Transgenic expression of antimicrobial peptides from insects as a tool for analysis of compatibility between plants and pathogens

Genetic engineering has proven to be a powerful tool for controlling plant diseases and to be an alternative to economically costly and environmentally undesirable chemical control. One promising approach to achieve enhanced disease-resistance has been through the expression of genes encoding antimicrobial peptides (AMPs) in transgenic plants. Hence, this study aimed to investigate the feasibility of using the novel insect AMP EtDef, a defensin from drone fly Eristalis tenax and the well-known AMP thanatin from spined soldier bug Podisus maculiventris to engineer disease resistance in the model plant Arabidopsis. A prerequisite for the utilization of these peptides is a precise knowledge about their biological activity. Thus, in vitro antifungal activity of the chemically synthesized EtDef and thanatin was evaluated against the devastating phytopathogens F. culmorum, B. cinerea and P. parasitica using spore germination inhibition assays. Results of these assays revealed that synthetic EtDef led to total inhibition of spore germination and mycelial growth of all tested fungi, with minimum inhibitory concentrations (MICs) varying between 1 – 2 for B. cinerea and 5 – 10 µM for F. culmorum and P. parasitica. Synthetic thanatin showed higher efficacy as compared to EtDef regarding inhibitory effects. There the MICs ranged between 0.5 – 1 µM for B. cinerea, 5 – 10 µM for F. culmorum, and 2 – 5 µM for P. parasitica. Concomitantly, a protocol for the production of recombinant EtDef in E. coli expression system was established by inserting the sequence for mature EtDef peptide in frame downstream of the multiple tag TrxA - His -S of pET32a(+) vector. The resulting recombinant THS-EtDef protein was then refolded and its in vitro biological activity was evaluated against B. cinerea using spore germination inhibition assay. It was observed that THS-EtDef showed also a similar antifungal activity to the chemically synthesized counterpart, with IC50 occurred at 0.5 µM. This indicates that the presence of the tag, which is bigger than the AMP peptide, didn´t much alter the activity of EtDef in vitro. Because of their promising antimicrobial properties, EtDef (with its putative signal peptide) and the chimeric thanatin (containing HvChi26 signal peptide) were introduced into Arabidopsis via Agrobacterium-mediated transformation and expressed under the control of the constitutive CaMV35S promoter. Molecular characterization analysis revealed that both EtDef and thanatin genes were efficiently transcribed into mRNA, although the levels of expression varied among transformants. Due to the signal peptides both AMPs are thought to enter the secretory pathway. Therefor intercellular washing fluids (IWFs) from individual transgenic plants expressing either EtDef or thanatin were isolated. Spore germination of B. cinerea was inhibited to various degrees, indicating that the expression of these peptides was functional and localized to extracellular space in all transgenic lines tested. The degree of resistance achieved by expressing either EtDef or thanatin were then evaluated in planta against the fungal pathogens G. orontii and B. cinerea and the bacterial pathogen P. syringae pv. tomato strain DC3000. EtDef and thanatin transgenic Arabidopsis plants displayed remarkably reduced conidial sporulation, hyphal spread and proliferation of G. orontii on the rosette leaves, mediating enhanced disease resistance in these plants. This suppressive effect against G. orontii was correlated with RNA expression level. Three independent EtDef transgenic lines namely 395, 396, and 405 and thanatin transgenic lines 407, 410, and 411 showed high RNA expression levels, and correspondingly high resistance degree to G. orontii. In contrast, transgenic expression of EtDef and thanatin in Arabidopsis was clearly less active against B. cinerea. Nevertheless, two EtDef transgenic lines 396 and 405 and two thanatin transgenic lines 410 and 411 were consistently more resistant against B. cinerea. When challenged with Pst, all transgenic EtDef lines were as sensitive as the control plants, except for transgenic line 405. Transgenic expression of thanatin in Arabidopsis could provide, however, a higher degree of resistance against Pst. Thanatin transgenic lines 407, 410, and 411, showed the highest resistance to Pst. In summary, plants of the EtDef transgenic lines 395, 396, 398 and 405 and those of Thanatin 407, 410 and 411 seem to be promising candidates to evaluate their potential in planta against other phytopathogens. Finally, data presented here indicate that transgenic expression of EtDef and Thanatin could be utilized to improve disease resistance of other economically important crops.Gentechnische Methoden haben sich als wichtiges Werkzeug zur Kontrolle von Pflanzenkrankheiten erwiesen und bilden eine Alternative zum kostenintensiven und ökologisch unerwünschten Einsatz von Chemikalien. Als viel versprechender Ansatz zur Steigerung der Resistenz gegen Krankheiten hat sich die Expression von Genen in transgenen Pflanzen erwiesen, die für antimikrobielle Peptide (AMPs) kodieren. Ziel der hier vorgestellten Studie war es daher zu untersuchen, inwieweit sich EtDef, ein neues Peptid aus der Schwebfliege Eristalis tenax, und das gut untersuchte AMP Thanatin aus der Raubwanze Podisus maculiventris zur Erhöhung der Krankheitsresistenz der Modellpflanze Arabidopsis thaliana nutzen lassen. Voraussetzung für den Einsatz dieser Peptide ist ein präzises Wissen um deren biologische Wirkung. Aus diesem Grund wurde zu Beginn in Sporenkeimungstests die antimykotische Wirkung von synthetisch hergestelltem EtDef und Thanatin auf die phytopathogenen Pilze F. culmorum, B. cinerea und P. parasitica untersucht. Synthetisches EtDef führte hierbei zu einer vollständigen Inhibierung der Sporenkeimung und des Myzelwachstums bei allen getesteten Pilzen mit miminimalen Hemm-Konzentrationen (MHK) von 1 – 2 µM für B. cinerea und 5 – 10 µM für F. culmorum und P. parasitica. Für synthetisches Thanatin wurde eine größere inhibitorische Wirksamkeit als für EtDef beobachtet. Die minimalen Konzentrationen zur vollständigen Hemmung lagen hier bei 0,5 – 1 µM für B. cinerea, 5 – 10 µM für F. culmorum und 2 – 5 µM für P. parasitica. Parallel zu diesen Experimenten wurde ein Protokoll zur Produktion von rekombinantem EtDef in E. coli etabliert. Hierzu wurde die Sequenz des EtDef Peptids in frame abwärts des TrxA - His – S Tags des pET32a(+) Vektors inseriert. Die biologische Aktivität des hergestellten THS-EtDef Proteins wurde in vitro überprüft, wofür wiederum die Inhibierung der Sporenkeimung bei B. cinerea untersucht wurde. Es konnte eine ähnliche antimykotische Wirkung für THS-EtDef wie bei synthetischem EtDef gezeigt werden. Das deutet darauf hin, dass die Aktivität von THS-EtDef in vitro nur gering durch den Tag, der größer als das AMP selbst ist, beeinflusst wird. Aufgrund der viel versprechenden antimikrobiellen Eigenschaften wurden mittels Agrobacterium-vermittelter Transformation Arabidopsis-Pflanzen erstellt, die EtDef (mit seinem putativen Signalpeptid) oder chimäres Thanatin (mit dem pflanzlichen Signalpeptid HvChi26) unter Kontrolle des konstitutiven CaMV35S Promotors exprimieren. Molekularbiologische Tests zeigten, dass sowohl das EtDef-, als auch das Thanatingen effizient in mRNA transkribiert wurden, wobei zwischen einzelnen Transformanten variierende Expressionslevel nachgewiesen wurden. Die vorgeschalteten Signalpeptide sollten zur Sekretion der AMPs in den Apoplasten führen. Deshalb wurden von individuellen transgenen Pflanzen, die entweder EtDef oder Thanatin exprimierten intercellular washing fluids (IWFs) isoliert. Die Sporenkeimung von B. cinerea wurde im Vergleich zur Kontrolle bei den verschiedenen Linien in unterschiedlichem Ausmaß inhibiert. Das deutet darauf hin, dass die Peptide in allen untersuchten Linien funktionell und im extrazellulären Raum lokalisiert waren. Im Folgenden wurde der Grad der Resistenz, der durch die Expression von entweder EtDef oder Thanatin hervorgerufen wurde, in planta untersucht. Hierfür wurden die pilzlichen Pathogene G. orontii und B. cinerea und das bakterielle Pathogen P. syringae pv. tomato DC3000 (Pst) verwendet. Transgene Arabidopsis Pflanzen mit entweder EtDef oder Thanatin zeigten eine deutlich verringerte Sporulation der Konidien, verringertes Myzelwachstum und Vermehrung von G. orontii auf Rosettenblättern, was zu einer erhöhten Resistenz der Pflanzen gegen diesen Pilz führte. Der Effekt auf G. orontii korrelierte mit der Transcriptmenge in den Pflanzen. Drei unabhängige transgene EtDef- (395, 396 und 405) und drei Thanatin-Linien (407, 410 und 411) wiesen hohe RNAExpressionslevel auf, was mit einem hohen Grad an Resistenz gegen G. orontii einherging. Im Gegensatz dazu zeigte die Expression von EtDef und Thanatin in Arabidopsis eine deutlich geringere Wirkung auf B. cinerea. Gleichwohl zeigten zwei transgene EtDef- (396 und405) und zwei transgene Thanatin-Linien (410 und 411) eine gesteigerte Resistenz gegen diesen Pilz. In den EtDef-Linien konnte eine erhöhte Resistenz gegenüber Pst nur in Linie 405 beobachtet werden. Die Sensitivität der übrigen Linien gegenüber dem Bakterium war nicht signifikant unterschiedlich zu den Kontrollpflanzen. Jedoch vermittelte die transgene Expression von Thanatin eine deutlich erhöhte Resistenz der Pflanzen gegen Pst. Hier zeigten wiederum die transgenen Linien 407, 410 und 411 die stärkste Wirkung. Zusammengefasst erscheinen die Linien 395, 396, 398 und 405 und 407, 410 und 411 am besten geeignet, um in weiteren Untersuchungen das Potential von EtDef bzw. Thanatin gegen andere Phytopathogene in planta zu testen. Die in dieser Studie präsentierten Ergebnisse deuten darauf hin, dass die transgene Expression von EtDef und Thanatin genutzt werden könnte, um eine gesteigerte Resistenz gegenüber Krankheiten auch in anderen, ökonomisch wichtigen, Pflanzen zu erzielen

Experimental and theoretical behaviour of reinforced concrete beams containing hybrid fibres

The use of fibres of different sizes are needed to improve the control of multi-level cracking of reinforced concrete (RC). There are several studies of the use of hybrid fibres for this purpose, however, there is limited work on the finite element modelling (FEM) of fibres in crack arresting of RC beams. In this study, fifteen RC beams containing silica fume, polyvinyl alcohol (PVA), polypropylene (PP), or hybrid fibres were experimentally tested and then finite element analysis (FEA) was conducted using ATENA 3D. The fibres were used up to 2.5% in the beams which were reinforced with and without shear reinforcement. All the beams were tested under four point bending with span to depth (a/d) ratio of 2.25. It was found that the PP, PVA fibres, and their hybrid in RC beams showed higher ductility in terms of multiple cracking before failure as compared with control beam without fibres. It was noticed also that PVA fibre showed a relatively greater flexural strength and recovery effect compared to PP fibre. Adding more than 1.5% PVA or hybrid fibres (1.5% PVA and 0.375% PP) without shear reinforcement contributed towards increasing shear capacity and ductility compared to the control beam containing shear reinforcement without fibres. A combination of small amount of hybrid fibres (0.75% PVA and 0.75% PP) and stirrups reinforcement resulted in a higher shear strength and higher ductility compared to other studied beams without shear reinforcement, which contain PVA, PP fibres up to 2.5% or hybrid fibres (1.5% PVA and 0.375% PP). A simple empirical equation based on the ACI-code 318-19 was used for predicting the shear behaviour of the studied beams taking in to consideration the effect of hybrid fibres for predicting the shear strength of the studied beams in a simple and accurate way. Based on the results of this investigation, it is recommended that a combination of hybrid fibres (0.75% PVA and 0.75% PP) and stirrups reinforcement (7.5 Ø 6 /m) should be used to achieve adequate shear behaviour of hybrid fibre reinforced concrete beams. The FEA results of all beams showed a good correlation with the experimental results in terms of the maximum load, load versus deflection and crack patterns

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

Background: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research