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Cereal products contribute significantly to dietary intake of essential minerals. In wheat, iron and zinc are stored in specific grain structures including the aleurone, scutellum and embryo. Wheat cell walls are resistant to digestion in the human gastrointestinal tract and therefore this study investigated the hypothesis that physical disruption of the cell walls would increase the bioaccessibility and bioavailability of iron and zinc from wheat-based foods. Flour was micronized using a combination of roller milling and a micro-mill and this reduced median particle size by two-thirds. Hydrothermally processed wheat flour doughs were subjected to in vitro digestion to determine mineral bioaccessibility. Mineral bioavailability from food digests was measured using human intestinal Caco-2 cells. Iron (but not zinc) bioavailability from wheat foods made using the micronized flour (2.5 ± 0.5 nmol/mg cell protein) was increased significantly compared with foods produced from standard milled flour (1.3 ± 0.1 nmol/mg cell protein; P = 0.031). Micronization of wheat flour has the potential to increase the absorption of the endogenous iron present in cereal foods and this might have health benefits for population groups with poor iron status., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective treatments. It is characterized by activating KRAS mutations and p53 alterations. However, how these mutations dysregulate cancer-cell-intrinsic gene programs to influence the immune landscape of the tumor microenvironment (TME) remains poorly understood. Here, we show that p53 R172H establishes an immunosuppressive TME, diminishes the efficacy of immune checkpoint inhibitors (ICIs), and enhances tumor growth. Our findings reveal that the upregulation of the immunosuppressive chemokine Cxcl1 mediates these pro-tumorigenic functions of p53 R172H . Mechanistically, we show that p53 R172H associates with the distal enhancers of the Cxcl1 gene, increasing enhancer activity and Cxcl1 expression. p53 R172H occupies these enhancers in an NF-κB-pathway-dependent manner, suggesting NF-κB's role in recruiting p53 R172H to the Cxcl1 enhancers. Our work uncovers how a common mutation in a tumor-suppressor transcription factor appropriates enhancers, stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC., Competing Interests: R.K.J. is a Consultant for Accurius, DynamiCure, and SynDevRx; owns equity in Accurius, Enlight, and SynDevRx; served on the Board of Trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, Tekla World Healthcare Fund and received a research Grant from Sanofi. No funding or reagents from these organizations were used in this study. S.S. is an SAB member for Related Sciences, Arcus Biosciences, Ankyra Therapeutics, Prox Bio, and Repertoire Immune Medicines. S.S. is a co-founder of Danger Bio. S.S. is a consultant for TAKEDA and Merck and receives funding for unrelated projects from Leap Therapeutics and iTeos Therapeutics. S.S.’s interests are reviewed and managed under MIT’s policies for potential conflicts of interest.

Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias toward retaining the competing 5' strands hinders efficiency and can cause indel errors. Using rational design of the constituent Cas9-nickase to reposition prime editor nicks, we discovered that competing 5' strands are destabilized to favor the edited 3' new strands. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next- generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 465:1., One Sentence Summary: Prime editors designed with repositioned DNA breaks nearly eliminate undesired genome editing errors.

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers 1,2 . Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations 3 . However, fundamental questions about the temporal regulation of transcription and enhancer-gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq-a new single-cell nascent RNA sequencing assay that uses click chemistry-and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO-seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO-seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression., (© 2024. The Author(s).)

Sequence-specific RNA-binding proteins (RBPs) play central roles in splicing decisions. Here, we describe a modular splicing architecture that leverages in vitro-derived RNA affinity models for 79 human RBPs and the annotated human genome to produce improved models of RBP binding and activity. Binding and activity are modeled by separate Motif and Aggregator components that can be mixed and matched, enforcing sparsity to improve interpretability. Training a new Adjusted Motif (AM) architecture on the splicing task not only yields better splicing predictions but also improves prediction of RBP-binding sites in vivo and of splicing activity, assessed using independent data., (© 2024. The Author(s).)

Competing Interests: The authors declare no conflict of interest.

Yams ( Dioscorea species) are an important food resource in Madagascar, where both cultivated winged yam ( D. alata ) and wild edible yams are consumed. However, there is limited knowledge on the nutrient composition of wild edible yams in Madagascar, and on how they compare with the cultivated winged yam. Therefore, in this study, nine wild edible yam species, one with two subspecies from Madagascar ( D. bako , D. buckleyana , D. irodensis , D. maciba , D. orangeana , D. pteropoda , D. sambiranensis subsp. bardotiae and subsp. sambiranensis , D. seriflora , and Dioscorea species Ovy valiha), were analyzed for their nutrient composition, compared with cultivated D. alata . They include 6/6 of the most favored wild edible yam species in Madagascar. New nutrient composition data (protein, carbohydrate/starch, energy, lipid, β-carotene, and minerals) are presented for these nine wild edible yam species. The results show that they contain comparable levels of lipids and starch to D. alata , but none are better sources of protein than D. alata . The results show that D. irodensis contains a significantly higher β-carotene content when compared to all other edible yams analyzed, and that D. buckleyana , D. irodensis , and D. sambiranensis subsp. bardotiae have a higher calcium content than cultivated D. alata , while all nine wild edible yam species analyzed contain a higher iron content, compared to cultivated D. alata . The nutrient composition data presented could provide new incentives to conserve wild edible yams and inform on strategies to select Dioscorea species for sustainable cultivation and use, providing opportunities to enhance future food security in Madagascar., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Food Science & Nutrition published by Wiley Periodicals LLC.)

Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers 1-5 . Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations 6-9 . However, fundamental questions in the temporal regulation of transcription and enhancer-gene synchrony remain unanswered primarily due to the absence of a single-cell perspective on active transcription. In this study, we present scGRO-seq - a novel single-cell nascent RNA sequencing assay using click-chemistry - and unveil the coordinated transcription throughout the genome. scGRO-seq demonstrates the episodic nature of transcription, and estimates burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells. It reveals the co-transcription of functionally related genes and leverages the replication-dependent non-polyadenylated histone genes transcription to elucidate cell-cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO-seq identifies networks of enhancers and genes and indicates that the bursting of transcription at super-enhancers precedes the burst from associated genes. By imparting insights into the dynamic nature of transcription and the origin and propagation of transcription signals, scGRO-seq demonstrates its unique ability to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression., Competing Interests: Competing interests US patent number US-11519027-B2 on ‘SINGLE-CELL RNA SEQUENCING USING CLICK-CHEMISTRY” was granted on December 6, 2022, to the Massachusetts Institute of Technology, Cambridge, MA (US), on which P.A.S. and D.B.M. are inventors. The authors declare no other competing interests.

Liver as iron storage organ is particularly susceptible to oxidative stress-induced injury from excess iron. Thus, antioxidant therapies are often used to reverse oxidative damage and protect cells and tissues. This study investigated the protective effects of phenolic acids; ferulic acid (FA) and its metabolite, ferulic acid 4-O-sulfate disodium salt (FAS) against oxidative stress under iron overload conditions in mouse and HepG2 cells. Cells were exposed to FA or FAS and then treated with iron-induced oxidative stress complex of 50 μmol/L FAC and 20 μmol/L of 8-hydroxyquinoline 8HQ (8HQ-FAC). Iron dextran was injected intraperitoneally on alternate days for 10 days to induce the iron overload condition in BALB/c mice. The study revealed that the phenolic acids were protective against ROS production, lipid peroxidation and antioxidant depletion in HepG2 cells and liver tissues of BALB/c mice during iron-induced oxidative stress. The protective function of phenolic acids was achieved by the transcriptional activation of nuclear factor erythroid-2-related factor 2 (Nrf2) to regulate antioxidant genes. In conclusion, the study provides evidence that FA has the potential as a therapeutic agent against iron-related diseases such as T2D., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Tugba Kose reports administrative support and equipment, drugs, or supplies were provided by King's College London., (© 2023 Published by Elsevier B.V.)

Cereal products provide 50 % of iron and 30 % of zinc in the UK diet. However, despite having high content, the bioavailability of minerals from cereals is low. This review discusses strategies to increase mineral bioavailability from cereal-based foods. Iron and zinc are localised to specific tissue structures within cereals; however, the cell walls of these structures are resistant to digestion in the human gastrointestinal tract and therefore the bioaccessibility of these essential minerals from foods for absorption in the intestine is limited. In addition, minerals are stored in cereals bound to phytate, which is the main dietary inhibitor of mineral absorption. Recent research has focused on ways to enhance mineral bioavailability from cereals. Current strategies include disruption of plant cell walls to increase mineral release (bioaccessibility) during digestion; increasing the mineral:phytate ratio either by increasing the mineral content through conventional breeding and/or agronomic biofortification, or by reducing phytate levels; and genetic biofortification to increase the mineral content in the starchy endosperm, which is used to produce white wheat flour. While much of this work is at an early stage, there is potential for these strategies to lead to the development of cereal-based foods with enhanced nutritional qualities that could address the low mineral status in the UK and globally.

Increasing numbers of individuals follow plant-based diets. This has sparked interest in the nutritional evaluation of the meat substitute sector. Nutritional understanding of these products is vital as plant-based eating becomes more common. For example, animal products are rich sources of iron and zinc, and plant-based foods could be inadequate in these minerals. The main aim was to analyse the mineral composition and absorption from a range of plant-based meat-free burgers and compare them to a typical beef burger. Total and bioaccessible mineral contents of plant-based burgers and a beef burger were determined using microwave digestion and in vitro simulated gastrointestinal digestion, respectively. Mineral bioavailability was analysed by in vitro simulated gastrointestinal digestion of foods, followed by exposure of Caco-2 cells to the sample digests and assessment of mineral uptake. Mineral quantification for all samples was achieved using inductively coupled ICP-optical emission spectrometry (ICP-OES). The content of minerals varied significantly amongst the burgers. Significantly greater quantities of Fe and Zn were found in the beef burger compared to most meat substitutes. Bioaccessible Fe was significantly higher in the beef compared to most of the plant-based meat alternatives; however, bioavailable Fe of most plant-based burgers was comparable to beef ( p > 0.05). Similarly, bioaccessible Zn was significantly ( p < 0.001) higher from the beef burger. Moreover, beef was superior regarding bioavailable Zn ( p ≤ 0.05-0.0001), with only the mycoprotein burger displaying comparable Zn bioavailability ( p > 0.05). Beef is an excellent source of bioaccessible Fe and Zn compared to most plant-based substitutes; however, these plant-based substitutes were superior sources of Ca, Cu, Mg and Mn. The quantity of bioaccessible and absorbable Fe varies dramatically among the meat alternatives. Plant-based burgers have the potential to provide adequate quantities of iron and zinc to those consuming such burgers as part of a varied diet. Thus, guiding consumer choices will depend on the variety of the vegetable constituents and their iron nutritional quality in different burgers.

Iron is an essential element involved in a variety of physiological functions. However, excess iron catalyzes the generation of reactive oxygen species (ROS) via the Fenton reaction. Oxidative stress, caused by an increase in intracellular ROS production, can be a contributory factor to metabolic syndromes such as dyslipidemia, hypertension, and type 2 diabetes (T2D). Accordingly, interest has grown recently in the role and use of natural antioxidants to prevent iron-induced oxidative damage. This study investigated the protective effect of the phenolic acids; ferulic acid (FA) and its metabolite ferulic acid 4-O-sulfate disodium salt (FAS) against excess iron-related oxidative stress in murine MIN6 cells and the pancreas of BALB/c mice. Rapid iron overload was induced with 50 μmol/L ferric ammonium citrate (FAC) and 20 μmol/L 8-hydroxyquinoline (8HQ) in MIN6 cells, while iron dextran (ID) was used to facilitate iron overload in mice. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, ROS levels were determined by dihydrodichlorofluorescein (H2DCF) cell-permeant probe, iron levels were measured by inductively coupled plasma mass spectrometry (ICP-MS), glutathione, SOD (superoxide dismutase) and lipid peroxidation, and mRNA were assayed with commercially available kits. The phenolic acids enhanced cell viability in iron-overloaded MIN6 cells in a dose-dependent manner. Furthermore, MIN6 cells exposed to iron showed elevated levels of ROS, glutathione (GSH) depletion and lipid peroxidation ( p < 0.05) compared to cells that were protected by treatment with FA or FAS. The treatment of BALB/c mice with FA or FAS following exposure to ID increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) gene levels in the pancreas. Consequently, levels of its downstream antioxidant genes, HO-1, NQO1, GCLC and GPX4, increased in the pancreas. In conclusion, this study shows that FA and FAS protect pancreatic cells and liver tissue from iron-induced damage via the Nrf2 antioxidant activation mechanism.

RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 ( CDK13 ) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.

CRISPR-Cas9 introduces targeted DNA breaks that engage competing DNA repair pathways, producing a spectrum of imprecise insertion/deletion mutations (indels) and precise templated mutations (precise edits). The relative frequencies of these pathways are thought to primarily depend on genomic sequence and cell state contexts, limiting control over mutational outcomes. Here, we report that engineered Cas9 nucleases that create different DNA break structures engage competing repair pathways at dramatically altered frequencies. We accordingly designed a Cas9 variant (vCas9) that produces breaks which suppress otherwise dominant nonhomologous end-joining (NHEJ) repair. Instead, breaks created by vCas9 are predominantly repaired by pathways utilizing homologous sequences, specifically microhomology-mediated end-joining (MMEJ) and homology-directed repair (HDR). Consequently, vCas9 enables efficient precise editing through HDR or MMEJ while suppressing indels caused by NHEJ in dividing and nondividing cells. These findings establish a paradigm of targeted nucleases custom-designed for specific mutational applications.

Dietary deficiencies of iron and zinc cause human malnutrition that can be mitigated by biofortified staple crops. Conventional breeding approaches to increase grain mineral concentrations in wheat (Triticum aestivum L.) have had only limited success, and our understanding of the genetic and physiological barriers to altering this trait is incomplete. Here we demonstrate that a transgenic approach combining endosperm-specific expression of the wheat VACUOLAR IRON TRANSPORTER gene TaVIT2-D with constitutive expression of the rice (Oryza sativa) NICOTIANAMINE SYNTHASE gene OsNAS2 significantly increases the total concentration of zinc and relocates iron to white-flour fractions. In two distinct bread wheat cultivars, we show that the so called VIT-NAS construct led to a two-fold increase in zinc in wholemeal flour, to ∼50 µg g-1. Total iron was not significantly increased, but redistribution within the grain resulted in a three-fold increase in iron in highly pure, roller-milled white flour, to ∼25 µg g-1. Interestingly, expression of OsNAS2 partially restored iron translocation to the aleurone, which is iron depleted in grain overexpressing TaVIT2 alone. A greater than three-fold increase in the level of the natural plant metal chelator nicotianamine in the grain of VIT-NAS lines corresponded with improved iron and zinc bioaccessibility in white flour. The growth of VIT-NAS plants in the greenhouse was indistinguishable from untransformed controls. Our results provide insights into mineral translocation and distribution in wheat grain and demonstrate that the individual and combined effects of the two transgenes can enhance the nutritional quality of wheat beyond what is possible by conventional breeding., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Ferroptosis is a regulated cell death process characterised by the iron-dependent accumulation of oxidised polyunsaturated fatty acid-containing phospholipids. Its initiation is complicated and involves reactive oxygen species (ROS) and a loss of the activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4). These play critical roles in the development of ferroptotic cell damage by lipid peroxidation. Antioxidant therapy is a promising therapeutic strategy to prevent or even reverse the progression of ferroptosis. This study was designed to demonstrate the protective effect of ferulic acid (FA) against oxidative stress and erastin-mediated ferroptosis in murine MIN6 cells. Cells were treated with FA or its metabolite ferulic acid 4-O-sulfate disodium salt (FAS) and 20 μM of erastin. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay, iron levels were measured by inductively coupled plasma mass spectrometry (ICP-MS), ROS levels were determined by a dihydrodichlorofluorescein (H2DCF) cell-permeant probe, and glutathione and lipid peroxidation were assayed with commercially available kits. The phenolic acids enhanced cell viability in erastin-treated MIN6 cells in a dose-dependent manner. Furthermore, MIN6 cells exposed to erastin alone showed elevated levels of iron and ROS, glutathione (GSH) depletion, and lipid peroxidation (p < 0.05) compared to cells that were protected by co-treatment with FA or FAS. The treatment of MIN6 cells with FA or FAS following exposure to erastin increased the nuclear translocation of nuclear factor erythroid-2-related factor 2 (Nrf2) protein levels. Consequently, levels of its downstream antioxidant proteins, HO-1, NQO1, GCLC, and GPX4, increased. FA and FAS greatly decreased erastin-induced ferroptosis in the presence of the Nrf2 inhibitor, ML385, through the regulation of Nrf2 response genes. In conclusion, these results show that FA and FAS protect MIN6 cells from erastin-induced ferroptosis by the Nrf2 antioxidant protective mechanism.

Gene expression heterogeneity underlies cell states and contributes to developmental robustness. While heterogeneity can arise from stochastic transcriptional processes, the extent to which it is regulated is unclear. Here, we characterize the regulatory program underlying heterogeneity in murine embryonic stem cell (mESC) states. We identify differentially active and transcribed enhancers (DATEs) across states. DATEs regulate differentially expressed genes and are distinguished by co-binding of transcription factors Klf4 and Zfp281. In contrast to other factors that interact in a positive feedback network stabilizing mESC cell-type identity, Klf4 and Zfp281 drive opposing transcriptional and chromatin programs. Abrogation of factor binding to DATEs dampens variation in gene expression, and factor loss alters kinetics of switching between states. These results show antagonism between factors at enhancers results in gene expression heterogeneity and formation of cell states, with implications for the generation of diverse cell types during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Zinc stimulates intestinal iron absorption via induction of divalent metal ion transporter (DMT1) and hephaestin (HEPH). While the increase in DMT1 is mediated via a PI3K/IPR2 axis, the mechanisms of Zn-induced HEPH expression downstream of PI3K remain elusive. In the current study we probed the role of Caudal-related homeobox transcription factor-2 (CDX2) on Zn-induced HEPH expression. Zn treatment of Caco-2 cells increased CDX2 phosphorylation and HEPH protein and mRNA expression. siRNA-silencing of CDX2 inhibited Zn-induced HEPH expression. LY294002, an antagonist of PI3K inhibited Zn-induced phosphorylation of CDX2, and downstream HEPH expression. These results suggest that increased expression of HEPH in intestinal cells following Zn treatment is mediated via a PI3K-CDX2 pathway., Competing Interests: Declaration of conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Trigonella foenum-graecum L. (fenugreek), a member of the legume family (Fabaceae), is a promising source of bioactive phytochemicals, which explains its traditional use for a variety of metabolic disorders including cancer. The current study aimed to evaluate extracts of fenugreek seeds and sprouts, and some of their constituents, to compare their cytotoxic and antiproliferative activities in MCF-7 breast cancer cells. The extracts were chemically characterised using high-resolution accurate mass liquid chromatography-mass spectrometry to reveal the detection of compounds assigned as flavone C -glycosides including those derived from apigenin and luteolin, in addition to isoflavones. Five different flavones or their glycosides (apigenin, vicenin-2, vitexin, luteolin and orientin) and two isoflavones (daidzein and formononetin) were quantified in the fenugreek extracts. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay using MCF-7 cells treated with fenugreek methanolic extracts showed dose- and time-dependent effects on cell viability. The MCF-7 cancer cells treated with the fenugreek methanolic extracts also displayed increased relative mitochondrial DNA damage as well as suppressed metastasis and proliferation. This study demonstrates the potential anti-cancer effects of fenugreek seeds and sprouts and reveals fenugreek sprouts as an untapped resource for bioactive compounds.

Macroscopic membraneless organelles containing RNA such as the nucleoli, germ granules, and the Cajal body have been known for decades. These biomolecular condensates are liquid-like bodies that can be formed by a phase transition. Recent evidence has revealed the presence of similar microscopic condensates associated with the transcription of genes. This brief article summarizes thoughts about the importance of condensates in the regulation of transcription and how RNA molecules, as components of such condensates, control the synthesis of RNA. Models and experimental data suggest that RNAs from enhancers facilitate the formation of a condensate that stabilizes the binding of transcription factors and accounts for a burst of transcription at the promoter. Termination of this burst is pictured as a nonequilibrium feedback loop where additional RNA destabilizes the condensate., (© 2022 Sharp et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Iron deficiency anaemia (IDA) is a common nutritional disorder worldwide. Sustainable food-based approaches are being advocated to use high and bioavailable dietary iron sources to prevent iron deficiency. The study investigated the bioaccessibility and bioavailability of iron from some plant products. Total iron levels in the samples were measured by inductively coupled plasma optical emission spectrometry (ICP-OES). Fractionation of the iron from the digested extracts was carried out by centrifugation and ultrafiltration. Iron bioavailability was determined using an in vitro simulated peptic-pancreatic digestion, followed by measurement of ferritin in Caco-2 cells. The highest amount of bioaccessible iron was obtained from moringa leaves (9.88% ± 0.45 and 8.44 ± 0.01 mg/100 g), but the highest percentage bioavailability was from baobab fruit pulp (99.7% ± 0.13 and 1.74 ± 0.01 mg/100 g) respectively. All the plant products, except for baobab, significantly inhibited iron uptake from FeSO 4 and FAC, with fenugreek sprout being the most inhibitory., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Regulation of biological processes typically incorporates mechanisms that initiate and terminate the process and, where understood, these mechanisms often involve feedback control. Regulation of transcription is a fundamental cellular process where the mechanisms involved in initiation have been studied extensively, but those involved in arresting the process are poorly understood. Modeling of the potential roles of RNA in transcriptional control suggested a non-equilibrium feedback control mechanism where low levels of RNA promote condensates formed by electrostatic interactions whereas relatively high levels promote dissolution of these condensates. Evidence from in vitro and in vivo experiments support a model where RNAs produced during early steps in transcription initiation stimulate condensate formation, whereas the burst of RNAs produced during elongation stimulate condensate dissolution. We propose that transcriptional regulation incorporates a feedback mechanism whereby transcribed RNAs initially stimulate but then ultimately arrest the process., Competing Interests: Declaration of Interests R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics, and Dewpoint Therapeutics. T.I.L. is a shareholder of Syros Pharmaceuticals and a consultant to Camp4 Therapeutics. A.K.C. and P.A.S. are shareholders and consultants to Dewpoint Therapeutics. A.K.C. is a SAB member of Omega Therapeutics. The Whitehead Institute has filed a patent application based on this study., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Liver iron excess is observed in several chronic liver diseases and is associated with the development of hepatocellular carcinoma (HCC). However, apart from oxidative stress, other cellular mechanisms by which excess iron may mediate/increase HCC predisposition/progression are not known. HCC pathology involves epithelial to mesenchymal transition (EMT), the basis of cancer phenotype acquisition. Here, the effect of excess iron (holo-transferrin 0-2 g/L for 24 and 48 h) on EMT biomarkers in the liver-derived HepG2 cells was investigated. Holo-transferrin substantially increased intracellular iron. Unexpectedly, mRNA and protein expression of the epithelial marker E-cadherin either remained unaltered or increased. The mRNA and protein levels of metastasis marker N-cadherin and mesenchymal marker vimentin increased significantly. While the mRNA expression of EMT transcription factors SNAI1 and SNAI2 increased and decreased, respectively after 24 h, both factors increased after 48 h. The mRNA expression of TGF-β (EMT-inducer) showed no significant alterations. In conclusion, data showed direct link between iron and EMT. Iron elevated mesenchymal and metastatic biomarkers in HepG2 cells without concomitant decrement in the epithelial marker E-cadherin and altered the expression of the key EMT-mediating transcription factors. Such studies can help identify molecular targets to devise iron-related adjunctive therapies to ameliorate HCC pathophysiology.

Introduction: Micronutrient deficiencies, commonly referred to as 'hidden hunger', affect more than two billion people worldwide, with zinc and iron-deficiency frequently reported. The aim of this study is to examine the impact of consuming zinc biofortified flour (Zincol-2016) on biochemical and functional measures of status in adolescent girls and children living in a low-resource setting in Pakistan., Methods and Analysis: We are conducting a pragmatic, cluster-randomised, double-blind, controlled trial. A total of 482 households have been recruited from two catchment areas approximately 30-40 km distance from Peshawar. Household inclusion criteria are the presence of both an adolescent girl, aged 10-16 years, and a child aged 1-5 years. The study duration is 12 months, divided into two 6-month phases. During phase 1, all households will be provided with locally procured flour from standard varieties of wheat. During phase 2, clusters will be paired, and randomised to either the control or intervention arm of the study. The intervention arm will be provided with zinc biofortified wheat flour, with a target zinc concentration of 40 mg/kg. The control arm will be provided with locally procured wheat flour from standard varieties with an expected zinc concentration of 20 mg/kg. The primary outcome measure is plasma zinc concentration. Secondary outcomes include anthropometric measurements, biomarkers of iron and zinc status, and the presence and duration of respiratory tract infections and diarrhoea., Ethics and Dissemination: Ethical approval was granted from the University of Central Lancashire STEMH Ethics Committee (reference number: STEMH 1014) and Khyber Medical University Ethics Committee (DIR/KMU-EB/BZ/000683). The final study methods will be published in peer-reviewed journals, alongside the study outcomes. In addition, findings will be disseminated to the scientific community via conference presentations and abstracts and communicated to the study participants through the village elders at an appropriate community forum., Trial Registration Number: ISRCTN17107812; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Tea polyphenolics have been suggested to possess blood glucose lowering properties by inhibiting sugar transporters in the small intestine and improving insulin sensitivity. In this report, we studied the effects of teas and tea catechins on the small intestinal sugar transporters, SGLT1 and GLUTs (GLUT1, 2 and 5). Green tea extract (GT), oolong tea extract (OT), and black tea extract (BT) inhibited glucose uptake into the intestinal Caco-2 cells with GT being the most potent inhibitor (IC 50 : 0.077 mg/mL), followed by OT (IC 50 : 0.136 mg/mL) and BT (IC 50 : 0.56 mg/mL). GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (K i ) = 0.0317 and 0.0571 mg/mL, respectively, whereas BT was pure non-competitive, K i  = 0.36 mg/mL. Oocytes injected to express small intestinal GLUTs were inhibited by teas, but SGLT1 was not. Furthermore, catechins present in teas were the predominant inhibitor of glucose uptake into Caco-2 cells, and gallated catechins the most potent: CG > ECG > EGCG ≥ GCG when compared to the non-gallated catechins (C, EC, GC, and EGC). In Caco-2 cells, individual tea catechins reduced the SGLT1 gene, but not protein expression levels. In contrast, GLUT2 gene and protein expression levels were reduced after 2 hours exposure to catechins but increased after 24 hours. These in vitro studies suggest teas containing catechins may be useful dietary supplements capable of blunting postprandial glycaemia in humans, including those with or at risk to Type 2 diabetes mellitus., (© 2020 Federation of American Societies for Experimental Biology.)

The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Iron deficiency is a global epidemic affecting a third of the world's population. Current efforts are focused on investigating sustainable ways to improve the bioavailability of iron in plant-based diets. Incorporating microgreens into the diet of at-risk groups in populations could be a useful tool in the management and prevention of iron deficiency. This study analysed and compared the mineral content and bioavailability of iron from microgreen and mature vegetables. The mineral content of rocket, broccoli and fenugreek microgreens and their mature counterparts was determined using microwave digestion and ICP-OES. Iron solubility and bioavailability from the vegetables were determined by a simulated gastrointestinal in vitro digestion and subsequent measurement of ferritin in Caco-2 cells as a surrogate marker of iron uptake. Iron contents of mature fenugreek and rocket were significantly higher than those of the microgreens. Mature fenugreek and broccoli showed significantly ( p < 0.001) higher bioaccessibility and low-molecular-weight iron than found in the microgreens. Moreover, iron uptake by Caco-2 cells was significantly higher only from fenugreek microgreens than the mature vegetable. While all vegetables except broccoli enhanced FeSO 4 uptake, the response to ferric ammonium citrate (FAC) was inhibitory apart from the mature rocket. Ascorbic acid significantly enhanced iron uptake from mature fenugreek and rocket. Microgreen fenugreek may be bred for a higher content of enhancers of iron availability as a strategy to improve iron nutrition in the populace., Competing Interests: The authors declare no conflict of interest.

Imprinted genes with parental-biased allelic expression are frequently co-regulated and enriched in common biological pathways. Here, we functionally characterize a large cluster of microRNAs (miRNAs) expressed from the maternally inherited allele ("maternally expressed") to explore the molecular and cellular consequences of imprinted miRNA activity. Using an induced neuron (iN) culture system, we show that maternally expressed miRNAs from the miR-379/410 cluster direct the RNA-induced silencing complex (RISC) to transcriptional and developmental regulators, including paternally expressed transcripts like Plagl1. Maternal deletion of this imprinted miRNA cluster resulted in increased protein levels of several targets and upregulation of a broader transcriptional program regulating synaptic transmission and neuronal function. A subset of the transcriptional changes resulting from miR-379/410 deletion can be attributed to de-repression of Plagl1. These data suggest maternally expressed miRNAs antagonize paternally driven gene programs in neurons., Competing Interests: Declaration of Interests The authors declare no competing interests., (Published by Elsevier Inc.)

Pluripotent embryonic stem cells (ESCs) contain the potential to form a diverse array of cells with distinct gene expression states, namely the cells of the adult vertebrate. Classically, diversity has been attributed to cells sensing their position with respect to external morphogen gradients. However, an alternative is that diversity arises in part from cooption of fluctuations in the gene regulatory network. Here we find ESCs exhibit intrinsic heterogeneity in the absence of external gradients by forming interconverting cell states. States vary in developmental gene expression programs and display distinct activity of microRNAs (miRNAs). Notably, miRNAs act on neighborhoods of pluripotency genes to increase variation of target genes and cell states. Loss of miRNAs that vary across states reduces target variation and delays state transitions, suggesting variable miRNAs organize and propagate variation to promote state transitions. Together these findings provide insight into how a gene regulatory network can coopt variation intrinsic to cell systems to form robust gene expression states. Interactions between intrinsic heterogeneity and environmental signals may help achieve developmental outcomes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

In previous studies we demonstrated that zinc stimulates iron uptake in intestinal Caco-2 cells via Zinc-PI3K-IRP2-DMT1 axis. In the current study we investigated the effect of zinc on basolateral iron release and characterized the associated mechanisms. In Caco-2 cells grown on permeable supports, zinc induced iron transport and expression of DMT1, HEPH mRNA and protein, but not that of FPN1. LY294002, an inhibitor of PI3K, inhibited the zinc-induced iron transport, DMT1, HEPH mRNA and protein expression. In addition, LY294002 also inhibited the basal expression of HEPH and FPN1 resulting in blockade of iron egress from cells. In addition, siRNA-silencing of HEPH led to inhibition of both zinc-induced and basal iron transport. Conversely, TPEN, a chelator of zinc, inhibited iron uptake, DMT1, HEPH and FPN1 mRNA and protein expression. These results suggest that intestinal cell zinc status is a critical determinant of iron absorption and effects are mediated via activation of PI3K. Further, PI3K pathway appears to selectively modulate the expression of iron transporters and iron absorption, therefore this might serve as a therapeutic target in iron overload disorders., Competing Interests: Declaration of competing interest The Authors declare that there are no competing interests associated with the manuscript., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3'-UTRs of about 2000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3'-UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality., (© 2019 Kelly et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Iron and zinc are essential micronutrients required for growth and health. Deficiencies of these nutrients are highly prevalent among populations, but can be alleviated by supplementation and food fortification. Cross-sectional studies in humans showed positive association of serum zinc levels with hemoglobin and markers of iron status. Dietary restriction of zinc or intestinal specific conditional knock out of ZIP4 (SLC39A4), an intestinal zinc transporter, in experimental animals demonstrated iron deficiency anemia and tissue iron accumulation. Similarly, increased iron accumulation has been observed in cultured cells exposed to zinc deficient media. These results together suggest a potential role of zinc in modulating intestinal iron absorption and mobilization from tissues. Studies in intestinal cell culture models demonstrate that zinc induces iron uptake and transcellular transport via induction of divalent metal iron transporter-1 (DMT1) and ferroportin (FPN1) expression, respectively. It is interesting to note that intestinal cells are exposed to very high levels of zinc through pancreatic secretions, which is a major route of zinc excretion from the body. Therefore, zinc appears to be modulating the iron metabolism possibly via regulating the DMT1 and FPN1 levels. Herein we critically reviewed the available evidence to hypothesize novel mechanism of Zinc-DMT1/FPN1 axis in regulating intestinal iron absorption and tissue iron accumulation to facilitate future research aimed at understanding the yet elusive mechanisms of iron and zinc interactions.

Enhancers are DNA elements that are bound by transcription factors (TFs), which recruit coactivators and the transcriptional machinery to genes. Phase-separated condensates of TFs and coactivators have been implicated in assembling the transcription machinery at particular enhancers, yet the role of DNA sequence in this process has not been explored. We show that DNA sequences encoding TF binding site number, density, and affinity above sharply defined thresholds drive condensation of TFs and coactivators. A combination of specific structured (TF-DNA) and weak multivalent (TF-coactivator) interactions allows for condensates to form at particular genomic loci determined by the DNA sequence and the complement of expressed TFs. DNA features found to drive condensation promote enhancer activity and transcription in cells. Our study provides a framework to understand how the genome can scaffold transcriptional condensates at specific loci and how the universal phenomenon of phase separation might regulate this process., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex 1-4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus 5,6 . Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain 7-12 . Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers 7,8 , and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9-12 . Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.