Discovery of Proline-Based p300/CBP Inhibitors Using DNA-Encoded Library Technology in Combination with High-Throughput Screening.

E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.

Enhancing the use of technology in the long-term care sector in Canada: Insights from citizen panels and a national stakeholder dialogue.

Enhancing the use of technology in long-term care has been identified as a key part of broader efforts to strengthen the sector in the wake of the COVID-19 pandemic. To inform such efforts, we convened a series of citizen panels, followed by a national stakeholder dialogue with system leaders focused on reimagining the long-term care sector using technology. Key actions prioritized through the deliberations convened included: developing an innovation roadmap/agenda (including national standards and guidelines); using co-design approaches for the strengthening the long-term care sector and for technological innovation; identifying and coordinating existing innovation projects to support scale and spread; enabling rapid-learning and improvement cycles to support the development, evaluation, and implementation of new technologies; and using funding models that enable the flexibility needed for such rapid-learning cycles.

The phylogeny of extant starfish (Asteroidea: Echinodermata) including Xyloplax, based on comparative transcriptomics.

Multi-locus phylogenetic studies of echinoderms based on Sanger and RNA-seq technologies and the fossil record have provided evidence for the Asterozoa-Echinozoa hypothesis. This hypothesis posits a sister relationship between asterozoan classes (Asteroidea and Ophiuroidea) and a similar relationship between echinozoan classes (Echinoidea and Holothuroidea). Despite this consensus around Asterozoa-Echinozoa, phylogenetic relationships within the class Asteroidea (sea stars or starfish) have been controversial for over a century. Open questions include relationships within asteroids and the status of the enigmatic taxon Xyloplax. Xyloplax is thought by some to represent a newly discovered sixth class of echinoderms - and by others to be an asteroid. To address these questions, we applied a novel workflow to a large RNA-seq dataset that encompassed a broad taxonomic and genomic sample. This study included 15 species sampled from all extant orders and 13 families, plus four ophiuroid species as an outgroup. To expand the taxonomic coverage, the study also incorporated five previously published transcriptomes and one previously published expressed sequence tags (EST) dataset. We developed and applied methods that used a range of alignment parameters with increasing permissiveness in terms of gap characters present within an alignment. This procedure facilitated the selection of phylogenomic data subsets from large amounts of transcriptome data. The results included 19 nested data subsets that ranged from 37 to 4,281loci. Tree searches on all data subsets reconstructed Xyloplax as a velatid asteroid rather than a new class. This result implies that asteroid morphology remains labile well beyond the establishment of the body plan of the group. In the phylogenetic tree with the highest average asteroid nodal support several monophyletic groups were recovered. In this tree, Forcipulatida and Velatida are monophyletic and form a clade that includes Brisingida as sister to Forcipulatida. Xyloplax is consistently recovered as sister to Pteraster. Paxillosida and Spinulosida are each monophyletic, with Notomyotida as sister to the Paxillosida. Valvatida is recovered as paraphyletic. The results from other data subsets are largely consistent with these results. Our results support the hypothesis that the earliest divergence event among extant asteroids separated Velatida and Forcipulatacea from Valvatacea and Spinulosida.

RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing.

BACKGROUND: Blueberries are a rich source of antioxidants and other beneficial compounds that can protect against disease. Identifying genes involved in synthesis of bioactive compounds could enable the breeding of berry varieties with enhanced health benefits. RESULTS: Toward this end, we annotated a previously sequenced draft blueberry genome assembly using RNA-Seq data from five stages of berry fruit development and ripening. Genome-guided assembly of RNA-Seq read alignments combined with output from ab initio gene finders produced around 60,000 gene models, of which more than half were similar to proteins from other species, typically the grape Vitis vinifera. Comparison of gene models to the PlantCyc database of metabolic pathway enzymes identified candidate genes involved in synthesis of bioactive compounds, including bixin, an apocarotenoid with potential disease-fighting properties, and defense-related cyanogenic glycosides, which are toxic. Cyanogenic glycoside (CG) biosynthetic enzymes were highly expressed in green fruit, and a candidate CG detoxification enzyme was up-regulated during fruit ripening. Candidate genes for ethylene, anthocyanin, and 400 other biosynthetic pathways were also identified. Homology-based annotation using Blast2GO and InterPro assigned Gene Ontology terms to around 15,000 genes. RNA-Seq expression profiling showed that blueberry growth, maturation, and ripening involve dynamic gene expression changes, including coordinated up- and down-regulation of metabolic pathway enzymes and transcriptional regulators. Analysis of RNA-seq alignments identified developmentally regulated alternative splicing, promoter use, and 3' end formation. CONCLUSIONS: We report genome sequence, gene models, functional annotations, and RNA-Seq expression data that provide an important new resource enabling high throughput studies in blueberry.

Arsenite elicits anomalous sulfur starvation responses in barley.

Treatment of barley (Hordeum vulgare) seedlings with arsenite (AsIII) rapidly induced physiological and transcriptional changes characteristic of sulfur deficiency, even in plants replete with sulfur. AsIII and sulfur deficiency induced 5- to 20-fold increases in the three genes responsible for sulfate reduction. Both treatments also caused up-regulation of a sulfate transporter, but only in the case of sulfur deficiency was there an increase in sulfate influx. Longer-term changes included reduction in transfer of sulfur from roots to shoots and an increase in root growth relative to shoot growth. Genes involved in complexation and compartmentation of arsenic were up-regulated by AsIII, but not by sulfur deficiency. The rate at which arsenic accumulated appeared to be controlled by the rate of thiol synthesis. Over a range of AsIII concentrations and growth periods, the ratio of thiols to arsenic was always close to 3:1, which is consistent with the formation of a stable complex between three glutathione molecules per AsIII. The greater toxicity of arsenic under sulfur-limiting conditions is likely to be due to an intensification of sulfur deficiency as a result of thiol synthesis, rather than to a direct toxicity to metabolism. Because influx of AsIII was nearly 20-fold faster than the rate of synthesis of thiols, it is questionable whether this complexation strategy can be effective in preventing arsenic toxicity, unless arsenic uptake becomes limited by diffusive resistances in the rhizosphere.

Influence of leaf tolerance mechanisms and rain on boron toxicity in barley and wheat.

Boron (B) toxicity is common in many areas of the world. Plant tolerance to high B varies widely and has previously been attributed to reduced uptake of B, most commonly as a result of B efflux from roots. In this study, it is shown that the expression of genes encoding B efflux transporters in leaves of wheat (Triticum aestivum) and barley (Hordeum vulgare) is associated with an ability of leaf tissues to withstand higher concentrations of B. In tolerant cultivars, necrosis in leaves occurred at B concentrations more than 2-fold higher than in sensitive cultivars. It is hypothesized that this leaf tolerance is achieved via redistribution of B by efflux transporters from sensitive symplastic compartments into the leaf apoplast. Measurements of B concentrations in leaf protoplasts, and of B released following infiltration of leaves, support this hypothesis. It was also shown that under B-toxic conditions, leaching of B from leaves by rain had a strong positive effect on growth of both roots and shoots. Measurements of rates of guttation and the concentration of B in guttation droplets indicated that the impact of guttation on the alleviation of B toxicity would be small.

The involvement of aquaglyceroporins in transport of boron in barley roots.

Boron (B) enters cells as the uncharged boric acid, a small neutral molecule with sufficient lipid solubility to cross cell membranes without the aid of transport proteins. The extent to which the observed uptake rates for B in plants can be explained by this simple physical process was examined by applying treatments expected to inhibit the membrane transporters most likely to be involved in B transport. These experiments established that at least 50% of B uptake could be facilitated by transporters. The B transport characteristics of two barley aquaglyceroporins, HvPIP1;3 and HvPIP1;4, were investigated using yeast complementation assays. Expression of both genes in yeast resulted in increased B sensitivity. Transport assays in yeast confirmed that HvPIP1;3 and HvPIP1;4 are both capable of transporting B. The physiological role of these HvPIP1 genes in B transport is uncertain since their expression was not responsive to B nutritional status, and they continued to be expressed under toxicity conditions.

Identification of boron transporter genes likely to be responsible for tolerance to boron toxicity in wheat and barley.

Tolerance to boron (B) toxicity in cereals is known to be associated with reduced tissue accumulation of B. Genes from roots of B-tolerant cultivars of wheat and barley with high similarities to previously reported B efflux transporters from Arabidopsis and rice were cloned. Expression of these genes was strongly correlated with the ability of tolerant genotypes to lower the concentration of B in roots. The gene from barley located to chromosome 4. Backcross lines containing a B tolerance locus on chromosome 4 showed tolerance in proportion to the level of expression of the transporter gene, whereas those lacking the locus were sensitive to B and had very low levels of gene expression. The results are consistent with a widespread mechanism of tolerance to high B based on efflux of B from root cells.

Measurement of trace metal influx in plants: a case study with Co.

The analysis of transport systems involved in the uptake of trace metals in plants is complicated by technical difficulties associated with measurement of uptake and by the likely presence of multiple transporters with broad specificity. In this study, influx of Co was used to illustrate the problems involved and potential solutions. Issues surrounding kinetic descriptions of transport, multiple uptake systems, specificity of transporters, pH effects and the role of membrane surface charge in determining fluxes are addressed. A list of criteria for validation of flux measurements is provided.