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In the context of a PRMT5 inhibitor program, we describe our efforts to develop a flexible and robust strategy to access tetrahydrofuro[3,4-b]furan nucleoside analogues. Ultimately, it was found that a Wolfe type carboetherification from an alkenol derived from d-glucofuranose diacetonide was capable of furnishing the B-ring and installing the desired heteroaryl group in a single step. Using this approach, key intermediate 1.3-A was delivered on a gram scale in a 62% yield and 9.1:1 dr in favor of the desired S-isomer. After deprotection of 1.3-A, a late-stage glycosylation was performed under Mitsunobu conditions to install the pyrrolopyrimidine base. This provided serviceable yields of nucleoside analogues in the range of 31-48% yield. Compound 1.1-C was profiled in biochemical and cellular assays and was demonstrated to be a potent and cellularly active PRMT5 inhibitor, with a PRMT5-MEP50 biochemical IC50 of 0.8 nM, a MCF-7 target engagement EC50 of 3 nM, and a Z138 cell proliferation EC50 of 15 nM. This work sets the stage for the development of new inhibitors of PRMT5 and novel nucleoside chemical matter for alternate drug discovery programs.

Background Non-ER nuclear receptor activity can alter estrogen receptor (ER) chromatin association and resultant ER-mediated transcription. Consistent with GR modulation of ER activity, high tumor glucocorticoid receptor (GR) expression correlates with improved relapse-free survival in ER+ breast cancer (BC) patients. Methods In vitro cell proliferation assays were used to assess ER-mediated BC cell proliferation following GR modulation. ER chromatin association following ER/GR co-liganding was measured using global ChIP sequencing and directed ChIP analysis of proliferative gene enhancers. Results We found that GR liganding with either a pure agonist or a selective GR modulator (SGRM) slowed estradiol (E2)-mediated proliferation in ER+ BC models. SGRMs that antagonized transcription of GR-unique genes both promoted GR chromatin association and inhibited ER chromatin localization at common DNA enhancer sites. Gene expression analysis revealed that ER and GR co-activation decreased proliferative gene activation (compared to ER activation alone), specifically reducing CCND1, CDK2, and CDK6 gene expression. We also found that ligand-dependent GR occupancy of common ER-bound enhancer regions suppressed both wild-type and mutant ER chromatin association and decreased corresponding gene expression. In vivo, treatment with structurally diverse SGRMs also reduced MCF-7 Y537S ER-expressing BC xenograft growth. Conclusion These studies demonstrate that liganded GR can suppress ER chromatin occupancy at shared ER-regulated enhancers, including CCND1 (Cyclin D1), regardless of whether the ligand is a classic GR agonist or antagonist. Resulting GR-mediated suppression of ER+ BC proliferative gene expression and cell division suggests that SGRMs could decrease ER-driven gene expression. Electronic supplementary material The online version of this article (10.1186/s13058-019-1164-6) contains supplementary material, which is available to authorized users.

Agricultural over-fertilization may adversely impact plant−microbial interactions affecting crop yield. It is unclear if soil microbiomes respond quickly to changes in fertilizer inputs once conditioned to specific nutrient regimes. We conducted a growth chamber study assessing the compositional and functional resilience of root-associated microbiomes of Medicago sativa to nutrient regime changes, and consequences for plant growth. Plants were grown with a common starting soil microbiome under four nutrient treatments: control (no fertilizer), organic phosphorus (compost added), low inorganic P (low triple superphosphate, TSP) and high inorganic P (high TSP). After several conditioning generations, in which microbiomes from rhizospheres of high biomass plants were transferred forward, microbiome composition was distinct across the four treatments. The resulting microbiomes were then transplanted into each of the nutrient treatments, leading generally to functional changes in hydrolytic enzyme activity and taxonomic convergence with other microbiomes transplanted into the same nutrient regime. However, high inorganic P-conditioned microbiomes were resistant to compositional change. Correspondingly, M. sativa grown with high inorganic P-conditioned microbiomes had lower biomass, fewer nodules, and lower %N than plants grown under the same nutrient regime with other microbiomes. These findings suggest that excessive inorganic P fertilization may change microbiomes such that they negatively affect plant growth.

Protein arginine methyltransferase 5 (PRMT5) is a type II arginine methyltransferase that catalyzes the post-translational symmetric dimethylation of protein substrates. PRMT5 plays a critical role in regulating biological processes including transcription, cell cycle progression, RNA splicing, and DNA repair. As such, dysregulation of PRMT5 activity is implicated in the development and progression of multiple cancers and is a target of growing clinical interest. Described herein are the structure-based drug designs, robust synthetic efforts, and lead optimization strategies toward the identification of two novel 5,5-fused bicyclic nucleoside-derived classes of potent and efficacious PRMT5 inhibitors. Utilization of compound docking and strain energy calculations inspired novel designs, and the development of flexible synthetic approaches enabled access to complex chemotypes with five contiguous stereocenters. Additional efforts in balancing bioavailability, solubility, potency, and CYP3A4 inhibition led to the identification of diverse lead compounds with favorable profiles, promising in vivo activity, and low human dose projections.

Multivalent oligonucleotide-based bonding elements have been synthesized and studied for the assembly and crystallization of gold nanoparticles. Through the use of organic branching points, divalent and trivalent DNA linkers were readily incorporated into the oligonucleotide shells that define DNA-nanoparticles and compared to monovalent linker systems. These multivalent bonding motifs enable the change of "bond strength" between particles and therefore modulate the effective "bond order." In addition, the improved accessibility of strands between neighboring particles, either due to multivalency or modifications to increase strand flexibility, gives rise to superlattices with less strain in the crystallites compared to traditional designs. Furthermore, the increased availability and number of binding modes also provide a new variable that allows previously unobserved crystal structures to be synthesized, as evidenced by the formation of a thorium phosphide superlattice.

Two feeding trials were conducted to investigate the effects of dietary pH (pH 2.5, 3.7, 4.6, 5.7-control, pH 7.0, and pH 8.0) and dietary acid source (acetic acid, citric acid, hydrochloric acid and control-no acid) on growth rate and feed utilization efficiency of the Nile tilapia fry. In addition, attractability of the diets at different pH and dietary acid sources were determined. Results of the first feeding trial showed that the feed with pH adjusted to 4.6 resulted in significantly highest final average body weight (FABW), weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) and most efficient food conversion ratio (FCR) in the Nile tilapia fry. Survival was 100% in diets with pH 3.7 up to 7.0; the diet with pH 2.5 exhibited significantly lowest survival followed by the pH 8.0 diet. In the second feeding trial, diets that contained hydrochloric, citric or acetic acids that were used to adjust the pH to the optimal 4.6 resulted in higher FABW, WG, SGR, and PER values but lower FCR values than did the control diet; these parameters were not significantly different among the dietary groups. This study demonstrated that the dietary pH promoted growth and efficiency in the Nile tilapia fry and that the three acids that were used were similar in their effects on growth and efficiency. Furthermore, it was demonstrated that either acidifying the diet to pH 4.6 by an inorganic acid such as HCl or the provision of a dietary acidifier such as citric acid or acetic acid could enhance growth, feed efficiency and survival of the Nile tilapia.

If a solution of DNA-coated nanoparticles is allowed to crystallize, the thermodynamic structure can be predicted by a set of structural design rules analogous to Pauling's rules for ionic crystallization. The details of the crystallization process, however, have proved more difficult to characterize as they depend on a complex interplay of many factors. Here, we report that this crystallization process is dictated by the individual DNA bonds and that the effect of changing structural or environmental conditions can be understood by considering the effect of these parameters on free oligonucleotides. Specifically, we observed the reorganization of nanoparticle superlattices using time-resolved synchrotron small-angle X-ray scattering in systems with different DNA sequences, salt concentrations, and densities of DNA linkers on the surface of the nanoparticles. The agreement between bulk crystallization and the behavior of free oligonucleotides may bear important consequences for constructing novel classes of crystals and incorporating new interparticle bonds in a rational manner.

Background: Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker's yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting that interspecies hybridization may also offer potential for biofuel research. Results: To investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose-fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in ammonia fiber expansion-pretreated corn stover hydrolysate and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose. Conclusions: This research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry. Fil: Peris, David. University of Wisconsin; Estados Unidos Fil: Moriarty, Ryan V.. University of Wisconsin; Estados Unidos Fil: Alexander, William G.. University of Wisconsin; Estados Unidos Fil: Baker, EmilyClare. University of Wisconsin; Estados Unidos Fil: Sylvester, Kayla. University of Wisconsin; Estados Unidos Fil: Sardi, Maria. University of Wisconsin; Estados Unidos Fil: Langdon, Quinn K.. University of Wisconsin; Estados Unidos Fil: Libkind Frati, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales. Universidad Nacional del Comahue. Instituto Andino Patagónico de Tecnologías Biológicas y Geoambientales.; Argentina Fil: Wang, Qi-Ming. University of Wisconsin; Estados Unidos. Institute Of Microbiology Chinese Academy Of Sciences; China Fil: Bai, Feng-Yan. Institute Of Microbiology Chinese Academy Of Sciences; China Fil: Leducq, Jean Baptiste. University of Montreal; Canadá. Laval University; Canadá Fil: Charron, Guillaume. Laval University; Canadá Fil: Landry, Christian R.. Laval University; Canadá Fil: Sampaio, José Paulo. New University Of Lisbon; Portugal Fil: Gonçalves, Paula. New University Of Lisbon; Portugal Fil: Hyma, Katie E.. Washington University in St. Louis; Estados Unidos Fil: Fay, Justin C.. Washington University in St. Louis; Estados Unidos Fil: Sato, Trey K.. University of Wisconsin; Estados Unidos Fil: Hittinger, Chris Todd. University of Wisconsin; Estados Unidos

Triblock peptide-oligonucleotide chimeras (POCs) consisting of peptides and oligonucleotides interlinked by an organic core are presented and their assembly behaviour is investigated. Several factors influence POC assembly, resulting in the formation of either vesicles or fibres. Design rules are introduced and used to predict and alter POC assembly morphology.

Evidence for microbes has been detected in extreme subsurface environments as deep as 2.5 km with temperatures as high as 90 °C, demonstrating that microbes can adapt and survive extreme environmental conditions. Deep subsurface shales are increasingly exploited for their energy applications, thus characterizing the prevalence and role of microbes in these ecosystems essential for understanding biogeochemical cycles and maximizing production from hydrocarbon-bearing formations. Here, we describe the distribution of bacterial ester-linked phospholipid fatty acids (PLFA) and diglyceride fatty acids (DGFA) in sidewall cores retrieved from three distinct geologic horizons collected to 2275 m below ground surface in a Marcellus Shale well, West Virginia, USA. We examined the abundance and variety of PLFA and DGFA prior to energy development within and above the Marcellus Shale Formation into the overlying Mahantango Formation of the Appalachian Basin. Lipid biomarkers in the cores suggest the presence of microbial communities comprising Gram (+), Gram (-) as well as stress indicative biomarkers. Microbial PLFA and DGFA degradation in the subsurface can be influenced by stressful environmental conditions associated with the subsurface. The PLFA concentration and variety were higher in the transition zone between the extremely low permeability Marcellus Shale Formation and the more permeable Mahantango Formation. In contrast to this distribution, more abundant and diverse DGFA membrane profiles were associated with the Mahantango Formation. The stress indicative biomarkers like the trans-membrane fatty acids, oxiranes, keto-, and dimethyl lipid fatty acids were present in all cores, potentially indicating that the bacterial communities had experienced physiological stress or nutrient deprivation during or after deposition. The DGFA profiles expressed more stress indicative biomarkers as opposed to the PLFA membrane profiles. These findings suggest the probable presence of indigenous microbial communities in the deep subsurface shale and also improves our understanding of microbial survival mechanisms in ancient deep subsurface environments.

The resurgence of Group A Streptococcus (GAS) infections in the past two decades has been a rising major public health concern. Due to a large number of GAS infections occurring in the skin, mast cells (MCs), innate immune cells known to localize to the dermis, could play an important role in controlling infection. MCs can exert their antimicrobial activities either early during infection, by degranulation and release of antimicrobial proteases and the cathelicidin-derived antimicrobial peptide LL-37, or by forming antibacterial MC extracellular traps (MCETs) in later stages of infection. We demonstrate that MCs do not directly degranulate in response to GAS, reducing their ability to control bacterial growth in early stages of infection. However, MC granule components are highly cytotoxic to GAS due to the pore-forming activity of LL-37, while MC granule proteases do not significantly affect GAS viability. We therefore confirmed the importance of MCETs by demonstrating their capacity to reduce GAS survival. The data therefore suggests that LL-37 from MC granules become embedded in MCETs, and are the primary effector molecule by which MCs control GAS infection. Our work underscores the importance of a non-traditional immune effector cell, utilizing a non-conventional mechanism, in the defense against an important human pathogen.

Chemical bonds are a key determinant of the structure and properties of a material. Thus, rationally designing arbitrary materials requires complete control over the bond. While atomic bonding is dictated by the identity of the atoms, nanoparticle superlattice engineering, where nano-particle “atoms” are held together by DNA “bonds”, offers a route to design crystal lattices in a way that nature cannot: through altering the oligonucleotide bond. Herein, the use of RNA, as opposed to DNA, is explored by synthesizing superlattices in which nanoparticles are bonded by DNA/DNA, RNA/RNA, and DNA/RNA duplexes. By moving beyond nanoparticle superlattices assembled only with DNA, a new degree of freedom is introduced, providing programmed responsiveness to enzymes and greater bond versatility. Therefore, the oligonucleotide bond can have programmable function beyond dictating the structure of the material and moves nanoparticle superlattices closer to naturally occurring biomaterials, where the line between structural and functional elements is blurred.

Complementary tetrahedral small molecule-DNA hybrid (SMDH) building blocks have been combined to form nucleic acid-based polymeric nanoparticles without the need for an underlying template or scaffold. The sizes of these particles can be tailored in a facile fashion by adjusting assembly conditions such as SMDH concentration, assembly time, and NaCl concentration. Notably, these novel particles can be stabilized and transformed into functionalized spherical nucleic acid (SNA) structures through the incorporation of capping DNA strands conjugated with functional groups. These results demonstrate a systematic, efficient strategy for the construction and surface functionalization of well-defined, size-tunable nucleic acid particles from readily accessible molecular building blocks. Furthermore, because these nucleic acid-based polymeric nanoparticles exhibited enhanced cellular internalization and resistance to DNase I compared to free synthetic nucleic acids, they should have a plethora of applications in diagnostics and therapeutics.

Growing interest in the utilization of black shales for hydrocarbon development and environmental applications has spurred investigations of microbial functional diversity in the deep subsurface shale ecosystem. Lipid biomarker analyses including phospholipid fatty acids (PLFAs) and diglyceride fatty acids (DGFAs) represent sensitive tools for estimating biomass and characterizing the diversity of microbial communities. However, complex shale matrix properties create immense challenges for microbial lipid extraction procedures. Here, we test three different lipid extraction methods: modified Bligh and Dyer (mBD), Folch (FOL), and microwave assisted extraction (MAE), to examine their ability in the recovery and reproducibility of lipid biomarkers in deeply buried shales. The lipid biomarkers were analyzed as fatty acid methyl esters (FAMEs) with the GC-MS, and the average PL-FAME yield ranged from 67 to 400 pmol/g, while the average DG-FAME yield ranged from 600 to 3,000 pmol/g. The biomarker yields in the intact phospholipid Bligh and Dyer treatment (mBD + Phos + POPC), the Folch, the Bligh and Dyer citrate buffer (mBD-Cit), and the MAE treatments were all relatively higher and statistically similar compared to the other extraction treatments for both PLFAs and DGFAs. The biomarker yields were however highly variable within replicates for most extraction treatments, although the mBD + Phos + POPC treatment had relatively better reproducibility in the consistent fatty acid profiles. This variability across treatments which is associated with the highly complex nature of deeply buried shale matrix, further necessitates customized methodological developments for the improvement of lipid biomarker recovery.

A gram-scale route to bryostatin Scientists once accumulated 14 tons of the red, bushy, tufted sea creature Bugula neritina to extract 18 grams of bryostatin 1. The macrocyclic organic compound is under study for treatment of HIV, cancer, and Alzheimer's disease but has proven frustratingly scarce. Wender et al. report a 29-step chemical synthesis of bryostatin 1 that proceeds in 4.8% overall yield and provides gram quantities of the compound (see the Perspective by Lanman). Intermediates along the pathway can be straightforwardly modified to produce analogs, two of which were prepared en route and studied in vitro. Science , this issue p. 218 ; see also p. 166

Metal contaminants in soils can persist for millennia, causing lasting negative impacts on local ecosystems. Long-term contaminant bioavailability is related to soil pH and to the strength and stability of solid-phase associations. We combined physical density separation with synchrotron-based microspectroscopy to reduce solid-phase complexity and to study Zn speciation in field-contaminated soils. We also investigated Zn uptake in two Zn-hyperaccumulating ecotypes of (Ganges and Prayon). Soils were either moderately contaminated (500-800 mg Zn kg via contaminated biosolids application) or grossly enriched (26,000 mg Zn kg via geogenic enrichment). Soils were separated using sodium polytungstate into three fractions: light fraction (LF) (1.6 g cm), medium fraction (MF) (1.6-2.8 g cm), and heavy fraction (HF) (2.8 g cm). Approximately 45% of the total Zn was associated with MF in biosolids-contaminated soils. From these data, we infer redistribution to the MF after biosolids application because Zn in biosolids is principally associated with HF and LF. Our results suggest that increasing proportions of HF-associated Zn in soils may be related to greater relative Zn removal by Zn hyperaccumulating plants. Using density fractions enabled assessment of Zn speciation on a microscale despite incomplete fractionation. Analyzing both density fractions and whole soils revealed certain phases (e.g., ZnS, Zn coprecipitated with Fe oxides) that were not obvious in all analyses, indicating multiple views of the same soils enable a more complete understanding of Zn speciation.

We describe here step-economical, function-oriented strategies towards the syntheses of potent kinase inhibitors inspired by the natural product staurosporine., The human kinome comprises over 500 protein kinases. When mutated or over-expressed, many play critical roles in abnormal cellular functions associated with cancer, cardiovascular disease and neurological disorders. Here we report a step-economical approach to designed kinase inhibitors inspired by the potent, but non-selective, natural product staurosporine, and synthetically enabled by a novel, complexity-increasing, serialized [5 + 2]/[4 + 2] cycloaddition strategy. This function-oriented synthesis approach rapidly affords tunable scaffolds, and produced a low nanomolar inhibitor of protein kinase C.

Multicomponent reactions allow for more bond-forming events per synthetic operation, enabling more step- and time-economical conversion of simple starting materials to complex and thus value-added targets. These processes invariably require that reactivity be relayed from intermediate to intermediate over several mechanistic steps until a termination event produces the final product. Here, we report a multicomponent process in which a novel 1,2,3-butatriene equivalent (TMSBO: TMSCH2C≡CCH2OH) engages chemospecifically as a two-carbon alkyne component in a metal-catalysed [5 + 2] cycloaddition with a vinylcyclopropane to produce an intermediate cycloadduct. Under the reaction conditions, this intermediate undergoes a remarkably rapid 1,4-Peterson elimination, producing a reactive four-carbon diene intermediate that is readily intercepted in either a metal-catalysed or thermal [4 + 2] cycloaddition. TMSBO thus serves as an yne-to-diene transmissive reagent coupling two powerful and convergent cycloadditions--the homologous Diels-Alder and Diels-Alder cycloadditions--through a vinylogous Peterson elimination, and enabling flexible access to diverse polycycles.

The human kinome comprises over 500 protein kinases. When mutated or over-expressed, many play critical roles in abnormal cellular functions associated with cancer, cardiovascular disease and neurological disorders. Here we report a step-economical approach to designed kinase inhibitors inspired by the potent, but non-selective, natural product staurosporine, and synthetically enabled by a novel, complexity-increasing, serialized [5 + 2]/[4 + 2] cycloaddition strategy. This function-oriented synthesis approach rapidly affords tunable scaffolds, and produced a low nanomolar inhibitor of protein kinase C.

A broad range of synthetically challenging-to-access small molecule–DNA hybrids can be readily synthesized in “one pot” and in high yields by coupling multi-azide cores to alkyne-modified DNAs on a solid support using click chemistry. The multi-functional products can be obtained in pure forms and on large scales (1 μmol) in a facile fashion. In addition, the distribution of the products can be controlled by changing the concentration of the azide core in solution and the strand-loading density on the solid-supports.

Reduction of a subbituminous coal by lithium in liquid ammonia yields a coal salt that can be reacted with 1-iodododecane to yield dodecylated coal that is partially soluble in organic solvents. The coal salt also acts as an initiator for the in situ oligomerization of alkenes onto the surface of the coal nanoplatelets. Atomic force microscopy and high-resolution transmission electron microscopy images of this material have been recorded. Energy-dispersive X-ray spectroscopy has been used to identify minerals that occur in the coal.

A facile route to water-soluble graphite nanoplatelets that uses graphite as the starting material is described. The method relies on the addition of phenyl radicals with subsequent sulfonation of the phenyl groups. Atomic force microscopy, high-resolution transmission electron microscopy, and scanning tunneling microscopy images show that a high degree of exfoliation occurs during the sulfonation step. The sheet resistance of the bulk films of the nanoplatelets prepared by vacuum filtration using an anodisc membrane was found to be 212 Ω/sq.

Background: A recent effort to distinguish early-stage TNBC based on outcome and to expose driver pathways using gene profiling/cluster analysis divided TNBC into four subtypes- basal-like, mesenchymal, immunomodulatory, and luminal androgen receptor (LAR). The clinical AR+ TNBC subtype (>10% AR by IHC) comprises up to 35% of metastatic disease. The discovery of this TNBC subtype and the concomitant development of potent new anti-androgens for prostate cancer have led to several clinical trials evaluating AR antagonists in TNBC. In parallel, glucocorticoid receptor (GR) expression and activation have been shown to mediate anti-apoptotic gene expression in TNBC. However, to our knowledge, no one has examined the coordinate expression of GR and AR activity and the potential crosstalk downstream of dual GR and AR activation. We hypothesized that AR/GR cooperative gene expression pathways may induce TNBC therapy resistance and tumor progression. Methods and Results: We analyzed GR+ TNBC primary tumor gene expression from TCGA samples and found a wide range of AR expression. To examine whether GR-associated gene expression differed in high versus low AR+ TNBC, we divided TCGA TNBC primary tumors (N=123) at the AR median into “high” versus “low” AR expression. We then identified relative GR target gene expression levels in those genes identified from a previous TNBC meta-analysis. Invasion, motility and proliferation pathways were found to be most significantly expressed among GR-associated genes in AR high TNBC (top 5 significant pathways). Similarly, we found that GR and AR co-activation in AR+ MDA-MB-453 TNBC cells significantly increased cell migration compared to GR or AR activation alone. In addition, when MDA-MB-453 cells were exposed to the AR antagonist enzalutamide (enza) for more than 30 days (long-term, LT), the magnitude of increase in cell migration following GR activation increased, suggesting that increased GR activity may compensate for long-term AR blockade. Furthermore, long-term enza exposure (LTenza) in AR+ MFM-223 and MDA-MB-453 cells resulted in increased GR mRNA and protein levels. The magnitude of GR-mediated target gene expression was also significantly increased in LTEnza versus control-treated cells. In vivo, LTenza-treated MDA-MB-453 xenografts were relatively insensitive to paclitaxel chemotherapy treatment, consistent with increased GR activity compared to control cells. Conclusions: We conclude that coordinate GR and AR expression in TNBC contributes to a particularly poor outcome in TNBC. Downstream AR/GR gene expression favoring cell survival and invasive phenotypes may contribute to this outcome. Consideration of dual AR/GR inhibition in TNBC may increase the effectiveness of anti-androgen therapy. Citation Format: Deniz N. Dolcen, Eva Tonsing-Carter, Ryan Harkless, Caroline Kim, Kathleen Bowie, Gini Fleming, Suzanne Conzen. Combined androgen and glucocorticoid receptor (AR/GR) activity drives TNBC progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2541.

Reduction of activated charcoal by lithium in liquid ammonia yields charcoal salts that can be reacted with dodecyl iodide to yield soluble dodecylated activated charcoal. Atomic force microscopy images reveal a heterogeneous size distribution of nearly spherical nanoparticles. High-resolution transmission electron microscopy images show a layered microcrystalline arrangement that becomes separated to form mostly single layered disordered structures after dodecylation. A 1H–13C cross polarization magic angle spinning spectrum of the charcoal revealed a broad, featureless signal from sp2 carbons and a much weaker broad signal from aliphatic carbons.

Maltose metabolism during the conversion of transitory (leaf) starch to sucrose requires a 4-alpha-glucanotransferase (EC 2.4.1.25) in the cytosol of leaf cells. This enzyme is called DPE2 because of its similarity to the disproportionating enzyme in plastids (DPE1). DPE1 does not use maltose; it primarily transfers a maltosyl unit from one maltotriose to a second maltotriose to make glucose and maltopentaose. DPE2 is a modular protein consisting of a family 77 glycosyl hydrolase domain, similar to DPE1, but unlike DPE1 the domain is interrupted by an insertion of approximately 150 amino acids as well as an N-terminal extension that consists of two carbohydrate binding modules. Phylogenetic analysis shows that the DPE2-type enzyme is present in a limited but highly diverse group of organisms. Here we show that DPE2 transfers the non-reducing glucosyl unit from maltose to glycogen by a ping-pong mechanism. The forward reaction (consumption of maltose) is specific for the beta-anomer of maltose, while the reverse reaction (production of maltose) is not stereospecific for the acceptor glucose. Additionally, through deletion mutants we show that the glycosyl hydrolase domain alone provides disproportionating activity with a much higher affinity for short maltodextrins than the complete wild-type enzyme, while absence of the carbohydrate binding modules completely abolishes activity with large complex carbohydrates, reflecting the presumed function of DPE2 in vivo.

Multicomponent reactions allow for more bond-forming events per synthetic operation, enabling more step- and time-economical conversion of simple starting materials to complex and thus value-added targets. These processes invariably require that reactivity be relayed from intermediate to intermediate over several mechanistic steps until a termination event produces the final product. Here, we report a multicomponent process in which a novel 1,2,3-butatriene equivalent (TMSBO: TMSCH2C≡CCH2OH) engages chemospecifically as a two-carbon alkyne component in a metal-catalysed [5 + 2] cycloaddition with a vinylcyclopropane to produce an intermediate cycloadduct. Under the reaction conditions, this intermediate undergoes a remarkably rapid 1,4-Peterson elimination, producing a reactive four-carbon diene intermediate that is readily intercepted in either a metal-catalysed or thermal [4 + 2] cycloaddition. TMSBO thus serves as an yne-to-diene transmissive reagent coupling two powerful and convergent cycloadditions--the homologous Diels-Alder and Diels-Alder cycloadditions--through a vinylogous Peterson elimination, and enabling flexible access to diverse polycycles.

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

The leader (L) protein of encephalomyocarditis virus (EMCV) shuts off host cell nucleocytoplasmic trafficking (NCT) by inducing hyperphosphorylation of nuclear pore proteins. This dramatic effect by a nonenzymatic protein of 6 kDa is not well understood but clearly involves L binding to cellular Ran GTPase, a critical factor of active NCT. Exogenous GDP and GTP are inhibitory to L-Ran binding, but the guanine-nucleotide exchange factor RCC1 can relieve this inhibition. In the presence of RCC1, L binds Ran with a K D (equilibrium dissociation constant) of ∼3 nM and reaches saturation within 20 min. The results of fluorescently tagged nucleotide experiments suggest that L-Ran interactions affect the nucleotide-binding pocket of Ran.

Dopamine (DA) is released in the medial preoptic area (MPOA) of male rats during copulation. DA agonists infused into the MPOA facilitate, and antagonists impair, copulatory behavior. Local administration of the nitric oxide (NO) precursor L-arginine also increases DA release in the MPOA. The present experiment used microdialysis to test whether NO promotes DA release during copulation. Males received either an NO synthesis inhibitor, nitro-L-arginine methyl ester (L-NAME, 400 microM), or its inactive isomer D-NAME (400 microM) into the MPOA via a microdialysis probe for 3 h prior to the introduction of a female. Following D-NAME administration, DA increased during copulation, while L-NAME prevented this increase. NO may therefore promote DA release in the MPOA of male rats, thereby facilitating copulation.

Broiler chickens exhibit a high frequency of leg problems, including fractures, associated with rapid growth. We hypothesized that administering a diet high in vitamin K and flaxseed oil rich in n-3 PUFA to chickens early in development would improve bone strength. Seventy-two day-old male broiler chicks were randomly assigned to one of four groups: 1) control diet (n-6/n-3=50:1) containing corn oil and normal vitamin K levels (menadione sodium bisulfite, 22.0 mg/kg diet); 2) high vitamin K diet containing corn oil and high vitamin K (3.3 g/kg diet); 3) flaxseed oil diet (n-6/n-3=5:1) containing flaxseed oil and normal levels of vitamin K and; 4) high vitamin K/flaxseed oil diet. Diets were administered six weeks. After the fourth week, six birds from each group were sacrificed each week for the next three weeks. The mechanical strength of the tibiotarsus was determined using a three-point bending test. BMC was measured using DEXA scanning and bone ashing. Results indicated no significant difference in bo...

Liposomes are currently well-established as biocompatible delivery vehicles for numerous compounds. However, conventional manufacturing tends to rely on time-consuming processes, costly equipment, unstable reaction parameters, and numerous pre- and post-processing steps. Herein, we demonstrate a microscope-slide-sized alternative: a double flow-focusing microfluidic geometry capable of sub-hour synthesis and controlled loading of tunable liposomes. Using phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine as the bilayer constituent, the effect of varying the dissolved lipid concentration and flow rate ratio on synthesized liposome diameters was investigated and the encapsulation of fluorescent hydrophobic drug model ergost-5,7,9(11),22-tetraen-3β-ol was performed to ascertain the potential of this device as a loading platform.

In the title compound, C20H15NOS, the dihedral angle between the phenyl rings is 74.25 (6)°. The six-membered 1,3-thiazine ring has an envelope conformation with the C atom at the 2-position forming the flap. The crystal structure features weak C—H...O interactions, which lead to the formation of a tape motif along [110].

Background Under acidic conditions of the stomach with gastric juice pH ranging from 1.7 to 2.6, nitrite is reduced to nitric oxide. When a diet rich in nitrate is consumed, it is partially reduced to nitrite in the mouth by oral bacteria followed by ingestion and partial reduction of nitrite to nitric oxide (NO) in the stomach. When the dietary nitrate is consumed along with protein, secondary amines from protein react with nitrite via the formation of reactive nitrogen species to form nitrosamines, which are carcinogenic. Ascorbic acid, also obtained from diet, can react with nitrite to form NO in the acidic gastric juice, which does not react with amines. Ascorbate has a protective effect on the formation of carcinogenic compounds like nitrosamines by reducing nitrite faster and out competing nitrosamine formation by reactive oxygen species and amines. Iodide can also reduce nitrite to NO in acidic pH, which is the basis of a nitrite assay used in nitric oxide analyzers. Although it has been suggested that dietary iodide would lead to increased nitrosamine formation the enhanced formation of NO from nitrite due to iodide suggests iodide would be protective against nitrosamine formation. Thus, the combined effect of ascorbate and sodium iodide in the presence of nitrite and amines under acidic conditions on nitrosamine formation was studied. Methods In this study the effect of ascorbic acid and sodium nitrite on nitrite reduction and nitrosamine formation was determined in vitro. Nitrite reduction to NO was determined under anaerobic conditions by ozone chemiluminescence by the Nitric Oxide Analyzer (NOA 280i, Sievers, GE Analytical Instruments, Boulder, CO). Nitrosamine concentrations were measured using absorption spectroscopy according to a modification of method by Mirvish et al., Science 175 (1972) 65–68. Results Both Iodide and ascorbic acid are effective in reducing nitrite to NO at the pH range that includes the pH of stomach. There is a synergistic effect of ascorbic acid and iodide on the rates of nitrite reduction to NO. Very low levels of nitrite are reduced to NO by either ascorbic acid or iodide alone. Nitrosomorpholine formation is reduced by iodide and ascorbate combined compared to ascorbic acid alone at pH 2. Conclusions The results indicate that, due to the increased rate of nitrite reduction byascorbic acid in the presence of iodide, nitrosation of morpholine to nitrosomorpholine can be reduced. Given that both nitrate and iodide are taken up by salivary glands and concentrated in the oral cavity, their combined effects on nitrosamine formation could have substantial impact. Disclosure This work was supported by NIH Grant HL058091.