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For patients with advanced non-small-cell lung cancer (NSCLC), dual immune checkpoint blockade (ICB) with CTLA4 inhibitors and PD-1 or PD-L1 inhibitors (hereafter, PD-(L)1 inhibitors) is associated with higher rates of anti-tumour activity and immune-related toxicities, when compared with treatment with PD-(L)1 inhibitors alone. However, there are currently no validated biomarkers to identify which patients will benefit from dual ICB 1,2 . Here we show that patients with NSCLC who have mutations in the STK11 and/or KEAP1 tumour suppressor genes derived clinical benefit from dual ICB with the PD-L1 inhibitor durvalumab and the CTLA4 inhibitor tremelimumab, but not from durvalumab alone, when added to chemotherapy in the randomized phase III POSEIDON trial 3 . Unbiased genetic screens identified loss of both of these tumour suppressor genes as independent drivers of resistance to PD-(L)1 inhibition, and showed that loss of Keap1 was the strongest genomic predictor of dual ICB efficacy-a finding that was confirmed in several mouse models of Kras-driven NSCLC. In both mouse models and patients, KEAP1 and STK11 alterations were associated with an adverse tumour microenvironment, which was characterized by a preponderance of suppressive myeloid cells and the depletion of CD8 + cytotoxic T cells, but relative sparing of CD4 + effector subsets. Dual ICB potently engaged CD4 + effector cells and reprogrammed the tumour myeloid cell compartment towards inducible nitric oxide synthase (iNOS)-expressing tumoricidal phenotypes that-together with CD4 + and CD8 + T cells-contributed to anti-tumour efficacy. These data support the use of chemo-immunotherapy with dual ICB to mitigate resistance to PD-(L)1 inhibition in patients with NSCLC who have STK11 and/or KEAP1 alterations., Competing Interests: Competing interests F.S. reports consulting for AstraZeneca, Amgen, Revolution Medicines, Novartis, BridgeBio, Beigene, BergenBio, Guardant Health, Calithera Biosciences, Tango Therapeutics, Hookipa Pharma, Novocure, Merck Sharp & Dohme, Roche; grant or research support from Amgen, Mirati Therapeutics, Revolution Medicines, Pfizer, Novartis, Merck & Co; stockholder in BioNTech, Moderna; and honoraria from ESMO, Japanese Lung Cancer Society, Medscape, Intellisphere, VSPO McGill Universite de Montreal, RV Mais Promocao Eventos, MJH Life Sciences, IDEOlogy Health, MI&T, PER, CURIO, DAVA Oncology, the American Association for Cancer Research and the International Association for the Study of Lung Cancer. M.M. reports stockholder in Roche Holdings. N.J. reports shareholder and former employee of Pionyr Immunotherapeutics. J.M.J. reports stock of D2G Oncology. C.M., Y.Y., X.P. and A.H. report employee of Tango Therapeutics. K.C.A. reports personal fees from Sanofi Genzyme and other support from Revolution Medicines, Genentech, and Mirati outside the submitted work. N.V. receives consulting fees from Sanofi Genzyme, Oncocyte, Eli Lilly, Regeneron, and research funding to the institution from Mirati, Oncocyte, and Circulogene, outside the submitted work. P.D.P. reports advisory fees from AstraZeneca and Jazz Pharmaceuticals. M.E.M. reports research funding from Eli Lilly (Inst), AstraZeneca (Inst), Merck (Inst), Genentech (Inst); consulting role with AstraZeneca, Novocure, Boehringer Ingelheim, Janssen, Takeda, Blueprint Pharmaceuticals, Bayer, Bristol Myers Squibb, Ikena; honorarium from Thermo Fisher Scientific; and stock in Merck, Johnson & Johnson. M.M.A. reports grants and personal fees from Genentech, Bristol Myers Squibb and AstraZeneca; grants from Lilly; and personal fees from Maverick, Blueprint Medicine, Syndax, Nektar, Gritstone, ArcherDX, Mirati, NextCure, Novartis, EMD Serono and Panvaxal/NovaRX, outside of the submitted work. J.C.M. reports consulting or honoraria: MJH Life Sciences, Johnson & Johnson and Doximity; and research funding (to institution): Merck via the Conquer Cancer Foundation. J.F.G. reports served as a compensated consultant or received honoraria from Bristol Myers Squibb, Genentech (Roche), Takeda, Loxo (Lilly), Blueprint Medicine, Gilead, Moderna, AstraZeneca, Mariana Therapeutics, Mirati, Jounce, Merus Pharmacueticals, Nuvalent, Pfizer, Novocure, AI Proteins, Novartis, Merck, iTeos, Karyopharm and Silverback Therapeutics; research support from Novartis, Genentech (Roche) and Takeda; institutional research support from Bristol Myers Squibb, Palleon, Tesaro, Moderna, Blueprint, Jounce, Array Biopharma, Merck, Adaptimmune, Novartis and Alexo; has equity in AI Proteins; and has an immediate family member who is an employee with equity at Ironwood Pharmaceuticals. A.D. reports honoraria: Intellisphere, Roche (Genentech); ad board: TP Therapeutics, Guardant Health, AnHeart Therapeutics, ChromaCode; clinical trial support: Syntrix Pharmaceuticals, Novartis, Merck, AnHeart Therapeutics, Sorrento Therapeutics, Guardant Health and Philogen. C. M. Bestvina reports consulting or advisory role: AstraZeneca, Genentech, AbbVie, Curio Science, OncLive Clinical Congress Consultants, Seagen, Creative Educational Concepts, Takeda, Janssen, CVS, Bristol Myers Squibb (Celgene), Jazz Pharmaceuticals, Novartis, Sanofi–Regeneron and Novocure; speakers’ bureau: Merck; research funding: Bristol Myers Squibb and AstraZeneca. C.A.S. reports advisory boards to AstraZeneca, Gilead, Janssen, Genentech, Mirati, Takeda and Arcus Biosciences. J.W.R. reports honoraria or consulting (self): Boehringer Ingelheim, BMS, Turning Point Roche (Genentech), Biodesix, EMD Serono, Daiichi Sankyo, Blueprint, Novartis, Regeneron, Sanofi, Janssen, Jazz Pharmaceuticals, Bayer, Beigene, Merus and Seagen; research support (to institution): ArriVent, Merck, Novartis, AstraZeneca, Spectrum, Revolution Medicines, IO Biotech and Kinnate. C. M. Blakely reports research funding: AstraZeneca, Novartis, Mirati, Spectrum, Takeda, Puma and Pfizer; and consulting: Janssen and Bayer. C.V.P. reports a founder and equity and intellectual property in EnFuego Therapeutics. F.T. reports speaker bureau or honoraria: Roche, AstraZeneca, Novartis and Takeda. M.S. reports institutional support: Dracen Pharmaceuticals; advisory board: Amgen, AstraZeneca, Boehringer Ingelheim, Janssen Pharmaceuticals, Novartis, Pfizer, Roche, Sanofi-Aventis, Siemens Healthineers, Takeda Pharmaceuticals and Bristol Myers Squibb; leadership or fiduciary role: ESMO and EORTC. S.D. reports independent image analysis for hospital-contracted clinical research trials programs for Merck, Pfizer, Bristol Myers Squibb, Novartis, Roche, Polaris, Cascadian, Abbvie, Gradalis, Bayer, Zai laboratories, Biengen, Resonance and Analise; research grants from Lunit, GE, Qure AI; and an honorarium from Siemens. M.J.M. reports AstraZeneca, Bristol Myers Squibb and Istari Oncology & Regeneron. A.G.S. reports AstraZeneca, Genentech (Roche) and Bristol Myers Squibb. A.N.S. reports serving as a consultant and/or advisory board member for Eli Lilly, Daiichi Sankyo and Zymeworks; and research funding from Eli Lilly, Novartis, Daiichi Sankyo, Genentech, AstraZeneca, Memgen, Mersana, Turning Point Therapeutics, BioAtla and Genmab to his institution. J.R. reports consulting fees or honoraria from AstraZeneca, Takeda, Sanofi Genzyme, Genentech, Guardant Health, Summit, G1 Therapeutics, BioAtla, Jazz, Amgen, and Janssen; contracted for research (institutional) with AstraZeneca, BioAtla, Blueprint Medicines, EpimAb Biotherapeutics, LOXO Oncology, Enliven, Redcloud and ORIC. T.S. reports advisory board or consulting fees for Seagen, Astellas and AstraZeneca; and research grant GRAIL. S.B.G. reports research funding from AstraZeneca, Boehringer Ingelheim and Mirati; and consulting or advisory board member for AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Amgen, Blueprint Medicine, Sanofi Genzyme, Daiichi Sankyo, Takeda, Janssen, Summit Therapeutics, Merck and Regeneron. J.K. reports for Arterys. K.S. reports consultant, advisor or speaker for Clinica Alemana Santiago, Shattuck Labs, AstraZeneca, EMD Serono, Takeda, Torque/Repertoire Therapeutics, CSRlife, Agenus, Genmab, OnCusp, Parthenon Therapeutics, Bristol Myers Squibb, Roche, Molecular Templates, Abbvie, Sanofi, Merck, PeerView, PER and Forefront Collaborative; research funding from Navigate BP, Tesaro/GSK, Moderna, Takeda, Surface Oncology, Pierre-Fabre, Merck, Bristol Myers Squibb, AstraZeneca, Ribon Therapeutics, Eli Lilly, Boehringer Ingelheim, Genentech (Roche) and Akoya Biosciences. K.D.D. reports consulting fees or other remuneration from Rain Therapeutics. V.A. reports research funding to Johns Hopkins University from AstraZeneca and Personal Genome Diagnostics, has received research funding to Johns Hopkins University from Bristol Myers Squibb and Delfi Diagnostics in the past five years and is an advisory board member for Neogenomics and AstraZeneca. V.A. is an inventor on patent applications (63/276,525, 17/779,936, 16/312,152, 16/341,862, 17/047,006 and 17/598,690) submitted by Johns Hopkins University related to cancer genomic analyses, ctDNA therapeutic response monitoring and immunogenomic features of response to immunotherapy that have been licensed to one or more entities. Under the terms of these license agreements, the University and inventors are entitled to fees and royalty distributions. K.A.M. reports consulting or advisory fees from AstraZeneca, Amgen, Janssen, Mirati Therapeutics, Daiichi Sankyo (Lilly) and Puma Biotechnology, as well as honoraria from AstraZeneca. K.A.M. receives research funding to Johns Hopkins University from Bristol Myers Squibb and Mirati Therapeutics. P.M.F. reports consulting fee (for example, advisory board): Ascendis, AstraZeneca, BMS, Curevac, Novartis, Regeneron, G1, Genelux, Genentech, Gritstone, Merck, Janssen, F-Star, Sanofi, Amgen, Fosun, Teva, Synthekine, Flame, Iteos, Tavotek and Teva; contracted research: AstraZeneca, BMS, Novartis, Regeneron and BioNTech. B.R. reports advisory board or consultant: Regeneron, AstraZeneca, Amgen; honoraria: Targeted Oncology. E.M.V.A reports advisory or consulting: Tango Therapeutics, Genome Medical, Genomic Life, Enara Bio, Manifold Bio, Monte Rosa, Novartis Institute for Biomedical Research, Riva Therapeutics and Serinus Bio; research support: Novartis, BMS and Sanofi; equity: Tango Therapeutics, Genome Medical, Genomic Life, Syapse, Enara Bio, Manifold Bio, Microsoft, Monte Rosa, Riva Therapeutics and Serinus Bio; patents: institutional patents filed on chromatin mutations and immunotherapy response, and methods for clinical interpretation; intermittent legal consulting on patents for Foaley & Hoag; editorial boards: JCO Precision Oncology and Science Advances. A.L.C. reports consulting for AstraZeneca, Merus and Tempus; research funds from AstraZeneca, Genentech (Roche), Merck and Novartis. J.W.G. reports research grants: AstraZeneca, BMS, Eli Lilly, Genentech, Merck and Pfizer; consulting fees: AstraZeneca, BMS, Genentech, Gilead, Gritstone, Eli Lilly, Pfizer and Regeneron. C.A. reports consulting or advisory role: Genentech, Lilly, Celgene, Merck, AstraZeneca, Blueprint Genetics, Shionogi, Daiichi Sankyo–AstraZeneca, Sanofi/Regeneron, Eisai, BeiGene, Turning Point Therapeutics, Pfizer, Janssen, Boehringer Ingelheim; research funding: Genentech/Roche (Inst), Incyte (Inst), Macrogenics (Inst), Merck Sharp & Dohme (Inst) and AstraZeneca–MedImmune (Inst). L.R. reports honoraria: Merck, AstraZeneca, Genzyme, EMD Serono and Astellas Pharmas; consulting or advisory role: Amgen. J.W.N. reports honoraria: CME Matters, Clinical Care Options CME, Research to Practice CME, Medscape CME, Biomedical Learning Institute CME, MLI Peerview CME, Prime Oncology CME, Projects in Knowledge CME, Rockpointe CME, MJH Life Sciences CME, Medical Educator Consortium, HMP education consulting or advisory role: AstraZeneca, Genentech (Roche), Exelixis, Takeda Pharmaceuticals, Eli Lilly, Amgen, Iovance Biotherapeutics, Blueprint Pharmaceuticals, Regeneron Pharmaceuticals, Natera, Sanofi, D2G Oncology, Surface Oncology, Turning Point Therapeutics, Mirati Therapeutics, Gilead Sciences, Abbive, Summit Therapeutics, Novartis, Novocure, Janssen Oncology, Anheart Therapeutics Research Funding: Genentech (Roche), Merck, Novartis, Boehringer Ingelheim, Exelixis, Nektart Therapeutics, Takeda Pharmaceuticals, Adaptimmune, GlaxoSmithKline, Janssen Pharmaceuticals, Abbvie Pharmaceuticals and Novocure. Y.Y.E. reports honoraria: CME Matters, Clinical Care Options CME, Research to Practice CME, Medscape CME, Biomedical Learning Institute CME, MLI Peerview CME, Prime Oncology CME, Projects in Knowledge CME, Rockpointe CME, MJH Life Sciences CME, Medical Educator Consortium, HMP education consulting or advisory role: AstraZeneca, Genentech (Roche), Exelixis, Takeda Pharmaceuticals, Eli Lilly, Amgen, Iovance Biotherapeutics, Blueprint Pharmaceuticals, Regeneron Pharmaceuticals, Natera, Sanofi, D2G Oncology, Surface Oncology, Turning Point Therapeutics, Mirati Therapeutics, Gilead Sciences, Abbive, Summit Therapeutics, Novartis, Novocure, Janssen Oncology, Anheart Therapeutics Reserach Funding: Genentech/Roche, Merck, Novartis, Boehringer Ingelheim, Exelixis, Nektart Therapeutics, Takeda Pharmaceuticals, Adaptimmune, GlaxoSmithKline, Janssen Pharmaceuticals, Abbvie Pharmaceuticals and Novocure. M.V.N. reports research funding to institution: Mirati, Novartis, Checkmate (ended), Alaunos, AstraZeneca, Pfizer, Genentech, Navire; consultant or advisory board: Mirati, Merck/MSD, Novartis and Genentech. X.L. reports receiving consulting or advisory fees from EMD Serono (Merck KGaA), AstraZeneca, Spectrum Pharmaceutics, Novartis, Eli Lilly, Boehringer Ingelheim, Hengrui Therapeutics, Janssen, Blueprint Medicines, Regeneron, Sensei Biotherapeutics, Abion and Abbvie, and research funding from Eli Lilly, EMD Serono, Janssen, ArriVent, Teligene, BlackDiamond, Regeneron and Boehringer Ingelheim. V.K.L. reports consultant or advisory role for Takeda, Seattle Genetics, BMS, AstraZeneca, Guardant Health and AnHeart Therapeutics and has received research funding from GSK, BMS, AstraZeneca, Merck and Seattle Genetics. W.E.L. reports advisory boards: Eisai and Takeda, Grand Rounds and BMS for repotrectinib. J.A.R. reports consultancy, stock, Genprex; patents issued and pending. T.C. reports speaker fees or honoraria from the Society for Immunotherapy of Cancer (SITC), Mark Foundation for Cancer Research, Bristol Myers Squibb, Roche, Medscape, IDEOlogy Health, Physicians’ Education Resource (PER), OncLive and PeerView; travel and/or food/beverage expenses from SITC, International Association for the Study of Lung Cancer, Parker Institute for Cancer Immunotherapy, PER, Dava Oncology, IDEOlogy Health, AstraZeneca and Bristol Myers Squibb; advisory role or consulting fees from MedImmune–AstraZeneca, Bristol Myers Squibb, Merck, Genentech, Arrowhead Pharmaceuticals, Pfizer and Regeneron; and institutional research funding from MedImmune–AstraZeneca, Bristol Myers Squibb and EMD Serono. J.Z. reports grants from Merck, grants and personal fees from Johnson & Johnson and Novartis, personal fees from Bristol Myers Squibb, AstraZeneca, GenePlus, Innovent and Hengrui outside the submitted work. G.R.B. reports grant or contract for clinical trial from MacroGenics; grants from Amgen, Bayer, Adaptimmune, Exelixis, Daiichi Sankyo, GlaxoSmithKline, Immatics, Immunocore, Incyte, Kite Pharma, MacroGenics, Torque, AstraZeneca, Bristol Myers Squibb, Celgene, Genentech, MedImmune, Merck, Novartis, Roche, Sanofi, Xcovery, Tmunity Therapeutics, Regeneron, BeiGene, Repertoire Immune Medicines, Verastem, CytomX Therapeutics and Duality Biologics; consulting fees from AbbVie, Adicet, Amgen, Ariad, Bayer, Clovis Oncology, AstraZeneca, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Instil Bio, Genentech, Genzyme, Gilead, Eli Lilly, Janssen, MedImmune, Merck, Novartis, Roche, Sanofi, Tyme Oncology, Xcovery, Virogin Biotech, Maverick Therapeutics, BeiGene, Regeneron, Cytomx Therapeutics, Intervenn Biosciences and Onconova Therapeutics; participation in advisory boards at Virogin Biotech and Maverick Therapeutics; stock in Virogin Biotech; and employment of family member at Johnson & Johnson/Janssen. C.G. reports member of the advisory board at Jazz Pharmaceuticals, AstraZeneca and Bristol Myers Squibb and served as speaker for AstraZeneca and BeiGene. L.A.B. reports consulting fees and research funding from AstraZeneca, GenMab and Sierra Oncology and research funding from Tolero Pharmaceuticals and served as advisor or consultant for PharmaMar, AbbVie, Bristol Myers Squibb, Alethia, Merck, Pfizer, Jazz Pharmaceuticals, Genentech and Debiopharm Group. D.L.G. reports AstraZeneca, Eli Lilly, Menarini Richerche, 4D Pharma, Onconova Therapeutics and Sanofi. A.T. reports Ariad, AstraZeneca, Bristol Myers Squibb, Boehringer Ingelheim, Eli Lilly, EMD Serono, Genentech, GlaxoSmithKline, Merck, Novartis, Pfizer, Roche, Seattle Genetics, Gilead Sciences and Summit Therapeutics. T.G.B. reports Chan-Zuckerberg Biohub. D.R.C. reports CoI Company sponsored trials at institution (PI roles): AZ, Roche (Genentech) advisory role: ad hoc advisory boards or consultations 2023: Abbvie (steering committee), Anheart, Beigene (IDMC), Eli Lilly (IDMC), Imagene, Immunocore, Janssen, Mirati (IDMC), Prelude, Seattle Genetics, Valencee; 2022: Abbvie, Anheart, Appolomics (SRC), AstraZeneca–Daiichi (ILD adjudication committee), Beigene (DSMB) Dizal, EMD Serono, Elevation, Hengrui, (DSMB), Hummingbird, Janssen, Medtronic, Mersana (ILD adjudication committee), Mirati, Nalo Therapeutics, Onkure, Regeneron, Roche, Sanofi, Takeda, Theseus, Xcovery; 2021: Abbvie, Amgen, Anheart, Apollomics (SRC), AstraZeneca (SRC/SC), Beigene (DSMC), Bio-Thera (DSMB), Blueprint, Daiichi Sankyo (ILD adjudication committee), Elevation (SRC), Eli Lilly (DSMB and NCCN), EMD Serono, Helsinn (DSMB), Hengrui (DSMC), Janssen, Kestrel (SAB, Shares), Mersana, Nuvalent (SAB), Puma (NCCN), Ribon, Roche (Genentech), Sanofi, Seattle Genetics, Takeda and Turning Point. J.E.G. reports honoraria: Jazz Pharmaceuticals, Merck and OncoCyte; consulting or advisory role: AstraZeneca, Blueprint Medicines, Bristol Myers Squibb, EMD Serono, Lilly, Sanofi, Merck Sharp & Dohme, Loxo, Jazz Pharmaceuticals, Novartis, AstraZeneca–MedImmune, Janssen Scientific Affairs, National Comprehensive Cancer Network, AbbVie and Regeneron; research funding: Merck (Inst), AstraZeneca (Inst), Bristol Myers Squibb (Inst), Boehringer Ingelheim (Inst), Genentech (Roche) (Inst), G1 Therapeutics (Inst), Novartis (Inst), Pfizer (Inst), Ludwig Institute for Cancer Research (Inst), SWOG (Inst), Array BioPharma (Inst) and ECOG-ACRIN (Inst). N.L. reports CME and research funding: AstraZeneca. B.L. reports Genentech (Roche), Eli Lilly, Merck, Pfizer, Janssen, Daiichi Sankyo, Takeda, Mirati, Novartis and Guardant 360. J.R.B. reports grant funding: AstraZeneca; advisory board: AstraZeneca, Amgen, Novartis, Incyte, Bristol Myers Squibb, Genentech, Janssen, Sanofi, Regeneron, Merck, GlaxoSmithKline; DSMB: Sanofi, Janssen and GlaxoSmithKline. M.C.G. reports AstraZeneca, Abion, MSD International, Bayer, BMS, Boehringer Ingelheim Italia S.p.A, Celgene, Eli Lilly, Incyte, Novartis, Pfizer, Roche, Takeda, Seattle Genetics, Mirati, Daiichi Sankyo, Regeneron, Merck, Blueprint, Janssen, Sanofi, AbbVie, BeiGenius, Oncohost and Medscape. D.R.G. reports institutional research grants from Amgen, AstraZeneca, Genentech and Merck; consultant or advisory board for Adagene, AstraZeneca, Roche (Genentech), Guardant, IO Biotech, Oncocyte, OncoHost, Lilly, Merck and Novartis. E.B.G. reports consulting or advisory role: Novartis, GlaxoSmithKline, Merck, Boehringer Ingelheim, Shionogi, Eisai, Bristol Myers Squibb, ABL Bio, Xilio Therapeutics, Natera, Sanofi–Regeneron, Lilly, Personalis, Gilead Sciences, AstraZeneca, AbbVie–Abbott, Arcus Biosciences, Seagan and Summit Therapeutics; research funding: Merck (Inst), Genentech (Inst), AstraZeneca (Inst), Novartis (Inst), Lilly (Inst), Bristol Myers Squibb (Inst), Mirati Therapeutics (Inst), Dynavax Technologies (Inst), Iovance Biotherapeutics (Inst), Neon Therapeutics (Inst), EMD Serono (Inst), ABL Bio (Inst) and Daiichi Sankyo–UCB Japan (Inst); patents, royalties, other intellectual property: diagnosistic and therapeutic use of ‘Motif Neoepitopes’ as defined by Cummings et al.76. N.A.R. reports an employee at Synthekine and holds equity at Synthekine and Gritstone. G.V.S. reports honoraria, research funding and personal fees from AstraZeneca, Bayer, BeiGene Switzerland, F. Hoffman–La Roche, Merck Sharpe & Dohme, Eli Lilly, Johnson & Johnson, Pfizer, Takeda Oncology, Tesaro and Verastem outside the submitted work. J.W. reports advisory boards and lecture fees: Amgen, AstraZeneca, Bayer, Blueprint, BMS, Boehringer Ingelheim, Chugai, Daiichi Sankyo, Ignyta, Janssen, Lilly, Loxo, Merck, Mirati, MSD (Merck Sharp & Dohme), Novartis, Nuvalent, Pfizer, Roche, Seattle Genetics, Takeda and Turning Point; research support (to institution): BMS, Janssen, Novartis, Pfizer and AstraZeneca. B.B. reports receiving grants from AbbVie, Amgen, AstraZeneca, Chugai, Daiichi Sankyo, Ellipse, EISAI, Genmab, Genzyme Corporation, Hedera Dx, Inivata, IPSEN, Janssen, MSD, PharmaMar, Roche (Genentech), Sanofi, Socar Research, Tahio Oncology and Turning Point Therapeutics. R.S.H. reports consulting roles with AbbVie Pharmaceuticals, ARMO Biosciences, AstraZeneca, Biodesix, Bolt Biotherapeutics, Bristol Myers Squibb, Cybrexa Therapeutics, eFFECTOR Therapeutics, Eli Lilly, EMD Serono, Genentech (Roche), Genmab, Halozyme Therapeutics, Heat Biologics, I-Mab Biopharma, Immunocore, Infinity Pharmaceuticals, Loxo Oncology, Merck, Mirati Therapeutics, Nektar, Neon Therapeutics, NextCure, Novartis, Oncternal Therapeutics, Pfizer, Sanofi, Seattle Genetics, Shire, Spectrum Pharmaceuticals, STCube Pharmaceuticals, Symphogen, Takeda, Tesaro, Tocagen and WindMIL Therapeutics; advisory board roles with AstraZeneca, Bolt Biotherapeutics, Cybrexa Therapeutics, EMD Serono, I-Mab Biopharma, Immunocore, Infinity Pharmaceuticals, Neon Therapeutics, Novartis and STCube Pharmaceuticals; research support from AstraZeneca, Eli Lilly, Genentech (Roche) and Merck; and non-executive board membership for Junshi Pharmaceuticals and Immunocore. H.A.W. reports grants or contracts from any entity: Bayer, Arrys Therapeutics, AstraZeneca–Medimmune, BMS, Clovis Oncology, Genentech (Roche), Merck, Novartis, Seagen, Xcovery and Helsinn; advisory board: Mirati, Merck and Genentech (Roche); leadership: International Association for the Study of Lung Cancer (IASLC), President; ECOG-ACRIN, executive committee. N.A.P. reports an employee at Synthekine and holds equity at Synthekine and Gritstone. A.T.S. is currently an employee of Novartis. P.A.J. reports stock and other ownership interests: Gatekeeper Pharmaceuticals and Loxo; consulting or advisory role: Pfizer, Boehringer Ingelheim, AstraZeneca, Merrimack, Chugai, Roche (Genentech), Loxo, Mirati Therapeutics, Araxes Pharma, Ignyta, Lilly, Takeda, Novartis, Biocartis, Voronoi Health Analytics, SFJ Pharmaceuticals Group, Sanofi, Daiichi Sankyo, Silicon Therapeutics, Nuvalent, Eisai, Bayer, Syndax, AbbVie, Allorion Therapeutics, Accutar Biotech, Transcenta, Monte Rosa Therapeutics, Scorpion Therapeutics, Merus, Frontier Medicines, Hongyun Biotech and Duality Biologics; research funding: AstraZeneca (Inst), Astellas Pharma (Inst), Daiichi Sankyo (Inst), Lilly (Inst), Boehringer Ingelheim (Inst), Puma Biotechnology (Inst), Takeda (Inst) and Revolution Medicines (Inst); patents, royalties, other intellectual property: I am a co-inventor on a DFCI owned patent on EGFR mutations licensed to Lab Corp. I receive post-marketing royalties from this invention. D.P.C. reports advisory boards or consulting: Abbvie, Arcus Biosciences, AstraZeneca, BMS Israel, G1 Therapeutics, Genentech, GlaxoSmithKline, InThought, Iovance Biotherapeutics, Janssen, Jazz, JNJ, Merck–EMD Serono, Merck KGaA, Mirati, MSD, Novartis, Novocure, OncoHost, Pfizer Egypt, Regeneron, Roche and Sanofi; KOL/presentation/education/forum: AstraZeneca, Curio Science, Intellisphere, Merck US, OncLive, Pfizer, PPD Development and Roche Taiwan. M.D.H. is an employee of and stockholder in AstraZeneca. C.M.R. reports consulted regarding oncology drug development with AbbVie, Amgen, AstraZeneca, D2G, Daiichi Sankyo, Epizyme, Genentech (Roche), Ipsen, Jazz, Kowa, Lilly, Merck and Syros. He serves on the scientific advisory boards of Auron, Bridge Medicines, DISCO, Earli and Harpoon Therapeutics. L.A. is an employee of Foundation Medicine and a stockholder of Roche Holding AG. H.M. reports full-time employee of AstraZeneca and stock/shares. Z.Z. reports employee of AstraZeneca as well as stockholder of AstraZeneca and Pfizer. Z.L. reports full-time employee and stock owner of AstraZeneca. R.S. reports other support from AstraZeneca during the conduct of the study, as well as other support from Pfizer outside the submitted work. S.P. reports consultation or advisory role: AbbVie, Amgen, Arcus, AstraZeneca, Bayer, Beigene, BerGenBio, Biocartis, BioInvent, Blueprint Medicines, Boehringer Ingelheim, Bristol Myers Squibb, Clovis, Daiichi Sankyo, Debiopharm, Eli Lilly, F-Star, Fishawack, Foundation Medicine, Genzyme, Gilead, GSK, Hutchmed, Illumina, Incyte, Ipsen, iTeos, Janssen, Merck Sharp and Dohme, Merck Serono, Merrimack, Mirati, Nykode Therapeutics, Novartis, Novocure, PharmaMar, Promontory Therapeutics, Pfizer, Regeneron, Roche (Genentech), Sanofi, Seattle Genetics and Takeda; Board of Directors position: Galenica; talk in a company’s organized public event: AstraZeneca, Boehringer Ingelheim, Bristol Myers Squibb, Eli Lilly, Foundation Medicine, GSK, Illumina, Ipsen, Merck Sharp and Dohme, Mirati, Novartis, Pfizer, Roche (Genentech), Sanofi and Takeda; receipt of grants or research support: principal investigator in trials (institutional financial support for clinical trials) sponsored by Amgen, Arcus, AstraZeneca, Beigene, Bristol Myers Squibb, GSK, iTeos, Merck Sharp and Dohme, Mirati, PharmaMar, Promontory Therapeutics, Roche (Genentech) and Seattle Genetics. J.M.J. and M.J.R. are employees and shareholders of D2G Oncology. I.P.W. is a co-founder, employee and shareholder of D2G Oncology. M.M.W. is a co-founder, shareholder, member of the board of directors and compensated scientific advisor of D2G Oncology. I.P.W. and M.M.W. are co-inventors of patents relating to technologies for an autochthonous mouse model of human cancer, which D2G Oncology has exclusively licensed from Stanford University. V.A.P. reports Pfizer employee and stockholder. K.K.W. is a founder and equity holder of G1 Therapeutics and has sponsored research agreements with Takeda, TargImmune, Bristol Myers Squibb, Mirati, Merus and Alkermes, and consulting and sponsored research agreements with AstraZeneca, Janssen, Pfizer, Novartis, Merck, Zentalis, BridgeBio and Blueprint. P.J. reports employee and stock or stock options of AstraZeneca. I.I.W. reports honoraria from Genentech (Roche), Bayer, Bristol Myers Squibb, AstraZeneca, Pfizer, Merck, Guardant Health, Flame, Novartis, Sanofi, Daiichi Sankyo, Amgen, Janssen, Merus, G1 Therapeutics, Abbvie, Catalyst Therapeutics, Regeneron, Oncocyte, Medscape, Platform Health and Physicians’ Education Resources; research support from Genentech, Merck, Bristol Myers Squibb, Medimmune, Adaptive, Adaptimmune, EMD Serono, Pfizer, Takeda, Amgen, Karus, Johnson & Johnson, Bayer, Iovance, 4D, Novartis and Akoya. A.G.P. reports Pfizer. J.V.H. reports Advisory Committees: Genentech, Mirati Therapeutics, Eli Lilly, Janssen, Boehringer Ingelheim, Regeneron, Takeda, BerGenBio, Jazz, Curio Science, Novartis, AstraZeneca, BioAlta, Sanofi, Spectrum, GlaxoSmithKline, EMD Serono, BluePrint Medicine and Chugai; research support: AstraZeneca, Boehringer Ingelheim, Spectrum, Mirati, Bristol Myers Squibb and Takeda; licensing or royalties: Spectrum. H.A.A., M.T.D., A.G.C., Y.Q., R.P., D.M., D.H.O., R.M., J.A.H., L.M., S.C.M.L., R.P.J., C.L., Z.W., M.G.W., D.V., H.S., M.K., S.K., Y.N., J.T.A., J.S., L.L., H.N.K., B.C., S.S., R.L., T.Z., J.J.L., D.P., M.L.J. T.T., A.P., Y.K. and J.L.: no disclosures were reported by these authors., (© 2024. The Author(s).)

The present study aims to synthesize and characterize two quaternary ammonium (QAM) based monomers such as - dimethyl-hexadecyl-methacryloxyethyl-ammonium iodide (DHMAI) and 2-dimethyl-2-dodecyl-1-methacryloxyethyl ammonium iodine (DDMAI) and assess their cytotoxicity and antimicrobial properties. The study also aims to incorporate the optimized concentration of these monomers as copolymerizing monomers into conventional Polymethyl methacrylate (PMMA) denture base resin and evaluate their suitability for prosthetic applications. DHMAI and DDMAI monomers were synthesized through a Menschutkin reaction and their chemical structure was characterized using FT-IR and 1 H-NMR spectroscopy. Cytotoxicity was determined using Methyl Thiazolyl Tetrazolium (MTT) assay whereas antimicrobial activity was assessed using the agar-disc diffusion method. Subsequently, optimized concentrations of DHMAI or DDMAI, based on the cytotoxicity results, were added to conventional PMMA resin. Antimicrobial activity, cytotoxicity, surface hardness, and water sorption of PMMA denture base rein incorporated with DHMAI or DDMAI were evaluated. FT-IR and 1 H-NMR results confirmed the structure of monomers and copolymerization of DHMAI and DDMAI with PMMA resin. DHMAI and DDMAI monomers were found to be cytocompatible with mouse fibroblast cells up to a concentration of 5 μg/mL and 20 μg/mL respectively. In addition, incorporation of DHMAI or DDMAI at 5 μg/mL and 20 μg/mL respectively into PMMA denture base material did not affect their cytocompatibility. PMMA denture base resin incorporated with DHMAI or DDMAI significantly reduced the adhesion of microbes. Further, an increase in the surface hardness and a reduction in the water sorption was observed. Hence DHMAI and DDMAI can be considered as potential candidates for imparting antimicrobial activity to polymeric denture base materials., Competing Interests: The authors declare no conflict of interest., (© 2022 The Author(s).)

Ion current rectification is highly reported in aqueous electrochemical systems and sensors but lacks exploration in organic systems due to the additional complexity introduced by non-aqueous solvents. Herein, a detailed study on ion current rectification with highly polar and mildly polar aprotic organic solvents as a function of tetraethylammonium tetrafluoroborate supporting electrolyte concentration is presented. To explain our experimental results, we introduce a previously unreported phenomenon: the formation of a double-junction diode within the nanopore that arises due to a complex interplay between ion and solvent enrichment effects. Finite element simulations are used to explore this phenomenon and the subsequent effect on the rectifying behavior of conical quartz nanopores.

A thorough understanding of nanoscale transport properties is vital for the development and optimization of nanopore sensors. The thickness of the electrical double layers (EDLs) at the internal walls of a nanopore, as well as the dimensions of the nanopore itself, plays a crucial role in determining transport properties. Herein, we demonstrate the effect of the electrolyte concentration, which is inversely proportional to the EDL thickness, and the effect of pore size, which controls the extent of the electrical double layer overlap, on the ion current rectification phenomenon observed for conical nanopores. Experimental and numerical results showed that as the electrolyte concentration is decreased, the rectification ratio reaches a maximum, then decreases, and eventually inverts below unity. We also show that as the pore size is decreased, the rectification maximum and the inversion take place at higher electrolyte concentrations. Numerical investigations revealed that both phenomena occur due to the shifting of ion enrichment distributions within the nanopore as the electrolyte concentration or the pore size is varied., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5- subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.

Proton computed tomography (pCT) is a promising tomographic imaging modality allowing direct reconstruction of proton relative stopping power (RSP) required for proton therapy dose calculation. In this review article, we aim at highlighting the role of Monte Carlo (MC) simulation in pCT studies. After describing the requirements for performing proton computed tomography and the various pCT scanners actively used in recent research projects, we present an overview of available MC simulation platforms. The use of MC simulations in the scope of investigations of image reconstruction, and for the evaluation of optimal RSP accuracy, precision and spatial resolution omitting detector effects is then described. In the final sections of the review article, we present specific applications of realistic MC simulations of an existing pCT scanner prototype, which we describe in detail., (Copyright © 2020. Published by Elsevier GmbH.)

Hierarchical genotyping approaches can provide insights into the source, geography and temporal distribution of bacterial pathogens. Multiple hierarchical SNP genotyping schemes have previously been developed so that new isolates can rapidly be placed within pre-computed population structures, without the need to rebuild phylogenetic trees for the entire dataset. This classification approach has, however, seen limited uptake in routine public health settings due to analytical complexity and the lack of standardized tools that provide clear and easy ways to interpret results. The BioHansel tool was developed to provide an organism-agnostic tool for hierarchical SNP-based genotyping. The tool identifies split k-mers that distinguish predefined lineages in whole genome sequencing (WGS) data using SNP-based genotyping schemes. BioHansel uses the Aho-Corasick algorithm to type isolates from assembled genomes or raw read sequence data in a matter of seconds, with limited computational resources. This makes BioHansel ideal for use by public health agencies that rely on WGS methods for surveillance of bacterial pathogens. Genotyping results are evaluated using a quality assurance module which identifies problematic samples, such as low-quality or contaminated datasets. Using existing hierarchical SNP schemes for Mycobacterium tuberculosis and Salmonella Typhi, we compare the genotyping results obtained with the k-mer-based tools BioHansel and SKA, with those of the organism-specific tools TBProfiler and genotyphi, which use gold-standard reference-mapping approaches. We show that the genotyping results are fully concordant across these different methods, and that the k-mer-based tools are significantly faster. We also test the ability of the BioHansel quality assurance module to detect intra-lineage contamination and demonstrate that it is effective, even in populations with low genetic diversity. We demonstrate the scalability of the tool using a dataset of ~8100 S . Typhi public genomes and provide the aggregated results of geographical distributions as part of the tool's output. BioHansel is an open source Python 3 application available on PyPI and Conda repositories and as a Galaxy tool from the public Galaxy Toolshed. In a public health context, BioHansel enables rapid and high-resolution classification of bacterial pathogens with low genetic diversity.

"Smart" polymeric nanoformulations are evolving as a promising therapeutic, diagnostic paradigm. The polymeric nanovehicles demonstrated excellent capability to encapsulate theranostic cargos and their successful delivery in physiological conditions and even to monitor the therapeutic response. Currently, polymer nanogels (NGs) are established as capable carriers toward triggered delivery of diverse therapeutic and diagnostic agents. Notably, biodegradable and "intelligent" NGs constructed from intelligent polymers are highly beneficial because of their responsiveness toward endogenous as well as exogenous stimuli like pH gradients, bioresponsiveness, photoresponsiveness, temperature, and so on. In the past decade, plenty of multifunctional NGs with excellent targetability and sensitivity were reported for a wide range of theragnostic applications. This mini-review briefly propounds the synthesis strategies of "smart" NGs and summarizes the notable applications like delivery of genetic materials, anticancer agents, photodynamic/photothermal therapies, imaging, and biosensing. Herein, we have also addressed the current clinical status of NGs and the major challenges that are essential to overcome for the further advancement of NGs for specific applications., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Proton CT (pCT) is a promising new imaging technique that can reconstruct relative stopping power (RSP) more accurately than x-ray CT in each cubic millimeter voxel of the patient. This, in turn, will result in better proton range accuracy and, therefore, smaller planned tumor volumes (PTV). The hardware description and some reconstructed images have previously been reported. In a series of two contributions, we focus on presenting the software algorithms that convert pCT detector data to the final reconstructed pCT images for application in proton treatment planning. There were several options on how to accomplish this, and we will describe our solutions at each stage of the data processing chain. In the first paper of this series, we present the data acquisition with the pCT tracking and energy-range detectors and how the data are preprocessed, including the conversion to the well-formatted track information from tracking data and water-equivalent path length from the data of a calibrated multi-stage energy-range detector. These preprocessed data are then used for the initial image formation with an FDK cone-beam CT algorithm. The output of data acquisition, preprocessing, and FDK reconstruction is presented along with illustrative imaging results for two phantoms, including a pediatric head phantom. The second paper in this series will demonstrate the use of iterative solvers in conjunction with the superiorization methodology to further improve the images resulting from the upfront FDK image reconstruction and the implementation of these algorithms on a hybrid CPU/GPU computer cluster.

Verotoxigenic Escherichia coli (VTEC) are food- and water-borne pathogens associated with both sporadic illness and outbreaks of enteric disease. While it is known that cattle are reservoirs of VTEC, little is known about the genomic variation of VTEC in cattle, and whether the variation in genomes reported for human outbreak strains is consistent with individual animal or group/herd sources of infection. A previous study of VTEC prevalence identified serotypes carried persistently by three consecutive cohorts of heifers within a closed herd of cattle. This present study aimed to: (i) determine whether the genomic relatedness of bovine isolates is similar to that reported for human strains associated with single source outbreaks, (ii) estimate the rates of genome change among dominant serotypes over time within a cattle herd, and (iii) identify genomic features of serotypes associated with persistence in cattle. Illumina MiSeq genome sequencing and genotyping based on allelic and single nucleotide variations were completed, while genome change over time was measured using Bayesian evolutionary analysis sampling trees. The accessory genome, including the non-protein-encoding intergenic regions (IGRs), virulence factors, antimicrobial-resistance genes and plasmid gene content of representative persistent and sporadic cattle strains were compared using Fisher's exact test corrected for multiple comparisons. Herd strains from serotypes O6:H34 ( n =22), O22:H8 ( n =30), O108:H8 ( n =39), O139:H19 ( n =44) and O157:H7 ( n =106) were readily distinguishable from epidemiologically unrelated strains of the same serotype using a similarity threshold of 10 or fewer allele differences between adjacent nodes. Temporal-cohort clustering within each serotype was supported by date randomization analysis. Substitutions per site per year were consistent with previously reported values for E. coli ; however, there was low branch support for these values. Acquisition of the phage-encoded Shiga toxin 2 gene in serotype O22:H8 was observed. Pan-genome analyses identified accessory regions that were more prevalent in persistent serotypes ( P ≤0.05) than in sporadic serotypes. These results suggest that VTEC serotypes from a specific cattle population are highly clonal with a similar level of relatedness as human single-source outbreak-associated strains, but changes in the genome occur gradually over time. Additionally, elements in the accessory genomes may provide a selective advantage for persistence of VTEC within cattle herds.

A previously isolated a bacteriophage, vB&#95;EcoS&#95;AKFV33 of T5virus, demonstrated great potential in biocontrol of Shiga toxigenic Escherichia coli (STEC) O157. This study further evaluated its potential as a biocontrol agent in broth culture against other important non-O157 serogroups of STEC and Salmonella. AKFV33 was capable of lysing isolates of STEC serogroups O26 (n = 1), O145 (n = 1) and Salmonella enterica serovars (n = 6). In a broth culture microplate system, efficacy of AKFV33 for killing STEC O26:H11, O145:NM and Salmonella was improved (P < 0.05) at a lower multiplicity of infection and sampling time (6-10 h), when STEC O157:H7 was also included in the culture. This phage was able to simultaneously reduce numbers of STEC and Salmonella in mixtures with enhanced activity (P < 0.05) against O157:H7 and O26:H11, offering great promise for control of multiple zoonotic pathogens at both pre and post-harvest.

Arenes undergo rearrangement of phenyl, alkyl, halogen and other groups through the intermediacy of ipso arenium ions in which a proton is attached at the same carbon as the migrating substituent. Interconversions among the six quaterphenyl isomers have been studied here as a model for rearrangements of linear polyphenyls. All reactions were carried out in 1 M CF 3 SO 3 H (TfOH) in dichloroethane at 150 °C in a microwave reactor for 30-60 min, with product formation assessed by high field NMR analysis. Under these reaction conditions, m,p' -quaterphenyl is the equilibrium product. This isomer is unchanged by the reaction conditions and all other quaterphenyl isomers rearrange to m,p' as the dominant or sole product. DFT computations with inclusion of implicit solvation support a complex network of phenyl and biphenyl shifts, with barriers to rearrangement in the range of 10-21 kcal/mol. Consistent with experiments, the lowest energy arenium ion located on this surface is due to protonation of m,p' -quaterphenyl. This supports thermodynamic control based on carbocation energies., (Copyright © 2019, Skraba-Joiner et al.; licensee Beilstein-Institut.)

We report high-quality closed reference genomes for 1 bovine strain and 10 human Shiga toxin (Stx)-producing Escherichia coli (STEC) strains from serogroups O26, O45, O91, O103, O104, O111, O113, O121, O145, and O157. We also report draft assemblies, with standardized metadata, for 360 STEC strains isolated from watersheds, animals, farms, food, and human infections., (© Crown copyright 2019.)

Calcium is the key macromineral having a role in skeletal structure and function, muscle contraction, and neurotransmission. Bone remodeling is maintained through a constant balance between calcium resorption and deposition. Calcium deficiency is resolved through calcium supplementation, and among the supplements, water-soluble organic molecules attracted great pharmaceutical interest. Calcium glucoheptonate is a highly water-soluble organic calcium salt having clinical use; however, detailed investigations on its biological effects are limited. We assessed the effects of calcium glucoheptonate on cell viability and proliferation of osteoblast-like MG-63 cells. Calcium uptake and mineralization were evaluated using Alizarin red staining of osteoblast-like MG-63 cells treated with calcium glucoheptonate. Expression of osteogenic markers were monitored by western blotting, immunofluorescence, and qRT-PCR assays. Increased proliferation and calcium uptake were observed in the MG-63 cells treated with calcium glucoheptonate. The treatment also increased the expression of osteopontin and osteogenic genes such as collagen-1, secreted protein acidic and cysteine rich (SPARC), and osteocalcin. Calcium glucoheptonate treatment did not exert any cytotoxicity on colorectal and renal epithelial cells, indicating the safety of the treatment. This is the first report with evidence for its beneficial effect for pharmaceutical use in addressing calcium deficiency conditions., Competing Interests: The authors have read the journal's policy and authors of this manuscript have the following competing interests: SV is employed in Global Calcium Pvt. Ltd. and his salary is paid by Global Calcium Pvt. Ltd. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products to declare.

Natural killer (NK) cells develop from common progenitors but diverge into distinct subsets, which differ in cytokine production, cytotoxicity, homing, and memory traits. Given their promise in adoptive cell therapies for cancer, a deeper understanding of regulatory modules controlling clinically beneficial NK phenotypes is of high priority. We report integrated "-omics" analysis of human NK subsets, which revealed super-enhancers associated with gene cohorts that may coordinate NK functions and localization. A transcription factor-based regulatory scheme also emerged, which is evolutionarily conserved and shared by innate and adaptive lymphocytes. For both NK and T lineages, a TCF1-LEF1-MYC axis dominated the regulatory landscape of long-lived, proliferative subsets that traffic to lymph nodes. In contrast, effector populations circulating between blood and peripheral tissues shared a PRDM1-dominant landscape. This resource defines transcriptional modules, regulated by feedback loops, which may be leveraged to enhance phenotypes for NK cell-based therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Shiga toxin-producing Escherichia coli (STEC) are important enteric pathogens responsible for sporadic cases and outbreaks of gastroenteritis. E.coli O157:H7/NM (STEC O157) are the most commonly known STEC serotypes but it is now increasingly apparent that non-O157 STEC serotypes have been underreported in the past because they were not part of routine screening in many front-line laboratories. The Canadian Public Health Laboratory Network (CPHLN) has identified the need for improved detection and surveillance of non-O157 STEC and has developed the following recommendations to assist in the decision-making process for clinical and reference microbiology laboratories. These recommendations should be followed to the best of a laboratory's abilities based on the availability of technology and resources. The CPHLN recommends that when screening for the agents of bacterial gastroenteritis from a stool sample, front-line laboratories use either a chromogenic agar culture or a culture-independent diagnostic test (CIDT). CIDT options include nucleic acid amplification tests (NAATs) to detect Shiga toxin genes or enzyme immunoassays (EIAs) to detect Shiga toxins. If either CIDT method is positive for possible STEC, laboratories must have a mechanism to culture and isolate STEC in order to support both provincial and national surveillance as well as outbreak investigations and response. These CPHLN recommendations should result in improved detection of STEC in patients presenting with diarrhea, especially when due to the non-O157 serotypes. These measures should enhance the overall quality of healthcare and food safety, and provide better protection of the public via improved surveillance and outbreak detection and response., Competing Interests: Conflict of interest: None.

Tetherin (BST-2 or CD317) is an interferon-inducible transmembrane protein that inhibits virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on simian immunodeficiency virus (SIV) infection, tetherin alleles were sequenced from 146 rhesus macaques, including 68 animals infected with wild-type SIV mac 239 and 47 animals infected with SIV mac 239Δ nef Since Nef is the viral gene product of SIV that counteracts restriction by tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 15 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Acute viremia did not differ significantly among the wild-type SIV-infected animals; however, differences in acute viral loads were associated with polymorphisms in tetherin among the animals infected with SIVΔ nef In particular, polymorphisms at positions 43 and 111 (P 43 and H 111 ) were associated with lower acute-phase viral loads for SIVΔ nef infection. These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef. IMPORTANCE As a consequence of ongoing evolutionary conflict with viral pathogens, tetherin has accumulated numerous species-specific differences that represent important barriers to the transmission of viruses between species. This study reveals extensive polymorphism in rhesus macaque tetherin and identifies specific alleles that are associated with lower viral loads during the first few weeks after infection with nef -deleted SIV. These observations suggest that the variable selective pressure of viral pathogens, in addition to driving the diversification of tetherin among species, also operates within certain species to maintain sequence variation in tetherin., (Copyright © 2018 American Society for Microbiology.)

In this study, fecal samples were collected from a closed beef herd in Alberta, Canada from 2012 to 2015. To limit serotype bias, which was observed in enrichment broth cultures, Verotoxigenic Escherichia coli (VTEC) were isolated directly from samples using a hydrophobic grid-membrane filter verotoxin immunoblot assay. Overall VTEC isolation rates were similar for three different cohorts of yearling heifers on both an annual (68.5 to 71.8%) and seasonal basis (67.3 to 76.0%). Across all three cohorts, O139:H19 (37.1% of VTEC-positive samples), O22:H8 (15.8%) and O?(O108):H8 (15.4%) were among the most prevalent serotypes. However, isolation rates for serotypes O139:H19, O130:H38, O6:H34, O91:H21, and O113:H21 differed significantly between cohort-years, as did isolation rates for some serotypes within a single heifer cohort. There was a high level of VTEC serotype diversity with an average of 4.3 serotypes isolated per heifer and 65.8% of the heifers classified as "persistent shedders" of VTEC based on the criteria of >50% of samples positive and ≥4 consecutive samples positive. Only 26.8% (90/336) of the VTEC isolates from yearling heifers belonged to the human disease-associated seropathotypes A (O157:H7), B (O26:H11, O111:NM), and C (O22:H8, O91:H21, O113:H21, O137:H41, O2:H6). Conversely, seropathotypes B (O26:NM, O111:NM) and C (O91:H21, O2:H29) strains were dominant (76.0%, 19/25) among VTEC isolates from month-old calves from this herd. Among VTEC from heifers, carriage rates of vt1 , vt2 , vt1+vt2 , eae , and hlyA were 10.7, 20.8, 68.5, 3.9, and 88.7%, respectively. The adhesin gene saa was present in 82.7% of heifer strains but absent from all of 13 eae +ve strains (from serotypes/intimin types O157:H7/γ1, O26:H11/β1, O111:NM/θ, O84:H2/ζ, and O182:H25/ζ). Phylogenetic relationships inferred from wgMLST and pan genome-derived core SNP analysis showed that strains clustered by phylotype and serotype. Further, VTEC strains of the same serotype usually shared the same suite of antibiotic resistance and virulence genes, suggesting the circulation of dominant clones within this distinct herd. This study provides insight into the diverse and dynamic nature of VTEC populations within groups of cattle and points to a broad spectrum of human health risks associated with these E. coli strains.

Cervicovaginal epithelium plays a critical role in determining the outcome of virus transmission in the female reproductive tract (FRT) by initiating or suppressing transmission-facilitating mucosal immune responses in naïve and SIVmac239Δnef-vaccinated animals, respectively. In this study, we examined the very early responses of cervical epithelium within 24 h after vaginal exposure to SIV in naive and SIVmac239Δnef-vaccinated rhesus macaques. Using both ex vivo and in vivo experimental systems, we found that vaginal exposure to SIV rapidly induces a broad spectrum of pro-inflammatory responses in the epithelium associated with a reciprocal regulation of NF-kB and glucocorticoid receptor (GR) signaling pathways. Conversely, maintenance of high-level GR expression and suppression of NF-kB expression in the epithelium were associated with an immunologically quiescent state in the FRT mucosa and protection against vaginal challenge in SIVmac239Δnef-vaccinated animals. We show that the immunologically quiescent state is induced by FCGR2B-immune complexes interactions that modify the reciprocal regulation of NF-kB and GR signaling pathways. Our results suggest that targeting the balance of NF-kB and GR signaling in early cervicovaginal epithelium responses could moderate mucosal inflammation and target cell availability after vaginal infection, thereby providing a complementary approach to current prevention strategies.

In 1 M triflic acid/dichloroethane, anthracene is protonated at C9, and the resulting 9-anthracenium ion is easily observed by NMR at ambient temperature. When heated as a dilute solution in triflic acid/dichloroethane, anthracene undergoes conversion to phenanthrene as the major volatile product. Minor dihydro and tetrahydro products are also observed. MALDI analysis supports the simultaneous formation of oligomers, which represent 10-60% of the product. Phenanthrene is nearly inert to the same superacid conditions. DFT and CCSD(T)//DFT computational models were constructed for isomerization and automerization mechanisms. These reactions are believed to occur by cationic ring pirouettes which pass through spirocyclic intermediates. The direct aryl pirouette mechanism for anthracene has a predicted DFT barrier of 33.6 kcal/mol; this is too high to be consistent with experiment. The ensemble of experimental and computational models supports a multistep isomerization process, which proceeds by reduction to 1,2,3,4-tetrahydroanthracene, acid-catalyzed isomerization to 1,2,3,4-tetrahydrophenanthrene with a predicted DFT barrier of 19.7 kcal/mol, and then reoxidation to phenanthrene. By contrast, DFT computations support a direct pirouette mechanism for automerization of outer ring carbons in phenanthrene, a reaction demonstrated previously by Balaban through isotopic labeling.

Produce has become a major source of foodborne illness, and may become contaminated through surface water irrigation. The objectives of this study were to (i) determine the frequency of verotoxigenic E. coli (VTEC), Listeria monocytogenes, and Salmonella in surface waters used for irrigation in the Lower Mainland of British Columbia, (ii) assess the suitability of fecal coliforms and generic E. coli as hygiene indicators, and (iii) investigate the correlations of environmental factors with pathogen occurrence. Water samples were collected semi-monthly for 18 months from seven irrigation ditches across the Serpentine and Sumas watersheds. VTEC colonies on water filters were detected using a verotoxin colony immunoblot, and the presence of virulence genes vt1 and vt2 was ascertained via multiplex PCR. Detection of L. monocytogenes and Salmonella was completed using standard, Health Canada Compendium of Analytical Methods. Fecal coliforms and generic E. coli were enumerated by 3M™ Petrifilm™ and filtration methods, and meteorological and geographic data were collected from government records. VTEC, L. monocytogenes, and Salmonella were detected in 4.93%, 10.3%, and 2.69% of 223 samples, respectively. L. monocytogenes occurrence was greatest in the Serpentine watershed (χ2; p < 0.05), and was most common during the winter and fall (Fisher exact test; p < 0.05). Site dependence of VTEC and Salmonella occurrence was observed within watersheds (Fisher's exact test; p < 0.10). Pathogen occurrence correlated with fecal coliform counts (r = 0.448), while VTEC occurrence also correlated with precipitation over the five days before sampling (r = 0.239). The density of upstream livestock correlated with VTEC (rs = 0.812), and L. monocytogenes (rs = 0.841) detection. These data show that foodborne pathogens are present in the waters used for irrigation in the Lower Mainland of British Columbia, but their frequency may depend on spatial and temporal factors.

A precise relative stopping power map of the patient is crucial for accurate particle therapy. Charged particle imaging determines the stopping power either tomographically - particle computed tomography (pCT) - or by combining prior knowledge from particle radiography (pRad) and x-ray CT. Generally, multiple Coulomb scattering limits the spatial resolution. Compared to protons, heavier particles scatter less due to their lower charge/mass ratio. A theoretical framework to predict the most likely trajectory of particles in matter was developed for light ions up to carbon and was found to be the most accurate for helium comparing for fixed initial velocity. To further investigate the potential of helium in particle imaging, helium computed tomography (HeCT) and radiography (HeRad) were studied at the Heidelberg Ion-Beam Therapy Centre (HIT) using a prototype pCT detector system registering individual particles, originally developed by the U.S. pCT collaboration. Several phantoms were investigated: modules of the Catphan QA phantom for analysis of spatial resolution and achievable stopping power accuracy, a paediatric head phantom (CIRS) and a custommade phantom comprised of animal meat enclosed in a 2 % agarose mixture representing human tissue. The pCT images were reconstructed applying the CARP iterative reconstruction algorithm. The MTF 10% was investigated using a sharp edge gradient technique. HeRad provides a spatial resolution above that of protons (MTF10 10% =6.07 lp/cm for HeRad versus MTF 10% =3.35 lp/cm for proton radiography). For HeCT, the spatial resolution was limited by the number of projections acquired (90 projections for a full scan). The RSP accuracy for all inserts of the Catphan CTP404 module was found to be 2.5% or better and is subject to further optimisation. In conclusion, helium imaging appears to offer higher spatial resolution compared to proton imaging. In future studies, the advantage of helium imaging compared to other imaging modalities in clinical applications will be further explored.

Apoptosis plays a central role in developmental and pathological angiogenesis and vessel regression. Bim is a pro-apoptotic Bcl-2 family member that plays a prominent role in both developmental and pathological ocular vessel regression, and neovascularization. Endothelial cells (EC) and pericytes (PC) each play unique roles during vascular development, maintenance and regression. We recently showed that germline deletion of Bim results in persistent hyaloid vasculature, increased retinal vascular density and prevents retinal vessel regression in response to hyperoxia. To determine whether retinal vascular regression is attributable to Bim expression in EC or PC we generated mice carrying a conditional Bim allele (BimFlox/Flox) and VE-cadherin-cre (BimEC mice) or Pdgfrb-cre (BimPC mice). BimEC and BimPC mice demonstrated attenuated hyaloid vessel regression and postnatal retinal vascular remodeling. We also observed decreased retinal vascular apoptosis and proliferation. Unlike global Bim -/- mice, mice conditionally lacking Bim in EC or PC underwent hyperoxia-mediated vessel obliteration and subsequent retinal neovascularization during oxygen-induced ischemic retinopathy similar to control littermates. Thus, understanding the cell autonomous role Bim plays in the retinal vascular homeostasis will give us new insight into how to modulate pathological retinal neovascularization and vessel regression to preserve vision.

The live attenuated vaccine (LAV) SIVmac239Δnef (SIVΔnef) confers the best protection among all the vaccine modalities tested in rhesus macaque model of HIV-1 infection. This vaccine has a unique feature of time-dependent protection: macaques are not protected at 3-5 weeks post vaccination (WPV), whereas immune protection emerges between 15 and 20 WPV. Although the exact mechanisms of the time-dependent protection remain incompletely understood, studies suggested that both cellular and humoral immunities contribute to this time-dependent protection. To further elucidate the mechanisms of protection induced by SIVΔnef, we longitudinally compared the global gene expression profiles of SIV Gag-CM9+ CD8+ (Gag-specific CD8+) T cells from peripheral blood of Mamu-A*01+ rhesus macaques at 3 and 20 WPV using rhesus microarray. We found that gene expression profiles of Gag-specific CD8+ T cells at 20 WPV are qualitatively different from those at 3 WPV. At 20 WPV, the most significant transcriptional changes of Gag-specific CD8+ T cells were genes involved in TCR signaling, differentiation and maturation toward central memory cells, with increased expression of CCR7, TCRα, TCRβ, CD28 and decreased expression of CTLA-4, IFN-γ, RANTES, granzyme A and B. Our study suggests that a higher quality of SIV-specific CD8+ T cells elicited by SIVΔnef over time contributes to the maturation of time-dependent protection.

Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4 + T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4 + T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4 + T cells, jejunal CCR5 + CD4 + T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G , MX2 , and TRIM25 , which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4 + T cell memory subsets at the peak of acute infection. Jejunal CCR5 + CD4 + T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4 + T cells to lentiviral infection. IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4 + T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4 + T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4 + T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4 + T cells to SIV infection., (Copyright © 2017 American Society for Microbiology.)

In the SIV (simian immunodeficiency virus)-rhesus macaque model of HIV-1 (human immunodeficiency virus type I) transmission to women, one hallmark of the mucosal response to exposure to high doses of SIV is CD4 T-cell recruitment that fuels local virus expansion in early infection. In this study, we systematically analyzed the cellular events and chemoattractant profiles in cervical tissues that precede CD4 T-cell recruitment. We show that vaginal exposure to the SIV inoculum rapidly induces chemokine expression in cervical epithelium including CCL3, CCL20, and CXCL8. The chemokine expression is associated with early recruitment of macrophages and plasmacytoid dendritic cells that are co-clustered underneath the cervical epithelium. Production of chemokines CCL3 and CXCL8 by these cells in turn generates a chemokine gradient that is spatially correlated with the recruitment of CD4 T cells. We further show that the protection of SIVmac239Δnef vaccination against vaginal challenge is correlated with the absence of this epithelium-innate immune cell-CD4 T-cell axis response in the cervical mucosa. Our results reveal a critical role for cervical epithelium in initiating early mucosal responses to vaginal infection, highlight an important role for macrophages in target cell recruitment, and provide further evidence of a paradoxical dampening effect of a protective vaccine on these early mucosal responses.

Objectives: Human infection with Escherichia coli O157:H7/NM has historically been associated with consumption of undercooked ground beef. The purpose of this paper is to investigate the correlation of the decline in E. coli O157:H7/NM infections in Canada with the introduction of control efforts in ground beef by industry., Methods: The human incidence of E. coli O157:H7/NM, prevalence in ground beef and interventions from 1996 to 2014 were analyzed. Pathogen prevalence data were obtained from federal government and industry surveillance and inspection/compliance programs. A survey of the largest ground beef producers in Canada was conducted to identify when interventions were implemented., Results: The incidence of E. coli O157:H7/NM infections in Canada declined from ≈4 cases/100 000 to ≈1 case/100000 from 2000 to 2010. Verotoxigenic Escherichia coli (VTEC) prevalence in ground beef sold at retail declined from about 30% around the year 2000 to <2% since 2012. Other measures of the prevalence of E. coli, VTEC, and E. coli O157:H7/NM in beef and ground beef also declined. The number and types of interventions implemented in the major beef processing establishments in Canada increased from 1996 to 2016., Conclusion: The observed decline in human illnesses and pathogen levels in relation to retail meats was associated with the introduction of control efforts by industry, federal and provincial/territorial governments, and the general population. Industry-led changes in beef processing along with the introduction of food safety policies, regulations, and public education have led to improved food safety in Canada.

Defining the correlates of immune protection conferred by SIVΔnef, the most effective vaccine against SIV challenge, could enable the design of a protective vaccine against HIV infection. Here we provide a comprehensive assessment of immune responses that protect against SIV infection through detailed analyses of cellular and humoral immune responses in the blood and tissues of rhesus macaques vaccinated with SIVΔnef and then vaginally challenged with wild-type SIV. Despite the presence of robust cellular immune responses, animals at 5 weeks after vaccination displayed only transient viral suppression of challenge virus, whereas all macaques challenged at weeks 20 and 40 post-SIVΔnef vaccination were protected, as defined by either apparent sterile protection or significant suppression of viremia in infected animals. Multiple parameters of CD8 T cell function temporally correlated with maturation of protection, including polyfunctionality, phenotypic differentiation, and redistribution to gut and lymphoid tissues. Importantly, we also demonstrate the induction of a tissue-resident memory population of SIV-specific CD8 T cells in the vaginal mucosa, which was dependent on ongoing low-level antigenic stimulation. Moreover, we show that vaginal and serum antibody titers inversely correlated with post-challenge peak viral load, and we correlate the accumulation and affinity maturation of the antibody response to the duration of the vaccination period as well as to the SIVΔnef antigenic load. In conclusion, maturation of SIVΔnef-induced CD8 T cell and antibody responses, both propelled by viral persistence in the gut mucosa and secondary lymphoid tissues, results in protective immune responses that are able to interrupt viral transmission at mucosal portals of entry as well as potential sites of viral dissemination., Competing Interests: The authors have declared that no competing interests exist.

Acute human immunodeficiency virus (HIV) infection represents a period of intense immune perturbation and activation of the host immune system. Study of the eclipse and viral expansion phases of infection is difficult in humans, but studies in nonprogressive and progressive nonhuman primate (NHP) infection models can provide significant insight into critical events occurring during this time. Cytokines, chemokines, and other soluble immune factors were measured in longitudinal samples from rhesus macaques infected with either SIVmac251 (progressive infection) or SIVmac239Δnef (attenuated/nonprogressive infection) and from African green monkeys infected with SIVsab9315BR (nonpathogenic infection). Levels of acute-phase peak viral replication were highest in SIVmac251 infection but correlated positively with viremia at 3 months postinfection in all three infection models. SIVmac251 infection was associated with stronger corresponding acute-phase cytokine/chemokine responses than the nonprogressive infections. The production of interleukin 15 (IL-15), IL-18, gamma interferon (IFN-γ), granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 1β (MIP-1β), and serum amyloid A protein (SAA) during acute SIVmac251 infection, but not during SIVmac239Δnef or SIVsab9315BR infection, correlated positively with chronic viremia at 3 months postinfection. Acute-phase production of MCP-1 correlated with viremia at 3 months postinfection in both nonprogressive infections. Finally, a positive correlation between the acute-phase area under the curve (AUC) for IL-6 and soluble CD40 ligand (sCD40L) and chronic viremia was observed only for the nonprogressive infection models. While we observed dynamic acute inflammatory immune responses in both progressive and nonprogressive SIV infections, the responses in the nonprogressive infections were not only lower in magnitude but also qualitatively different biomarkers of disease progression., Importance: NHP models of HIV infection constitute a powerful tool with which to study viral pathogenesis in order to gain critical information for a better understanding of HIV infection in humans. Here we studied progressive and nonprogressive simian immunodeficiency virus (SIV) infection models in both natural and nonnatural host NHP species. Regardless of the pathogenicity of the virus infection and regardless of the NHP species studied, the magnitude of viremia, as measured by area under the curve, during the first 4 weeks of infection correlated positively with viremia in chronic infection. The magnitude of cytokine and chemokine responses during primary infection also correlated positively with both acute-phase and chronic viremia. However, the pattern and levels of specific cytokines and chemokines produced differed between nonprogressive and progressive SIV infection models. The qualitative differences in the early immune response in pathogenic and nonpathogenic infections identified here may be important determinants of the subsequent disease course., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Vaccination with SIVmac239Δnef provides robust protection against subsequent challenge with wild-type simian immunodeficiency virus (SIV), but safety issues have precluded designing an HIV-1 vaccine based on a live-attenuated virus concept. Safe immunogens and adjuvants that could reproduce identified immune correlates of SIVmac239Δnef protection therefore offer an alternative path for development of an HIV vaccine. Here we describe SIV envelope trimeric gp41 (gp41t) immunogens based on a protective correlate of antibodies to gp41t concentrated on the path of virus entry by the neonatal Fc receptor (FcRn) in cervical vaginal epithelium. We developed a gp41t immunogen-monophosphoryl lipid A adjuvant liposomal nanoparticle for intramuscular (i.m.) immunization and a gp41t-Fc immunogen for intranasal immunization for pilot studies in mice, rabbits, and rhesus macaques. Repeated immunizations to mimic persistent antigen exposure in infection elicited gp41t antibodies in rhesus macaques that were detectable in FcRn+ cervical vaginal epithelium, thus recapitulating one key feature of SIVmac239Δnef vaccinated and protected animals. Although this strategy did not reproduce the system of local production of antibody in SIVmac239Δnef-vaccinated animals, passive immunization experiments supported the concept that sufficiently high levels of antibody can be concentrated by the FcRn at mucosal frontlines, thus setting the stage for assessing protection against vaginal challenge by gp41t immunization., Competing Interests: and Source of Funding: The authors declare no conflicts of interest.

Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain several drug resistance genes., (© Crown copyright 2016.)

Unlabelled: Bacteriophages present huge potential both as a resource for developing novel tools for bacterial diagnostics and for use in phage therapy. This potential is also valid for bacteriophages specific for Yersinia enterocolitica To increase our knowledge of Y. enterocolitica-specific phages, we characterized two novel yersiniophages. The genomes of the bacteriophages vB&#95;YenM&#95;TG1 (TG1) and vB&#95;YenM&#95;ϕR1-RT (ϕR1-RT), isolated from pig manure in Canada and from sewage in Finland, consist of linear double-stranded DNA of 162,101 and 168,809 bp, respectively. Their genomes comprise 262 putative coding sequences and 4 tRNA genes and share 91% overall nucleotide identity. Based on phylogenetic analyses of their whole-genome sequences and large terminase subunit protein sequences, a genus named Tg1virus within the family Myoviridae is proposed, with TG1 and ϕR1-RT (R1RT in the ICTV database) as member species. These bacteriophages exhibit a host range restricted to Y. enterocolitica and display lytic activity against the epidemiologically significant serotypes O:3, O:5,27, and O:9 at and below 25°C. Adsorption analyses of lipopolysaccharide (LPS) and OmpF mutants demonstrate that these phages use both the LPS inner core heptosyl residues and the outer membrane protein OmpF as phage receptors. Based on RNA sequencing and quantitative proteomics, we also demonstrate that temperature-dependent infection is due to strong repression of OmpF at 37°C. In addition, ϕR1-RT was shown to be able to enter into a pseudolysogenic state. Together, this work provides further insight into phage-host cell interactions by highlighting the importance of understanding underlying factors which may affect the abundance of phage host receptors on the cell surface., Importance: Only a small number of bacteriophages infecting Y. enterocolitica, the predominant causative agent of yersiniosis, have been previously described. Here, two newly isolated Y. enterocolitica phages were studied in detail, with the aim of elucidating the host cell receptors required for infection. Our research further expands the repertoire of phages available for consideration as potential antimicrobial agents or as diagnostic tools for this important bacterial pathogen., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Pooled feces collected over two years from 1749 transport trailers hauling western-Canadian slaughter cattle were analysed by PCR for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157. Sequential immunomagnetic separation was then used to collect bacterial isolates (n = 1035) from feces positive for target serogroups. Isolated bacteria were tested by PCR to confirm serogroup and the presence of eae, ehxA, stx1, and stx2 virulence genes. Based on PCR screening, serogroup prevalence in feces ranged from 7.0% (O145) to 94.4% (O103) with at least 3 serogroups present in 79.5% of samples. Origin of cattle affected serogroup PCR prevalence and O157 was most prevalent in feces from south-west Alberta (P < 0.001). All serogroups demonstrated seasonal variations in PCR prevalence, with O26, O45, O103, O121, and O157 least prevalent (P < 0.001) in cooler winter months, while uncommon serogroups O111 and O145 increased in prevalence during winter (P < 0.001). However, isolates collected during winter were predominantly from serogroups O103 and O45. No seasonal variation was noted in proportion of isolates which were Shiga toxin containing E. coli (STEC; P = 0.18) or positive for Shiga toxin and eae (enterohemorrhagic E. coli; EHEC; P = 0.29). Isolates of serogroups O111, O145, and O157 were more frequently EHEC than were others, although 37.6-54.3% of isolates from other serogroups were also EHEC. Shiga-toxin genes present also varied by geographic origin of cattle (P < 0.05) in all serogroups except O157. As cattle within feedlots are sourced from multiple regions, locational differences in serogroup prevalence and virulence genes imply existence of selection pressures for E. coli and their virulence in western-Canadian cattle. Factors which reduce carriage or expression of virulence genes, particularly in non-O157 serogroups, should be investigated.

A hydrophobic grid membrane filtration-Shiga toxin immunoblot method was used to examine the prevalence of Shiga toxin-producing Escherichia coli (STEC) in four watersheds located in the Lower Mainland of British Columbia, Canada, a region characterized by rapid urbanization and intensive agricultural activity. STEC were recovered from 21.6, 23.2, 19.5, and 9.2% of surface water samples collected monthly from five sites in each watershed over a period of 1 year. Overall prevalence was subject to seasonal variation however, ranging between 13.3% during fall months and 34.3% during winter months. STEC were also recovered from 23.8% of sediment samples collected in one randomly selected site. One hundred distinct STEC isolates distributed among 29 definitive and 4 ambiguous or indeterminate serotypes were recovered from water and sediments, including isolates from Canadian "priority" serogroups O157 (3), O26 (4), O103 (5), and O111 (7). Forty seven isolates were further characterized by analysis of whole genome sequences to detect Shiga toxin gene (stx 1 and stx 2), intimin gene (eaeA) allelic variants and acquired virulence factors. These analyses collectively showed that surface waters from the region support highly diverse STEC populations that include strains with virulence factors commonly associated with human pathotypes. The present work served to characterize the microbiological hazard implied by STEC to support future assessments of risks to public health arising from non-agricultural and agricultural uses of surface water resources in the region.

Background: Salmonellae are major worldwide zoonotic pathogens infecting a wide range of vertebrate species including humans. Consumption of contaminated dairy products and contact with dairy cattle represent a common source of non-typhoidal Salmonella infection in humans. Despite a large number of small-scale dairy farms in Addis Ababa and its surrounding districts, little is known about the status of Salmonella in these farms., Results: Salmonella was recovered from the feces of at least one animal in 7.6% (10/132) of the dairy farms. Out of 1203 fecal samples examined, 30 were positive for Salmonella resulting in a weighted animal level prevalence of 2.3%. Detection of diarrhea in an animal and in a farm was significantly associated with animal level (p = 0.012) and herd level (p < 0.001) prevalence of Salmonella. Animal level prevalence of Salmonella was significantly associated with age (p = 0.023) and study location; it was highest among those under 6 months of age and in farms from Adaa district and Addis Ababa (p < 0.001). Nine different serotypes were identified using standard serological agglutination tests. The most frequently recovered serotypes were Salmonella Typhimurium (23.3%), S. Saintpaul (20%), S. Kentucky (16.7%) and S. Virchow (16.7%). All isolates were resistant or intermediately resistant to at least one of the 18 drugs tested. Twenty-six (86.7%), 19 (63.3 %), 18 (60%), 16 (53.3%) of the isolates were resistant to streptomycin, nitrofurantoin, sulfisoxazole and tetracycline , respectively. Resistance to 2 drugs was detected in 27 (90%) of the isolates. Resistance to 3 or more drugs was detected in 21 (70%) of the isolates, while resistance to 7 or more drugs was detected in 11 (36.7%) of the isolates. The rate of occurrence of multi-drug resistance (MDR) in Salmonella strains isolated from dairy farms in Addis Ababa was significantly higher than those isolated from farms outside of Addis Ababa (p = 0.009). MDR was more common in S. Kentucky, S. Virchow and S. Saintpaul., Conclusion: Isolation of Salmonella serotypes commonly known for causing human salmonellosis that are associated with an MDR phenotype in dairy farms in close proximity with human population is a major public health concern. These findings imply the need for a strict pathogen reduction strategy.

Isolates of Salmonella enterica subsp. enterica serovar Heidelberg are often associated with poultry products and may cause severe human illness. Here, we report the fully assembled genome and plasmid sequences of three S. Heidelberg strains with phage types 9, 29, and 41., (Copyright © 2016 Labbé et al.)

The conceptual dehydrogenation of pericyclic reactions yields dehydropericyclic processes, which usually lead to strained or reactive intermediates. This is a simple scheme for inventing new chemical reactions. Computational results on two novel dehydropericyclic reactions are presented here. Conjugated enynes undergo a singlet-state photoisomerization that transposes the methylene carbon. We previously suggested excited-state closure to 1,2-cyclobutadiene followed by thermal ring opening. CCSD(T)//DFT computations show two minima of similar energy corresponding to 1,2-cyclobutadiene, one chiral and closed shell and the second a planar diradical. The chiral structure has a low barrier to ring opening and may best explain results on enyne photoisomerization. The first examples of 1,3-diyne + yne cycloadditions to give o-benzynes were reported in 1997. Computations on intramolecular versions of this tridehydro (-3H2) Diels-Alder reaction support a concerted mechanism for the parent triyne (1,3,8-nonatriyne); however, a slight electronic advantage in the concerted path may be outweighed by the difference in entropy of activation for sequential vs simultaneous formation of two new ring bonds.

Bcl-2 is an anti-apoptotic protein with important roles in vascular homeostasis and angiogenesis. Mice globally lacking Bcl-2 (Bcl-2 -/-) are small in stature and succumb to renal failure shortly after weaning as a result of renal hypoplasia/cystic dysplasia. We have shown that Bcl-2 -/- mice displayed attenuated retinal vascular development and neovascularization. In vitro studies indicated that in addition to modulating apoptosis, Bcl-2 expression also impacts endothelial and epithelial cell adhesion, migration and extracellular matrix production. However, studies delineating the cell autonomous role Bcl-2 expression plays in the endothelium during vascular development, pruning and remodeling, and neovascularization are lacking. Here we generated mice carrying a conditional Bcl-2 allele (Bcl-2Flox/Flox) and VE-cadherin-cre (Bcl-2EC mice). Bcl-2EC mice were of normal stature and lifespan and displayed some but not all of the retinal vascular defects previously observed in global Bcl-2 deficient mice. Bcl-2EC mice had decreased numbers of endothelial cells, decreased retinal arteries and premature primary branching of the retinal vasculature, but unlike the global knockout mice, spreading of the retinal superficial vascular layer proceeded normally. Choroidal neovascularization was attenuated in Bcl-2EC mice, although retinal neovascularization accompanying oxygen-induced ischemic retinopathy was not. Thus, Bcl-2 expression in the endothelium plays a significant role during postnatal retinal vascularization, and pathological choroidal but not retinal neovascularization, suggesting vascular bed specific Bcl-2 function in the endothelium.