The effect of nitrogen source and levels on hybrid aspen tree physiology and wood formation.

Nitrogen can be taken up by trees in the form of nitrate, ammonium and amino acids, but the influence of the different forms on tree growth and development is poorly understood in angiosperm species like Populus. We studied the effects of both organic and inorganic forms of nitrogen on growth and wood formation of hybrid aspen trees in experimental conditions that allowed growth under four distinct steady-state nitrogen levels. Increased nitrogen availability had a positive influence on biomass accumulation and the radial dimensions of both xylem vessels and fibers, and a negative influence on wood density. An optimal level of nitrogen availability was identified where increases in biomass accumulation outweighed decreases in wood density. None of these responses depended on the source of nitrogen except for shoot biomass accumulation, which was stimulated more by treatments complemented with nitrate than by ammonium alone or the organic source arginine. The most striking difference between the nitrogen sources was the effect on lignin composition, whereby the abundance of H-type lignin increased only in the presence of nitrate. The differential effect of nitrate is possibly related to the well-known role of nitrate as a signaling compound. RNA-sequencing revealed that while the lignin-biosynthetic genes did not significantly (FDR <0.01) respond to added NO3 - , the expression of several laccases, catalysing lignin polymerization, was dependent on N-availability. These results reveal a unique role of nitrate in wood formation and contribute to the knowledge basis for decision-making in utilizing hybrid aspen as a bioresource.

Development of LB244, an Irreversible STING Antagonist.

The cGMP-AMP Synthase (cGAS)-Stimulator of Interferon Genes (STING) pathway plays a critical role in sensing dsDNA localized to the cytosol, resulting in the activation of a robust inflammatory response. While cGAS-STING signaling is essential for antiviral immunity, aberrant STING activation is observed in amyotrophic lateral sclerosis (ALS), lupus, and autoinflammatory diseases such as Aicardi-Goutières syndrome (AGS) and STING associated vasculopathy with onset in infancy (SAVI). Significant efforts have therefore focused on the development of STING inhibitors. In a concurrent submission, we reported that BB-Cl-amidine inhibits STING-dependent signaling in the nanomolar range, both in vitro and in vivo. Considering this discovery, we sought to generate analogs with higher potency and proteome-wide selectivity. Herein, we report the development of LB244, which displays nanomolar potency and inhibits STING signaling with markedly enhanced proteome-wide selectivity. Moreover, LB244 mirrored the efficacy of BB-Cl-amidine in vivo. In summary, our data identify novel chemical entities that inhibit STING signaling and provide a scaffold for the development of therapeutics for treating STING-dependent inflammatory diseases.

Simple monocyclic pyrimidine analogs as microtubule targeting agents binding to the colchicine site.

Complex natural products that bind to tubulin/microtubules come under the broad category of microtubule binding agents. The design of simplified analogs of previously reported bicyclic, microtubule depolymerizer, pyrrolo[2,3-d]pyrimidine, provided valuable structure-activity relationship data and led to the identification of novel monocyclic pyrimidine analogs of which 12 was 47-fold more potent (EC50 123 nM) for cellular microtubule depolymerization activity and 7.5-fold more potent (IC50 24.4 nM) at inhibiting the growth of MDA-MB-435 cancer cells, suggesting significantly better binding of the target within the colchicine site of tubulin compared to lead compound 1. This compound and others of this series of monocyclic pyrimidine analogs were able to overcome multidrug resistance due to the expression of the ßIII-isotype of tubulin and P-glycoprotein. In vivo evaluation of the most potent analog 12 in an MDA-MB-435 xenograft mouse model indicated, along with paclitaxel, that both compounds showed a trend towards lower tumor volume however neither compound showed significant antitumor activity in the trial. To our knowledge these are the first examples of simple substituted monocyclic pyrimidines as colchicine site binding antitubulin compounds with potent antitumor activity.

The Influence of a Wearable-Based Reward Program on Health Care Costs: Retrospective, Propensity Score-Matched Cohort Study.

BACKGROUND: Mobile health (mHealth) technology holds great promise as an easily accessible and effective solution to improve population health at scale. Despite the abundance of mHealth offerings, only a minority are grounded in evidence-based practice, whereas even fewer have line of sight into population-level health care spending, limiting the clinical utility of such tools. OBJECTIVE: This study aimed to explore the influence of a health plan-sponsored, wearable-based, and reward-driven digital health intervention (DHI) on health care spending over 1 year. The DHI was delivered through a smartphone-based mHealth app available only to members of a large commercial health plan and leveraged a combination of behavioral economics, user-generated sensor data from the connected wearable device, and claims history to create personalized, evidence-based recommendations for each user. METHODS: This study deployed a propensity score-matched, 2-group, and pre-post observational design. Adults (≥18 years of age) enrolled in a large, national commercial health plan and self-enlisted in the DHI for ≥7 months were allocated to the intervention group (n=56,816). Members who were eligible for the DHI but did not enlist were propensity score-matched to the comparison group (n=56,816). Average (and relative change from baseline) medical and pharmacy spending per user per month was computed for each member of the intervention and comparison groups during the pre- (ie, 12 months) and postenlistment (ie, 7-12 months) periods using claims data. RESULTS: Baseline characteristics and medical spending were similar between groups (P=.89). On average, the total included sample population (N=113,632) consisted of young to middle-age (mean age 38.81 years), mostly White (n=55,562, 48.90%), male (n=46,731, 41.12%) and female (n=66,482, 58.51%) participants. Compared to a propensity score-matched cohort, DHI users demonstrated approximately US $10 per user per month lower average medical spending (P=.02) with a concomitant increase in preventive care activities and decrease in nonemergent emergency department admissions. These savings translated to approximately US $6.8 million in avoidable health care costs over the course of 1 year. CONCLUSIONS: This employer-sponsored, digital health engagement program has a high likelihood for return on investment within 1 year owing to clinically meaningful changes in health-seeking behaviors and downstream medical cost savings. Future research should aim to elucidate health behavior-related mechanisms in support of these findings and continue to explore novel strategies to ensure equitable access of DHIs to underserved populations that stand to benefit the most.

Transcriptome-Wide Mapping of Small-Molecule RNA-Binding Sites in Cells Informs an Isoform-Specific Degrader of QSOX1 mRNA.

The interactions between cellular RNAs in MDA-MB-231 triple negative breast cancer cells and a panel of small molecules appended with a diazirine cross-linking moiety and an alkyne tag were probed transcriptome-wide in live cells. The alkyne tag allows for facile pull-down of cellular RNAs bound by each small molecule, and the enrichment of each RNA target defines the compound's molecular footprint. Among the 34 chemically diverse small molecules studied, six bound and enriched cellular RNAs. The most highly enriched interaction occurs between the novel RNA-binding compound F1 and a structured region in the 5' untranslated region of quiescin sulfhydryl oxidase 1 isoform a (QSOX1-a), not present in isoform b. Additional studies show that F1 specifically bound RNA over DNA and protein; that is, we studied the entire DNA, RNA, and protein interactome. This interaction was used to design a ribonuclease targeting chimera (RIBOTAC) to locally recruit Ribonuclease L to degrade QSOX1 mRNA in an isoform-specific manner, as QSOX1-a, but not QSOX1-b, mRNA and protein levels were reduced. The RIBOTAC alleviated QSOX1-mediated phenotypes in cancer cells. This approach can be broadly applied to discover ligands that bind RNA in cells, which could be bioactive themselves or augmented with functionality such as targeted degradation.

Potential of substituted quinazolines to interact with multiple targets in the treatment of cancer.

The efficacy of quinazoline-based antiglioma agents has been attributed to their effects on microtubule dynamics.1,2 The design, synthesis and biological evaluation of quinazolines as potent inhibitors of multiple intracellular targets, including microtubules and multiple RTKs, is described. In addition to the known ability of quinazolines 1 and 2 to cause microtubule depolymerization, they were found to be low nanomolar inhibitors of EGFR, VEGFR-2 and PDGFR-ß. Low nanomolar inhibition of EGFR was observed for 1-3 and 9-10. Compounds 1 and 4 inhibited VEGFR-2 kinase with activity better than or equal to that of sunitinib. In addition, compounds 1 and 2 had similar potency to sunitinib in the CAM angiogenesis assay. Multitarget activities of compounds in the present study demonstrates that the quinazolines can affect multiple pathways and could lead to these agents having antitumor potential caused by their activity against multiple targets.

Machine Learning for Health: Algorithm Auditing & Quality Control.

Developers proposing new machine learning for health (ML4H) tools often pledge to match or even surpass the performance of existing tools, yet the reality is usually more complicated. Reliable deployment of ML4H to the real world is challenging as examples from diabetic retinopathy or Covid-19 screening show. We envision an integrated framework of algorithm auditing and quality control that provides a path towards the effective and reliable application of ML systems in healthcare. In this editorial, we give a summary of ongoing work towards that vision and announce a call for participation to the special issue  Machine Learning for Health: Algorithm Auditing & Quality Control in this journal to advance the practice of ML4H auditing.

Macrocyclization of a Ligand Targeting a Toxic RNA Dramatically Improves Potency.

RNA molecules both contribute to and are causative of many human diseases. One method to perturb RNA function is to target its structure with small molecules. However, discovering bioactive ligands for RNA targets is challenging. Here, we show that the bioactivity of a linear dimeric ligand that inactivates the RNA trinucleotide repeat expansion that causes myotonic dystrophy type 1 [DM1; r(CUG)exp ] can be improved by macrocyclization. Indeed, the macrocyclic compound is ten times more potent than the linear compound for improving DM1-associated defects in cells, including in patient-derived myotubes (muscle cells). This enhancement in potency is due to the macrocycle's increased affinity and selectively for the target, which inhibit r(CUG)exp 's toxic interaction with muscleblind-like 1 (MBNL1), and its superior cell permeability. Macrocyclization could prove to be an effective way to enhance the bioactivity of modularly assembled ligands targeting RNA.

Exploring microRNA profiles for circadian clock and flowering development regulation in Himalayan Rhododendron.

miRNA is a non-coding, yet crucial entity in remodeling the genetic architecture. Rhododendron arboreum of Himalayas grows and even flower under fluctuating climate. sRNA from leaves of vegetative and reproductive periods was sequenced to elucidate its seasonal associations. Conserved (256) and novel (210) miRNAs and their precursors were located based on homology with plant databases and transcriptome of the species. 27,139 predicted targets were involved with metabolism, reproduction, and response to abiotic stimuli. A comparative analysis showed differential expression of 198 miRNAs with season-specific abundance of 103 miRNAs. Specific isoforms of 11 miRNA families exhibited a temporal expression and targeted different genes implying a complex regulation. The variable miRNA expression among the tissues of different conditions can be associated with the adaptability of the species, which will prove essential for further study on miRNAs mediating seasonal response. Moreover, exogenous cues also mediate phase transition via networking of flowering pathways and their components. In this context, 18 known families and 77 novel miRNAs modulating 117 genes crucial in circadian entrainment were filtered. A negative correlation was obtained between the expression of 18 of these miRNAs and their targets when tested through quantitative-PCR. It highlighted the role of miRNA-target pairs in perceiving environmental variabilities and monitoring flowering growth. Furthermore, a phylogenetic clustering was performed, which supported the lineage-specific evolution and function of putative miR156 sequence in the species. This documentation of genome-wide profiling of miRNA, their targets, and expression will enhance the understanding of developmental and climate-tolerance strategies in high-altitude trees.

Comparative transcriptome profiling reveals the reprogramming of gene networks under arsenic stress in Indian mustard.

Arsenic is a widespread toxic metalloid that is classified as a class I carcinogen known to cause adverse health effects in humans. In the present study, we investigated arsenic accumulation potential and comparative gene expression in Indian mustard. The amount of arsenic accumulated in shoots varied in the range of 15.99-1138.70 mg/kg on a dry weight basis among five cultivars. Comparative expression analysis revealed 10 870 significantly differentially expressed genes mostly belonging to response to stress, metabolic processes, signal transduction, transporter activity, and transcription regulator activity to be up-regulated, while most of the genes involved in photosynthesis, developmental processes, and cell growth were found to be down-regulated in arsenic-treated tissues. Further, pathway analysis using the KEGG Automated Annotation server (KAAS) revealed a large-scale reprogramming of genes involved in genetic and environmental information processing pathways. Top pathways with maximum KEGG orthology hits included carbon metabolism (2.5%), biosynthesis of amino acids (2.1%), plant hormone signal transduction (1.4%), and glutathione metabolism (0.6%). A transcriptomic investigation to understand the arsenic accumulation and detoxification in Indian mustard will not only help to improve its phytoremediation efficiency but also add to the control measures required to check bioaccumulation of arsenic in the food chain.

RNAseq-based phylogenetic reconstruction of Taxaceae and Cephalotaxaceae.

Taxaceae and Cephalotaxaceae are the two economically important conifer families. Over the years there has been much controversy over the issue of merging these families. The position of Amentotaxus and Torreya is also ambiguous. Some authors consider them closer to Taxaceae while others deemed them to fit within Cephalotaxaceae. Still, others prefer to raise them to their own tribe. Different morphological, anatomical, embryological and phylogenetic evidence supports one or the other view, making the precise delineation between them unresolved. Here we used an RNAseq-based approach to obtain orthologous genes across the selected species to reconstruct a more robust phylogeny of these families. A total of 233.123 million raw reads were de novo assembled to generate nine different transcript assemblies for the corresponding species. Of the 940 191 assembled transcripts across nine species, we generated 409 734 unigenes, which were clustered into orthologous groups. A total of 331 single-copy complete orthologous groups were selected for phylogenetic analysis. Maximum-likelihood, maximum-parsimony and Bayesian phylogenetic trees showed a sister relationship between Taxaceae and Cephalotaxaceae. Our analysis supports their distinctiveness at the family level and also shows that Amentotaxus and Torreya fit within Cephalotaxaceae.

Molecular basis of transitivity in plant RNA silencing.

The discovery of small RNAs has offered exciting opportunities in manipulating gene expression. The non-coding RNAs cause target gene inactivation at the transcriptional, post-transcriptional or translational level. In addition to the default silencing approach, they provide another mode of gene regulation by transitivity. Here, gradual amplification in effector RNAs number allows regulation of genes other than the original target and causes the outspread of silencing from its origin to aid a robust response. Unlike the short-range cell-to-cell movement of silencing signal (through plasmodesmata), little is known of the mediators of systemic silencing (usually through phloem). Through the present review, we combine the reports available so far to better understand the characteristics of secondary silencing, factors involved, and summarize the instances where it has been employed in plants. Understanding the molecular mechanism behind transitivity has led to the designing of efficient transgenes for targeted gene inactivation, utilized in silencing of a multigene family, and in the field of functional genomics. Studies uncovering the origin of distinct secondary silencing pathways in plants have been exploited for developing artificial RNA silencing methods such as hairpin RNA, artificial microRNA, intrinsic direct repeat, inverted repeat, artificial trans-acting siRNA, phased siRNA, and virus-induced gene silencing. The techniques have facilitated the spread of traits such as pathogenic resistance or alter plant growth and development features. The mechanism of reprogramming in the silencing machinery and the consequent genetic manipulation through transitive RNA is still not completely understood and its exploitation in crop improvement programmes is still in a developing phase.

Transcriptome characterization and screening of molecular markers in ecologically important Himalayan species (Rhododendron arboreum).

Rhododendron arboreum is an ecologically prominent species, which also lends commercial and medicinal benefits in the form of palatable juices and useful herbal drugs. Local abundance and survival of the species under a highly fluctuating climate make it an ideal model for genetic structure and functional analysis. However, a lack of genomic data has hampered additional research. In the present study, cDNA libraries from floral and foliar tissues of the species were sequenced to provide a foundation for understanding the functional aspects of the genome and to construct an enriched repository that will promote genomics studies in the genera. Illumina's platform facilitated the generation of ∼100 million high-quality paired-end reads. De novo assembly, clustering, and filtering out of shorter transcripts predicted 113 167 non-redundant transcripts with an average length of 1164.6 bases. Of these, 71 961 transcripts were categorized based on functional annotations in the Gene Ontology database, whereby 5710 were grouped into 141 pathways and 23 746 encoded for different transcription factors. Transcriptome screening further identified 35 419 microsatellite regions, of which, 43 polymorphic loci were characterized on 30 genotypes. Seven hundred and nineteen transcripts had 811 high-quality single-nucleotide polymorphic variants with a minimum coverage of 10, a total score of 20, and SNP% of 50.

Design, synthesis and preclinical evaluation of 5-methyl-N4-aryl-furo[2,3-d]pyrimidines as single agents with combination chemotherapy potential.

The design, synthesis and biological evaluation of 4-substituted 5-methyl-furo[2,3-d]pyrimidines is described. The Ullmann coupling of 5-methyl-furo[2,3-d]pyrimidine with aryl iodides was successfully optimized to synthesize these analogs. Compounds 6-10 showed single-digit nanomolar inhibition of EGFR kinase. Compounds 1 and 6-10 inhibited VEGFR-2 kinase better than or equal to sunitinib. Compounds 1 and 3-10 were more potent inhibitors of PDGFR-ß kinase than sunitinib. In addition, compounds 4-11 had higher potency in the CAM angiogenesis assay than sunitinib. Compound 1 showed in vivo efficacy in an A498 renal xenograft model in mice. Multiple RTK and tubulin inhibitory attributes of 1, 4, 6 and 8 indicates that these compounds may be valuable preclinical single agents targeting multiple intracellular targets.

Structure based drug design and in vitro metabolism study: Discovery of N-(4-methylthiophenyl)-N,2-dimethyl-cyclopenta[d]pyrimidine as a potent microtubule targeting agent.

We report a series of tubulin targeting agents, some of which demonstrate potent antiproliferative activities. These analogs were designed to optimize the antiproliferative activity of 1 by varying the heteroatom substituent at the 4'-position, the basicity of the 4-position amino moiety, and conformational restriction. The potential metabolites of the active compounds were also synthesized. Some compounds demonstrated single digit nanomolar IC50 values for antiproliferative effects in MDA-MB-435 melanoma cells. Particularly, the S-methyl analog 3 was more potent than 1 in MDA-MB-435 cells (IC50 = 4.6 nM). Incubation of 3 with human liver microsomes showed that the primary metabolite of the S-methyl moiety of 3 was the methyl sulfinyl group, as in analog 5. This metabolite was equipotent with the lead compound 1 in MDA-MB-435 cells (IC50 = 7.9 nM). Molecular modeling and electrostatic surface area were determined to explain the activities of the analogs. Most of the potent compounds overcome multiple mechanisms of drug resistance and compound 3 emerged as the lead compound for further SAR and preclinical development.

Design, synthesis, and structure-activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells.

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2-8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2-8. Compounds 2 and 6 had two-digit nanomolar potency (IC50) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the ßIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.

Discovery and preclinical evaluation of 7-benzyl-N-(substituted)-pyrrolo[3,2-d]pyrimidin-4-amines as single agents with microtubule targeting effects along with triple-acting angiokinase inhibition as antitumor agents.

The utility of cytostatic antiangiogenic agents (AA) in cancer chemotherapy lies in their combination with cytotoxic chemotherapeutic agents. Clinical combinations of AA with microtubule targeting agents (MTAs) have been particularly successful. The discovery, synthesis and biological evaluations of a series of 7-benzyl-N-substituted-pyrrolo[3,2-d]pyrimidin-4-amines are reported. Novel compounds which inhibit proangiogenic receptor tyrosine kinases (RTKs) including vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-ß (PDGFR-ß) and epidermal growth factor receptor (EGFR), along with microtubule targeting in single molecules are described. These compounds also inhibited blood vessel formation in the chicken chorioallantoic membrane (CAM) assay, and some potently inhibited tubulin assembly (with activity comparable to that of combretastatin A-4 (CA)). In addition, some of the analogs circumvent the most clinically relevant tumor resistance mechanisms (P-glycoprotein and ß-III tubulin expression) to microtubule targeting agents (MTA). These MTAs bind at the colchicine site on tubulin. Two analogs displayed two to three digit nanomolar GI50 values across the entire NCI 60 tumor cell panel and one of these, compound 7, freely water soluble as its HCl salt, afforded excellent in vivo antitumor activity against an orthotopic triple negative 4T1 breast cancer model and was superior to doxorubicin.

Janus Compounds, 5-Chloro-N4-methyl-N4-aryl-9H-pyrimido[4,5-b]indole-2,4-diamines, Cause Both Microtubule Depolymerizing and Stabilizing Effects.

While evaluating a large library of compounds designed to inhibit microtubule polymerization, we identified four compounds that have unique effects on microtubules. These compounds cause mixed effects reminiscent of both microtubule depolymerizers and stabilizers. Immunofluorescence evaluations showed that each compound initially caused microtubule depolymerization and, surprisingly, with higher concentrations, microtubule bundles were also observed. There were subtle differences in the propensity to cause these competing effects among the compounds with a continuum of stabilizing and destabilizing effects. Tubulin polymerization experiments confirmed the differential effects and, while each of the compounds increased the initial rate of tubulin polymerization at high concentrations, total tubulin polymer was not enhanced at equilibrium, likely because of the dueling depolymerization effects. Modeling studies predict that the compounds bind to tubulin within the colchicine site and confirm that there are differences in their potential interactions that might underlie their distinct effects on microtubules. Due to their dual properties of microtubule stabilization and destabilization, we propose the name Janus for these compounds after the two-faced Roman god. The identification of synthetically tractable, small molecules that elicit microtubule stabilizing effects is a significant finding with the potential to identify new mechanisms of microtubule stabilization.

Catalytic Isonitrile Insertions and Condensations Initiated by RNC-X Complexation.

Isonitriles are delicately poised chemical entities capable of being coaxed to react as nucleophiles or electrophiles. Directing this tunable reactivity with metal and non-metal catalysts provides rapid access to a large array of complex nitrogenous structures ideally functionalized for medicinal applications. Isonitrile insertion into transition metal complexes has featured in numerous synthetic and mechanistic studies, leading to rapid deployment of isonitriles in numerous catalytic processes, including multicomponent reactions (MCR). Covering the literature from 1990-2014, the present review collates reaction types to highlight reactivity trends and allow catalyst comparison.