Heterologous production of the insecticidal pea seed albumin PA1 protein by Pichia pastoris and protein engineering to potentiate aphicidal activity via fusion to snowdrop lectin Galanthus nivalis agglutinin; GNA)

BackgroundNew bioinsecticides with novel modes of action are urgently needed to minimise the environmental and safety hazards associated with the use of synthetic chemical pesticides and to combat growing levels of pesticide resistance. The pea seed albumin PA1b knottin peptide is the only known proteinaceous inhibitor of insect vacuolar adenosine triphosphatase (V-ATPase) rotary proton pumps. Oral toxicity towards insect pests and an absence of activity towards mammals makes Pa1b an attractive candidate for development as a bioinsecticide. The purpose of this study was to investigate if Pichia pastoris could be used to express a functional PA1b peptide and if it’s insecticidal activity could be enhanced via engineering to produce a fusion protein comprising the pea albumin protein fused to the mannose-specific snowdrop lectin (Galanthus nivalis agglutinin; GNA).ResultsWe report the production of a recombinant full-length pea albumin protein (designated PAF) and a fusion protein (PAF/GNA) comprised of PAF fused to the N-terminus of GNA in the yeast Pichia pastoris. PAF was orally toxic to pea (Acyrthosiphon pisum) and peach potato (Myzus persicae) aphids with respective, Day 5 LC50 values of 54 µM and 105 µM derived from dose–response assays. PAF/GNA was significantly more orally toxic as compared to PAF, with LC50 values tenfold (5 µM) and 3.3-fold (32 µM) lower for pea and peach potato aphids, respectively. By contrast, no phenotypic effects were observed for worker bumble bees (Bombus terristrus) fed PAF, GNA or PAF/GNA in acute toxicity assays. Confocal microscopy of pea aphid guts after pulse-chase feeding fluorescently labelled proteins provides evidence that enhanced efficacy of the fusion protein is attributable to localisation and retention of PAF/GNA to the gut epithelium. In contact assays the fusion protein was also found to be significantly more toxic towards A. pisum as compared to PAF, GNA or a combination of the two proteins.ConclusionsOur results suggest that GNA mediated binding to V-type ATPase pumps acts to potentiate the oral and contact aphicidal activity of PAF. This work highlights potential for the future commercial development of plant protein-based bioinsecticides that offer enhanced target specificity as compared to chemical pesticides, and compatibility with integrated pest management strategies

Antiparasitic and Antiproliferative Effects of Indoleamine 2,3-dioxygenase Enzyme Expression in Human Fibroblasts.

Studies were carried out to evaluate the proposed role of indoleamine 2,3-dioxygenase (INDO) induction in the antimicrobial and antiproliferative effects of gamma interferon (IFN-gamma) in human fibroblasts. The INDO cDNA coding region was cloned in the pMEP4 expression vector, containing the metallothionein (MTII) promoter in the sense (+ve) or the antisense (-ve) orientation. Human fibroblasts (GM637) stably transfected with the sense construct expressed INDO activity after treatment with CdCl2 or ZnSO4, but cells transfected with the antisense construct did not. The growth of Chlamydia psittaci was strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+ or Zn2+. The inhibition correlated with the level of INDO activity induced and could be reversed by the addition of excess tryptophan to the medium. The growth of Toxoplasma gondii was also strongly inhibited in INDO +ve cells but not in INDO -ve cells after treatment with Cd2+. Expression of Cd(2+)-induced INDO activity also inhibited thymidine incorporation and led to cytotoxicity in INDO +ve cells but not in INDO -ve cells. Thus, the induction of INDO activity by IFN-gamma may be an important factor in the antimicrobial and antiproliferative effects of IFN-gamma in human fibroblasts

p63 Mediates an Apoptotic Response to Pharmacological and Disease-Related ER Stress in the Developing Epidermis

SummaryEndoplasmic reticulum (ER) stress triggers tissue-specific responses that culminate in either cellular adaptation or apoptosis, but the genetic networks distinguishing these responses are not well understood. Here we demonstrate that ER stress induced in the developing zebrafish causes rapid apoptosis in the brain, spinal cord, tail epidermis, lens, and epiphysis. Focusing on the tail epidermis, we uncover an apoptotic response that depends on Puma, but not on p53 or Chop. puma is transcriptionally activated during this ER stress response in a p53-independent manner, and is an essential mediator of epidermal apoptosis. We demonstrate that the p63 transcription factor is upregulated to initiate this apoptotic pathway and directly activates puma transcription in response to ER stress. We also show that a mutation of human Connexin 31, which causes erythrokeratoderma variabilis, induces ER stress and p63-dependent epidermal apoptosis in the zebrafish embryo, thus implicating this pathway in the pathogenesis of inherited disease

Optics of Nonuniformly Moving Media

A moving dielectric appears to light as an effective gravitational field. At low flow velocities the dielectric acts on light in the same way as a magnetic field acts on a charged matter wave. We develop in detail the geometrical optics of moving dispersionless media. We derive a Hamiltonian and a Lagrangian to describe ray propagation. We elucidate how the gravitational and the magnetic model of light propagation are related to each other. Finally, we study light propagation around a vortex flow. The vortex shows an optical Aharonov--Bohm effect at large distances from the core, and, at shorter ranges, the vortex may resemble an optical black hole.Comment: Physical Review A (submitted

Transforming growth factor- directly induces p53-up-regulated modulator of apoptosis (PUMA) during the rapid induction of apoptosis in myc-driven B-cell lymphomas

Background: TGF-β induces apoptosis in Burkitt's lymphoma cells. Results: PUMA is a direct target gene of TGF-β signaling and is required for rapid apoptosis. Conclusion: TGF-β-mediated direct induction of PUMA contributes to apoptosis in human and murine c-Myc-driven lymphomas. Significance: These studies link TGF-β signaling and transcriptional activation of PUMA, two factors with critical roles in regulating B-cell survival

Development of a Management Algorithm for Post-operative Pain (MAPP) after total knee and total hip replacement: study rationale and design.

BACKGROUND: Evidence from clinical practice and the extant literature suggests that post-operative pain assessment and treatment is often suboptimal. Poor pain management is likely to persist until pain management practices become consistent with guidelines developed from the best available scientific evidence. This work will address the priority in healthcare of improving the quality of pain management by standardising evidence-based care processes through the incorporation of an algorithm derived from best evidence into clinical practice. In this paper, the methodology for the creation and implementation of such an algorithm that will focus, in the first instance, on patients who have undergone total hip or knee replacement is described. METHODS: In partnership with clinicians, and based on best available evidence, the aim of the Management Algorithm for Post-operative Pain (MAPP) project is to develop, implement, and evaluate an algorithm designed to support pain management decision-making for patients after orthopaedic surgery. The algorithm will provide guidance for the prescription and administration of multimodal analgesics in the post-operative period, and the treatment of breakthrough pain. The MAPP project is a multisite study with one coordinating hospital and two supporting (rollout) hospitals. The design of this project is a pre-implementation-post-implementation evaluation and will be conducted over three phases. The Promoting Action on Research Implementation in Health Services (PARiHS) framework will be used to guide implementation. Outcome measurements will be taken 10 weeks post-implementation of the MAPP. The primary outcomes are: proportion of patients prescribed multimodal analgesics in accordance with the MAPP; and proportion of patients with moderate to severe pain intensity at rest. These data will be compared to the pre-implementation analgesic prescribing practices and pain outcome measures. A secondary outcome, the efficacy of the MAPP, will be measured by comparing pain intensity scores of patients where the MAPP guidelines were or were not followed. DISCUSSION: The outcomes of this study have relevance for nursing and medical professionals as well as informing health service evaluation. In establishing a framework for the sustainable implementation and evaluation of a standardised approach to post-operative pain management, the findings have implications for clinicians and patients within multiple surgical contexts

Overexpression of the aphid-induced serine protease inhibitor <i>CI2c </i>gene in barley affects the generalist green peach aphid, not the specialist bird cherry-oat aphid

<div><p>Aphids are serious pests in crop plants. In an effort to identify plant genes controlling resistance against aphids, we have here studied a protease inhibitor, CI2c in barley (<i>Hordeum vulgare</i> L.). The <i>CI2c</i> gene was earlier shown to be upregulated by herbivory of the bird cherry-oat aphid <i>(Rhopalosiphum padi</i> L.<i>)</i> in barley genotypes with moderate resistance against this aphid, but not in susceptible lines. We hypothesized that CI2c contributes to the resistance. To test this idea, cDNA encoding <i>CI2c</i> was overexpressed in barley and bioassays were carried out with <i>R</i>. <i>padi</i>. For comparison, tests were carried out with the green peach aphid (<i>Myzus persicae</i> Sulzer), for which barley is a poor host. The performance of <i>R</i>. <i>padi</i> was not different on the <i>CI2c</i>-overexpressing lines in comparison to controls in test monitoring behavior and fecundity. <i>M</i>. <i>persicae</i> preference was affected as shown in the choice test, this species moved away from control plants, but remained on the <i>CI2c</i>-overexpressing lines. <i>R</i>. <i>padi</i>-induced responses related to defense were repressed in the overexpressing lines as compared to in control plants or the moderately resistant genotypes. A putative susceptibility gene, coding for a β-1,3-glucanase was more strongly induced by aphids in one of the <i>CI2c</i>-overexpressing lines. The results indicate that the CI2c inhibitor in overexpressing lines affects aphid-induced responses by suppressing defense. This is of little consequence to the specialist <i>R</i>.<i>padi</i>, but causes lower non-host resistance towards the generalist <i>M</i>. <i>persicae</i> in barley.</p></div

SNW1 Is a Critical Regulator of Spatial BMP Activity, Neural Plate Border Formation, and Neural Crest Specification in Vertebrate Embryos

In frog and fish embryos, SNW1 is a protein required for the spatio-temporal activity of BMP signaling necessary for neural plate border formation and specification of neural crest tissue

Identification of Early Requirements for Preplacodal Ectoderm and Sensory Organ Development

Preplacodal ectoderm arises near the end of gastrulation as a narrow band of cells surrounding the anterior neural plate. This domain later resolves into discrete cranial placodes that, together with neural crest, produce paired sensory structures of the head. Unlike the better-characterized neural crest, little is known about early regulation of preplacodal development. Classical models of ectodermal patterning posit that preplacodal identity is specified by readout of a discrete level of Bmp signaling along a DV gradient. More recent studies indicate that Bmp-antagonists are critical for promoting preplacodal development. However, it is unclear whether Bmp-antagonists establish the proper level of Bmp signaling within a morphogen gradient or, alternatively, block Bmp altogether. To begin addressing these issues, we treated zebrafish embryos with a pharmacological inhibitor of Bmp, sometimes combined with heat shock-induction of Chordin and dominant-negative Bmp receptor, to fully block Bmp signaling at various developmental stages. We find that preplacodal development occurs in two phases with opposing Bmp requirements. Initially, Bmp is required before gastrulation to co-induce four transcription factors, Tfap2a, Tfap2c, Foxi1, and Gata3, which establish preplacodal competence throughout the nonneural ectoderm. Subsequently, Bmp must be fully blocked in late gastrulation by dorsally expressed Bmp-antagonists, together with dorsally expressed Fgf and Pdgf, to specify preplacodal identity within competent cells abutting the neural plate. Localized ventral misexpression of Fgf8 and Chordin can activate ectopic preplacodal development anywhere within the zone of competence, whereas dorsal misexpression of one or more competence factors can activate ectopic preplacodal development in the neural plate. Conversely, morpholino-knockdown of competence factors specifically ablates preplacodal development. Our work supports a relatively simple two-step model that traces regulation of preplacodal development to late blastula stage, resolves two distinct phases of Bmp dependence, and identifies the main factors required for preplacodal competence and specification