripening, shelf life, and the biotic and abiotic stress that impacts fruit quality were made possible. Utilizing genomic assisted breeding methods such as genome wide association (GWAS), genomic selection (GS) and genetic modifications using CRISPR/Cas9 and transgenics has paved the way to studying gene function and developing cultivars with desirable fruit traits by overcoming long breeding cycles. Such comprehensive multi-omics approaches related to fruit characters in tropical fruits and their applications in breeding strategies and crop improvement are reviewed, discussed and presented here.

Maternal Heat Stress Alters Expression of Genes Associated with Nutrient Transport Activity and Metabolism in Female Placentae from Mid-Gestating Pigs.

Placental insufficiency is a known consequence of maternal heat stress during gestation in farm animals. The molecular regulation of placentae during the stress response is little known in pigs. This study aims to identify differential gene expression in pig placentae caused by maternal heat exposure during early to mid-gestation. RNA sequencing (RNA-seq) was performed on female placental samples from pregnant pigs exposed to thermoneutral control (CON; constant 20 °C; n = 5) or cyclic heat stress (HS; cyclic 28 to 33 °C; n = 5) conditions between d40 and d60 of gestation. On d60 of gestation, placental efficiency (fetal/placental weight) was decreased (p = 0.023) by maternal HS. A total of 169 genes were differentially expressed (FDR ≤ 0.1) between CON and HS placentae of female fetuses, of which 35 genes were upregulated and 134 genes were downregulated by maternal HS. The current data revealed transport activity (FDR = 0.027), glycoprotein biosynthetic process (FDR = 0.044), and carbohydrate metabolic process (FDR = 0.049) among the terms enriched by the downregulated genes (HS vs. CON). In addition, solute carrier (SLC)-mediated transmembrane transport (FDR = 0.008) and glycosaminoglycan biosynthesis (FDR = 0.027), which modulates placental stroma synthesis, were identified among the pathways enriched by the downregulated genes. These findings provide evidence that heat-stress induced placental inefficiency may be underpinned by altered expression of genes associated with placental nutrient transport capacity and metabolism. A further understanding of the molecular mechanism contributes to the identification of placental gene signatures of summer infertility in pigs.

Nutrient-sensing components of the mouse stomach and the gastric ghrelin cell.

BACKGROUND: The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells. METHODS: Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin. KEY RESULTS: Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. CONCLUSIONS AND INFERENCES: The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake.

Transcriptome Analysis Reveals Important Candidate Genes Related to Nutrient Reservoir, Carbohydrate Metabolism, and Defence Proteins during Grain Development of Hexaploid Bread Wheat and Its Diploid Progenitors.

Wheat grain development after anthesis is an important biological process, in which major components of seeds are synthesised, and these components are further required for germination and seed vigour. We have made a comparative RNA-Seq analysis between hexaploid wheat and its individual diploid progenitors to know the major differentially expressed genes (DEGs) involved during grain development. Two libraries from each species were generated with an average of 55.63, 55.23, 68.13, and 103.81 million reads, resulting in 79.3K, 113.7K, 90.6K, and 121.3K numbers of transcripts in AA, BB, DD, and AABBDD genome species respectively. Number of expressed genes in hexaploid wheat was not proportional to its genome size, but marginally higher than that of its diploid progenitors. However, to capture all the transcripts in hexaploid wheat, sufficiently higher number of reads was required. Functional analysis of DEGs, in all the three comparisons, showed their predominance in three major classes of genes during grain development, i.e., nutrient reservoirs, carbohydrate metabolism, and defence proteins; some of them were subsequently validated through real time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Further, developmental stage-specific gene expression showed most of the defence protein genes expressed during initial developmental stages in hexaploid contrary to the diploids at later stages. Genes related to carbohydrates anabolism expressed during early stages, whereas catabolism genes expressed at later stages in all the species. However, no trend was observed in case of different nutrient reservoirs gene expression. This data could be used to study the comparative gene expression among the three diploid species and homeologue-specific expression in hexaploid.

Nutrigenetics and nutrigenomics: ready for clinical use or still a way to go?

Nutritional Genomics or nutrigenetics/nutrigenomics is an emerging area of research aiming to delineate the interplay between nutrients intake and the reciprocal pathologies with the human genome. Coupled with other omics disciplines, such as metabolomics, proteomics and transcriptomics, nutrigenomics aspires to individualize nutrition, reminiscent of pharmacogenomics and the individualization of drug use. Here, we provide an overview of a session focused on nutrigenomics, organized in conjunction with the Panhellenic Bioscientists Association during the First Greek National Personalised Medicine Conference in Athens, Greece on 15 December 2019.

The Paralogous Transcription Factors Stp1 and Stp2 of Candida albicans Have Distinct Functions in Nutrient Acquisition and Host Interaction.

Nutrient acquisition is a central challenge for all organisms. For the fungal pathogen Candida albicans, utilization of amino acids has been shown to be critical for survival, immune evasion, and escape, while the importance of catabolism of host-derived proteins and peptides in vivo is less well understood. Stp1 and Stp2 are paralogous transcription factors (TFs) regulated by the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing system and have been proposed to have distinct, if uncertain, roles in protein and amino acid utilization. We show here that Stp1 is required for proper utilization of peptides but has no effect on amino acid catabolism. In contrast, Stp2 is critical for utilization of both carbon sources. Commensurate with this observation, we found that Stp1 controls a very limited set of genes, while Stp2 has a much more extensive regulon that is partly dependent on the Ssy1 amino acid sensor (amino acid uptake and catabolism) and partly Ssy1 independent (genes associated with filamentous growth, including the regulators UME6 and SFL2). The ssy1Δ/Δ and stp2Δ/Δ mutants showed reduced fitness in a gastrointestinal (GI) colonization model, yet induced greater damage to epithelial cells and macrophages in a manner that was highly dependent on the growth status of the fungal cells. Surprisingly, the stp1Δ/Δ mutant was better able to colonize the gut but the mutation had no effect on host cell damage. Thus, proper protein and amino acid utilization are both required for normal host interaction and are controlled by an interrelated network that includes Stp1 and Stp2.

Overexpression of the High-Affinity Nitrate Transporter OsNRT2.3b Driven by Different Promoters in Barley Improves Yield and Nutrient Uptake Balance.

Improving nitrogen use efficiency (NUE) is very important for crops throughout the world. Rice mainly utilizes ammonium as an N source, but it also has four NRT2 genes involved in nitrate transport. The OsNRT2.3b transporter is important for maintaining cellular pH under mixed N supplies. Overexpression of this transporter driven by a ubiquitin promoter in rice greatly improved yield and NUE. This strategy for improving the NUE of crops may also be important for other cereals such as wheat and barley, which also face the challenges of nutrient uptake balance. To test this idea, we constructed transgenic barley lines overexpressing OsNRT2.3b. These transgenic barley lines overexpressing the rice transporter exhibited improved growth, yield, and NUE. We demonstrated that NRT2 family members and the partner protein HvNAR2.3 were also up-regulated by nitrate treatment (0.2 mM) in the transgenic lines. This suggests that the expression of OsNRT2.3b and other HvNRT2 family members were all up-regulated in the transgenic barley to increase the efficiency of N uptake and usage. We also compared the ubiquitin (Ubi) and a phloem-specific (RSs1) promoter-driven expression of OsNRT2.3b. The Ubi promoter failed to improve nutrient uptake balance, whereas the RSs1 promoter succeed in increasing the N, P, and Fe uptake balance. The nutrient uptake enhancement did not include Mn and Mg. Surprisingly, we found that the choice of promoter influenced the barley phenotype, not only increasing NUE and grain yield, but also improving nutrient uptake balance.

Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum.

Malaria is a deadly, infectious disease caused by the parasite Plasmodium, leading to millions of deaths worldwide. Plasmodium requires a coordinated pattern of sequential gene expression for surviving in both invertebrate and vertebrate host environments. As parasites largely depend on host resources, they also develop efficient mechanisms to sense and adapt to variable nutrient conditions in the environment and modulate their virulence. Earlier we have shown that PfGCN5, a histone acetyltransferase, binds to the stress-responsive and virulence-related genes in a poised state and regulates their expression under temperature and artemisinin treatment conditions in P. falciparum. In this study, we show upregulation of PfGCN5 upon nutrient stress condition. With the help of chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq) and transcriptomic (RNA-sequencing) analyses, we show that PfGCN5 is associated with the genes that are important for the maintenance of parasite cellular homeostasis upon nutrient stress condition. Furthermore, we identified various metabolic enzymes as interacting partners of PfGCN5 by immunoprecipitation coupled with mass spectroscopy, possibly acting as a sensor of nutrient conditions in the environment. We also demonstrated that PfGCN5 interacts and acetylates PfGAPDH in vitro. Collectively, our data provides important insights into transcriptional deregulation upon nutrient stress condition and elucidate the role of PfGCN5 during nutrient stress condition.

Optimizing the procedure of grain nutrient predictions in barley via hyperspectral imaging.

Hyperspectral imaging enables researchers and plant breeders to analyze various traits of interest like nutritional value in high throughput. In order to achieve this, the optimal design of a reliable calibration model, linking the measured spectra with the investigated traits, is necessary. In the present study we investigated the impact of different regression models, calibration set sizes and calibration set compositions on prediction performance. For this purpose, we analyzed concentrations of six globally relevant grain nutrients of the wild barley population HEB-YIELD as case study. The data comprised 1,593 plots, grown in 2015 and 2016 at the locations Dundee and Halle, which have been entirely analyzed through traditional laboratory methods and hyperspectral imaging. The results indicated that a linear regression model based on partial least squares outperformed neural networks in this particular data modelling task. There existed a positive relationship between the number of samples in a calibration model and prediction performance, with a local optimum at a calibration set size of ~40% of the total data. The inclusion of samples from several years and locations could clearly improve the predictions of the investigated nutrient traits at small calibration set sizes. It should be stated that the expansion of calibration models with additional samples is only useful as long as they are able to increase trait variability. Models obtained in a certain environment were only to a limited extent transferable to other environments. They should therefore be successively upgraded with new calibration data to enable a reliable prediction of the desired traits. The presented results will assist the design and conceptualization of future hyperspectral imaging projects in order to achieve reliable predictions. It will in general help to establish practical applications of hyperspectral imaging systems, for instance in plant breeding concepts.

PARP1 and Poly(ADP-ribosyl)ation Signaling during Autophagy in Response to Nutrient Deprivation.

Autophagy is considered to be the primary degradative pathway that takes place in all eukaryotic cells. Morphologically, the autophagy pathway refers to a process by which cytoplasmic portions are delivered to double-membrane organelles, called autophagosomes, to fuse with lysosomes for bulk degradation. Autophagy, as a prosurvival mechanism, can be stimulated by different types of cellular stress such as nutrient deprivation, hypoxia, ROS, pH, DNA damage, or ER stress, promoting adaptation of the cell to the changing and hostile environment. The functional relevance of autophagy in many diseases such as cancer or neurodegenerative diseases remains controversial, preserving organelle function and detoxification and promoting cell growth, although in other contexts, autophagy could suppress cell expansion. Poly(ADP-ribosyl)ation (PARylation) is a covalent and reversible posttranslational modification (PTM) of proteins mediated by Poly(ADP-ribose) polymerases (PARPs) with well-described functions in DNA repair, replication, genome integrity, cell cycle, and metabolism. Herein, we review the current state of PARP1 activation and PARylation in starvation-induced autophagy.

Reciprocal regulation of TORC signaling and tRNA modifications by Elongator enforces nutrient-dependent cell fate.

Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.

Determinants of nutrient limitation in cancer.

Proliferation requires that cells accumulate sufficient biomass to grow and divide. Cancer cells within tumors must acquire a variety of nutrients, and tumor growth slows or stops if necessary metabolites are not obtained in sufficient quantities. Importantly, the metabolic demands of cancer cells can be different from those of untransformed cells, and nutrient accessibility in tumors is different than in many normal tissues. Thus, cancer cell survival and proliferation may be limited by different metabolic factors than those that are necessary to maintain noncancerous cells. Understanding the variables that dictate which nutrients are critical to sustain tumor growth may identify vulnerabilities that could be used to treat cancer. This review examines the various cell-autonomous, local, and systemic factors that determine which nutrients are limiting for tumor growth.

Genetic Ablation of the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses.

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. SIGNIFICANCE: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

Complex Gene Regulation Underlying Mineral Nutrient Homeostasis in Soybean Root Response to Acidity Stress.

Proton toxicity is one of the major environmental stresses limiting crop production and becomes increasingly serious because of anthropogenic activities. To understand acid tolerance mechanisms, the plant growth, mineral nutrients accumulation, and global transcriptome changes in soybean (Glycine max) in response to long-term acidity stress were investigated. Results showed that acidity stress significantly inhibited soybean root growth but exhibited slight effects on the shoot growth. Moreover, concentrations of essential mineral nutrients were significantly affected by acidity stress, mainly differing among soybean organs and mineral nutrient types. Concentrations of phosphorus (P) and molybdenum (Mo) in both leaves and roots, nitrogen (N), and potassium (K) in roots and magnesium (Mg) in leaves were significantly decreased by acidity stress, respectively. Whereas, concentrations of calcium (Ca), sulfate (S), and iron (Fe) were increased in both leaves and roots. Transcriptome analyses in soybean roots resulted in identification of 419 up-regulated and 555 down-regulated genes under acid conditions. A total of 38 differentially expressed genes (DEGs) were involved in mineral nutrients transportation. Among them, all the detected five GmPTs, four GmZIPs, two GmAMTs, and GmKUPs, together with GmIRT1, GmNramp5, GmVIT2.1, GmSKOR, GmTPK5, and GmHKT1, were significantly down-regulated by acidity stress. Moreover, the transcription of genes encoding transcription factors (e.g., GmSTOP2s) and associated with pH stat metabolic pathways was significantly up-regulated by acidity stress. Taken together, it strongly suggests that maintaining pH stat and mineral nutrient homeostasis are adaptive strategies of soybean responses to acidity stress, which might be regulated by a complex signaling network.

The Influence of Diet and Obesity on Gene Expression in SLE.

This review provides an overview of the known effects of diet, obesity, and the intake of different nutrients on systemic lupus erythematosus (SLE). It summarizes and discusses the studies in rodents that identified how different diets can regulate gene expression in the disease, together with a description of the effects of diet on lupus patients' inflammatory state and disease severity. The identification of selected dietary candidates that can modulate SLE onset and progression is analyzed in relation to possible targeted approaches that could ultimately ameliorate the management and prognosis of this disease.

Autophagy in Paracoccidioides brasiliensis under normal mycelia to yeast transition and under selective nutrient deprivation.

Paracoccidioides spp. is a thermally dimorphic fungus endemic to Latin America and the etiological agent of paracoccidioidomycosis (PCM), a granulomatous disease acquired through fungal propagule inhalation by its mammalian host. The infection is established after successful mycelia to yeast transition in the host pulmonary alveoli. The challenging environment inside the host exposes the fungus to the need of adaptation in order to circumvent nutritional, thermal, oxidative, immunological and other stresses that can directly affect their survival. Considering that autophagy is a response to abrupt environmental changes and is induced by stress conditions, this study hypothesizes that this process might be crucially involved in the adaptation of Paracoccidioides spp. to the host and, therefore, it is essential for the proper establishment of the disease. By labelling autophagous vesicles with monodansylcadaverine, autophagy was observed as an early event in cells during the normal mycelium to yeast transition, as well as in yeast cells of P. brasiliensis under glucose deprivation, and under either rapamycin or 3-methyladenine (3-MA). Findings in this study demonstrated that autophagy is triggered in P. brasiliensis during the thermal-induced mycelium to yeast transition and by glucose-limited conditions in yeasts, both of which modulated by rapamycin or 3-MA. Certainly, further genetic and in vivo analyses are needed in order to finally address the contribution of autophagy for adaptation. Yet, our data propose that autophagy possibly plays an important role in Paracoccidioides brasiliensis virulence and pathogenicity.

Human-Induced Pluripotent Stem Cells Generate Light Responsive Retinal Organoids with Variable and Nutrient-Dependent Efficiency.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.

Identification of Novel Genomic Loci Associated with Soybean Shoot Tissue Macro- and Micronutrient Concentrations.

The mineral composition of crop shoot tissues is important for yield formation and nutrient remobilization to seeds. The natural diversity that exists within crop species can be used to investigate mechanisms that define plant mineral composition and to identify important genomic loci for these processes. The objective of this study was to determine shoot mineral nutrient concentrations in genetically diverse soybean [ (L.) Merr.] genotypes and to identify genomic regions associated with concentrations of different nutrients in shoot tissue. The genotypes were grown at two locations in 2 yr and characterized for macronutrient (Ca, Mg, P, K, and S) and micronutrient (B, Cu, Fe, Mn, and Zn) concentrations in shoot tissues. Genome-wide association studies were conducted with 31,748 single nucleotide polymorphisms (SNPs) via a unified mixed model to identify SNPs associated with macro- and micronutrient concentrations. The number of putative loci identified for the macronutrients ranged from 11 for Ca to 20 for K. For the micronutrients, the number ranged from 10 for Mn to 24 for Fe. In addition to colocated loci for multiple nutrients, 22 individual SNPs were associated with more than one nutrient such that 11 different nutrient combinations were encompassed by these SNPs. Ultimately, the putative loci identified in this study will need to be confirmed and are expected to aid in the identification of new sources of variation for use in soybean breeding programs as well as for mechanistic studies aimed at understanding the regulation of mineral nutrient uptake, translocation, and shoot tissue concentrations.