Characterization of antifungal natural products isolated from endophytic fungi of finger millet (Eleusine coracana) Annex 6

W.K.M. was supported by generous scholarships from the Government of Egypt, the University of Guelph International Graduate Student Scholarship (2012, 2014). This research was supported by grants to M.N.R. by the Ontario Ministry of Agriculture and Food (OMAF), Grain Farmers of Ontario (GFO) and the Canadian International Development Research Centre (IDRC) and Global Affairs Canada as part of the CIFSRF program.Finger millet is an ancient African-Indian crop that is resistant to many pathogens including the fungus, Fusarium graminearum. We previously reported the first isolation of putative fungal endophytes from finger millet and showed that the crude extracts of four strains had anti-Fusarium activity. However, active compounds were isolated from only one strain. The objectives of this study were to confirm the endophytic lifestyle of the three remaining anti-Fusarium isolates, to identify the major underlying antifungal compounds, and to initially characterize the mode(s) of action of each compound. Results of confocal microscopy and a plant disease assay were consistent with the three fungal strains behaving as endophytes. Using bio-assay guided fractionation and spectroscopic structural elucidation, three anti-Fusarium secondary metabolites were purified and characterized. These molecules were not previously reported to derive from fungi nor have antifungal activity. The purified antifungal compounds were: 5-hydroxy 2(3H)-benzofuranone, dehydrocostus lactone (guaianolide sesquiterpene lactone), and harpagoside (an iridoide glycoside). Light microscopy and vitality staining were used to visualize the in vitro interactions between each compound and Fusarium; the results suggested a mixed fungicidal/fungistatic mode of action. We conclude that finger millet possesses fungal endophytes that can synthesize anti-fungal compounds not previously reported as bio-fungicides against F. graminearum

MUSiC : a model-unspecific search for new physics in proton-proton collisions at root s=13TeV

Results of the Model Unspecific Search in CMS (MUSiC), using proton-proton collision data recorded at the LHC at a centre-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 35.9 fb(-1), are presented. The MUSiC analysis searches for anomalies that could be signatures of physics beyond the standard model. The analysis is based on the comparison of observed data with the standard model prediction, as determined from simulation, in several hundred final states and multiple kinematic distributions. Events containing at least one electron or muon are classified based on their final state topology, and an automated search algorithm surveys the observed data for deviations from the prediction. The sensitivity of the search is validated using multiple methods. No significant deviations from the predictions have been observed. For a wide range of final state topologies, agreement is found between the data and the standard model simulation. This analysis complements dedicated search analyses by significantly expanding the range of final states covered using a model independent approach with the largest data set to date to probe phase space regions beyond the reach of previous general searches.Peer reviewe

Search for long-lived particles decaying to jets with displaced vertices in proton-proton collisions at root s=13 Te V

A search is presented for long-lived particles produced in pairs in proton-proton collisions at the LHC operating at a center-of-mass energy of 13 TeV. The data were collected with the CMS detector during the period from 2015 through 2018, and correspond to a total integrated luminosity of 140 fb(-1). This search targets pairs of long-lived particles with mean proper decay lengths between 0.1 and 100 mm, each of which decays into at least two quarks that hadronize to jets, resulting in a final state with two displaced vertices. No significant excess of events with two displaced vertices is observed. In the context of R-parity violating supersymmetry models, the pair production of long-lived neutralinos, gluinos, and top squarks is excluded at 95% confidence level for cross sections larger than 0.08 fb, masses between 800 and 3000 GeV, and mean proper decay lengths between 1 and 25 mm.Peer reviewe

Search for lepton-flavor violating decays of the Higgs boson in the mu tau and e tau final states in proton-proton collisions at root s=13 TeV

A search is presented for lepton-flavor violating decays of the Higgs boson to mu t and et. The dataset corresponds to an integrated luminosity of 137 fb(-1) collected at the LHC in proton-proton collisions at a center-of-mass energy of 13 TeV. No significant excess has been found, and the results are interpreted in terms of upper limits on lepton-flavor violating branching fractions of the Higgs boson. The observed (expected) upper limits on the branching fractions are, respectively, B(H -> mu t) e tau) < 0.22(0.16)% at 95% confidence level.Peer reviewe

Measurement of the top quark Yukawa coupling from t(t)over-bar kinematic distributions in the dilepton final state in proton-proton collisions at root s=13 TeV

Peer reviewe

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Biodiversity of genes encoding anti-microbial traits within plant associated microbes

The plant is an attractive versatile home for diverse associated microbes. A subset of these microbes produce a diversity of anti-microbial natural products including polyketides, non-ribosomal peptides, terpenoids, heterocylic nitrogenous compounds, volatile compounds, bacteriocins and lytic enzymes. In recent years, detailed molecular analysis has led to a better understanding of the underlying genetic mechanisms. New genomic and bioinformatic tools have permitted comparisons of orthologous genes between species, leading to predictions of the associated evolutionary mechanisms responsible for diversification at the genetic and corresponding biochemical levels. The purpose of this review is to describe the biodiversity of biosynthetic genes of plant-associated bacteria and fungi that encode selected examples of antimicrobial natural products. For each compound, the target pathogen and biochemical mode of action are described, in order to draw attention to the complexity of these phenomena. We review recent information of the underlying molecular diversity and draw lessons through comparative genomic analysis of the orthologous genes. We conclude by discussing emerging themes and gaps, discuss the metabolic pathways in the context of the phylogeny and ecology of their microbial hosts, and discuss potential evolutionary mechanisms that led to the diversification of biosynthetic gene clusters

The diversity of anti-microbial secondary metabolites produced by fungal endophytes: An interdisciplinary perspective

Endophytes are microbes that inhabit host plants without causing disease and are reported to be reservoirs of metabolites that combat microbes and other pathogens. Here we review diverse classes of secondary metabolites, focusing on anti-microbial compounds, synthesized by fungal endophytes including terpenoids, alkaloids, phenylpropanoids, aliphatic compounds, polyketides and peptides from the interdisciplinary perspectives of biochemistry, genetics, fungal biology, host plant biology, human and plant pathology. Several trends were apparent. First, host plants are often investigated for endophytes when there is prior indigenous knowledge concerning human medicinal uses (e.g. Chinese herbs). However, within their native ecosystems, and where investigated, endophytes were shown to produce compounds that target pathogens of the host plant. In a few examples, both fungal endophytes and their hosts were reported to produce the same compounds. Terpenoids and polyketides are the most purified anti-microbial secondary metabolites from endophytes, while flavonoids and lignans are rare. Examples are provided where fungal genes encoding anti-microbial compounds are clustered on chromosomes. As different genera of fungi can produce the same metabolite, genetic clustering may facilitate sharing of anti-microbial secondary metabolites between fungi. We discuss gaps in the literature and how more interdisciplinary research may lead to new opportunities to develop bio-based commercial products to combat global crop and human pathogens

Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation

Wild maize (teosinte) has been reported to be less susceptible to pests than their modern maize (corn) relatives. Endophytes, defined as microbes that inhabit plants without causing disease, are known for their ability to antagonize plant pests and pathogens. We hypothesized that the wild relatives of modern maize may host endophytes that combat pathogens. Fusarium graminearum is the fungus that causes Gibberella Ear Rot (GER) in modern maize and produces the mycotoxin, deoxynivalenol (DON). In this study, 215 bacterial endophytes, previously isolated from diverse maize genotypes including wild teosintes, traditional landraces and modern varieties, were tested for their ability to antagonize F. graminearum in vitro. Candidate endophytes were then tested for their ability to suppress GER in modern maize in independent greenhouse trials. The results revealed that three candidate endophytes derived from wild teosintes were most potent in suppressing F. graminearum in vitro and GER in a modern maize hybrid. These wild teosinte endophytes could suppress a broad spectrum of fungal pathogens of modern crops in vitro. The teosinte endophytes also suppressed DON mycotoxin during storage to below acceptable safety threshold levels. A fourth, less robust anti-fungal strain was isolated from a modern maize hybrid. Three of the anti-fungal endophytes were predicted to be Paenibacillus polymyxa, along with one strain of Citrobacter. Microscopy studies suggested a fungicidal mode of action by all four strains. Molecular and biochemical studies showed that the P. polymyxa strains produced the previously characterized anti-Fusarium compound, fusaricidin. Our results suggest that the wild relatives of modern crops may serve as a valuable reservoir for endophytes in the ongoing fight against serious threats to modern agriculture. We discuss the possible impact of crop evolution and domestication on endophytes in the context of plant defense