Cytokinin‐dependent specification of the functional megaspore in the Arabidopsis female gametophyte

The life cycle of higher plants alternates between the diploid sporophytic and the haploid gametophytic phases. In angiosperms, male and female gametophytes develop within the sporophyte. During female gametophyte (FG) development, a single archesporial cell enlarges and differentiates into a megaspore mother cell, which then undergoes meiosis to give rise to four megaspores. In most species of higher plants, including Arabidopsis thaliana, the megaspore closest to the chalaza develops into the functional megaspore (FM), and the remaining three megaspores degenerate. Here, we examined the role of cytokinin signaling in FG development. We characterized the FG phenotype in three triple mutants harboring non‐overlapping T–DNA insertions in cytokinin AHK receptors. We demonstrate that even the strongest mutant is not a complete null for the cytokinin receptors. Only the strongest mutant displayed a near fully penetrant disruption of FG development, and the weakest triple ahk mutant had only a modest FG phenotype. This suggests that cytokinin signaling is essential for FG development, but that only a low threshold of signaling activity is required for this function. Furthermore, we demonstrate that there is elevated cytokinin signaling localized in the chalaza of the ovule, which is enhanced by the asymmetric localization of cytokinin biosynthetic machinery and receptors. We show that an FM‐specific marker is absent in the multiple ahk ovules, suggesting that disruption of cytokinin signaling elements in Arabidopsis blocks the FM specification. Together, this study reveals a chalazal‐localized sporophytic cytokinin signal that plays an important role in FM specification in FG development.     

Design,synthesis, and evaluation of curcumin-derived arylheptanoids for glioblastoma and neuroblastoma cytotoxicity

Using an innovative approach toward multiple carbon–carbon bond-formations that relies on the multifaceted catalytic properties of titanocene complexes we constructed a series of C1–C7 analogs of curcumin for evaluation as brain and peripheral nervous system anti-cancer agents. C2-Arylated analogs proved efficacious against neuroblastoma (SK-N-SH & SK-N-FI) and glioblastoma multiforme (U87MG) cell lines. Similar inhibitory activity was also evident in p53 knockdown U87MG GBM cells. Furthermore, lead compounds showed limited growth inhibition in vitro against normal primary human CD34+hematopoietic progenitor cells. Taken together, the present findings indicate that these curcumin analogs are viable lead compounds for the development of new central and peripheral nervous system cancer chemotherapeutics with the potential for little effects on normal hematopoietic progenitor cells.     

Pharmacological properties and predicted binding mode of arylmethylene quinuclidine-like derivatives at the α3β4 nicotinic acetylcholine receptor (nAChR)

We have carried out a pharmacological evaluation of arylmethylene quinuclidine derivatives interactions with human α3β4 nAChRs subtype, using cell-based receptor binding, calcium-influx, electrophysiological patch-clamp assays and molecular modeling techniques. We have found that the compounds bind competitively to the α3β4 receptor with micromolar affinities and some of the compounds behave as non-competitive antagonists (compounds 1, 2 and 3), displaying submicromolar IC₅₀ values. These evidences suggest a mixed mode of action for these compounds, having interactions at the orthosteric site and more pronounced interactions at an allosteric site to block agonist effects. One of the compounds, 1-benzyl-3-(diphenylmethylene)-1-azoniabicyclo[2.2.2]octane chloride (compound 3), exhibited poorly reversible use-dependent block of α3β4 channels. We also found that removal of a phenyl group from compound 1 confers a partial agonism to the derived analog (compound 6). Introducing a hydrogen-bond acceptor into the 3-benzylidene quinuclidine derivative (compound 7) increases agonism potency at the α3β4 receptor subtype. Docking into the orthosteric binding site of a α3β4 protein structure derived by comparative modeling accurately predicted the experimentally-observed trend in binding affinity. Results supported the notion that binding requires a hydrogen bond formation between the ligand basic nitrogen and the backbone carbonyl oxygen atom of the conserved Trp-149.     

Nonbenzamidine acylsulfonamide tissue factor–factor VIIa inhibitors

Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P′ binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.     

A convenient synthesis of GDP-d-rhamnose: The donor substrate for d-rhamnosyltransferase WbpZ from Pseudomonas aeruginosa PAO1

Gram negative bacteria have lipopolysaccharides (LPS) that are critical for their survival. LPS molecules are composed of antigenic exopolysaccharide chains (O antigens). We are interested in discovering the enzymes involved in the biosynthesis of O antigens in Pseudomonas aeruginosa. The common polysaccharide antigen contains α-linked d-rhamnose residues. We have now synthesized GDP-d-rhamnose by a convenient synthesis in aqueous solution, and have shown that it can be used without extensive purification as the donor substrate for d-rhamnosyltransferase (WbpZ) from the P. aeruginosa strain PAO1. The availability of this nucleotide sugar preparation allows for characterization of d-rhamnosyltransferases.     

Discovery of 2,5-diarylnicotinamides as selective orexin-2 receptor antagonists (2-SORAs)

The orexin (or hypocretin) system has been identified as a novel target for the treatment of insomnia due to the wealth of biological and genetic data discovered over the past decade. Recently, clinical proof-of-concept was achieved for the treatment of primary insomnia using dual (OX₁R/OX₂R) orexin receptor antagonists. However, elucidation of the pharmacology associated with selective orexin-2 receptor antagonists (2-SORAs) has been hampered by the lack of orally bioavailable, highly selective small molecule probes. Herein, the discovery and optimization of a novel series of 2,5-diarylnicotinamides as potent and orally bioavailable orexin-2 receptor selective antagonists is described. A compound from this series demonstrated potent sleep promotion when dosed orally to EEG telemetrized rats.     

Using biogeographical history to inform conservation: the case of Preble's meadow jumping mouse

The last Pleistocene deglaciation shaped temperate and boreal communities in North America. Rapid northward expansion into high latitudes created distinctive spatial genetic patterns within species that include closely related groups of populations that are now widely spread across latitudes, while longitudinally adjacent populations, especially those near the southern periphery, often are distinctive due to long‐term disjunction. Across a spatial expanse that includes both recently colonized and long‐occupied regions, we analysed molecular variation in zapodid rodents to explore how past climate shifts influenced diversification in this group. By combining molecular analyses with species distribution modelling and tests of ecological interchangeability, we show that the lineage including the Preble's meadow jumping mouse (Zapus hudsonius preblei), a US federally listed taxon of conservation concern, is not restricted to the southern Rocky Mountains. Rather, populations along the Front Range are part of a single lineage that is ecologically indistinct and extends to the far north. Of the 21 lineages identified, this Northern lineage has the largest geographical range and low measures of intralineage genetic differentiation, consistent with recent northward expansion. Comprehensive sampling combined with coalescent‐based analyses and niche modelling leads to a radically different view of geographical structure within jumping mice and indicates the need to re‐evaluate their taxonomy and management. This analysis highlights a premise in conservation biology that biogeographical history should play a central role in establishing conservation priorities.     

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long‐lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of ‘cosmopolitan’ species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re‐instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re‐evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re‐affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.     

Variation at phenological candidate genes correlates with timing of dispersal and plumage morph in a sedentary bird of prey

Polymorphic genes involved in the conserved molecular signalling of circadian and circannual clocks may play important roles in governing the timing of breeding and dispersal and thereby affect fitness in vertebrates. However, relatively few studies have explored associations between phenological candidate genes and behaviour, and these are somewhat biased towards particular taxonomic groups such as passerine birds and salmonid fish. Consequently, we assayed microsatellite polymorphisms within the exonic and 3′ untranslated regions of the regulatory genes CLOCK, NPAS2, ADCYAP1 and CREB1 in the common buzzard (Buteo buteo), a polymorphic raptor species with three plumage morphs that differ in key life history traits including lifetime reproductive success. In contrast to studies of passerines, CLOCK poly‐glutamine (poly‐Q) was found to be monomorphic in 976 common buzzard nestlings as well as in three other Buteo species. Moreover, none of the candidate genes were significantly associated with fledging dates, although intermediately melanized females were found to lay earlier on average than light or dark morph individuals, and their offspring carried longer ADCYAP1 alleles. In contrast, all three candidate genes explained significant variation in one or more measures of juvenile buzzard dispersal (resighting probability, timing of dispersal and distance dispersed). Our findings contribute towards a broader body of work on the adaptive significance of CLOCK polymorphism, while also building upon previous studies that have documented links between ADCYAP1 variability and the timing of migration.