Structure and functional motifs of GCR1, the only plant protein with a GPCR fold?

Whether GPCRs exist in plants is a fundamental biological question. Interest in deorphanizing new G protein coupled receptors (GPCRs), arises because of their importance in signaling. Within plants, this is controversial as genome analysis has identified 56 putative GPCRs, including GCR1 which is reportedly a remote homologue to class A, B and E GPCRs. Of these, GCR2, is not a GPCR; more recently it has been proposed that none are, not even GCR1. We have addressed this disparity between genome analysis and biological evidence through a structural bioinformatics study, involving fold recognition methods, from which only GCR1 emerges as a strong candidate. To further probe GCR1, we have developed a novel helix alignment method, which has been benchmarked against the the class A – class B - class F GPCR alignments. In addition, we have presented a mutually consistent set of alignments of GCR1 homologues to class A, class B and class F GPCRs, and shown that GCR1 is closer to class A and /or class B GPCRs than class A, class B or class F GPCRs are to each other. To further probe GCR1, we have aligned transmembrane helix 3 of GCR1 to each of the 6 GPCR classes. Variability comparisons provide additional evidence that GCR1 homologues have the GPCR fold. From the alignments and a GCR1 comparative model we have identified motifs that are common to GCR1, class A, B and E GPCRs. We discuss the possibilities that emerge from this controversial evidence that GCR1 has a GPCR fol

Diffuse retro-reflective imaging for improved mosquito tracking around human baited bednets

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterisation of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues whilst requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although, noise removal algorithms are needed to reduce the effects of shot noise particularly across low intensity bednet regions. By systematically introducing mosquitoes in front and behind the bednet in lab experiments we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging setup delivers mosquito segmentation rates in excess of 90% compared to less than 70% with back-lit systems

Retinoid isomerase inhibitors impair but do not block mammalian cone photoreceptor function

Visual function in vertebrates critically depends on the continuous regeneration of visual pigments in rod and cone photoreceptors. RPE65 is a well-established retinoid isomerase in the pigment epithelium that regenerates rhodopsin during the rod visual cycle; however, its contribution to the regeneration of cone pigments remains obscure. In this study, we use potent and selective RPE65 inhibitors in rod- and cone-dominant animal models to discern the role of this enzyme in cone-mediated vision. We confirm that retinylamine and emixustat-family compounds selectively inhibit RPE65 over DES1, the putative retinoid isomerase of the intraretinal visual cycle. In vivo and ex vivo electroretinography experiments in Gnat1-/- mice demonstrate that acute administration of RPE65 inhibitors after a bleach suppresses the late, slow phase of cone dark adaptation without affecting the initial rapid portion, which reflects intraretinal visual cycle function. Acute administration of these compounds does not affect the light sensitivity of cone photoreceptors in mice during extended exposure to background light, but does slow all phases of subsequent dark recovery. We also show that cone function is only partially suppressed in cone-dominant ground squirrels and wild-type mice by multiday administration of an RPE65 inhibitor despite profound blockade of RPE65 activity. Complementary experiments in these animal models using the DES1 inhibitor fenretinide show more modest effects on cone recovery. Collectively, these studies demonstrate a role for continuous RPE65 activity in mammalian cone pigment regeneration and provide further evidence for RPE65-independent regeneration mechanisms

Matching roots to their environment

Background Plants form the base of the terrestrial food chain and provide medicines, fuel, fibre and industrial materials to humans. Vascular land plants rely on their roots to acquire the water and mineral elements necessary for their survival in nature or their yield and nutritional quality in agriculture. Major biogeochemical fluxes of all elements occur through plant roots, and the roots of agricultural crops have a significant role to play in soil sustainability, carbon sequestration, reducing emissions of greenhouse gasses, and in preventing the eutrophication of water bodies associated with the application of mineral fertilisers. ● Scope This article provides the context for a Special Issue of Annals of Botany on ‘Matching Roots to Their Environment’. It first examines how land plants and their roots evolved, describes how the ecology of roots and their rhizospheres contributes to the acquisition of soil resources, and discusses the influence of plant roots on biogeochemical cycles. It then describes the role of roots in overcoming the constraints to crop production imposed by hostile or infertile soils, illustrates root phenotypes that improve the acquisition of mineral elements and water, and discusses high-throughput methods to screen for these traits in the laboratory, glasshouse and field. Finally, it considers whether knowledge of adaptations improving the acquisition of resources in natural environments can be used to develop root systems for sustainable agriculture in the future

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis. Keywords: embryonic stem cells; human genome engineering; shelterin; telomerase telomere maintenanceNational Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869)National Institutes of Health (U.S.) (Grant RO1-HD045022

Potent and Broad Inhibition of HIV-1 by a Peptide from the gp41 Heptad Repeat-2 Domain Conjugated to the CXCR4 Amino Terminus.

HIV-1 entry can be inhibited by soluble peptides from the gp41 heptad repeat-2 (HR2) domain that interfere with formation of the 6-helix bundle during fusion. Inhibition has also been seen when these peptides are conjugated to anchoring molecules and over-expressed on the cell surface. We hypothesized that potent anti-HIV activity could be achieved if a 34 amino acid peptide from HR2 (C34) were brought to the site of virus-cell interactions by conjugation to the amino termini of HIV-1 coreceptors CCR5 or CXCR4. C34-conjugated coreceptors were expressed on the surface of T cell lines and primary CD4 T cells, retained the ability to mediate chemotaxis in response to cognate chemokines, and were highly resistant to HIV-1 utilization for entry. Notably, C34-conjugated CCR5 and CXCR4 each exhibited potent and broad inhibition of HIV-1 isolates from diverse clades irrespective of tropism (i.e., each could inhibit R5, X4 and dual-tropic isolates). This inhibition was highly specific and dependent on positioning of the peptide, as HIV-1 infection was poorly inhibited when C34 was conjugated to the amino terminus of CD4. C34-conjugated coreceptors could also inhibit HIV-1 isolates that were resistant to the soluble HR2 peptide inhibitor, enfuvirtide. When introduced into primary cells, CD4 T cells expressing C34-conjugated coreceptors exhibited physiologic responses to T cell activation while inhibiting diverse HIV-1 isolates, and cells containing C34-conjugated CXCR4 expanded during HIV-1 infection in vitro and in a humanized mouse model. Notably, the C34-conjugated peptide exerted greater HIV-1 inhibition when conjugated to CXCR4 than to CCR5. Thus, antiviral effects of HR2 peptides can be specifically directed to the site of viral entry where they provide potent and broad inhibition of HIV-1. This approach to engineer HIV-1 resistance in functional CD4 T cells may provide a novel cell-based therapeutic for controlling HIV infection in humans

The role of discharge variability in determining alluvial stratigraphy

We illustrate the potential for using physics-based modeling to link alluvial stratigraphy to large river morphology and dynamics. Model simulations, validated using ground penetrating radar data from the Río Paraná, Argentina, demonstrate a strong relationship between bar-scale set thickness and channel depth, which applies across a wide range of river patterns and bar types. We show that hydrologic regime, indexed by discharge variability and flood duration, exerts a first-order influence on morphodynamics and hence bar set thickness, and that planform morphology alone may be a misleading variable for interpreting deposits. Indeed, our results illustrate that rivers evolving under contrasting hydrologic regimes may have very similar morphology, yet be characterized by marked differences in stratigraphy. This realization represents an important limitation on the application of established theory that links river topography to alluvial deposits, and highlights the need to obtain field evidence of discharge variability when developing paleoenvironmental reconstructions. Model simulations demonstrate the potential for deriving such evidence using metrics of paleocurrent variance