Heat Stress Triggers Differential Protein Accumulation in the Extracellular Matrix of Sorghum Cell Suspension Cultures

Plants reprogram gene expression as an adaptive response to survive high temperatures. While the identity and functions of intracellular heat stress-responsive proteins have been extensively studied, the heat response of proteins secreted to the extracellular matrix is unknown. Here, we used Sorghum bicolor, a species adapted for growth in hot climates, to investigate the extracellular heat-induced responses. When exposed to 40 C for 72 h, heat-sensitive Arabidopsis cell suspension cultures died, while ICSB338 sorghum cell cultures survived by activation of a transcriptional response characterized by the induction of HSP70 and HSP90 genes. Quantitative proteomic analysis of proteins recovered from cell culture medium revealed specific heat stress-induced protein accumulation within the sorghum secretome. Of the 265 secreted proteins identified, 31 responded to heat (2-fold change), with 84% possessing a predicted signal peptide for targeting to the classical secretory pathway. The differentially accumulated proteins have putative functions in metabolism, detoxification, and protein modifications. A germin (SORBI_3003G427700) was highly heat-inducible at both protein and gene level. Overall, our study reveals new insights into sorghum responses to heat and provides a useful resource of extracellular proteins that could serve as targets for developing thermotolerant crops. Data are available via ProteomeXchange with identifier PXD021536

Canonical Quantization of the Electromagnetic Field on the Kerr Background

We investigate the canonical quantization of the electromagnetic field on the Kerr background. We give new expressions for the expectation value of the electromagnetic stress-energy tensor in various vacua states and give a physical interpretation of the separate terms appearing in them. We numerically calculate the luminosity in these states. We also study the form of the renormalized stress-energy tensor close to the horizon when the electromagnetic field is in the past Boulware state.Comment: 27 zipped, postscript figure file

Resource use data by patient report or hospital records: Do they agree?

Background: Economic evaluations alongside clinical trials are becoming increasingly common. Cost data are often collected through the use of postal questionnaires; however, the accuracy of this method is uncertain. We compared postal questionnaires with hospital records for collecting data on physiotherapy service use. Methods: As part of a randomised trial of orthopaedic medicine compared with orthopaedic surgery we collected physiotherapy use data on a group of patients from retrospective postal questionnaires and from hospital records. Results: 315 patients were referred for physiotherapy. Hospital data on attendances was available for 30% (n = 96), compared with 48% (n = 150) of patients completing questionnaire data (95% Cl for difference = 10% to 24%); 19% (n = 59) had data available from both sources. The two methods produced an intraclass correlation coefficient of 0.54 (95% Cl 0.31 to 0.70). However, the two methods produced significantly different estimates of resource use with patient self report recalling a mean of 1.3 extra visits (95% Cl 0.4 to 2.2) compared with hospital records. Conclusions: Using questionnaires in this study produced data on a greater number of patients compared with examination of hospital records. However, the two data sources did differ in the quantity of physiotherapy used and this should be taken into account in any analysi

Exogenous abscisic acid treatment regulates protein secretion in sorghum cell suspension cultures.

Drought stress adversely affects plant growth, often leading to total crop failure. Upon sensing soil water deficits, plants switch on biosynthesis of abscisic acid (ABA), a stress hormone for drought adaptation. Here, we used exogenous ABA application to dark-grown sorghum cell suspension cultures as an experimental system to understand how a drought-tolerant crop responds to ABA. We evaluated intracellular and secreted proteins using isobaric tags for relative and absolute quantification. While the abundance of only ~ 7% (46 proteins) intracellular proteins changed in response to ABA, ~32% (82 proteins) of secreted proteins identified in this study were ABA responsive. This shows that the extracellular matrix is disproportionately targeted and suggests it plays a vital role in sorghum adaptation to drought. Extracellular proteins responsive to ABA were predominantly defense/detoxification and cell wall-modifying enzymes. We confirmed that sorghum plants exposed to drought stress activate genes encoding the same proteins identified in the cell culture system with ABA. Our results suggest that ABA activates defense and cell wall remodeling systems during stress response. This could underpin the success of sorghum adaptation to drought stress

Isolation of Arabidopsis extracellular ATP‐binding proteins by affinity proteomics and identification of PHOSPHOLIPASE C‐LIKE 1 as an extracellular protein essential for fumonisin B1 toxicity

ATP is secreted to the extracellular matrix where it activates plasma membrane receptors for controlling plant growth and stress‐adaptive processes. DOES NOT RESPOND TO NUCLEOTIDES 1 (DORN1), the first plant ATP receptor was identified, but key downstream proteins are sought after. Here, we identified 120 proteins secreted by Arabidopsis cell cultures and screened them for putative stress‐responsive proteins using ATP‐affinity purification. We report three Arabidopsis proteins isolated by ATP‐affinity: PEROXIDASE 52, SUBTILASE‐LIKE SERINE PROTEASE 1.7, and PHOSPHOLIPASE C‐LIKE 1. In wildtype Arabidopsis, expression of genes encoding all three proteins responded to fumonisin B1, a cell death‐activating mycotoxin. Expression of PEROXIDASE 52 and PHOSPHOLIPASE C‐LIKE 1 genes was altered in fumonisin B1‐resistant salicylic acid induction‐deficient (sid2) mutants. Exposure to fumonisin B1 suppressed PHOSPHOLIPASE C‐LIKE 1 expression in sid2 mutants, suggesting that inactivation of this gene might provide mycotoxin tolerance. Accordingly, gene knockout mutants of PHOSPHOLIPASE C‐LIKE 1 were resistant to fumonisin B1‐induced death. Activation of PHOSPHOLIPASE C‐LIKE 1 gene expression by exogenous ATP was not blocked in dorn1 loss‐of‐function mutants, indicating that DORN1 is not required. Furthermore, exogenous ATP rescued both wildtype and dorn1 mutants from fumonisin B1 toxicity, suggesting that different ATP receptor(s) are operational in this process. Our results point to the existence of additional plant ATP receptor(s) and provide crucial downstream targets for use in designing screens to identify these receptors. Finally, PHOSPHOLIPASE C‐LIKE 1 serves as a convergence point for fumonisin B1 and extracellular ATP signalling, and functions in Arabidopsis stress response to fumonisin B1

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

An investigation of three-dimensional scanning of human body surfaces and its use in the design and manufacture of prostheses

The capture of highly accurate data describing the complex surfaces of the human body may prove extremely useful in many medical situations. The data provide a method of measuring and recording changes to the surface of a patient's soft tissue. The data may be applied to computer-controlled manufacturing techniques, such as rapid prototyping (RP). This enables accurate physical replicas of the patient topography to be produced. Such models may be used as an aid in the design and manufacture of prostheses. This paper describes an investigation aimed at identifying problems that may be encountered when scanning patients and describes the application of the resulting data in the design and manufacture of facial prostheses. The results of the experiment are presented together with a discussion of the accuracy and potential advantages afforded by this approach

GALA: an international multicentre randomised trial comparing general anaesthesia versus local anaesthesia for carotid surgery

Background: Patients who have severe narrowing at or near the origin of the internal carotid artery as a result of atherosclerosis have a high risk of ischaemic stroke ipsilateral to the arterial lesion. Previous trials have shown that carotid endarterectomy improves long-term outcomes, particularly when performed soon after a prior transient ischaemic attack or mild ischaemic stroke. However, complications may occur during or soon after surgery, the most serious of which is stroke, which can be fatal. It has been suggested that performing the operation under local anaesthesia, rather than general anaesthesia, may be safer. Therefore, a prospective, randomised trial of local versus general anaesthesia for carotid endarterectomy was proposed to determine whether type of anaesthesia influences peri-operative morbidity and mortality, quality of life and longer term outcome in terms of stroke-free survival. Methods/design: A two-arm, parallel group, multicentre randomised controlled trial with a recruitment target of 5000 patients. For entry into the study, in the opinion of the responsible clinician, the patient requiring an endarterectomy must be suitable for either local or general anaesthesia, and have no clear indication for either type. All patients with symptomatic or asymptomatic internal carotid stenosis for whom open surgery is advised are eligible. There is no upper age limit. Exclusion criteria are: no informed consent; definite preference for local or general anaesthetic by the clinician or patient; patient unlikely to be able to co-operate with awake testing during local anaesthesia; patient requiring simultaneous bilateral carotid endarterectomy; carotid endarterectomy combined with another operation such as coronary bypass surgery; and, the patient has been randomised into the trial previously. Patients are randomised to local or general anaesthesia by the central trial office. The primary outcome is the proportion of patients alive, stroke free ( including retinal infarction) and without myocardial infarction 30 days post-surgery. Secondary outcomes include the proportion of patients alive and stroke free at one year; health related quality of life at 30 days; surgical adverse events, re-operation and re-admission rates; the relative cost of the two methods of anaesthesia; length of stay and intensive and high dependency bed occupancy

Discovery of a meteoritic ejecta layer containing unmelted impactor fragments at the base of Paleocene lavas, Isle of Skye, Scotland

Evidence for meteorite impacts in the geological record may include the presence of shocked minerals, spherule layers, and geochemical anomalies. However, it is highly unusual to nd unmelted crystals from the actual impactor within an ejecta layer. Here we detail the rst recorded occurrence of vanadium-rich osbornite (TiVN) on Earth, from two sites on Skye, northwest Scotland, which are interpreted as part of a meteoritic ejecta layer. TiVN has only previously been reported as dust from comet Wild 2, but on Skye it has been identi ed as an unmelted phase. Both ejecta layer sites also contain niobium-rich osbornite (TiNbN), which has not previously been reported. An extraterrestrial origin for these deposits is strongly supported by the presence of reidite (a high-pressure zircon polymorph), which is only found naturally at sites of meteorite impact. Barringerite [(Fe,Ni)2P], baddeleyite (ZrO2), alabandite (MnS), and carbon-bearing native iron spherules, together with planar deformation features and diaplectic glass in quartz, further support this thesis. We demonstrate through eld relationships and Ar-Ar dating that the meteorite strike occurred during the mid-Paleocene. This is the rst recorded mid-Paleocene impact event in the region and is coincident with the onset of magmatism in the British Palaeogene Igneous Province (BPIP). The Skye ejecta layer deposits provoke important questions regarding their lateral extent at the base of the BPIP and the possibility of their presence elsewhere beneath the much larger North Atlantic Igneous Province