Showing papers by "Phillip A. Sharp published in 2020"

Partitioning of Cancer Therapeutics in Nuclear Condensates

Abstract: 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.

Inhibition of the facilitative sugar transporters (GLUTs) by tea extracts and catechins.

Abstract: 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 (IC50 : 0077 mg/mL), followed by OT (IC50 : 0136 mg/mL) and BT (IC50 : 056 mg/mL) GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (Ki ) = 00317 and 00571 mg/mL, respectively, whereas BT was pure non-competitive, Ki = 036 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

MicroRNAs organize intrinsic variation into stem cell states

Abstract: 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.

Biofortification of wheat with zinc for eliminating deficiency in Pakistan: study protocol for a cluster-randomised, double-blind, controlled effectiveness study (BIZIFED2).

Abstract: 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 483 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 2-5 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. Registration details: The trial has been registered with the ISRCTN registry, study ID ISRCTN17107812.

Iron elevates mesenchymal and metastatic biomarkers in HepG2 cells.

Abstract: 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.

In Vitro Bioaccessibility and Bioavailability of Iron from Mature and Microgreen Fenugreek, Rocket and Broccoli

Abstract: 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 FeSO4 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.

Agronomic Biofortification of Maize with Zinc Fertilizers Increases Zinc Uptake from Maize Flour by Human Intestinal Caco-2 Cells

Abstract: Zinc deficiency is prevalent in many low- and middle-income countries. Maize is an important staple food source in many of these regions, however, it contains low levels of nutritionally important minerals including zinc. The purpose of this study was to test the hypothesis that application of zinc-containing fertilisers would increase both the zinc content of, and amount of zinc available for absorption from, maize flour. Maize was grown in pots in zinc-deficient soils (<1.22 mg/L) at the Bako Agricultural Research Centre, Ethiopia. Zinc (ZnSO4) was applied via several routes: directly to the soil; in the side dressing; as a foliar application; or through seed priming. Zinc content of maize flour samples was measured by ICP-OES. Flour samples were then subjected to simulated in vitro gastrointestinal digestion and zinc uptake by human intestinal Caco-2 cells was measured using ICP-MS. Addition of zinc to the soil alone did not increase zinc uptake by Caco-2 cells compared with untreated control maize. In contrast, addition of ZnSO4, to the side dressing (3.6-fold increase), through foliar application (2.9-fold increase), or through seed priming (4.2-fold increase) significantly increased zinc uptake compared with untreated controls and the soil zinc treatment groups (1-way ANOVA and Tukey's test; P < 0.05). Zinc uptake by Caco-2 cells was correlated significantly with the zinc content of the flour (r2 = 0.24; P < 0.02). Together, these data demonstrate that several fertilisation methods can be used to increase zinc content of maize flour. This additional zinc is, at least partly, available for absorption by human intestinal epithelial cells. Agronomic biofortification is therefore a promising strategy to combat zinc deficiency in vulnerable populations. This work was funded in part by a grant from the Bill & Melinda Gates Foundation.

Methods and assays for modulating gene transcription by modulating condensates

Abstract: Described herein are compositions and methods for modulating gene regulation by modulating condensate formation, composition, maintenance, dissolution and regulation.