tznurmin/tea_curated · Datasets at Hugging Face

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5388809896c7b2a9adf5d708f6ea4b08	[ "Introduction", "Cationic", "antimicrobial", "peptides", "(CAMPs)", "are", "short-length,", "amphiphilic,", "rich", "in", "basic", "residues", "of", "the", "innate", "immune", "system", "of", "complex", "organisms,", "serving", "as", "the", "first", "defense", "line", "against", "pathogens.", "CAMPs", "can", "potentially", "give", "rise", "to", "a", "new", "generation", "of", "drugs", "due", "to", "their", "broad", "spectrum", "of", "antimicrobial", "activity", "against", "bacteria,", "rare", "cases", "of", "the", "appearance", "of", "AMP-resistant", "bacteria,", "selectivity,", "and", "rapid", "effects.", "Due", "to", "their", "potential", "advantage", "as", "antimicrobials,", "new", "AMPs", "are", "increasingly", "being", "isolated", "or", "synthesized", "and", "their", "properties", "investigated.", "Although", "increasing", "evidence", "shows", "that", "some", "AMPs", "may", "also", "act", "intracellularly,", "inhibiting", "proteins", "1", ",", "nucleic", "acids", "1", "–", "4", "and", "cell-wall", "synthesis", "1", "–", "5", "and", "inducing", "cell", "apoptosis", "1", ",", "3", ",", "5", ",", "most", "of", "them", "kill", "cells", "by", "disturbing", "the", "membrane’s", "integrity", "1", ",", "2", ",", "6", ",", "7", ".", "Several", "CAMPs", "seem", "to", "disturb", "the", "membrane", "of", "cells", "by", "self-aggregation", "on", "the", "membrane", "and", "forming", "pores", "beyond", "a", "peptide/lipid", "(P/L)", "ratio", "threshold", "1", ",", "2", ".", "BP100", "(H-KKLFKKILKYL-NH", "2", "),", "is", "a", "hybrid", "CAMP", "synthesized", "by", "combinatorial", "chemistry", "involving", "parts", "of", "two", "natural", "AMPs,", "melittin,", "and", "cecropin", "A", "8", ".", "BP100", "shows", "high", "antimicrobial", "activity", "and", "selectivity", "against", "some", "Gram-negative", "bacteria,", "with", "a", "promising", "minimal", "inhibitory", "concentration", "ranging", "from", "2.5", "to", "5", "µM", "and", "low", "cytotoxicity", "against", "mammalian", "cells", "making", "this", "peptide", "an", "exciting", "candidate", "for", "therapeutic", "use", "8", ".", "BP100", "conformation", "and", "activity", "on", "model", "membranes", "have", "been", "extensively", "analyzed", "with", "experimental", "techniques", "9", "–", "13", ".", "BP100", "has", "a", "random", "coil", "conformation", "in", "solution", "and", "zwitterionic", "bilayers", "but", "acquires", "predominantly", "alpha-helical", "and", "amphipathic", "conformation", "in", "the", "presence", "of", "negatively", "charged", "membranes.", "BP100", "is", "highly", "mobile", "on", "the", "membrane", "surface", "and", "orients", "parallel", "in", "predominantly", "negatively", "charged", "vesicles", "9", ",", "10", ".", "BP100", "secondary", "structure", "formation", "and", "bacteria", "killing", "performance", "seem", "to", "be", "directly", "related", "to", "negative", "charge", "content", "in", "membranes,", "thus", "explaining", "its", "high", "selectivity", "towards", "negative", "bacterial", "membrane", "and", "low", "cytotoxicity", "against", "predominantly", "less", "negatively", "charged", "mammalian", "cell", "membranes", "11", ",", "14", ".", "Experimental", "approaches", "to", "investigate", "peptide/membrane", "interactions", "mainly", "focus", "macroscopic-averaged", "insight", "on", "a", "mesoscopic", "scale.", "A", "detailed", "description", "of", "peptide/membrane", "interactions", "at", "the", "atomic", "scale", "may", "help", "to", "gain", "information", "on", "CAMPs", "disruption", "mechanisms,", "permitting", "the", "rational", "design", "of", "new", "derivatives", "and,", "therefore,", "the", "better", "therapeutic", "use", "of", "these", "novel", "antimicrobials.", "Molecular", "Dynamics", "(MD)", "simulations", "provide", "atomic-level", "insights", "and", "have", "been", "widely", "used", "to", "study", "the", "interaction", "of", "AMPs", "with", "membranes", "15", "–", "18", ".", "MD", "simulations", "were", "also", "utilized", "to", "study", "BP100’s", "structure", "and", "activity:", "Wang", "Y.", "et", "al.", "17", "performed", "an", "8", "µs-long", "MD", "simulation", "of", "BP100", "in", "DMPC", "bilayer", "and", "found", "that", "BP100", "remained", "in", "the", "surface-bound-state", "inserting", "through", "its", "C-terminus.", "They", "also", "reported", "the", "unfolding", "of", "BP100", "N-terminus", "(Lys1", "and", "Lys2)", "after", "1.5", "µs,", "resulting", "in", "a", "tilt", "angle", "of", "97°", "and", "81%", "helicity", "10", ",", "17", ".", "In", "a", "study", "with", "Brownian", "Dynamics", "simulations", "with", "a", "coarse-grained", "peptide", "model,", "Alves", "et", "al.", "18", "simulated", "BP100", "confined", "in", "model", "membranes", "at", "293", "K", "for", "1", "µs", "each", "and", "reported", "the", "high", "stability", "of", "BP100", "pre-folded", "alpha", "helix", "and", "a", "threefold", "decrease", "in", "its", "lateral", "diffusion", "in", "POPC:", "POPG", "membranes", "relative", "to", "that", "in", "POPC", "membrane", "18", ".", "In", "this", "work", "we", "report", "simulations,", "in", "solution", "and", "membranes,", "extending", "for", "more", "than", "25", "µs", "of", "BP100", "and", "membranes", "of", "1,2-dipalmitoyl-sn-glycerol-3-phosphocholine", "(DPPC),", "1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol", "(DPPG),", "and", "DPPC:", "DPPG", "mixtures", "(1:1).", "We", "systematically", "investigated", "the", "dependence", "of", "BP100", "secondary", "structure", "on", "DPPG", "lipids", "which", "agrees", "with", "experimental", "findings", "11", ".", "We", "also", "analyzed", "the", "contribution", "of", "PG-containing", "lipid", "rafts", "on", "BP100", "conformation", "and", "behavior.", "In", "most", "simulations", "containing", "DPPG", "lipids,", "we", "observed", "the", "occurrence", "of", "a", "dynamic", "transition", "of", "the", "peptide", "adsorbed", "at", "the", "membrane", "interface,", "where", "the", "apolar", "facet", "of", "the", "BP100", "(Fig.", "1c", "),", "initially", "exposed", "to", "water,", "rotates", "toward", "the", "membrane", "interior,", "leaving", "the", "polar", "facet", "of", "the", "peptide", "exposed", "to", "water.", "This", "process,", "defined", "here", "as", "peptide", "flip,", "could", "be", "part", "of", "CAMPs", "primary", "antimicrobial", "mechanism.", "Constraining", "four", "residues", "of", "the", "peptide", "into", "an", "alpha", "helix", "turn,", "we", "observed", "that", "the", "unconstrained", "regions", "of", "the", "peptide", "rapidly", "acquired", "an", "alpha-helix", "conformation", "in", "DPPG-containing", "membranes,", "suggesting", "that", "the", "formation", "of", "these", "small", "alpha-helical", "nuclei", "in", "peptide/membrane", "interactions", "may", "be", "the", "initial", "step", "for", "the", "peptide", "insertion", "into", "the", "hydrophobic", "core", "of", "the", "membranes.", "Figure", "1", "BP100", "initial", "structures:", "random", "coil", "L", "-BP100", "(", "a", ")", "and", "alpha-helical", "α", "-BP100", "(", "b", ").", "Helical", "wheel", "projection", "(", "c", "),", "Lys5", "and", "Ile7", "were", "used", "as", "representative", "residues", "of", "polar", "and", "apolar", "moieties,", "respectively.", "In", "(", "d", ")", "DPPC", "(in", "blue)", "and", "DPPG", "(in", "yellow).", "Atom", "names", "are", "shown", "for", "peptide", "and", "lipids;", "they", "were", "used", "in", "our", "peptide-lipid", "pair", "analysis.", "Methods", "All", "simulations", "sets", "were", "assembled,", "carried", "out", "and", "analyzed", "using", "GROMACS", "5.0.2", "software", "package.", "For", "trajectories", "visual", "inspections", "and", "image", "rendering,", "VMD", "1.9.2", "software", "was", "used.", "Table", "1", "shows", "all", "simulation", "systems", "and", "its", "simulation", "time.", "The", "program", "used", "to", "analyze", "the", "hydration", "shell", "of", "the", "peptide", "using", "the", "Minimum", "Distance", "Distribution", "Function", "was", "developed", "in", "our", "group.", "Table", "1", "Table", "containing", "all", "MD", "simulation", "sets", "carried", "out", "in", "this", "work.", "For", "bilayers,", "we", "used", "Slipids", "forcefield", "and", "ff99sb-ildn-NMR", "for", "BP100.", "All", "sets", "were", "simulated", "at", "323", "K", "(above", "DPPC", "and", "DPPG", "transition", "temperatures)", "and", "1", "bar.", "L", "-BP100", "indicate", "constraint-free", "simulations", "using", "random", "coil", "BP100", "as", "peptide", "starting", "configuration", "and", "α", "-BP100,", "as", "alpha", "helix.", "Res1–5,", "res4–8", "and", "res7–11", "indicate", "simulations", "with", "BP100", "with", "permanent", "constraints", "on", "the", "referred", "residues.", "Simulation", "sets", "Simulations", "Peptide/Lipid", "Waters/Lipid", "Time", "(ns)", "Bilayers", "DPPC", "0/128", "54", "860", "DPPG", "53", "1100", "PCPG-R", "0/[64/64]", "54", "1200", "PCPG-NR", "52", "1200", "Peptide", "in", "water", "L", "-BP100", "in", "water", "1/0", "—", "2000", "α", "-BP100", "in", "water", "—", "2850", "Peptide", "with", "bilayers", "L", "-BP100", "in", "DPPC", "1/128", "54", "1700", "α", "-BP100", "in", "DPPC", "54", "2000", "L", "-BP100", "in", "DPPG", "1/128", "53", "1700", "α", "-BP100", "in", "DPPG", "53", "2000", "L", "-BP100", "in", "PCPG-R", "1/[64/64]", "54", "1600", "α", "-BP100", "in", "PCPG-R", "54", "2000", "L", "-BP100", "in", "PCPG-NR", "1/[64/64]", "52", "1700", "α", "-BP100", "in", "PCPG-NR", "52", "2000", "Partially", "constrained", "peptide", "in", "DPPG", "α-", "BP100", "(res1–5)", "in", "DPPG", "1/128", "54", "600", "α-", "BP100", "(res4–8)", "in", "DPPG", "54", "600", "α-", "BP100", "(res7–11)", "in", "DPPG", "54", "600", "Lipid", "bilayers", "We", "used", "Slipids", "forcefield", "19", "parameters", "were", "chosen", "to", "simulate", "DPPC,", "DPPG,", "and", "DPPC/DPPG", "mixed", "bilayers.", "64-lipids-per-monolayer", "DPPC", "and", "DPPG", "pure", "membrane", "topologies", "were", "taken", "from", "the", "Slipids", "developer", "group", "website", "(http://www.fos.su.se/~sasha/Slipids/Downloads.html).", "Mixed", "bilayers", "of", "DPPC", "and", "DPPG", "containing", "32", "DPPCs", "and", "32", "DPPGs", "in", "each", "leaflet", "were", "constructed", "using", "the", "PACKMOL", "20", "software.", "One", "membrane", "was", "composed", "of", "randomly", "distributed", "lipids", "(PCPG-NR)", "on", "both", "leaflets", "and", "the", "other,", "a", "4", "×", "4", "DPPG", "raft", "was", "placed", "in", "the", "center", "of", "both", "monolayers", "(PCPG-R)", "and", "the", "remaining", "48", "lipids", "were", "randomly", "distributed", "(Fig.", "2", ").", "Figure", "2", "Initial", "membrane", "configurations", "membranes.", "TIP3P", "water", "molecules", "were", "used", "to", "solvate", "our", "systems,", "with", "an", "approximately", "53", "water/lipid", "ratio.", "Na", "+", "ions", "(Aqvist", "21", ")", "were", "included", "to", "counter-balance", "charges", "of", "membranes", "containing", "DPPG.", "All", "bilayer", "sets", "were", "minimized", "and", "equilibrated", "for", "20", "ps", "in", "a", "NVT", "ensemble", "and", "thereafter,", "in", "NPT", "conditions", "for", "50", "ns.", "After", "that,", "the", "bilayers", "were", "simulated", "for", "860", "ns", "(DPPC),", "1100", "ns", "(DPPG)", "and", "1200", "ns", "(for", "both", "PCPG-R", "and", "PCPG-NR).", "The", "area", "per", "lipid", "and", "membrane", "thickness", "were", "calculated", "for", "all", "membranes", "and", "thNen", "compared", "with", "experimental", "data", "and", "those", "obtained", "by", "Slipids", "authors", "(Tables", "S1", "and", "S2", ").", "The", "last", "trajectory", "frames", "of", "the", "membrane", "simulations", "were", "used", "to", "build", "our", "peptide-lipid", "systems.", "Peptide-in-solution", "and", "peptide-lipid", "systems", "Following", "a", "study", "by", "Beauchamp", "et", "al.", "22", ",", "ff99sb-ildn-NMR", "force", "field", "was", "chosen", "to", "characterize", "BP100", "in", "our", "modeling", "due", "to", "its", "good", "performance", "in", "simulating", "peptides", "and", "proteins", "structures", "in", "TIP3P", "water", "when", "compared", "with", "NMR", "measurements.", "BP100", "(H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH", "2", ")", "has", "an", "amidated", "C-terminal", "and", "a", "+6", "charge", "at", "physiological", "pH,", "with", "all", "its", "Lysines", "and", "N-terminal", "amine", "protonated.", "To", "study", "secondary", "structure", "behavior", "of", "BP100", "both", "in", "solution", "and", "in", "bilayers,", "two", "initial", "BP100", "conformations", "were", "used:", "linear", "BP100", "(", "L", "-BP100)", "and", "alpha-helical", "BP100", "(", "α", "-BP100)", "(Fig.", "1", "a,b", ").", "In", "the", "peptide/water", "system,", "one", "peptide", "(", "α", "-BP100", "or", "L-BP100", "initial", "conformation)", "were", "simulated", "in", "a", "cubic", "box", "with", "3975", "TIP3P", "water", "molecules", "for", "more", "than", "1", "microsecond", "with", "its", "+6", "charges", "neutralized", "with", "6", "Cl", "−", "ions", "(Dang", "23", ").", "Simulations", "containing", "peptides", "with", "membranes", "had", "a", "peptide/lipid", "ratio", "of", "1/128", "and", "the", "peptide", "was", "initially", "positioned", "in", "parallel", "orientation", "with", "the", "membrane", "surface", "approximately", "2", "nm", "away.", "For", "the", "α", "-BP100,", "additionally,", "we", "tested", "two", "parallel", "orientations,", "where", "the", "polar", "amino", "acids", "were", "facing", "the", "membrane", "and", "a", "random", "orientation.", "For", "the", "perpendicular", "orientation", "we", "tested", "two", "ways", "with", "either", "the", "C-terminal", "or", "N-terminal", "facing", "the", "interface.", "For", "all", "these", "initial", "conditions,", "we", "obtained", "the", "same", "final", "location", "and", "orientation", "of", "the", "peptide", "that", "will", "be", "discussed", "in", "the", "results", "section.", "To", "investigate", "the", "influence", "of", "small", "core", "alpha-helical", "sequences", "in", "BP100", "to", "its", "stability", "when", "interacting", "with", "bilayers,", "constrained", "simulations", "of", "BP100", "in", "DPPG", "membranes", "were", "performed.", "The", "alpha-helix", "dihedral", "angles", "(ϕ", "=", "−60°;", "ψ", "=", "−40°)", "in", "3", "different", "regions", "were", "fixed:", "1–5", "(Lys-Lys-Leu-Phe-Lys),", "4–8", "(Phe-Lys-Lys-Ile-Leu)", "and", "7–11", "(Ile-Leu-Lys-Tyr-Leu)", "residues", "(Fig.", "3", ").", "We", "maintained", "the", "constrained", "regions", "during", "the", "simulations.", "Figure", "3", "Starting", "structures", "for", "peptides", "with", "permanent", "constrained", "dihedral", "angles.", "Following", "the", "previous", "lipid", "membrane", "systems,", "energy-minimization,", "NVT,", "and", "NPT", "ensembles", "were", "used", "in", "the", "equilibration", "stage", "of", "the", "MD", "simulation.", "Simulation", "details", "MD", "simulations", "were", "carried", "out", "with", "a", "2", "fs", "time-step", "with", "a", "Leap-Frog", "integrator", "24", ",", "in", "isobaric-isothermal", "(NPT)", "ensemble,", "at", "323", "K", "(50", "°C)", "with", "temperature", "coupling", "treated", "separately", "for", "BP100,", "lipids,", "water", "and", "ions", "with", "the", "V-rescale", "thermostat", "25", ".", "Atmospheric", "pressure", "of", "1", "bar", "was", "kept", "with", "the", "Berendsen", "barostat", "26", "and", "semi-isotropic", "pressure", "coupling", "was", "used", "for", "all", "systems.", "All", "bonds", "were", "constrained", "with", "LINCS", "27", "algorithm.", "Long-range", "electrostatic", "correction", "was", "treated", "by", "a", "Particle-mesh-Ewald", "method", "28", "with", "a", "real-space", "cut-off", "of", "1.5", "nm.", "van", "der", "Waals", "interactions", "were", "truncated", "at", "1.5", "nm", "distance", "with", "a", "switch", "function", "from", "1.4", "nm.", "Simulation", "analyses", "Lipid/peptide", "contacts", "For", "the", "computation", "of", "lipid", "contacts", "with", "BP100", "and", "pair", "frequency,", "we", "labeled", "some", "lipid(s)", "and", "BP100", "atoms", "as", "“polar”", "or", "“apolar”,", "based", "on", "charges.", "Then,", "the", "radial", "distribution", "function", "(RDF)", "between", "each", "pair", "of", "apolar", "(or", "polar)", "atoms", "of", "the", "lipids/BP100", "system", "were", "computed.", "The", "time", "window", "selected", "for", "computing", "these", "RDFs", "includes", "that", "of", "the", "membrane", "bound", "peptide", "250", "ns-before", "and", "250", "ns-after", "the", "peptide", "flip.", "The", "pairs", "with", "distinguishable", "RDF", "peaks", "and", "following", "minimum", "were", "used", "to", "analyze", "the", "number", "of", "polar", "and", "apolar", "contacts", "between", "lipids", "and", "peptide.", "A", "cutoff", "distance", "for", "each", "pair", "was", "defined", "from", "the", "position", "of", "the", "first", "minimum", "in", "the", "RDF", "curve,", "and", "a", "contact", "was", "defined", "as", "a", "pair", "of", "atoms", "at", "smaller", "distance", "than", "their", "corresponding", "cutoff", "distance.", "The", "simulations", "were", "analyzed", "and", "the", "total", "number", "of", "polar", "and", "apolar", "contacts", "as", "a", "function", "of", "time", "was", "obtained.", "Additionally,", "some", "polar", "and", "apolar", "pairs", "were", "analyzed", "individually.", "Peptide", "hydration", "The", "time", "evolution", "of", "the", "solvent", "shell", "surrounding", "the", "peptide", "in", "its", "α", "and", "L", "conformations", "in", "all", "simulated", "membranes", "was", "analyzed", "using", "the", "minimum", "distance", "distribution", "function", "(MDDF)", "29", "between", "the", "peptide", "and", "the", "water", "molecules,", "as", "well", "as", "the", "average", "number", "of", "water", "molecules", "N(r),", "within", "a", "distance", "r", "=", "0.5", "nm,", "named", "here", "as", "N(0.5).", "For", "comparison", "the", "MDDF", "of", "BP100", "in", "water", "was", "used", "to", "define", "the", "bulk", "water", "hydration", "shell", "at", "the", "same", "distance.", "Results", "The", "17", "sets", "of", "simulations", "(Table", "1", ")", "were", "run", "for", "more", "than", "25", "µs", "of", "simulation", "time,", "and", "each", "unconstrained", "peptide/membrane", "simulation", "had", "at", "least", "1.6", "µs", "of", "simulation.", "After", "comparing", "the", "simulated", "membrane", "properties", "with", "experimental", "and", "other", "computational", "studies", "(Tables", "S1", "and", "S2", "),", "we", "systematically", "analyzed", "the", "peptide", "conformation", "in", "solution", "and", "on", "bilayers,", "peptide", "trajectory,", "and", "dynamics", "on", "membranes", "and", "the", "effect", "of", "BP100", "on", "model", "bilayers.", "In", "the", "following", "sections,", "we", "describe", "our", "results.", "Secondary", "structure", "Peptide", "in", "solution", "To", "study", "BP100", "secondary", "structure", "in", "water,", "we", "did", "simulations", "for", "more", "than", "2", "µs", "with", "both", "α-BP100", "and", "L-BP100", "initial", "conformations.", "Figure", "4", "shows", "that", "BP100", "conformation", "in", "solution", "was,", "predominantly,", "random", "coil.", "The", "peptide", "lost", "the", "initial", "alpha", "helix", "conformation", "in", "approximately", "40", "ns", "when", "starting", "with", "an", "alpha-helix", "configuration", "and", "occasionally", "formed", "an", "alpha-helix", "turn", "in", "the", "5–9", "residue", "region", "(Fig.", "4a", ").", "For", "L-BP100", "in", "solution,", "we", "observed", "a", "similar", "pattern", "(Fig.", "4b", ").", "Circular", "Dichroism", "(CD)", "shows", "that", "BP100", "is", "random", "in", "solution", "10", ",", "11", ",", "30", ",", "and", "our", "simulations", "reproduced", "these", "data.", "Figure", "4", "Secondary", "structure", "analyses", "graphs", "of", "α", "-BP100", "(", "a", ")", "and", "L", "-BP100", "(", "b", ")", "in", "solution.", "In", "both", "cases,", "the", "peptide", "shows", "predominance", "in", "random", "coil", "structure", "(orange)", "with", "occasional", "formation", "of", "1", "alpha", "helix", "turn", "(blue)", "in", "residues", "5", "to", "9.", "Peptide", "in", "bilayers", "BP100", "conformation,", "antimicrobial", "activity,", "and", "PG", "content", "are", "related", "11", ".", "We", "simulated", "α-BP100", "and", "L-BP100", "on", "DPPC,", "DPPG,", "PCPG-R,", "and", "PCPG-NR", "at", "323", "K,", "above", "DPPG", "and", "DPPC", "transition", "temperatures.", "In", "all", "cases,", "the", "peptide", "was", "rapidly", "adsorbed", "on", "the", "membranes", "and", "diffused", "laterally.", "Figure", "5", "shows", "that", "the", "BP100", "alpha-helical", "conformation", "was", "proportional", "to", "PG", "content,", "as", "the", "alpha-helix", "percentage", "in", "DPPC", "is", "considerably", "smaller", "than", "that", "observed", "in", "DPPG-containing", "membranes.", "Alpha-helix", "formed", "in", "the", "5", "to", "9", "residues", "region", "(Lys-Lys-Ile-Leu-Lys)", "was", "highly", "stable", "in", "all", "simulations.", "Figure", "5", "Secondary", "structure", "analyses", "of", "α", "-BP100", "in", "DPPC", "(", "a", "),", "DPPG", "(", "b", "),", "PCPG-R", "(", "c", ")", "and", "PCPG-NR", "(", "d", ").", "In", "DPPC,", "α-BP100", "alpha-helix", "conformation", "reached", "a", "plateau", "at", "38%", "helicity", "throughout", "the", "simulation,", "being", "relatively", "less", "stable", "than", "α-BP100", "in", "bilayers", "containing", "DPPG,", "where", "a", "higher", "propensity", "of", "maintaining", "its", "conformation", "was", "observed.", "For", "pure", "DPPG", "(Fig.", "5b", ")", "we", "found", "73%", "(8", "out", "of", "11", "residues),", "and", "for", "both", "PCPG", "(1:1)", "membranes", "we", "report", "54%", "(6", "out", "of", "11)", "of", "helicity", "(Fig.", "5c,d", ").", "No", "alpha-helix", "formation", "was", "detected", "in", "L-BP100", "on", "bilayers", "simulations", "(Fig.", "S3", ").", "Therefore,", "the", "1.8", "µs", "simulation", "was", "not", "enough", "to", "present", "a", "spontaneous", "peptide", "folding.", "The", "alpha-helix", "formed", "in", "the", "region", "of", "the", "residues", "5", "to", "9", "showed", "higher", "stability", "in", "all", "simulations", "with", "α-BP100", "in", "bilayers", "(Fig.", "5", ").", "This", "secondary", "structure", "pattern", "led", "us", "to", "suggest", "that", "the", "residues", "5", "to", "9", "are", "related", "to", "helix", "nucleation,", "in", "which", "the", "formation", "of", "this", "helix", "core", "could", "be", "the", "first", "step", "in", "the", "AMP", "actions.", "Following", "this", "assumption,", "we", "tested", "three", "conformations", "with", "α-BP100", "torsionally", "constrained", "in", "three", "regions:", "res1–5,", "res4–8,", "res7–11", "(Fig.", "3", ")", "and", "simulated", "with", "pure", "DPPG", "membranes.", "Figure", "6", "shows", "the", "evolution", "of", "the", "secondary", "structure", "in", "each", "simulation.", "Figure", "6", "Secondary", "structure", "analyses", "graphs", "of", "BP100", "in", "DPPG", "with", "one", "turn", "constraints", "on", "res1–5", "(", "a", "),", "res", "4–8", "(", "b", ")", "and", "res7–11", "(", "c", ").", "α-BP100", "with", "constraints", "on", "residues", "1–5", "(Lys-Lys-Leu-Phe-Lys)", "acquired", "identical", "helicity", "(73%)", "to", "α-BP100", "with", "no", "constraints", "at", "the", "same", "conditions", "(Fig.", "5b", "),", "after", "50", "ns", "of", "simulation", "(Fig.", "6a", "),", "with", "a", "helix", "spanning", "from", "Lys2", "to", "Lys9", "(Fig.", "6a", ").", "Constraints", "on", "residues", "4–8", "(Phe-Lys-Lys-Ile-Leu)", "showed", "an", "overall", "52%", "of", "helicity", "(Fig.", "6b", "),", "with", "a", "helix", "spanning", "from", "Phe4", "to", "Lys9", "(Fig.", "6b", ").", "Constraints", "on", "residues", "7–11", "(Ile-Leu-Lys-Tyr-Leu)", "did", "not", "affect", "the", "rest", "of", "the", "peptide", "conformation", "(Fig.", "6c", ")", "in", "600", "ns", "of", "simulation.", "Peptide", "flip", "Simulation", "trajectories", "analyses", "(Fig.", "7", ")", "revealed", "that", "the", "peptide", "in", "alpha-helix", "conformation", "initially", "adsorbed", "at", "the", "DPPG-containing", "membranes", "interface", "with", "its", "hydrophilic", "facet", "in", "contact", "with", "the", "membrane,", "leaving", "the", "hydrophobic", "facet", "exposed", "to", "water.", "We", "observed", "that", "the", "alpha-helix", "rotated,", "inserting", "the", "hydrophobic", "facet", "into", "the", "hydrophobic", "region", "of", "the", "membrane.", "We", "define", "this", "dynamic", "phenomenon,", "observed", "in", "our", "simulations,", "as", "peptide", "flip.", "Although", "this", "final", "peptide", "orientation", "is", "known", "31", "–", "34", ",", "no", "MD", "simulation", "on", "peptide", "flip", "has", "been", "reported.", "Figure", "7", "Peptide", "flip", "observed", "from", "above", "in", "α", "-BP100", "in", "DPPG", "set", "after", "circa", "1400", "ns", "of", "simulation.", "In", "blue,", "polar", "facet", "and", "in", "green,", "the", "apolar", "facet.", "Peptide", "flip", "was", "also", "observed", "in", "α", "-BP100", "in", "DPPC,", "α", "-BP100", "in", "PCPG-R,", "and", "α", "-BP100", "res(1–5)", "in", "DPPG", "simulations.", "The", "peptide", "flip", "caused", "dehydration", "(Fig.", "8", ").", "When", "in", "contact", "with", "membranes,", "depending", "on", "the", "secondary", "structure", "of", "the", "peptide", "and", "the", "lipid", "nature,", "the", "hydration", "of", "the", "peptide", "was", "slightly", "different.", "However,", "when", "the", "peptide", "flip", "took", "place,", "its", "hydration", "was", "drastically", "altered.", "Figure", "8", "Average", "number", "of", "water", "molecules", "up", "to", "a", "distance", "of", "0.5", "nm,", "N(0.5),", "obtained", "from", "the", "minimum-distance", "distribution", "function", "(MDDF),", "between", "L-BP100", "(", "a", ")", "and", "α", "-BP100", "(", "b", ")", "and", "water", "molecules.", "Vertical", "lines", "indicate", "when", "peptide", "flip", "occurred.", "In", "the", "peptide/membrane", "simulations", "with", "L-BP100", "as", "a", "starting", "configuration", "(Fig.", "8a", "),", "no", "significant", "change", "in", "the", "number", "of", "water", "molecules", "surrounding", "the", "peptide", "was", "observed", "in", "comparison", "the", "150", "obtained", "in", "the", "bulk", "aqueous", "solution.", "In", "the", "last", "500", "ns", "of", "simulation,", "N(0.5)", "was", "approximately", "178", "±", "11", "in", "DPPC,", "145", "±", "7", "in", "DPPG,", "145", "±", "13", "in", "PCPG-NR,", "and", "148", "±", "3", "in", "PCPG-R.", "No", "peptide", "flip", "was", "detected", "in", "the", "trajectories", "for", "those", "simulations.", "However,", "in", "the", "peptide/membrane", "simulations", "with", "α-BP100", "as", "a", "starting", "configuration", "(Fig.", "8b", "),", "N", "(0.5)", "lowered", "in", "DPPC", "(starting", "at", "~700", "ns),", "DPPG", "(~1500", "ns)", "and", "PCPG-R", "(~390", "ns).", "Alongside", "with", "dehydration,", "a", "peptide-flip", "was", "observed", "in", "DPPG", "and", "PCPG-R.", "For", "the", "α-", "BP100", "in", "DPPC", "simulation,", "we", "observed", "a", "semi-flip,", "as", "the", "peptide", "alpha-helical", "conformation", "was", "only", "38%", "(Fig.", "5a", "),", "suggesting", "that", "the", "peptide", "overall", "polar/apolar", "segregation", "(Fig.", "2c", ")", "was", "lost.", "The", "peptide", "flip", "transition", "time", "varied", "from", "100", "to", "400", "ns.", "During", "the", "peptide-flip,", "in", "the", "simulations", "of", "α-BP100", "in", "DPPG", "and", "PCPG-R,", "the", "mean", "number", "N(0.5)", "was", "reduced", "from", "147", "±", "5", "and", "142", "±", "7", "to", "76", "±", "4", "and", "75", "±", "4,", "respectively.", "These", "represent", "on", "average", "a", "reduction", "of", "approximately", "48%", "in", "the", "number", "of", "water", "molecules", "in", "the", "hydration", "shell", "of", "α-BP100.", "In", "DPPC,", "we", "detected", "a", "smaller", "decrease", "in", "the", "hydration", "of", "α-BP100,", "around", "45%", "(from", "172", "±", "8", "to", "94", "±", "2),", "having", "around", "20", "water", "molecules", "more", "after", "the", "semi-flip.", "Figure", "9", "shows", "the", "density", "profile", "before", "and", "after", "peptide", "flip", "for", "α-BP100", "in", "DPPG", "and", "PCPG-R.", "We", "chose", "residues", "Lys5", "and", "Ile7", "to", "illustrate", "the", "changes", "in", "the", "mean", "position", "of", "hydrophobic", "and", "hydrophilic", "residues", "with", "respect", "to", "the", "membrane", "due", "to", "the", "peptide", "flip.", "These", "residues", "are", "polar", "and", "apolar,", "respectively,", "and", "located", "opposite", "to", "each", "other", "when", "in", "an", "alpha-helix", "conformation", "(Fig.", "2c", ").", "Trajectory", "visualization", "and", "density", "profile", "(Figs", "7", "and", "9", ")", "clearly", "show", "the", "distinct", "orientation", "in", "BP100", "hydrophobic", "facet", "before", "and", "after", "peptide", "flip.", "During", "the", "initial", "phase", "of", "approaching", "towards", "the", "membrane,", "BP100", "polar", "side", "faces", "the", "membrane", "with", "its", "N-terminal", "residues", "(Lys1", "and", "Lys2)", "interacting", "with", "the", "bilayer", "headgroups.", "After", "the", "flip,", "the", "apolar", "residues", "facet", "turns", "to", "the", "membrane,", "and", "simultaneously", "inserts", "into", "the", "bilayer,", "below", "the", "carbonyl", "carbons", "(C31)", "of", "the", "lipids", "(Fig.", "9", ").", "Figure", "9", "Peptide", "flip", "observed", "through", "number", "density", "analysis.", "(", "a", "and", "b", ")", "show", "respectively", "before", "and", "after", "the", "flip", "in", "α", "-BP100", "in", "DPPG", "simulation;", "c)", "and", "d)", "demonstrate", "the", "flip", "for", "α", "-BP100", "in", "PCPG-R", "simulation.", "Lys5", "represents", "the", "polar", "side", "of", "BP100", "and", "Ile7,", "the", "apolar", "facet", "(Fig.", "2c", ").", "OC3", "(DPPG", "head", "group", "further", "out", "glycerol", "Oxygen),", "N", "chol", "(DPPC", "head", "group", "choline", "Nitrogen),", "P", "(Phosphorus)", "and", "C31", "(first", "carbon", "from", "sn-1", "acyl", "chain)", "are", "shown", "to", "display", "BP100", "insertion", "into", "the", "bilayer", "after", "the", "flip.", "Peptide", "flip", "was", "not", "observed", "in", "α-BP100", "in", "PCPG-NR,", "where", "DPPG", "lipids", "were", "initially", "scattered", "randomly.", "PG", "content", "might", "be", "related", "to", "the", "occurrence", "of", "the", "flip,", "but", "also,", "the", "PG", "content", "in", "the", "vicinity", "of", "BP100.", "Several", "studies", "suggest", "membrane-active", "peptides", "cause", "anionic", "lipid", "clustering,", "or", "subdomain", "formation,", "upon", "binding", "to", "bilayers", "containing", "neutral", "and", "charged", "phospholipids", "35", "–", "37", ".", "The", "same", "mechanism", "could", "be", "involved", "in", "BP100/bilayers", "interactions.", "Lipid", "clustering", "The", "distribution", "of", "the", "lipids", "in", "the", "vicinity", "of", "BP100", "was", "evaluated", "by", "computing", "the", "total", "number", "of", "lipids", "in", "contact", "with", "α-BP100", "as", "a", "function", "of", "time", "(Fig.", "S5", "),", "the", "time-averaged", "values", "(Table", "2", ",", "Fig.", "10", "),", "and", "the", "lipid", "raft", "size", "distribution", "(Fig.", "S6", ").", "Table", "2", "Average", "number", "of", "lipids", "in", "contact", "with", "BP100", "in", "simulations", "using", "α", "-BP100", "as", "the", "peptide", "starting", "configuration,", "except", "α", "-BP100", "(1–5),", "which", "had", "permanent", "constraints", "on", "residues", "1", "to", "5.", "Standard", "deviations", "are", "shown", "in", "parenthesis.", "Average", "Number", "of", "Lipids", "in", "Contact", "with", "BP100", "Simulation", "Lipid", "in", "Contact", "Average", "α", "-BP100", "in", "DPPC", "DPPC", "10.7", "(±2.6)", "α", "-BP100", "in", "DPPG", "DPPG", "13.1", "(±1.4)", "α", "-BP100", "(1–5)", "in", "DPPG", "DPPG", "13.5", "(±1.9)", "α", "-BP100", "in", "PCPG-R", "Total", "12.3", "(±1.7)", "DPPC", "3.6", "(±1.5)", "DPPG", "8.7", "(±1.2)", "α", "-BP100", "in", "PCPG-NR", "Total", "11.0", "(±1.7)", "DPPC", "2.7", "(±1.3)", "DPPG", "8.3", "(±1.6)", "Figure", "10", "Average", "Number", "of", "DPPCs", "(", "a", ")", "and", "DPPGs", "(", "b", ")", "in", "contact", "with", "BP100", "analyzed", "for", "all", "constraint-free", "simulations", "containing", "BP100", "in", "an", "initially", "alpha", "helical", "conformation", "in", "DPPC,", "DPPG,", "PCPG-R", "and", "PCPG-NR", "membranes.", "Vertical", "lines", "represent", "the", "average", "time", "where", "peptide", "flip", "was", "observed.", "Standard", "deviation", "error", "bars", "are", "represented", "as", "fillings.", "α-BP100", "in", "DPPC", "simulation", "had", "on", "average", "10.7", "peptide/lipid", "contacts", "(Table", "2", ")", "and", "a", "steady", "increase", "in", "the", "number", "of", "contact", "lipids", "(Figs", "10b", "and", "S5a", ").", "This", "increase", "is", "due", "to", "the", "peptide", "losing", "its", "alpha-helical", "conformation", "(Fig.", "5a", "),", "expanding", "the", "available", "peptide", "interaction", "area", "with", "the", "membrane.", "Lipid", "raft", "size", "distribution", "also", "shows", "a", "broader", "distribution", "of", "DPPC", "raft", "size,", "supporting", "this", "observation", "(Fig.", "S6a", ").", "A", "semi-peptide", "flip", "was", "observed", "at", "~700", "ns", "of", "simulation", "and,", "concomitantly,", "while", "BP100", "inserts", "into", "the", "bilayer", "as", "the", "density", "profile", "shows", "(Fig.", "S4a,b", "),", "the", "number", "of", "DPPCs", "in", "contact", "with", "BP100", "increased", "(Fig.", "S5a", ").", "α-BP100", "in", "DPPG", "simulation", "shows", "on", "average", "13.1", "DPPGs", "in", "close", "contact", "with", "the", "peptide", "(Table", "2", ").", "The", "number", "of", "DPPGs", "in", "contact", "rose", "after", "the", "flip,", "approximately", "in", "1500", "ns", "of", "simulation", "(Figs", "10a", "and", "S5b", ")", "and", "concurrently,", "BP100", "inserted", "deeply", "into", "the", "membrane", "(Fig.", "7a,b", ").", "Constrained", "α-BP100", "(res1–5)", "in", "DPPG,", "which", "showed", "identical", "alpha-helix", "content", "with", "α-BP100", "in", "DPPG", "(73%,", "Figs", "5b", "and", "6a", "),", "not", "only", "exhibits", "peptide", "flip", "in", "approximately", "300", "ns", "of", "simulation", "but", "also", "has,", "on", "average,", "13.5", "DPPGs", "in", "contact", "with", "BP100", "(Table", "2", ").", "In", "the", "simulation", "with", "α-BP100", "in", "PCPG-NR,", "the", "number", "of", "DPPG", "in", "contact", "with", "the", "peptide", "showed", "a", "slight", "increase", "(Fig.", "10b", "),", "reaching", "the", "average", "value", "observed", "in", "the", "simulation", "with", "PCPG-R", "(Table", "2", ").", "Also,", "lipid", "raft", "size", "probability", "analysis", "(Fig.", "S6d", ")", "showed", "a", "narrower", "peak", "distribution", "for", "DPPGs", "in", "contact", "with", "BP100", "in", "PCPG-R", "system,", "compared", "to", "PCPG-NR", "(Fig.", "S6e", "),", "indicating", "that", "DPPG", "lipid", "raft", "size", "was", "stable.", "Peptide", "flip", "was", "observed", "in", "the", "PCPG-R", "set", "after", "~390", "ns", "of", "simulation", "and", "no", "significant", "variation", "of", "the", "number", "of", "DPPG", "in", "contact", "with", "peptide", "was", "observed.", "Polar", "and", "apolar", "interaction", "analysis", "Peptide", "contact", "analysis", "revealed", "the", "increase", "of", "lipid/peptide", "contacts", "after", "peptide", "flip", "and", "DPPG", "clustering", "on", "PCPG", "membranes.", "We", "analyzed", "the", "nature", "of", "the", "intermolecular", "interactions", "that", "determine", "peptide-membrane", "interactions", "throughout", "the", "simulations.", "We", "calculated", "the", "number", "of", "polar", "and", "apolar", "pair", "contacts", "between", "peptide", "atoms", "and", "membrane", "atoms", "(Figs", "S7", "–", "S13", ").", "Using", "α-BP100", "in", "PCPG-R", "system", "as", "an", "example,", "Fig.", "11", "shows", "the", "sum", "of", "the", "10", "most", "frequent", "apolar/polar", "pairs", "between", "BP100", "with", "DPPC", "and", "DPPG.", "Figure", "11", "BP100", "pair", "occurrence", "with", "DPPC", "(", "a", ")", "and", "DPPG", "(", "b", ")", "for", "α", "-BP100", "in", "PCPG-R", "simulation.", "Graphs", "show", "the", "sum", "of", "the", "ten", "most", "frequent", "apolar/polar", "pairs", "during", "the", "simulation.", "Pairs", "were", "calculated", "using", "the", "monolayer", "facing", "the", "peptide.", "Green", "vertical", "lines", "indicate", "when", "peptide", "flip", "occurred", "(circa", "390", "ns).", "The", "total", "number", "of", "pairs,", "regardless", "if", "it", "is", "polar", "or", "apolar,", "between", "BP100", "and", "DPPG", "is", "higher", "than", "that", "with", "DPPC", "(Fig.", "11", "),", "which", "directly", "reflects", "on", "the", "number", "of", "lipids", "in", "contact", "with", "BP100", "(Fig.", "10", ").", "During", "the", "initial", "approach", "of", "the", "peptide", "towards", "the", "bilayer,", "polar", "interactions", "between", "charged", "Lysines", "and", "DPPG", "headgroup", "dominate", "the", "interaction", "(Figs", "11", "b,", "S9", ",", "S10", ",", "S12", "and", "S13", ").", "The", "primary", "interaction", "was", "between", "positively", "charged", "(+1)", "Lysine", "side", "chain", "amino", "group", "(NZ)", "and", "DPPG", "head", "phosphoglycerol", "group", "oxygens", "(OC2,", "OC3),", "even", "after", "peptide", "flip", "(Figs", "S9", ",", "S10", ",", "S12", "and", "S13", ").", "Before", "the", "peptide", "flip,", "the", "number", "of", "polar", "contacts", "is", "higher", "than", "the", "apolar", "contacts", "for", "DPPG/BP100.", "As", "the", "peptide", "starts", "to", "flip,", "the", "number", "of", "apolar", "contacts", "increase,", "while", "the", "polar", "contacts", "decrease.", "After", "the", "peptide", "flip", "is", "completed,", "apolar", "contacts", "are", "as", "numerous", "as", "the", "polar", "contacts", "(Fig.", "11a,b", "and", "S7–10,13", ").", "Also,", "after", "the", "flip,", "apolar", "contacts", "between", "BP100", "and", "DPPG", "were", "more", "frequent", "than", "with", "DPPC", "(Fig.", "11b", ").", "The", "higher", "peptide/DPPG", "apolar", "contact", "frequency", "after", "the", "flip", "is", "also", "observed", "for", "other", "simulations", "with", "peptide", "flip", "(Figs", "S7", "–", "10", ",", "13).", "Discussion", "Peptide", "secondary", "structure", "CD", "spectra", "of", "BP100", "in", "solution", "and", "POPC", "LUVs", "show", "no", "secondary", "structure", "10", ",", "11", ",", "30", "Our", "simulations", "with", "BP100", "in", "solution,", "and", "DPPC", "membrane", "exhibited", "the", "same", "pattern.", "α-BP100", "and", "L-BP100", "in", "solution", "showed", "similar", "behavior,", "with", "a", "prevalence", "of", "random", "conformation", "(Fig.", "4", ").", "In", "a", "related", "MD", "study", "of", "AMPs", "interacting", "with", "membranes", "17", ",", "an", "8", "µs-long", "all-atom", "MD", "simulation", "of", "BP100", "in", "DMPC", "at", "35◽", "the", "authors", "reported", "a", "full", "helix", "length", "during", "1.5", "µs", "and", "a", "subsequent", "unfolding", "event", "of", "Lysines", "at", "positions", "1", "and", "2,", "resulting", "in", "81%", "of", "helicity", "on", "a", "pure", "zwitterionic", "bilayer.", "However,", "as", "reported", "for", "other", "simulated", "peptides", "in", "the", "same", "work,", "the", "forcefield", "used", "could", "result", "in", "an", "over-helical", "artifact", "17", ".", "A", "Brownian", "Dynamics", "simulation", "study", "of", "BP100", "confined", "inside", "an", "implicit", "POPC", "membrane", "reports", "a", "stable", "alpha-helix", "18", ".", "However,", "no", "experimental", "data", "confirms", "that", "BP100", "diffuses", "freely", "in", "the", "membrane", "hydrophobic", "space", "and", "no", "secondary", "structure", "formation", "is", "reported", "for", "pure", "POPC", "vesicles", "11", ".", "In", "our", "simulations", "of", "α-BP100", "and", "L-BP100", "in", "DPPC", "membrane,", "the", "peptide", "quickly", "approached", "the", "membrane", "and", "remained", "on", "the", "surface", "during", "all", "simulation.", "Peptide-lipid", "pair", "analysis", "(Fig.", "S7", ")", "showed", "that", "cationic", "Lysine", "residues", "interacted", "mainly", "with", "phosphate", "groups", "of", "the", "DPPC", "bilayer,", "evidencing", "that", "electrostatic", "interactions", "were", "present.", "In", "the", "case", "of", "L-BP100", "in", "DPPC,", "no", "alpha-helical", "structure", "formation", "was", "observed", "(Fig.", "S3", "),", "and", "for", "α-BP100,", "the", "peptide", "alpha-helix", "conformation", "was", "mostly", "lost,", "although", "one", "helix", "turn", "from", "residues", "5", "to", "9", "remained", "(Fig.", "5A", ").", "The", "minimum", "alpha-helix", "length", "detected", "by", "CD", "spectroscopy", "is", "proposed", "to", "encompass", "seven", "to", "eleven", "residues", "38", ",", "hence", "the", "alpha-helix", "turn", "in", "residues", "5–9", "might", "not", "be", "detected", "by", "CD.", "Alternatively,", "this", "could", "be", "an", "over-helical", "forcefield", "artifact", "22", ",", "but", "it", "also", "indicates", "the", "high", "stability", "of", "the", "alpha-helix", "formed", "by", "the", "(5–9)", "Lys-Lys-Ile-Leu-Lys", "sequence.", "This", "stability", "leads", "us", "to", "conjecture", "that", "BP100", "alpha-helix", "formation", "could", "be", "initiated", "through", "a", "core", "sequence,", "a", "nascent", "helix", "39", ".", "The", "negatively", "charged", "membrane", "interface", "could", "promote", "the", "initial", "peptide", "folding.", "To", "support", "this,", "we", "performed", "three", "600", "ns-long", "simulations", "with", "constrained", "alpha", "helices", "of", "BP100", "on", "three", "regions:", "residues", "1", "to", "5", "(Lys-Lys-Leu-Phe-Lys),", "4", "to", "8", "(Phe-Lys-Lys-Ile-Leu)", "and", "7", "to", "11", "(Ile-Leu-Lys-Tyr-Leu)", "in", "pure", "DPPG", "bilayers.", "The", "secondary", "structure", "analysis", "of", "the", "constrained", "BP100", "simulations", "yielded", "different", "outcomes.", "Constraint", "on", "residues", "1", "to", "5", "rapidly", "induced", "almost", "full", "alpha-helix", "content", "from", "Lys2", "to", "Lys9", "(Fig.", "6A", "),", "reaching", "73%", "of", "helicity,", "similar", "to", "α-BP100", "in", "DPPG", "without", "constraints", "(Fig.", "5B", ").", "Constraint", "on", "residues", "4", "to", "8", "also", "showed", "a", "helix", "formation", "tendency", "(overall", "63%", "helicity,", "5", "out", "of", "11", "residues,", "from", "Phe4", "to", "Lys9).", "In", "sharp", "contrast,", "constraints", "on", "residues", "7", "to", "11", "(36%", "of", "helicity,", "4", "out", "of", "11", "residues,", "from", "Lys7", "to", "Tyr10)", "had", "no", "effects", "on", "the", "secondary", "structure", "formation", "of", "BP100", "during", "600", "ns", "simulation", "(Fig.", "6C", "),", "indicating", "that", "this", "region", "has", "little", "influence", "on", "initial", "peptide", "folding.", "Pace", "and", "Scholtz", "40", ",", "based", "on", "experimental", "studies,", "proposed", "a", "helix", "propensity", "scale", "as", "Δ(ΔG)", "values", "relative", "to", "Alanine", "set", "to", "zero.", "On", "the", "Pace", "and", "Scholtz", "scale,", "the", "5–9", "region", "has", "the", "highest", "helix", "promoting", "residues", "(overall", "helix", "propensity", "of", "1.4", "kcal/mol).", "We", "find", "1.53", "kcal/mol", "for", "residues", "1", "to", "5", "(Lys-Lys-Leu-Phe-Lys),", "1.68", "kcal/mol", "for", "residues", "4", "to", "8", "(Phe-Lys-Lys-Ile-Leu),", "and", "1.62", "kcal/mol", "for", "residues", "7–11", "(Ile-Leu-Lys-Tyr-Leu).", "Our", "secondary", "structure", "data", "indicates", "that", "BP100", "nascent", "helix", "could", "be", "formed", "either", "in", "the", "region", "between", "residues", "1", "to", "5", "or", "between", "residues", "5", "to", "9.", "We", "favor", "the", "latter", "due", "to", "the", "presence", "of", "positively", "charged", "Lysine", "residues", "in", "the", "N-terminal", "side", "(Lys1", "and", "Lys2)", "which", "could", "produce", "an", "energetically", "unfavorable", "barrier", "to", "initial", "peptide", "folding.", "The", "5–9", "region", "shows", "high", "stability", "in", "all", "constraint-free", "α-BP100/membranes", "simulations", "and", "has", "the", "overall", "lowest", "energy", "barrier", "in", "the", "helix", "propensity", "scale", "40", ".", "DPPG", "seems", "to", "have", "a", "helix-stabilizing", "effect", "on", "BP100.", "All", "the", "simulations", "with", "α-BP100", "in", "DPPG-containing-membranes", "showed", "high", "alpha-helical", "content,", "including", "the", "region", "of", "residues", "5", "to", "9.", "Our", "helicity", "results", "from", "the", "simulations", "with", "α-BP100", "in", "pure", "DPPG", "(73%)", "and", "in", "PCPG", "(50:50)", "mixed", "bilayers", "(54%)", "are", "remarkably", "close", "to", "those", "reported", "in", "experimental", "studies:", "54%", "in", "POPC:POPG", "(50:50)", "11", "and", "61%", "in", "DMPC/DMPG", "(75:25)", "LUVs", "10", ",", "and", "maximum", "observable", "alpha-helix", "spectrum", "for", "BP100", "in", "pure", "POPG", "vesicles", "11", "proving", "that", "BP100", "alpha-helical", "structure", "is", "dependent", "on", "PG", "content", "11", ".", "For", "L-BP100", "simulations", "with", "DPPG,", "PCPG-R,", "and", "PCPG-NR,", "no", "secondary", "structure", "formation", "was", "detected", "(Fig.", "S3", "),", "with", "the", "peptide", "being", "in", "a", "random", "conformation.", "Non-biased", "random", "peptide", "simulations", "require", "much", "longer", "simulation", "time", "to", "sample", "the", "peptide", "conformational", "potential", "landscape", "or", "might", "dwell", "in", "several", "local", "minimum-energy", "states.", "Peptide", "flip", "BP100", "is", "too", "short", "to", "span", "the", "bilayer", "thickness,", "especially", "in", "the", "alpha-helical", "conformation,", "and", "thus", "single", "molecule", "pore", "formation", "is", "an", "unlikely", "scenario.", "NMR", "data", "of", "BP100", "in", "DMPC:", "DMPG", "(3:1)", "10", "shows", "that", "upon", "binding", "to", "negatively", "charged", "membranes,", "alpha-helical", "BP100", "positions", "parallel", "to", "the", "membrane", "as", "in", "a", "surface-bound", "orientation,", "and", "has", "an", "azimuthal", "rotation", "angle", "close", "to", "160°,", "with", "Lysine", "residues", "facing", "the", "solvent.", "This", "last", "arrangement", "is", "commonly", "observed", "with", "other", "CAMPs", "30", "–", "33", ".", "Peptide/membrane", "simulations", "with", "initially", "alpha-helical", "BP100", "had", "its", "polar", "moiety", "facing", "the", "membrane,", "for", "being", "the", "most", "probable", "scenario", "in", "a", "physical", "environment.", "In", "the", "α-BP100", "in", "DPPG", "(Fig.", "7", "),", "α-BP100", "in", "DPPC,", "α-BP100", "in", "PCPG-R", "and", "α-BP100", "(res1–5)", "in", "DPPG", "simulations", "trajectories,", "we", "observed", "the", "binding", "of", "BP100", "from", "bulk", "solution", "to", "the", "membranes", "with", "its", "Lysines", "residues", "pointing", "towards", "the", "bilayer,", "and", "then,", "the", "peptide", "rotated,", "leaving", "its", "apolar", "facet", "facing", "the", "interior", "of", "the", "bilayer.", "We", "termed", "this", "transition", "peptide", "flip.", "Although", "BP100", "surface-bound", "state", "is", "reported", "12", ",", "17", ",", "no", "previous", "peptide/membrane", "MD", "simulation", "work", "has", "reported", "a", "peptide", "flip", "transition.", "Peptide", "flip", "brings", "significant", "outcomes", "regarding", "BP100", "localization", "in", "the", "membrane,", "in", "peptide/membrane", "interaction", "and", "the", "peptide", "hydration,", "possibly", "being", "a", "crucial", "step", "of", "carpet", "mechanism", "for", "BP100", "and", "other", "CAMPs.", "Through", "the", "density", "profile,", "we", "observed", "deep", "penetration", "of", "the", "peptide", "into", "the", "membrane", "through", "its", "apolar", "residues", "with", "larger", "side-chains,", "after", "the", "flip", "(Fig.", "7", ").", "BP100", "hydration", "analysis", "also", "shows", "a", "decrease", "in", "the", "overall", "peptide", "hydration", "after", "the", "flip", "(Fig.", "8", ").", "Apolar/polar", "pair", "interaction", "analysis", "(Fig.", "11", ")", "revealed", "that", "after", "the", "flip,", "the", "number", "of", "contacts", "between", "apolar", "residues", "from", "the", "lipids", "and", "the", "peptide", "increases", "significantly,", "while", "the", "number", "of", "contacts", "between", "polar", "groups", "of", "the", "lipids", "and", "the", "peptides", "slightly", "decreases.", "In", "other", "words,", "peptide", "flip", "intensifies", "peptide", "binding", "to", "membranes", "burying", "the", "apolar", "residues", "of", "the", "peptide", "deeper", "into", "the", "membrane", "core.", "We", "investigated", "what", "factors", "could", "lead", "to", "the", "occurrence", "of", "BP100", "peptide", "flip.", "Membrane", "composition", "appears", "to", "have", "a", "significant", "role.", "For", "BP100", "alpha-helical", "structure", "formation", "is", "favored", "in", "negatively", "charged", "membranes", "8", ".", "In", "our", "simulations,", "BP100", "entirely", "flipped", "in", "DPPG", "enriched", "regions", "of", "the", "membrane.", "Peptide", "flip", "was", "detected", "in", "α-BP100", "in", "DPPG,", "α-BP100", "(res1–5)", "in", "DPPG", "and", "α-BP100", "in", "PCPG-R", "systems.", "No", "peptide", "flip", "was", "detected", "in", "the", "other", "constrained", "BP100", "simulations", "(α-BP100", "(res4–8)", "in", "DPPG", "and", "α-BP100", "(res7–11)", "in", "DPPG).", "However,", "it", "is", "hasty", "to", "affirm", "that", "it", "is", "unlikely", "to", "expect", "peptide", "flip", "to", "happen", "in", "those", "systems", "with", "600", "ns", "of", "simulation", "time.", "For", "instance,", "peptide", "flip", "was", "observed", "after", "1.4", "µs", "of", "simulation", "in", "α-BP100", "in", "DPPG.", "Hydration", "analysis", "(Fig.", "8b", ")", "and", "density", "profile", "(Fig.", "S4A,B", ")", "for", "α-BP100", "in", "DPPC", "show", "also", "that", "BP100", "flipped.", "However,", "the", "alpha-helix", "secondary", "structure", "is", "mostly", "lost", "(Fig.", "5a", ").", "For", "this", "reason,", "BP100", "amphiphilic", "apolar-polar", "facet", "separation", "(Fig.", "2c", ")", "is", "lost.", "Additionally,", "the", "apolar", "moiety", "of", "the", "peptide", "could", "have", "a", "crucial", "role", "in", "peptide", "flip.", "Hydration", "analysis", "(Fig.", "8", ")", "suggests", "peptide", "flip", "results", "in", "peptide", "dehydration", "and", "concomitantly", "enhances", "peptide/lipid", "interaction", "(Fig.", "11", "),", "increasing", "specially", "apolar", "contacts", "(Fig.", "11", ").", "Similar", "behavior", "was", "observed", "in", "a", "study", "of", "the", "interaction", "of", "micelles", "with", "polyatomic", "counterions", "with", "apolar", "and", "polar", "moieties.", "Experimental", "results", "and", "MD", "simulations", "showed", "that", "counterions", "with", "the", "same", "apolar", "groups", "but", "different", "polar", "groups", "resulted", "in", "similar", "ionic", "affinities", "for", "dodecyltrimethylammonium", "micelles,", "and", "the", "simulations", "showed", "that", "counterion", "adsorption", "to", "the", "micelles", "was", "related", "with", "the", "dehydration", "of", "the", "apolar", "group", "of", "the", "counterions", "rather", "than", "by", "the", "polar", "group", "41", ".", "These", "factors", "could", "help", "to", "explain", "BP100", "selectivity", "towards", "negatively", "charged", "bacterial", "membranes", "and", "low", "cytotoxicity.", "Results", "of", "leakage", "from", "the", "inner", "compartment", "of", "LUVs", "upon", "addition", "of", "BP100", "showed", "that", "BP100", "leakage", "ability", "is", "proportional", "to", "PG", "content", "while", "the", "peptide", "has", "small", "effects", "on", "zwitterionic", "PC", "vesicles,", "observed", "through", "studies", "of", "zeta", "potential", "and", "vesicle", "leakage", "11", ".", "BP100", "does", "not", "perturb", "PC", "bilayers", "even", "at", "high", "peptide/lipid", "ratio", "and", "causes", "membrane", "thinning", "on", "mixed", "DMPC/DMPG", "bilayers", "where", "a", "stronger", "effect", "appears", "in", "anionic", "DMPG", "lipids", "compared", "to", "zwitterionic", "DMPC", "lipids", "12", ".", "In", "zwitterionic-lipid-enriched", "mammalian", "membranes,", "BP100", "would", "bind", "to", "membranes", "through", "electrostatic", "interactions", "and", "the", "absence", "of", "a", "helix-promoting", "environment", "would", "prevent", "helix", "formation", "and", "even", "with", "peptide", "flip,", "as", "in", "our", "α-BP100", "in", "DPPC", "simulation,", "it", "could", "lead", "to", "mild", "membrane", "thinning", "11", ",", "12", ".", "On", "the", "other", "hand,", "bacterial", "membranes", "with", "high", "content", "of", "anionic", "lipids", "on", "the", "outer", "part", "of", "the", "membrane,", "could", "promote", "peptide", "binding,", "alpha-helix", "nucleation,", "and", "propagation,", "leading", "to", "peptide", "flip", "and", "more", "pronounced", "membrane", "thinning,", "which", "is", "consistent", "with", "our", "observations", "from", "α-BP100", "in", "DPPG,", "α-BP100", "in", "PCPG-R", "and", "α-BP100", "(res1–5)", "in", "DPPG", "sets.", "Although", "we", "could", "not", "detect", "membrane", "thinning", "in", "our", "simulations", "(Table", "S2", "),", "it", "should", "be", "noted", "that", "our", "simulations", "had", "only", "one", "peptide", "on", "128-lipid-bilayers.", "A", "more", "pronounced", "effect", "on", "the", "membrane", "might", "be", "observed", "by", "increasing", "the", "number", "of", "peptides", "in", "the", "simulations.", "DPPG", "subdomain", "formation", "Lipid", "clustering,", "or", "subdomain", "formation,", "induced", "by", "CAMPs", "is", "well", "established", "5", ",", "14", ",", "35", "–", "37", ",", "42", "–", "45", ".", "Through", "electrostatic", "interactions,", "CAMPs", "clusters", "anionic", "phosphatidylglycerol", "(PG)", "or", "cardiolipin", "(CL)", "lipids", "on", "the", "outer", "membrane", "of", "bacteria,", "inducing", "phase", "separation", "and", "phase", "boundary", "defects", "between", "lipid", "domains", "and", "the", "rest", "of", "the", "membrane,", "increasing", "membrane", "permeability", "36", ".", "Epand", "et", "al.", "first", "proposed", "it", "45", ",", "in", "a", "study", "with", "oligomers", "of", "lysine", "(OAK).", "Upon", "the", "addition", "of", "OAK", "in", "1-palmitoyl-2-oleoyl", "phosphatidylethanolamine", "(POPE)", "and", "tetraoleoyl", "cardiolipin", "(TOCL)", "mixed", "vesicles,", "transition", "temperature", "shift", "was", "observed", "through", "differential", "scanning", "calorimetry,", "indicating", "lipid", "phase", "separation", "45", ".", "Lipid", "phase", "separation", "was", "later", "observed", "for", "other", "cAMPs", "and", "cell-penetrating-peptides", "37", ",", "45", ",", "46", ".", "Although", "not", "so", "pronounced,", "DSC", "also", "detected", "BP100", "ability", "to", "promote", "lipid", "phase", "separation", "on", "POPE/TOCL", "(75:25)", "vesicles", "37", ".", "Lipid", "phase", "separation", "promoted", "by", "CAMPs", "has", "also", "been", "observed", "in", "computational", "modeling", "studies.", "In", "a", "coarse-grained", "MD", "simulation", "study", "35", ",", "lipid", "cluster", "promotion", "was", "seen", "in", "POPE/POPG", "(70:30)", "membranes", "by", "Ltc1,", "a", "linear", "alpha-helical", "cAMP", "with", "overall", "+10", "charge.", "Simulations", "showed", "Ltc1", "induced", "POPG", "domains", "in", "small", "(256", "lipids)", "and", "large", "membranes", "(512", "or", "2048", "lipids).", "Also,", "due", "to", "peptide-POPG", "charge", "neutralization,", "Ltc1-induced", "anionic", "domains", "seem", "to", "facilitate", "peptide", "oligomerization,", "enhancing", "peptide", "activity", "on", "bacterial", "model", "membranes", "35", ".", "Likewise,", "our", "simulations", "suggest", "that", "BP100", "promotes", "PG", "aggregation.", "Two", "DPPC:", "DPPG", "(50:50)", "mixed", "bilayers", "were", "designed", "to", "explore", "lipid", "aggregation", "by", "the", "peptide.", "One", "membrane", "was", "composed", "of", "randomly", "mixed", "(PCPG-NR)", "and", "in", "the", "other,", "a", "4", "×", "4", "DPPG", "raft", "(PCPG-R)", "was", "positioned", "in", "the", "middle", "of", "both", "monolayers", "(see", "Methods", "1", "and", "Fig.", "1", ").", "BP100", "was", "able", "to", "cluster", "DPPGs", "in", "PCPG-NR,", "which", "can", "be", "observed", "by", "the", "amount", "of", "DPPG", "in", "contact", "with", "BP100", "(Table", "2", ").", "From", "a", "total", "of", "11", "lipids", "in", "the", "closer", "contact", "with", "BP100,", "8.3", "in", "average", "are", "DPPG", "corresponding", "to", "75%", "of", "the", "lipid", "in", "the", "vicinity", "of", "the", "peptide.", "Additionally,", "this", "DPPG", "clustering", "can", "also", "be", "observed", "in", "lipid", "raft", "size", "distribution", "(Fig.", "S6E", "),", "reaching", "similar", "values", "of", "DPPG/peptide", "contact", "in", "α-BP100", "in", "PCPG-R", "simulation", "(Fig.", "10", ")", "and", "DPPG", "lipid", "raft", "size", "probability", "(Fig.", "S6D", ").", "These", "data", "suggest", "that", "even", "a", "short-lengthened", "CAMP", "as", "BP100", "can", "promote", "anionic", "lipid", "clustering", "and", "also", "maintain", "it,", "supporting", "the", "experimental", "findings", "37", ".", "BP100", "also", "adsorbs", "on", "pure", "zwitterionic", "PC", "membranes", "and", "promotes", "DPPC", "clustering", "as", "observed", "in", "Figs", "S5", "A", "and", "S6A", ".", "However,", "possibly", "due", "to", "the", "presence", "of", "positively", "charged", "choline", "headgroups", "(Fig.", "1d", "-", "left),", "secondary", "structure", "is", "lost", "(Fig.", "5a", ")", "and", "the", "peptide", "turns", "into", "random", "coil,", "resulting", "in", "an", "increase", "of", "available", "interaction", "surface", "area", "with", "the", "DPPC", "headgroups,", "which", "is", "reflected", "in", "a", "broad", "distribution", "in", "lipid", "raft", "size", "distribution", "(Fig.", "S5A", ").", "Polar/Apolar", "pair", "analysis", "for", "α-BP100", "in", "PCPG-R", "(Fig.", "11", ")", "helps", "to", "explain", "lipid", "aggregation.", "We", "computed", "the", "ten", "most", "frequent", "apolar", "and", "polar", "pairs", "between", "peptide", "and", "lipids.", "The", "number", "of", "polar", "pairs", "between", "the", "peptide", "and", "DPPG", "is", "higher", "than", "those", "with", "DPPC", "and", "is", "maintained", "even", "after", "the", "flip", "(~390", "ns).", "As", "expected,", "+1", "charged", "amino", "group", "(NZ)", "from", "Lysines", "side", "chains", "are", "the", "main", "participants", "in", "polar", "pairings", "(Figs", "S7", "–", "S13", "),", "interacting", "with", "DPPG", "headgroup", "glycerol", "(OC2,", "OC3,", "Fig.", "1d", "-", "right),", "phosphate", "group", "(P),", "and", "carbonyl", "groups", "(C21,", "C31,", "O21,", "O22,", "O31,", "O32)", "from", "chains", "sn1", "and", "sn2", "(Figs", "S9", ",", "S10", ",", "S12", ",", "S13", ").", "Peptide", "flip", "seems", "to", "contribute", "to", "lipid", "clustering.", "Polar/apolar", "pairs", "analysis", "(Fig.", "11", ")", "and", "pair", "frequency", "(Figs", "S8", ",", "S9", ")", "for", "the", "previous", "simulation,", "reveals", "that", "apolar", "pairs", "between", "BP100", "and", "both", "lipids", "rise", "after", "peptide", "flip", "occurs", "(~390", "ns),", "intensifying", "peptide-membrane", "interaction.", "The", "same", "phenomena", "were", "observed", "for", "other", "simulations", "with", "peptide", "flip", "(Figs", "S7", ",", "S10", "and", "S13", ").", "As", "long", "as", "peptide", "flip", "had", "occurred,", "apolar", "residue", "side", "chains", "(from", "Leu3,", "Phe4,", "Ile7,", "Leu8", "and", "Tyr10,", "and", "Leu11)", "were", "in", "close", "contact", "mainly", "with", "groups", "of", "the", "lipid", "tails", "(Figs", "S7", "–", "S10", ",", "S13", ").", "The", "mechanisms", "of", "bacterial", "toxicity", "via", "lipid", "clustering", "induced", "by", "CAMPs", "still", "has", "to", "be", "investigated.", "Moreover,", "possibly", "more", "than", "one", "mechanism", "could", "be", "involved", "in", "the", "process", "of", "bacterial", "killing.", "Evidence", "suggest", "lipid", "clustering", "by", "CAMPs", "could", "lead", "to", "negative", "charge", "concentration", "on", "membranes,", "promoting", "CAMPs", "attachment,", "leading", "to", "pore", "formation", "5", ".", "Anionic", "lipid", "clustering", "would", "have", "surrounding", "interfaces", "with", "the", "rest", "of", "the", "membrane", "which", "could", "lead", "to", "phase", "boundary", "defects,", "leading", "to", "slow", "leakage", "of", "inner", "cell", "content", "32", 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0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]	{"negatives/white": ["144+1", "1172+1", "1993+1", "2830+1", "3917+1", "4922+1", "5674+1", "6385+1", "6565+1", "6525+1", "6725+1"]}	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	Original work titled: 'Binding and Flip as Initial Steps for BP-100 Antimicrobial Actions' (Scientific Reports/Nature Publishing Group UK) by the following authors: Peter Park; Leandro R. Franco; Hernan Chaimovich; Kaline Coutinho; Iolanda M. Cuccovia; Filipe S. Lima DOI: 10.1038/s41598-019-45075-5
f7083ca86a7871cc8d04d2500480cbf3	[ "Introduction", "Colorectal", "cancer", "(CRC)", "is", "the", "third", "most", "common", "malignant", "tumor", "worldwide", "[", "1", "].", "With", "the", "development", "of", "early", "diagnosis", "methods", "and", "molecular", "targeted", "therapy,", "the", "progression", "free", "survival", "(PFS)", "and", "the", "overall", "survival", "(OS)", "of", "patients", "with", "CRC", "have", "increased", "[", "2", "].", "However,", "metastatic", "colorectal", "cancer", "(mCRC)", "is", "associated", "with", "a", "poor", "prognosis", "[", "3", ",", "4", "].", "Chemotherapy", "is", "a", "strongly", "recommended", "therapeutic", "regimen", "to", "prevent", "and", "control", "mCRC,", "and", "it", "has", "been", "associated", "with", "a", "prolonged", "life", "[", "5", ",", "6", "].", "Adjuvant", "chemotherapy", "may", "confer", "a", "survival", "advantage", "in", "resected", "CRC", "patients", "with", "stage", "III", "or", "high-risk", "stage", "II", "disease", "[", "7", ",", "8", "].", "Cytotoxic", "chemotherapeutic", "agents", "inhibit", "the", "proliferation", "of", "cancer", "cells,", "but,", "at", "the", "same", "time,", "they", "produce", "toxicity", "and", "side", "effects", "on", "other", "tissues", "and", "organs.", "How", "to", "effectively", "and", "efficiently", "avoid", "or", "to", "alleviate", "the", "toxicity", "and", "side", "effects", "is", "a", "critical", "clinical", "problem", "that", "should", "be", "solved.", "CID", "is", "a", "common", "side", "effect", "of", "the", "digestive", "system", "in", "the", "antitumor", "treatment", "process", "of", "cytotoxic", "drugs.", "The", "typical", "clinical", "characteristics", "of", "CID", "are", "as", "follows:", "symptoms", "range", "from", "loose", "stool", "without", "pain", "to", "severe,", "watery", "diarrhea", "with", "abdominal", "pain;", "symptoms", "typically", "last", "5", "to", "7", "days", "and", "may", "occur", "during", "or", "after", "chemotherapy;", "and", "patients", "with", "CID", "have", "a", "poor", "treatment", "response", "to", "gentamicin,", "berberine,", "and", "furoxone", "[", "9", "].", "CID", "can", "lead", "to", "weakness,", "electrolyte", "disorders,", "renal", "failure,", "blood", "volume", "reductions,", "shock,", "and", "even", "death", "[", "10", "].", "Its", "occurrence", "can", "result", "in", "delaying", "chemotherapy,", "increasing", "hospitalization", "time", "and", "costs,", "aggravating", "the", "psychological", "stress", "of", "patients,", "reducing", "the", "treatment", "compliance", "of", "patients", "and", "even", "altering", "the", "entire", "chemotherapy", "plan", "[", "11", "].", "A", "unique", "microecosystem", "and", "characteristic", "microbial", "community", "lives", "in", "the", "colorectal", "regions", "owing", "to", "their", "function", "of", "storing", "feces", "[", "12", "].", "Accumulating", "evidence", "points", "to", "the", "gut", "microbiome", "being", "involved", "in", "CRC", "[", "13", "].", "For", "example,", "some", "researchers", "have", "reported", "that", "Streptococcus", "bovis", "[", "14", "]", "and", "Streptococcus", "gallolyticus", "[", "15", "]", "are", "specific", "bacteria", "involved", "in", "colorectal", "cancer;", "Castellarin", "et", "al.", "[", "16", "]", "found", "that", "Fusobacterium", "nucleatum", "infection", "is", "prevalent", "in", "CRC", "tissue", "specimens", "and", "Kostic", "et", "al.", "[", "17", "],", "found", "that", "Fusobacterium", "nucleatum", "can", "generate", "a", "proinflammatory", "microenvironment", "that", "is", "conducive", "to", "the", "progression", "of", "CRC", "through", "recruitment", "of", "tumor-infiltrating", "immune", "cells.", "Moreover,", "many", "enzymes,", "peptides", "and", "small", "molecules", "secreted", "by", "the", "intestinal", "gut", "microbiome", "are", "involved", "in", "activating", "and", "regulating", "important", "signaling", "molecules", "and", "signaling", "pathways", "involved", "in", "the", "progression", "of", "CRC", "[", "18", ",", "19", "].", "Diarrhea", "and", "gut", "microbiome", "disorders", "interact", "as", "both", "cause", "and", "effect.", "Diarrhea", "can", "disrupt", "the", "balance", "of", "the", "gut", "microenvironment.", "Meanwhile,", "the", "invasion", "of", "exogenous", "pathogenic", "microorganisms", "and", "the", "imbalance", "of", "intestinal", "microbes", "can", "lead", "to", "diarrhea", "[", "20", "].", "For", "example,", "the", "invasion", "of", "some", "pathogenic", "bacteria", "including", "Shigella,", "Salmonella", "and", "Klebsiella", "[", "21", ",", "22", "],", "and", "the", "abundance", "changes", "of", "intestinal", "parasitic", "microbes", "include", "Candida", "albicans,", "Escherichia", "coli,", "and", "Aeromonas", "can", "induce", "diarrhea", "[", "23", "–", "25", "].", "Chemotherapy", "has", "been", "widely", "used", "in", "the", "clinic", "as", "an", "effective", "therapy", "for", "colorectal", "cancer,", "but", "diarrhea", "caused", "by", "chemotherapeutic", "drugs", "often", "affects", "the", "implementation", "of", "chemotherapy", "regimens", "[", "26", "].", "Many", "probiotics", "and", "antibiotics", "play", "a", "positive", "role", "in", "the", "treatment", "of", "infectious", "diarrhea", "by", "regulating", "the", "gut", "microbiome", "[", "27", ",", "28", "].", "The", "study", "of", "relationships", "between", "gut", "microbiome", "and", "CID", "may", "provide", "a", "new", "direction", "for", "solving", "this", "clinical", "problem.", "In", "the", "present", "study,", "we", "aimed", "to", "explore", "the", "association", "between", "the", "gut", "microorganisms", "and", "CID", "of", "the", "CapeOX", "regimen", "in", "resected", "stage", "III", "CRC", "and", "to", "provide", "some", "research", "methods", "and", "research", "ideas", "for", "further", "exploring", "the", "relationship", "between", "intestinal", "microbes", "and", "CID.", "The", "results", "may", "provide", "a", "fresh", "approach", "to", "the", "prevention", "and", "treatment", "of", "CID", "from", "a", "microbiological", "perspective.", "Subjects", "and", "methods", "Subjects", "Patients", "with", "CRC", "treated", "at", "Huzhou", "Central", "Hospital", "from", "January", "2016", "to", "January", "2018", "were", "studied.", "The", "clinical", "protocols", "involving", "the", "patients", "and", "the", "informed", "consent", "form", "were", "approved", "by", "the", "Ethics", "Committee", "of", "Huzhou", "Central", "Hospital", "(No.", "201601023).", "According", "to", "the", "American", "Joint", "Committee", "on", "Cancer", "(AJCC)", "staging", "manual", "system,", "stage", "III", "CRC", "is", "defined", "as", "when", "colorectal", "cancer", "is", "diagnosed", "by", "pathology", "with", "lymphatic", "metastasis", "and", "without", "distant", "metastasis", "[", "29", "].", "The", "inclusion", "criteria", "were", "as", "follows.", "①", "Cancer", "was", "confirmed", "by", "pathologic", "diagnosis,", "and", "the", "patients", "volunteered", "to", "participate", "in", "the", "study.", "②", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "③", "Patients", "voluntarily", "accepted", "and", "completed", "8", "cycles", "of", "the", "CapeOX", "regimen", "[capecitabine", "(1000", "mg/m", "2", "twice", "daily)", "combined", "with", "oxaliplatin", "(130", "mg/m", "2", "every", "3", "weeks)].", "The", "exclusion", "criteria", "were", "as", "follows.", "①", "Patients", "with", "diarrhea", "before", "chemotherapy.", "②", "Patients", "with", "other", "intestinal", "diseases,", "such", "as", "ulcerative", "colitis", "and", "Crohn’s", "disease.", "③", "Alternation", "of", "chemotherapy", "regimen", "and", "chemotherapeutic", "dosage", "or", "the", "occurrence", "of", "progression", "of", "the", "disease", "(PD)", "during", "the", "8", "cycles", "of", "the", "CapeOX", "regimen.", "④", "Patients", "who", "accepted", "neo-adjuvant", "chemotherapy.", "⑤", "Patients", "with", "a", "history", "of", "the", "use", "of", "oral", "microbial", "agents", "within", "1", "month", "before", "chemotherapy.", "⑥", "Patients", "with", "another", "primary", "cancer.", "⑦", "Patients", "with", "known", "primary", "organ", "failure.", "⑧", "Patients", "who", "could", "not", "obtained", "the", "stool", "samples", "(n", "=", "15)", "or", "obtained", "unqualified", "stool", "samples.", "All", "of", "the", "subjects", "signed", "informed", "consent", "under", "the", "guidelines", "approved", "by", "the", "Ethics", "Committee", "of", "Huzhou", "Central", "Hospital.", "CRC", "patients", "with", "and", "without", "CID", "were", "including", "in", "the", "experimental", "group", "and", "control", "group,", "respectively.", "According", "to", "National", "Cancer", "Institute", "Common", "Toxicity", "Criteria", "(NCI", "CTC)", "V3.0", "[", "30", "]", "CID", "grade", "1", "is", "defined", "as", "the", "number", "of", "defecations", "increased", "to", "less", "than", "4", "times", "a", "day", "and", "an", "excretion", "volume", "slightly", "increased.", "During", "a", "course", "of", "8", "cycles", "of", "chemotherapy", "administered", "over", "nearly", "half", "a", "year,", "there", "are", "many", "factors", "that", "can", "cause", "diarrhea,", "and", "these", "factors", "interfere", "with", "the", "diagnosis", "of", "CID.", "Clinical", "intervention", "measures,", "such", "as", "using", "antibiotics", "and", "antidiarrheal", "drugs,", "may", "be", "performed", "and", "the", "chemotherapy", "regimen", "may", "be", "changed", "in", "patients", "with", "CID", "grade", "3", "and", "grade", "4.", "Therefore,", "we", "did", "not", "include", "patients", "with", "grade", "1,", "grade", "3", "and", "grade", "4", "in", "the", "experimental", "group.", "CID", "grade", "2", "is", "defined", "as", "the", "number", "of", "defecations", "increased", "to", "4–6", "times", "per", "day", "or", "nocturnal", "stools,", "with", "the", "amount", "of", "excretion", "increased", "but", "with", "no", "interference", "in", "daily", "life.", "All", "of", "the", "subjects", "recruited", "into", "the", "experimental", "group", "were", "CID", "grade", "2", "in", "the", "present", "study.", "Collection", "of", "clinical", "data", "and", "stool", "samples", "Basic", "information", "and", "clinical", "serological", "indicators", "were", "obtained", "from", "the", "medical", "record", "management", "system", "of", "Huzhou", "Central", "Hospital", "with", "informed", "consent", "from", "patients.", "Stool", "samples", "of", "the", "patients", "were", "collected", "in", "the", "2", "weeks", "after", "the", "8", "cycles", "of", "chemotherapy.", "Stool", "samples", "were", "collected", "in", "the", "morning", "prior", "to", "breakfast.", "An", "approximately", "5–10", "g", "stool", "sample", "was", "collected", "after", "defecation", "without", "the", "use", "of", "a", "purgative", "or", "lubricant.", "Within", "half", "an", "hour,", "the", "stool", "samples", "were", "stored", "in", "an", "ultra-low", "temperature", "freezer.", "The", "sample", "preservation", "time", "was", "not", "beyond", "1", "month.", "Finally,", "17", "stool", "samples", "from", "CRC", "patients", "were", "analyzed", "after", "the", "patients", "had", "signed", "informed", "consent", "forms", "and", "the", "unqualified", "specimens", "were", "eliminated.", "Intestinal", "microorganism", "detection", "DNA", "extraction", "and", "PCR", "amplification", "A", "E.Z.N.A.", "®", "Stool", "DNA", "Kit", "(Omega", "Bio-Tek,", "Norcross,", "GA,", "U.S.)", "was", "used", "to", "extract", "total", "DNA", "from", "the", "stool", "samples", "according", "to", "the", "manufacturer’s", "protocols.", "The", "nanodrop", "ND-1000", "spectrophotometer", "(LabTech,", "Washington,", "DC,", "USA)", "with", "the", "absorbances", "at", "260", "nm", "and", "280", "nm", "(A260/A280)", "was", "used", "to", "determine", "the", "quality", "and", "quantity", "of", "the", "purified", "DNA.", "DNA", "integrity", "was", "further", "verified", "by", "electrophoresis", "through", "a", "2.0%", "(w/v)", "agarose", "gel.", "PCR", "(95", "°C", "for", "3", "min,", "followed", "by", "25", "cycles", "at", "95", "°C", "for", "30", "s,", "55", "°C", "for", "30", "s,", "and", "72", "°C", "for", "45", "s", "and", "a", "final", "extension", "at", "72", "°C", "for", "5", "min)", "was", "used", "to", "amplify", "the", "V3–V4", "region", "of", "the", "bacterial", "16S", "ribosomal", "RNA", "gene", "(the", "primers", "of", "16S", "V3–V4", "rDNA", "are", "as", "follows:", "forward,", "CCTACGGGNGGCWGCAG", "and", "reverse,", "GACTACHVGGGTATCTAATCC).", "PCR", "amplifications", "were", "performed", "in", "triplicate", "in", "a", "25", "μL", "mixture", "containing", "5", "μL", "of", "DNA", "template,", "2", "μL", "of", "Nextera", "XT", "Index", "Primer", "1", "(10", "M),", "2", "μL", "of", "Nextera", "XT", "Index", "Primer", "2", "(10", "M)", "and", "16", "μL", "ddH", "2", "O.", "Amplicons", "were", "extracted", "from", "2%", "agarose", "gels,", "purified", "using", "the", "AxyPrep", "DNA", "Gel", "Extraction", "Kit", "(Axygen", "Biosciences,", "Union", "City,", "CA,", "USA)", "and", "quantified", "using", "QuantiFluor™-ST", "(Promega,", "U.S.)", "according", "to", "the", "manufacturer’s", "instructions.", "Library", "construction", "and", "sequencing", "The", "MiSeq", "library", "was", "constructed", "as", "follows:", "purified", "PCR", "products", "were", "quantified", "by", "Qubit", "®", "3.0", "(Life", "Technologies,", "Invitrogen).", "PCR", "products", "were", "ligated", "with", "Y", "adapter.", "Magnetic", "nanoparticles", "were", "used", "to", "take", "out", "the", "self-ligated", "Y", "adapters.", "An", "Illumina", "Pair-End", "library", "was", "constructed", "using", "pooled", "DNA", "products,", "and", "the", "amplicon", "library", "was", "pair-end", "sequenced", "(2", "×", "250)", "on", "an", "Illumina", "MiSeq", "platform", "(Shanghai", "BIOZERON", "Co.,", "Ltd.)", "according", "to", "standard", "protocols", "(MiSeq", "V3", "or", "V4", "sequencing", "kit,", "Hua", "Ya", "Regenerative", "Medicine", "Biological", "Engineering", "Technology", "CO.,", "LTD,", "Shanghai,", "China).", "Sequencing", "data", "bioinformatics", "analysis", "Sequencing", "data", "processing", "and", "optimization", "were", "performed", "according", "to", "the", "criteria", "at", "http://en.wikipedia.org/wiki/Fastq", ".", "Cutadapt", "(version", "1.11)", "was", "used", "to", "demultiplex", "and", "quality", "filter", "the", "raw", "fastq", "files.", "Pandaseq", "(version", "2.9)", "was", "used", "to", "assemble", "PE", "reads;", "only", "sequences", "that", "overlapped", "longer", "than", "10", "bp", "were", "assembled", "according", "to", "their", "overlap", "sequence.", "Mosaic", "sequences", "and", "sequences", "longer", "than", "300–480", "bp", "were", "discarded.", "In", "addition,", "the", "reads", "that", "receiving", "an", "average", "quality", "score", "<", "20", "were", "discarded.", "The", "Silva", "database", "was", "used", "for", "comparison", "of", "16S", "rRNA", "gene", "sequences", "[", "31", "]", "and", "the", "GeneBank", "database", "was", "used", "to", "analyze", "functional", "genes", "[", "32", "].", "Mothur", "software", "[", "33", "]", "(", "http://www.mothur.org/wiki/Main_Page", ")", "was", "used", "to", "analyze", "the", "microbial", "diversity.", "OTU", "(operational", "taxonomic", "units),", "as", "the", "artificial", "classification", "unit,", "was", "used", "to", "estimate", "the", "number", "of", "species", "in", "each", "sample,", "and", "the", "similarity", "threshold", "was", "97%.", "The", "coverage", "(", "http://www.mothur.org/wiki/Coverage", ")", "was", "used", "as", "the", "sequencing", "depth", "index.", "The", "ACE", "estimator", "(", "http://www.mothur.org/wiki/Ace", ")", "and", "Chao", "estimator", "(", "http://www.mothur.org/wiki/Chao", ")", "were", "used", "to", "estimate", "the", "gut", "microbial", "community", "richness.", "The", "Shannon", "index", "(", "http://www.mothur.org/wiki/Shannon", ")", "and", "Simpson", "index", "(", "http://www.mothur.org/wiki/Simpson", ")", "were", "used", "to", "estimate", "the", "gut", "microbial", "community", "diversity.", "The", "pie", "charts", "were", "used", "to", "describe", "the", "distribution", "of", "microorganisms", "at", "the", "species", "level.", "Proportions", "less", "than", "1%", "were", "included", "in", "“Others”,", "considering", "the", "best", "visual", "presentation.", "Venn", "diagrams", "were", "implemented", "by", "Venn", "diagram.", "We", "performed", "clustering", "on", "genera", "obtained", "from", "the", "RDP", "Classifier", "by", "means", "of", "the", "complete", "linkage", "hierarchical", "clustering", "technique", "using", "the", "R", "package", "HCLUST", "(", "http://sekhon.berkeley.edu/stats/html/hclust.html", ").", "The", "basic", "process", "of", "LDA", "analysis", "[", "34", "]", "was", "as", "follows:", "First,", "non-parametric", "Kruskal–Wallis", "rank", "sum", "test", "was", "used", "to", "detect", "the", "significant", "difference", "of", "abundance", "among", "species", "between", "the", "two", "groups", "of", "samples.", "Then", "Wilcoxon", "rank", "sum", "test", "was", "used", "to", "analyze", "the", "difference", "between", "groups", "based", "on", "the", "significant", "difference", "species.", "Finally,", "LDA", "was", "used", "to", "reduce", "the", "dimension", "of", "data", "and", "evaluate", "the", "influence", "of", "significantly", "different", "species", "(LDA", "score).", "LDA", "effect", "size", "more", "than", "2", "was", "included", "in", "the", "figure.", "The", "genes", "of", "the", "16S", "corresponding", "species", "and", "their", "copy", "number", "have", "been", "generally", "known,", "and", "these", "genes", "can", "be", "compared", "with", "databases", "(e.g.", "KEGG,", "NOG)", "to", "predict", "the", "possible", "metabolic", "capacity", "of", "these", "species.", "The", "Picrust", "software", "was", "used", "to", "analyze", "the", "pathway", "based", "on", "the", "KEGG", "database.", "The", "specific", "methods", "of", "function", "prediction", "analysis", "are", "shown", "in", "the", "following", "website", "(", "http://picrust.github.io/picrust/tutorials/algorithm_description.html", ").", "Statistical", "analysis", "SPSS", "software", "(V:", "16.0;", "SPSS", "Inc.,", "Chicago,", "IL)", "was", "used", "to", "analyze", "the", "data.", "The", "data", "are", "expressed", "as", "the", "mean", "±", "standard", "deviation", "(SD).", "Statistically", "significant", "differences", "between", "two", "groups", "were", "analyzed", "using", "the", "independent-samples", "t-test.", "Frequency", "table", "data", "were", "analyzed", "with", "the", "Chi-square", "test", "or", "Fisher’s", "exact", "test.", "All", "tests", "were", "performed", "at", "the", "0.05", "level", "of", "significance.", "Results", "Basic", "characteristics", "of", "the", "patients", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Overall,", "68", "resected", "stage", "III", "CRC", "patients", "accepted", "the", "CapeOX", "regimen", "in", "Huzhou", "Central", "Hospital", "from", "January", "2016", "to", "January", "2018.", "However,", "51", "patients", "were", "excluded", "from", "this", "study", "for", "the", "following", "reasons:", "patients", "with", "diarrhea", "before", "chemotherapy", "(n", "=", "3),", "terminated", "chemotherapy", "because", "of", "PD", "and", "side", "effects", "(n", "=", "7),", "accepted", "neo-adjuvant", "chemotherapy", "(n", "=", "4),", "could", "not", "obtain", "the", "stool", "samples", "(n", "=", "15),", "unqualified", "stool", "samples", "(n", "=", "14),", "and", "used", "oral", "probiotics", "including", "Bifid-triple", "Viable", "Capsule", "(n", "=", "6)", "and", "Clostridium", "Butyricum", "Capsule", "(n", "=", "2)", "before", "chemotherapy.", "After", "screening", "and", "identification,", "finally,", "17", "stage", "III", "CRC", "patients", "were", "enrolled", "in", "the", "present", "study.", "The", "study", "strategy", "is", "shown", "in", "Fig.", "1", ".", "The", "pathology", "type", "of", "all", "of", "the", "patients", "with", "resected", "stage", "III", "colorectal", "cancer", "was", "moderately", "differentiated", "adenocarcinoma.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "The", "17", "CRC", "patients", "were", "divided", "into", "experimental", "group", "(CID+)", "and", "control", "group", "(CID−)", "according", "to", "whether", "they", "had", "diarrhea", "or", "not.", "The", "patient’s", "clinical", "information", "is", "shown", "in", "Table", "1", ".", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "Blood", "samples", "of", "patients", "before", "chemotherapy", "were", "collected", "for", "serological", "examination.", "There", "was", "no", "significant", "difference", "between", "the", "two", "groups", "in", "terms", "of", "age,", "sex,", "BMI,", "or", "smoking,", "drinking", "history", "and", "clinical", "serological", "indicators", "including", "hemoglobin,", "alanine", "transaminase,", "glutamic", "oxaloacetic", "transaminase,", "albumin,", "creatinine,", "carbohydrate", "antigen", "199,", "carbohydrate", "antigen", "742", "and", "carcino", "embryonic", "antigen.", "Fig.", "1", "Study", "strategy.", "After", "screening", "and", "identification,", "finally,", "17", "stage", "III", "CRC", "patients", "undergoing", "the", "CapeOX", "regimen", "after", "curative", "resection", "were", "recruited", "into", "the", "present", "study.", "Stool", "samples", "were", "collected", "from", "the", "recruited", "patients.", "The", "bacterial", "16S", "ribosomal", "RNA", "from", "the", "samples", "were", "sequenced.", "Bioinformatics", "analysis", "was", "used", "to", "analyze", "the", "distinctive", "gut", "microbiome", "and", "functional", "metabolism", "Table", "1", "Characteristics", "of", "the", "study", "participants", "Experimental", "group", "Control", "group", "p", "value", "Cases,", "n", "4", "13", "–", "Rectal", "cancer,", "n", "2", "6", "0.89", "Males,", "n", "3", "8", "0.62", "Age", "(years)", "63.88", "±", "8.61", "64.23", "±", "8.76", "0.94", "BMI", "(kg/m", "2", ")", "22.74", "±", "2.64", "23.28", "±", "2.43", "0.71", "Long-term", "smoking", "history,", "n", "0", "1", "0.57", "Long-term", "drinking", "history,", "n", "1", "4", "0.82", "Known", "diabetes,", "n", "0", "1", "0.57", "Known", "hypertension,", "n", "0", "0", "/", "Hemoglobin", "(g/L)", "134.25", "±", "5.06", "124.62", "±", "13.71", "0.20", "Alanine", "transaminase", "(U/L)", "29.00", "±", "30.97", "19.80", "±", "8.94", "0.33", "Glutamic", "oxaloacetic", "transaminase", "(U/L)", "27.40", "±", "15.78", "24.56", "±", "8.72", "0.64", "Albumin", "(g/L)", "42.03", "±", "4.57", "38.11", "±", "3.02", "0.06", "Creatinine", "(μmol/L)", "83.10", "±", "33.74", "68.95", "±", "14.66", "0.24", "Carbohydrate", "antigen", "199", "(U/mL)", "5.46", "±", "3.61", "12.24", "±", "9.36", "0.18", "Carbohydrate", "antigen", "742", "(U/mL)", "8.58", "±", "3.61", "9.89", "±", "19.71", "0.90", "Carcino", "embryonic", "antigen", "(ng/mL)", "24.34", "±", "41.47", "3.98", "±", "3.70", "0.08", "Overall,", "17", "resected", "stage", "III", "CRC", "patients", "undergoing", "the", "CapeOX", "regimen", "were", "recruited.", "These", "patients", "were", "divided", "into", "experimental", "group", "(CID+)", "and", "control", "group", "(CID−)", "according", "to", "whether", "they", "had", "diarrhea", "or", "not.", "The", "patient’s", "clinical", "information", "was", "shown", "in", "thus", "table.", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "Blood", "samples", "of", "patients", "before", "chemotherapy", "were", "collected", "for", "serological", "examination", "Gut", "microbial", "richness", "and", "diversity", "associated", "with", "CID", "during", "the", "Cape", "OX", "regimen", "for", "CRC", "As", "shown", "in", "Table", "2", ",", "the", "coverage", "index", "suggested", "that", "the", "coverage", "of", "the", "genomic", "library", "of", "the", "sequencing", "samples", "was", "more", "than", "99%.", "The", "statistically", "significant", "differences", "of", "the", "ACE", "estimator", "and", "Chao", "estimator", "between", "the", "experimental", "group", "and", "control", "group", "indicated", "that", "the", "community", "richness", "of", "the", "gut", "microbiome", "was", "lower", "in", "the", "patients", "with", "chemotherapy-induced", "diarrhea", "(p", "<", "0.05,", "vs", "control", "group).", "The", "smaller", "Shannon", "index", "and", "larger", "Simpson", "index", "indicate", "that", "the", "gut", "microbial", "community", "diversity", "was", "lower", "in", "the", "patients", "with", "chemotherapy-induced", "diarrhea", "(p", "<", "0.01,", "vs", "control", "group).", "The", "alpha", "diversity", "analysis", "is", "shown", "in", "Additional", "file", "1", ".", "Table", "2", "Gut", "microbial", "richness", "and", "diversity", "associated", "with", "chemotherapy-induced", "diarrhea", "of", "the", "CapeOX", "regimen", "in", "CRC", "Group", "n", "Coverage", "OTU", "Ace", "Chao", "Shannon", "Simpson", "Experimental", "group", "4", "0.9994", "±", "0.0008", "119.75", "±", "53.44", "159.50", "±", "100.09", "150.75", "±", "77.58", "2.21", "±", "0.47", "0.21", "±", "0.08", "Control", "group", "13", "0.9985", "±", "0.0012", "273.38", "±", "93.68", "336.31", "±", "117.73", "332.46", "±", "113.34", "3.36", "±", "0.59", "0.09", "±", "0.07", "t", "value", "–", "–", "−", "3.08", "−", "2.70", "−", "2.97", "−", "3.54", "2.30", "p", "value", "–", "–", "0.008", "0.016", "0.010", "0.003", "0.009", "Mothur", "software", "(", "http://www.mothur.org/wiki/Main_Page", ")", "was", "used", "to", "analyze", "the", "microbial", "diversity.", "Operational", "taxonomic", "units", "(OTU),", "as", "the", "artificial", "classification", "unit,", "was", "used", "to", "estimate", "the", "number", "of", "species", "in", "each", "sample,", "and", "the", "similarity", "threshold", "was", "97%.", "The", "alpha", "diversity", "analysis", "was", "shown", "in", "Additional", "file", "1", "Gut", "microbial", "community", "structure", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC", "A", "pie", "chart", "was", "constructed", "for", "the", "microorganisms", "that", "accounted", "for", "more", "than", "1%", "in", "the", "two", "groups", "of", "samples", "to", "show", "the", "microbial", "community", "structure", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC.", "The", "distribution", "of", "microorganisms", "from", "the", "same", "group", "was", "merged", "into", "a", "whole.", "As", "shown", "in", "Fig.", "2", ",", "the", "stacked", "bar", "graph", "showed", "that", "the", "percent", "of", "Klebsiella", "pneumoniae", "was", "35%", "and", "it", "occupied", "the", "highest", "proportion", "in", "the", "experimental", "group", "(CID+).", "But", "the", "rate", "was", "only", "3%", "in", "the", "control", "group", "(CID−).", "Fig.", "2", "Microbial", "community", "structure", "analysis.", "The", "distribution", "of", "microorganisms", "from", "the", "same", "group", "was", "merged", "into", "the", "whole.", "The", "stacked", "bar", "graph", "was", "used", "to", "describe", "the", "distribution", "of", "microorganisms", "at", "the", "species", "level.", "CID-", "and", "CID+", "represent", "the", "control", "group", "and", "experimental", "group,", "respectively", "The", "difference", "in", "microbial", "abundance", "associated", "with", "CID", "of", "the", "CapeOX", "regimen", "for", "CRC", "The", "samples", "were", "divided", "into", "two", "groups", "according", "to", "patients", "with", "or", "without", "CID.", "As", "shown", "in", "Fig.", "3", "a,", "there", "were", "220", "shared", "OTUs", "and", "that", "the", "numbers", "of", "unique", "OTUs", "in", "the", "control", "group", "and", "experimental", "group", "were", "645", "and", "34,", "respectively.", "LDA", "effect", "size", "analysis", "was", "used", "to", "compare", "the", "differences", "in", "microbial", "abundance", "between", "the", "two", "groups.", "LDA", "Effect", "Size", "analysis", "(Fig.", "3", "b)", "showed", "that", "there", "were", "75", "microorganisms", "with", "significant", "differences", "at", "the", "genus", "level", "between", "the", "two", "groups.", "The", "length", "of", "the", "histogram", "indicates", "that", "some", "microorganisms", "such", "as", "Proteobacteria,", "Enterobacteriales,", "Gammaproteobacteria,", "Enterobacteriaceae,", "Klebsiella,", "Clostridiales,", "Clostridia,", "Ruminococcaceae,", "Bacteroidetes,", "Bacteroidia,", "Bacteroidales,", "Bacteroides", "and", "Bacteroidaceae", "have", "a", "larger", "influence.", "The", "cladogram", "in", "Fig.", "3", "c", "displays", "the", "composition", "and", "proportion", "of", "microorganisms", "at", "different", "taxonomic", "levels.", "The", "cladogram", "showed", "that", "the", "abundance", "of", "these", "bacteria", "including", "Actinomycetaceae,", "Micrococcaceae,", "Bacteroidaceae,", "Porphyromonadaceae,", "Ruminococcaceae,", "Erysipelotrichaceae,", "Leptotrichiaceae,", "Enterobacteriaceae", "(Klebsiella", "belongs", "to", "Enterobacteriaceae)", "and", "Synergistaceae", "had", "significant", "differences", "at", "family", "level", "between", "the", "two", "groups.", "Fig.", "3", "Differential", "microbes", "analysis.", "The", "samples", "were", "divided", "into", "two", "groups", "according", "to", "patients", "with", "or", "without", "CID.", "The", "number", "of", "OTUs", "that", "were", "exclusive", "or", "shared", "by", "the", "two", "groups", "were", "visualized", "by", "a", "Venn", "Chart", "(", "a", ").", "The", "strip", "figure", "in", "b", "displays", "the", "LDA", "effect", "size", "analysis.", "LDA", "effect", "size", "analysis", "was", "used", "to", "compare", "the", "differences", "in", "microbial", "abundance", "between", "the", "two", "groups", "(", "b", ").", "LDA", "scores", "over", "2", "mean", "that", "the", "differences", "are", "statistically", "significant", "(p", "<", "0.05).", "The", "cladogram", "in", "c", "displays", "the", "composition", "and", "proportion", "of", "microorganisms", "at", "different", "taxonomic", "levels.", "The", "innermost", "layer", "shows", "the", "taxonomic", "tree.", "The", "circle", "from", "the", "inside", "to", "the", "outside", "represents", "different", "taxon", "levels", "from", "phylum", "to", "genus.", "Yellow", "circles", "represent", "no", "statistical", "difference", "in", "species", "between", "the", "two", "groups,", "and", "red", "circles", "and", "green", "circles", "represent", "the", "higher", "abundance", "of", "genus", "in", "the", "control", "group", "and", "the", "chemotherapy-induced", "diarrhea", "group,", "respectively.", "The", "lowercase", "English", "letters", "corresponding", "to", "the", "different", "bacteria", "are", "shown", "in", "the", "legends", "The", "functional", "analysis", "of", "16S", "rRNA", "genes", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC", "It", "has", "been", "confirmed", "that", "many", "gene", "function", "annotation", "databases", "are", "associated", "with", "corresponding", "16S", "rRNA", "gene", "sequences.", "Functional", "analysis", "of", "enzymes", "and", "genes", "would", "be", "conducive", "to", "understand", "the", "whole", "microecological", "environment.", "As", "shown", "in", "Fig.", "4", ",", "the", "figure", "shows", "the", "results", "of", "the", "functional", "analysis.", "Overall,", "23", "pathways", "(p", "<", "0.001)", "associated", "with", "statistically", "significant", "differences", "might", "be", "involved", "in", "CID", "during", "the", "CapeOX", "regimen", "for", "adjuvant", "chemotherapy", "in", "resected", "stage", "III", "colorectal", "cancer.", "These", "differential", "pathways", "are", "related", "to", "microbial", "metabolites,", "cell", "proliferation", "and", "death,", "immune", "system,", "and", "many", "other", "aspects.", "Fig.", "4", "Functional", "analysis.", "The", "Picrust", "software", "was", "used", "to", "analyze", "the", "pathway", "based", "on", "the", "KEGG", "database.", "The", "significant", "differences", "in", "functional", "metabolism", "are", "shown", "in", "the", "figure", "Discussion", "The", "CapeOX", "regimen", "is", "recommended", "as", "the", "postoperative", "adjuvant", "chemotherapy", "regimen", "in", "resected", "CRC", "patients", "with", "stage", "III", "or", "high-risk", "stage", "II", "[", "35", "].", "The", "high-risk", "factors", "in", "stage", "II", "may", "affect", "the", "accuracy", "of", "experimental", "results,", "Thus,", "CRC", "patients", "with", "stage", "III", "were", "recruited", "in", "the", "present", "study.", "According", "to", "the", "NCCN", "guidelines,", "the", "CapeOX", "regimen", "for", "resected", "stage", "III", "CRC", "has", "been", "defined", "as", "8", "cycles", "of", "capecitabine", "(1000", "mg/m", "2", "twice", "daily)", "combined", "with", "oxaliplatin", "(130", "mg/m", "2", "every", "3", "weeks)", "[", "36", "].", "CID", "is", "one", "of", "the", "most", "common", "side", "effects", "of", "the", "CapeOX", "regimen", "[", "37", "].", "The", "degree", "of", "diarrhea", "caused", "by", "different", "chemotherapeutic", "agents", "varies.", "Among", "the", "chemotherapeutic", "drugs", "for", "colorectal", "cancer,", "the", "most", "common", "drugs", "that", "cause", "diarrhea", "are", "fluorouracil,", "irinotecan", "and", "platinum,", "and", "the", "incidence", "of", "diarrhea", "associated", "with", "these", "drugs", "can", "be", "as", "high", "as", "50–80%", "[", "38", "].", "Our", "previous", "clinical", "investigation", "found", "that", "the", "probability", "of", "CID", "increased", "with", "an", "increase", "of", "chemotherapeutic", "frequency", "and", "chemotherapeutic", "dosage", "and", "most", "cases", "of", "CID", "occur", "within", "2", "weeks", "after", "chemotherapy.", "Therefore,", "in", "the", "present", "study,", "stool", "samples", "from", "the", "patients", "were", "collected", "in", "2", "weeks", "after", "the", "8", "cycles", "of", "chemotherapy", "of", "the", "CapeOX", "regimen", "were", "completed.", "Studies", "of", "Carroll", "et", "al.", "[", "39", "]", "suggested", "that", "the", "richness", "of", "16S", "rRNA", "sequences", "was", "significantly", "decreased", "in", "patients", "with", "diarrhea-predominant", "irritable", "bowel", "syndrome.", "Lee", "[", "40", "]", "has", "reported", "the", "community", "diversity", "of", "gut", "microbiome", "is", "lower", "in", "patients", "with", "kidney", "posttransplant", "diarrhea.", "Similar", "results", "have", "been", "reported", "in", "dogs", "with", "acute", "diarrhea", "[", "41", "].", "The", "research", "on", "these", "diarrhea", "diseases", "is", "consistent", "with", "our", "findings.", "We", "found", "that", "the", "gut", "microbial", "community", "richness", "and", "community", "diversity", "was", "lower", "in", "CRC", "patients", "with", "CID", "in", "the", "present", "study.", "The", "decrease", "of", "microbial", "diversity", "may", "be", "related", "to", "the", "imbalance", "of", "gut", "microbiome.", "The", "dominant", "pathogenic", "bacteria", "leads", "to", "the", "reduction", "of", "the", "resident", "normal", "microbiota", "through", "the", "plunder", "of", "nutrients", "or", "the", "killing", "effect", "of", "bacterial", "toxic", "metabolites.", "Our", "results", "from", "the", "microbial", "community", "structure", "analysis", "showed", "Klebsiella", "pneumoniae", "occupied", "the", "highest", "proportion", "(31.22%)", "of", "gut", "microbiome", "in", "CRC", "patients", "with", "CID.", "Lu", "et", "al.", "[", "42", "]", "reported", "that", "the", "rate", "of", "Klebsiella", "pneumoniae", "was", "approximately", "0.5%", "in", "Beijing", "by", "culturing", "the", "stool", "pathogens", "from", "outpatients", "with", "diarrhea", "syndromes", "using", "the", "Vitek2", "Compact", "instrument.", "Zhang", "et", "al.", "[", "22", "]", "only", "isolated", "43", "Klebsiella", "pneumoniae", "strains", "from", "551", "stool", "specimens", "from", "diarrhea", "patients.", "Although", "the", "study", "may", "have", "be", "interfered", "by", "other", "factors", "such", "as", "geographical", "location,", "nosocomial", "infection,", "or", "pollution", "contamination", "in", "the", "fecal", "collection", "process.", "The", "increased", "proportion", "of", "Klebsiella", "pneumoniae", "in", "patients", "with", "CID", "remains", "a", "phenomenon", "of", "concern.", "Microorganisms", "can", "adjust", "and", "modify", "their", "surroundings", "due", "to", "their", "metabolic", "products.", "A", "series", "of", "enzymes", "and", "genes", "participate", "in", "the", "process", "of", "metabolism", "[", "12", "].", "Functional", "analysis", "of", "these", "enzymes", "and", "genes", "would", "be", "conducive", "to", "understand", "the", "whole", "microecological", "environment.", "In", "the", "present", "study,", "we", "screened", "75", "differentiated", "microorganisms", "at", "the", "species", "level", "by", "using", "LDA", "effect", "size", "analysis", "as", "well", "as", "23", "pathways", "associated", "with", "differential", "microorganisms", "by", "using", "KEGG", "databases.", "These", "differential", "pathways", "are", "related", "to", "microbial", "metabolites,", "cell", "proliferation", "and", "death,", "immune", "system,", "and", "many", "other", "aspects.", "These", "differentiated", "microorganisms,", "their", "metabolic", "products", "and", "the", "relevant", "pathways", "make", "up", "the", "intestinal", "microecosystem", "that", "causes", "CID.", "These", "results", "may", "provide", "characteristic", "microorganisms", "or", "potential", "molecular", "targets", "for", "the", "treatment", "and", "prevention", "of", "CID.", "It", "must", "be", "emphasized", "that", "small", "sample", "size", "and", "potential", "sample", "contamination", "may", "affect", "the", "accuracy", "of", "the", "present", "study.", "Strict", "inclusion", "and", "exclusion", "criteria", "was", "performed", "in", "this", "study.", "Subjects", "in", "the", "experimental", "group", "were", "only", "included", "the", "resected", "stage", "III", "colorectal", "cancer", "(CRC)", "patients", "who", "voluntarily", "accepted", "and", "completed", "8", "cycles", "of", "the", "CapeOX", "regimen", "and", "accompanied", "with", "grade", "2", "of", "CID.", "Thus,", "the", "small", "sample", "size", "limits", "the", "applicability", "of", "the", "findings.", "A", "multi-center,", "large", "study", "will", "provide", "more", "powerful", "data", "to", "support", "the", "clarification", "of", "the", "microbial", "differences", "in", "CRC", "patients", "with", "CID.", "Conclusion", "Chemotherapy", "induced", "diarrhea", "(CID)", "is", "a", "common", "side", "effect", "in", "patients", "receiving", "chemotherapy", "for", "cancer.", "In", "the", "present", "study,", "We", "found", "that", "the", "gut", "microbial", "community", "structure", "and", "community", "diversity", "have", "changed", "in", "CRC", "patients", "with", "CID.", "The", "gut", "microbial", "community", "richness", "and", "community", "diversity", "were", "lower", "in", "CID.", "Klebsiella", "pneumoniae", "was", "the", "most", "predominant", "species", "among", "the", "gut", "microbiome", "in", "CRC", "patients", "with", "CID.", "It", "may", "provide", "novel", "insights", "into", "the", "prevention", "and", "treatment", "of", "patients", "with", "CID", "to", "improve", "patients’", "quality", "of", "life", "and", "chemotherapy", "tolerance." ]	[ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 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]	{"negatives/white": ["2767+1", "1193+1", "1660+1", "1959+1", "2046+1", "3276+1", "3671+1", "3730+1", "4060+1"], "pathogens/yellow": ["353+2", "371+2", "495+2", "497+2"]}	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	Original work titled: 'Gut microbiome associated with chemotherapy-induced diarrhea from the CapeOX regimen as adjuvant chemotherapy in resected stage III colorectal cancer' (Gut Pathogens/BioMed Central) by the following authors: Zuo Fei; Yin Lijuan; Yang Xi; Wu Wei; Zhong Jing; Da Miao; Han Shuwen DOI: 10.1186/s13099-019-0299-4
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"crop", "species", "have", "shown", "that", "many", "alleles", "representing", "broad", "genetic", "diversity", "and", "phenotypic", "variation", "reside", "in", "wild", "germplasm", "[", "8", "].", "Exploring", "novel", "disease", "resistance", "alleles", "from", "exotic", "libraries", "has", "been", "a", "useful", "strategy", "for", "genetic", "improvement", "in", "many", "crops", "[", "3", ",", "9", "].", "In", "lentil,", "Podder", "et", "al.", "[", "7", "]", "evaluated", "resistance", "to", "SB", "among", "70", "lentil", "accessions", "representing", "seven", "species", "in", "the", "genus", "Lens", "and", "found", "that", "L.", "ervoides", "(Brign.)", "Grande", "exhibited", "superior", "SB", "resistance", "in", "a", "much", "higher", "frequency", "than", "other", "species.", "Facilitated", "by", "the", "sophisticated", "ovule", "rescue", "technique", "that", "overcomes", "the", "interspecific", "reproductive", "barriers", "between", "L.", "ervoides", "and", "L.", "culinaris", "[", "10", "],", "interest", "in", "introgressing", "useful", "genes", "from", "L.", "ervoides", "to", "elite", "cultivars", "has", "been", "increasing", "strongly,", "in", "particular", "because", "resistance", "to", "other", "diseases", "such", "as", "ascochyta", "blight", "(caused", "by", "Ascochyta", "lentis", "Vassiljevsky)", "and", "anthracnose", "(caused", "by", "Colletotrichum", "lentis", "Damm)", "has", "also", "been", "identified", "in", "this", "species", "[", "11", "].", "To", "date,", "detailed", "knowledge", "of", "the", "molecular", "bases", "underlying", "SB", "resistance", "in", "L.", "ervoides", "is", "still", "lacking", "and", "the", "only", "available", "information", "is", "the", "presence", "of", "three", "quantitative", "trait", "loci", "(QTLs)", "associated", "with", "SB", "on", "a", "single", "nucleotide", "polymorphism", "(SNP)", "-based", "linkage", "map", "of", "an", "F", "9", "recombinant", "inbred", "line", "(RIL)", "population", "of", "L.", "ervoides", "[", "11", "].", "Despite", "the", "identification", "and", "localization", "of", "these", "QTLs,", "their", "direct", "use", "in", "marker-assisted", "selection", "is", "limited", "and", "identification", "of", "candidate", "resistance", "genes", "is", "difficult", "because", "those", "QTL", "intervals", "(2-LOD)", "are", "relatively", "large", "(6", "to", "21", "MB)", "and", "contain", "hundreds", "of", "genes", "[", "11", "].", "A", "functional", "understanding", "of", "SB", "resistance", "has", "been", "largely", "developed", "in", "model", "plants", "such", "as", "tomato", "(Solanum", "lycopersicum", "L.)", "and", "Arabidopsis", "thaliana", "(L.)", "Heynh.", "In", "tomato,", "Yang", "et", "al.", "[", "12", "]", "mapped", "a", "single", "dominant", "locus", "in", "a", "260", "Kb", "chromosomal", "region", "conferring", "resistance", "to", "SB", "(caused", "by", "S.", "lycopersici", "(Enjoji)", "W.", "Yamam.)", "and", "identified", "two", "putative", "resistance", "genes", "coding", "for", "a", "cysteine-rich", "receptor-like", "kinase", "(CRK)", "and", "a", "receptor-like", "kinase", "(RLK).", "This", "indicated", "that", "resistance", "in", "tomato", "was", "likely", "mediated", "via", "the", "recognition", "between", "plant", "and", "pathogen.", "In", "A.", "thaliana,", "Di", "et", "al.", "[", "13", "]", "reported", "that", "a", "gene", "encoding", "polygalactoronase-inhibiting", "protein", "(PGIP)", "was", "upregulated", "during", "the", "S.", "solani", "infection", "period.", "As", "PGIP", "belongs", "to", "a", "large", "family", "of", "leucine-rich", "repeat", "(LRR)", "proteins,", "it", "was", "suspected", "that", "a", "hypersensitive", "response", "(HR)", "may", "affect", "SB", "resistance", "in", "A.", "thaliana", "[", "13", "].", "However,", "due", "to", "the", "highly", "complex", "nature", "of", "resistances", "to", "necrotrophic", "pathogens,", "it", "is", "still", "unknown", "if", "these", "mechanisms", "can", "be", "extended", "to", "other", "plant", "species.", "Therefore,", "a", "comprehensive", "molecular", "study", "of", "SB", "resistance", "in", "L.", "ervoides", "would", "not", "only", "benefit", "lentil", "breeding", "but", "also", "broaden", "our", "knowledge", "of", "SB", "resistance", "in", "systems", "other", "than", "model", "plant", "systems.", "In", "recent", "years,", "the", "study", "of", "RNA", "has", "been", "significantly", "improved", "by", "the", "advancement", "in", "high-throughput", "next-generation", "sequencing", "techniques", "that", "generate", "massive", "amounts", "of", "data", "suitable", "for", "in-depth", "quantification", "of", "genome-wide", "gene", "expression", "across", "treatments,", "time", "points", "and", "genotypes", "[", "14", "].", "In", "lentil,", "several", "RNA-sequencing", "(RNA-Seq)", "studies", "were", "performed", "to", "profile", "transcriptomes,", "develop", "molecular", "markers", "or", "investigate", "plant", "responses", "to", "various", "biotic", "and", "abiotic", "stresses", "[", "15", "–", "19", "].", "Most", "of", "these", "studies", "were", "conducted", "by", "de", "novo", "assembly", "without", "the", "aid", "of", "a", "reference", "genome.", "The", "common", "issues", "with", "de", "novo", "assembly", "have", "been", "alignment", "errors,", "problems", "in", "reconstruction", "of", "full-length", "transcripts,", "and", "chimerism", "errors", "[", "20", "].", "As", "the", "L.", "culinaris", "genome", "was", "recently", "constructed", "by", "Bett", "et", "al.", "[", "21", "],", "reference-based", "transcriptome", "assembly", "is", "possible", "and", "encouraged", "to", "improve", "downstream", "analyses.", "As", "RNA", "transcription", "is", "a", "highly", "dynamic", "process,", "the", "appropriate", "sampling", "time", "is", "of", "high", "importance", "to", "capture", "genes", "of", "interest,", "and", "to", "achieve", "this,", "the", "quantitative", "assessments", "of", "fungal", "development", "implemented", "by", "microscopic", "observation", "and", "quantitative", "polymerase", "chain", "reaction", "(qPCR)", "are", "promising", "options.", "Several", "studies", "proved", "that", "these", "two", "methods", "well", "integrate", "both", "visual", "and", "digital", "assessments", "of", "fungal", "biomass", "and", "development", "in", "a", "range", "of", "hosts", "[", "22", "–", "24", "].", "In", "this", "study,", "we", "recruited", "those", "methods", "and", "RILs", "from", "the", "F", "9", "L.", "ervoides", "RIL", "population", "LR-66", "[", "11", "]", "to", "study", "SB", "resistance", "in", "L.", "ervoides.", "Firstly,", "we", "performed", "microscopic", "and", "qPCR", "studies", "to", "quantify", "the", "development", "of", "S.", "botryosum", "in", "plants", "during", "the", "first", "10", "days", "post-inoculation.", "Secondly,", "we", "conducted", "a", "time-series", "RNA-Seq", "experiment", "on", "two", "SB", "infected", "RILs", "that", "displayed", "contrasting", "SB", "susceptibilities.", "Gene", "expression", "data", "were", "submitted", "to", "differentially", "expressed", "gene", "analyses", "(DEG)", "to", "understand", "the", "regulatory", "pathways", "that", "were", "involved", "in", "SB", "defense", "responses.", "To", "further", "screen", "the", "candidate", "genes", "governing", "resistance", "and", "susceptibility", "to", "SB,", "we", "then", "performed", "bulk", "segregation", "gene", "expression", "analysis", "using", "pooled", "susceptible", "and", "resistant", "RILs", "from", "the", "LR-66", "population", "to", "verify", "the", "expression", "of", "those", "DEGs", "that", "were", "located", "in", "identified", "QTLs", "[", "11", "].", "The", "objectives", "of", "this", "study", "were", "to", "extend", "our", "understanding", "of", "SB", "resistance", "and", "identify", "putative", "resistant", "genes", "to", "facilitate", "use", "of", "L.", "ervoides", "in", "commercial", "lentil", "breeding.", "Results", "Quantification", "of", "fungal", "development", "Our", "inoculation", "experiment", "with", "S.", "botryosum", "isolate", "SB19", "resulted", "in", "distinctly", "more", "disease", "in", "L.", "ervoides", "RIL", "LR-66-577", "than", "LR-66-637", "(Fig.", "1", ").", "The", "percentage", "of", "conidial", "germination", "and", "penetration,", "and", "germ", "tube", "lengths", "were", "recorded", "for", "the", "first", "48", "h", "post-inoculation", "(hpi)", "(Fig.", "2", "a).", "Results", "showed", "that", "the", "germination", "of", "conidia", "increased", "over", "time", "and", "reached", "its", "highest", "level", "(94.4%", "for", "LR-66-637,", "98.5%", "for", "LR-66-577)", "at", "48", "hpi.", "Approximate", "50%", "of", "conidia", "had", "germinated", "with", "germ", "tubes", "that", "had", "successfully", "penetrated", "into", "the", "plant", "epidermal", "cells", "at", "6", "hpi", "(both", "RILs),", "which", "increased", "to", "69.6%", "(LR-66-637)", "or", "67.2%", "(LR-66-577)", "at", "12", "hpi.", "Germ", "tube", "length", "on", "the", "leaf", "surface", "was", "quantified", "from", "12", "to", "24", "hpi", "and", "revealed", "a", "substantial", "increase", "from", "approximate", "200", "μm", "(both", "RILs)", "at", "12", "hpi", "to", "395", "μm", "(LR-66-637)", "or", "486", "μm", "(LR-66-577)", "at", "24", "hpi.", "However,", "there", "was", "no", "statistical", "difference", "between", "the", "resistant", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "for", "these", "fungal", "growth", "parameters.", "At", "48", "hpi,", "we", "started", "to", "observe", "a", "few", "chlorotic", "spots", "on", "leaflets.", "As", "the", "infected", "plants", "displayed", "evidence", "of", "leaf", "necrosis", "at", "72", "hpi,", "we", "decided", "to", "record", "the", "percentage", "of", "necrotic", "leaf", "tissue", "from", "72", "to", "144", "hpi", "at", "24-h-intervals.", "Results", "showed", "that", "necrosis", "increased", "with", "incubation", "time", "in", "both", "RILs,", "and", "the", "susceptible", "RIL", "LR-66-577", "had", "a", "significantly", "higher", "percentage", "of", "necrotic", "leaf", "tissue", "than", "the", "resistant", "RIL", "LR-66-637", "throughout", "this", "period.", "Fig.", "1", "The", "distinct", "differences", "in", "the", "level", "of", "resistance", "to", "Stemphylium", "botryosum", "(isolate", "SB19)", "between", "the", "Lens", "ervoides", "RILs", "LR-66-637", "and", "LR-66-577.", "a", "Lateral", "view", "of", "the", "resistant", "RIL", "LR-66-637", "(left)", "and", "the", "susceptible", "RIL", "LR-66-577", "(right)", "at", "144", "hpi.", "b", "Histopathological", "overview", "of", "SB19", "progression", "at", "48,", "96", "and", "144", "hpi", "in", "the", "two", "RILs.", "Photos", "were", "taken", "at", "200x", "magnification", "with", "bright", "field", "microscopy", "Fig.", "2", "Quantification", "of", "Stemphylium", "botryosum", "(isolate", "SB19)", "developmental", "stages", "in", "the", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577.", "a", "Assessments", "through", "histopathology", "of", "SB19", "development", "in", "two", "RILs", "from", "24", "to", "144", "hpi.", "b", "qPCR", "quantification", "of", "relative", "SB19", "biomass", "in", "two", "RILs", "from", "6", "to", "240", "hpi.", "SB19", "biomass", "was", "determined", "by", "the", "ratio", "of", "SB19", "over", "L.", "ervoides", "gDNA", "using", "qPCR", "primers", "of", "RPL-4", "(SB19", "specific)", "and", "LcEF1a", "(L.", "ervoides", "specific).", "Asterisks", "indicate", "significant", "differences", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "errors", "of", "the", "mean", "To", "confirm", "this", "trend,", "we", "then", "used", "qPCR", "to", "assess", "the", "relative", "fungal", "biomass", "(Fig.", "2", "b).", "Results", "showed", "that", "fungal", "growth", "was", "slow", "before", "48", "hpi", "but", "accelerated", "after", "48", "hpi.", "No", "statistical", "differences", "were", "seen", "between", "RILs", "before", "48", "hpi,", "which", "agrees", "with", "our", "histopathological", "observations,", "but", "significantly", "more", "fungal", "biomass", "developed", "in", "the", "susceptible", "LR-66-577", "up", "to", "the", "end", "of", "observations", "at", "240", "hpi.", "Interestingly,", "the", "relative", "fungal", "biomass", "decreased", "after", "144", "hpi", "in", "both", "resistant", "and", "susceptible", "RILs,", "indicating", "that", "expansion", "and", "growth", "of", "lentil", "plants", "outpaced", "fungal", "growth", "as", "a", "secondary", "cycle", "of", "infection", "through", "a", "new-generation", "of", "air-borne", "conidia", "of", "SB19", "was", "largely", "immobilized", "due", "to", "lack", "of", "air", "currents", "inside", "the", "bags", "that", "enclosed", "single", "pots", "to", "maintain", "high", "air", "humidity.", "Based", "on", "these", "observations,", "we", "decided", "to", "collect", "samples", "at", "48,", "96,", "and", "144", "hpi", "for", "further", "analysis.", "Analyses", "of", "variability", "among", "RILs", "samples", "at", "different", "time", "points", "To", "assess", "the", "variability", "of", "gene", "expression", "of", "RIL", "samples", "at", "different", "time", "points,", "we", "performed", "principal", "component", "analysis", "(PCA)", "to", "reduce", "the", "data", "dimensionality", "for", "ease", "of", "visualization.", "This", "revealed", "that", "the", "three", "replicates", "of", "samples", "clustered", "closely", "together", "after", "excluding", "one", "replicate", "of", "LR-66-637", "at", "0", "hpi", "which", "deviated", "from", "other", "replicates.", "This", "sample", "was", "considered", "as", "an", "outlier", "and", "was", "therefore", "discarded", "from", "the", "subsequent", "analyses.", "After", "removing", "this", "outlier,", "the", "PCA", "plot", "clearly", "separated", "samples", "of", "the", "same", "RIL", "at", "0", "and", "48", "hpi,", "but", "not", "at", "96", "and", "144", "hpi", "which", "were", "located", "together", "(Fig.", "3", "a).", "PCA", "also", "separated", "LR-66-637", "from", "LR-66-577", "samples,", "especially", "at", "96", "and", "144", "hpi.", "Fig.", "3", "Overview", "of", "transcriptome", "variabilities", "among", "24", "Stemphylium", "botryosum", "(isolate", "SB19)-infected", "samples", "of", "Lens", "ervoides", "RILs", "LR-66-637", "and", "RIL", "LR-66-577.", "a", "Principal", "component", "analysis", "of", "samples.", "Each", "point", "on", "the", "PCA", "plot", "represents", "an", "individual", "sample.", "b", "Partition", "of", "main", "effects", "of", "hours", "post", "inoculation", "(hpi),", "RILs,", "and", "the", "interaction", "of", "RIL", "×", "hpi", "using", "principal", "variation", "component", "analysis", "(PVCA)", "To", "further", "attribute", "the", "proportion", "of", "variance", "to", "known", "sources", "of", "variation,", "we", "conducted", "a", "principal", "variance", "component", "analysis", "(PVCA)", "on", "these", "samples.", "Results", "showed", "that", "hpi,", "RILs,", "and", "the", "interaction", "of", "RILs", "×", "hpi", "together", "accounted", "for", "85%", "of", "the", "total", "variance", "(Fig.", "3", "b).", "Among", "these", "effects,", "the", "largest", "variance", "proportion", "was", "contributed", "by", "hpi", "(61%),", "followed", "by", "RILs", "(15%)", "and", "the", "interaction", "of", "RILs", "×", "hpi", "(9%).", "A", "very", "small", "part", "of", "the", "variance", "(1%)", "was", "partitioned", "to", "replicates,", "which", "was", "expected.", "The", "remaining", "14%", "of", "variance", "proportion", "cannot", "be", "explained", "by", "the", "effects", "of", "the", "model", "and", "were", "attributed", "to", "the", "residual.", "Analyses", "of", "common", "disease-responsive", "genes", "The", "variable", "transcriptome", "profiles", "between", "LR-66-637", "and", "LR-66-577", "at", "0", "hpi", "indicated", "that", "differentially", "expressed", "genes", "between", "RILs", "also", "correlated", "with", "plant", "characteristics", "other", "than", "disease", "defense", "responses.", "As", "such,", "in", "order", "to", "limit", "the", "differential", "gene", "expression", "(DGE)", "analyses", "to", "those", "genes", "that", "are", "likely", "involved", "in", "disease", "defense", "responses,", "we", "first", "compared", "the", "genes", "expression", "at", "48,", "96,", "and", "144", "hpi", "with", "that", "at", "0", "hpi,", "which", "resulted", "in", "a", "total", "of", "8810", "genes", "that", "displayed", "differential", "gene", "expression", "(fold", "change", ">", "2", "or", "FDR", "<", "0.05)", "during", "the", "SB19", "infection", "process", "between", "48", "and", "144", "hpi.", "The", "expression", "heatmap", "showed", "that", "LR-66-637", "and", "LR-66-577", "exhibited", "some", "similar", "features", "in", "their", "expression", "patterns", "(Additional", "file", "1", ":", "Figure", "S1).", "These", "data", "indicated", "that", "a", "large", "proportion", "of", "disease-responsive", "genes", "reprogrammed", "in", "a", "similar", "manner", "in", "the", "resistant", "and", "susceptible", "RILs", "after", "the", "initial", "challenge", "with", "SB19.", "To", "identify", "these", "genes,", "we", "performed", "DGE", "analyses", "between", "RILs", "at", "48,", "96,", "and", "144", "hpi", "and", "found", "that", "the", "vast", "majority", "of", "the", "8810", "disease-responsive", "genes", "(7526", "genes)", "were", "not", "differentially", "expressed", "(fold", "change", "<", "2", "or", "FDR", ">", "0.05)", "between", "RILs.", "Those", "genes", "may", "be", "involved", "in", "non-host", "resistance", "of", "plants,", "which", "is", "a", "non-specific", "and", "broad-spectrum", "defense", "mechanism", "that", "is", "universally", "present", "in", "plants", "as", "a", "defense", "against", "microorganisms", "[", "25", "].", "Data", "of", "these", "7526", "genes", "were", "submitted", "to", "K-mean", "clustering", "(Fig.", "4", "a)", "and", "Gene", "Ontology", "(GO)", "mapping", "analyses", "(Fig.", "4", "b).", "As", "a", "result,", "three", "distinct", "gene", "clusters", "were", "obtained.", "Cluster", "1", "consisted", "of", "3155", "genes", "which", "were", "primarily", "downregulated", "at", "96", "and", "144", "hpi.", "The", "GO", "mapping", "analysis", "showed", "that", "the", "genes", "in", "this", "cluster", "were", "mostly", "enriched", "in", "development-related", "GO", "terms", "such", "as", "“tissue", "development”,", "“system", "development”,", "“organ", "development”,", "“nuclear", "division”,", "and", "“anatomical", "structure", "formation", "involved", "in", "morphogenesis”,", "and", "energy", "synthesis-related", "GO", "terms", "such", "as", "“photosynthesis”", "and", "“carbohydrate", "metabolic", "process”", "(Fig.", "4", "b,", "Additional", "file", "2", ":", "Table", "S1", "and", "Additional", "file", "3", ":", "Table", "S2).", "Fig.", "4", "K-mean", "clustering", "and", "GO", "enrichment", "analyses", "of", "7526", "common", "disease-responsive", "genes", "between", "the", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "a", "Gene", "expression", "heatmap", "of", "three", "hierarchically", "clustered", "(Ward", "method)", "genes.", "The", "averaged", "FPKM", "of", "three", "biological", "replicates", "was", "used", "to", "calculate", "Z-score.", "b", "Top", "10", "significant", "(FDR", "<", "0.05)", "associated", "GO", "terms", "for", "three", "gene", "clusters", "Clusters", "2", "and", "3", "contained", "1670", "and", "2701", "genes,", "respectively.", "Cluster", "2", "showed", "a", "peak", "of", "gene", "expression", "at", "48", "hpi,", "whereas", "Cluster", "3", "displayed", "peaks", "at", "96", "and", "144", "hpi.", "The", "most", "enriched", "GO", "terms", "for", "Cluster", "2", "were", "cell", "wall-related", "processes", "such", "as", "‘hemicellulose", "metabolic", "process’,", "‘cell", "wall", "polysaccharide", "metabolic", "process’,", "‘fatty", "acid", "metabolic", "process’", "and", "‘cell", "wall", "biogenesis’,", "indicating", "that", "both", "RILs", "underwent", "dramatic", "cell", "wall", "modifications", "during", "the", "early", "infection", "period.", "Disease-responsive", "genes", "in", "Cluster", "3", "relating", "to", "“response", "to", "oxidative", "stress”,", "“response", "to", "oxygen-containing", "compound”,", "and", "‘oxidation-reduction", "process’", "indicated", "that", "reactive", "oxygen", "species", "(ROS)", "were", "promoted", "in", "both", "RILs", "during", "the", "late", "SB19", "infection", "process.", "Analyses", "of", "genes", "differentially", "expressed", "between", "RILs", "Among", "the", "8810", "disease-responsive", "genes,", "1284", "genes", "were", "differentially", "expressed", "(fold", "change", ">", "2,", "FDR", "<", "0.05)", "between", "LR-66-637", "and", "LR-66-577", "at", "48,", "96", "and", "144", "hpi", "(Additional", "file", "4", ":", "Table", "S3).", "We", "arbitrarily", "selected", "eight", "of", "these", "DEGs", "upregulated", "in", "LR-66-637", "and", "another", "eight", "DEGs", "upregulated", "in", "LR-66-577", "for", "qPCR", "verification", "in", "an", "independent", "SB19", "inoculation", "experiment", "(Fig.", "5", ").", "All", "but", "one", "(94%)", "exhibited", "expression", "trends", "consistent", "with", "those", "of", "the", "RNA-Seq", "experiment,", "indicating", "that", "expression", "of", "DEGs", "is", "reproducible", "across", "independent", "experiments.", "Fig.", "5", "qPCR", "validation", "of", "arbitrarily", "selected", "16", "differential", "expressed", "genes", "identified", "in", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "Asterisks", "indicate", "the", "significant", "difference", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "error", "For", "a", "better", "visualization", "of", "genes,", "the", "1284", "DEGs", "were", "hierarchically", "clustered", "into", "five", "expression", "clusters", "(Fig.", "6", ").", "Clusters", "1", "(306", "genes)", "and", "2", "(406", "genes)", "comprised", "genes", "that", "were", "upregulated", "in", "the", "resistant", "RIL", "LR-66-637,", "whereas", "310", "genes", "in", "Cluster", "3,", "161", "in", "Cluster", "4", "and", "101", "genes", "in", "Cluster", "5", "were", "upregulated", "in", "the", "susceptible", "RIL", "LR-66-577.", "Based", "on", "their", "expression", "patterns,", "it", "was", "hypothesized", "that", "the", "712", "genes", "in", "Clusters", "1", "and", "2", "are", "correlated", "with", "the", "enhanced", "resistance", "to", "S.", "botryosum", "in", "LR-66-637,", "and", "the", "572", "genes", "in", "Clusters", "3,", "4", "and", "5", "are", "associated", "with", "susceptibility", "to", "the", "pathogen", "in", "LR-66-577.", "Fig.", "6", "K-mean", "clustering", "(Ward", "method)", "of", "1284", "differentially", "expressed", "genes", "between", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "The", "identified", "five", "major", "gene", "clusters", "are", "visualized", "in", "heatmap", "(left)", "and", "line", "graph", "(right)", "To", "understand", "their", "biological", "relevance,", "we", "performed", "GO", "enrichment", "analyses", "for", "those", "DEGs", "in", "each", "of", "the", "clusters", "(Fig.", "7", ",", "Additional", "file", "5", ":", "Table", "S4", "and", "Additional", "file", "6", ":", "Table", "S5).", "Genes", "in", "Cluster", "1", "were", "primarily", "enriched", "in", "those", "involved", "in", "a", "variety", "of", "primary", "metabolic", "and", "transportation", "processes", "including", "‘carbohydrate", "metabolic", "process’,", "‘cellular", "carbohydrate", "metabolic", "process’,", "‘polysaccharide", "metabolic", "process’,", "‘transport’,", "‘establishment", "of", "localization’,", "and", "‘transmembrane", "transport’.", "In", "Cluster", "2,", "a", "large", "number", "of", "genes", "were", "enriched", "in", "several", "cell", "wall", "related", "processes", "such", "as", "‘cell", "wall", "organization", "or", "biogenesis’,", "‘cell", "wall", "polysaccharide", "metabolic", "process’,", "‘pectin", "catabolic", "process’,", "and", "‘hemicellulose", "metabolic", "process’.", "In", "Cluster", "3,", "the", "majority", "of", "genes", "were", "enriched", "for", "those", "associated", "with", "immune", "responses", "such", "as", "‘oxidation-reduction", "process’,", "‘response", "to", "stimulus’,", "and", "‘response", "to", "chemical’.", "For", "the", "remaining", "262", "genes", "in", "Clusters", "4", "and", "5,", "HR-associated", "GO", "function", "of", "‘oxidation-reduction", "process’,", "and", "two", "PCD-related", "GO", "terms", "of", "‘asparagine", "metabolic", "process’", "and", "‘asparagine", "biosynthetic", "process’", "were", "identified,", "indicating", "a", "higher", "level", "of", "HR", "activity", "in", "LR-66-577", "during", "the", "early", "SB19", "infection", "[", "26", "].", "Fig.", "7", "Significant", "(FDR", "<", "0.1)", "associated", "GO", "terms", "for", "five", "differentially", "expressed", "genes", "clusters", "identified", "from", "samples", "of", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19)", "Screening", "of", "candidate", "resistance", "genes", "using", "QTL", "and", "bulk", "segregant", "gene", "expression", "analyses", "Previously,", "three", "significant", "QTL", "intervals", "associated", "with", "SB19", "resistance", "were", "identified", "in", "a", "SNP-based", "linkage", "map", "of", "RIL", "population", "LR-66", "[", "11", "].", "To", "narrow", "down", "the", "resistance", "or", "susceptibility", "gene", "candidates", "based", "on", "RNA-Seq", "here,", "the", "1284", "DEGs", "were", "compared", "to", "those", "in", "the", "QTL", "confidence", "intervals", "(2-LOD),", "which", "resulted", "in", "the", "identification", "of", "nine", "genes", "that", "were", "up-", "or", "downregulated", "in", "LR-66-637", "at", "48,", "96", "and", "144", "hpi.", "Among", "these", "nine", "genes,", "six", "genes", "were", "localized", "in", "the", "QTL", "qSB-2.2", "interval", "and", "three", "in", "the", "QTL", "qSB-3", "interval.", "Expression", "analysis", "of", "those", "genes", "in", "pools", "of", "the", "five", "most", "SB-resistant", "and", "the", "five", "most", "susceptible", "RILs", "revealed", "that", "two", "genes", "(Lc05858:", "calcium-transporting", "ATPase;", "Lc07593:", "uncharacterized", "protein)", "present", "in", "qSB-2.2", "and", "one", "gene", "(Lc12983:", "glutamate", "receptor3.2)", "in", "qSB-3", "were", "expressed", "in", "a", "manner", "similar", "to", "that", "in", "resistant", "RIL", "LR-66-637", "and", "susceptible", "LR-66-577", "based", "on", "RNA-Seq", "analysis", "(Fig.", "8", "and", "Additional", "file", "7", ":", "Table", "S6).", "Quantitative", "PCR", "for", "another", "two", "of", "these", "genes", "(Lc09908", "and", "Lc07065)", "was", "inconclusive,", "and", "four", "genes", "(Lc06015,", "Lc10098,", "Lc11261,", "and", "Lc13725)", "displayed", "similar", "expression", "between", "the", "two", "pools,", "indicating", "that", "they", "are", "more", "likely", "associated", "with", "other", "differences", "specific", "to", "LR-66-637", "and", "LR-66-577.", "Fig.", "8", "Bulk", "segregation", "gene", "expression", "analysis", "of", "three", "genes", "that", "co-localize", "within", "two", "putative", "Stemphylium", "botryosum", "(isolate", "SB19)", "resistance", "QTL", "intervals", "on", "the", "genetic", "map", "of", "Lens", "ervoides.", "a", "Projection", "of", "QTL", "brackets", "from", "linkage", "group", "to", "the", "physical", "chromosomal", "regions.", "The", "linkage", "maps", "(left)", "show", "the", "arrangement", "of", "SNP", "markers", "distributing", "in", "2-LOD", "QTL", "intervals", "(Bhadauria", "et", "al.,", "2017).", "The", "right", "column", "shows", "the", "projected", "QTL", "putative", "regions", "(yellow)", "in", "the", "corresponding", "physical", "chromosomal", "regions.", "b", "Expression", "profiling", "of", "three", "genes", "between", "pools", "of", "SB-resistant", "and", "susceptible", "RILs.", "Lc05858", "and", "Lc07593", "are", "in", "qSB-2.2", "QTL", "region,", "and", "Lc12983", "is", "in", "qSB-3", "interval.", "Asterisks", "indicate", "the", "significant", "difference", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "error", "For", "Lc07593,", "it", "was", "observed", "that", "this", "gene", "was", "highly", "expressed", "in", "the", "SB19-susceptible", "RILs", "compared", "to", "the", "resistant", "RILs,", "suggesting", "that", "Lc07593", "could", "be", "a", "susceptibility", "gene,", "upregulation", "of", "which", "seemed", "to", "increase", "SB", "susceptibility.", "On", "the", "other", "hand,", "Lc05858", "and", "Lc12983", "could", "be", "disease", "resistance", "genes,", "as", "they", "displayed", "a", "reverse", "expression", "trend", "to", "that", "of", "Lc07593.", "Discussion", "A", "thorough", "understanding", "of", "how", "pathogens", "interact", "with", "their", "hosts", "is", "essential", "to", "identify", "appropriate", "times", "during", "the", "infection", "process", "for", "the", "profiling", "of", "the", "transcriptome.", "In", "the", "present", "study,", "the", "difference", "in", "SB19", "development", "between", "the", "resistant", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "was", "negligible", "before", "24", "hpi,", "indicating", "that", "the", "resistance", "toward", "SB19", "in", "RIL", "LR-66-637", "was", "probably", "based", "on", "post-penetration", "inhibition", "of", "the", "fungus.", "Similar", "observations", "were", "also", "reported", "for", "S.", "botryosum", "infecting", "alfalfa", "where", "Cowling", "and", "Gilchrist", "[", "27", "]", "found", "that", "the", "development", "of", "S.", "botryosum", "did", "not", "differ", "among", "the", "resistant,", "moderately", "resistant", "and", "susceptible", "alfalfa", "clones", "during", "the", "early", "period", "of", "infection.", "During", "the", "period", "from", "48", "to", "144", "hpi,", "fungal", "biomass", "started", "to", "accumulate", "in", "RILs,", "suggesting", "that", "S.", "botryosum", "initiated", "the", "release", "of", "phytotoxins", "to", "disrupt", "host", "cells", "around", "48", "hpi.", "Biomass", "accumulation", "was", "significantly", "higher", "in", "LR-66-577", "than", "in", "LR-66-637,", "possibly", "as", "a", "result", "of", "enhanced", "tolerance", "to", "phytotoxins", "in", "LR-66-637", "compared", "to", "LR-66-577", "[", "28", "].", "As", "such,", "48,", "96", "and", "144", "hpi", "were", "considered", "to", "be", "interesting", "time", "points", "warranting", "further", "exploration", "of", "gene", "expression.", "PCA", "and", "PVCA", "are", "useful", "tools", "to", "visualize", "the", "overall", "gene", "expression", "variability", "and", "to", "detect", "outliers", "among", "samples", "[", "29", "].", "After", "excluding", "an", "outlier", "in", "the", "PCA", "plot", "here,", "separation", "of", "those", "samples", "collected", "at", "48", "hpi", "from", "others", "gathered", "at", "96", "and", "144", "hpi", "was", "evident,", "which", "clearly", "reflected", "that", "gene", "reprogramming", "at", "96", "and", "144", "hpi", "was", "similar,", "but", "was", "different", "from", "48", "hpi.", "PCA", "also", "separated", "LR-66-637", "from", "LR-66-577", "samples,", "indicating", "that", "the", "transcriptome", "responses", "of", "LR-66-637", "and", "LR-66-577", "were", "different", "after", "challenge", "with", "SB19.", "As", "this", "separation", "was", "more", "evident", "at", "96", "or", "144", "hpi", "than", "at", "48", "hpi,", "a", "significant", "interaction", "effect", "of", "hpi", "×", "RIL", "was", "expected.", "These", "results", "confirmed", "that", "the", "transcriptomes", "of", "RILs", "responded", "to", "SB19", "infection", "in", "a", "time-dependent", "manner", "as", "has", "been", "observed", "in", "other", "transcriptome", "studies", "[", "30", "–", "32", "].", "Non-host", "resistance", "is", "a", "non-specific", "and", "broad-spectrum", "defense", "mechanism", "that", "is", "universally", "present", "in", "plants", "as", "a", "defense", "against", "microorganisms", "[", "25", ",", "33", "].", "By", "analyzing", "those", "disease-responsive", "genes", "of", "both", "RILs,", "we", "observed", "that", "a", "large", "number", "of", "genes", "downregulated", "in", "both", "RILs", "were", "enriched", "in", "a", "series", "of", "development-related", "and", "energy", "synthesis-related", "GO", "terms.", "These", "results", "seemed", "to", "indicate", "that", "SB19", "infection", "disrupted", "the", "normal", "growth", "and", "energy", "synthesis", "of", "both", "RILs", "regardless", "of", "their", "varying", "levels", "of", "resistance", "to", "the", "pathogen.", "As", "the", "resistance", "towards", "necrotrophs", "in", "plants", "is", "always", "in", "an", "incomplete", "form,", "the", "withdrawal", "of", "nutrients", "by", "S.", "botryosum", "from", "plants", "normally", "occurs", "in", "all", "individuals", "of", "the", "host", "species", "[", "34", "].", "Pathogen", "infection", "leads", "to", "a", "decrease", "in", "photosynthesis", "at", "the", "infection", "sites,", "which", "negatively", "impacts", "the", "energy", "assimilation", "and", "interferes", "with", "the", "normal", "development", "and", "growth", "of", "the", "plant", "[", "35", "].", "This", "has", "been", "extensively", "reported", "in", "a", "variety", "of", "diseased", "plants", "[", "36", "–", "38", "].", "On", "the", "other", "hand,", "there", "were", "also", "genes", "that", "were", "upregulated", "after", "inoculation,", "either", "at", "48", "hpi", "or", "at", "96", "and", "144", "hpi.", "As", "these", "genes", "were", "mostly", "enriched", "in", "cell", "wall-related", "and", "oxidation-reduction", "processes,", "it", "seemed", "that", "cell", "wall", "modifications", "and", "ROS", "were", "promoted", "at", "certain", "time", "points", "during", "the", "SB19", "infection", "process.", "Any", "type", "of", "interaction", "between", "pathogen", "and", "host", "will", "trigger", "some", "degrees", "of", "cell", "wall", "modifications", "[", "25", ",", "33", ",", "34", "].", "When", "plants", "are", "stressed", "by", "pathogens,", "variable", "amounts", "of", "ROS", "are", "released", "to", "activate", "the", "downstream", "signal", "transduction", "pathways", "and", "expression", "of", "a", "series", "of", "defense", "genes", "[", "39", "–", "41", "].", "The", "activation", "of", "ROS", "is", "a", "common", "form", "of", "non-host", "resistance", "that", "universally", "occurs", "in", "plants,", "regardless", "of", "their", "difference", "in", "the", "level", "of", "disease", "resistance", "[", "42", "–", "44", "].", "In", "addition", "to", "these", "common", "disease-responsive", "genes,", "another", "1284", "disease-responsive", "genes", "were", "differentially", "expressed", "between", "LR-66-637", "and", "LR-66-577.", "Some", "of", "these", "genes", "may", "contribute", "to", "the", "difference", "in", "the", "level", "of", "disease", "resistance", "between", "the", "two", "RILs.", "According", "to", "the", "expression", "patterns", "and", "enriched", "GO", "terms", "of", "these", "genes,", "we", "found", "that", "LR-66-637", "mainly", "upregulated", "a", "variety", "of", "carbohydrate", "metabolic,", "transportation", "and", "cell", "wall", "related", "processes.", "Other", "gene", "expression", "data", "on", "the", "lentil", "-", "S.", "botryosum", "system", "have", "not", "been", "reported", "to", "date,", "but", "stronger", "upregulation", "of", "a", "series", "of", "metabolic", "processes", "(organic", "substance", "metabolic,", "single-organism", "metabolic,", "or", "cellular", "glucan", "metabolic)", "based", "on", "RNA-Seq", "experiments", "were", "reported", "in", "the", "resistant", "lentil", "genotypes", "CDC-Robin,", "964a-46,", "or", "ILL7537", "after", "inoculation", "of", "Ascochyta", "lentis,", "another", "lentil", "pathogen", "presumed", "to", "be", "a", "necrotroph", "[", "15", ",", "16", "].", "In", "addition,", "Yang", "et", "al.", "[", "45", "]", "studied", "the", "tomato", "–", "S.", "lycopersici", "system", "and", "found", "that", "several", "GO", "terms", "of", "‘localization’,", "‘transporter", "activity’", "and", "‘molecular", "transducer", "activity’", "were", "activated", "in", "the", "resistant", "tomato", "genotype,", "proposing", "that", "the", "upregulation", "of", "these", "activities", "were", "related", "to", "S.", "lycopersici", "resistance.", "Carbohydrates", "and", "transporters", "are", "considered", "essential", "parts", "of", "plant", "immune", "systems,", "with", "the", "former", "generating", "energy", "to", "sustain", "the", "continuous", "production", "of", "a", "series", "of", "defense-related", "metabolites", "and", "carbon-based", "polymers,", "and", "the", "latter", "fulfilling", "signal", "transduction,", "energy", "translocation", "and", "also", "extrusion", "of", "toxic", "compounds", "[", "46", "–", "48", "].", "Theoretically,", "the", "promotion", "of", "these", "processes", "is", "in", "accordance", "with", "elevated", "disease", "resistance", "against", "necrotrophs", "[", "35", ",", "48", ",", "49", "].", "Morkunas", "et", "al.", "[", "50", "]", "found", "that", "a", "high", "level", "of", "sucrose", "accounted", "for", "the", "accumulation", "of", "a", "variety", "of", "flavonoids", "which", "resulted", "in", "enhanced", "resistance", "in", "yellow", "lupin", "to", "Fusarium", "oxysporum.", "Likewise,", "it", "was", "reported", "that", "the", "upregulation", "of", "the", "two", "plant", "ATP", "binding", "cassette", "transporters", "NpPDR1", "and", "PEN3,", "could", "enhance", "disease", "resistance", "to", "the", "necrotrophic", "pathogens", "Botrytis", "cinerea", "and", "Plectosphaerella", "cucumerina", "in", "Arabidopsis,", "respectively", "[", "49", ",", "51", "].", "The", "promotion", "of", "several", "cell", "wall", "related", "processes", "suggested", "that", "LR-66-637", "initiated", "additional", "cell", "wall", "modification", "compared", "to", "LR-66-577.", "In", "the", "lentil", "–", "A.", "lentis", "system,", "Khorramdelazad", "et", "al.", "[", "15", "]", "also", "reported", "the", "significant", "enrichment", "for", "the", "GO", "term", "‘cell", "wall’", "in", "the", "resistant", "genotype", "ILL7537.", "During", "attack", "by", "necrotrophic", "pathogens,", "the", "cell", "wall", "of", "a", "host", "is", "damaged", "by", "a", "series", "of", "cell", "wall-degrading", "enzymes", "released", "by", "the", "pathogen.", "If", "such", "cell", "wall", "degradation", "can", "be", "arrested", "by", "the", "host,", "a", "resistant", "phenotype", "would", "be", "expected", "[", "52", "].", "In", "tomato,", "Miedes", "and", "Lorences", "[", "53", "]", "found", "that", "the", "appropriate", "level", "of", "xyloglucan", "(a", "form", "of", "hemicellulose)", "was", "essential", "for", "maintaining", "cell", "wall", "integrity", "when", "under", "attack", "from", "Penicillium", "expansum.", "In", "Arabidopsis,", "Lionetti", "et", "al.", "[", "54", "]", "found", "that", "B.", "cinerea", "resistance", "was", "enhanced", "through", "an", "increase", "of", "pectin", "methyl", "esterification", "which", "disrupted", "the", "ability", "of", "microbes", "to", "degrade", "the", "plant", "cell", "wall.", "As", "such,", "we", "hypothesize", "that", "the", "upregulation", "of", "genes", "in", "cell", "wall", "processes,", "to", "a", "certain", "degree,", "may", "compensate", "for", "the", "cell", "wall", "damage", "through", "infection", "and", "thus", "improve", "SB", "resistance", "in", "LR-66-637.", "In", "contrast,", "LR-66-577", "appeared", "to", "respond", "to", "SB19", "invasion", "in", "a", "completely", "different", "form", "considering", "that", "the", "majority", "of", "genes", "upregulated", "in", "this", "RIL", "were", "enriched", "for", "those", "involved", "in", "‘oxidation-reduction", "process’.", "This", "suggests", "that", "redox", "homeostasis", "was", "substantially", "altered", "in", "LR-66-577", "after", "SB19", "invasion,", "which", "was", "reported", "for", "other", "systems", "previously", "[", "55", "].", "A", "change", "in", "redox", "status", "in", "the", "challenged", "cells", "could", "release", "a", "variety", "of", "reactive", "oxygen", "species", "including", "singlet", "oxygen,", "superoxide", "radicals", "and", "hydrogen", "peroxide,", "accumulation", "of", "which", "could", "trigger", "the", "plant’s", "hypersensitive", "response", "and", "programmed", "cell", "death", "(PCD)", "to", "cause", "leaf", "necrosis", "and,", "eventually,", "leaf", "dehiscence", "[", "56", ",", "57", "].", "As", "necrotrophs", "can", "thrive", "in", "dead", "tissues,", "several", "studies", "have", "reported", "that", "necrotrophs", "are", "able", "to", "hijack", "the", "plant", "immune", "system", "and", "use", "the", "host", "HR", "machinery", "to", "boost", "their", "virulence", "[", "39", ",", "58", ",", "59", "].", "From", "these", "results,", "we", "hypothesize", "that", "HR", "and", "PCD", "were", "significantly", "more", "promoted", "in", "LR-66-577", "than", "LR-66-637", "during", "the", "SB19", "infection", "process,", "resulting", "in", "susceptibility", "in", "LR-66-577.", "Previous", "evidences", "has", "shown", "that", "HR", "and", "PCD", "are", "largely", "induced", "through", "the", "activation", "of", "R", "genes", "[", "56", "–", "59", "].", "In", "the", "present", "study,", "we", "observed", "that", "a", "number", "of", "TIR-NBS-LRR", "and", "CC-NBS-LRR", "genes", "were", "stronger", "upregulated", "in", "LR-66-577", "than", "LR-66-637", "during", "the", "infection", "process", "(Additional", "file", "5", ":", "Table", "S4).", "In", "the", "lentil", "–", "A.", "lentis", "system,", "Sari", "et", "al.", "[", "16", "]", "found", "a", "TIR-NBS-LRR", "gene", "that", "was", "also", "significantly", "upregulated", "in", "two", "susceptible", "lentil", "genotypes", "after", "inoculation", "of", "A.", "lentis.", "Such", "results", "corroborate", "an", "association", "of", "R", "genes,", "cell", "death", "and", "susceptibility", "after", "infection", "by", "necrotrophic", "pathogen", "[", "39", "].", "However,", "there", "was", "another", "set", "of", "TIR-NBS-LRR,", "NBS-LRR,", "and", "NB-ARC", "genes", "that", "had", "higher", "expression", "in", "LR-66-637", "than", "LR-66-577,", "as", "would", "be", "expected", "for", "resistant", "genotypes.", "Involvement", "of", "such", "R", "genes", "was", "also", "reported", "in", "the", "lentil", "–", "A.", "lentis", "system,", "and", "Khorramdelazad", "et", "al.", "[", "15", "]", "speculated", "that", "a", "stronger", "upregulation", "of", "a", "NB-ARC", "gene", "conferred", "enhanced", "resistance", "in", "ILL", "7537.", "Taken", "together,", "this", "supports", "the", "emerging", "picture", "of", "R-genes", "as", "true", "resistance", "genes", "as", "well", "as", "their", "possible", "role", "as", "susceptibility", "factors", "[", "34", "].", "Comparing", "DEGs", "with", "previously", "identified", "resistance", "QTLs", "of", "LR-66", "[", "11", "],", "we", "identified", "nine", "resistance", "gene", "candidates", "and", "explored", "them", "further", "through", "bulk", "segregation", "analysis.", "In", "a", "bulk", "segregation", "analysis", "(BSA),", "the", "phenotypic", "extremes", "of", "two", "pools", "tend", "to", "either", "differ", "in", "the", "presence", "of", "the", "gene(s)", "characterizing", "the", "phenotype,", "or", "differ", "in", "the", "expression", "level", "of", "the", "gene(s)", "responsible", "for", "the", "trait", "[", "60", "].", "BSA", "coupled", "with", "gene", "expression", "studies", "has", "been", "used", "frequently", "to", "identify", "candidate", "genes", "in", "varieties", "of", "plant", "species", "(e.g.", "[", "61", "–", "63", "]).", "BSA", "was", "used", "here", "and", "revealed", "a", "calcium-transporting", "ATPase", "(Lc05858)", "and", "a", "receptor3.2", "(Lc12983)", "with", "a", "high", "expression", "in", "the", "resistant", "pool", "indicating", "that", "they", "could", "be", "putative", "resistance", "genes.", "A", "calcium", "transporting", "ATPase,", "such", "as", "Lc05858,", "could", "play", "an", "important", "role", "in", "calcium", "influx", "and", "efflux", "regulation", "to", "maintain", "an", "appropriate", "cellular", "calcium", "homeostasis,", "the", "change", "of", "which", "is", "thought", "to", "initiate", "calcium", "signaling", "that", "triggers", "a", "series", "of", "downstream", "responses", "influencing", "plant", "growth,", "development", "and", "responses", "to", "biotic", "and", "abiotic", "stresses", "[", "64", "–", "66", "].", "Zhu", "et", "al.", "[", "67", "]", "reported", "that", "the", "expression", "of", "a", "gene", "coding", "calcium", "ATPase", "decreased", "HR", "in", "Pseudomonas", "syringae-infected", "tomatoes", "as", "NbCA1", "(ER-localized", "type", "IIB", "calcium", "ATPase)-silenced", "plants", "exhibited", "accelerated", "programmed", "cell", "death", "(PCD)", "compared", "to", "the", "wild", "type.", "Similarly,", "Boursiac", "et", "al.", "[", "68", "]", "found", "that", "the", "knockout", "of", "ACA4", "and", "ACA11", "(two", "calcium-ATPases)", "induced", "a", "higher", "frequency", "of", "HR-like", "lesions", "by", "deactivating", "the", "calcium", "signaling", "in", "Arabidopsis", "inoculated", "with", "P.", "syringae.", "Based", "on", "this", "it", "can", "be", "speculated", "that", "upregulation", "of", "Lc05858", "during", "SB19", "infection", "results", "in", "the", "activation", "of", "calcium", "transportation", "and", "the", "suppression", "of", "HR", "and", "PCD.", "Upregulation", "of", "the", "glutamate", "receptor3.2", "Lc12983", "in", "the", "resistant", "RILs", "compared", "to", "the", "susceptible", "RILs", "at", "48", "and", "144", "hpi", "may", "also", "affect", "calcium", "regulation,", "highlighting", "its", "potential", "importance", "in", "the", "lentil", "–S.", "botryosum", "interaction.", "A", "previous", "study", "showed", "that", "a", "glutamate", "receptor", "is", "mainly", "responsible", "for", "glutamate-dependent", "membrane", "depolarization", "and", "calcium", "transportation", "[", "69", "],", "and", "Kang", "et", "al.", "[", "70", "]", "and", "Manzoor", "et", "al.", "[", "71", "]", "observed", "that", "the", "upregulation", "of", "glutamate-like", "receptors", "AtGLR3.3", "or", "AtGluR3.2", "elevated", "calcium", "influx", "and", "conferred", "enhanced", "resistance", "in", "Arabidopsis", "against", "the", "necrotroph", "B.", "cinerea.", "However,", "further", "functional", "studies", "are", "required", "to", "confirm", "the", "role", "of", "these", "genes", "in", "resistance.", "One", "gene", "encoding", "an", "uncharacterized", "protein", "(Lc07593)", "was", "highly", "expressed", "in", "the", "SB19-susceptible", "RILs", "compared", "to", "the", "resistant", "RILs,", "suggesting", "that", "Lc07593", "could", "be", "a", "susceptibility", "gene.", "However,", "this", "gene", "is", "not", "homologous", "to", "any", "well", "characterized", "genes", "in", "the", "database", "and", "thus", "its", "function", "is", "not", "understood.", "Future", "work", "involving", "protein", "purification,", "structural", "modeling,", "and", "protein-protein", "interaction", "analyses", "may", "be", "needed", "to", "understand", "its", "biological", "function.", "Methods", "Plant", "material", "and", "inoculum", "preparation", "RILs", "LR-66-577", "(susceptible", "to", "SB)", "and", "LR-66-637", "(resistant", "to", "SB)", "used", "in", "this", "study", "were", "transgressive", "segregants", "chosen", "from", "the", "L.", "ervoides", "RIL", "population", "LR-66", "derived", "from", "the", "cross", "of", "L01-827A", "×", "IG", "72815", "developed", "at", "the", "Crop", "Development", "Centre", "at", "the", "University", "of", "Saskatchewan", "(Saskatoon,", "SK,", "Canada)", "[", "11", "].", "Evident", "transgressive", "segregation", "and", "disease", "severity", "in", "RILs", "ranging", "from", "11", "to", "80%", "in", "this", "population", "indicated", "that", "there", "was", "fluent", "genetic", "variability", "in", "SB", "resistance", "[", "11", "].", "Prior", "to", "planting,", "seeds", "of", "RILs", "were", "scarified", "to", "facilitate", "germination.", "The", "scarified", "seeds", "were", "sowed", "in", "10-cm", "plastic", "pots", "filled", "with", "Fafard®", "Germination", "Mix", "(Sungro", "Horticulture®", "Ltd.,", "Vancouver,", "BC,", "Canada).", "All", "plants", "were", "grown", "in", "a", "growth", "chamber", "with", "a", "constant", "temperature", "of", "23", "°C", "and", "a", "day", "length", "of", "16", "h", "light/8", "h", "dark.", "Only", "four", "plants", "per", "pot", "were", "kept", "for", "the", "subsequent", "inoculation", "experiment.", "The", "inoculum", "was", "prepared", "from", "the", "aggressive", "S.", "botryosum", "isolate", "SB19", "which", "originated", "from", "the", "southeast", "of", "Saskatchewan,", "Canada.", "The", "cryopreserved", "SB19", "spores", "were", "revitalized", "on", "oatmeal", "V8", "agar", "medium", "[150", "mL", "V8", "juice", "(Campbell", "Co.,", "Canada),", "10", "g", "Difco™", "Potato", "Dextrose", "Agar", "(Becton", "Dickinson", "and", "Co.,", "Franklin", "Lake,", "NJ,", "USA),", "3", "g", "calcium", "carbonate,", "850", "mL", "sterile", "water].", "After", "seven", "days", "of", "incubation", "at", "room", "temperature,", "conidia", "were", "collected", "from", "Petri", "dishes", "by", "flooding", "and", "scraping", "the", "surface", "of", "the", "plates.", "The", "conidial", "suspension", "was", "filtered", "through", "two", "layers", "of", "miracloth", "and", "adjusted", "to", "1", "×", "10", "5", "condia", "mL", "−", "1", "using", "a", "hemocytometer.", "Two", "droplets", "of", "Tween", "20", "(Sigma,", "Saint", "Louis,", "MO,", "USA)", "were", "added", "to", "every", "1000", "mL", "of", "suspension", "before", "inoculation.", "Pathogen", "inoculation", "and", "experimental", "design", "Four-week", "old", "plants", "were", "sprayed", "with", "the", "conidial", "suspension", "until", "run", "off,", "which", "was", "equivalent", "to", "approximate", "2", "to", "2.5", "mL", "per", "plant.", "Four", "plants", "within", "a", "pot", "represented", "one", "of", "three", "biological", "replicates", "and", "were", "pooled", "to", "generate", "a", "biological", "replicate.", "Pots", "were", "arranged", "in", "a", "completely", "randomized", "design", "in", "a", "misting", "chamber", "at", "100%", "humidity", "for", "the", "first", "48", "h", "to", "favor", "germination", "of", "conidia.", "After", "48", "hpi,", "those", "inoculated", "plants", "were", "removed", "from", "the", "misting", "chamber,", "each", "pot", "was", "enclosed", "in", "a", "translucent", "plastic", "bag,", "and", "pots", "were", "maintained", "at", "23", "°C", "and", "16", "h", "photoperiod.", "Leaves", "were", "sampled", "at", "0,", "6,", "12,", "24,", "48,", "72,", "96,", "120,", "144,", "196,", "and", "240", "hpi,", "immediately", "frozen", "in", "liquid", "nitrogen", "and", "then", "stored", "in", "a", "-80", "°C", "freezer", "for", "future", "uses.", "Determination", "of", "fungal", "development", "Microscopy-based", "methods", "and", "qPCR", "were", "used", "to", "quantify", "growth", "of", "SB19", "in", "plants.", "For", "qPCR,", "the", "genes", "targeting", "RNA", "polymerase", "II", "second", "largest", "subunit", "(RPL-4)", "to", "amplify", "gDNA", "of", "SB19", "and", "elongation", "factor", "(LcEF1α)", "to", "quantify", "L.", "ervoides", "were", "used", "(Additional", "file", "8", ":", "Table", "S7).", "The", "SDS", "method", "[", "72", "]", "was", "used", "to", "extract", "gDNA", "from", "both", "organisms", "and", "a", "spectrophotometer", "(NanoDrop™", "8000,", "Thermo", "Scientific,", "Waltham,", "USA)", "was", "employed", "to", "quantify", "the", "DNA", "concentration,", "which", "was", "adjusted", "to", "25", "ng", "μl", "−", "1", "for", "all", "samples.", "Each", "qPCR", "reaction", "contained", "2", "μl", "DNA", "template,", "5", "μl", "SYBR®", "Green", "(catalog", "no.", "4309155,", "Thermo", "Scientific),", "0.2", "μl", "each", "of", "10", "μM", "forward", "and", "reverse", "primers,", "and", "2.6", "μl", "molecular", "grade", "water.", "A", "default", "fast-run", "program", "was", "used", "to", "perform", "qPCR", "amplification", "in", "a", "QuantStudio™", "3", "System", "(Applied", "Biosystems", "Inc.,", "Foster", "City,", "CA,", "USA).", "According", "to", "the", "criteria", "proposed", "by", "Weβling", "and", "Panstruga", "[", "24", "],", "the", "relative", "fungal", "biomass", "was", "estimated", "as", "the", "proportion", "of", "gDNA", "amplified", "between", "RPL-4", "and", "LcEF1α.", "For", "microscopic", "quantification,", "six", "leaflets", "per", "biological", "replicate", "were", "collected", "at", "each", "of", "6,", "12,", "24,", "48,", "72,", "96,", "120,", "144,", "196,", "and", "240", "hpi.", "The", "collected", "leaflets", "were", "cleared", "in", "CMAA", "fixation", "solution", "(60%", "methanol,", "30%", "chloroform,", "10%", "acetic", "acid)", "for", "at", "least", "24", "h", "at", "room", "temperature", "to", "eliminate", "chlorophyll", "in", "cells.", "Once", "treated,", "they", "were", "immersed", "in", "a", "series", "of", "decreasing", "ethanol", "concentrations", "of", "70%", "for", "1", "h,", "50%", "for", "1.5", "h", "and", "30%", "for", "1.5", "h.", "Staining", "was", "performed", "by", "immersing", "leaflets", "in", "0.05%", "trypan", "blue", "overnight.", "Well", "stained", "leaflets", "were", "mounted", "on", "glass", "slides", "in", "a", "droplet", "of", "50%", "glycerol", "and", "evaluated", "under", "a", "Zeiss", "Axioskop", "40", "microscope", "(Carl", "Zeiss,", "Göttingen,", "Germany).", "High-quality", "photos", "were", "generated", "using", "a", "Pixelink", "A686C", "camera", "(Pixelink,", "Rochester,", "NY,", "USA)", "and", "Zeiss", "Axiovision", "software", "(Carl", "Zeiss,", "Oberkochen,", "Germany).", "To", "quantify", "fungal", "development", "during", "the", "incubation", "process,", "we", "record", "percentage", "of", "conidial", "germination,", "percentage", "of", "conidia", "resulting", "in", "successful", "penetration", "(%", "conidial", "penetration),", "and", "germ", "tube", "length.", "Furthermore,", "the", "percentage", "area", "of", "dead", "tissue", "per", "leaflet", "was", "visually", "estimated", "(%", "leaf", "necrosis).", "Data", "were", "analyzed", "using", "SAS", "9.3", "(SAS", "Institute,", "Inc.,", "Cary,", "NC,", "USA).", "Normality", "of", "errors", "was", "assessed", "with", "the", "Shapiro-Wilk", "test", "and", "homogeneity", "of", "variance", "with", "the", "Levene’s", "tests.", "The", "mean", "separation", "between", "RILs", "were", "conducted", "using", "Student’s", "t-test", "method", "(P", "<", "0.05).", "RNA", "sequencing", "and", "raw", "data", "processing", "Samples", "were", "collected", "at", "0,", "48,", "96,", "and", "144", "hpi", "with", "three", "biological", "replicates", "for", "LR-66-570", "and", "LR-66-629,", "for", "a", "total", "of", "24", "samples.", "Total", "RNA", "extraction", "was", "performed", "from", "the", "frozen", "leaves", "using", "the", "RNeasy", "Plant", "Mini", "Kit", "(Cat", "no.", "74904,", "Qiagen", "Company,", "Hilden,", "Germany)", "following", "the", "manufacturer’s", "instructions.", "The", "quality", "and", "concentration", "of", "RNA", "was", "assessed", "in", "an", "Agilent", "2100", "Bioanalyzer", "(Agilent", "Technologies,", "Santa", "Clara,", "CA,", "USA).", "Only", "high-quality", "RNA", "samples", "(RNA", "integrity", "number", ">", "7)", "were", "used", "for", "library", "construction", "using", "TruSeq", "Stranded", "Total", "RNA", "(Illumina,", "Inc.,", "San", "Diego,", "CA,", "USA).", "The", "constructed", "libraries", "were", "then", "loaded", "into", "a", "HiSeq", "2500", "system", "using", "TruSeq", "SBS", "KIT-HS", "V4", "(Illumina)", "for", "125", "bp", "pair-ended", "sequencing.", "The", "quality", "control", "of", "returned", "raw", "reads", "was", "implemented", "in", "Trimmomatic", "(version", "0.36)", "[", "73", "]", "to", "remove", "low-quality", "reads", "and", "adaptors", "under", "the", "following", "parameters:", "TruSeq3-PE-2.fa:2:30:10,", "leading:3,", "trailing:3,", "slidingwindow:4:15", "and", "minlen:36.", "The", "cleaned", "reads", "were", "aligned", "against", "the", "reference", "genome", "of", "Lens", "culinaris", "V1.2", "[", "21", "]", "using", "STAR", "(default", "settings,", "version", "2.6.1a)", "[", "74", "].", "As", "a", "result,", "approximate", "90%", "of", "reads", "were", "uniquely", "aligned", "to", "the", "reference", "genome", "for", "all", "samples,", "confirming", "the", "high", "quality", "of", "sequencing", "and", "mapping", "(Additional", "file", "9", ":", "Figure", "S2).", "Gene", "counting", "was", "performed", "using", "STAR", "quantmode", "during", "the", "mapping", "process.", "The", "resulting", "gene", "expression", "data", "for", "each", "sample", "were", "first", "normalized", "using", "the", "fragments", "per", "kilobase", "of", "transcript", "per", "million", "(FPKM)", "method", "before", "proceeding", "to", "principal", "component", "analysis", "(PCA)", "and", "principal", "variance", "component", "analysis", "(PVCA)", "in", "the", "statistical", "software", "JMP", "Genomics", "8.0", "(JMP", "Genomics®,", "SAS", "Institute).", "Identification", "of", "differentially", "expressed", "genes", "(DEGs)", "Before", "comparing", "the", "gene", "expression", "between", "RILs,", "we", "first", "conducted", "comparisons", "of", "gene", "expression", "in", "samples", "from", "0", "hpi", "with", "those", "collected", "at", "48,", "96,", "and", "144", "hpi", "for", "each", "RIL", "to", "identify", "those", "genes", "that", "significantly", "responded", "to", "SB19", "infection.", "These", "comparisons", "were", "performed", "in", "the", "R", "package", "DESeq2", "[", "75", "]", "using", "the", "thresholds", "of", "false", "discovery", "rate", "(FDR)", "<", "0.05", "and", "gene", "expression", "fold", "change", ">", "2.", "Among", "the", "identified", "disease-responsive", "genes,", "we", "then", "conducted", "pair-wise", "comparisons", "between", "LR-66-637", "and", "LR-66-577", "samples", "from", "each", "time", "point", "(48,", "96", "and", "144", "hpi).", "The", "same", "thresholds", "used", "previously", "were", "applied", "here", "to", "declare", "significant", "DEGs.", "The", "resulting", "DEGs", "were", "submitted", "to", "JMP", "Genomics", "8.0", "(JMP", "Genomics®,", "SAS", "Institute)", "for", "K-means", "clustering", "analysis.", "The", "genes", "of", "each", "cluster", "with", "known", "Medicago", "truncatula", "orthologs", "were", "then", "mapped", "to", "the", "Gene", "Ontology", "(GO)", "database", "using", "the", "PANTHER", "(version", "14.1)", "(", "http://www.pantherdb.org", ").", "qPCR", "validation", "To", "verify", "the", "repeatability", "of", "gene", "expression", "captured", "in", "the", "RNA-Seq", "experiment,", "an", "independently", "inoculated", "experiment", "with", "LR-66-570", "and", "LR-66-629", "was", "conducted", "following", "the", "same", "experimental", "conditions", "and", "setup", "as", "described", "before.", "Infected", "leaf", "samples", "were", "collected", "and", "processed", "as", "described", "before,", "and", "16", "DEGs", "identified", "from", "RNA-Seq", "data", "analysis", "and", "one", "reference", "gene", "(LcEF1α)", "were", "used", "for", "qPCR", "amplification", "(Additional", "file", "10", ":", "Table", "S8).", "Each", "PCR", "reaction", "consisted", "of", "2", "μl", "DNA", "template,", "5", "μl", "SYBR", "Green,", "0.2", "μl", "of", "each", "10", "μM", "forward", "and", "reverse", "primers,", "and", "2.6", "μl", "molecular", "grade", "water.", "qPCR", "was", "performed", "in", "a", "QuantStudio™", "3", "System", "(Applied", "Biosystems", "Inc.)", "using", "the", "fast-run", "program", "with", "default", "settings.", "The", "relative", "expression", "of", "each", "gene", "of", "interest", "was", "calculated", "as", "2", "−", "CT", "gene", 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0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]	{"negatives/white": ["21+1", "210+1", "392+1", "591+1", "631+1", "719+1", "884+1", "1011+1", "1405+1", "1833+1", "2333+1", "2966+1", "3221+1", "4294+1", "4189+1", "4159+1", "5377+1", "6533+1", "6064+1", "5943+1"]}	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	Original work titled: 'Using a transcriptome sequencing approach to explore candidate resistance genes against stemphylium blight in the wild lentil species Lens ervoides' (BMC Plant Biology/BioMed Central) by the following authors: Zhe Cao; Li Li; Karan Kapoor; Sabine Banniza DOI: 10.1186/s12870-019-2013-6
26a1f557bd4198a14b3936b03e73878b	["Main","text","Metagenomic","next-generation","sequencing","(mNGS)","is","a","highly","sensitive","(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"943+1\", \"972+1\", \"1068+1\", \"1607+1\", \"1736+1\", \"1795+1\", \"154+(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Towards precision quantification of contamination in metagenomic sequencing (...TRUNCATED)
a7ada41b8b96e164fffd552e396bccdc	["Introduction","In","general,","a","calorimeter","is","capable","of","detecting","the","reaction","(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"356+1\", \"416+1\", \"2625+1\", \"2900+1\", \"5043+1\", \"6067+1\", \"7292(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'How to speed up the detection of aerobic microbial contaminations by using i(...TRUNCATED)
ff90a552716af1a13c336d1a7608a904	["Introduction","Several","experiences","during","the","perinatal","period,","including","stress,","(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"89+1\", \"347+1\", \"476+1\", \"533+1\", \"641+1\", \"663+1\", \"706+1\", (...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Neonatal infection leads to increased susceptibility to Aβ oligomer-induced(...TRUNCATED)
14dd5e79c50801a2cb4c3d4dc03381e7	["Introduction","Nucleotide-binding","oligomerization","domain-like","receptors","(NLRs,","e.g.,","N(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"230+1\", \"329+1\", \"534+1\", \"786+1\", \"1062+1\", \"1858+1\", \"2193+1(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Omnipresence of inflammasome activities in inflammatory bone diseases' (Semi(...TRUNCATED)
ad03adbf592115b4d12541751416fe37	["Highlights","We","studied","standardized","municipality","tuberculosis","case","notification","rat(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"3232+1\", \"1247+1\", \"1060+1\", \"761+1\", \"417+1\", \"263+1\", \"577+1(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Spatial and temporal analysis of tuberculosis in an area of social inequalit(...TRUNCATED)
d979be5a3c35292ae9e3e7ec627532b4	["Introduction","Microbes","are","ubiquitous","and","are","essential","components","of","ecosystems,(...TRUNCATED)	[0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"186+1\", \"5241+1\", \"3940+1\", \"4058+1\", \"4401+1\", \"3753+1\", \"300(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Camouflaging bacteria by wrapping with cell membranes' (Nature Communication(...TRUNCATED)
fa7330abf4e6708b485c5a7e682d423a	["Background","Malaria","has","an","overwhelming","impact","on","people’s","health","and","livelih(...TRUNCATED)	[0,0,0,0,7,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0,0(...TRUNCATED)	"{\"negatives/white\": [\"4+1\", \"220+1\", \"1469+1\", \"1521+1\", \"1943+1\", \"2504+1\", \"2711+1(...TRUNCATED)	CC-BY	4.0	http://creativecommons.org/licenses/by/4.0/	"Original work titled: 'Species composition, blood meal hosts and Plasmodium infection rates of Anop(...TRUNCATED)

5388809896c7b2a9adf5d708f6ea4b08

[ "Introduction", "Cationic", "antimicrobial", "peptides", "(CAMPs)", "are", "short-length,", "amphiphilic,", "rich", "in", "basic", "residues", "of", "the", "innate", "immune", "system", "of", "complex", "organisms,", "serving", "as", "the", "first", "defense", "line", "against", "pathogens.", "CAMPs", "can", "potentially", "give", "rise", "to", "a", "new", "generation", "of", "drugs", "due", "to", "their", "broad", "spectrum", "of", "antimicrobial", "activity", "against", "bacteria,", "rare", "cases", "of", "the", "appearance", "of", "AMP-resistant", "bacteria,", "selectivity,", "and", "rapid", "effects.", "Due", "to", "their", "potential", "advantage", "as", "antimicrobials,", "new", "AMPs", "are", "increasingly", "being", "isolated", "or", "synthesized", "and", "their", "properties", "investigated.", "Although", "increasing", "evidence", "shows", "that", "some", "AMPs", "may", "also", "act", "intracellularly,", "inhibiting", "proteins", "1", ",", "nucleic", "acids", "1", "–", "4", "and", "cell-wall", "synthesis", "1", "–", "5", "and", "inducing", "cell", "apoptosis", "1", ",", "3", ",", "5", ",", "most", "of", "them", "kill", "cells", "by", "disturbing", "the", "membrane’s", "integrity", "1", ",", "2", ",", "6", ",", "7", ".", "Several", "CAMPs", "seem", "to", "disturb", "the", "membrane", "of", "cells", "by", "self-aggregation", "on", "the", "membrane", "and", "forming", "pores", "beyond", "a", "peptide/lipid", "(P/L)", "ratio", "threshold", "1", ",", "2", ".", "BP100", "(H-KKLFKKILKYL-NH", "2", "),", "is", "a", "hybrid", "CAMP", "synthesized", "by", "combinatorial", "chemistry", "involving", "parts", "of", "two", "natural", "AMPs,", "melittin,", "and", "cecropin", "A", "8", ".", "BP100", "shows", "high", "antimicrobial", "activity", "and", "selectivity", "against", "some", "Gram-negative", "bacteria,", "with", "a", "promising", "minimal", "inhibitory", "concentration", "ranging", "from", "2.5", "to", "5", "µM", "and", "low", "cytotoxicity", "against", "mammalian", "cells", "making", "this", "peptide", "an", "exciting", "candidate", "for", "therapeutic", "use", "8", ".", "BP100", "conformation", "and", "activity", "on", "model", "membranes", "have", "been", "extensively", "analyzed", "with", "experimental", "techniques", "9", "–", "13", ".", "BP100", "has", "a", "random", "coil", "conformation", "in", "solution", "and", "zwitterionic", "bilayers", "but", "acquires", "predominantly", "alpha-helical", "and", "amphipathic", "conformation", "in", "the", "presence", "of", "negatively", "charged", "membranes.", "BP100", "is", "highly", "mobile", "on", "the", "membrane", "surface", "and", "orients", "parallel", "in", "predominantly", "negatively", "charged", "vesicles", "9", ",", "10", ".", "BP100", "secondary", "structure", "formation", "and", "bacteria", "killing", "performance", "seem", "to", "be", "directly", "related", "to", "negative", "charge", "content", "in", "membranes,", "thus", "explaining", "its", "high", "selectivity", "towards", "negative", "bacterial", "membrane", "and", "low", "cytotoxicity", "against", "predominantly", "less", "negatively", "charged", "mammalian", "cell", "membranes", "11", ",", "14", ".", "Experimental", "approaches", "to", "investigate", "peptide/membrane", "interactions", "mainly", "focus", "macroscopic-averaged", "insight", "on", "a", "mesoscopic", "scale.", "A", "detailed", "description", "of", "peptide/membrane", "interactions", "at", "the", "atomic", "scale", "may", "help", "to", "gain", "information", "on", "CAMPs", "disruption", "mechanisms,", "permitting", "the", "rational", "design", "of", "new", "derivatives", "and,", "therefore,", "the", "better", "therapeutic", "use", "of", "these", "novel", "antimicrobials.", "Molecular", "Dynamics", "(MD)", "simulations", "provide", "atomic-level", "insights", "and", "have", "been", "widely", "used", "to", "study", "the", "interaction", "of", "AMPs", "with", "membranes", "15", "–", "18", ".", "MD", "simulations", "were", "also", "utilized", "to", "study", "BP100’s", "structure", "and", "activity:", "Wang", "Y.", "et", "al.", "17", "performed", "an", "8", "µs-long", "MD", "simulation", "of", "BP100", "in", "DMPC", "bilayer", "and", "found", "that", "BP100", "remained", "in", "the", "surface-bound-state", "inserting", "through", "its", "C-terminus.", "They", "also", "reported", "the", "unfolding", "of", "BP100", "N-terminus", "(Lys1", "and", "Lys2)", "after", "1.5", "µs,", "resulting", "in", "a", "tilt", "angle", "of", "97°", "and", "81%", "helicity", "10", ",", "17", ".", "In", "a", "study", "with", "Brownian", "Dynamics", "simulations", "with", "a", "coarse-grained", "peptide", "model,", "Alves", "et", "al.", "18", "simulated", "BP100", "confined", "in", "model", "membranes", "at", "293", "K", "for", "1", "µs", "each", "and", "reported", "the", "high", "stability", "of", "BP100", "pre-folded", "alpha", "helix", "and", "a", "threefold", "decrease", "in", "its", "lateral", "diffusion", "in", "POPC:", "POPG", "membranes", "relative", "to", "that", "in", "POPC", "membrane", "18", ".", "In", "this", "work", "we", "report", "simulations,", "in", "solution", "and", "membranes,", "extending", "for", "more", "than", "25", "µs", "of", "BP100", "and", "membranes", "of", "1,2-dipalmitoyl-sn-glycerol-3-phosphocholine", "(DPPC),", "1,2-dipalmitoyl-sn-glycerol-3-phosphoglycerol", "(DPPG),", "and", "DPPC:", "DPPG", "mixtures", "(1:1).", "We", "systematically", "investigated", "the", "dependence", "of", "BP100", "secondary", "structure", "on", "DPPG", "lipids", "which", "agrees", "with", "experimental", "findings", "11", ".", "We", "also", "analyzed", "the", "contribution", "of", "PG-containing", "lipid", "rafts", "on", "BP100", "conformation", "and", "behavior.", "In", "most", "simulations", "containing", "DPPG", "lipids,", "we", "observed", "the", "occurrence", "of", "a", "dynamic", "transition", "of", "the", "peptide", "adsorbed", "at", "the", "membrane", "interface,", "where", "the", "apolar", "facet", "of", "the", "BP100", "(Fig.", "1c", "),", "initially", "exposed", "to", "water,", "rotates", "toward", "the", "membrane", "interior,", "leaving", "the", "polar", "facet", "of", "the", "peptide", "exposed", "to", "water.", "This", "process,", "defined", "here", "as", "peptide", "flip,", "could", "be", "part", "of", "CAMPs", "primary", "antimicrobial", "mechanism.", "Constraining", "four", "residues", "of", "the", "peptide", "into", "an", "alpha", "helix", "turn,", "we", "observed", "that", "the", "unconstrained", "regions", "of", "the", "peptide", "rapidly", "acquired", "an", "alpha-helix", "conformation", "in", "DPPG-containing", "membranes,", "suggesting", "that", "the", "formation", "of", "these", "small", "alpha-helical", "nuclei", "in", "peptide/membrane", "interactions", "may", "be", "the", "initial", "step", "for", "the", "peptide", "insertion", "into", "the", "hydrophobic", "core", "of", "the", "membranes.", "Figure", "1", "BP100", "initial", "structures:", "random", "coil", "L", "-BP100", "(", "a", ")", "and", "alpha-helical", "α", "-BP100", "(", "b", ").", "Helical", "wheel", "projection", "(", "c", "),", "Lys5", "and", "Ile7", "were", "used", "as", "representative", "residues", "of", "polar", "and", "apolar", "moieties,", "respectively.", "In", "(", "d", ")", "DPPC", "(in", "blue)", "and", "DPPG", "(in", "yellow).", "Atom", "names", "are", "shown", "for", "peptide", "and", "lipids;", "they", "were", "used", "in", "our", "peptide-lipid", "pair", "analysis.", "Methods", "All", "simulations", "sets", "were", "assembled,", "carried", "out", "and", "analyzed", "using", "GROMACS", "5.0.2", "software", "package.", "For", "trajectories", "visual", "inspections", "and", "image", "rendering,", "VMD", "1.9.2", "software", "was", "used.", "Table", "1", "shows", "all", "simulation", "systems", "and", "its", "simulation", "time.", "The", "program", "used", "to", "analyze", "the", "hydration", "shell", "of", "the", "peptide", "using", "the", "Minimum", "Distance", "Distribution", "Function", "was", "developed", "in", "our", "group.", "Table", "1", "Table", "containing", "all", "MD", "simulation", "sets", "carried", "out", "in", "this", "work.", "For", "bilayers,", "we", "used", "Slipids", "forcefield", "and", "ff99sb-ildn-NMR", "for", "BP100.", "All", "sets", "were", "simulated", "at", "323", "K", "(above", "DPPC", "and", "DPPG", "transition", "temperatures)", "and", "1", "bar.", "L", "-BP100", "indicate", "constraint-free", "simulations", "using", "random", "coil", "BP100", "as", "peptide", "starting", "configuration", "and", "α", "-BP100,", "as", "alpha", "helix.", "Res1–5,", "res4–8", "and", "res7–11", "indicate", "simulations", "with", "BP100", "with", "permanent", "constraints", "on", "the", "referred", "residues.", "Simulation", "sets", "Simulations", "Peptide/Lipid", "Waters/Lipid", "Time", "(ns)", "Bilayers", "DPPC", "0/128", "54", "860", "DPPG", "53", "1100", "PCPG-R", "0/[64/64]", "54", "1200", "PCPG-NR", "52", "1200", "Peptide", "in", "water", "L", "-BP100", "in", "water", "1/0", "—", "2000", "α", "-BP100", "in", "water", "—", "2850", "Peptide", "with", "bilayers", "L", "-BP100", "in", "DPPC", "1/128", "54", "1700", "α", "-BP100", "in", "DPPC", "54", "2000", "L", "-BP100", "in", "DPPG", "1/128", "53", "1700", "α", "-BP100", "in", "DPPG", "53", "2000", "L", "-BP100", "in", "PCPG-R", "1/[64/64]", "54", "1600", "α", "-BP100", "in", "PCPG-R", "54", "2000", "L", "-BP100", "in", "PCPG-NR", "1/[64/64]", "52", "1700", "α", "-BP100", "in", "PCPG-NR", "52", "2000", "Partially", "constrained", "peptide", "in", "DPPG", "α-", "BP100", "(res1–5)", "in", "DPPG", "1/128", "54", "600", "α-", "BP100", "(res4–8)", "in", "DPPG", "54", "600", "α-", "BP100", "(res7–11)", "in", "DPPG", "54", "600", "Lipid", "bilayers", "We", "used", "Slipids", "forcefield", "19", "parameters", "were", "chosen", "to", "simulate", "DPPC,", "DPPG,", "and", "DPPC/DPPG", "mixed", "bilayers.", "64-lipids-per-monolayer", "DPPC", "and", "DPPG", "pure", "membrane", "topologies", "were", "taken", "from", "the", "Slipids", "developer", "group", "website", "(http://www.fos.su.se/~sasha/Slipids/Downloads.html).", "Mixed", "bilayers", "of", "DPPC", "and", "DPPG", "containing", "32", "DPPCs", "and", "32", "DPPGs", "in", "each", "leaflet", "were", "constructed", "using", "the", "PACKMOL", "20", "software.", "One", "membrane", "was", "composed", "of", "randomly", "distributed", "lipids", "(PCPG-NR)", "on", "both", "leaflets", "and", "the", "other,", "a", "4", "×", "4", "DPPG", "raft", "was", "placed", "in", "the", "center", "of", "both", "monolayers", "(PCPG-R)", "and", "the", "remaining", "48", "lipids", "were", "randomly", "distributed", "(Fig.", "2", ").", "Figure", "2", "Initial", "membrane", "configurations", "membranes.", "TIP3P", "water", "molecules", "were", "used", "to", "solvate", "our", "systems,", "with", "an", "approximately", "53", "water/lipid", "ratio.", "Na", "+", "ions", "(Aqvist", "21", ")", "were", "included", "to", "counter-balance", "charges", "of", "membranes", "containing", "DPPG.", "All", "bilayer", "sets", "were", "minimized", "and", "equilibrated", "for", "20", "ps", "in", "a", "NVT", "ensemble", "and", "thereafter,", "in", "NPT", "conditions", "for", "50", "ns.", "After", "that,", "the", "bilayers", "were", "simulated", "for", "860", "ns", "(DPPC),", "1100", "ns", "(DPPG)", "and", "1200", "ns", "(for", "both", "PCPG-R", "and", "PCPG-NR).", "The", "area", "per", "lipid", "and", "membrane", "thickness", "were", "calculated", "for", "all", "membranes", "and", "thNen", "compared", "with", "experimental", "data", "and", "those", "obtained", "by", "Slipids", "authors", "(Tables", "S1", "and", "S2", ").", "The", "last", "trajectory", "frames", "of", "the", "membrane", "simulations", "were", "used", "to", "build", "our", "peptide-lipid", "systems.", "Peptide-in-solution", "and", "peptide-lipid", "systems", "Following", "a", "study", "by", "Beauchamp", "et", "al.", "22", ",", "ff99sb-ildn-NMR", "force", "field", "was", "chosen", "to", "characterize", "BP100", "in", "our", "modeling", "due", "to", "its", "good", "performance", "in", "simulating", "peptides", "and", "proteins", "structures", "in", "TIP3P", "water", "when", "compared", "with", "NMR", "measurements.", "BP100", "(H-Lys-Lys-Leu-Phe-Lys-Lys-Ile-Leu-Lys-Tyr-Leu-NH", "2", ")", "has", "an", "amidated", "C-terminal", "and", "a", "+6", "charge", "at", "physiological", "pH,", "with", "all", "its", "Lysines", "and", "N-terminal", "amine", "protonated.", "To", "study", "secondary", "structure", "behavior", "of", "BP100", "both", "in", "solution", "and", "in", "bilayers,", "two", "initial", "BP100", "conformations", "were", "used:", "linear", "BP100", "(", "L", "-BP100)", "and", "alpha-helical", "BP100", "(", "α", "-BP100)", "(Fig.", "1", "a,b", ").", "In", "the", "peptide/water", "system,", "one", "peptide", "(", "α", "-BP100", "or", "L-BP100", "initial", "conformation)", "were", "simulated", "in", "a", "cubic", "box", "with", "3975", "TIP3P", "water", "molecules", "for", "more", "than", "1", "microsecond", "with", "its", "+6", "charges", "neutralized", "with", "6", "Cl", "−", "ions", "(Dang", "23", ").", "Simulations", "containing", "peptides", "with", "membranes", "had", "a", "peptide/lipid", "ratio", "of", "1/128", "and", "the", "peptide", "was", "initially", "positioned", "in", "parallel", "orientation", "with", "the", "membrane", "surface", "approximately", "2", "nm", "away.", "For", "the", "α", "-BP100,", "additionally,", "we", "tested", "two", "parallel", "orientations,", "where", "the", "polar", "amino", "acids", "were", "facing", "the", "membrane", "and", "a", "random", "orientation.", "For", "the", "perpendicular", "orientation", "we", "tested", "two", "ways", "with", "either", "the", "C-terminal", "or", "N-terminal", "facing", "the", "interface.", "For", "all", "these", "initial", "conditions,", "we", "obtained", "the", "same", "final", "location", "and", "orientation", "of", "the", "peptide", "that", "will", "be", "discussed", "in", "the", "results", "section.", "To", "investigate", "the", "influence", "of", "small", "core", "alpha-helical", "sequences", "in", "BP100", "to", "its", "stability", "when", "interacting", "with", "bilayers,", "constrained", "simulations", "of", "BP100", "in", "DPPG", "membranes", "were", "performed.", "The", "alpha-helix", "dihedral", "angles", "(ϕ", "=", "−60°;", "ψ", "=", "−40°)", "in", "3", "different", "regions", "were", "fixed:", "1–5", "(Lys-Lys-Leu-Phe-Lys),", "4–8", "(Phe-Lys-Lys-Ile-Leu)", "and", "7–11", "(Ile-Leu-Lys-Tyr-Leu)", "residues", "(Fig.", "3", ").", "We", "maintained", "the", "constrained", "regions", "during", "the", "simulations.", "Figure", "3", "Starting", "structures", "for", "peptides", "with", "permanent", "constrained", "dihedral", "angles.", "Following", "the", "previous", "lipid", "membrane", "systems,", "energy-minimization,", "NVT,", "and", "NPT", "ensembles", "were", "used", "in", "the", "equilibration", "stage", "of", "the", "MD", "simulation.", "Simulation", "details", "MD", "simulations", "were", "carried", "out", "with", "a", "2", "fs", "time-step", "with", "a", "Leap-Frog", "integrator", "24", ",", "in", "isobaric-isothermal", "(NPT)", "ensemble,", "at", "323", "K", "(50", "°C)", "with", "temperature", "coupling", "treated", "separately", "for", "BP100,", "lipids,", "water", "and", "ions", "with", "the", "V-rescale", "thermostat", "25", ".", "Atmospheric", "pressure", "of", "1", "bar", "was", "kept", "with", "the", "Berendsen", "barostat", "26", "and", "semi-isotropic", "pressure", "coupling", "was", "used", "for", "all", "systems.", "All", "bonds", "were", "constrained", "with", "LINCS", "27", "algorithm.", "Long-range", "electrostatic", "correction", "was", "treated", "by", "a", "Particle-mesh-Ewald", "method", "28", "with", "a", "real-space", "cut-off", "of", "1.5", "nm.", "van", "der", "Waals", "interactions", "were", "truncated", "at", "1.5", "nm", "distance", "with", "a", "switch", "function", "from", "1.4", "nm.", "Simulation", "analyses", "Lipid/peptide", "contacts", "For", "the", "computation", "of", "lipid", "contacts", "with", "BP100", "and", "pair", "frequency,", "we", "labeled", "some", "lipid(s)", "and", "BP100", "atoms", "as", "“polar”", "or", "“apolar”,", "based", "on", "charges.", "Then,", "the", "radial", "distribution", "function", "(RDF)", "between", "each", "pair", "of", "apolar", "(or", "polar)", "atoms", "of", "the", "lipids/BP100", "system", "were", "computed.", "The", "time", "window", "selected", "for", "computing", "these", "RDFs", "includes", "that", "of", "the", "membrane", "bound", "peptide", "250", "ns-before", "and", "250", "ns-after", "the", "peptide", "flip.", "The", "pairs", "with", "distinguishable", "RDF", "peaks", "and", "following", "minimum", "were", "used", "to", "analyze", "the", "number", "of", "polar", "and", "apolar", "contacts", "between", "lipids", "and", "peptide.", "A", "cutoff", "distance", "for", "each", "pair", "was", "defined", "from", "the", "position", "of", "the", "first", "minimum", "in", "the", "RDF", "curve,", "and", "a", "contact", "was", "defined", "as", "a", "pair", "of", "atoms", "at", "smaller", "distance", "than", "their", "corresponding", "cutoff", "distance.", "The", "simulations", "were", "analyzed", "and", "the", "total", "number", "of", "polar", "and", "apolar", "contacts", "as", "a", "function", "of", "time", "was", "obtained.", "Additionally,", "some", "polar", "and", "apolar", "pairs", "were", "analyzed", "individually.", "Peptide", "hydration", "The", "time", "evolution", "of", "the", "solvent", "shell", "surrounding", "the", "peptide", "in", "its", "α", "and", "L", "conformations", "in", "all", "simulated", "membranes", "was", "analyzed", "using", "the", "minimum", "distance", "distribution", "function", "(MDDF)", "29", "between", "the", "peptide", "and", "the", "water", "molecules,", "as", "well", "as", "the", "average", "number", "of", "water", "molecules", "N(r),", "within", "a", "distance", "r", "=", "0.5", "nm,", "named", "here", "as", "N(0.5).", "For", "comparison", "the", "MDDF", "of", "BP100", "in", "water", "was", "used", "to", "define", "the", "bulk", "water", "hydration", "shell", "at", "the", "same", "distance.", "Results", "The", "17", "sets", "of", "simulations", "(Table", "1", ")", "were", "run", "for", "more", "than", "25", "µs", "of", "simulation", "time,", "and", "each", "unconstrained", "peptide/membrane", "simulation", "had", "at", "least", "1.6", "µs", "of", "simulation.", "After", "comparing", "the", "simulated", "membrane", "properties", "with", "experimental", "and", "other", "computational", "studies", "(Tables", "S1", "and", "S2", "),", "we", "systematically", "analyzed", "the", "peptide", "conformation", "in", "solution", "and", "on", "bilayers,", "peptide", "trajectory,", "and", "dynamics", "on", "membranes", "and", "the", "effect", "of", "BP100", "on", "model", "bilayers.", "In", "the", "following", "sections,", "we", "describe", "our", "results.", "Secondary", "structure", "Peptide", "in", "solution", "To", "study", "BP100", "secondary", "structure", "in", "water,", "we", "did", "simulations", "for", "more", "than", "2", "µs", "with", "both", "α-BP100", "and", "L-BP100", "initial", "conformations.", "Figure", "4", "shows", "that", "BP100", "conformation", "in", "solution", "was,", "predominantly,", "random", "coil.", "The", "peptide", "lost", "the", "initial", "alpha", "helix", "conformation", "in", "approximately", "40", "ns", "when", "starting", "with", "an", "alpha-helix", "configuration", "and", "occasionally", "formed", "an", "alpha-helix", "turn", "in", "the", "5–9", "residue", "region", "(Fig.", "4a", ").", "For", "L-BP100", "in", "solution,", "we", "observed", "a", "similar", "pattern", "(Fig.", "4b", ").", "Circular", "Dichroism", "(CD)", "shows", "that", "BP100", "is", "random", "in", "solution", "10", ",", "11", ",", "30", ",", "and", "our", "simulations", "reproduced", "these", "data.", "Figure", "4", "Secondary", "structure", "analyses", "graphs", "of", "α", "-BP100", "(", "a", ")", "and", "L", "-BP100", "(", "b", ")", "in", "solution.", "In", "both", "cases,", "the", "peptide", "shows", "predominance", "in", "random", "coil", "structure", "(orange)", "with", "occasional", "formation", "of", "1", "alpha", "helix", "turn", "(blue)", "in", "residues", "5", "to", "9.", "Peptide", "in", "bilayers", "BP100", "conformation,", "antimicrobial", "activity,", "and", "PG", "content", "are", "related", "11", ".", "We", "simulated", "α-BP100", "and", "L-BP100", "on", "DPPC,", "DPPG,", "PCPG-R,", "and", "PCPG-NR", "at", "323", "K,", "above", "DPPG", "and", "DPPC", "transition", "temperatures.", "In", "all", "cases,", "the", "peptide", "was", "rapidly", "adsorbed", "on", "the", "membranes", "and", "diffused", "laterally.", "Figure", "5", "shows", "that", "the", "BP100", "alpha-helical", "conformation", "was", "proportional", "to", "PG", "content,", "as", "the", "alpha-helix", "percentage", "in", "DPPC", "is", "considerably", "smaller", "than", "that", "observed", "in", "DPPG-containing", "membranes.", "Alpha-helix", "formed", "in", "the", "5", "to", "9", "residues", "region", "(Lys-Lys-Ile-Leu-Lys)", "was", "highly", "stable", "in", "all", "simulations.", "Figure", "5", "Secondary", "structure", "analyses", "of", "α", "-BP100", "in", "DPPC", "(", "a", "),", "DPPG", "(", "b", "),", "PCPG-R", "(", "c", ")", "and", "PCPG-NR", "(", "d", ").", "In", "DPPC,", "α-BP100", "alpha-helix", "conformation", "reached", "a", "plateau", "at", "38%", "helicity", "throughout", "the", "simulation,", "being", "relatively", "less", "stable", "than", "α-BP100", "in", "bilayers", "containing", "DPPG,", "where", "a", "higher", "propensity", "of", "maintaining", "its", "conformation", "was", "observed.", "For", "pure", "DPPG", "(Fig.", "5b", ")", "we", "found", "73%", "(8", "out", "of", "11", "residues),", "and", "for", "both", "PCPG", "(1:1)", "membranes", "we", "report", "54%", "(6", "out", "of", "11)", "of", "helicity", "(Fig.", "5c,d", ").", "No", "alpha-helix", "formation", "was", "detected", "in", "L-BP100", "on", "bilayers", "simulations", "(Fig.", "S3", ").", "Therefore,", "the", "1.8", "µs", "simulation", "was", "not", "enough", "to", "present", "a", "spontaneous", "peptide", "folding.", "The", "alpha-helix", "formed", "in", "the", "region", "of", "the", "residues", "5", "to", "9", "showed", "higher", "stability", "in", "all", "simulations", "with", "α-BP100", "in", "bilayers", "(Fig.", "5", ").", "This", "secondary", "structure", "pattern", "led", "us", "to", "suggest", "that", "the", "residues", "5", "to", "9", "are", "related", "to", "helix", "nucleation,", "in", "which", "the", "formation", "of", "this", "helix", "core", "could", "be", "the", "first", "step", "in", "the", "AMP", "actions.", "Following", "this", "assumption,", "we", "tested", "three", "conformations", "with", "α-BP100", "torsionally", "constrained", "in", "three", "regions:", "res1–5,", "res4–8,", "res7–11", "(Fig.", "3", ")", "and", "simulated", "with", "pure", "DPPG", "membranes.", "Figure", "6", "shows", "the", "evolution", "of", "the", "secondary", "structure", "in", "each", "simulation.", "Figure", "6", "Secondary", "structure", "analyses", "graphs", "of", "BP100", "in", "DPPG", "with", "one", "turn", "constraints", "on", "res1–5", "(", "a", "),", "res", "4–8", "(", "b", ")", "and", "res7–11", "(", "c", ").", "α-BP100", "with", "constraints", "on", "residues", "1–5", "(Lys-Lys-Leu-Phe-Lys)", "acquired", "identical", "helicity", "(73%)", "to", "α-BP100", "with", "no", "constraints", "at", "the", "same", "conditions", "(Fig.", "5b", "),", "after", "50", "ns", "of", "simulation", "(Fig.", "6a", "),", "with", "a", "helix", "spanning", "from", "Lys2", "to", "Lys9", "(Fig.", "6a", ").", "Constraints", "on", "residues", "4–8", "(Phe-Lys-Lys-Ile-Leu)", "showed", "an", "overall", "52%", "of", "helicity", "(Fig.", "6b", "),", "with", "a", "helix", "spanning", "from", "Phe4", "to", "Lys9", "(Fig.", "6b", ").", "Constraints", "on", "residues", "7–11", "(Ile-Leu-Lys-Tyr-Leu)", "did", "not", "affect", "the", "rest", "of", "the", "peptide", "conformation", "(Fig.", "6c", ")", "in", "600", "ns", "of", "simulation.", "Peptide", "flip", "Simulation", "trajectories", "analyses", "(Fig.", "7", ")", "revealed", "that", "the", "peptide", "in", "alpha-helix", "conformation", "initially", "adsorbed", "at", "the", "DPPG-containing", "membranes", "interface", "with", "its", "hydrophilic", "facet", "in", "contact", "with", "the", "membrane,", "leaving", "the", "hydrophobic", "facet", "exposed", "to", "water.", "We", "observed", "that", "the", "alpha-helix", "rotated,", "inserting", "the", "hydrophobic", "facet", "into", "the", "hydrophobic", "region", "of", "the", "membrane.", "We", "define", "this", "dynamic", "phenomenon,", "observed", "in", "our", "simulations,", "as", "peptide", "flip.", "Although", "this", "final", "peptide", "orientation", "is", "known", "31", "–", "34", ",", "no", "MD", "simulation", "on", "peptide", "flip", "has", "been", "reported.", "Figure", "7", "Peptide", "flip", "observed", "from", "above", "in", "α", "-BP100", "in", "DPPG", "set", "after", "circa", "1400", "ns", "of", "simulation.", "In", "blue,", "polar", "facet", "and", "in", "green,", "the", "apolar", "facet.", "Peptide", "flip", "was", "also", "observed", "in", "α", "-BP100", "in", "DPPC,", "α", "-BP100", "in", "PCPG-R,", "and", "α", "-BP100", "res(1–5)", "in", "DPPG", "simulations.", "The", "peptide", "flip", "caused", "dehydration", "(Fig.", "8", ").", "When", "in", "contact", "with", "membranes,", "depending", "on", "the", "secondary", "structure", "of", "the", "peptide", "and", "the", "lipid", "nature,", "the", "hydration", "of", "the", "peptide", "was", "slightly", "different.", "However,", "when", "the", "peptide", "flip", "took", "place,", "its", "hydration", "was", "drastically", "altered.", "Figure", "8", "Average", "number", "of", "water", "molecules", "up", "to", "a", "distance", "of", "0.5", "nm,", "N(0.5),", "obtained", "from", "the", "minimum-distance", "distribution", "function", "(MDDF),", "between", "L-BP100", "(", "a", ")", "and", "α", "-BP100", "(", "b", ")", "and", "water", "molecules.", "Vertical", "lines", "indicate", "when", "peptide", "flip", "occurred.", "In", "the", "peptide/membrane", "simulations", "with", "L-BP100", "as", "a", "starting", "configuration", "(Fig.", "8a", "),", "no", "significant", "change", "in", "the", "number", "of", "water", "molecules", "surrounding", "the", "peptide", "was", "observed", "in", "comparison", "the", "150", "obtained", "in", "the", "bulk", "aqueous", "solution.", "In", "the", "last", "500", "ns", "of", "simulation,", "N(0.5)", "was", "approximately", "178", "±", "11", "in", "DPPC,", "145", "±", "7", "in", "DPPG,", "145", "±", "13", "in", "PCPG-NR,", "and", "148", "±", "3", "in", "PCPG-R.", "No", "peptide", "flip", "was", "detected", "in", "the", "trajectories", "for", "those", "simulations.", "However,", "in", "the", "peptide/membrane", "simulations", "with", "α-BP100", "as", "a", "starting", "configuration", "(Fig.", "8b", "),", "N", "(0.5)", "lowered", "in", "DPPC", "(starting", "at", "~700", "ns),", "DPPG", "(~1500", "ns)", "and", "PCPG-R", "(~390", "ns).", "Alongside", "with", "dehydration,", "a", "peptide-flip", "was", "observed", "in", "DPPG", "and", "PCPG-R.", "For", "the", "α-", "BP100", "in", "DPPC", "simulation,", "we", "observed", "a", "semi-flip,", "as", "the", "peptide", "alpha-helical", "conformation", "was", "only", "38%", "(Fig.", "5a", "),", "suggesting", "that", "the", "peptide", "overall", "polar/apolar", "segregation", "(Fig.", "2c", ")", "was", "lost.", "The", "peptide", "flip", "transition", "time", "varied", "from", "100", "to", "400", "ns.", "During", "the", "peptide-flip,", "in", "the", "simulations", "of", "α-BP100", "in", "DPPG", "and", "PCPG-R,", "the", "mean", "number", "N(0.5)", "was", "reduced", "from", "147", "±", "5", "and", "142", "±", "7", "to", "76", "±", "4", "and", "75", "±", "4,", "respectively.", "These", "represent", "on", "average", "a", "reduction", "of", "approximately", "48%", "in", "the", "number", "of", "water", "molecules", "in", "the", "hydration", "shell", "of", "α-BP100.", "In", "DPPC,", "we", "detected", "a", "smaller", "decrease", "in", "the", "hydration", "of", "α-BP100,", "around", "45%", "(from", "172", "±", "8", "to", "94", "±", "2),", "having", "around", "20", "water", "molecules", "more", "after", "the", "semi-flip.", "Figure", "9", "shows", "the", "density", "profile", "before", "and", "after", "peptide", "flip", "for", "α-BP100", "in", "DPPG", "and", "PCPG-R.", "We", "chose", "residues", "Lys5", "and", "Ile7", "to", "illustrate", "the", "changes", "in", "the", "mean", "position", "of", "hydrophobic", "and", "hydrophilic", "residues", "with", "respect", "to", "the", "membrane", "due", "to", "the", "peptide", "flip.", "These", "residues", "are", "polar", "and", "apolar,", "respectively,", "and", "located", "opposite", "to", "each", "other", "when", "in", "an", "alpha-helix", "conformation", "(Fig.", "2c", ").", "Trajectory", "visualization", "and", "density", "profile", "(Figs", "7", "and", "9", ")", "clearly", "show", "the", "distinct", "orientation", "in", "BP100", "hydrophobic", "facet", "before", "and", "after", "peptide", "flip.", "During", "the", "initial", "phase", "of", "approaching", "towards", "the", "membrane,", "BP100", "polar", "side", "faces", "the", "membrane", "with", "its", "N-terminal", "residues", "(Lys1", "and", "Lys2)", "interacting", "with", "the", "bilayer", "headgroups.", "After", "the", "flip,", "the", "apolar", "residues", "facet", "turns", "to", "the", "membrane,", "and", "simultaneously", "inserts", "into", "the", "bilayer,", "below", "the", "carbonyl", "carbons", "(C31)", "of", "the", "lipids", "(Fig.", "9", ").", "Figure", "9", "Peptide", "flip", "observed", "through", "number", "density", "analysis.", "(", "a", "and", "b", ")", "show", "respectively", "before", "and", "after", "the", "flip", "in", "α", "-BP100", "in", "DPPG", "simulation;", "c)", "and", "d)", "demonstrate", "the", "flip", "for", "α", "-BP100", "in", "PCPG-R", "simulation.", "Lys5", "represents", "the", "polar", "side", "of", "BP100", "and", "Ile7,", "the", "apolar", "facet", "(Fig.", "2c", ").", "OC3", "(DPPG", "head", "group", "further", "out", "glycerol", "Oxygen),", "N", "chol", "(DPPC", "head", "group", "choline", "Nitrogen),", "P", "(Phosphorus)", "and", "C31", "(first", "carbon", "from", "sn-1", "acyl", "chain)", "are", "shown", "to", "display", "BP100", "insertion", "into", "the", "bilayer", "after", "the", "flip.", "Peptide", "flip", "was", "not", "observed", "in", "α-BP100", "in", "PCPG-NR,", "where", "DPPG", "lipids", "were", "initially", "scattered", "randomly.", "PG", "content", "might", "be", "related", "to", "the", "occurrence", "of", "the", "flip,", "but", "also,", "the", "PG", "content", "in", "the", "vicinity", "of", "BP100.", "Several", "studies", "suggest", "membrane-active", "peptides", "cause", "anionic", "lipid", "clustering,", "or", "subdomain", "formation,", "upon", "binding", "to", "bilayers", "containing", "neutral", "and", "charged", "phospholipids", "35", "–", "37", ".", "The", "same", "mechanism", "could", "be", "involved", "in", "BP100/bilayers", "interactions.", "Lipid", "clustering", "The", "distribution", "of", "the", "lipids", "in", "the", "vicinity", "of", "BP100", "was", "evaluated", "by", "computing", "the", "total", "number", "of", "lipids", "in", "contact", "with", "α-BP100", "as", "a", "function", "of", "time", "(Fig.", "S5", "),", "the", "time-averaged", "values", "(Table", "2", ",", "Fig.", "10", "),", "and", "the", "lipid", "raft", "size", "distribution", "(Fig.", "S6", ").", "Table", "2", "Average", "number", "of", "lipids", "in", "contact", "with", "BP100", "in", "simulations", "using", "α", "-BP100", "as", "the", "peptide", "starting", "configuration,", "except", "α", "-BP100", "(1–5),", "which", "had", "permanent", "constraints", "on", "residues", "1", "to", "5.", "Standard", "deviations", "are", "shown", "in", "parenthesis.", "Average", "Number", "of", "Lipids", "in", "Contact", "with", "BP100", "Simulation", "Lipid", "in", "Contact", "Average", "α", "-BP100", "in", "DPPC", "DPPC", "10.7", "(±2.6)", "α", "-BP100", "in", "DPPG", "DPPG", "13.1", "(±1.4)", "α", "-BP100", "(1–5)", "in", "DPPG", "DPPG", "13.5", "(±1.9)", "α", "-BP100", "in", "PCPG-R", "Total", "12.3", "(±1.7)", "DPPC", "3.6", "(±1.5)", "DPPG", "8.7", "(±1.2)", "α", "-BP100", "in", "PCPG-NR", "Total", "11.0", "(±1.7)", "DPPC", "2.7", "(±1.3)", "DPPG", "8.3", "(±1.6)", "Figure", "10", "Average", "Number", "of", "DPPCs", "(", "a", ")", "and", "DPPGs", "(", "b", ")", "in", "contact", "with", "BP100", "analyzed", "for", "all", "constraint-free", "simulations", "containing", "BP100", "in", "an", "initially", "alpha", "helical", "conformation", "in", "DPPC,", "DPPG,", "PCPG-R", "and", "PCPG-NR", "membranes.", "Vertical", "lines", "represent", "the", "average", "time", "where", "peptide", "flip", "was", "observed.", "Standard", "deviation", "error", "bars", "are", "represented", "as", "fillings.", "α-BP100", "in", "DPPC", "simulation", "had", "on", "average", "10.7", "peptide/lipid", "contacts", "(Table", "2", ")", "and", "a", "steady", "increase", "in", "the", "number", "of", "contact", "lipids", "(Figs", "10b", "and", "S5a", ").", "This", "increase", "is", "due", "to", "the", "peptide", "losing", "its", "alpha-helical", "conformation", "(Fig.", "5a", "),", "expanding", "the", "available", "peptide", "interaction", "area", "with", "the", "membrane.", "Lipid", "raft", "size", "distribution", "also", "shows", "a", "broader", "distribution", "of", "DPPC", "raft", "size,", "supporting", "this", "observation", "(Fig.", "S6a", ").", "A", "semi-peptide", "flip", "was", "observed", "at", "~700", "ns", "of", "simulation", "and,", "concomitantly,", "while", "BP100", "inserts", "into", "the", "bilayer", "as", "the", "density", "profile", "shows", "(Fig.", "S4a,b", "),", "the", "number", "of", "DPPCs", "in", "contact", "with", "BP100", "increased", "(Fig.", "S5a", ").", "α-BP100", "in", "DPPG", "simulation", "shows", "on", "average", "13.1", "DPPGs", "in", "close", "contact", "with", "the", "peptide", "(Table", "2", ").", "The", "number", "of", "DPPGs", "in", "contact", "rose", "after", "the", "flip,", "approximately", "in", "1500", "ns", "of", "simulation", "(Figs", "10a", "and", "S5b", ")", "and", "concurrently,", "BP100", "inserted", "deeply", "into", "the", "membrane", "(Fig.", "7a,b", ").", "Constrained", "α-BP100", "(res1–5)", "in", "DPPG,", "which", "showed", "identical", "alpha-helix", "content", "with", "α-BP100", "in", "DPPG", "(73%,", "Figs", "5b", "and", "6a", "),", "not", "only", "exhibits", "peptide", "flip", "in", "approximately", "300", "ns", "of", "simulation", "but", "also", "has,", "on", "average,", "13.5", "DPPGs", "in", "contact", "with", "BP100", "(Table", "2", ").", "In", "the", "simulation", "with", "α-BP100", "in", "PCPG-NR,", "the", "number", "of", "DPPG", "in", "contact", "with", "the", "peptide", "showed", "a", "slight", "increase", "(Fig.", "10b", "),", "reaching", "the", "average", "value", "observed", "in", "the", "simulation", "with", "PCPG-R", "(Table", "2", ").", "Also,", "lipid", "raft", "size", "probability", "analysis", "(Fig.", "S6d", ")", "showed", "a", "narrower", "peak", "distribution", "for", "DPPGs", "in", "contact", "with", "BP100", "in", "PCPG-R", "system,", "compared", "to", "PCPG-NR", "(Fig.", "S6e", "),", "indicating", "that", "DPPG", "lipid", "raft", "size", "was", "stable.", "Peptide", "flip", "was", "observed", "in", "the", "PCPG-R", "set", "after", "~390", "ns", "of", "simulation", "and", "no", "significant", "variation", "of", "the", "number", "of", "DPPG", "in", "contact", "with", "peptide", "was", "observed.", "Polar", "and", "apolar", "interaction", "analysis", "Peptide", "contact", "analysis", "revealed", "the", "increase", "of", "lipid/peptide", "contacts", "after", "peptide", "flip", "and", "DPPG", "clustering", "on", "PCPG", "membranes.", "We", "analyzed", "the", "nature", "of", "the", "intermolecular", "interactions", "that", "determine", "peptide-membrane", "interactions", "throughout", "the", "simulations.", "We", "calculated", "the", "number", "of", "polar", "and", "apolar", "pair", "contacts", "between", "peptide", "atoms", "and", "membrane", "atoms", "(Figs", "S7", "–", "S13", ").", "Using", "α-BP100", "in", "PCPG-R", "system", "as", "an", "example,", "Fig.", "11", "shows", "the", "sum", "of", "the", "10", "most", "frequent", "apolar/polar", "pairs", "between", "BP100", "with", "DPPC", "and", "DPPG.", "Figure", "11", "BP100", "pair", "occurrence", "with", "DPPC", "(", "a", ")", "and", "DPPG", "(", "b", ")", "for", "α", "-BP100", "in", "PCPG-R", "simulation.", "Graphs", "show", "the", "sum", "of", "the", "ten", "most", "frequent", "apolar/polar", "pairs", "during", "the", "simulation.", "Pairs", "were", "calculated", "using", "the", "monolayer", "facing", "the", "peptide.", "Green", "vertical", "lines", "indicate", "when", "peptide", "flip", "occurred", "(circa", "390", "ns).", "The", "total", "number", "of", "pairs,", "regardless", "if", "it", "is", "polar", "or", "apolar,", "between", "BP100", "and", "DPPG", "is", "higher", "than", "that", "with", "DPPC", "(Fig.", "11", "),", "which", "directly", "reflects", "on", "the", "number", "of", "lipids", "in", "contact", "with", "BP100", "(Fig.", "10", ").", "During", "the", "initial", "approach", "of", "the", "peptide", "towards", "the", "bilayer,", "polar", "interactions", "between", "charged", "Lysines", "and", "DPPG", "headgroup", "dominate", "the", "interaction", "(Figs", "11", "b,", "S9", ",", "S10", ",", "S12", "and", "S13", ").", "The", "primary", "interaction", "was", "between", "positively", "charged", "(+1)", "Lysine", "side", "chain", "amino", "group", "(NZ)", "and", "DPPG", "head", "phosphoglycerol", "group", "oxygens", "(OC2,", "OC3),", "even", "after", "peptide", "flip", "(Figs", "S9", ",", "S10", ",", "S12", "and", "S13", ").", "Before", "the", "peptide", "flip,", "the", "number", "of", "polar", "contacts", "is", "higher", "than", "the", "apolar", "contacts", "for", "DPPG/BP100.", "As", "the", "peptide", "starts", "to", "flip,", "the", "number", "of", "apolar", "contacts", "increase,", "while", "the", "polar", "contacts", "decrease.", "After", "the", "peptide", "flip", "is", "completed,", "apolar", "contacts", "are", "as", "numerous", "as", "the", "polar", "contacts", "(Fig.", "11a,b", "and", "S7–10,13", ").", "Also,", "after", "the", "flip,", "apolar", "contacts", "between", "BP100", "and", "DPPG", "were", "more", "frequent", "than", "with", "DPPC", "(Fig.", "11b", ").", "The", "higher", "peptide/DPPG", "apolar", "contact", "frequency", "after", "the", "flip", "is", "also", "observed", "for", "other", "simulations", "with", "peptide", "flip", "(Figs", "S7", "–", "10", ",", "13).", "Discussion", "Peptide", "secondary", "structure", "CD", "spectra", "of", "BP100", "in", "solution", "and", "POPC", "LUVs", "show", "no", "secondary", "structure", "10", ",", "11", ",", "30", "Our", "simulations", "with", "BP100", "in", "solution,", "and", "DPPC", "membrane", "exhibited", "the", "same", "pattern.", "α-BP100", "and", "L-BP100", "in", "solution", "showed", "similar", "behavior,", "with", "a", "prevalence", "of", "random", "conformation", "(Fig.", "4", ").", "In", "a", "related", "MD", "study", "of", "AMPs", "interacting", "with", "membranes", "17", ",", "an", "8", "µs-long", "all-atom", "MD", "simulation", "of", "BP100", "in", "DMPC", "at", "35◽", "the", "authors", "reported", "a", "full", "helix", "length", "during", "1.5", "µs", "and", "a", "subsequent", "unfolding", "event", "of", "Lysines", "at", "positions", "1", "and", "2,", "resulting", "in", "81%", "of", "helicity", "on", "a", "pure", "zwitterionic", "bilayer.", "However,", "as", "reported", "for", "other", "simulated", "peptides", "in", "the", "same", "work,", "the", "forcefield", "used", "could", "result", "in", "an", "over-helical", "artifact", "17", ".", "A", "Brownian", "Dynamics", "simulation", "study", "of", "BP100", "confined", "inside", "an", "implicit", "POPC", "membrane", "reports", "a", "stable", "alpha-helix", "18", ".", "However,", "no", "experimental", "data", "confirms", "that", "BP100", "diffuses", "freely", "in", "the", "membrane", "hydrophobic", "space", "and", "no", "secondary", "structure", "formation", "is", "reported", "for", "pure", "POPC", "vesicles", "11", ".", "In", "our", "simulations", "of", "α-BP100", "and", "L-BP100", "in", "DPPC", "membrane,", "the", "peptide", "quickly", "approached", "the", "membrane", "and", "remained", "on", "the", "surface", "during", "all", "simulation.", "Peptide-lipid", "pair", "analysis", "(Fig.", "S7", ")", "showed", "that", "cationic", "Lysine", "residues", "interacted", "mainly", "with", "phosphate", "groups", "of", "the", "DPPC", "bilayer,", "evidencing", "that", "electrostatic", "interactions", "were", "present.", "In", "the", "case", "of", "L-BP100", "in", "DPPC,", "no", "alpha-helical", "structure", "formation", "was", "observed", "(Fig.", "S3", "),", "and", "for", "α-BP100,", "the", "peptide", "alpha-helix", "conformation", "was", "mostly", "lost,", "although", "one", "helix", "turn", "from", "residues", "5", "to", "9", "remained", "(Fig.", "5A", ").", "The", "minimum", "alpha-helix", "length", "detected", "by", "CD", "spectroscopy", "is", "proposed", "to", "encompass", "seven", "to", "eleven", "residues", "38", ",", "hence", "the", "alpha-helix", "turn", "in", "residues", "5–9", "might", "not", "be", "detected", "by", "CD.", "Alternatively,", "this", "could", "be", "an", "over-helical", "forcefield", "artifact", "22", ",", "but", "it", "also", "indicates", "the", "high", "stability", "of", "the", "alpha-helix", "formed", "by", "the", "(5–9)", "Lys-Lys-Ile-Leu-Lys", "sequence.", "This", "stability", "leads", "us", "to", "conjecture", "that", "BP100", "alpha-helix", "formation", "could", "be", "initiated", "through", "a", "core", "sequence,", "a", "nascent", "helix", "39", ".", "The", "negatively", "charged", "membrane", "interface", "could", "promote", "the", "initial", "peptide", "folding.", "To", "support", "this,", "we", "performed", "three", "600", "ns-long", "simulations", "with", "constrained", "alpha", "helices", "of", "BP100", "on", "three", "regions:", "residues", "1", "to", "5", "(Lys-Lys-Leu-Phe-Lys),", "4", "to", "8", "(Phe-Lys-Lys-Ile-Leu)", "and", "7", "to", "11", "(Ile-Leu-Lys-Tyr-Leu)", "in", "pure", "DPPG", "bilayers.", "The", "secondary", "structure", "analysis", "of", "the", "constrained", "BP100", "simulations", "yielded", "different", "outcomes.", "Constraint", "on", "residues", "1", "to", "5", "rapidly", "induced", "almost", "full", "alpha-helix", "content", "from", "Lys2", "to", "Lys9", "(Fig.", "6A", "),", "reaching", "73%", "of", "helicity,", "similar", "to", "α-BP100", "in", "DPPG", "without", "constraints", "(Fig.", "5B", ").", "Constraint", "on", "residues", "4", "to", "8", "also", "showed", "a", "helix", "formation", "tendency", "(overall", "63%", "helicity,", "5", "out", "of", "11", "residues,", "from", "Phe4", "to", "Lys9).", "In", "sharp", "contrast,", "constraints", "on", "residues", "7", "to", "11", "(36%", "of", "helicity,", "4", "out", "of", "11", "residues,", "from", "Lys7", "to", "Tyr10)", "had", "no", "effects", "on", "the", "secondary", "structure", "formation", "of", "BP100", "during", "600", "ns", "simulation", "(Fig.", "6C", "),", "indicating", "that", "this", "region", "has", "little", "influence", "on", "initial", "peptide", "folding.", "Pace", "and", "Scholtz", "40", ",", "based", "on", "experimental", "studies,", "proposed", "a", "helix", "propensity", "scale", "as", "Δ(ΔG)", "values", "relative", "to", "Alanine", "set", "to", "zero.", "On", "the", "Pace", "and", "Scholtz", "scale,", "the", "5–9", "region", "has", "the", "highest", "helix", "promoting", "residues", "(overall", "helix", "propensity", "of", "1.4", "kcal/mol).", "We", "find", "1.53", "kcal/mol", "for", "residues", "1", "to", "5", "(Lys-Lys-Leu-Phe-Lys),", "1.68", "kcal/mol", "for", "residues", "4", "to", "8", "(Phe-Lys-Lys-Ile-Leu),", "and", "1.62", "kcal/mol", "for", "residues", "7–11", "(Ile-Leu-Lys-Tyr-Leu).", "Our", "secondary", "structure", "data", "indicates", "that", "BP100", "nascent", "helix", "could", "be", "formed", "either", "in", "the", "region", "between", "residues", "1", "to", "5", "or", "between", "residues", "5", "to", "9.", "We", "favor", "the", "latter", "due", "to", "the", "presence", "of", "positively", "charged", "Lysine", "residues", "in", "the", "N-terminal", "side", "(Lys1", "and", "Lys2)", "which", "could", "produce", "an", "energetically", "unfavorable", "barrier", "to", "initial", "peptide", "folding.", "The", "5–9", "region", "shows", "high", "stability", "in", "all", "constraint-free", "α-BP100/membranes", "simulations", "and", "has", "the", "overall", "lowest", "energy", "barrier", "in", "the", "helix", "propensity", "scale", "40", ".", "DPPG", "seems", "to", "have", "a", "helix-stabilizing", "effect", "on", "BP100.", "All", "the", "simulations", "with", "α-BP100", "in", "DPPG-containing-membranes", "showed", "high", "alpha-helical", "content,", "including", "the", "region", "of", "residues", "5", "to", "9.", "Our", "helicity", "results", "from", "the", "simulations", "with", "α-BP100", "in", "pure", "DPPG", "(73%)", "and", "in", "PCPG", "(50:50)", "mixed", "bilayers", "(54%)", "are", "remarkably", "close", "to", "those", "reported", "in", "experimental", "studies:", "54%", "in", "POPC:POPG", "(50:50)", "11", "and", "61%", "in", "DMPC/DMPG", "(75:25)", "LUVs", "10", ",", "and", "maximum", "observable", "alpha-helix", "spectrum", "for", "BP100", "in", "pure", "POPG", "vesicles", "11", "proving", "that", "BP100", "alpha-helical", "structure", "is", "dependent", "on", "PG", "content", "11", ".", "For", "L-BP100", "simulations", "with", "DPPG,", "PCPG-R,", "and", "PCPG-NR,", "no", "secondary", "structure", "formation", "was", "detected", "(Fig.", "S3", "),", "with", "the", "peptide", "being", "in", "a", "random", "conformation.", "Non-biased", "random", "peptide", "simulations", "require", "much", "longer", "simulation", "time", "to", "sample", "the", "peptide", "conformational", "potential", "landscape", "or", "might", "dwell", "in", "several", "local", "minimum-energy", "states.", "Peptide", "flip", "BP100", "is", "too", "short", "to", "span", "the", "bilayer", "thickness,", "especially", "in", "the", "alpha-helical", "conformation,", "and", "thus", "single", "molecule", "pore", "formation", "is", "an", "unlikely", "scenario.", "NMR", "data", "of", "BP100", "in", "DMPC:", "DMPG", "(3:1)", "10", "shows", "that", "upon", "binding", "to", "negatively", "charged", "membranes,", "alpha-helical", "BP100", "positions", "parallel", "to", "the", "membrane", "as", "in", "a", "surface-bound", "orientation,", "and", "has", "an", "azimuthal", "rotation", "angle", "close", "to", "160°,", "with", "Lysine", "residues", "facing", "the", "solvent.", "This", "last", "arrangement", "is", "commonly", "observed", "with", "other", "CAMPs", "30", "–", "33", ".", "Peptide/membrane", "simulations", "with", "initially", "alpha-helical", "BP100", "had", "its", "polar", "moiety", "facing", "the", "membrane,", "for", "being", "the", "most", "probable", "scenario", "in", "a", "physical", "environment.", "In", "the", "α-BP100", "in", "DPPG", "(Fig.", "7", "),", "α-BP100", "in", "DPPC,", "α-BP100", "in", "PCPG-R", "and", "α-BP100", "(res1–5)", "in", "DPPG", "simulations", "trajectories,", "we", "observed", "the", "binding", "of", "BP100", "from", "bulk", "solution", "to", "the", "membranes", "with", "its", "Lysines", "residues", "pointing", "towards", "the", "bilayer,", "and", "then,", "the", "peptide", "rotated,", "leaving", "its", "apolar", "facet", "facing", "the", "interior", "of", "the", "bilayer.", "We", "termed", "this", "transition", "peptide", "flip.", "Although", "BP100", "surface-bound", "state", "is", "reported", "12", ",", "17", ",", "no", "previous", "peptide/membrane", "MD", "simulation", "work", "has", "reported", "a", "peptide", "flip", "transition.", "Peptide", "flip", "brings", "significant", "outcomes", "regarding", "BP100", "localization", "in", "the", "membrane,", "in", "peptide/membrane", "interaction", "and", "the", "peptide", "hydration,", "possibly", "being", "a", "crucial", "step", "of", "carpet", "mechanism", "for", "BP100", "and", "other", "CAMPs.", "Through", "the", "density", "profile,", "we", "observed", "deep", "penetration", "of", "the", "peptide", "into", "the", "membrane", "through", "its", "apolar", "residues", "with", "larger", "side-chains,", "after", "the", "flip", "(Fig.", "7", ").", "BP100", "hydration", "analysis", "also", "shows", "a", "decrease", "in", "the", "overall", "peptide", "hydration", "after", "the", "flip", "(Fig.", "8", ").", "Apolar/polar", "pair", "interaction", "analysis", "(Fig.", "11", ")", "revealed", "that", "after", "the", "flip,", "the", "number", "of", "contacts", "between", "apolar", "residues", "from", "the", "lipids", "and", "the", "peptide", "increases", "significantly,", "while", "the", "number", "of", "contacts", "between", "polar", "groups", "of", "the", "lipids", "and", "the", "peptides", "slightly", "decreases.", "In", "other", "words,", "peptide", "flip", "intensifies", "peptide", "binding", "to", "membranes", "burying", "the", "apolar", "residues", "of", "the", "peptide", "deeper", "into", "the", "membrane", "core.", "We", "investigated", "what", "factors", "could", "lead", "to", "the", "occurrence", "of", "BP100", "peptide", "flip.", "Membrane", "composition", "appears", "to", "have", "a", "significant", "role.", "For", "BP100", "alpha-helical", "structure", "formation", "is", "favored", "in", "negatively", "charged", "membranes", "8", ".", "In", "our", "simulations,", "BP100", "entirely", "flipped", "in", "DPPG", "enriched", "regions", "of", "the", "membrane.", "Peptide", "flip", "was", "detected", "in", "α-BP100", "in", "DPPG,", "α-BP100", "(res1–5)", "in", "DPPG", "and", "α-BP100", "in", "PCPG-R", "systems.", "No", "peptide", "flip", "was", "detected", "in", "the", "other", "constrained", "BP100", "simulations", "(α-BP100", "(res4–8)", "in", "DPPG", "and", "α-BP100", "(res7–11)", "in", "DPPG).", "However,", "it", "is", "hasty", "to", "affirm", "that", "it", "is", "unlikely", "to", "expect", "peptide", "flip", "to", "happen", "in", "those", "systems", "with", "600", "ns", "of", "simulation", "time.", "For", "instance,", "peptide", "flip", "was", "observed", "after", "1.4", "µs", "of", "simulation", "in", "α-BP100", "in", "DPPG.", "Hydration", "analysis", "(Fig.", "8b", ")", "and", "density", "profile", "(Fig.", "S4A,B", ")", "for", "α-BP100", "in", "DPPC", "show", "also", "that", "BP100", "flipped.", "However,", "the", "alpha-helix", "secondary", "structure", "is", "mostly", "lost", "(Fig.", "5a", ").", "For", "this", "reason,", "BP100", "amphiphilic", "apolar-polar", "facet", "separation", "(Fig.", "2c", ")", "is", "lost.", "Additionally,", "the", "apolar", "moiety", "of", "the", "peptide", "could", "have", "a", "crucial", "role", "in", "peptide", "flip.", "Hydration", "analysis", "(Fig.", "8", ")", "suggests", "peptide", "flip", "results", "in", "peptide", "dehydration", "and", "concomitantly", "enhances", "peptide/lipid", "interaction", "(Fig.", "11", "),", "increasing", "specially", "apolar", "contacts", "(Fig.", "11", ").", "Similar", "behavior", "was", "observed", "in", "a", "study", "of", "the", "interaction", "of", "micelles", "with", "polyatomic", "counterions", "with", "apolar", "and", "polar", "moieties.", "Experimental", "results", "and", "MD", "simulations", "showed", "that", "counterions", "with", "the", "same", "apolar", "groups", "but", "different", "polar", "groups", "resulted", "in", "similar", "ionic", "affinities", "for", "dodecyltrimethylammonium", "micelles,", "and", "the", "simulations", "showed", "that", "counterion", "adsorption", "to", "the", "micelles", "was", "related", "with", "the", "dehydration", "of", "the", "apolar", "group", "of", "the", "counterions", "rather", "than", "by", "the", "polar", "group", "41", ".", "These", "factors", "could", "help", "to", "explain", "BP100", "selectivity", "towards", "negatively", "charged", "bacterial", "membranes", "and", "low", "cytotoxicity.", "Results", "of", "leakage", "from", "the", "inner", "compartment", "of", "LUVs", "upon", "addition", "of", "BP100", "showed", "that", "BP100", "leakage", "ability", "is", "proportional", "to", "PG", "content", "while", "the", "peptide", "has", "small", "effects", "on", "zwitterionic", "PC", "vesicles,", "observed", "through", "studies", "of", "zeta", "potential", "and", "vesicle", "leakage", "11", ".", "BP100", "does", "not", "perturb", "PC", "bilayers", "even", "at", "high", "peptide/lipid", "ratio", "and", "causes", "membrane", "thinning", "on", "mixed", "DMPC/DMPG", "bilayers", "where", "a", "stronger", "effect", "appears", "in", "anionic", "DMPG", "lipids", "compared", "to", "zwitterionic", "DMPC", "lipids", "12", ".", "In", "zwitterionic-lipid-enriched", "mammalian", "membranes,", "BP100", "would", "bind", "to", "membranes", "through", "electrostatic", "interactions", "and", "the", "absence", "of", "a", "helix-promoting", "environment", "would", "prevent", "helix", "formation", "and", "even", "with", "peptide", "flip,", "as", "in", "our", "α-BP100", "in", "DPPC", "simulation,", "it", "could", "lead", "to", "mild", "membrane", "thinning", "11", ",", "12", ".", "On", "the", "other", "hand,", "bacterial", "membranes", "with", "high", "content", "of", "anionic", "lipids", "on", "the", "outer", "part", "of", "the", "membrane,", "could", "promote", "peptide", "binding,", "alpha-helix", "nucleation,", "and", "propagation,", "leading", "to", "peptide", "flip", "and", "more", "pronounced", "membrane", "thinning,", "which", "is", "consistent", "with", "our", "observations", "from", "α-BP100", "in", "DPPG,", "α-BP100", "in", "PCPG-R", "and", "α-BP100", "(res1–5)", "in", "DPPG", "sets.", "Although", "we", "could", "not", "detect", "membrane", "thinning", "in", "our", "simulations", "(Table", "S2", "),", "it", "should", "be", "noted", "that", "our", "simulations", "had", "only", "one", "peptide", "on", "128-lipid-bilayers.", "A", "more", "pronounced", "effect", "on", "the", "membrane", "might", "be", "observed", "by", "increasing", "the", "number", "of", "peptides", "in", "the", "simulations.", "DPPG", "subdomain", "formation", "Lipid", "clustering,", "or", "subdomain", "formation,", "induced", "by", "CAMPs", "is", "well", "established", "5", ",", "14", ",", "35", "–", "37", ",", "42", "–", "45", ".", "Through", "electrostatic", "interactions,", "CAMPs", "clusters", "anionic", "phosphatidylglycerol", "(PG)", "or", "cardiolipin", "(CL)", "lipids", "on", "the", "outer", "membrane", "of", "bacteria,", "inducing", "phase", "separation", "and", "phase", "boundary", "defects", "between", "lipid", "domains", "and", "the", "rest", "of", "the", "membrane,", "increasing", "membrane", "permeability", "36", ".", "Epand", "et", "al.", "first", "proposed", "it", "45", ",", "in", "a", "study", "with", "oligomers", "of", "lysine", "(OAK).", "Upon", "the", "addition", "of", "OAK", "in", "1-palmitoyl-2-oleoyl", "phosphatidylethanolamine", "(POPE)", "and", "tetraoleoyl", "cardiolipin", "(TOCL)", "mixed", "vesicles,", "transition", "temperature", "shift", "was", "observed", "through", "differential", "scanning", "calorimetry,", "indicating", "lipid", "phase", "separation", "45", ".", "Lipid", "phase", "separation", "was", "later", "observed", "for", "other", "cAMPs", "and", "cell-penetrating-peptides", "37", ",", "45", ",", "46", ".", "Although", "not", "so", "pronounced,", "DSC", "also", "detected", "BP100", "ability", "to", "promote", "lipid", "phase", "separation", "on", "POPE/TOCL", "(75:25)", "vesicles", "37", ".", "Lipid", "phase", "separation", "promoted", "by", "CAMPs", "has", "also", "been", "observed", "in", "computational", "modeling", "studies.", "In", "a", "coarse-grained", "MD", "simulation", "study", "35", ",", "lipid", "cluster", "promotion", "was", "seen", "in", "POPE/POPG", "(70:30)", "membranes", "by", "Ltc1,", "a", "linear", "alpha-helical", "cAMP", "with", "overall", "+10", "charge.", "Simulations", "showed", "Ltc1", "induced", "POPG", "domains", "in", "small", "(256", "lipids)", "and", "large", "membranes", "(512", "or", "2048", "lipids).", "Also,", "due", "to", "peptide-POPG", "charge", "neutralization,", "Ltc1-induced", "anionic", "domains", "seem", "to", "facilitate", "peptide", "oligomerization,", "enhancing", "peptide", "activity", "on", "bacterial", "model", "membranes", "35", ".", "Likewise,", "our", "simulations", "suggest", "that", "BP100", "promotes", "PG", "aggregation.", "Two", "DPPC:", "DPPG", "(50:50)", "mixed", "bilayers", "were", "designed", "to", "explore", "lipid", "aggregation", "by", "the", "peptide.", "One", "membrane", "was", "composed", "of", "randomly", "mixed", "(PCPG-NR)", "and", "in", "the", "other,", "a", "4", "×", "4", "DPPG", "raft", "(PCPG-R)", "was", "positioned", "in", "the", "middle", "of", "both", "monolayers", "(see", "Methods", "1", "and", "Fig.", "1", ").", "BP100", "was", "able", "to", "cluster", "DPPGs", "in", "PCPG-NR,", "which", "can", "be", "observed", "by", "the", "amount", "of", "DPPG", "in", "contact", "with", "BP100", "(Table", "2", ").", "From", "a", "total", "of", "11", "lipids", "in", "the", "closer", "contact", "with", "BP100,", "8.3", "in", "average", "are", "DPPG", "corresponding", "to", "75%", "of", "the", "lipid", "in", "the", "vicinity", "of", "the", "peptide.", "Additionally,", "this", "DPPG", "clustering", "can", "also", "be", "observed", "in", "lipid", "raft", "size", "distribution", "(Fig.", "S6E", "),", "reaching", "similar", "values", "of", "DPPG/peptide", "contact", "in", "α-BP100", "in", "PCPG-R", "simulation", "(Fig.", "10", ")", "and", "DPPG", "lipid", "raft", "size", "probability", "(Fig.", "S6D", ").", "These", "data", "suggest", "that", "even", "a", "short-lengthened", "CAMP", "as", "BP100", "can", "promote", "anionic", "lipid", "clustering", "and", "also", "maintain", "it,", "supporting", "the", "experimental", "findings", "37", ".", "BP100", "also", "adsorbs", "on", "pure", "zwitterionic", "PC", "membranes", "and", "promotes", "DPPC", "clustering", "as", "observed", "in", "Figs", "S5", "A", "and", "S6A", ".", "However,", "possibly", "due", "to", "the", "presence", "of", "positively", "charged", "choline", "headgroups", "(Fig.", "1d", "-", "left),", "secondary", "structure", "is", "lost", "(Fig.", "5a", ")", "and", "the", "peptide", "turns", "into", "random", "coil,", "resulting", "in", "an", "increase", "of", "available", "interaction", "surface", "area", "with", "the", "DPPC", "headgroups,", "which", "is", "reflected", "in", "a", "broad", "distribution", "in", "lipid", "raft", "size", "distribution", "(Fig.", "S5A", ").", "Polar/Apolar", "pair", "analysis", "for", "α-BP100", "in", "PCPG-R", "(Fig.", "11", ")", "helps", "to", "explain", "lipid", "aggregation.", "We", "computed", "the", "ten", "most", "frequent", "apolar", "and", "polar", "pairs", "between", "peptide", "and", "lipids.", "The", "number", "of", "polar", "pairs", "between", "the", "peptide", "and", "DPPG", "is", "higher", "than", "those", "with", "DPPC", "and", "is", "maintained", "even", "after", "the", "flip", "(~390", "ns).", "As", "expected,", "+1", "charged", "amino", "group", "(NZ)", "from", "Lysines", "side", "chains", "are", "the", "main", "participants", "in", "polar", "pairings", "(Figs", "S7", "–", "S13", "),", "interacting", "with", "DPPG", "headgroup", "glycerol", "(OC2,", "OC3,", "Fig.", "1d", "-", "right),", "phosphate", "group", "(P),", "and", "carbonyl", "groups", "(C21,", "C31,", "O21,", "O22,", "O31,", "O32)", "from", "chains", "sn1", "and", "sn2", "(Figs", "S9", ",", "S10", ",", "S12", ",", "S13", ").", "Peptide", "flip", "seems", "to", "contribute", "to", "lipid", "clustering.", "Polar/apolar", "pairs", "analysis", "(Fig.", "11", ")", "and", "pair", "frequency", "(Figs", "S8", ",", "S9", ")", "for", "the", "previous", "simulation,", "reveals", "that", "apolar", "pairs", "between", "BP100", "and", "both", "lipids", "rise", "after", "peptide", "flip", "occurs", "(~390", "ns),", "intensifying", "peptide-membrane", "interaction.", "The", "same", "phenomena", "were", "observed", "for", "other", "simulations", "with", "peptide", "flip", "(Figs", "S7", ",", "S10", "and", "S13", ").", "As", "long", "as", "peptide", "flip", "had", "occurred,", "apolar", "residue", "side", "chains", "(from", "Leu3,", "Phe4,", "Ile7,", "Leu8", "and", "Tyr10,", "and", "Leu11)", "were", "in", "close", "contact", "mainly", "with", "groups", "of", "the", "lipid", "tails", "(Figs", "S7", "–", "S10", ",", "S13", ").", "The", "mechanisms", "of", "bacterial", "toxicity", "via", "lipid", "clustering", "induced", "by", "CAMPs", "still", "has", "to", "be", "investigated.", "Moreover,", "possibly", "more", "than", "one", "mechanism", "could", "be", "involved", "in", "the", "process", "of", "bacterial", "killing.", "Evidence", "suggest", "lipid", "clustering", "by", "CAMPs", "could", "lead", "to", "negative", "charge", "concentration", "on", "membranes,", "promoting", "CAMPs", "attachment,", "leading", "to", "pore", "formation", "5", ".", "Anionic", "lipid", "clustering", "would", "have", "surrounding", "interfaces", "with", "the", "rest", "of", "the", "membrane", "which", "could", "lead", "to", "phase", "boundary", "defects,", "leading", "to", "slow", "leakage", "of", "inner", "cell", "content", "32", ".", "Also,", "CAMPs", "and", "cell-penetrating", "peptides", "could", "induce", "negative", "membrane", "curvature,", "enabling", "the", "internalization", "of", "peptides", "into", "cells,", "leading", "to", "intracellular", "killing", "mechanisms.", "The", "formation", "of", "lipid", "clusters", "patches", "would", "also", "lead", "to", "anomalies", "in", "membrane", "protein", "function,", "membrane", "fluidity,", "and", "membrane", "polarization.", "In", "a", "recent", "study", "5", "with", "cWFW,", "a", "cyclic", "antimicrobial", "peptide,", "it", "was", "shown", "in", "vitro", "and", "in", "vivo", "that", "cWFW", "promotes", "lipid", "phase", "separation,", "a", "sharp", "decrease", "in", "membrane", "fluidity.", "The", "significant", "phase", "separation", "appears", "to", "cause", "disorganization", "of", "membrane", "proteins,", "inhibiting", "cell", "wall", "synthesis", "and", "inducing", "autolysis.", "BP100", "mode", "of", "action", "on", "membranes", "is", "dependent", "on", "peptide/lipid", "ratio", "11", ",", "47", ".", "As", "our", "studies", "comprised", "of", "low", "peptide/lipid", "concentration", "(1/128),", "we", "suggest", "that", "at", "low", "peptide/lipid", "concentrations,", "BP100", "causes", "lipid", "aggregation", "on", "negatively", "charged", "membranes,", "leading", "to", "phase", "boundary", "defects", "between", "negatively", "charged", "lipid", "rafts", "and", "the", "rest", "of", "the", "membrane.", "Through", "this,", "membrane", "permeability", "would", "increase,", "leading", "to", "slow", "leakage", "of", "the", "cell", "inner", "content." ]

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{"negatives/white": ["144+1", "1172+1", "1993+1", "2830+1", "3917+1", "4922+1", "5674+1", "6385+1", "6565+1", "6525+1", "6725+1"]}

CC-BY

4.0

http://creativecommons.org/licenses/by/4.0/

Original work titled: 'Binding and Flip as Initial Steps for BP-100 Antimicrobial Actions' (Scientific Reports/Nature Publishing Group UK) by the following authors: Peter Park; Leandro R. Franco; Hernan Chaimovich; Kaline Coutinho; Iolanda M. Cuccovia; Filipe S. Lima DOI: 10.1038/s41598-019-45075-5

f7083ca86a7871cc8d04d2500480cbf3

[ "Introduction", "Colorectal", "cancer", "(CRC)", "is", "the", "third", "most", "common", "malignant", "tumor", "worldwide", "[", "1", "].", "With", "the", "development", "of", "early", "diagnosis", "methods", "and", "molecular", "targeted", "therapy,", "the", "progression", "free", "survival", "(PFS)", "and", "the", "overall", "survival", "(OS)", "of", "patients", "with", "CRC", "have", "increased", "[", "2", "].", "However,", "metastatic", "colorectal", "cancer", "(mCRC)", "is", "associated", "with", "a", "poor", "prognosis", "[", "3", ",", "4", "].", "Chemotherapy", "is", "a", "strongly", "recommended", "therapeutic", "regimen", "to", "prevent", "and", "control", "mCRC,", "and", "it", "has", "been", "associated", "with", "a", "prolonged", "life", "[", "5", ",", "6", "].", "Adjuvant", "chemotherapy", "may", "confer", "a", "survival", "advantage", "in", "resected", "CRC", "patients", "with", "stage", "III", "or", "high-risk", "stage", "II", "disease", "[", "7", ",", "8", "].", "Cytotoxic", "chemotherapeutic", "agents", "inhibit", "the", "proliferation", "of", "cancer", "cells,", "but,", "at", "the", "same", "time,", "they", "produce", "toxicity", "and", "side", "effects", "on", "other", "tissues", "and", "organs.", "How", "to", "effectively", "and", "efficiently", "avoid", "or", "to", "alleviate", "the", "toxicity", "and", "side", "effects", "is", "a", "critical", "clinical", "problem", "that", "should", "be", "solved.", "CID", "is", "a", "common", "side", "effect", "of", "the", "digestive", "system", "in", "the", "antitumor", "treatment", "process", "of", "cytotoxic", "drugs.", "The", "typical", "clinical", "characteristics", "of", "CID", "are", "as", "follows:", "symptoms", "range", "from", "loose", "stool", "without", "pain", "to", "severe,", "watery", "diarrhea", "with", "abdominal", "pain;", "symptoms", "typically", "last", "5", "to", "7", "days", "and", "may", "occur", "during", "or", "after", "chemotherapy;", "and", "patients", "with", "CID", "have", "a", "poor", "treatment", "response", "to", "gentamicin,", "berberine,", "and", "furoxone", "[", "9", "].", "CID", "can", "lead", "to", "weakness,", "electrolyte", "disorders,", "renal", "failure,", "blood", "volume", "reductions,", "shock,", "and", "even", "death", "[", "10", "].", "Its", "occurrence", "can", "result", "in", "delaying", "chemotherapy,", "increasing", "hospitalization", "time", "and", "costs,", "aggravating", "the", "psychological", "stress", "of", "patients,", "reducing", "the", "treatment", "compliance", "of", "patients", "and", "even", "altering", "the", "entire", "chemotherapy", "plan", "[", "11", "].", "A", "unique", "microecosystem", "and", "characteristic", "microbial", "community", "lives", "in", "the", "colorectal", "regions", "owing", "to", "their", "function", "of", "storing", "feces", "[", "12", "].", "Accumulating", "evidence", "points", "to", "the", "gut", "microbiome", "being", "involved", "in", "CRC", "[", "13", "].", "For", "example,", "some", "researchers", "have", "reported", "that", "Streptococcus", "bovis", "[", "14", "]", "and", "Streptococcus", "gallolyticus", "[", "15", "]", "are", "specific", "bacteria", "involved", "in", "colorectal", "cancer;", "Castellarin", "et", "al.", "[", "16", "]", "found", "that", "Fusobacterium", "nucleatum", "infection", "is", "prevalent", "in", "CRC", "tissue", "specimens", "and", "Kostic", "et", "al.", "[", "17", "],", "found", "that", "Fusobacterium", "nucleatum", "can", "generate", "a", "proinflammatory", "microenvironment", "that", "is", "conducive", "to", "the", "progression", "of", "CRC", "through", "recruitment", "of", "tumor-infiltrating", "immune", "cells.", "Moreover,", "many", "enzymes,", "peptides", "and", "small", "molecules", "secreted", "by", "the", "intestinal", "gut", "microbiome", "are", "involved", "in", "activating", "and", "regulating", "important", "signaling", "molecules", "and", "signaling", "pathways", "involved", "in", "the", "progression", "of", "CRC", "[", "18", ",", "19", "].", "Diarrhea", "and", "gut", "microbiome", "disorders", "interact", "as", "both", "cause", "and", "effect.", "Diarrhea", "can", "disrupt", "the", "balance", "of", "the", "gut", "microenvironment.", "Meanwhile,", "the", "invasion", "of", "exogenous", "pathogenic", "microorganisms", "and", "the", "imbalance", "of", "intestinal", "microbes", "can", "lead", "to", "diarrhea", "[", "20", "].", "For", "example,", "the", "invasion", "of", "some", "pathogenic", "bacteria", "including", "Shigella,", "Salmonella", "and", "Klebsiella", "[", "21", ",", "22", "],", "and", "the", "abundance", "changes", "of", "intestinal", "parasitic", "microbes", "include", "Candida", "albicans,", "Escherichia", "coli,", "and", "Aeromonas", "can", "induce", "diarrhea", "[", "23", "–", "25", "].", "Chemotherapy", "has", "been", "widely", "used", "in", "the", "clinic", "as", "an", "effective", "therapy", "for", "colorectal", "cancer,", "but", "diarrhea", "caused", "by", "chemotherapeutic", "drugs", "often", "affects", "the", "implementation", "of", "chemotherapy", "regimens", "[", "26", "].", "Many", "probiotics", "and", "antibiotics", "play", "a", "positive", "role", "in", "the", "treatment", "of", "infectious", "diarrhea", "by", "regulating", "the", "gut", "microbiome", "[", "27", ",", "28", "].", "The", "study", "of", "relationships", "between", "gut", "microbiome", "and", "CID", "may", "provide", "a", "new", "direction", "for", "solving", "this", "clinical", "problem.", "In", "the", "present", "study,", "we", "aimed", "to", "explore", "the", "association", "between", "the", "gut", "microorganisms", "and", "CID", "of", "the", "CapeOX", "regimen", "in", "resected", "stage", "III", "CRC", "and", "to", "provide", "some", "research", "methods", "and", "research", "ideas", "for", "further", "exploring", "the", "relationship", "between", "intestinal", "microbes", "and", "CID.", "The", "results", "may", "provide", "a", "fresh", "approach", "to", "the", "prevention", "and", "treatment", "of", "CID", "from", "a", "microbiological", "perspective.", "Subjects", "and", "methods", "Subjects", "Patients", "with", "CRC", "treated", "at", "Huzhou", "Central", "Hospital", "from", "January", "2016", "to", "January", "2018", "were", "studied.", "The", "clinical", "protocols", "involving", "the", "patients", "and", "the", "informed", "consent", "form", "were", "approved", "by", "the", "Ethics", "Committee", "of", "Huzhou", "Central", "Hospital", "(No.", "201601023).", "According", "to", "the", "American", "Joint", "Committee", "on", "Cancer", "(AJCC)", "staging", "manual", "system,", "stage", "III", "CRC", "is", "defined", "as", "when", "colorectal", "cancer", "is", "diagnosed", "by", "pathology", "with", "lymphatic", "metastasis", "and", "without", "distant", "metastasis", "[", "29", "].", "The", "inclusion", "criteria", "were", "as", "follows.", "①", "Cancer", "was", "confirmed", "by", "pathologic", "diagnosis,", "and", "the", "patients", "volunteered", "to", "participate", "in", "the", "study.", "②", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "③", "Patients", "voluntarily", "accepted", "and", "completed", "8", "cycles", "of", "the", "CapeOX", "regimen", "[capecitabine", "(1000", "mg/m", "2", "twice", "daily)", "combined", "with", "oxaliplatin", "(130", "mg/m", "2", "every", "3", "weeks)].", "The", "exclusion", "criteria", "were", "as", "follows.", "①", "Patients", "with", "diarrhea", "before", "chemotherapy.", "②", "Patients", "with", "other", "intestinal", "diseases,", "such", "as", "ulcerative", "colitis", "and", "Crohn’s", "disease.", "③", "Alternation", "of", "chemotherapy", "regimen", "and", "chemotherapeutic", "dosage", "or", "the", "occurrence", "of", "progression", "of", "the", "disease", "(PD)", "during", "the", "8", "cycles", "of", "the", "CapeOX", "regimen.", "④", "Patients", "who", "accepted", "neo-adjuvant", "chemotherapy.", "⑤", "Patients", "with", "a", "history", "of", "the", "use", "of", "oral", "microbial", "agents", "within", "1", "month", "before", "chemotherapy.", "⑥", "Patients", "with", "another", "primary", "cancer.", "⑦", "Patients", "with", "known", "primary", "organ", "failure.", "⑧", "Patients", "who", "could", "not", "obtained", "the", "stool", "samples", "(n", "=", "15)", "or", "obtained", "unqualified", "stool", "samples.", "All", "of", "the", "subjects", "signed", "informed", "consent", "under", "the", "guidelines", "approved", "by", "the", "Ethics", "Committee", "of", "Huzhou", "Central", "Hospital.", "CRC", "patients", "with", "and", "without", "CID", "were", "including", "in", "the", "experimental", "group", "and", "control", "group,", "respectively.", "According", "to", "National", "Cancer", "Institute", "Common", "Toxicity", "Criteria", "(NCI", "CTC)", "V3.0", "[", "30", "]", "CID", "grade", "1", "is", "defined", "as", "the", "number", "of", "defecations", "increased", "to", "less", "than", "4", "times", "a", "day", "and", "an", "excretion", "volume", "slightly", "increased.", "During", "a", "course", "of", "8", "cycles", "of", "chemotherapy", "administered", "over", "nearly", "half", "a", "year,", "there", "are", "many", "factors", "that", "can", "cause", "diarrhea,", "and", "these", "factors", "interfere", "with", "the", "diagnosis", "of", "CID.", "Clinical", "intervention", "measures,", "such", "as", "using", "antibiotics", "and", "antidiarrheal", "drugs,", "may", "be", "performed", "and", "the", "chemotherapy", "regimen", "may", "be", "changed", "in", "patients", "with", "CID", "grade", "3", "and", "grade", "4.", "Therefore,", "we", "did", "not", "include", "patients", "with", "grade", "1,", "grade", "3", "and", "grade", "4", "in", "the", "experimental", "group.", "CID", "grade", "2", "is", "defined", "as", "the", "number", "of", "defecations", "increased", "to", "4–6", "times", "per", "day", "or", "nocturnal", "stools,", "with", "the", "amount", "of", "excretion", "increased", "but", "with", "no", "interference", "in", "daily", "life.", "All", "of", "the", "subjects", "recruited", "into", "the", "experimental", "group", "were", "CID", "grade", "2", "in", "the", "present", "study.", "Collection", "of", "clinical", "data", "and", "stool", "samples", "Basic", "information", "and", "clinical", "serological", "indicators", "were", "obtained", "from", "the", "medical", "record", "management", "system", "of", "Huzhou", "Central", "Hospital", "with", "informed", "consent", "from", "patients.", "Stool", "samples", "of", "the", "patients", "were", "collected", "in", "the", "2", "weeks", "after", "the", "8", "cycles", "of", "chemotherapy.", "Stool", "samples", "were", "collected", "in", "the", "morning", "prior", "to", "breakfast.", "An", "approximately", "5–10", "g", "stool", "sample", "was", "collected", "after", "defecation", "without", "the", "use", "of", "a", "purgative", "or", "lubricant.", "Within", "half", "an", "hour,", "the", "stool", "samples", "were", "stored", "in", "an", "ultra-low", "temperature", "freezer.", "The", "sample", "preservation", "time", "was", "not", "beyond", "1", "month.", "Finally,", "17", "stool", "samples", "from", "CRC", "patients", "were", "analyzed", "after", "the", "patients", "had", "signed", "informed", "consent", "forms", "and", "the", "unqualified", "specimens", "were", "eliminated.", "Intestinal", "microorganism", "detection", "DNA", "extraction", "and", "PCR", "amplification", "A", "E.Z.N.A.", "®", "Stool", "DNA", "Kit", "(Omega", "Bio-Tek,", "Norcross,", "GA,", "U.S.)", "was", "used", "to", "extract", "total", "DNA", "from", "the", "stool", "samples", "according", "to", "the", "manufacturer’s", "protocols.", "The", "nanodrop", "ND-1000", "spectrophotometer", "(LabTech,", "Washington,", "DC,", "USA)", "with", "the", "absorbances", "at", "260", "nm", "and", "280", "nm", "(A260/A280)", "was", "used", "to", "determine", "the", "quality", "and", "quantity", "of", "the", "purified", "DNA.", "DNA", "integrity", "was", "further", "verified", "by", "electrophoresis", "through", "a", "2.0%", "(w/v)", "agarose", "gel.", "PCR", "(95", "°C", "for", "3", "min,", "followed", "by", "25", "cycles", "at", "95", "°C", "for", "30", "s,", "55", "°C", "for", "30", "s,", "and", "72", "°C", "for", "45", "s", "and", "a", "final", "extension", "at", "72", "°C", "for", "5", "min)", "was", "used", "to", "amplify", "the", "V3–V4", "region", "of", "the", "bacterial", "16S", "ribosomal", "RNA", "gene", "(the", "primers", "of", "16S", "V3–V4", "rDNA", "are", "as", "follows:", "forward,", "CCTACGGGNGGCWGCAG", "and", "reverse,", "GACTACHVGGGTATCTAATCC).", "PCR", "amplifications", "were", "performed", "in", "triplicate", "in", "a", "25", "μL", "mixture", "containing", "5", "μL", "of", "DNA", "template,", "2", "μL", "of", "Nextera", "XT", "Index", "Primer", "1", "(10", "M),", "2", "μL", "of", "Nextera", "XT", "Index", "Primer", "2", "(10", "M)", "and", "16", "μL", "ddH", "2", "O.", "Amplicons", "were", "extracted", "from", "2%", "agarose", "gels,", "purified", "using", "the", "AxyPrep", "DNA", "Gel", "Extraction", "Kit", "(Axygen", "Biosciences,", "Union", "City,", "CA,", "USA)", "and", "quantified", "using", "QuantiFluor™-ST", "(Promega,", "U.S.)", "according", "to", "the", "manufacturer’s", "instructions.", "Library", "construction", "and", "sequencing", "The", "MiSeq", "library", "was", "constructed", "as", "follows:", "purified", "PCR", "products", "were", "quantified", "by", "Qubit", "®", "3.0", "(Life", "Technologies,", "Invitrogen).", "PCR", "products", "were", "ligated", "with", "Y", "adapter.", "Magnetic", "nanoparticles", "were", "used", "to", "take", "out", "the", "self-ligated", "Y", "adapters.", "An", "Illumina", "Pair-End", "library", "was", "constructed", "using", "pooled", "DNA", "products,", "and", "the", "amplicon", "library", "was", "pair-end", "sequenced", "(2", "×", "250)", "on", "an", "Illumina", "MiSeq", "platform", "(Shanghai", "BIOZERON", "Co.,", "Ltd.)", "according", "to", "standard", "protocols", "(MiSeq", "V3", "or", "V4", "sequencing", "kit,", "Hua", "Ya", "Regenerative", "Medicine", "Biological", "Engineering", "Technology", "CO.,", "LTD,", "Shanghai,", "China).", "Sequencing", "data", "bioinformatics", "analysis", "Sequencing", "data", "processing", "and", "optimization", "were", "performed", "according", "to", "the", "criteria", "at", "http://en.wikipedia.org/wiki/Fastq", ".", "Cutadapt", "(version", "1.11)", "was", "used", "to", "demultiplex", "and", "quality", "filter", "the", "raw", "fastq", "files.", "Pandaseq", "(version", "2.9)", "was", "used", "to", "assemble", "PE", "reads;", "only", "sequences", "that", "overlapped", "longer", "than", "10", "bp", "were", "assembled", "according", "to", "their", "overlap", "sequence.", "Mosaic", "sequences", "and", "sequences", "longer", "than", "300–480", "bp", "were", "discarded.", "In", "addition,", "the", "reads", "that", "receiving", "an", "average", "quality", "score", "<", "20", "were", "discarded.", "The", "Silva", "database", "was", "used", "for", "comparison", "of", "16S", "rRNA", "gene", "sequences", "[", "31", "]", "and", "the", "GeneBank", "database", "was", "used", "to", "analyze", "functional", "genes", "[", "32", "].", "Mothur", "software", "[", "33", "]", "(", "http://www.mothur.org/wiki/Main_Page", ")", "was", "used", "to", "analyze", "the", "microbial", "diversity.", "OTU", "(operational", "taxonomic", "units),", "as", "the", "artificial", "classification", "unit,", "was", "used", "to", "estimate", "the", "number", "of", "species", "in", "each", "sample,", "and", "the", "similarity", "threshold", "was", "97%.", "The", "coverage", "(", "http://www.mothur.org/wiki/Coverage", ")", "was", "used", "as", "the", "sequencing", "depth", "index.", "The", "ACE", "estimator", "(", "http://www.mothur.org/wiki/Ace", ")", "and", "Chao", "estimator", "(", "http://www.mothur.org/wiki/Chao", ")", "were", "used", "to", "estimate", "the", "gut", "microbial", "community", "richness.", "The", "Shannon", "index", "(", "http://www.mothur.org/wiki/Shannon", ")", "and", "Simpson", "index", "(", "http://www.mothur.org/wiki/Simpson", ")", "were", "used", "to", "estimate", "the", "gut", "microbial", "community", "diversity.", "The", "pie", "charts", "were", "used", "to", "describe", "the", "distribution", "of", "microorganisms", "at", "the", "species", "level.", "Proportions", "less", "than", "1%", "were", "included", "in", "“Others”,", "considering", "the", "best", "visual", "presentation.", "Venn", "diagrams", "were", "implemented", "by", "Venn", "diagram.", "We", "performed", "clustering", "on", "genera", "obtained", "from", "the", "RDP", "Classifier", "by", "means", "of", "the", "complete", "linkage", "hierarchical", "clustering", "technique", "using", "the", "R", "package", "HCLUST", "(", "http://sekhon.berkeley.edu/stats/html/hclust.html", ").", "The", "basic", "process", "of", "LDA", "analysis", "[", "34", "]", "was", "as", "follows:", "First,", "non-parametric", "Kruskal–Wallis", "rank", "sum", "test", "was", "used", "to", "detect", "the", "significant", "difference", "of", "abundance", "among", "species", "between", "the", "two", "groups", "of", "samples.", "Then", "Wilcoxon", "rank", "sum", "test", "was", "used", "to", "analyze", "the", "difference", "between", "groups", "based", "on", "the", "significant", "difference", "species.", "Finally,", "LDA", "was", "used", "to", "reduce", "the", "dimension", "of", "data", "and", "evaluate", "the", "influence", "of", "significantly", "different", "species", "(LDA", "score).", "LDA", "effect", "size", "more", "than", "2", "was", "included", "in", "the", "figure.", "The", "genes", "of", "the", "16S", "corresponding", "species", "and", "their", "copy", "number", "have", "been", "generally", "known,", "and", "these", "genes", "can", "be", "compared", "with", "databases", "(e.g.", "KEGG,", "NOG)", "to", "predict", "the", "possible", "metabolic", "capacity", "of", "these", "species.", "The", "Picrust", "software", "was", "used", "to", "analyze", "the", "pathway", "based", "on", "the", "KEGG", "database.", "The", "specific", "methods", "of", "function", "prediction", "analysis", "are", "shown", "in", "the", "following", "website", "(", "http://picrust.github.io/picrust/tutorials/algorithm_description.html", ").", "Statistical", "analysis", "SPSS", "software", "(V:", "16.0;", "SPSS", "Inc.,", "Chicago,", "IL)", "was", "used", "to", "analyze", "the", "data.", "The", "data", "are", "expressed", "as", "the", "mean", "±", "standard", "deviation", "(SD).", "Statistically", "significant", "differences", "between", "two", "groups", "were", "analyzed", "using", "the", "independent-samples", "t-test.", "Frequency", "table", "data", "were", "analyzed", "with", "the", "Chi-square", "test", "or", "Fisher’s", "exact", "test.", "All", "tests", "were", "performed", "at", "the", "0.05", "level", "of", "significance.", "Results", "Basic", "characteristics", "of", "the", "patients", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Overall,", "68", "resected", "stage", "III", "CRC", "patients", "accepted", "the", "CapeOX", "regimen", "in", "Huzhou", "Central", "Hospital", "from", "January", "2016", "to", "January", "2018.", "However,", "51", "patients", "were", "excluded", "from", "this", "study", "for", "the", "following", "reasons:", "patients", "with", "diarrhea", "before", "chemotherapy", "(n", "=", "3),", "terminated", "chemotherapy", "because", "of", "PD", "and", "side", "effects", "(n", "=", "7),", "accepted", "neo-adjuvant", "chemotherapy", "(n", "=", "4),", "could", "not", "obtain", "the", "stool", "samples", "(n", "=", "15),", "unqualified", "stool", "samples", "(n", "=", "14),", "and", "used", "oral", "probiotics", "including", "Bifid-triple", "Viable", "Capsule", "(n", "=", "6)", "and", "Clostridium", "Butyricum", "Capsule", "(n", "=", "2)", "before", "chemotherapy.", "After", "screening", "and", "identification,", "finally,", "17", "stage", "III", "CRC", "patients", "were", "enrolled", "in", "the", "present", "study.", "The", "study", "strategy", "is", "shown", "in", "Fig.", "1", ".", "The", "pathology", "type", "of", "all", "of", "the", "patients", "with", "resected", "stage", "III", "colorectal", "cancer", "was", "moderately", "differentiated", "adenocarcinoma.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "The", "17", "CRC", "patients", "were", "divided", "into", "experimental", "group", "(CID+)", "and", "control", "group", "(CID−)", "according", "to", "whether", "they", "had", "diarrhea", "or", "not.", "The", "patient’s", "clinical", "information", "is", "shown", "in", "Table", "1", ".", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "Blood", "samples", "of", "patients", "before", "chemotherapy", "were", "collected", "for", "serological", "examination.", "There", "was", "no", "significant", "difference", "between", "the", "two", "groups", "in", "terms", "of", "age,", "sex,", "BMI,", "or", "smoking,", "drinking", "history", "and", "clinical", "serological", "indicators", "including", "hemoglobin,", "alanine", "transaminase,", "glutamic", "oxaloacetic", "transaminase,", "albumin,", "creatinine,", "carbohydrate", "antigen", "199,", "carbohydrate", "antigen", "742", "and", "carcino", "embryonic", "antigen.", "Fig.", "1", "Study", "strategy.", "After", "screening", "and", "identification,", "finally,", "17", "stage", "III", "CRC", "patients", "undergoing", "the", "CapeOX", "regimen", "after", "curative", "resection", "were", "recruited", "into", "the", "present", "study.", "Stool", "samples", "were", "collected", "from", "the", "recruited", "patients.", "The", "bacterial", "16S", "ribosomal", "RNA", "from", "the", "samples", "were", "sequenced.", "Bioinformatics", "analysis", "was", "used", "to", "analyze", "the", "distinctive", "gut", "microbiome", "and", "functional", "metabolism", "Table", "1", "Characteristics", "of", "the", "study", "participants", "Experimental", "group", "Control", "group", "p", "value", "Cases,", "n", "4", "13", "–", "Rectal", "cancer,", "n", "2", "6", "0.89", "Males,", "n", "3", "8", "0.62", "Age", "(years)", "63.88", "±", "8.61", "64.23", "±", "8.76", "0.94", "BMI", "(kg/m", "2", ")", "22.74", "±", "2.64", "23.28", "±", "2.43", "0.71", "Long-term", "smoking", "history,", "n", "0", "1", "0.57", "Long-term", "drinking", "history,", "n", "1", "4", "0.82", "Known", "diabetes,", "n", "0", "1", "0.57", "Known", "hypertension,", "n", "0", "0", "/", "Hemoglobin", "(g/L)", "134.25", "±", "5.06", "124.62", "±", "13.71", "0.20", "Alanine", "transaminase", "(U/L)", "29.00", "±", "30.97", "19.80", "±", "8.94", "0.33", "Glutamic", "oxaloacetic", "transaminase", "(U/L)", "27.40", "±", "15.78", "24.56", "±", "8.72", "0.64", "Albumin", "(g/L)", "42.03", "±", "4.57", "38.11", "±", "3.02", "0.06", "Creatinine", "(μmol/L)", "83.10", "±", "33.74", "68.95", "±", "14.66", "0.24", "Carbohydrate", "antigen", "199", "(U/mL)", "5.46", "±", "3.61", "12.24", "±", "9.36", "0.18", "Carbohydrate", "antigen", "742", "(U/mL)", "8.58", "±", "3.61", "9.89", "±", "19.71", "0.90", "Carcino", "embryonic", "antigen", "(ng/mL)", "24.34", "±", "41.47", "3.98", "±", "3.70", "0.08", "Overall,", "17", "resected", "stage", "III", "CRC", "patients", "undergoing", "the", "CapeOX", "regimen", "were", "recruited.", "These", "patients", "were", "divided", "into", "experimental", "group", "(CID+)", "and", "control", "group", "(CID−)", "according", "to", "whether", "they", "had", "diarrhea", "or", "not.", "The", "patient’s", "clinical", "information", "was", "shown", "in", "thus", "table.", "The", "clinical", "stages", "conformed", "to", "the", "criterion", "of", "stage", "III", "CRC", "according", "to", "AJCC.", "Smoking", "and", "drinking", "history", "over", "the", "course", "of", "1", "year", "were", "collected.", "Blood", "samples", "of", "patients", "before", "chemotherapy", "were", "collected", "for", "serological", "examination", "Gut", "microbial", "richness", "and", "diversity", "associated", "with", "CID", "during", "the", "Cape", "OX", "regimen", "for", "CRC", "As", "shown", "in", "Table", "2", ",", "the", "coverage", "index", "suggested", "that", "the", "coverage", "of", "the", "genomic", "library", "of", "the", "sequencing", "samples", "was", "more", "than", "99%.", "The", "statistically", "significant", "differences", "of", "the", "ACE", "estimator", "and", "Chao", "estimator", "between", "the", "experimental", "group", "and", "control", "group", "indicated", "that", "the", "community", "richness", "of", "the", "gut", "microbiome", "was", "lower", "in", "the", "patients", "with", "chemotherapy-induced", "diarrhea", "(p", "<", "0.05,", "vs", "control", "group).", "The", "smaller", "Shannon", "index", "and", "larger", "Simpson", "index", "indicate", "that", "the", "gut", "microbial", "community", "diversity", "was", "lower", "in", "the", "patients", "with", "chemotherapy-induced", "diarrhea", "(p", "<", "0.01,", "vs", "control", "group).", "The", "alpha", "diversity", "analysis", "is", "shown", "in", "Additional", "file", "1", ".", "Table", "2", "Gut", "microbial", "richness", "and", "diversity", "associated", "with", "chemotherapy-induced", "diarrhea", "of", "the", "CapeOX", "regimen", "in", "CRC", "Group", "n", "Coverage", "OTU", "Ace", "Chao", "Shannon", "Simpson", "Experimental", "group", "4", "0.9994", "±", "0.0008", "119.75", "±", "53.44", "159.50", "±", "100.09", "150.75", "±", "77.58", "2.21", "±", "0.47", "0.21", "±", "0.08", "Control", "group", "13", "0.9985", "±", "0.0012", "273.38", "±", "93.68", "336.31", "±", "117.73", "332.46", "±", "113.34", "3.36", "±", "0.59", "0.09", "±", "0.07", "t", "value", "–", "–", "−", "3.08", "−", "2.70", "−", "2.97", "−", "3.54", "2.30", "p", "value", "–", "–", "0.008", "0.016", "0.010", "0.003", "0.009", "Mothur", "software", "(", "http://www.mothur.org/wiki/Main_Page", ")", "was", "used", "to", "analyze", "the", "microbial", "diversity.", "Operational", "taxonomic", "units", "(OTU),", "as", "the", "artificial", "classification", "unit,", "was", "used", "to", "estimate", "the", "number", "of", "species", "in", "each", "sample,", "and", "the", "similarity", "threshold", "was", "97%.", "The", "alpha", "diversity", "analysis", "was", "shown", "in", "Additional", "file", "1", "Gut", "microbial", "community", "structure", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC", "A", "pie", "chart", "was", "constructed", "for", "the", "microorganisms", "that", "accounted", "for", "more", "than", "1%", "in", "the", "two", "groups", "of", "samples", "to", "show", "the", "microbial", "community", "structure", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC.", "The", "distribution", "of", "microorganisms", "from", "the", "same", "group", "was", "merged", "into", "a", "whole.", "As", "shown", "in", "Fig.", "2", ",", "the", "stacked", "bar", "graph", "showed", "that", "the", "percent", "of", "Klebsiella", "pneumoniae", "was", "35%", "and", "it", "occupied", "the", "highest", "proportion", "in", "the", "experimental", "group", "(CID+).", "But", "the", "rate", "was", "only", "3%", "in", "the", "control", "group", "(CID−).", "Fig.", "2", "Microbial", "community", "structure", "analysis.", "The", "distribution", "of", "microorganisms", "from", "the", "same", "group", "was", "merged", "into", "the", "whole.", "The", "stacked", "bar", "graph", "was", "used", "to", "describe", "the", "distribution", "of", "microorganisms", "at", "the", "species", "level.", "CID-", "and", "CID+", "represent", "the", "control", "group", "and", "experimental", "group,", "respectively", "The", "difference", "in", "microbial", "abundance", "associated", "with", "CID", "of", "the", "CapeOX", "regimen", "for", "CRC", "The", "samples", "were", "divided", "into", "two", "groups", "according", "to", "patients", "with", "or", "without", "CID.", "As", "shown", "in", "Fig.", "3", "a,", "there", "were", "220", "shared", "OTUs", "and", "that", "the", "numbers", "of", "unique", "OTUs", "in", "the", "control", "group", "and", "experimental", "group", "were", "645", "and", "34,", "respectively.", "LDA", "effect", "size", "analysis", "was", "used", "to", "compare", "the", "differences", "in", "microbial", "abundance", "between", "the", "two", "groups.", "LDA", "Effect", "Size", "analysis", "(Fig.", "3", "b)", "showed", "that", "there", "were", "75", "microorganisms", "with", "significant", "differences", "at", "the", "genus", "level", "between", "the", "two", "groups.", "The", "length", "of", "the", "histogram", "indicates", "that", "some", "microorganisms", "such", "as", "Proteobacteria,", "Enterobacteriales,", "Gammaproteobacteria,", "Enterobacteriaceae,", "Klebsiella,", "Clostridiales,", "Clostridia,", "Ruminococcaceae,", "Bacteroidetes,", "Bacteroidia,", "Bacteroidales,", "Bacteroides", "and", "Bacteroidaceae", "have", "a", "larger", "influence.", "The", "cladogram", "in", "Fig.", "3", "c", "displays", "the", "composition", "and", "proportion", "of", "microorganisms", "at", "different", "taxonomic", "levels.", "The", "cladogram", "showed", "that", "the", "abundance", "of", "these", "bacteria", "including", "Actinomycetaceae,", "Micrococcaceae,", "Bacteroidaceae,", "Porphyromonadaceae,", "Ruminococcaceae,", "Erysipelotrichaceae,", "Leptotrichiaceae,", "Enterobacteriaceae", "(Klebsiella", "belongs", "to", "Enterobacteriaceae)", "and", "Synergistaceae", "had", "significant", "differences", "at", "family", "level", "between", "the", "two", "groups.", "Fig.", "3", "Differential", "microbes", "analysis.", "The", "samples", "were", "divided", "into", "two", "groups", "according", "to", "patients", "with", "or", "without", "CID.", "The", "number", "of", "OTUs", "that", "were", "exclusive", "or", "shared", "by", "the", "two", "groups", "were", "visualized", "by", "a", "Venn", "Chart", "(", "a", ").", "The", "strip", "figure", "in", "b", "displays", "the", "LDA", "effect", "size", "analysis.", "LDA", "effect", "size", "analysis", "was", "used", "to", "compare", "the", "differences", "in", "microbial", "abundance", "between", "the", "two", "groups", "(", "b", ").", "LDA", "scores", "over", "2", "mean", "that", "the", "differences", "are", "statistically", "significant", "(p", "<", "0.05).", "The", "cladogram", "in", "c", "displays", "the", "composition", "and", "proportion", "of", "microorganisms", "at", "different", "taxonomic", "levels.", "The", "innermost", "layer", "shows", "the", "taxonomic", "tree.", "The", "circle", "from", "the", "inside", "to", "the", "outside", "represents", "different", "taxon", "levels", "from", "phylum", "to", "genus.", "Yellow", "circles", "represent", "no", "statistical", "difference", "in", "species", "between", "the", "two", "groups,", "and", "red", "circles", "and", "green", "circles", "represent", "the", "higher", "abundance", "of", "genus", "in", "the", "control", "group", "and", "the", "chemotherapy-induced", "diarrhea", "group,", "respectively.", "The", "lowercase", "English", "letters", "corresponding", "to", "the", "different", "bacteria", "are", "shown", "in", "the", "legends", "The", "functional", "analysis", "of", "16S", "rRNA", "genes", "associated", "with", "CID", "during", "the", "CapeOX", "regimen", "for", "CRC", "It", "has", "been", "confirmed", "that", "many", "gene", "function", "annotation", "databases", "are", "associated", "with", "corresponding", "16S", "rRNA", "gene", "sequences.", "Functional", "analysis", "of", "enzymes", "and", "genes", "would", "be", "conducive", "to", "understand", "the", "whole", "microecological", "environment.", "As", "shown", "in", "Fig.", "4", ",", "the", "figure", "shows", "the", "results", "of", "the", "functional", "analysis.", "Overall,", "23", "pathways", "(p", "<", "0.001)", "associated", "with", "statistically", "significant", "differences", "might", "be", "involved", "in", "CID", "during", "the", "CapeOX", "regimen", "for", "adjuvant", "chemotherapy", "in", "resected", "stage", "III", "colorectal", "cancer.", "These", "differential", "pathways", "are", "related", "to", "microbial", "metabolites,", "cell", "proliferation", "and", "death,", "immune", "system,", "and", "many", "other", "aspects.", "Fig.", "4", "Functional", "analysis.", "The", "Picrust", "software", "was", "used", "to", "analyze", "the", "pathway", "based", "on", "the", "KEGG", "database.", "The", "significant", "differences", "in", "functional", "metabolism", "are", "shown", "in", "the", "figure", "Discussion", "The", "CapeOX", "regimen", "is", "recommended", "as", "the", "postoperative", "adjuvant", "chemotherapy", "regimen", "in", "resected", "CRC", "patients", "with", "stage", "III", "or", "high-risk", "stage", "II", "[", "35", "].", "The", "high-risk", "factors", "in", "stage", "II", "may", "affect", "the", "accuracy", "of", "experimental", "results,", "Thus,", "CRC", "patients", "with", "stage", "III", "were", "recruited", "in", "the", "present", "study.", "According", "to", "the", "NCCN", "guidelines,", "the", "CapeOX", "regimen", "for", "resected", "stage", "III", "CRC", "has", "been", "defined", "as", "8", "cycles", "of", "capecitabine", "(1000", "mg/m", "2", "twice", "daily)", "combined", "with", "oxaliplatin", "(130", "mg/m", "2", "every", "3", "weeks)", "[", "36", "].", "CID", "is", "one", "of", "the", "most", "common", "side", "effects", "of", "the", "CapeOX", "regimen", "[", "37", "].", "The", "degree", "of", "diarrhea", "caused", "by", "different", "chemotherapeutic", "agents", "varies.", "Among", "the", "chemotherapeutic", "drugs", "for", "colorectal", "cancer,", "the", "most", "common", "drugs", "that", "cause", "diarrhea", "are", "fluorouracil,", "irinotecan", "and", "platinum,", "and", "the", "incidence", "of", "diarrhea", "associated", "with", "these", "drugs", "can", "be", "as", "high", "as", "50–80%", "[", "38", "].", "Our", "previous", "clinical", "investigation", "found", "that", "the", "probability", "of", "CID", "increased", "with", "an", "increase", "of", "chemotherapeutic", "frequency", "and", "chemotherapeutic", "dosage", "and", "most", "cases", "of", "CID", "occur", "within", "2", "weeks", "after", "chemotherapy.", "Therefore,", "in", "the", "present", "study,", "stool", "samples", "from", "the", "patients", "were", "collected", "in", "2", "weeks", "after", "the", "8", "cycles", "of", "chemotherapy", "of", "the", "CapeOX", "regimen", "were", "completed.", "Studies", "of", "Carroll", "et", "al.", "[", "39", "]", "suggested", "that", "the", "richness", "of", "16S", "rRNA", "sequences", "was", "significantly", "decreased", "in", "patients", "with", "diarrhea-predominant", "irritable", "bowel", "syndrome.", "Lee", "[", "40", "]", "has", "reported", "the", "community", "diversity", "of", "gut", "microbiome", "is", "lower", "in", "patients", "with", "kidney", "posttransplant", "diarrhea.", "Similar", "results", "have", "been", "reported", "in", "dogs", "with", "acute", "diarrhea", "[", "41", "].", "The", "research", "on", "these", "diarrhea", "diseases", "is", "consistent", "with", "our", "findings.", "We", "found", "that", "the", "gut", "microbial", "community", "richness", "and", "community", "diversity", "was", "lower", "in", "CRC", "patients", "with", "CID", "in", "the", "present", "study.", "The", "decrease", "of", "microbial", "diversity", "may", "be", "related", "to", "the", "imbalance", "of", "gut", "microbiome.", "The", "dominant", "pathogenic", "bacteria", "leads", "to", "the", "reduction", "of", "the", "resident", "normal", "microbiota", "through", "the", "plunder", "of", "nutrients", "or", "the", "killing", "effect", "of", "bacterial", "toxic", "metabolites.", "Our", "results", "from", "the", "microbial", "community", "structure", "analysis", "showed", "Klebsiella", "pneumoniae", "occupied", "the", "highest", "proportion", "(31.22%)", "of", "gut", "microbiome", "in", "CRC", "patients", "with", "CID.", "Lu", "et", "al.", "[", "42", "]", "reported", "that", "the", "rate", "of", "Klebsiella", "pneumoniae", "was", "approximately", "0.5%", "in", "Beijing", "by", "culturing", "the", "stool", "pathogens", "from", "outpatients", "with", "diarrhea", "syndromes", "using", "the", "Vitek2", "Compact", "instrument.", "Zhang", "et", "al.", "[", "22", "]", "only", "isolated", "43", "Klebsiella", "pneumoniae", "strains", "from", "551", "stool", "specimens", "from", "diarrhea", "patients.", "Although", "the", "study", "may", "have", "be", "interfered", "by", "other", "factors", "such", "as", "geographical", "location,", "nosocomial", "infection,", "or", "pollution", "contamination", "in", "the", "fecal", "collection", "process.", "The", "increased", "proportion", "of", "Klebsiella", "pneumoniae", "in", "patients", "with", "CID", "remains", "a", "phenomenon", "of", "concern.", "Microorganisms", "can", "adjust", "and", "modify", "their", "surroundings", "due", "to", "their", "metabolic", "products.", "A", "series", "of", "enzymes", "and", "genes", "participate", "in", "the", "process", "of", "metabolism", "[", "12", "].", "Functional", "analysis", "of", "these", "enzymes", "and", "genes", "would", "be", "conducive", "to", "understand", "the", "whole", "microecological", "environment.", "In", "the", "present", "study,", "we", "screened", "75", "differentiated", "microorganisms", "at", "the", "species", "level", "by", "using", "LDA", "effect", "size", "analysis", "as", "well", "as", "23", "pathways", "associated", "with", "differential", "microorganisms", "by", "using", "KEGG", "databases.", "These", "differential", "pathways", "are", "related", "to", "microbial", "metabolites,", "cell", "proliferation", "and", "death,", "immune", "system,", "and", "many", "other", "aspects.", "These", "differentiated", "microorganisms,", "their", "metabolic", "products", "and", "the", "relevant", "pathways", "make", "up", "the", "intestinal", "microecosystem", "that", "causes", "CID.", "These", "results", "may", "provide", "characteristic", "microorganisms", "or", "potential", "molecular", "targets", "for", "the", "treatment", "and", "prevention", "of", "CID.", "It", "must", "be", "emphasized", "that", "small", "sample", "size", "and", "potential", "sample", "contamination", "may", "affect", "the", "accuracy", "of", "the", "present", "study.", "Strict", "inclusion", "and", "exclusion", "criteria", "was", "performed", "in", "this", "study.", "Subjects", "in", "the", "experimental", "group", "were", "only", "included", "the", "resected", "stage", "III", "colorectal", "cancer", "(CRC)", "patients", "who", "voluntarily", "accepted", "and", "completed", "8", "cycles", "of", "the", "CapeOX", "regimen", "and", "accompanied", "with", "grade", "2", "of", "CID.", "Thus,", "the", "small", "sample", "size", "limits", "the", "applicability", "of", "the", "findings.", "A", "multi-center,", "large", "study", "will", "provide", "more", "powerful", "data", "to", "support", "the", "clarification", "of", "the", "microbial", "differences", "in", "CRC", "patients", "with", "CID.", "Conclusion", "Chemotherapy", "induced", "diarrhea", "(CID)", "is", "a", "common", "side", "effect", "in", "patients", "receiving", "chemotherapy", "for", "cancer.", "In", "the", "present", "study,", "We", "found", "that", "the", "gut", "microbial", "community", "structure", "and", "community", "diversity", "have", "changed", "in", "CRC", "patients", "with", "CID.", "The", "gut", "microbial", "community", "richness", "and", "community", "diversity", "were", "lower", "in", "CID.", "Klebsiella", "pneumoniae", "was", "the", "most", "predominant", "species", "among", "the", "gut", "microbiome", "in", "CRC", "patients", "with", "CID.", "It", "may", "provide", "novel", "insights", "into", "the", "prevention", "and", "treatment", "of", "patients", "with", "CID", "to", "improve", "patients’", "quality", "of", "life", "and", "chemotherapy", "tolerance." ]

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0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 7, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0 ]

{"negatives/white": ["2767+1", "1193+1", "1660+1", "1959+1", "2046+1", "3276+1", "3671+1", "3730+1", "4060+1"], "pathogens/yellow": ["353+2", "371+2", "495+2", "497+2"]}

CC-BY

4.0

http://creativecommons.org/licenses/by/4.0/

Original work titled: 'Gut microbiome associated with chemotherapy-induced diarrhea from the CapeOX regimen as adjuvant chemotherapy in resected stage III colorectal cancer' (Gut Pathogens/BioMed Central) by the following authors: Zuo Fei; Yin Lijuan; Yang Xi; Wu Wei; Zhong Jing; Da Miao; Han Shuwen DOI: 10.1186/s13099-019-0299-4

0ce28549ffb17c27829b0d628ff2e0c4

[ "Background", "The", "cultivated", "lentil", "(Lens", "culinaris", "Medikus", "ssp.", "culinaris)", "is", "one", "of", "the", "most", "agronomically", "important", "grain", "legumes", "with", "a", "global", "production", "of", "6.3", "million", "tons", "in", "2016", "which", "was", "primarily", "contributed", "by", "Canada", "(50.8%),", "India", "(16.8%),", "Turkey", "(5.8%)", "and", "the", "USA", "(4.0%)", "[", "1", "].", "Abiotic", "and", "biotic", "stresses", "are", "reducing", "the", "yield", "potential", "of", "lentil,", "so", "breeding", "efforts", "have", "focused", "on", "the", "development", "of", "varieties", "with", "resistance", "to", "these", "stresses.", "The", "main", "breeding", "strategy", "has", "been", "relying", "on", "selection", "of", "superior", "individuals", "derived", "from", "crosses", "between", "elite", "breeding", "lines", "or", "cultivars.", "Due", "to", "the", "intensive", "selection", "driven", "by", "certain", "traits", "of", "interests", "and", "hybridization", "of", "closely", "related", "breeding", "lines,", "the", "genetic", "variability", "of", "lentil", "germplasm", "inevitably", "has", "been", "narrow", "[", "2", "].", "Associated", "with", "limited", "genetic", "variability", "in", "crops", "is", "a", "heightened", "risk", "for", "crop", "vulnerability", "to", "biotic", "and", "abiotic", "stresses,", "a", "major", "concern", "shared", "among", "plant", "breeders", "and", "growers", "alike", "[", "3", "].", "Stemphylium", "blight", "(SB),", "caused", "by", "the", "necrotrophic", "ascomycete", "Stemphylium", "botryosum", "Wallr.,", "is", "a", "devastating", "disease", "of", "lentil", "in", "several", "lentil-producing", "countries,", "including", "Canada,", "India,", "the", "USA,", "and", "Australia", "which", "together", "account", "for", "over", "70%", "of", "global", "production", "[", "4", ",", "5", "].", "This", "pathogen", "infects", "plants", "by", "airborne", "conidia", "that", "develop", "successive", "cycles", "of", "conidia", "on", "plants.", "In", "the", "early", "stage", "of", "SB,", "disease", "symptoms", "manifest", "as", "tan", "to", "light", "brown", "spots", "on", "lentil", "leaves.", "As", "the", "infection", "progresses,", "those", "initial", "spots", "expand", "to", "the", "entire", "leaves,", "resulting", "in", "the", "complete", "drying", "of", "leaves", "followed", "by", "defoliation.", "In", "the", "final", "stages,", "SB", "symptoms", "are", "also", "found", "on", "stems", "and", "hamper", "nutrient", "and", "water", "transport,", "which", "eventually", "kills", "the", "host.", "The", "economic", "losses", "caused", "by", "this", "pathogen", "can", "be", "up", "to", "80%", "[", "6", "].", "A", "few", "SB", "resistant", "commercial", "cultivars", "have", "been", "developed", "in", "some", "of", "the", "lentil-producing", "regions,", "especially", "in", "Bangladesh", "(reviewed", "in", "[", "7", "]).", "Studies", "in", "a", "range", "of", "crop", "species", "have", "shown", "that", "many", "alleles", "representing", "broad", "genetic", "diversity", "and", "phenotypic", "variation", "reside", "in", "wild", "germplasm", "[", "8", "].", "Exploring", "novel", "disease", "resistance", "alleles", "from", "exotic", "libraries", "has", "been", "a", "useful", "strategy", "for", "genetic", "improvement", "in", "many", "crops", "[", "3", ",", "9", "].", "In", "lentil,", "Podder", "et", "al.", "[", "7", "]", "evaluated", "resistance", "to", "SB", "among", "70", "lentil", "accessions", "representing", "seven", "species", "in", "the", "genus", "Lens", "and", "found", "that", "L.", "ervoides", "(Brign.)", "Grande", "exhibited", "superior", "SB", "resistance", "in", "a", "much", "higher", "frequency", "than", "other", "species.", "Facilitated", "by", "the", "sophisticated", "ovule", "rescue", "technique", "that", "overcomes", "the", "interspecific", "reproductive", "barriers", "between", "L.", "ervoides", "and", "L.", "culinaris", "[", "10", "],", "interest", "in", "introgressing", "useful", "genes", "from", "L.", "ervoides", "to", "elite", "cultivars", "has", "been", "increasing", "strongly,", "in", "particular", "because", "resistance", "to", "other", "diseases", "such", "as", "ascochyta", "blight", "(caused", "by", "Ascochyta", "lentis", "Vassiljevsky)", "and", "anthracnose", "(caused", "by", "Colletotrichum", "lentis", "Damm)", "has", "also", "been", "identified", "in", "this", "species", "[", "11", "].", "To", "date,", "detailed", "knowledge", "of", "the", "molecular", "bases", "underlying", "SB", "resistance", "in", "L.", "ervoides", "is", "still", "lacking", "and", "the", "only", "available", "information", "is", "the", "presence", "of", "three", "quantitative", "trait", "loci", "(QTLs)", "associated", "with", "SB", "on", "a", "single", "nucleotide", "polymorphism", "(SNP)", "-based", "linkage", "map", "of", "an", "F", "9", "recombinant", "inbred", "line", "(RIL)", "population", "of", "L.", "ervoides", "[", "11", "].", "Despite", "the", "identification", "and", "localization", "of", "these", "QTLs,", "their", "direct", "use", "in", "marker-assisted", "selection", "is", "limited", "and", "identification", "of", "candidate", "resistance", "genes", "is", "difficult", "because", "those", "QTL", "intervals", "(2-LOD)", "are", "relatively", "large", "(6", "to", "21", "MB)", "and", "contain", "hundreds", "of", "genes", "[", "11", "].", "A", "functional", "understanding", "of", "SB", "resistance", "has", "been", "largely", "developed", "in", "model", "plants", "such", "as", "tomato", "(Solanum", "lycopersicum", "L.)", "and", "Arabidopsis", "thaliana", "(L.)", "Heynh.", "In", "tomato,", "Yang", "et", "al.", "[", "12", "]", "mapped", "a", "single", "dominant", "locus", "in", "a", "260", "Kb", "chromosomal", "region", "conferring", "resistance", "to", "SB", "(caused", "by", "S.", "lycopersici", "(Enjoji)", "W.", "Yamam.)", "and", "identified", "two", "putative", "resistance", "genes", "coding", "for", "a", "cysteine-rich", "receptor-like", "kinase", "(CRK)", "and", "a", "receptor-like", "kinase", "(RLK).", "This", "indicated", "that", "resistance", "in", "tomato", "was", "likely", "mediated", "via", "the", "recognition", "between", "plant", "and", "pathogen.", "In", "A.", "thaliana,", "Di", "et", "al.", "[", "13", "]", "reported", "that", "a", "gene", "encoding", "polygalactoronase-inhibiting", "protein", "(PGIP)", "was", "upregulated", "during", "the", "S.", "solani", "infection", "period.", "As", "PGIP", "belongs", "to", "a", "large", "family", "of", "leucine-rich", "repeat", "(LRR)", "proteins,", "it", "was", "suspected", "that", "a", "hypersensitive", "response", "(HR)", "may", "affect", "SB", "resistance", "in", "A.", "thaliana", "[", "13", "].", "However,", "due", "to", "the", "highly", "complex", "nature", "of", "resistances", "to", "necrotrophic", "pathogens,", "it", "is", "still", "unknown", "if", "these", "mechanisms", "can", "be", "extended", "to", "other", "plant", "species.", "Therefore,", "a", "comprehensive", "molecular", "study", "of", "SB", "resistance", "in", "L.", "ervoides", "would", "not", "only", "benefit", "lentil", "breeding", "but", "also", "broaden", "our", "knowledge", "of", "SB", "resistance", "in", "systems", "other", "than", "model", "plant", "systems.", "In", "recent", "years,", "the", "study", "of", "RNA", "has", "been", "significantly", "improved", "by", "the", "advancement", "in", "high-throughput", "next-generation", "sequencing", "techniques", "that", "generate", "massive", "amounts", "of", "data", "suitable", "for", "in-depth", "quantification", "of", "genome-wide", "gene", "expression", "across", "treatments,", "time", "points", "and", "genotypes", "[", "14", "].", "In", "lentil,", "several", "RNA-sequencing", "(RNA-Seq)", "studies", "were", "performed", "to", "profile", "transcriptomes,", "develop", "molecular", "markers", "or", "investigate", "plant", "responses", "to", "various", "biotic", "and", "abiotic", "stresses", "[", "15", "–", "19", "].", "Most", "of", "these", "studies", "were", "conducted", "by", "de", "novo", "assembly", "without", "the", "aid", "of", "a", "reference", "genome.", "The", "common", "issues", "with", "de", "novo", "assembly", "have", "been", "alignment", "errors,", "problems", "in", "reconstruction", "of", "full-length", "transcripts,", "and", "chimerism", "errors", "[", "20", "].", "As", "the", "L.", "culinaris", "genome", "was", "recently", "constructed", "by", "Bett", "et", "al.", "[", "21", "],", "reference-based", "transcriptome", "assembly", "is", "possible", "and", "encouraged", "to", "improve", "downstream", "analyses.", "As", "RNA", "transcription", "is", "a", "highly", "dynamic", "process,", "the", "appropriate", "sampling", "time", "is", "of", "high", "importance", "to", "capture", "genes", "of", "interest,", "and", "to", "achieve", "this,", "the", "quantitative", "assessments", "of", "fungal", "development", "implemented", "by", "microscopic", "observation", "and", "quantitative", "polymerase", "chain", "reaction", "(qPCR)", "are", "promising", "options.", "Several", "studies", "proved", "that", "these", "two", "methods", "well", "integrate", "both", "visual", "and", "digital", "assessments", "of", "fungal", "biomass", "and", "development", "in", "a", "range", "of", "hosts", "[", "22", "–", "24", "].", "In", "this", "study,", "we", "recruited", "those", "methods", "and", "RILs", "from", "the", "F", "9", "L.", "ervoides", "RIL", "population", "LR-66", "[", "11", "]", "to", "study", "SB", "resistance", "in", "L.", "ervoides.", "Firstly,", "we", "performed", "microscopic", "and", "qPCR", "studies", "to", "quantify", "the", "development", "of", "S.", "botryosum", "in", "plants", "during", "the", "first", "10", "days", "post-inoculation.", "Secondly,", "we", "conducted", "a", "time-series", "RNA-Seq", "experiment", "on", "two", "SB", "infected", "RILs", "that", "displayed", "contrasting", "SB", "susceptibilities.", "Gene", "expression", "data", "were", "submitted", "to", "differentially", "expressed", "gene", "analyses", "(DEG)", "to", "understand", "the", "regulatory", "pathways", "that", "were", "involved", "in", "SB", "defense", "responses.", "To", "further", "screen", "the", "candidate", "genes", "governing", "resistance", "and", "susceptibility", "to", "SB,", "we", "then", "performed", "bulk", "segregation", "gene", "expression", "analysis", "using", "pooled", "susceptible", "and", "resistant", "RILs", "from", "the", "LR-66", "population", "to", "verify", "the", "expression", "of", "those", "DEGs", "that", "were", "located", "in", "identified", "QTLs", "[", "11", "].", "The", "objectives", "of", "this", "study", "were", "to", "extend", "our", "understanding", "of", "SB", "resistance", "and", "identify", "putative", "resistant", "genes", "to", "facilitate", "use", "of", "L.", "ervoides", "in", "commercial", "lentil", "breeding.", "Results", "Quantification", "of", "fungal", "development", "Our", "inoculation", "experiment", "with", "S.", "botryosum", "isolate", "SB19", "resulted", "in", "distinctly", "more", "disease", "in", "L.", "ervoides", "RIL", "LR-66-577", "than", "LR-66-637", "(Fig.", "1", ").", "The", "percentage", "of", "conidial", "germination", "and", "penetration,", "and", "germ", "tube", "lengths", "were", "recorded", "for", "the", "first", "48", "h", "post-inoculation", "(hpi)", "(Fig.", "2", "a).", "Results", "showed", "that", "the", "germination", "of", "conidia", "increased", "over", "time", "and", "reached", "its", "highest", "level", "(94.4%", "for", "LR-66-637,", "98.5%", "for", "LR-66-577)", "at", "48", "hpi.", "Approximate", "50%", "of", "conidia", "had", "germinated", "with", "germ", "tubes", "that", "had", "successfully", "penetrated", "into", "the", "plant", "epidermal", "cells", "at", "6", "hpi", "(both", "RILs),", "which", "increased", "to", "69.6%", "(LR-66-637)", "or", "67.2%", "(LR-66-577)", "at", "12", "hpi.", "Germ", "tube", "length", "on", "the", "leaf", "surface", "was", "quantified", "from", "12", "to", "24", "hpi", "and", "revealed", "a", "substantial", "increase", "from", "approximate", "200", "μm", "(both", "RILs)", "at", "12", "hpi", "to", "395", "μm", "(LR-66-637)", "or", "486", "μm", "(LR-66-577)", "at", "24", "hpi.", "However,", "there", "was", "no", "statistical", "difference", "between", "the", "resistant", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "for", "these", "fungal", "growth", "parameters.", "At", "48", "hpi,", "we", "started", "to", "observe", "a", "few", "chlorotic", "spots", "on", "leaflets.", "As", "the", "infected", "plants", "displayed", "evidence", "of", "leaf", "necrosis", "at", "72", "hpi,", "we", "decided", "to", "record", "the", "percentage", "of", "necrotic", "leaf", "tissue", "from", "72", "to", "144", "hpi", "at", "24-h-intervals.", "Results", "showed", "that", "necrosis", "increased", "with", "incubation", "time", "in", "both", "RILs,", "and", "the", "susceptible", "RIL", "LR-66-577", "had", "a", "significantly", "higher", "percentage", "of", "necrotic", "leaf", "tissue", "than", "the", "resistant", "RIL", "LR-66-637", "throughout", "this", "period.", "Fig.", "1", "The", "distinct", "differences", "in", "the", "level", "of", "resistance", "to", "Stemphylium", "botryosum", "(isolate", "SB19)", "between", "the", "Lens", "ervoides", "RILs", "LR-66-637", "and", "LR-66-577.", "a", "Lateral", "view", "of", "the", "resistant", "RIL", "LR-66-637", "(left)", "and", "the", "susceptible", "RIL", "LR-66-577", "(right)", "at", "144", "hpi.", "b", "Histopathological", "overview", "of", "SB19", "progression", "at", "48,", "96", "and", "144", "hpi", "in", "the", "two", "RILs.", "Photos", "were", "taken", "at", "200x", "magnification", "with", "bright", "field", "microscopy", "Fig.", "2", "Quantification", "of", "Stemphylium", "botryosum", "(isolate", "SB19)", "developmental", "stages", "in", "the", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577.", "a", "Assessments", "through", "histopathology", "of", "SB19", "development", "in", "two", "RILs", "from", "24", "to", "144", "hpi.", "b", "qPCR", "quantification", "of", "relative", "SB19", "biomass", "in", "two", "RILs", "from", "6", "to", "240", "hpi.", "SB19", "biomass", "was", "determined", "by", "the", "ratio", "of", "SB19", "over", "L.", "ervoides", "gDNA", "using", "qPCR", "primers", "of", "RPL-4", "(SB19", "specific)", "and", "LcEF1a", "(L.", "ervoides", "specific).", "Asterisks", "indicate", "significant", "differences", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "errors", "of", "the", "mean", "To", "confirm", "this", "trend,", "we", "then", "used", "qPCR", "to", "assess", "the", "relative", "fungal", "biomass", "(Fig.", "2", "b).", "Results", "showed", "that", "fungal", "growth", "was", "slow", "before", "48", "hpi", "but", "accelerated", "after", "48", "hpi.", "No", "statistical", "differences", "were", "seen", "between", "RILs", "before", "48", "hpi,", "which", "agrees", "with", "our", "histopathological", "observations,", "but", "significantly", "more", "fungal", "biomass", "developed", "in", "the", "susceptible", "LR-66-577", "up", "to", "the", "end", "of", "observations", "at", "240", "hpi.", "Interestingly,", "the", "relative", "fungal", "biomass", "decreased", "after", "144", "hpi", "in", "both", "resistant", "and", "susceptible", "RILs,", "indicating", "that", "expansion", "and", "growth", "of", "lentil", "plants", "outpaced", "fungal", "growth", "as", "a", "secondary", "cycle", "of", "infection", "through", "a", "new-generation", "of", "air-borne", "conidia", "of", "SB19", "was", "largely", "immobilized", "due", "to", "lack", "of", "air", "currents", "inside", "the", "bags", "that", "enclosed", "single", "pots", "to", "maintain", "high", "air", "humidity.", "Based", "on", "these", "observations,", "we", "decided", "to", "collect", "samples", "at", "48,", "96,", "and", "144", "hpi", "for", "further", "analysis.", "Analyses", "of", "variability", "among", "RILs", "samples", "at", "different", "time", "points", "To", "assess", "the", "variability", "of", "gene", "expression", "of", "RIL", "samples", "at", "different", "time", "points,", "we", "performed", "principal", "component", "analysis", "(PCA)", "to", "reduce", "the", "data", "dimensionality", "for", "ease", "of", "visualization.", "This", "revealed", "that", "the", "three", "replicates", "of", "samples", "clustered", "closely", "together", "after", "excluding", "one", "replicate", "of", "LR-66-637", "at", "0", "hpi", "which", "deviated", "from", "other", "replicates.", "This", "sample", "was", "considered", "as", "an", "outlier", "and", "was", "therefore", "discarded", "from", "the", "subsequent", "analyses.", "After", "removing", "this", "outlier,", "the", "PCA", "plot", "clearly", "separated", "samples", "of", "the", "same", "RIL", "at", "0", "and", "48", "hpi,", "but", "not", "at", "96", "and", "144", "hpi", "which", "were", "located", "together", "(Fig.", "3", "a).", "PCA", "also", "separated", "LR-66-637", "from", "LR-66-577", "samples,", "especially", "at", "96", "and", "144", "hpi.", "Fig.", "3", "Overview", "of", "transcriptome", "variabilities", "among", "24", "Stemphylium", "botryosum", "(isolate", "SB19)-infected", "samples", "of", "Lens", "ervoides", "RILs", "LR-66-637", "and", "RIL", "LR-66-577.", "a", "Principal", "component", "analysis", "of", "samples.", "Each", "point", "on", "the", "PCA", "plot", "represents", "an", "individual", "sample.", "b", "Partition", "of", "main", "effects", "of", "hours", "post", "inoculation", "(hpi),", "RILs,", "and", "the", "interaction", "of", "RIL", "×", "hpi", "using", "principal", "variation", "component", "analysis", "(PVCA)", "To", "further", "attribute", "the", "proportion", "of", "variance", "to", "known", "sources", "of", "variation,", "we", "conducted", "a", "principal", "variance", "component", "analysis", "(PVCA)", "on", "these", "samples.", "Results", "showed", "that", "hpi,", "RILs,", "and", "the", "interaction", "of", "RILs", "×", "hpi", "together", "accounted", "for", "85%", "of", "the", "total", "variance", "(Fig.", "3", "b).", "Among", "these", "effects,", "the", "largest", "variance", "proportion", "was", "contributed", "by", "hpi", "(61%),", "followed", "by", "RILs", "(15%)", "and", "the", "interaction", "of", "RILs", "×", "hpi", "(9%).", "A", "very", "small", "part", "of", "the", "variance", "(1%)", "was", "partitioned", "to", "replicates,", "which", "was", "expected.", "The", "remaining", "14%", "of", "variance", "proportion", "cannot", "be", "explained", "by", "the", "effects", "of", "the", "model", "and", "were", "attributed", "to", "the", "residual.", "Analyses", "of", "common", "disease-responsive", "genes", "The", "variable", "transcriptome", "profiles", "between", "LR-66-637", "and", "LR-66-577", "at", "0", "hpi", "indicated", "that", "differentially", "expressed", "genes", "between", "RILs", "also", "correlated", "with", "plant", "characteristics", "other", "than", "disease", "defense", "responses.", "As", "such,", "in", "order", "to", "limit", "the", "differential", "gene", "expression", "(DGE)", "analyses", "to", "those", "genes", "that", "are", "likely", "involved", "in", "disease", "defense", "responses,", "we", "first", "compared", "the", "genes", "expression", "at", "48,", "96,", "and", "144", "hpi", "with", "that", "at", "0", "hpi,", "which", "resulted", "in", "a", "total", "of", "8810", "genes", "that", "displayed", "differential", "gene", "expression", "(fold", "change", ">", "2", "or", "FDR", "<", "0.05)", "during", "the", "SB19", "infection", "process", "between", "48", "and", "144", "hpi.", "The", "expression", "heatmap", "showed", "that", "LR-66-637", "and", "LR-66-577", "exhibited", "some", "similar", "features", "in", "their", "expression", "patterns", "(Additional", "file", "1", ":", "Figure", "S1).", "These", "data", "indicated", "that", "a", "large", "proportion", "of", "disease-responsive", "genes", "reprogrammed", "in", "a", "similar", "manner", "in", "the", "resistant", "and", "susceptible", "RILs", "after", "the", "initial", "challenge", "with", "SB19.", "To", "identify", "these", "genes,", "we", "performed", "DGE", "analyses", "between", "RILs", "at", "48,", "96,", "and", "144", "hpi", "and", "found", "that", "the", "vast", "majority", "of", "the", "8810", "disease-responsive", "genes", "(7526", "genes)", "were", "not", "differentially", "expressed", "(fold", "change", "<", "2", "or", "FDR", ">", "0.05)", "between", "RILs.", "Those", "genes", "may", "be", "involved", "in", "non-host", "resistance", "of", "plants,", "which", "is", "a", "non-specific", "and", "broad-spectrum", "defense", "mechanism", "that", "is", "universally", "present", "in", "plants", "as", "a", "defense", "against", "microorganisms", "[", "25", "].", "Data", "of", "these", "7526", "genes", "were", "submitted", "to", "K-mean", "clustering", "(Fig.", "4", "a)", "and", "Gene", "Ontology", "(GO)", "mapping", "analyses", "(Fig.", "4", "b).", "As", "a", "result,", "three", "distinct", "gene", "clusters", "were", "obtained.", "Cluster", "1", "consisted", "of", "3155", "genes", "which", "were", "primarily", "downregulated", "at", "96", "and", "144", "hpi.", "The", "GO", "mapping", "analysis", "showed", "that", "the", "genes", "in", "this", "cluster", "were", "mostly", "enriched", "in", "development-related", "GO", "terms", "such", "as", "“tissue", "development”,", "“system", "development”,", "“organ", "development”,", "“nuclear", "division”,", "and", "“anatomical", "structure", "formation", "involved", "in", "morphogenesis”,", "and", "energy", "synthesis-related", "GO", "terms", "such", "as", "“photosynthesis”", "and", "“carbohydrate", "metabolic", "process”", "(Fig.", "4", "b,", "Additional", "file", "2", ":", "Table", "S1", "and", "Additional", "file", "3", ":", "Table", "S2).", "Fig.", "4", "K-mean", "clustering", "and", "GO", "enrichment", "analyses", "of", "7526", "common", "disease-responsive", "genes", "between", "the", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "a", "Gene", "expression", "heatmap", "of", "three", "hierarchically", "clustered", "(Ward", "method)", "genes.", "The", "averaged", "FPKM", "of", "three", "biological", "replicates", "was", "used", "to", "calculate", "Z-score.", "b", "Top", "10", "significant", "(FDR", "<", "0.05)", "associated", "GO", "terms", "for", "three", "gene", "clusters", "Clusters", "2", "and", "3", "contained", "1670", "and", "2701", "genes,", "respectively.", "Cluster", "2", "showed", "a", "peak", "of", "gene", "expression", "at", "48", "hpi,", "whereas", "Cluster", "3", "displayed", "peaks", "at", "96", "and", "144", "hpi.", "The", "most", "enriched", "GO", "terms", "for", "Cluster", "2", "were", "cell", "wall-related", "processes", "such", "as", "‘hemicellulose", "metabolic", "process’,", "‘cell", "wall", "polysaccharide", "metabolic", "process’,", "‘fatty", "acid", "metabolic", "process’", "and", "‘cell", "wall", "biogenesis’,", "indicating", "that", "both", "RILs", "underwent", "dramatic", "cell", "wall", "modifications", "during", "the", "early", "infection", "period.", "Disease-responsive", "genes", "in", "Cluster", "3", "relating", "to", "“response", "to", "oxidative", "stress”,", "“response", "to", "oxygen-containing", "compound”,", "and", "‘oxidation-reduction", "process’", "indicated", "that", "reactive", "oxygen", "species", "(ROS)", "were", "promoted", "in", "both", "RILs", "during", "the", "late", "SB19", "infection", "process.", "Analyses", "of", "genes", "differentially", "expressed", "between", "RILs", "Among", "the", "8810", "disease-responsive", "genes,", "1284", "genes", "were", "differentially", "expressed", "(fold", "change", ">", "2,", "FDR", "<", "0.05)", "between", "LR-66-637", "and", "LR-66-577", "at", "48,", "96", "and", "144", "hpi", "(Additional", "file", "4", ":", "Table", "S3).", "We", "arbitrarily", "selected", "eight", "of", "these", "DEGs", "upregulated", "in", "LR-66-637", "and", "another", "eight", "DEGs", "upregulated", "in", "LR-66-577", "for", "qPCR", "verification", "in", "an", "independent", "SB19", "inoculation", "experiment", "(Fig.", "5", ").", "All", "but", "one", "(94%)", "exhibited", "expression", "trends", "consistent", "with", "those", "of", "the", "RNA-Seq", "experiment,", "indicating", "that", "expression", "of", "DEGs", "is", "reproducible", "across", "independent", "experiments.", "Fig.", "5", "qPCR", "validation", "of", "arbitrarily", "selected", "16", "differential", "expressed", "genes", "identified", "in", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "Asterisks", "indicate", "the", "significant", "difference", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "error", "For", "a", "better", "visualization", "of", "genes,", "the", "1284", "DEGs", "were", "hierarchically", "clustered", "into", "five", "expression", "clusters", "(Fig.", "6", ").", "Clusters", "1", "(306", "genes)", "and", "2", "(406", "genes)", "comprised", "genes", "that", "were", "upregulated", "in", "the", "resistant", "RIL", "LR-66-637,", "whereas", "310", "genes", "in", "Cluster", "3,", "161", "in", "Cluster", "4", "and", "101", "genes", "in", "Cluster", "5", "were", "upregulated", "in", "the", "susceptible", "RIL", "LR-66-577.", "Based", "on", "their", "expression", "patterns,", "it", "was", "hypothesized", "that", "the", "712", "genes", "in", "Clusters", "1", "and", "2", "are", "correlated", "with", "the", "enhanced", "resistance", "to", "S.", "botryosum", "in", "LR-66-637,", "and", "the", "572", "genes", "in", "Clusters", "3,", "4", "and", "5", "are", "associated", "with", "susceptibility", "to", "the", "pathogen", "in", "LR-66-577.", "Fig.", "6", "K-mean", "clustering", "(Ward", "method)", "of", "1284", "differentially", "expressed", "genes", "between", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19).", "The", "identified", "five", "major", "gene", "clusters", "are", "visualized", "in", "heatmap", "(left)", "and", "line", "graph", "(right)", "To", "understand", "their", "biological", "relevance,", "we", "performed", "GO", "enrichment", "analyses", "for", "those", "DEGs", "in", "each", "of", "the", "clusters", "(Fig.", "7", ",", "Additional", "file", "5", ":", "Table", "S4", "and", "Additional", "file", "6", ":", "Table", "S5).", "Genes", "in", "Cluster", "1", "were", "primarily", "enriched", "in", "those", "involved", "in", "a", "variety", "of", "primary", "metabolic", "and", "transportation", "processes", "including", "‘carbohydrate", "metabolic", "process’,", "‘cellular", "carbohydrate", "metabolic", "process’,", "‘polysaccharide", "metabolic", "process’,", "‘transport’,", "‘establishment", "of", "localization’,", "and", "‘transmembrane", "transport’.", "In", "Cluster", "2,", "a", "large", "number", "of", "genes", "were", "enriched", "in", "several", "cell", "wall", "related", "processes", "such", "as", "‘cell", "wall", "organization", "or", "biogenesis’,", "‘cell", "wall", "polysaccharide", "metabolic", "process’,", "‘pectin", "catabolic", "process’,", "and", "‘hemicellulose", "metabolic", "process’.", "In", "Cluster", "3,", "the", "majority", "of", "genes", "were", "enriched", "for", "those", "associated", "with", "immune", "responses", "such", "as", "‘oxidation-reduction", "process’,", "‘response", "to", "stimulus’,", "and", "‘response", "to", "chemical’.", "For", "the", "remaining", "262", "genes", "in", "Clusters", "4", "and", "5,", "HR-associated", "GO", "function", "of", "‘oxidation-reduction", "process’,", "and", "two", "PCD-related", "GO", "terms", "of", "‘asparagine", "metabolic", "process’", "and", "‘asparagine", "biosynthetic", "process’", "were", "identified,", "indicating", "a", "higher", "level", "of", "HR", "activity", "in", "LR-66-577", "during", "the", "early", "SB19", "infection", "[", "26", "].", "Fig.", "7", "Significant", "(FDR", "<", "0.1)", "associated", "GO", "terms", "for", "five", "differentially", "expressed", "genes", "clusters", "identified", "from", "samples", "of", "resistant", "Lens", "ervoides", "RIL", "LR-66-637", "and", "susceptible", "RIL", "LR-66-577", "after", "infection", "with", "Stemphylium", "botryosum", "(isolate", "SB19)", "Screening", "of", "candidate", "resistance", "genes", "using", "QTL", "and", "bulk", "segregant", "gene", "expression", "analyses", "Previously,", "three", "significant", "QTL", "intervals", "associated", "with", "SB19", "resistance", "were", "identified", "in", "a", "SNP-based", "linkage", "map", "of", "RIL", "population", "LR-66", "[", "11", "].", "To", "narrow", "down", "the", "resistance", "or", "susceptibility", "gene", "candidates", "based", "on", "RNA-Seq", "here,", "the", "1284", "DEGs", "were", "compared", "to", "those", "in", "the", "QTL", "confidence", "intervals", "(2-LOD),", "which", "resulted", "in", "the", "identification", "of", "nine", "genes", "that", "were", "up-", "or", "downregulated", "in", "LR-66-637", "at", "48,", "96", "and", "144", "hpi.", "Among", "these", "nine", "genes,", "six", "genes", "were", "localized", "in", "the", "QTL", "qSB-2.2", "interval", "and", "three", "in", "the", "QTL", "qSB-3", "interval.", "Expression", "analysis", "of", "those", "genes", "in", "pools", "of", "the", "five", "most", "SB-resistant", "and", "the", "five", "most", "susceptible", "RILs", "revealed", "that", "two", "genes", "(Lc05858:", "calcium-transporting", "ATPase;", "Lc07593:", "uncharacterized", "protein)", "present", "in", "qSB-2.2", "and", "one", "gene", "(Lc12983:", "glutamate", "receptor3.2)", "in", "qSB-3", "were", "expressed", "in", "a", "manner", "similar", "to", "that", "in", "resistant", "RIL", "LR-66-637", "and", "susceptible", "LR-66-577", "based", "on", "RNA-Seq", "analysis", "(Fig.", "8", "and", "Additional", "file", "7", ":", "Table", "S6).", "Quantitative", "PCR", "for", "another", "two", "of", "these", "genes", "(Lc09908", "and", "Lc07065)", "was", "inconclusive,", "and", "four", "genes", "(Lc06015,", "Lc10098,", "Lc11261,", "and", "Lc13725)", "displayed", "similar", "expression", "between", "the", "two", "pools,", "indicating", "that", "they", "are", "more", "likely", "associated", "with", "other", "differences", "specific", "to", "LR-66-637", "and", "LR-66-577.", "Fig.", "8", "Bulk", "segregation", "gene", "expression", "analysis", "of", "three", "genes", "that", "co-localize", "within", "two", "putative", "Stemphylium", "botryosum", "(isolate", "SB19)", "resistance", "QTL", "intervals", "on", "the", "genetic", "map", "of", "Lens", "ervoides.", "a", "Projection", "of", "QTL", "brackets", "from", "linkage", "group", "to", "the", "physical", "chromosomal", "regions.", "The", "linkage", "maps", "(left)", "show", "the", "arrangement", "of", "SNP", "markers", "distributing", "in", "2-LOD", "QTL", "intervals", "(Bhadauria", "et", "al.,", "2017).", "The", "right", "column", "shows", "the", "projected", "QTL", "putative", "regions", "(yellow)", "in", "the", "corresponding", "physical", "chromosomal", "regions.", "b", "Expression", "profiling", "of", "three", "genes", "between", "pools", "of", "SB-resistant", "and", "susceptible", "RILs.", "Lc05858", "and", "Lc07593", "are", "in", "qSB-2.2", "QTL", "region,", "and", "Lc12983", "is", "in", "qSB-3", "interval.", "Asterisks", "indicate", "the", "significant", "difference", "between", "means", "at", "P", "<", "0.05.", "Error", "bars", "represent", "standard", "error", "For", "Lc07593,", "it", "was", "observed", "that", "this", "gene", "was", "highly", "expressed", "in", "the", "SB19-susceptible", "RILs", "compared", "to", "the", "resistant", "RILs,", "suggesting", "that", "Lc07593", "could", "be", "a", "susceptibility", "gene,", "upregulation", "of", "which", "seemed", "to", "increase", "SB", "susceptibility.", "On", "the", "other", "hand,", "Lc05858", "and", "Lc12983", "could", "be", "disease", "resistance", "genes,", "as", "they", "displayed", "a", "reverse", "expression", "trend", "to", "that", "of", "Lc07593.", "Discussion", "A", "thorough", "understanding", "of", "how", "pathogens", "interact", "with", "their", "hosts", "is", "essential", "to", "identify", "appropriate", "times", "during", "the", "infection", "process", "for", "the", "profiling", "of", "the", "transcriptome.", "In", "the", "present", "study,", "the", "difference", "in", "SB19", "development", "between", "the", "resistant", "RIL", "LR-66-637", "and", "the", "susceptible", "RIL", "LR-66-577", "was", "negligible", "before", "24", "hpi,", "indicating", "that", "the", "resistance", "toward", "SB19", "in", "RIL", "LR-66-637", "was", "probably", "based", "on", "post-penetration", "inhibition", "of", "the", "fungus.", "Similar", "observations", "were", "also", "reported", "for", "S.", "botryosum", "infecting", "alfalfa", "where", "Cowling", "and", "Gilchrist", "[", "27", "]", "found", "that", "the", "development", "of", "S.", "botryosum", "did", "not", "differ", "among", "the", "resistant,", "moderately", "resistant", "and", "susceptible", "alfalfa", "clones", "during", "the", "early", "period", "of", "infection.", "During", "the", "period", "from", "48", "to", "144", "hpi,", "fungal", "biomass", "started", "to", "accumulate", "in", "RILs,", "suggesting", "that", "S.", "botryosum", "initiated", "the", "release", "of", "phytotoxins", "to", "disrupt", "host", "cells", "around", "48", "hpi.", "Biomass", "accumulation", "was", "significantly", "higher", "in", "LR-66-577", "than", "in", "LR-66-637,", "possibly", "as", "a", "result", "of", "enhanced", "tolerance", "to", "phytotoxins", "in", "LR-66-637", "compared", "to", "LR-66-577", "[", "28", "].", "As", "such,", "48,", "96", "and", "144", "hpi", "were", "considered", "to", "be", "interesting", "time", "points", "warranting", "further", "exploration", "of", "gene", "expression.", "PCA", "and", "PVCA", "are", "useful", "tools", "to", "visualize", "the", "overall", "gene", "expression", "variability", "and", "to", "detect", "outliers", "among", "samples", "[", "29", "].", "After", "excluding", "an", "outlier", "in", "the", "PCA", "plot", "here,", "separation", "of", "those", "samples", "collected", "at", "48", "hpi", "from", "others", "gathered", "at", "96", "and", "144", "hpi", "was", "evident,", "which", "clearly", "reflected", "that", "gene", "reprogramming", "at", "96", "and", "144", "hpi", "was", "similar,", "but", "was", "different", "from", "48", "hpi.", "PCA", "also", "separated", "LR-66-637", "from", "LR-66-577", "samples,", "indicating", "that", "the", "transcriptome", "responses", "of", "LR-66-637", "and", "LR-66-577", "were", "different", "after", "challenge", "with", "SB19.", "As", "this", "separation", "was", "more", "evident", "at", "96", "or", "144", "hpi", "than", "at", "48", "hpi,", "a", "significant", "interaction", "effect", "of", "hpi", "×", "RIL", "was", "expected.", "These", "results", "confirmed", "that", "the", "transcriptomes", "of", "RILs", "responded", "to", "SB19", "infection", "in", "a", "time-dependent", "manner", "as", "has", "been", "observed", "in", "other", "transcriptome", "studies", "[", "30", "–", "32", "].", "Non-host", "resistance", "is", "a", "non-specific", "and", "broad-spectrum", "defense", "mechanism", "that", "is", "universally", "present", "in", "plants", "as", "a", "defense", "against", "microorganisms", "[", "25", ",", "33", "].", "By", "analyzing", "those", "disease-responsive", "genes", "of", "both", "RILs,", "we", "observed", "that", "a", "large", "number", "of", "genes", "downregulated", "in", "both", "RILs", "were", "enriched", "in", "a", "series", "of", "development-related", "and", "energy", "synthesis-related", "GO", "terms.", "These", "results", "seemed", "to", "indicate", "that", "SB19", "infection", "disrupted", "the", "normal", "growth", "and", "energy", "synthesis", "of", "both", "RILs", "regardless", "of", "their", "varying", "levels", "of", "resistance", "to", "the", "pathogen.", "As", "the", "resistance", "towards", "necrotrophs", "in", "plants", "is", "always", "in", "an", "incomplete", "form,", "the", "withdrawal", "of", "nutrients", "by", "S.", "botryosum", "from", "plants", "normally", "occurs", "in", "all", "individuals", "of", "the", "host", "species", "[", "34", "].", "Pathogen", "infection", "leads", "to", "a", "decrease", "in", "photosynthesis", "at", "the", "infection", "sites,", "which", "negatively", "impacts", "the", "energy", "assimilation", "and", "interferes", "with", "the", "normal", "development", "and", "growth", "of", "the", "plant", "[", "35", "].", "This", "has", "been", "extensively", "reported", "in", "a", "variety", "of", "diseased", "plants", "[", "36", "–", "38", "].", "On", "the", "other", "hand,", "there", "were", "also", "genes", "that", "were", "upregulated", "after", "inoculation,", "either", "at", "48", "hpi", "or", "at", "96", "and", "144", "hpi.", "As", "these", "genes", "were", "mostly", "enriched", "in", "cell", "wall-related", "and", "oxidation-reduction", "processes,", "it", "seemed", "that", "cell", "wall", "modifications", "and", "ROS", "were", "promoted", "at", "certain", "time", "points", "during", "the", "SB19", "infection", "process.", "Any", "type", "of", "interaction", "between", "pathogen", "and", "host", "will", "trigger", "some", "degrees", "of", "cell", "wall", "modifications", "[", "25", ",", "33", ",", "34", "].", "When", "plants", "are", "stressed", "by", "pathogens,", "variable", "amounts", "of", "ROS", "are", "released", "to", "activate", "the", "downstream", "signal", "transduction", "pathways", "and", "expression", "of", "a", "series", "of", "defense", "genes", "[", "39", "–", "41", "].", "The", "activation", "of", "ROS", "is", "a", "common", "form", "of", "non-host", "resistance", "that", "universally", "occurs", "in", "plants,", "regardless", "of", "their", "difference", "in", "the", "level", "of", "disease", "resistance", "[", "42", "–", "44", "].", "In", "addition", "to", "these", "common", "disease-responsive", "genes,", "another", "1284", "disease-responsive", "genes", "were", "differentially", "expressed", "between", "LR-66-637", "and", "LR-66-577.", "Some", "of", "these", "genes", "may", "contribute", "to", "the", "difference", "in", "the", "level", "of", "disease", "resistance", "between", "the", "two", "RILs.", "According", "to", "the", "expression", "patterns", "and", "enriched", "GO", "terms", "of", "these", "genes,", "we", "found", "that", "LR-66-637", "mainly", "upregulated", "a", "variety", "of", "carbohydrate", "metabolic,", "transportation", "and", "cell", "wall", "related", "processes.", "Other", "gene", "expression", "data", "on", "the", "lentil", "-", "S.", "botryosum", "system", "have", "not", "been", "reported", "to", "date,", "but", "stronger", "upregulation", "of", "a", "series", "of", "metabolic", "processes", "(organic", "substance", "metabolic,", "single-organism", "metabolic,", "or", "cellular", "glucan", "metabolic)", "based", "on", "RNA-Seq", "experiments", "were", "reported", "in", "the", "resistant", "lentil", "genotypes", "CDC-Robin,", "964a-46,", "or", "ILL7537", "after", "inoculation", "of", "Ascochyta", "lentis,", "another", "lentil", "pathogen", "presumed", "to", "be", "a", "necrotroph", "[", "15", ",", "16", "].", "In", "addition,", "Yang", "et", "al.", "[", "45", "]", "studied", "the", "tomato", "–", "S.", "lycopersici", "system", "and", "found", "that", "several", "GO", "terms", "of", "‘localization’,", "‘transporter", "activity’", "and", "‘molecular", "transducer", "activity’", "were", "activated", "in", "the", "resistant", "tomato", "genotype,", "proposing", "that", "the", "upregulation", "of", "these", "activities", "were", "related", "to", "S.", "lycopersici", "resistance.", "Carbohydrates", "and", "transporters", "are", "considered", "essential", "parts", "of", "plant", "immune", "systems,", "with", "the", "former", "generating", "energy", "to", "sustain", "the", "continuous", "production", "of", "a", "series", "of", "defense-related", "metabolites", "and", "carbon-based", "polymers,", "and", "the", "latter", "fulfilling", "signal", "transduction,", "energy", "translocation", "and", "also", "extrusion", "of", "toxic", "compounds", "[", "46", "–", "48", "].", "Theoretically,", "the", "promotion", "of", "these", "processes", "is", "in", "accordance", "with", "elevated", "disease", "resistance", "against", "necrotrophs", "[", "35", ",", "48", ",", "49", "].", "Morkunas", "et", "al.", "[", "50", "]", "found", "that", "a", "high", "level", "of", "sucrose", "accounted", "for", "the", "accumulation", "of", "a", "variety", "of", "flavonoids", "which", "resulted", "in", "enhanced", "resistance", "in", "yellow", "lupin", "to", "Fusarium", "oxysporum.", "Likewise,", "it", "was", "reported", "that", "the", "upregulation", "of", "the", "two", "plant", "ATP", "binding", "cassette", "transporters", "NpPDR1", "and", "PEN3,", "could", "enhance", "disease", "resistance", "to", "the", "necrotrophic", "pathogens", "Botrytis", "cinerea", "and", "Plectosphaerella", "cucumerina", "in", "Arabidopsis,", "respectively", "[", "49", ",", "51", "].", "The", "promotion", "of", "several", "cell", "wall", "related", "processes", "suggested", "that", "LR-66-637", "initiated", "additional", "cell", "wall", "modification", "compared", "to", "LR-66-577.", "In", "the", "lentil", "–", "A.", "lentis", "system,", "Khorramdelazad", "et", "al.", "[", "15", "]", "also", "reported", "the", "significant", "enrichment", "for", "the", "GO", "term", "‘cell", "wall’", "in", "the", "resistant", "genotype", "ILL7537.", "During", "attack", "by", "necrotrophic", "pathogens,", "the", "cell", "wall", "of", "a", "host", "is", "damaged", "by", "a", "series", "of", "cell", "wall-degrading", "enzymes", "released", "by", "the", "pathogen.", "If", "such", "cell", "wall", "degradation", "can", "be", "arrested", "by", "the", "host,", "a", "resistant", "phenotype", "would", "be", "expected", "[", "52", "].", "In", "tomato,", "Miedes", "and", "Lorences", "[", "53", "]", "found", "that", "the", "appropriate", "level", "of", "xyloglucan", "(a", "form", "of", "hemicellulose)", "was", "essential", "for", "maintaining", "cell", "wall", "integrity", "when", "under", "attack", "from", "Penicillium", "expansum.", "In", "Arabidopsis,", "Lionetti", "et", "al.", "[", "54", "]", "found", "that", "B.", "cinerea", "resistance", "was", "enhanced", "through", "an", "increase", "of", "pectin", "methyl", "esterification", "which", "disrupted", "the", "ability", "of", "microbes", "to", "degrade", "the", "plant", "cell", "wall.", "As", "such,", "we", "hypothesize", "that", "the", "upregulation", "of", "genes", "in", "cell", "wall", "processes,", "to", "a", "certain", "degree,", "may", "compensate", "for", "the", "cell", "wall", "damage", "through", "infection", "and", "thus", "improve", "SB", "resistance", "in", "LR-66-637.", "In", "contrast,", "LR-66-577", "appeared", "to", "respond", "to", "SB19", "invasion", "in", "a", "completely", "different", "form", "considering", "that", "the", "majority", "of", "genes", "upregulated", "in", "this", "RIL", "were", "enriched", "for", "those", "involved", "in", "‘oxidation-reduction", "process’.", "This", "suggests", "that", "redox", "homeostasis", "was", "substantially", "altered", "in", "LR-66-577", "after", "SB19", "invasion,", "which", "was", "reported", "for", "other", "systems", "previously", "[", "55", "].", "A", "change", "in", "redox", "status", "in", "the", "challenged", "cells", "could", "release", "a", "variety", "of", "reactive", "oxygen", "species", "including", "singlet", "oxygen,", "superoxide", "radicals", "and", "hydrogen", "peroxide,", "accumulation", "of", "which", "could", "trigger", "the", "plant’s", "hypersensitive", "response", "and", "programmed", "cell", "death", "(PCD)", "to", "cause", "leaf", "necrosis", "and,", "eventually,", "leaf", "dehiscence", "[", "56", ",", "57", "].", "As", "necrotrophs", "can", "thrive", "in", "dead", "tissues,", "several", "studies", "have", "reported", "that", "necrotrophs", "are", "able", "to", "hijack", "the", "plant", "immune", "system", "and", "use", "the", "host", "HR", "machinery", "to", "boost", "their", "virulence", "[", "39", ",", "58", ",", "59", "].", "From", "these", "results,", "we", "hypothesize", "that", "HR", "and", "PCD", "were", "significantly", "more", "promoted", "in", "LR-66-577", "than", "LR-66-637", "during", "the", "SB19", "infection", "process,", "resulting", "in", "susceptibility", "in", "LR-66-577.", "Previous", "evidences", "has", "shown", "that", "HR", "and", "PCD", "are", "largely", "induced", "through", "the", "activation", "of", "R", "genes", "[", "56", "–", "59", "].", "In", "the", "present", "study,", "we", "observed", "that", "a", "number", "of", "TIR-NBS-LRR", "and", "CC-NBS-LRR", "genes", "were", "stronger", "upregulated", "in", "LR-66-577", "than", "LR-66-637", "during", "the", "infection", "process", "(Additional", "file", "5", ":", "Table", "S4).", "In", "the", "lentil", "–", "A.", "lentis", "system,", "Sari", "et", "al.", "[", "16", "]", "found", "a", "TIR-NBS-LRR", "gene", "that", "was", "also", "significantly", "upregulated", "in", "two", "susceptible", "lentil", "genotypes", "after", "inoculation", "of", "A.", "lentis.", "Such", "results", "corroborate", "an", "association", "of", "R", "genes,", "cell", "death", "and", "susceptibility", "after", "infection", "by", "necrotrophic", "pathogen", "[", "39", "].", "However,", "there", "was", "another", "set", "of", "TIR-NBS-LRR,", "NBS-LRR,", "and", "NB-ARC", "genes", "that", "had", "higher", "expression", "in", "LR-66-637", "than", "LR-66-577,", "as", "would", "be", "expected", "for", "resistant", "genotypes.", "Involvement", "of", "such", "R", "genes", "was", "also", "reported", "in", "the", "lentil", "–", "A.", "lentis", "system,", "and", "Khorramdelazad", "et", "al.", "[", "15", "]", "speculated", "that", "a", "stronger", "upregulation", "of", "a", "NB-ARC", "gene", "conferred", "enhanced", "resistance", "in", "ILL", "7537.", "Taken", "together,", "this", "supports", "the", "emerging", "picture", "of", "R-genes", "as", "true", "resistance", "genes", "as", "well", "as", "their", "possible", "role", "as", "susceptibility", "factors", "[", "34", "].", "Comparing", "DEGs", "with", "previously", "identified", "resistance", "QTLs", "of", "LR-66", "[", "11", "],", "we", "identified", "nine", "resistance", "gene", "candidates", "and", "explored", "them", "further", "through", "bulk", "segregation", "analysis.", "In", "a", "bulk", "segregation", "analysis", "(BSA),", "the", "phenotypic", "extremes", "of", "two", "pools", "tend", "to", "either", "differ", "in", "the", "presence", "of", "the", "gene(s)", "characterizing", "the", "phenotype,", "or", "differ", "in", "the", "expression", "level", "of", "the", "gene(s)", "responsible", "for", "the", "trait", "[", "60", "].", "BSA", "coupled", "with", "gene", "expression", "studies", "has", "been", "used", "frequently", "to", "identify", "candidate", "genes", "in", "varieties", "of", "plant", "species", "(e.g.", "[", "61", "–", "63", "]).", "BSA", "was", "used", "here", "and", "revealed", "a", "calcium-transporting", "ATPase", "(Lc05858)", "and", "a", "receptor3.2", "(Lc12983)", "with", "a", "high", "expression", "in", "the", "resistant", "pool", "indicating", "that", "they", "could", "be", "putative", "resistance", "genes.", "A", "calcium", "transporting", "ATPase,", "such", "as", "Lc05858,", "could", "play", "an", "important", "role", "in", "calcium", "influx", "and", "efflux", "regulation", "to", "maintain", "an", "appropriate", "cellular", "calcium", "homeostasis,", "the", "change", "of", "which", "is", "thought", "to", "initiate", "calcium", "signaling", "that", "triggers", "a", "series", "of", "downstream", "responses", "influencing", "plant", "growth,", "development", "and", "responses", "to", "biotic", "and", "abiotic", "stresses", "[", "64", "–", "66", "].", "Zhu", "et", "al.", "[", "67", "]", "reported", "that", "the", "expression", "of", "a", "gene", "coding", "calcium", "ATPase", "decreased", "HR", "in", "Pseudomonas", "syringae-infected", "tomatoes", "as", "NbCA1", "(ER-localized", "type", "IIB", "calcium", "ATPase)-silenced", "plants", "exhibited", "accelerated", "programmed", "cell", "death", "(PCD)", "compared", "to", "the", "wild", "type.", "Similarly,", "Boursiac", "et", "al.", "[", "68", "]", "found", "that", "the", "knockout", "of", "ACA4", "and", "ACA11", "(two", "calcium-ATPases)", "induced", "a", "higher", "frequency", "of", "HR-like", "lesions", "by", "deactivating", "the", "calcium", "signaling", "in", "Arabidopsis", "inoculated", "with", "P.", "syringae.", "Based", "on", "this", "it", "can", "be", "speculated", "that", "upregulation", "of", "Lc05858", "during", "SB19", "infection", "results", "in", "the", "activation", "of", "calcium", "transportation", "and", "the", "suppression", "of", "HR", "and", "PCD.", "Upregulation", "of", "the", "glutamate", "receptor3.2", "Lc12983", "in", "the", "resistant", "RILs", "compared", "to", "the", "susceptible", "RILs", "at", "48", "and", "144", "hpi", "may", "also", "affect", "calcium", "regulation,", "highlighting", "its", "potential", "importance", "in", "the", "lentil", "–S.", "botryosum", "interaction.", "A", "previous", "study", "showed", "that", "a", "glutamate", "receptor", "is", "mainly", "responsible", "for", "glutamate-dependent", "membrane", "depolarization", "and", "calcium", "transportation", "[", "69", "],", "and", "Kang", "et", "al.", "[", "70", "]", "and", "Manzoor", "et", "al.", "[", "71", "]", "observed", "that", "the", "upregulation", "of", "glutamate-like", "receptors", "AtGLR3.3", "or", "AtGluR3.2", "elevated", "calcium", "influx", "and", "conferred", "enhanced", "resistance", "in", "Arabidopsis", "against", "the", "necrotroph", "B.", "cinerea.", "However,", "further", "functional", "studies", "are", "required", "to", "confirm", "the", "role", "of", "these", "genes", "in", "resistance.", "One", "gene", "encoding", "an", "uncharacterized", "protein", "(Lc07593)", "was", "highly", "expressed", "in", "the", "SB19-susceptible", "RILs", "compared", "to", "the", "resistant", "RILs,", "suggesting", "that", "Lc07593", "could", "be", "a", "susceptibility", "gene.", "However,", "this", "gene", "is", "not", "homologous", "to", "any", "well", "characterized", "genes", "in", "the", "database", "and", "thus", "its", "function", "is", "not", "understood.", "Future", "work", "involving", "protein", "purification,", "structural", "modeling,", "and", "protein-protein", "interaction", "analyses", "may", "be", "needed", "to", "understand", "its", "biological", "function.", "Methods", "Plant", "material", "and", "inoculum", "preparation", "RILs", "LR-66-577", "(susceptible", "to", "SB)", "and", "LR-66-637", "(resistant", "to", "SB)", "used", "in", "this", "study", "were", "transgressive", "segregants", "chosen", "from", "the", "L.", "ervoides", "RIL", "population", "LR-66", "derived", "from", "the", "cross", "of", "L01-827A", "×", "IG", "72815", "developed", "at", "the", "Crop", "Development", "Centre", "at", "the", "University", "of", "Saskatchewan", "(Saskatoon,", "SK,", "Canada)", "[", "11", "].", "Evident", "transgressive", "segregation", "and", "disease", "severity", "in", "RILs", "ranging", "from", "11", "to", "80%", "in", "this", "population", "indicated", "that", "there", "was", "fluent", "genetic", "variability", "in", "SB", "resistance", "[", "11", "].", "Prior", "to", "planting,", "seeds", "of", "RILs", "were", "scarified", "to", "facilitate", "germination.", "The", "scarified", "seeds", "were", "sowed", "in", "10-cm", "plastic", "pots", "filled", "with", "Fafard®", "Germination", "Mix", "(Sungro", "Horticulture®", "Ltd.,", "Vancouver,", "BC,", "Canada).", "All", "plants", "were", "grown", "in", "a", "growth", "chamber", "with", "a", "constant", "temperature", "of", "23", "°C", "and", "a", "day", "length", "of", "16", "h", "light/8", "h", "dark.", "Only", "four", "plants", "per", "pot", "were", "kept", "for", "the", "subsequent", "inoculation", "experiment.", "The", "inoculum", "was", "prepared", "from", "the", "aggressive", "S.", "botryosum", "isolate", "SB19", "which", "originated", "from", "the", "southeast", "of", "Saskatchewan,", "Canada.", "The", "cryopreserved", "SB19", "spores", "were", "revitalized", "on", "oatmeal", "V8", "agar", "medium", "[150", "mL", "V8", "juice", "(Campbell", "Co.,", "Canada),", "10", "g", "Difco™", "Potato", "Dextrose", "Agar", "(Becton", "Dickinson", "and", "Co.,", "Franklin", "Lake,", "NJ,", "USA),", "3", "g", "calcium", "carbonate,", "850", "mL", "sterile", "water].", "After", "seven", "days", "of", "incubation", "at", "room", "temperature,", "conidia", "were", "collected", "from", "Petri", "dishes", "by", "flooding", "and", "scraping", "the", "surface", "of", "the", "plates.", "The", "conidial", "suspension", "was", "filtered", "through", "two", "layers", "of", "miracloth", "and", "adjusted", "to", "1", "×", "10", "5", "condia", "mL", "−", "1", "using", "a", "hemocytometer.", "Two", "droplets", "of", "Tween", "20", "(Sigma,", "Saint", "Louis,", "MO,", "USA)", "were", "added", "to", "every", "1000", "mL", "of", "suspension", "before", "inoculation.", "Pathogen", "inoculation", "and", "experimental", "design", "Four-week", "old", "plants", "were", "sprayed", "with", "the", "conidial", "suspension", "until", "run", "off,", "which", "was", "equivalent", "to", "approximate", "2", "to", "2.5", "mL", "per", "plant.", "Four", "plants", "within", "a", "pot", "represented", "one", "of", "three", "biological", "replicates", "and", "were", "pooled", "to", "generate", "a", "biological", "replicate.", "Pots", "were", "arranged", "in", "a", "completely", "randomized", "design", "in", "a", "misting", "chamber", "at", "100%", "humidity", "for", "the", "first", "48", "h", "to", "favor", "germination", "of", "conidia.", "After", "48", "hpi,", "those", "inoculated", "plants", "were", "removed", "from", "the", "misting", "chamber,", "each", "pot", "was", "enclosed", "in", "a", "translucent", "plastic", "bag,", "and", "pots", "were", "maintained", "at", "23", "°C", "and", "16", "h", "photoperiod.", "Leaves", "were", "sampled", "at", "0,", "6,", "12,", "24,", "48,", "72,", "96,", "120,", "144,", "196,", "and", "240", "hpi,", "immediately", "frozen", "in", "liquid", "nitrogen", "and", "then", "stored", "in", "a", "-80", "°C", "freezer", "for", "future", "uses.", "Determination", "of", "fungal", "development", "Microscopy-based", "methods", "and", "qPCR", "were", "used", "to", "quantify", "growth", "of", "SB19", "in", "plants.", "For", "qPCR,", "the", "genes", "targeting", "RNA", "polymerase", "II", "second", "largest", "subunit", "(RPL-4)", "to", "amplify", "gDNA", "of", "SB19", "and", "elongation", "factor", "(LcEF1α)", "to", "quantify", "L.", "ervoides", "were", "used", "(Additional", "file", "8", ":", "Table", "S7).", "The", "SDS", "method", "[", "72", "]", "was", "used", "to", "extract", "gDNA", "from", "both", "organisms", "and", "a", "spectrophotometer", "(NanoDrop™", "8000,", "Thermo", "Scientific,", "Waltham,", "USA)", "was", "employed", "to", "quantify", "the", "DNA", "concentration,", "which", "was", "adjusted", "to", "25", "ng", "μl", "−", "1", "for", "all", "samples.", "Each", "qPCR", "reaction", "contained", "2", "μl", "DNA", "template,", "5", "μl", "SYBR®", "Green", "(catalog", "no.", "4309155,", "Thermo", "Scientific),", "0.2", "μl", "each", "of", "10", "μM", "forward", "and", "reverse", "primers,", "and", "2.6", "μl", "molecular", "grade", "water.", "A", "default", "fast-run", "program", "was", "used", "to", "perform", "qPCR", "amplification", "in", "a", "QuantStudio™", "3", "System", "(Applied", "Biosystems", "Inc.,", "Foster", "City,", "CA,", "USA).", "According", "to", "the", "criteria", "proposed", "by", "Weβling", "and", "Panstruga", "[", "24", "],", "the", "relative", "fungal", "biomass", "was", "estimated", "as", "the", "proportion", "of", "gDNA", "amplified", "between", "RPL-4", "and", "LcEF1α.", "For", "microscopic", "quantification,", "six", "leaflets", "per", "biological", "replicate", "were", "collected", "at", "each", "of", "6,", "12,", "24,", "48,", "72,", "96,", "120,", "144,", "196,", "and", "240", "hpi.", "The", "collected", "leaflets", "were", "cleared", "in", "CMAA", "fixation", "solution", "(60%", "methanol,", "30%", "chloroform,", "10%", "acetic", "acid)", "for", "at", "least", "24", "h", "at", "room", "temperature", "to", "eliminate", "chlorophyll", "in", "cells.", "Once", "treated,", "they", "were", "immersed", "in", "a", "series", "of", "decreasing", "ethanol", "concentrations", "of", "70%", "for", "1", "h,", "50%", "for", "1.5", "h", "and", "30%", "for", "1.5", "h.", "Staining", "was", "performed", "by", "immersing", "leaflets", "in", "0.05%", "trypan", "blue", "overnight.", "Well", "stained", "leaflets", "were", "mounted", "on", "glass", "slides", "in", "a", "droplet", "of", "50%", "glycerol", "and", "evaluated", "under", "a", "Zeiss", "Axioskop", "40", "microscope", "(Carl", "Zeiss,", "Göttingen,", "Germany).", "High-quality", "photos", "were", "generated", "using", "a", "Pixelink", "A686C", "camera", "(Pixelink,", "Rochester,", "NY,", "USA)", "and", "Zeiss", "Axiovision", "software", "(Carl", "Zeiss,", "Oberkochen,", "Germany).", "To", "quantify", "fungal", "development", "during", "the", "incubation", "process,", "we", "record", "percentage", "of", "conidial", "germination,", "percentage", "of", "conidia", "resulting", "in", "successful", "penetration", "(%", "conidial", "penetration),", "and", "germ", "tube", "length.", "Furthermore,", "the", "percentage", "area", "of", "dead", "tissue", "per", "leaflet", "was", "visually", "estimated", "(%", "leaf", "necrosis).", "Data", "were", "analyzed", "using", "SAS", "9.3", "(SAS", "Institute,", "Inc.,", "Cary,", "NC,", "USA).", "Normality", "of", "errors", "was", "assessed", "with", "the", "Shapiro-Wilk", "test", "and", "homogeneity", "of", "variance", "with", "the", "Levene’s", "tests.", "The", "mean", "separation", "between", "RILs", "were", "conducted", "using", "Student’s", "t-test", "method", "(P", "<", "0.05).", "RNA", "sequencing", "and", "raw", "data", "processing", "Samples", "were", "collected", "at", "0,", "48,", "96,", "and", "144", "hpi", "with", "three", "biological", "replicates", "for", "LR-66-570", "and", "LR-66-629,", "for", "a", "total", "of", "24", "samples.", "Total", "RNA", "extraction", "was", "performed", "from", "the", "frozen", "leaves", "using", "the", "RNeasy", "Plant", "Mini", "Kit", "(Cat", "no.", "74904,", "Qiagen", "Company,", "Hilden,", "Germany)", "following", "the", "manufacturer’s", "instructions.", "The", "quality", "and", "concentration", "of", "RNA", "was", "assessed", "in", "an", "Agilent", "2100", "Bioanalyzer", "(Agilent", "Technologies,", "Santa", "Clara,", "CA,", "USA).", "Only", "high-quality", "RNA", "samples", "(RNA", "integrity", "number", ">", "7)", "were", "used", "for", "library", "construction", "using", "TruSeq", "Stranded", "Total", "RNA", "(Illumina,", "Inc.,", "San", "Diego,", "CA,", "USA).", "The", "constructed", "libraries", "were", "then", "loaded", "into", "a", "HiSeq", "2500", "system", "using", "TruSeq", "SBS", "KIT-HS", "V4", "(Illumina)", "for", "125", "bp", "pair-ended", "sequencing.", "The", "quality", "control", "of", "returned", "raw", "reads", "was", "implemented", "in", "Trimmomatic", "(version", "0.36)", "[", "73", "]", "to", "remove", "low-quality", "reads", "and", "adaptors", "under", "the", "following", "parameters:", "TruSeq3-PE-2.fa:2:30:10,", "leading:3,", "trailing:3,", "slidingwindow:4:15", "and", "minlen:36.", "The", "cleaned", "reads", "were", "aligned", "against", "the", "reference", "genome", "of", "Lens", "culinaris", "V1.2", "[", "21", "]", "using", "STAR", "(default", "settings,", "version", "2.6.1a)", "[", "74", "].", "As", "a", "result,", "approximate", "90%", "of", "reads", "were", "uniquely", "aligned", "to", "the", "reference", "genome", "for", "all", "samples,", "confirming", "the", "high", "quality", "of", "sequencing", "and", "mapping", "(Additional", "file", "9", ":", "Figure", "S2).", "Gene", "counting", "was", "performed", "using", "STAR", "quantmode", "during", "the", "mapping", "process.", "The", "resulting", "gene", "expression", "data", "for", "each", "sample", "were", "first", "normalized", "using", "the", "fragments", "per", "kilobase", "of", "transcript", "per", "million", "(FPKM)", "method", "before", "proceeding", "to", "principal", "component", "analysis", "(PCA)", "and", "principal", "variance", "component", "analysis", "(PVCA)", "in", "the", "statistical", "software", "JMP", "Genomics", "8.0", "(JMP", "Genomics®,", "SAS", "Institute).", "Identification", "of", "differentially", "expressed", "genes", "(DEGs)", "Before", "comparing", "the", "gene", "expression", "between", "RILs,", "we", "first", "conducted", "comparisons", "of", "gene", "expression", "in", "samples", "from", "0", "hpi", "with", "those", "collected", "at", "48,", "96,", "and", "144", "hpi", "for", "each", "RIL", "to", "identify", "those", "genes", "that", "significantly", "responded", "to", "SB19", "infection.", "These", "comparisons", "were", "performed", "in", "the", "R", "package", "DESeq2", "[", "75", "]", "using", "the", "thresholds", "of", "false", "discovery", "rate", "(FDR)", "<", "0.05", "and", "gene", "expression", "fold", "change", ">", "2.", "Among", "the", "identified", "disease-responsive", "genes,", "we", "then", "conducted", "pair-wise", "comparisons", "between", "LR-66-637", "and", "LR-66-577", "samples", "from", "each", "time", "point", "(48,", "96", "and", "144", "hpi).", "The", "same", "thresholds", "used", "previously", "were", "applied", "here", "to", "declare", "significant", "DEGs.", "The", "resulting", "DEGs", "were", "submitted", "to", "JMP", "Genomics", "8.0", "(JMP", "Genomics®,", "SAS", "Institute)", "for", "K-means", "clustering", "analysis.", "The", "genes", "of", "each", "cluster", "with", "known", "Medicago", "truncatula", "orthologs", "were", "then", "mapped", "to", "the", "Gene", "Ontology", "(GO)", "database", "using", "the", "PANTHER", "(version", "14.1)", "(", "http://www.pantherdb.org", ").", "qPCR", "validation", "To", "verify", "the", "repeatability", "of", "gene", "expression", "captured", "in", "the", "RNA-Seq", "experiment,", "an", "independently", "inoculated", "experiment", "with", "LR-66-570", "and", "LR-66-629", "was", "conducted", "following", "the", "same", "experimental", "conditions", "and", "setup", "as", "described", "before.", "Infected", "leaf", "samples", "were", "collected", "and", "processed", "as", "described", "before,", "and", "16", "DEGs", "identified", "from", "RNA-Seq", "data", "analysis", "and", "one", "reference", "gene", "(LcEF1α)", "were", "used", "for", "qPCR", "amplification", "(Additional", "file", "10", ":", "Table", "S8).", "Each", "PCR", "reaction", "consisted", "of", "2", "μl", "DNA", "template,", "5", "μl", "SYBR", "Green,", "0.2", "μl", "of", "each", "10", "μM", "forward", "and", "reverse", "primers,", "and", "2.6", "μl", "molecular", "grade", "water.", "qPCR", "was", "performed", "in", "a", "QuantStudio™", "3", "System", "(Applied", "Biosystems", "Inc.)", "using", "the", "fast-run", "program", "with", "default", "settings.", "The", "relative", "expression", "of", "each", "gene", "of", "interest", "was", "calculated", "as", "2", "−", "CT", "gene", "of", "interest", "−", "CT", "reference", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$", "{2}^{-\\left({CT}_{gene\\", "of\\", "interest}-{CT}_{reference}\\right)}", "$$\\end{document}", "recommended", "by", "Livak", "and", "Schmittgen", "[", "76", "].", "Mean", "separation", "between", "RILs", "was", "conducted", "using", "the", "Student’s", "t-test", "method", "(P", "<", "0.05).", "Bulk", "segregant", "gene", "expression", "analysis", "Based", "on", "the", "results", "of", "the", "phenotypic", "evaluation", "of", "the", "LR-66", "population", "[", "11", "],", "the", "five", "top", "SB-resistant", "RILs", "(LR-66-526,", "LR-66-543,", "LR-66-643,", "LR-66-658,", "and", "LR-66-712)", "and", "the", "five", "most", "SB-susceptible", "RILs", "(LR-66-594,", "LR-66-605,", "LR-66-697,", "LR-66-706,", "and", "LR-66-727)", "were", "selected", "to", "construct", "resistant", "and", "susceptible", "bulks,", "respectively.", "An", "inoculation", "experiment", "was", "performed", "on", "these", "RILs", "following", "the", "same", "experimental", "and", "environmental", "settings,", "and", "leaflets", "of", "RILs", "were", "collected", "at", "0,", "48,", "96", "and", "144", "hpi", "as", "described", "before.", "Total", "RNA", "extraction", "and", "quantification", "for", "each", "RIL,", "as", "well", "as", "qPCR", "were", "conducted", "using", "the", "same", "method", "described", "above.", "Each", "individual", "RIL", "was", "treated", "as", "one", "biological", "replicate", "in", "each", "of", "the", "two", "pools,", "thus", "each", "pool", "consisted", "of", "five", "biological", "replicates.", "qPCR", "reaction", "and", "gene", "expression", "estimation", "were", "conducted", "for", "Lc09908,", "Lc07593,", "Lc05858,", "Lc06015,", "Lc10098,", "Lc7065,", "Lc12983,", "Lc11261,", "and", "Lc13725", "which", "were", "differentially", "expressed", "genes", "that", "co-localized", "with", "previously", "described", "quantitative", "trait", "loci", "(QTLs).", "The", "mean", "separation", "between", "pools", "was", "conducted", "using", "the", "Student’s", "t-test", "method", "(P", "<", "0.05)." ]

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Original work titled: 'Using a transcriptome sequencing approach to explore candidate resistance genes against stemphylium blight in the wild lentil species Lens ervoides' (BMC Plant Biology/BioMed Central) by the following authors: Zhe Cao; Li Li; Karan Kapoor; Sabine Banniza DOI: 10.1186/s12870-019-2013-6

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Datasets:

tznurmin
/

tea_curated

Dataset Summary

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