Structure-based tailoring of compound libraries for high-throughput screening: discovery of novel EphB4 kinase inhibitors

High-throughput docking is a computational tool frequently used to discover small-molecule inhibitors of enzymes or receptors of known three-dimensional structure. Because of the large number of molecules in chemical libraries, automatic procedures to prune multimillion compound collections are useful for high-throughput docking and necessary for in vitro screening. Here, we propose an anchor-based library tailoring approach (termed ALTA) to focus a chemical library by docking and prioritizing molecular fragments according to their binding energy which includes continuum electrostatics solvation. In principle, ALTA does not require prior knowledge of known inhibitors, but receptor-based pharmacophore information (hydrogen bonds with the hinge region) is additionally used here to identify molecules with optimal anchor fragments for the ATP-binding site of the EphB4 receptor tyrosine kinase. The 21,418 molecules of the focused library (from an initial collection of about 730,000) are docked into EphB4 and ranked by force-field-based energy including electrostatic solvation. Among the 43 compounds tested in vitro, eight molecules originating from two different anchors show low-micromolar activity in a fluorescence-based enzymatic assay. Four of them are active in a cell-based assay and are potential anti-angiogenic compounds.     

A role for fibronectin-leucine-rich transmembrane cell-surface proteins in homotypic cell adhesion

The fibronectin-leucine-rich transmembrane (FLRT) family of leucine-rich repeat (LRR) proteins is implicated in fibroblast growth factor (FGF) signalling, early embryonic development and neurite outgrowth. Here, we have analysed whether FLRTs may also function in cell adhesion. We find that FLRT proteins can physically interact and that FLRT-transfected cultured cells sort out from non-transfected cells, suggesting a change in adhesive properties. A similar sorting effect is also observed in Xenopus embryos and tissue aggregates. FLRT-mediated cell sorting is calcium dependent and substrate independent. Deletion analysis indicates that cell sorting requires the LRR domains, which are dispensable for FLRT-mediated FGF signalling. Conversely, sorting is independent of the cytoplasmic domain, which is essential for FLRT-induced signalling. Therefore, FLRT-mediated FGF signal transduction and homotypic cell sorting can be molecularly uncoupled. The results indicate that FLRT proteins have a dual role, promoting FGF signalling and modulating homotypic cell adhesion.     

Familiar soil conditions help Pinus ponderosa seedlings cope with warming and drying climate

Changes in temperature and moisture as a result of climate forcing can impact performance of planted trees. Tree performance may also be sensitive to new soil conditions, for example, brought about by seeds germinating in soils different from those colonized by ancestral populations. Such “edaphic constraint” may occur with natural migration or human‐assisted movement. Pinus ponderosa seedlings, sourced from one location (“home” site), were grown across a field environmental gradient in either their original home soil or in soils from two different “away” sites. Seedlings were inoculated with site‐specific soil organisms by germinating seeds in living soil. After 6 months, the inoculated seedlings were transplanted into sterilized soils from the home or away sites. This experimental design allowed us to uncouple the importance of abiotic and biotic soil properties and test (1) how biotic and abiotic soil properties interact with climate to influence plant growth and stress tolerance, and (2) the role of soil biota in facilitating growth in novel environments. Seedlings grew least in hotter and drier away sites with away soil biota. Home soil biota ameliorated negative impacts on growth of hotter and drier away sites. Measurements of photosynthetic rate, stomatal conductance, and chlorophyll florescence (Fv/Fm) suggest that edaphic constraint reduced growth by increasing tree water stress. Results suggest that success of Ponderosa pine plantings into warming environments will be enhanced by pre‐inoculation with native soil biota of the seed source.     

Terrestrial carbon balance in a drier world: the effects of water availability in southwestern North America

Global modeling efforts indicate semiarid regions dominate the increasing trend and interannual variation of net CO₂ exchange with the atmosphere, mainly driven by water availability. Many semiarid regions are expected to undergo climatic drying, but the impacts on net CO₂ exchange are poorly understood due to limited semiarid flux observations. Here we evaluated 121 site‐years of annual eddy covariance measurements of net and gross CO₂ exchange (photosynthesis and respiration), precipitation, and evapotranspiration (ET) in 21 semiarid North American ecosystems with an observed range of 100 – 1000 mm in annual precipitation and records of 4–9 years each. In addition to evaluating spatial relationships among CO₂ and water fluxes across sites, we separately quantified site‐level temporal relationships, representing sensitivity to interannual variation. Across the climatic and ecological gradient, photosynthesis showed a saturating spatial relationship to precipitation, whereas the photosynthesis–ET relationship was linear, suggesting ET was a better proxy for water available to drive CO₂ exchanges after hydrologic losses. Both photosynthesis and respiration showed similar site‐level sensitivity to interannual changes in ET among the 21 ecosystems. Furthermore, these temporal relationships were not different from the spatial relationships of long‐term mean CO₂ exchanges with climatic ET. Consequently, a hypothetical 100‐mm change in ET, whether short term or long term, was predicted to alter net ecosystem production (NEP) by 64 gCm^?2 yr^?1. Most of the unexplained NEP variability was related to persistent, site‐specific function, suggesting prioritization of research on slow‐changing controls. Common temporal and spatial sensitivity to water availability increases our confidence that site‐level responses to interannual weather can be extrapolated for prediction of CO₂ exchanges over decadal and longer timescales relevant to societal response to climate change.     

Thallium ions can replace both sodium and potassium ions in the glutamate transporter excitatory amino acid carrier 1

The excitatory amino acid carrier EAAC1 belongs to a family of glutamate transporters that use the electrochemical transmembrane gradients of sodium and potassium to mediate uphill transport of glutamate into the cell. While the sites of cation interaction with EAAC1 are unknown, two cation binding sites were observed in the crystal structure of the bacterial glutamate transporter homologue GltPh. Although occupied by Tl(+) in the crystal structure, these sites were proposed to be Na(+) binding sites. Therefore, we tested whether Tl(+) has the ability to replace Na(+) also in the mammalian transporters. Our data demonstrate that Tl(+) can bind to EAAC1 with high affinity and mediate a host of different functions. Tl(+) can functionally replace potassium when applied to the cytoplasm and can support glutamate transport current. When applied extracellularly, Tl(+) induces some behavior that mimics that of the Na(+)-bound transporter, such as activation of the cation-induced anion conductance and creation of a substrate binding site, but it cannot replace Na(+) in supporting glutamate transport current. Moreover, our data show a differential effect of mutations to two acidic amino acids potentially involved in cation binding (D367 and D454) on Na(+) and Tl(+) affinity. Overall, our results demonstrate that the ability of the glutamate transporters to interact with Tl(+) is conserved between GltPh and a mammalian member of the transporter family. However, in contrast to GltPh, which does not bind K(+), Tl(+) is more efficient in mimicking K(+) than Na(+) when interacting with the mammalian protein.     

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.     

Organization of the outer plexiform layer of the primate retina: electron microscopy of Golgi-impregnated cells

Golgi-impregnated retinae of rhesus monkeys have been examined by serial section electron microscopy to establish in a quantitative manner the neural connexions in the outer plexiform layer. The results have shown that there are two types of midget bipolar cell, here called the invaginating midget bipolar and the flat midget bipolar. Both types of midget bipolar are exclusive to a single cone. The invaginating midget bipolar has been found to fit a dendritic terminal process into every invagination in the cone pedicle base. The flat midget bipolar has dendritic terminals that make superficial contact on the cone pedicle base. There are twice as many dendritic terminals and points of contact with the cone pedicle on a flat midget bipolar top as compared with an invaginating midget bipolar top. These observations, together with light microscope counts of the numbers of the two types of midget bipolars, suggest that there are two midget bipolars per cone. The diffuse cone bipolar (the flat bipolar) also makes superficial contacts on the cone pedicle base, and serial sections have shown that a flat bipolar contacts about six cones. Rod bipolars connect exclusively to rods and their dendritic terminals always end as one of the central processes that penetrate the invagination. Horizontal cell dendrites end exclusively in cone pedicles and their axon terminals end in rod spherules. The point of contact with both the types of receptor is as the lateral elements of the invaginations. A single small horizontal cell contacts about seven cones and a large horizontal cell contacts about twelve cones. The numbers of contacts per cone pedicle decrease from the centre to the periphery of the horizontal cell's dendritic field, suggesting there is an overlap of four to six horizontal cells onto a single cone pedicle. The horizontal cell axon terminals are too numerous to assess in absolute numbers but there is only one terminal to a given rod spherule from any particular axon.