Functional analysis of filipin tailoring genes from Streptomyces filipinensis reveals alternative routes in filipin III biosynthesis and yields bioactive derivatives

Background: Streptomyces filipinensis is the industrial producer of filipin, a pentaene macrolide, archetype of non-glycosylated polyenes, and widely used for the detection and the quantitation of cholesterol in biological membranes and as a tool for the diagnosis of Niemann-Pick type C disease. Genetic manipulations of polyene biosynthetic pathways have proven useful for the discovery of products with improved properties. Here, we describe the late biosynthetic steps for filipin III biosynthesis and strategies for the generation of bioactive filipin III derivatives at high yield. Results: A region of 13,778 base pairs of DNA from the S. filipinensis genome was isolated, sequenced, and characterized. Nine complete genes and two truncated ORFs were located. Disruption of genes proved that this genomic region is part of the biosynthetic cluster for the 28-membered ring of the polyene macrolide filipin. This set of genes includes two cytochrome P450 monooxygenase encoding genes, filC and filD, which are proposed to catalyse specific hydroxylations of the macrolide ring at C26 and C1' respectively. Gene deletion and complementation experiments provided evidence for their role during filipin III biosynthesis. Filipin III derivatives were accumulated by the recombinant mutants at high yield. These have been characterized by mass spectrometry and nuclear magnetic resonance following high-performance liquid chromatography purification thus revealing the post-polyketide steps during polyene biosynthesis. Two alternative routes lead to the formation of filipin III from the initial product of polyketide synthase chain assembly and cyclization filipin I, one trough filipin II, and the other one trough 1'-hydroxyfilipin I, all filipin III intermediates being biologically active. Moreover, minimal inhibitory concentration values against Candida utilis and Saccharomyces cerevisiae were obtained for all filipin derivatives, finding that 1'-hydroxyfilipin and especially filipin II show remarkably enhanced antifungal bioactivity. Complete nuclear magnetic resonance assignments have been obtained for the first time for 1'-hydroxyfilipin I. Conclusions: This report reveals the existence of two alternative routes for filipin III formation and opens new possibilities for the generation of biologically active filipin derivatives at high yield and with improved propertiesThis work was supported by the Spanish Ministerio de Economía y Competitividad (Grants BIO2010-19911 and BIO2013-42983-P to JFA), F.P.U. fellowships of the Ministerio de Educación, Cultura y Deporte (AP2005-3644 to JSA, AP2007-02055 to TDP, FPU13/01537 to AP), a contract from the Junta de Castilla y León cofinanced by the European Social Fund (to EGB), and a fellowship from the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/64006/2009 to CMV

A European-Japanese study on peach allergy: IgE to Pru p 7 associates with severity

Background: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. Methods: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. Results: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73–0.74). Pru p 3 tended to be a risk factor in South Europe only. Conclusions: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone

A European-Japanese study on peach allergy : IgE to Pru p 7 associates with severity

Funding Information: M. Fernández‐Rivas received grants or contracts from Instituto de Salud Carlos III, Spanish Government, Aimmune Therapeutics, Diater, and Novartis; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Aimmune Therapeutics, Ediciones Mayo S.A., Diater, Ga2LEN, HAL Allergy, GSK, MEDSCAPE, NOVARTIS, and EPG Health; is member of the Data Safety Monitoring Board at DBV and advisory board at Aimmune Therapeutics, Novartis, Reacta Healthcare, and SPRIM. B. Ballmer‐Weber received consulting fees from ALK, Allergopharma, Menarini, Sanofi, Novartis, Thermofisher and Aimune and payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from ALK, Menarini, Sanofi, Novartis, and Thermofisher. F. De Blay received grants or contract from Aimmune, Stallergenes Greer, GSK, ALK, Chiesi, and Regeneron. Y. Fukutomi received payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Thermo Fisher Diagnostics KK. K. Hoffmann‐Sommergruber received funding from Danube Allergy Research Cluster funded by the Country of Lower Austria to (P07) KHS; was Member of the EAACI board until 2022/07. J. Lidholm is employee at Thermo Fisher Scientific. E.N.C Mills received grants or has contracts from Food Standards Agency Patterns and prevalence of adult food allergy (FS101174), European Food Safety Authority (ThRAll; allergenicity prediction [with EuroFIR]) and from Innovate (ML for food allergy); has applied for a patent on oral food challenge meal formulations for diagnosis of food allergy; is member of the Advisory Board of Novartis and Advisory Committee on Novel Foods and Processes; and is shareholder of Reacta Healthcare Ltd. N.G. Papadopoulos received grants or contracts from Capricare, Nestle, Numil, Vianex; received consultancy fees from Abbott, Abbvie, Astra Zeneca, GSK, HAL, Medscape, Menarini/Faes Farma, Mylan, Novartis, Nutricia, OM Pharma, and Regeneron/Sanofi. S. Vieths received royalties or licenses from Schattauer Allergologie Handbuch, Elsevier Nahrungsmittelallergien and Intoleranzen and Karger Food Allergy: Molecular Basis and Clinical Practice; support for attending meetings and/or travel as Associate Editor of the Journal of Allergy and Clinical Immunology. R. van Ree received consulting fees from HAL Allergy, Citeq, Angany, Reacta Healthcare, Mission MightyMe, and Ab Enzymes; received payment of honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from HAL Allergy, Thermo Fisher Scientific and ALK; received payment for expert testimony from AB Enzymes; has stock option at Angany. The rest of the authors declare that they have no relevant conflicts of interest. Funding Information: This work was funded by the European Commission under the 6th Framework Programme through EuroPrevall (FP6‐FOOD‐CT‐2005‐514000), and the 7th Framework Programme iFAAM (grant agreement no. 31214). Funding Information: We thank all the patients for their participation in the study. We would like to thank ALK Abello (Madrid, Spain) for their generous gift of SPT reagents. We thank Angelica Ehrenberg, Jonas Östling and Lars Mattsson (Uppsala) for preparing recombinant Cup s 7 and custom ImmunoCAP tests for this study. We acknowledge the support by the 6th and 7th Framework Programmes of the EU, for EuroPrevall (FP6‐FOOD‐CT‐2005‐514000) and iFAAM (Grant agreement no. 312147), respectively. We thank Alejandro Gonzalo Fernández (Hospital Clinico San Carlos, IdISSC, Madrid) for implementing the FASS in the data set. Publisher Copyright: © 2023 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.BACKGROUND: Pru p 3 and Pru p 7 have been implicated as risk factors for severe peach allergy. This study aimed to establish sensitization patterns to five peach components across Europe and in Japan, to explore their relation to pollen and foods and to predict symptom severity. METHODS: In twelve European (EuroPrevall project) and one Japanese outpatient clinic, a standardized clinical evaluation was conducted in 1231 patients who reported symptoms to peach and/or were sensitized to peach. Specific IgE against Pru p 1, 2, 3, 4 and 7 and against Cup s 7 was measured in 474 of them. Univariable and multivariable Lasso regression was applied to identify combinations of parameters predicting severity. RESULTS: Sensitization to Pru p 3 dominated in Southern Europe but was also quite common in Northern and Central Europe. Sensitization to Pru p 7 was low and variable in the European centers but very dominant in Japan. Severity could be predicted by a model combining age of onset of peach allergy, probable mugwort, Parietaria pollen and latex allergy, and sensitization to Japanese cedar pollen, Pru p 4 and Pru p 7 which resulted in an AUC of 0.73 (95% CI 0.73-0.74). Pru p 3 tended to be a risk factor in South Europe only. CONCLUSIONS: Pru p 7 was confirmed as a significant risk factor for severe peach allergy in Europe and Japan. Combining outcomes from clinical and demographic background with serology resulted in a model that could better predict severity than CRD alone.Peer reviewe

Rigid amphipathic fusion inhibitors demonstrate antiviral activity against African swine fever virus

Rigid amphipathic fusion inhibitors (RAFIs) are a family of nucleoside derivatives that inhibit the infectivity of several enveloped viruses by interacting with virion envelope lipids and inhibiting fusion between viral and cellular membranes. Here we tested the antiviral activity of two RAFIs, 5-(Perylen-3-ylethynyl)-arabino-uridine (aUY11) and 5-(Perylen-3-ylethynyl)uracil-1-acetic acid (cm1UY11) against African swine fever virus (ASFV), for which no effective vaccine is available. Both compounds displayed a potent, dose-dependent inhibitory effect on ASFV infection in Vero cells. The major antiviral effect was observed when aUY11 and cm1UY11 were added at early stages of infection and maintained during the complete viral cycle. Furthermore, virucidal assay revealed a significant extracellular anti-ASFV activity for both compounds. We also found decrease in the synthesis of early and late viral proteins in Vero cells treated with cm1UY11. Finally, the inhibitory effect of aUY11 and cm1UY11 on ASFV infection in porcine alveolar macrophages was confirmed. Overall, our study has identified novel anti-ASFV compounds with potential for future therapeutic developments