Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+?breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.     

A barley stripe mosaic virus-based guide RNA delivery system for targeted mutagenesis in wheat and maize

Plant RNA virus-based guide RNA (gRNA) delivery has substantial advantages compared to that of the conventional constitutive promoter-driven expression due to the rapid and robust amplification of gRNAs during virus replication and movement. To date, virus-induced genome editing tools have not been developed for wheat and maize. In this study, we engineered a barley stripe mosaic virus (BSMV)-based gRNA delivery system for clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-mediated targeted mutagenesis in wheat and maize. BSMV-based delivery of single gRNAs for targeted mutagenesis was first validated in Nicotiana benthamiana. To extend this work, we transformed wheat and maize with the Cas9 nuclease gene and selected the wheat TaGASR7 and maize ZmTMS5 genes as targets to assess the feasibility and efficiency of BSMV-mediated mutagenesis. Positive targeted mutagenesis of the TaGASR7 and ZmTMS5 genes was achieved for wheat and maize with efficiencies of up to 78% and 48%. Our results provide a useful tool for fast and efficient delivery of gRNAs into economically important crops.     

Application of Human‐Induced Pluripotent Stem Cells (hiPSCs) to Study Synaptopathy of Neurodevelopmental Disorders

Synapses are the basic structural and functional units for information processing and storage in the brain. Their diverse properties and functions ultimately underlie the complexity of human behavior. Proper development and maintenance of synapses are essential for normal functioning of the nervous system. Disruption in synaptogenesis and the consequent alteration in synaptic function have been strongly implicated to cause neurodevelopmental disorders such as autism spectrum disorders (ASDs) and schizophrenia (SCZ). The introduction of human‐induced pluripotent stem cells (hiPSCs) provides a new path to elucidate disease mechanisms and potential therapies. In this review, we will discuss the advantages and limitations of using hiPSC‐derived neurons to study synaptic disorders. Many mutations in genes encoding for proteins that regulate synaptogenesis have been identified in patients with ASDs and SCZ. We use Methyl‐CpG binding protein 2 (MECP2), SH3 and multiple ankyrin repeat domains 3 (SHANK3) and Disrupted in schizophrenia 1 (DISC1) as examples to illustrate the promise of using hiPSCs as cellular models to elucidate the mechanisms underlying disease‐related synaptopathy.     

DHHC4 and DHHC5 Facilitate Fatty Acid Uptake by Palmitoylating and Targeting CD36 to the Plasma Membrane

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Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes

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SPE-HPLC method for the determination of four flavonols in rat plasma and urine after oral administration of Abelmoschus manihot extract

A SPE-HPLC method was developed and validated for the simultaneous determination of flavonols, isoquercitrin (1), hibifolin (2), myricetin (3), quercetin-3'-O-d-glucoside (4) and quercetin (5) in rat plasma and urine after oral administration of the total flavonoids from Abelmoschus manihot (TFA). The astragalin (6) and kaempferol (7) were used as internal standards (IS). Plasma and urine samples were pretreated by solid-phase extraction using Winchem C(18) reversed-phase cartridges. Analysis of the plasma and urinary extract was performed on YMC-Pack ODS-A C(18) and Thermo ODS-2HYEPRSIL C(18) reversed-phase column, respectively and a mobile phase of acetonitrile-0.1% phosphoric acid was employed. HPLC analysis was conducted with different elution gradients. The flow rate was 1.0 mL/min and the detection wavelength was set at 370 nm. Calibration ranges in plasma for flavonols 2-5 were at 0.011-2.220, 0.014-2.856, 0.022-4.320, and 0.028-5.600 microg/mL, respectively. In urine calibration ranges for flavonols 1, 2, 4 and 5 were at 2.00-16.00, 8.56-102.72, 2.70-21.60, and 3.00-24.00 microg/mL, respectively. The RSD of intra- and inter-day was less than 5.40% and 4.89% in plasma, and less than 3.96% and 6.85% in urine for all the analyses. A preliminary experiment to investigate the plasma concentration and urinary excretion of the flavonols after oral administration of TFA to rats demonstrated that the present method was suitable for determining the flavonols in rat plasma and urine.     

Protein oxidation implicated as the primary determinant of bacterial radioresistance

In the hierarchy of cellular targets damaged by ionizing radiation (IR), classical models of radiation toxicity place DNA at the top. Yet, many prokaryotes are killed by doses of IR that cause little DNA damage. Here we have probed the nature of Mn-facilitated IR resistance in Deinococcus radiodurans, which together with other extremely IR-resistant bacteria have high intracellular Mn/Fe concentration ratios compared to IR-sensitive bacteria. For in vitro and in vivo irradiation, we demonstrate a mechanistic link between Mn(II) ions and protection of proteins from oxidative modifications that introduce carbonyl groups. Conditions that inhibited Mn accumulation or Mn redox cycling rendered D. radiodurans radiation sensitive and highly susceptible to protein oxidation. X-ray fluorescence microprobe analysis showed that Mn is globally distributed in D. radiodurans, but Fe is sequestered in a region between dividing cells. For a group of phylogenetically diverse IR-resistant and IR-sensitive wild-type bacteria, our findings support the idea that the degree of resistance is determined by the level of oxidative protein damage caused during irradiation. We present the case that protein, rather than DNA, is the principal target of the biological action of IR in sensitive bacteria, and extreme resistance in Mn-accumulating bacteria is based on protein protection.     

Species identification and phylogenetic analysis of Leishmania isolated from patients,vectors and hares in the Xinjiang Autonomous Region,The People’s Republic of China

BackgroundVisceral leishmaniasis (VL) has been declared as one of the six major tropical diseases by the World Health Organization. This disease has been successfully controlled in China, except for some areas in the western region, such as the Xinjiang Autonomous Region, where both anthroponotic VL (AVL) and desert type zoonotic VL (DT-ZVL) remain endemic with sporadic epidemics.Methodology/Principal findingsHere, an eleven-year survey (2004–2014) of Leishmania species, encompassing both VL types isolated from patients, sand-fly vectors and Tarim hares (Lepus yarkandensis) from the Xinjiang Autonomous Region was conducted, with a special emphasis on the hares as a potential reservoir animal for DT-ZVL. Key diagnostic genes, ITS1, hsp70 and nagt (encoding N-acetylglucosamine-1-phosphate transferase) were used for phylogenetic analyses, placing all Xinjiang isolates into one clade of the L. donovani complex. Unexpectedly, AVL isolates were found to be closely related to L. infantum, while DT-ZVL isolates were closer to L. donovani. Unrooted parsimony networks of haplotypes for these isolates also revealed their relationship.Conclusions/SignificanceThe above analyses of the DT-ZVL isolates suggested their geographic isolation and independent evolution. The sequence identity of isolates from patients, vectors and the Tarim hares in a single DT-ZVL site provides strong evidence in support of this species as an animal reservoir.     

Regulation by prostaglandin E2 of interleukin release by T lymphocytes in mucosa

Regulation of immune cell activation in lymphocyte-bearing human tissues is a pivotal host function, and metabolites of arachidonic acid (prostaglandin E₂ in particular) have been reported to serve this function at non-mucosal sites. However, it is unknown whether prostaglandin E₂ is immunoregulatory for the large lymphocyte population in the lamina propria of intestine; whether low (nM) concentrations of prostaglandin E₂ modulate immune responses occurring there; and whether adjacent inflammation per se abrogates prostaglandin E₂'s regulatory effects. To address these issues, intestine-derived lymphocytes and T hybridoma cells were assessed, T cell activation was monitored by release of independently quantitated lymphokines, and dose-response studies were performed over an 8-log prostaglandin E₂concentration range. IL-3 release by normal intestinal lamina propria mononuclear cells was reduced (up to 78%) in a dose-dependent manner by prostaglandin E₂, when present in as low a concentration as 10⁻¹⁰M. PGE₂ also inhibited(by ≥ 60%) mucosal T lymphocytes' ability to destabilize the barrier function of human epithelial monolayers. Further, with an intestine-derived T lymphocyte hybridoma cell line, a prostaglandin E₂ dose-dependent reduction in IL-3 and IL-2 (90 and 95%, respectively) was found; this was true for both mitogen- and antigen-driven T cell lymphokine release. Concomitant [³H] thymidine uptake studies suggested this was not due to a prostaglandin E²-induced reduction in T cell proliferation or viability. In contrast, cells from chronically inflamed intestinal mucosa were substantially less sensitive to prostaglandin E², e.g., high concentrations (10⁻⁶ M) of prostaglandin E² inhibited IL-3 release by only 41%. We conclude that prostaglandin E² in nM concentrations is an important modulator of cytokine release from T lymphocytes derived from the gastrointestinal tract, and it may play a central role in regulation of lamina propria immunocyte populations residing there. © 1996 Wiley-Liss, Inc.