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[ "Introduction", "Recent", "reports", "have", "shown", "that", "nonsynonymous", "coding", "mutations", "may", "increase", "tumor", "immunogenicity.", "In", "selected", "tumor", "types", "such", "as", "melanoma,", "lung", "cancer,", "and", "colorectal", "tumors,", "the", "somatic", "mutational", "burden", "correlates", "with", "the", "probability", "to", "generate", "immunogenic", "peptides", "that", "are", "presented", "to", "CD8", "+", "T", "cells", "on", "restricted", "HLA-I", "subtypes", "1", "–", "4", ".", "However,", "in", "most", "solid", "tumors,", "T-cell", "immunity", "and", "a", "productive", "response", "to", "immune", "therapies", "have", "been", "reported", "only", "in", "a", "minority", "of", "patients", "and", "the", "identity", "of", "immunogenic", "tumor", "antigens,", "also", "referred", "to", "as", "neoantigens,", "remains", "unknown.", "High-grade", "glioma", "is", "the", "most", "frequent", "type", "of", "primary", "brain", "tumor,", "with", "grade", "IV", "glioma", "(glioblastoma,", "GBM)", "being", "an", "invariably", "lethal", "tumor", "type", "with", "median", "survival", "below", "15", "months", "5", ",", "6", ".", "In", "the", "case", "of", "glioma", "higher", "mutational", "load", "is", "associated", "with", "increased", "tumor", "aggressiveness", "7", ".", "Consequently,", "the", "role,", "if", "any,", "of", "mutation-generated", "neoantigens", "as", "inducers", "of", "immunogenic", "responses", "in", "GBM", "has", "remained", "elusive.", "A", "further", "element", "that", "limits", "a", "productive", "anti-tumor", "immunity", "in", "GBM", "is", "the", "tumor", "microenvironment,", "which", "is", "dominated", "by", "myeloid-derived", "cells,", "mostly", "blood-derived", "macrophages", "and", "resident", "microglia", "8", "–", "11", "actively", "operating", "to", "exclude", "T", "lymphocytes", "and", "undermine", "their", "function", "12", ".", "Accordingly,", "GBM", "typically", "lack", "significant", "number", "of", "T", "lymphocyte", "infiltrates", "13", ".", "The", "recognized", "unique", "genetic", "landscape", "and", "the", "biological", "features", "of", "the", "GBM", "microenvironment", "led", "to", "the", "exclusion", "of", "high-grade", "glioma", "patients", "from", "several", "multi-cancer", "studies", "that", "have", "characterized", "tumor", "immunity", "and", "reinforced", "the", "notion", "that", "a", "lymphocyte", "depleted", "and", "immunosuppressive", "microenvironment", "is", "a", "distinctive", "feature", "of", "malignant", "gliomas", "14", ".", "In", "this", "manuscript,", "we", "present", "the", "application", "of", "a", "neoantigen", "quality", "model", "for", "the", "accurate", "prediction", "of", "immunogenic", "neoantigens", "in", "IDH", "wild-type", "GBM,", "the", "largest", "and", "most", "aggressive", "group", "of", "high-grade", "gliomas.", "We", "found", "that", "in", "IDH", "wild-type", "GBM", "the", "production", "of", "high-quality", "neoantigens", "and", "infiltration", "of", "T", "lymphocytes", "are", "distinctive", "features", "of", "patients", "with", "the", "longest", "survival.", "The", "unique", "immunogenic", "attributes", "of", "this", "GBM", "subgroup", "informs", "on", "a", "cohort", "of", "patients", "who", "are", "optimally", "outfitted", "to", "mount", "the", "most", "effective", "responses", "following", "immunotherapy", "treatments.", "Results", "Neoantigen", "quantity", "fails", "to", "predict", "survival", "of", "gliomas", "To", "define", "the", "importance", "of", "neoantigens", "in", "human", "glioma,", "we", "designed", "a", "stringent", "neoantigen", "prediction", "algorithm", "that", "considers", "the", "differential", "binding", "affinity", "of", "mutant", "and", "wild-type", "9-mer", "peptides", "to", "HLA-I", "(neoantigen", "quantity", "model,", "Supplementary", "Fig.", "1", ").", "Binding", "affinity", "was", "determined", "by", "netMHCpan-4.0", "15", ".", "Only", "mutant", "peptides", "with", "binding", "affinity", "IC50", "<", "500", "nM", "for", "the", "restricted", "HLA-I", "subtype", "and", "binding", "affinity", "of", "the", "corresponding", "wild-type", "peptide", "IC50", ">", "500", "nM", "for", "all", "HLA-I", "subtypes", "were", "retained", "as", "neoantigens", "(Supplementary", "Figs.", "1", "–", "3", ").", "HLA-I", "subtyping", "for", "each", "glioma", "patient", "was", "done", "through", "the", "implementation", "of", "four", "different", "algorithms", "(PolySolver", "16", ",", "OptiType", "17", ",", "PHLAT", "18", ",", "and", "seq2HLA", "19", ").", "We", "applied", "the", "neoantigen", "prediction", "algorithm", "to", "the", "ATLAS-TCGA", "pan-glioma", "cohort", "that", "includes", "303", "GBM", "and", "509", "lower-grade", "glioma", "(LGG)", "profiled", "by", "whole", "exome", "sequence", "(WES),", "RNA-seq,", "and", "Agilent", "transcriptomic", "array.", "Clinical", "annotations", "were", "also", "available", "for", "this", "cohort", "(Supplementary", "Data", "1", ").", "The", "pan-glioma", "cohort", "had", "been", "previously", "stratified", "on", "the", "basis", "of", "histology,", "genetic", "alterations,", "DNA", "methylation,", "and", "transcriptome", "clustering", "resulting", "in", "19", "glioma", "subgroups", "with", "distinct", "overlaps", "(Supplementary", "Data", "1", ")", "20", ".", "First,", "we", "calculated", "the", "HLA-I", "allele", "frequency", "in", "each", "glioma", "subtype", "and", "found", "that", "different", "glioma", "subtypes", "have", "similar", "HLA-I", "alleles", "frequency", "(Benjamini–Hochberg", "corrected", "Fisher", "exact", "test", "p", "value", ">", "0.05).", "We", "characterized", "neoantigens", "and", "immune", "landscape", "for", "each", "glioma", "subgroup,", "stratified", "tumors", "into", "high", "and", "low-neoantigen", "groups", "on", "the", "basis", "of", "the", "mean", "value", "of", "the", "neoantigen", "load", "and", "compared", "survival", "by", "Kaplan–Meier", "analysis.", "As", "experimental", "validation", "of", "the", "neoantigen", "prediction,", "we", "used", "a", "homogenous,", "proximity-based", "luminescent", "oxygen", "channeling", "immunoassay", "to", "determine", "the", "affinity", "kinetics", "of", "the", "predicted", "glioma", "neoantigens", "for", "binding", "to", "HLA-I", "subtypes", "21", ".", "This", "analysis", "including", "14", "matched", "glioma", "neoantigens", "and", "corresponding", "wild-type", "peptides", "revealed", "that", "each", "mutant", "peptide", "bound", "with", "higher", "affinity", "to", "HLA-I", "than", "the", "wild-type", "counterpart,", "thus", "validating", "the", "stringency", "of", "our", "approach", "(Fig.", "1a–c", ",", "Supplementary", "Fig.", "4", ").", "However,", "neoantigen", "load,", "which", "correlated", "with", "mutational", "load", "across", "glioma", "subtypes", "(Fig.", "1d–f", "),", "did", "not", "distinguish", "patients", "according", "to", "clinical", "outcome", "in", "the", "cohort", "of", "GBM", "IDH", "wild-type,", "GBM,", "glioma", "IDH", "wild-type", "or", "the", "most", "aggressive", "form", "of", "glioma", "(mesenchymal", "and", "classical),", "but", "a", "higher", "neoantigen", "load", "was", "associated", "with", "worse", "prognosis", "in", "lower", "grade", "gliomas", "(with", "or", "without", "co-deletion", "of", "chromosome", "1p", "and", "chromosome", "19q", "and", "regardless", "of", "histology)", "and", "in", "glioma", "of", "the", "proneural", "and", "neural", "subtype", "(Fig.", "1g–i", ",", "Supplementary", "Fig.", "5", ").", "Recently,", "it", "has", "been", "proposed", "that", "the", "difference", "in", "binding", "affinity", "between", "any", "wild-type", "and", "mutant", "peptide", "(termed", "differential", "agretopicity", "index,", "DAI", "22", ",", "23", ")", "is", "a", "more", "accurate", "indicator", "of", "peptide", "immunogenicity", "than", "the", "binding", "affinity", "of", "the", "mutant", "peptide", "and", "it", "has", "been", "shown", "that", "the", "mean", "DAI", "of", "all", "tumor", "peptide", "pairs", "was", "a", "predictor", "of", "survival", "in", "melanoma", "and", "non-small", "cell", "lung", "cancer", "24", ".", "We", "calculated", "mean", "DAI", "for", "each", "glioma", "in", "the", "TCGA", "cohort", "and", "determined", "that", "similar", "to", "the", "results", "obtained", "from", "the", "quantity", "model,", "patients", "in", "different", "glioma", "sub-groups", "with", "high", "(above", "mean)", "or", "low", "(below", "mean)", "DAI", "had", "similar", "prognosis", "(Supplementary", "Fig.", "6", ").", "In", "some", "glioma", "sub-types", "and", "the", "aggregated", "cohort", "of", "all", "gliomas,", "high", "DAI", "was", "associated", "with", "a", "worse", "clinical", "outcome", "(Supplementary", "Fig.", "6", ").", "Together,", "these", "findings", "suggest", "that", "in", "contrast", "to", "other", "cancer", "types", "25", ",", "26", ",", "the", "neoantigen", "load", "is", "only", "a", "representation", "of", "the", "tumor", "mutation", "burden", "and", "is", "not", "associated", "with", "better", "survival.", "Fig.", "1", "Neoantigen", "quantity", "is", "not", "prognostic", "of", "survival", "in", "glioma.", "a", "–", "c", "In", "vitro", "binding", "affinity", "kinetics", "of", "neoantigens", "and", "corresponding", "wild-type", "peptides", "for", "their", "restricted", "HLA", "class", "I", "allele.", "Representative", "results", "for", "a", ",", "GBM", "IDH", "wild-type;", "b", ",", "GBM;", "c", ",", "glioma", "IDH", "wild-type.", "Data", "are", "shown", "as", "counts", "per", "second", "with", "increasing", "peptide", "concentration", "(log", "10", "mM).", "Data", "are", "mean", "of", "n", "=", "4", "technical", "replicates", "from", "two", "independent", "experiments", "for", "wild-type", "peptide;", "n", "=", "2", "technical", "replicates", "for", "mutant", "peptide", "(", "a", ",", "c", ");", "n", "=", "4", "technical", "replicates", "from", "two", "independent", "experiments", "for", "wild-type", "peptide;", "n", "=", "6", "technical", "replicates", "from", "two", "independent", "experiments", "for", "mutant", "peptide", "(", "b", ").", "d", "–", "f", "Analysis", "of", "the", "correlation", "between", "neoantigen", "quantity", "and", "missense", "mutation", "load;", "d", ",", "GBM", "IDH", "wild-type;", "e", ",", "GBM;", "f", ",", "glioma", "IDH", "wild-type.", "g", "–", "i", "Stratification", "of", "survival", "according", "to", "neoantigen", "quantity", "score;", "dashed", "line:", "high-quantity", "neoantigens;", "dotted", "line:", "low-quantity", "neoantigens.", "g", ",", "GBM", "IDH", "wild-type;", "h", ",", "GBM;", "i", ",", "glioma", "IDH", "wild-type.", "n", "is", "the", "number", "of", "patients.", "p-value", "was", "determined", "using", "a", "log-rank", "test", "High-quality", "neoantigens", "predict", "better", "survival", "in", "GBM", "Similarity", "to", "known", "pathogen", "immunogens", "has", "been", "used", "to", "define", "neoantigen", "fitness", "(neoantigen", "quality", "model)", "and", "appears", "to", "provide", "a", "more", "accurate", "prediction", "of", "anti-tumor", "immunity", "and", "patients", "survival", "than", "the", "neoantigen", "load", "27", ".", "We", "postulated", "that", "T", "cell", "receptors", "(TCRs)", "that", "can", "recognize", "pathogenic", "antigens", "can", "also", "recognize", "similar", "non-pathogenic", "neoantigens", "generated", "by", "glioma", "cells", "and", "applied", "a", "neoantigen", "fitness", "model", "to", "glioma.", "In", "this", "model", "the", "candidate", "neoantigens", "are", "used", "to", "compute", "the", "Neoantigen", "Recognition", "Potential", "(NRP)", "which", "is", "the", "product", "of", "two", "terms", "(Supplementary", "Fig.", "7", "and", "Methods).", "The", "first", "term", "approximates", "the", "probability", "that", "a", "presented", "neoantigen", "will", "be", "recognized", "by", "the", "TCR", "repertoire", "and", "it", "depends", "on", "the", "similarity", "of", "the", "mutant", "peptide", "to", "human", "infectious", "disease-derived", "peptide", "sequences", "with", "positive", "immune", "assays", "in", "the", "Immune", "Epitope", "Database", "(IEDB)", "28", ".", "The", "second", "term,", "defined", "as", "the", "amplitude", "and", "similar", "to", "the", "DAI,", "accounts", "for", "the", "ratio", "between", "the", "binding", "probabilities", "of", "wild-type", "and", "mutant", "peptides.", "This", "model", "was", "successfully", "applied", "to", "predict", "response", "to", "checkpoint", "blockade", "immune-therapy", "in", "melanoma", "and", "lung", "cancer", "29", ".", "The", "first", "term", "of", "NRP", "depends", "on", "the", "parameters", "of", "a", "logistic", "model", "that", "were", "optimized", "for", "best", "stability", "and", "robustness", "across", "the", "major", "subtypes", "of", "high-grade", "gliomas", "(Supplementary", "Fig.", "8", ").", "We", "discovered", "that", "NRP", "is", "significantly", "higher", "in", "IDH", "wild-type", "gliomas", "when", "compared", "with", "IDH", "mutant", "gliomas", "(Mann–Whitney", "U", "test", "p-value", "=", "2.82e–13,", "Supplementary", "Fig.", "9a", ").", "The", "application", "of", "the", "quality", "model", "analysis,", "which", "is", "independent", "of", "mutation", "burden", "(Fig.", "2a", "),", "to", "268", "IDH", "wild-type", "GBM", "patients,", "revealed", "that", "high-quality", "neoantigen", "score", "(above", "the", "mean", "values)", "was", "associated", "with", "a", "significantly", "longer", "survival", "(Fig.", "2b", ").", "This", "effect", "was", "independent", "of", "age,", "gender,", "and", "mutation", "load", "(Supplementary", "Table", "1", ").", "Similarly,", "stratification", "by", "the", "mean", "value", "of", "total", "neoantigen", "quality", "separated", "the", "cohorts", "of", "GBM", "(including", "292", "patients)", "and", "IDH", "wild-type", "gliomas", "(including", "363", "patients)", "into", "two", "distinct", "prognostic", "sub-groups", "(Supplementary", "Fig.", "10a,", "b", ").", "A", "non-statistically", "significant", "trend", "to", "better", "survival", "for", "high-quality", "neoantigens", "was", "also", "observed", "in", "classical,", "classic-like,", "and", "mesenchymal", "glioma", "(Supplementary", "Fig.", "10c–e", ").", "We", "also", "determined", "that", "neoantigens", "with", "high-quality", "score", "were", "not", "restricted", "to", "specific", "HLA-I", "subtypes", "in", "IDH", "wild-type", "GBM", "patients", "(Benjamini–Hochberg", "corrected", "proportion", "test,", "p", "value", ">", "0.05).", "As", "an", "independent", "validation", "of", "the", "quality", "model,", "we", "used", "WES", "from", "46", "primary", "GBMs", "from", "a", "recently", "published", "cohort", "for", "which", "we", "obtained", "most", "updated", "survival", "data", "30", ".", "We", "confirmed", "that", "the", "15", "patients", "with", "tumors", "predicted", "to", "contain", "high-quality", "neoantigens", "had", "a", "significantly", "better", "survival", "(log", "rank", "p", "=", "0.0339,", "Supplementary", "Fig.", "11", ").", "Fig.", "2", "High-quality", "neoantigens", "are", "prognostic", "of", "better", "survival", "in", "IDH", "wild-type", "GBM.", "a", "Lack", "of", "correlation", "between", "neoantigen", "quality", "and", "missense", "mutation", "load", "(R", "=", "0.050,", "p", "=", "0.417).", "b", "Stratification", "of", "survival", "according", "to", "neoantigen", "quality", "score", "(log", "rank", "test", "p", "=", "0.0035)", "As", "IDH", "wild-type", "tumors", "compose", "the", "largest", "and", "most", "homogeneous", "subgroup", "of", "GBM", "and", "exhibit", "optimal", "performance", "in", "the", "glioma", "fitness", "model", "analysis,", "we", "focused", "our", "subsequent", "analyses", "on", "this", "group", "of", "tumors.", "HLA", "class", "I", "molecules", "are", "highly", "polymorphic", "with", "variation", "located", "in", "the", "peptide-binding", "region,", "with", "each", "variant", "binding", "only", "to", "a", "highly", "restricted", "set", "of", "peptide", "ligands", "31", ".", "Compared", "to", "heterozygous", "HLA-I", "carriers,", "homozygosity", "for", "HLA-I", "loci", "is", "predicted", "to", "present", "a", "smaller", "and", "less", "diverse", "repertoire", "of", "tumor-derived", "neoantigens", "to", "cytotoxic", "T", "lymphocytes", "(CTLs)", "32", ".", "We", "therefore", "asked", "whether", "greater", "diversity", "(heterozygosity)", "in", "the", "repertoire", "of", "antigen-presenting", "HLA-I", "molecules", "is", "associated", "with", "more", "efficient", "recognition", "of", "high-quality", "neoantigens.", "The", "analysis", "of", "the", "variations", "at", "each", "of", "the", "HLA-I", "genes", "(HLA-A,", "HLA-B,", "and", "HLA-C)", "in", "the", "high", "and", "low-quality", "neoantigen", "cohorts", "revealed", "that", "at", "least", "one", "HLA-I", "allele", "was", "homozygous", "in", "25.0%", "(53", "of", "212)", "of", "IDH", "wild-type", "GBM", "containing", "only", "low-quality", "neoantigens", "but", "the", "frequency", "of", "HLA-I", "homozygosity", "was", "significantly", "lower", "in", "the", "high-quality", "neoantigen", "group", "(10.7%", "or", "6", "of", "56,", "One-sided", "Fisher", "exact", "test", "p", "=", "0.0136,", "Supplementary", "Table", "2", ").", "Although", "not", "reaching", "statistical", "significance,", "also", "the", "amplitude", "term", "of", "neoantigens", "in", "HLA-I", "homozygosity", "was", "inferior", "to", "the", "amplitude", "term", "of", "neoantigens", "in", "HLA-I", "heterozygosity", "(median", "amplitude", "term:", "1.583", "and", "1.785", "for", "HLA-I", "homozygosity", "and", "HLA-I", "heterozygosity,", "respectively;", "Mann–Whitney", "U", "test", "p", "value", "=", "0.176,", "Supplementary", "Fig.", "12a", ").", "We", "also", "determined", "that", "the", "number", "of", "neoantigens", "in", "HLA-I", "homozygous", "IDH", "wild-type", "GBM", "is", "significantly", "lower", "than", "HLA-I", "heterozygous", "patients", "(Mann–Whitney", "U", "test", "p", "value", "=", "0.0001,", "Supplementary", "Fig.", "12b", ").", "Together,", "these", "results", "indicate", "that", "homozygosity", "for", "HLA-I", "alleles", "hinders", "the", "ability", "to", "generate", "and", "recognize", "high-quality", "neoantigens.", "As", "the", "IEDB", "database", "also", "contains", "a", "collection", "of", "human", "immune", "epitopes", "tested", "with", "experimental", "assays", "and", "annotated", "for", "their", "ability", "to", "trigger", "an", "immune", "response,", "we", "sought", "to", "provide", "an", "independent", "validation", "of", "the", "quality", "of", "the", "computationally", "identified", "neoantigens", "in", "human", "gliomas.", "We", "aligned", "the", "sequences", "of", "the", "neoantigens", "detected", "in", "the", "high", "and", "low-quality", "neoantigen", "group", "of", "IDH", "wild-type", "GBM", "with", "experimentally", "validated", "human", "epitopes", "(allergy", "and", "autoimmune-derived)", "in", "IEDB.", "The", "analysis", "showed", "that,", "in", "comparison", "with", "low-quality", "neoantigens,", "high-quality", "neoantigens", "exhibited", "greater", "similarity", "to", "human", "peptides", "in", "IEDB", "that", "score", "as", "highly", "immunogenic", "(“positive", "high”)", "in", "T", "cell", "and", "MHC", "ligand", "assays", "(p", "=", "0.0002", "and", "p", "=", "0.0023,", "respectively;", "Fig.", "3a,", "b", ").", "Conversely,", "we", "found", "no", "difference", "between", "low", "and", "high-quality", "neoantigens", "in", "the", "alignment", "with", "human", "peptides", "in", "IEDB", "that", "score", "negative", "in", "validation", "immune", "assays", "(p", "=", "0.3804", "and", "0.2271,", "respectively,", "Fig.", "3c,", "d", ").", "Taken", "together,", "neoantigen", "quality", "rather", "than", "neoantigen", "load", "correlates", "with", "immunogenicity", "and", "predicts", "survival", "in", "IDH", "wild-type", "GBM.", "Fig.", "3", "High-quality", "neoantigens", "are", "associated", "with", "immunogenicity", "in", "IDH", "wild-type", "GBM.", "Comparison", "of", "the", "similarity", "between", "neoantigens", "and", "human", "immune", "epitopes", "scored", "as", "immunogenic", "(positive", "high)", "or", "non-immunogenic", "(negative)", "in", "high", "and", "low-quality", "neoantigen", "groups", "of", "IDH", "wild-type", "GBM.", "a", ",", "c", "T", "cell", "assays;", "b", ",", "d", "MHC", "ligand", "assays.", "n", "number", "of", "neoantigens.", "p-value", "was", "determined", "using", "two-sided", "Mann–Whitney", "U", "test", "High-quality", "neoantigens", "and", "CD8", "+", "T", "cells", "identify", "the", "longest", "survivors", "Having", "established", "that", "high-quality", "neoantigens", "improve", "survival", "of", "IDH", "wild-type", "GBM", "patients,", "we", "turned", "our", "attention", "to", "the", "role", "of", "the", "non-tumor", "cells", "that", "compose", "the", "GBM", "microenvironment.", "In", "particular,", "we", "sought", "to", "determine", "whether", "the", "presence", "of", "any", "of", "the", "non-tumor", "cell", "populations", "(immune", "and", "non-immune)", "that", "infiltrate", "human", "GBM", "impact", "survival", "and/or", "cooperate", "with", "high-quality", "neoantigens", "to", "determine", "a", "more", "favorable", "outcome", "of", "GBM", "patients.", "The", "GBM", "tumor", "microenvironment", "is", "dominated", "by", "myeloid-derived", "cells", "that", "can", "be", "separated", "into", "blood-derived", "macrophages", "and", "microglia.", "We", "recently", "reported", "the", "single", "cell", "transcriptome", "of", "8", "GBM", "33", ".", "The", "analysis", "of", "cells", "of", "the", "tumor", "microenvironment", "from", "this", "study", "using", "single", "cell", "MWW-Gene", "Set", "Test", "(MWW-GST)", "34", ",", "35", "led", "to", "the", "identification", "of", "6", "distinct", "cell", "populations", "(blood-derived", "macrophages,", "microglia,", "CD8", "+", "T", "lymphocytes,", "oligodendrocytes,", "endothelial", "cells", "and", "pericytes).", "For", "each", "cell", "type,", "we", "selected", "a", "specific", "signature", "of", "30", "genes", "and", "calculated", "the", "median", "enrichment", "score", "in", "order", "to", "separate", "IDH", "wild-type", "GBM", "patients", "into", "groups", "with", "high", "or", "low", "infiltration", "of", "individual", "cell", "populations", "and", "asked", "whether", "the", "enrichment", "of", "specific", "cell", "types", "was", "associated", "with", "changes", "in", "survival.", "We", "also", "computed", "the", "individual", "signature", "of", "12", "previously", "identified", "tumor", "infiltrating", "immune", "cells,", "thus", "including", "a", "total", "of", "18", "cell", "type-specific", "signatures", "plus", "two", "signatures", "for", "the", "interferon", "gamma", "response", "pathway", "(Supplementary", "Data", "2", ")", "33", ",", "36", "–", "39", ".", "The", "analysis", "was", "performed", "on", "132", "and", "248", "TCGA-derived", "GBM", "that", "had", "been", "analyzed", "by", "RNA-seq", "or", "Agilent", "expression", "arrays,", "respectively.", "Whereas", "we", "detected", "a", "significant", "effect", "on", "survival", "of", "some", "cell", "populations", "in", "one", "of", "the", "cohorts,", "none", "of", "the", "20", "signatures", "concordantly", "differentiated", "the", "survival", "of", "GBM", "patients", "in", "both", "cohorts", "(Supplementary", "Table", "3", ").", "Infiltration", "of", "CD8", "+", "T", "lymphocytes", "that", "has", "been", "shown", "to", "predict", "a", "favorable", "prognosis", "in", "several", "tumor", "types", "40", "–", "43", "was", "associated", "only", "with", "a", "non-statistically", "significant", "trend", "to", "a", "better", "clinical", "outcome", "in", "the", "RNAseq", "and", "Agilent", "microarray", "cohorts", "(p", "=", "0.134", "and", "p", "=", "0.136,", "respectively;", "Fig.", "4b,", "e", ").", "Confirming", "the", "lack", "of", "significance", "of", "CD8", "+", "T", "cell", "enrichment", "score", "for", "survival,", "we", "found", "a", "lower", "CD8", "+", "T", "cell", "enrichment", "score", "in", "the", "more", "favorable", "IDH", "mutant", "when", "compared", "with", "IDH", "wild-type", "gliomas", "(Wilcoxon", "p-value", "2.26E–16,", "Supplementary", "Fig.", "9b", ").", "When", "IDH", "wild-type", "GBM", "patients", "were", "stratified", "according", "to", "the", "enrichment", "of", "each", "non-tumor", "cell", "type", "in", "combination", "with", "high", "or", "low-quality", "neoantigens,", "elevated", "CD8", "+", "T", "lymphocytes", "and", "high-quality", "neoantigens", "emerged", "as", "the", "only", "features", "that", "induced", "a", "synergistic", "effect", "on", "survival", "and", "classified", "a", "subgroup", "of", "approximately", "10%", "of", "IDH", "wild-type", "GBM", "patients", "with", "the", "longest", "survival", "in", "both", "RNAseq", "and", "Agilent", "microarray", "datasets", "(p", "=", "0.0016", "and", "p", "=", "0.0048,", "respectively;", "Fig.", "4a–f", "and", "Supplementary", "Table", "3", ").", "The", "positive", "role", "of", "CD8", "+", "T", "cells", "in", "the", "synergy", "with", "high-quality", "neoantigens", "was", "recapitulated", "by", "the", "trend", "for", "a", "similar", "cooperation", "towards", "a", "better", "clinical", "outcome", "between", "high-quality", "neoantigens", "and", "the", "interferon", "gamma", "signature,", "which", "is", "used", "as", "a", "surrogate", "of", "CD8", "+", "T", "cell", "function", "in", "transcriptomic", "analyses", "38", ",", "39", ",", "44", "(Supplementary", "Table", "3", ").", "The", "synergistic", "effect", "of", "high-quality", "neoantigens", "and", "CD8", "+", "T", "lymphocyte", "activation", "was", "still", "significant", "in", "a", "multivariate", "regression", "model", "including", "age,", "gender", "and", "mutational", "load", "as", "additional", "covariates", "(Supplementary", "Table", "4", ").", "To", "determine", "the", "relationship", "between", "T", "cell", "infiltration,", "high-quality", "neoantigens", "and", "tumor", "purity,", "we", "used", "the", "tumor", "purity", "values", "calculated", "by", "ABSOLUTE", "45", ",", "a", "validated", "computational", "approach", "for", "the", "inference", "of", "the", "fraction", "of", "stromal/immune", "cells", "and", "consequently", "tumor", "cell", "purity.", "As", "expected,", "we", "found", "a", "negative", "correlation", "between", "CD8", "+", "T", "cell", "enrichment", "score", "and", "tumor", "purity", "(correlation", "coefficient", "=", "−0.457,", "p", "=", "3.695e–8,", "Supplementary", "Fig.", "13a", ").", "However,", "there", "was", "no", "correlation", "between", "neoantigen", "quality", "score", "and", "tumor", "purity", "(correlation", "coefficient", "=", "0.0839,", "p", "=", "0.186,", "Supplementary", "Fig.", "13b", "),", "indicating", "that", "high-quality", "neoantigens", "are", "independent", "of", "broad", "immune", "cell", "infiltration.", "Aberrant", "DNA", "methylation", "of", "genes", "expressed", "by", "immune", "cells", "were", "reported", "to", "regulate", "the", "extent", "of", "immune", "infiltration", "in", "solid", "tumors", "46", "–", "48", ".", "Therefore,", "we", "examined", "whether", "the", "enrichment", "of", "CD8", "+", "T", "cells", "in", "IDH", "wild-type", "GBM", "is", "associated", "with", "differential", "DNA", "methylation.", "Towards", "this", "aim,", "we", "performed", "an", "integrated", "analysis", "of", "gene", "expression", "and", "DNA", "methylation.", "From", "this", "analysis,", "diverse", "immune", "response", "categories", "emerged", "as", "significantly", "hypo-methylated", "and", "upregulated", "in", "IDH", "wild-type", "GBM", "patients", "with", "high", "CD8", "+", "T", "cells", "for", "both", "RNAseq", "(Supplementary", "Fig.", "14a,", "b", ";", "Supplementary", "Data", "3", ",", "5", ")", "and", "Agilent", "expression", "data", "(Supplementary", "Fig.", "14c,", "d", ";", "Supplementary", "Data", "4", ",", "6", ").", "Taken", "together,", "the", "combination", "of", "productive", "neoantigen", "T", "cell", "recognition", "and", "epigenetically", "directed", "intra-tumor", "infiltration", "of", "CD8", "+", "T", "lymphocytes", "characterizes", "a", "sizable", "group", "of", "IDH", "wild-type", "GBM", "patients", "who", "experience", "a", "more", "favorable", "prognosis.", "Fig.", "4", "Synergistic", "effect", "of", "CD8", "+", "T", "cells", "and", "high-quality", "neoantigens", "on", "survival", "of", "IDH", "wild-type", "GBM.", "a", "–", "c", "Analysis", "of", "the", "cohort", "for", "which", "WES", "and", "RNAseq", "were", "available.", "a", "Survival", "of", "patients", "stratified", "according", "to", "neoantigen", "quality", "score.", "b", "Survival", "of", "patients", "stratified", "according", "to", "CD8", "+", "T", "lymphocyte", "enrichment", "score.", "c", "Survival", "of", "patients", "stratified", "by", "neoantigen", "quality", "and", "CD8", "+", "T", "lymphocytes", "infiltration", "score.", "d", "–", "f", "Analysis", "of", "the", "cohort", "for", "which", "WES", "and", "Agilent", "microarray", "were", "available.", "d", "Survival", "of", "patients", "stratified", "according", "to", "neoantigen", "quality", "score.", "e", "Survival", "of", "patient", "stratified", "according", "to", "CD8", "+", "T", "lymphocyte", "enrichment", "score.", "f", "Survival", "of", "patients", "stratified", "by", "neoantigen", "quality", "and", "CD8", "+", "T", "lymphocytes", "infiltration", "score.", "Black", "dashed", "and", "dotted", "lines", "represent", "samples", "with", "high", "and", "low-quality", "neoantigens,", "respectively.", "Blue", "dashed", "and", "dotted", "lines", "represent", "patients", "with", "high", "and", "low", "CD8", "+", "T", "lymphocytes,", "respectively.", "Pink", "dashed", "and", "dotted", "lines", "represent", "patients", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "lymphocytes", "and", "low-quality", "neoantigens", "and", "low", "CD8", "+", "T", "cells,", "respectively.", "n", "number", "of", "patients.", "p-value", "was", "determined", "by", "the", "log-rank", "test", "High-quality", "neoantigens", "and", "CD8", "+", "T", "cells", "activate", "the", "immune", "response", "To", "identify", "the", "transcriptomic", "features", "of", "IDH", "wild-type", "GBM", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells,", "we", "used", "the", "combination", "of", "the", "easy", "ensemble", "(ee)", "undersampling", "technique", "and", "Mann–Whitney–Wilcoxon", "(MWW)", "test", "statistics", "(ee-MWW)", "we", "recently", "developed", "34", "and", "generated", "a", "ranked", "list", "of", "genes", "from", "RNAseq", "and", "Agilent", "microarray", "datasets", "of", "IDH", "wild-type", "GBM", "discriminating", "tumors", "with", "high-quality", "neoantigens/high", "CD8", "+", "T", "cells", "from", "those", "with", "low-quality", "neoantigens/low", "CD8", "+", "T", "cells.", "The", "gene", "ontology", "enrichment", "map", "network", "(Q", "<", "0.00001,", "normalized", "enrichment", "score,", "NES", ">", "0.6)", "revealed", "that", "the", "most", "significant", "biological", "processes", "enriched", "in", "IDH", "wild-type", "GBM", "from", "both", "RNAseq", "and", "Agilent", "microarray", "datasets", "were", "immune", "response", "categories,", "thus", "providing", "additional", "evidence", "for", "the", "specific", "immune", "functions", "implemented", "within", "the", "tumors", "that", "contain", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells", "(Fig.", "5a,", "b", ").", "Fig.", "5", "Gene", "ontology", "enrichment", "networks", "and", "genetic", "characteristics", "of", "IDH", "wild-type", "GBM", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells.", "a", "Enrichment", "map", "network", "of", "statistically", "significant", "GO", "categories", "in", "the", "patient", "cohort", "analyzed", "by", "WES", "and", "RNAseq.", "b", "Enrichment", "map", "network", "of", "statistically", "significant", "GO", "categories", "in", "the", "patient", "cohort", "with", "WES", "and", "Agilent", "data", "available", "(normalized", "enrichment", "score,", "NES", ">", "0.6,", "and", "q-value", "<", "0.00001).", "Nodes", "represent", "GO", "terms", "and", "lines", "their", "connectivity.", "Node", "size", "is", "proportional", "to", "the", "number", "of", "genes", "in", "the", "GO", "category", "and", "line", "thickness", "indicates", "the", "fraction", "of", "genes", "shared", "between", "groups.", "c", "Landscape", "of", "somatic", "genomic", "alterations", "(non-synonymous", "mutations,", "copy", "number", "alterations)", "in", "IDH", "wild-type", "GBM", "(GBM", "cohort", "analyzed", "by", "WES", "and", "RNAseq).", "d", "Landscape", "of", "somatic", "genomic", "alterations", "(non-synonymous", "mutations,", "CNVs)", "in", "IDH", "wild-type", "GBM", "(GBM", "cohort", "analyzed", "by", "WES", "and", "Agilent", "microarrays).", "Rows", "and", "columns", "represent", "genes", "and", "tumor", "samples,", "respectively.", "Genomic", "alterations", "are", "indicated.", "Genes", "are", "sorted", "according", "to", "frequency", "(%", "patients)", "in", "patients", "having", "both", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "lymphocytes", "or", "patients", "having", "both", "low-quality", "neoantigens", "and", "low", "CD8", "+", "T", "lymphocytes,", "respectively", "GBM", "with", "high-quality", "neoantigens", "and", "CD8", "+", "T", "cells", "harbor", "distinct", "genetic", "lesions", "Next,", "we", "sought", "to", "identify", "the", "genetic", "features", "(mutations", "and", "copy", "number", "variations,", "CNVs)", "that", "distinguish", "GBM", "that", "generate", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "lymphocyte", "signatures.", "We", "failed", "to", "find", "recurrent", "genes", "harboring", "mutations", "that", "produce", "neoantigens", "in", "the", "high-quality", "neoantigens/high", "CD8", "+", "T", "cell", "group.", "Similarly,", "this", "group", "lacked", "specific", "recurrent", "mutations.", "In", "contrast,", "we", "found", "that", "tumors", "without", "high-quality", "neoantigens", "and", "CD8", "+", "T", "cells", "harbored", "a", "number", "of", "recurrently", "mutated", "genes", "(somatic", "mutations", "and", "CNVs;", "Fig.", "5c,", "d", "),", "some", "of", "which", "are", "important", "cancer", "drivers.", "In", "particular,", "we", "found", "that", "genetic", "alterations", "of", "PIK3CA,", "RB1", "and", "MDM2", "were", "present", "in", "24–28%", "of", "GBM", "unable", "to", "generate", "high-quality", "neoantigens", "and", "attract", "CD8", "+", "T", "lymphocytes", "but", "only", "in", "0–8%", "of", "GBM", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "lymphocytes", "(RNA-seq,", "p", "=", "0.06;", "Agilent,", "p", "=", "0.02;", "Supplementary", "Table", "5", ").", "Together", "with", "the", "other", "genes", "that", "were", "exclusively", "mutated", "in", "the", "low-quality", "neoantigens/low", "CD8", "+", "T", "cell", "group,", "these", "findings", "point", "to", "the", "set", "of", "genetic", "determinants", "that", "should", "support", "the", "prospective", "exclusion", "of", "patients", "with", "GBM", "from", "the", "high-quality", "neoantigens/high", "CD8", "+", "T", "lymphocytes", "group.", "Discussion", "With", "neoantigens", "emerging", "as", "attractive", "targets", "in", "the", "development", "of", "personalized", "immunotherapies,", "strategies", "for", "the", "rapid", "identification", "of", "relevant", "neoantigens", "have", "become", "a", "major", "priority", "24", ",", "27", ",", "29", ".", "This", "study", "describes", "such", "strategy", "using", "a", "computational", "approach", "for", "the", "identification", "of", "GBM", "patients", "harboring", "high-quality", "neoantigens", "that,", "together", "with", "CD8", "+", "T", "lymphocyte", "infiltrates,", "perform", "optimally", "in", "identifying", "patients", "with", "the", "longest", "survival", "and", "a", "functionally", "activated", "tumor", "immune", "microenvironment.", "This", "information", "might", "be", "of", "clinical", "importance", "for", "the", "accurate", "stratification", "of", "the", "subgroup", "of", "GBM", "patients", "having", "the", "best", "probability", "to", "benefit", "from", "immunotherapies.", "GBM", "are", "classified", "as", "lymphocyte-depleted", "tumors", "49", "lacking", "naïve", "T", "cells", "that", "are", "instead", "found", "sequestered", "in", "large", "numbers", "in", "the", "bone", "marrow", "50", ".", "Nevertheless,", "recent", "studies", "based", "on", "single", "cell", "profiling", "have", "shown", "that", "a", "small", "proportion", "of", "GBM", "show", "CD8", "+", "T", "lymphocyte", "infiltrates", "33", ".", "Progression", "from", "low-grade", "to", "high-grade", "glioma", "evolves", "through", "increasing", "mutational", "burden", "7", ".", "Even", "among", "GBM", "patients,", "a", "higher", "number", "of", "mutations", "is", "associated", "with", "a", "more", "aggressive", "disease", "and", "worse", "survival", "7", ".", "Therefore,", "whereas", "in", "several", "tumor", "types", "the", "tumor", "mutational", "burden", "is", "associated", "with", "activation", "of", "an", "immune", "response", "and", "better", "survival,", "GBM", "displays", "an", "opposite", "behavior", "1", ",", "3", ",", "26", ",", "51", "–", "53", ".", "Consistent", "with", "this", "notion,", "we", "found", "that", "the", "simple", "estimate", "of", "the", "mutation-derived", "neoantigen", "load", "using", "a", "quantity", "model", "and", "the", "DAI", "index", "failed", "to", "segregate", "sub-groups", "of", "patients", "with", "distinct", "clinical", "outcome.", "Conversely,", "a", "high-quality", "neoantigen", "model", "that", "evaluates", "the", "similarity", "of", "tumor", "antigens", "with", "highly", "immunogenic", "pathogen-derived", "antigens", "emerged", "from", "our", "work", "as", "the", "exclusive", "parameter", "that", "was", "able", "to", "identify", "patients", "with", "IDH", "wild-type", "GBM", "who", "display", "a", "more", "favorable", "clinical", "outcome.", "Results", "from", "the", "previous", "studies", "that", "have", "analyzed", "the", "role", "of", "CD8", "+", "T", "lymphocytes", "for", "GBM", "survival", "have", "been", "conflicting", "54", ",", "55", ".", "Some", "of", "the", "studies", "reported", "that", "the", "presence", "of", "T", "lymphocyte", "infiltrates", "was", "associated", "with", "a", "more", "favorable", "clinical", "course", "of", "the", "disease", "56", "–", "58", ",", "but", "others", "reached", "opposite", "conclusions", "54", ",", "59", ".", "In", "our", "analysis,", "which", "is", "based", "on", "mRNA", "expression", "profile", "from", "GBM-derived", "single", "T", "lymphocytes,", "enrichment", "of", "the", "T", "cell-specific", "signature", "was", "associated", "with", "a", "weak", "positive", "effect", "on", "survival.", "However,", "when", "combined", "with", "the", "presence", "of", "high-quality", "neoantigens,", "CD8", "+", "T", "lymphocyte", "infiltrates", "provided", "the", "best", "predictive", "model", "for", "the", "identification", "of", "the", "longest", "survivors", "among", "IDH", "wild-type", "GBM.", "We", "suggest", "that", "the", "combination", "of", "high-quality", "neoantigen", "fitness", "model", "and", "elevated", "T", "lymphocyte-specific", "gene", "signature", "together", "with", "histopathological", "verification", "of", "tumor", "infiltration", "by", "CD8", "+", "T", "cells", "should", "be", "used", "in", "current", "clinical", "trials", "of", "IDH", "wild-type", "GBM", "for", "the", "identification", "of", "those", "patients", "who", "have", "the", "highest", "likelihood", "of", "clinical", "response", "to", "immune", "therapy.", "Methods", "Data", "preparation", "and", "preprocessing", "The", "patient", "cohort", "is", "from", "the", "ATLAS-TCGA", "pan-glioma", "study", "20", ",", "which", "includes", "1122", "glioma", "patients", "with", "clinical", "information.", "For", "812", "glioma", "patients", "with", "tumor", "and", "matched", "normal", "samples", "30,729", "somatic", "mutations", "were", "called", "using", "exome-seq", "data", "bam", "files", "from", "TCGA", "Data", "Portal", "(", "http://tcga-data.nci.nih.gov/tcga/", ")", "20", "using", "at", "least", "two", "of", "three", "methods,", "MuTect", "60", ",", "VarScan", "61", ",", "and", "RADIA", "62", ".", "RNA-seq", "raw", "counts", "of", "667", "cases", "(513", "LGG", "and", "154", "GBM)", "were", "downloaded,", "normalized,", "and", "filtered", "using", "the", "Bioconductor", "package", "TCGAbiolinks", "63", "including", "TCGAquery,", "TCGAdownload,", "and", "TCGAprepare", "for", "level", "3", "data", "from", "platform", "“IlluminaHiSeq_RNASeqV2”.", "The", "union", "of", "the", "two", "matrices", "(LGG", "and", "GBM)", "was", "then", "normalized", "using", "within-lane", "normalization", "to", "adjust", "for", "GC-content", "effect", "on", "read", "counts", "and", "upper-quantile", "between-lane", "normalization", "for", "distributional", "differences", "between", "lanes", "by", "applying", "the", "TCGAanalyze_Normalization", "function", "encompassing", "EDASeq", "protocol", "20", ".", "Gene", "expression", "microarray", "data", "with", "Agilent", "chip", "(G4502A)", "at", "level", "3", "were", "downloaded", "from", "TCGA", "Data", "Portal.", "The", "gene", "expression", "data", "matrix", "includes", "583", "samples", "(573", "GBM", "and", "10", "normal", "brains)", "and", "17,814", "genes.", "Data", "from", "1084", "glioma", "patients", "with", "tumor", "and", "normal", "samples", "that", "had", "been", "profiled", "on", "Affymetrix", "SNP6.0", "GeneChip", "arrays,", "processed", "into", "genome", "segmentation", "files", "64", "and", "analyzed", "by", "GISTIC2.0", "to", "identify", "focal", "copy", "number", "changes", "20", ",", "65", "were", "downloaded", "from", "TCGA", "Data", "Portal.", "We", "used", "TCGAbiolinks", "63", "using", "TCGAquery,", "TCGAdownload", "to", "obtain", "data", "from", "140", "GBM", "samples", "that", "had", "been", "profiled", "using", "Illumina", "platform", "HumanMethylation450,", "which", "interrogates", "485,421", "CpG", "sites", "(data", "level", "=", "3,", "platform", "type", "=", "“HumanMethylation450”).", "We", "removed", "from", "the", "analysis", "data", "point", "with", "a", "corresponding", "p-value", "greater", "than", "0.01,", "which", "were", "deemed", "not", "to", "carry", "statistically", "significant", "difference", "from", "background", "and", "defined", "as", "“NA”", "in", "TCGA", "level", "3", "data.", "HLA-I", "typing", "The", "four-digit", "resolution", "HLA-I", "type", "of", "812", "patients", "(including", "LGG", "and", "GBM)", "with", "exome", "sequence", "bam", "files", "available", "in", "the", "TCGA", "cohort", "was", "determined", "using", "POLYSOLVER", "(POLYmorphic", "loci", "reSOLVER)", "16", ".", "For", "647", "out", "of", "812", "patients", "(including", "LGG", "and", "GBM)", "having", "RNAseq", "bam", "files", "available,", "OptiType", "17", ",", "PHLAT", "18", ",", "and", "seq2HLA", "19", "were", "further", "applied", "to", "infer", "their", "four-digit", "resolution", "HLA-I", "types.", "The", "four-digit", "HLA-I", "type", "was", "determined", "if", "the", "predictions", "were", "consistent", "in", "any", "one", "of", "the", "following", "analyses:", "POLYSOLVER", "and", "OptiType;", "POLYSOLVER", "and", "PHLAT;", "POLYSOLVER", "and", "seq2HLA;", "OptiType", "and", "PHLAT;", "OptiType", "and", "seq2HLA.", "Neoantigen", "prediction", "Missense", "mutations", "were", "used", "to", "generate", "a", "list", "of", "all", "possible", "9-mer", "peptides.", "Binding", "affinities", "of", "mutant", "and", "corresponding", "wild-type", "9-mer", "peptides,", "relevant", "to", "the", "patient’s", "HLA-I", "alleles,", "were", "predicted", "using", "netMHCpan-4.0", "15", ".", "High-affinity", "binders", "were", "defined", "as", "those", "with", "IC", "50", "equal", "or", "less", "than", "500", "nM.", "Low-affinity", "wild-type", "binders", "were", "defined", "as", "having", "IC", "50", "greater", "than", "500", "nM.", "More", "stringent", "criteria", "were", "used", "to", "infer", "neoantigens.", "A", "mutant-specific", "binder,", "relevant", "to", "the", "restricted", "HLA-I", "allele,", "was", "referred", "to", "as", "neoantigen", "when", "the", "mutant", "IC", "50", "was", "less", "than", "500", "nM", "and", "IC", "50", "of", "the", "corresponding", "wild-type", "binder,", "relevant", "to", "all", "HLA-I", "alleles", "of", "the", "patient,", "more", "than", "500", "nM.", "All", "the", "downstream", "analyses", "were", "based", "on", "the", "inferred", "neoantigens", "(the", "mutant", "peptides)", "and", "their", "corresponding", "wild-type", "peptides.", "In", "the", "neoantigen", "fitness", "model,", "we", "calculated", "the", "neoantigen", "recognition", "potential", "(NRP)", "for", "each", "neoantigen", "using", "a", "recently", "developed", "method", "27", ",", "29", ".", "Briefly,", "each", "neoantigen", "was", "associated", "with", "a", "fitness", "cost", "designated", "as", "recognition", "potential,", "which", "is", "the", "likelihood", "that", "it", "is", "effectively", "recognized", "by", "the", "TCR", "repertoire.", "Given", "a", "neoantigen,", "the", "recognition", "potential", "was", "calculated", "as", "A", "×", "R.", "A,", "the", "amplitude,", "is", "the", "ratio", "of", "the", "relative", "probability", "that", "a", "neoantigen", "is", "bound", "to", "a", "class", "I", "MHC", "times", "the", "relative", "probability", "that", "the", "wild-type", "counterpart", "of", "the", "neoantigen", "is", "not", "bound", "A", "=", "1", "K", "d", "MT", "⋅", "K", "d", "WT", "∕", "L", "+", "ϵ", "(", "1", "+", "K", "d", "WT", "∕", "L", ")", "1", "+", "ϵ", "(", "1", "+", "K", "d", "WT", "∕", "L", ")", "≈", "K", "d", "WT", "K", "d", "MT", "⋅", "1", "1", "+", "(", "ϵ", "∕", "L", ")", "K", "d", "WT", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$A", "=", "{\\frac{1}{{K_d^{{\\mathrm{MT}}}}}}\\cdot{\\frac{{K_d^{{\\mathrm{WT}}}/\\left[", "L", "\\right]", "+", "\\epsilon(1", "+", "K_d^{{\\mathrm{WT}}}/\\left[", "L", "\\right])}}{{1", "+", "\\epsilon", "(1", "+", "K_d^{{\\mathrm{WT}}}/\\left[", "L", "\\right])}}}\\;", "\\approx", "\\;{\\frac{{K_d^{{\\mathrm{WT}}}}}{{K_d^{{\\mathrm{MT}}}}}}\\cdot{\\frac{1}{{1", "+", "(\\epsilon/\\left[", "L", "\\right])K_d^{{\\mathrm{WT}}}}}}$$\\end{document}", "ε", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$\\varepsilon$$\\end{document}", "is", "a", "pseudo-count,", "K", "d", "is", "the", "original", "dissociation", "constant,", "and", "[L]", "the", "peptide", "concentration", "29", ".", "We", "set", "ε", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$\\varepsilon$$\\end{document}", "∕", "L", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$/\\left[", "L", "\\right]$$\\end{document}", "to", "be", "0.0003", "29", ".", "R", "is", "the", "probability", "that", "a", "presented", "neoantigen", "will", "be", "recognized", "by", "the", "TCR", "repertoire.", "We", "estimated", "R", "using", "a", "sigmoid", "function", "applied", "to", "the", "score", "of", "the", "local", "alignment", "between", "the", "peptide", "sequences", "and", "the", "set", "of", "2552", "unique", "epitopes", "in", "the", "IEDB", "database", "29", ".", "The", "sigmoid", "function", "has", "two", "parameters", "a", "and", "k.", "These", "parameters", "define", "the", "shape", "of", "the", "sigmoid", "function", "and", "are", "optimized", "as", "explained", "below.", "In", "particular,", "a", "representing", "the", "horizontal", "displacement", "of", "the", "binding", "curve,", "and", "k", "is", "the", "steepness", "of", "the", "curve", "at", "a.", "A", "×", "R", "represents", "the", "neoantigen", "recognition", "potential", "(NRP).", "For", "each", "patient,", "NRP", "was", "first", "calculated", "for", "each", "neoantigen.", "The", "total", "neoantigen", "quality", "of", "a", "specific", "patient", "is", "equal", "to", "the", "mean", "value", "of", "NRPs", "of", "all", "neoantigens", "inferred", "for", "this", "patient.", "Parameter", "training", "To", "choose", "the", "optimal", "model", "parameters", "a", "and", "k,", "we", "generated", "4000", "different", "a", "and", "k", "settings", "with", "a", "increasing", "from", "1", "to", "40", "at", "the", "incremental", "step", "of", "1", "and", "k", "increasing", "from", "0.1", "to", "10", "at", "the", "incremental", "step", "of", "0.1.", "We", "selected", "the", "a", "and", "k", "that", "maximize", "the", "log-rank", "test", "scores", "of", "the", "survival", "analysis", "of", "a", "given", "patient", "cohort.", "Leave", "one", "out", "cross", "validation", "(LOOCV)", "Given", "a", "patient", "cohort", "of", "n", "samples,", "each", "sample", "was", "sequentially", "removed", "from", "the", "set", "and", "the", "remaining", "samples", "(n–1)", "were", "used", "as", "training", "set", "on", "which", "the", "quality", "model", "was", "reoptimized.", "The", "excluded", "sample", "sequentially", "became", "the", "test", "set", "and", "was", "classified", "as", "high", "or", "low-quality", "neoantigen", "group.", "After", "n", "samples", "were", "sequentially", "classified,", "Kaplan–Meier", "analysis", "was", "applied", "to", "determine", "whether", "there", "was", "a", "statistically", "significant", "survival", "difference", "between", "high", "and", "low-quality", "neoantigen", "sub-groups.", "Random", "subsampling", "validation", "Given", "a", "patient", "cohort,", "we", "randomly", "split", "samples", "into", "two", "groups", "in", "ratio", "of", "4", "vs.", "1", "in", "100", "runs.", "The", "larger", "group", "were", "used", "as", "training", "set", "and", "the", "smaller", "group", "as", "test", "set.", "The", "parameter", "settings", "(a,", "k)", "of", "the", "quality", "model", "were", "trained", "on", "the", "training", "set", "and", "tested", "on", "the", "test", "set.", "If", "samples", "in", "the", "test", "sets", "were", "separated", "into", "high", "and", "low-quality", "neoantigens", "groups", "with", "significant", "survival", "difference,", "the", "parameter", "setting", "(a,", "k)", "was", "successfully", "retained.", "The", "process", "was", "repeated", "100", "times", "to", "calculate", "the", "percentage", "of", "success", "for", "each", "parameter", "setting", "(a,", "k).", "To", "calculate", "the", "significance", "for", "the", "success", "rate", "of", "each", "parameter", "setting", "(a,", "k),", "we", "performed", "10,000", "permutations", "of", "neoantigen", "qualities", "of", "patients", "across", "samples", "in", "the", "cohort", "under", "each", "parameter", "setting", "(a,", "k)", "with", "the", "random", "split", "of", "4", "versus", "1.", "For", "each", "parameter", "setting", "(a,", "k),", "the", "success", "rate", "under", "each", "permutation", "was", "then", "compared", "with", "the", "success", "rate", "for", "the", "actual", "patient", "data.", "p-value", "was", "calculated", "as", "the", "percentage", "of", "permutations", "under", "which", "success", "rate", "was", "equal", "to", "or", "larger", "than", "the", "success", "rate", "of", "actual", "data.", "Neoantigen", "quantity", "model", "In", "neoantigen", "quantity", "model,", "the", "number", "of", "inferred", "neoantigens", "was", "counted", "for", "each", "patient.", "Patients", "of", "a", "given", "cohort", "were", "stratified", "according", "to", "the", "mean", "value", "of", "the", "number", "of", "neoantigens", "into", "high", "and", "low", "neoantigen", "quantity", "groups.", "Gene", "signatures", "of", "immune", "cells", "Gene", "signatures", "and", "relative", "sources", "of", "the", "distinct", "cell", "populations", "are", "reported", "in", "Supplementary", "Data", "2", ".", "Stratification", "of", "patients", "based", "on", "immune", "cell", "NES", "and", "NRP", "To", "evaluate", "the", "enrichment", "of", "each", "immune", "cell", "type", "in", "TCGA", "glioma", "samples", "we", "used", "Normalized", "Enrichment", "Score", "(NES)", "of", "the", "MWW", "Gene", "Set", "test", "34", ".", "NES", "is", "an", "estimate", "of", "the", "probability", "that", "the", "expression", "of", "a", "gene", "in", "the", "gene", "set", "is", "greater", "than", "the", "expression", "of", "a", "gene", "outside", "this", "set.", "Specifically,", "N", "E", "S", "=", "1", "-", "U", "m", "n", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$${NES}", "=", "1", "-", "\\frac{U}{mn}$$\\end{document}", "where", "m", "is", "the", "number", "of", "genes", "in", "a", "gene", "set,", "n", "the", "number", "of", "those", "outside", "the", "gene", "set,", "U", "=", "n", "m", "+", "m", "(", "m", "+", "1", ")", "2", "-", "T", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$U", "=", "nm", "+", "\\frac{{m(m", "+", "1)}}{2}", "-", "T$$\\end{document}", ",", "and", "T", "is", "the", "sum", "of", "the", "ranks", "of", "the", "genes", "in", "the", "gene", "set.", "For", "each", "immune", "cell", "signature,", "the", "survival", "analysis", "was", "performed", "by", "dividing", "the", "patient", "cohort", "by", "the", "median", "value", "of", "NES", "score", "into", "low", "and", "high-immune", "cell", "groups.", "By", "intersecting", "high", "and", "low-quality", "neoantigen", "groups", "with", "low", "and", "high-immune", "cell", "groups", "information,", "patients", "were", "stratified", "into", "four", "groups", "(high-quality", "neoantigens", "and", "high-immune", "cells;", "high-quality", "neoantigens", "and", "low-immune", "cells;", "low-quality", "neoantigens", "and", "high-immune", "cells;", "low-quality", "neoantigens", "and", "low", "immune", "cells)", "and", "Kaplan–Meier", "analysis", "was", "performed", "to", "evaluate", "their", "relationship", "with", "survival.", "ee-MWW", "based", "GO", "enrichment", "analysis", "To", "extract", "enriched", "GO", "categories", "in", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells", "compared", "with", "low-quality", "neoantigens", "and", "low", "CD8", "+", "T", "cells,", "we", "used", "our", "recently", "developed", "ee-MWW", "method", "34", "for", "comparing", "unbalanced", "datasets.", "The", "group", "with", "higher", "number", "of", "samples", "(majority", "class)", "is", "subsampled", "in", "order", "to", "have", "the", "same", "size", "of", "the", "subgroup", "with", "less", "samples", "(minority", "class),", "this", "process", "is", "repeated", "several", "times", "(K", "=", "10,000)", "and", "the", "MWW", "test", "statistics", "is", "averaged", "across", "the", "samplings.", "In", "particular,", "the", "MWW", "gene-wise", "was", "applied", "to", "the", "two", "class", "of", "samples.", "For", "each", "sample", "subset", "k", "and", "the", "gene", "j,", "the", "U", "jk", "value", "of", "the", "test", "statistic", "was", "retained.", "The", "value", "associate", "with", "each", "gene", "is", "the", "mean", "U", "jk", "values", "across", "the", "K", "random", "subsets,", "U", "j", "¯", "=", "1", "K", "∑", "k", "=", "1", "K", "U", "jk", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$\\overline", "{U_{\\mathrm{j}}}", "=", "\\frac{1}{K}{\\sum}_{k", "=", "1}^{K}", "U_{{\\mathrm{jk}}}$$\\end{document}", ".", "The", "collection", "of", "U", "j", "¯", "\\documentclass[12pt]{minimal}", "\\usepackage{amsmath}", "\\usepackage{wasysym}", "\\usepackage{amsfonts}", "\\usepackage{amssymb}", "\\usepackage{amsbsy}", "\\usepackage{mathrsfs}", "\\usepackage{upgreek}", "\\setlength{\\oddsidemargin}{-69pt}", "\\begin{document}$$\\overline", "{U_j}$$\\end{document}", "values", "is", "a", "set", "of", "values", "that", "are", "the", "input", "of", "enrichment", "method", "NES", "34", ".", "Identification", "of", "differentially", "expressed", "genes", "To", "identify", "genes", "differentially", "expressed", "between", "tumors", "in", "the", "group", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells", "and", "those", "in", "the", "group", "with", "low-quality", "neoantigens", "and", "low", "CD8", "+", "T", "cells", "from", "RNAseq", "data,", "we", "performed", "TCGAanalyze_DEA", "implementing", "the", "EdgeR", "protocol", "66", ".", "Multiple", "testing", "using", "the", "Benjamini–Hochberg", "procedure", "was", "applied", "to", "generate", "FDR.", "Genes", "with", "fold", "change", ">1.5", "and", "FDR", "<", "0.05", "were", "considered", "as", "differentially", "expressed", "genes.", "To", "identify", "genes", "differentially", "expressed", "between", "tumors", "in", "the", "group", "with", "high-quality", "neoantigens", "and", "high", "CD8", "+", "T", "cells", "and", "those", "in", "the", "group", "with", "low-quality", "neoantigens", "and", "low", "CD8", "+", "T", "cells", "from", "Agilent", "microarray", "data,", "we", "used", "the", "Wilcoxon", "test", "followed", "by", "multiple", "testing", "using", "the", "Benjamini–Hochberg", "method", "for", "FDR", "estimation.", "Genes", "with", "fold", "change", ">1.5", "and", "FDR", "<", "0.05", "were", "considered", "as", "differentially", "expressed", "genes.", "Analysis", "of", "mutations", "and", "DNA", "copy", "number", 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CC-BY
4.0
http://creativecommons.org/licenses/by/4.0/
Original work titled: 'The combination of neoantigen quality and T lymphocyte infiltrates identifies glioblastomas with the longest survival' (Communications Biology/Nature Publishing Group UK) by the following authors: Jing Zhang; Francesca P. Caruso; Jason K. Sa; Sune Justesen; Do-Hyun Nam; Peter Sims; Michele Ceccarelli; Anna Lasorella; Antonio Iavarone DOI: 10.1038/s42003-019-0369-7
db6df9c8ee71fd8c2c6ebd3a02d0d3a5
[ "Introduction", "Tumor", "microenvironment", "favors", "tumor", "cells", "to", "promote", "tumor", "growth,", "migration,", "invasion,", "and", "angiogenesis.", "One", "of", "the", "main", "features", "of", "tumor", "microenvironment", "is", "inflammation", "accompanied", "with", "the", "activation", "of", "immune", "cells", "and", "the", "production", "of", "pro-inflammatory", "cytokines", "1", ",", "2", ".", "IL-1β", "is", "one", "of", "the", "critical", "cytokines", "to", "promote", "tumor", "growth", "and", "metastasis", "by", "acting", "on", "its", "receptor,", "IL-1R,", "and", "to", "culminate", "in", "the", "secondary", "activation", "of", "immune", "cells", "and", "the", "expression", "of", "immune", "cytokines", "3", ".", "IL-1β", "requires", "maturation", "process", "of", "cleavage", "from", "pro-IL-1β", "transcribed", "via", "inflammatory", "signals", "and", "is", "secreted", "to", "extracellular", "matrix", "to", "act", "on", "its", "receptor", "on", "cellular", "membrane.", "Caspase-1", "is", "the", "intracellular", "enzyme", "responsible", "for", "cleavage", "of", "pro-IL-1β", "to", "IL-1β.", "The", "activation", "of", "caspase-1", "occurs", "upon", "the", "activation", "of", "inflammasomes", "which", "induces", "the", "degradation", "of", "pro-caspase-1", "(pro-form)", "to", "caspase-1", "(active", "form)", "4", ".", "Therefore,", "the", "regulation", "of", "inflammasomes", "by", "pharmacological", "inhibitors", "is", "considered", "as", "an", "efficient", "strategy", "to", "reduce", "IL-1β", "levels", "in", "various", "inflammatory", "and", "immune", "diseases", "5", ",", "6", ".", "NACHT,", "LRR", "and", "PYD", "domains-containing", "protein", "3", "(NLRP3)", "inflammasome", "is", "a", "protein", "complex", "composing", "of", "the", "NLRP3,", "an", "adaptor", "protein,", "and", "pro-caspase-1.", "NLRP3", "inflammasome", "is", "activated", "by", "recognizing", "a", "diverse", "set", "of", "inflammation-inducing", "stimuli", "that", "include", "pathogen-associated", "molecular", "patterns", "(PAMPs)", "and", "damage-associated", "molecular", "patterns", "(DAMPs)", "7", ".", "The", "activation", "of", "the", "NLRP3", "inflammasome", "by", "the", "DAMPs", "leads", "to", "the", "activation", "of", "intracellular", "signaling", "pathways", "such", "as", "increase", "in", "potassium", "efflux,", "production", "of", "reactive", "oxygen", "species,", "and", "lysosomal", "damage", "8", ".", "In", "particular,", "adenosine", "triphosphate", "(ATP)", "is", "the", "representative", "DAMPs", "that", "are", "endogenous", "substances", "derived", "from", "the", "host", "to", "activate", "NLRP3", "inflammasome", "9", ".", "ATP", "is", "released", "from", "damaged", "and", "dying", "cells", "to", "extracellular", "matrix,", "therefore,", "accumulated", "in", "high", "levels", "in", "inflamed", "site", "and", "tumor", "microenvironment", "10", ".", "These", "suggest", "that", "ATP", "accumulated", "in", "tumor", "microenvironment", "would", "activate", "NLRP3", "inflammasome", "resulting", "in", "the", "increased", "secretion", "of", "IL-1β", "from", "immune", "cells.", "In", "this", "study,", "we", "investigated", "whether", "the", "inhibition", "of", "NLRP3", "inflammasome", "in", "macrophages", "resulted", "in", "the", "regulation", "of", "metastatic", "potential", "of", "cancer", "cells.", "The", "results", "would", "provide", "an", "effective", "strategy", "to", "prevent", "tumor", "progression", "by", "modulating", "cytokine", "expression", "in", "tumor", "microenvironment.", "The", "suppression", "of", "NLRP3", "inflammasome", "in", "immune", "cells", "could", "be", "a", "novel", "strategy", "for", "anti-cancer", "therapy", "by", "reducing", "IL-1β", "secretion.", "Results", "Deficiency", "of", "NLRP3", "inflammasome", "in", "macrophages", "results", "in", "reduction", "of", "metastatic", "potential", "of", "cancer", "cells", "Since", "IL-1β", "is", "the", "major", "cytokine", "secreted", "upon", "NLRP3", "inflammasome", "activation", "in", "macrophages,", "we", "investigated", "whether", "IL-1β", "would", "increase", "the", "metastatic", "potential", "of", "cancer", "cells.", "Treatment", "of", "recombinant", "IL-1β", "increased", "the", "number", "of", "migrated", "melanoma", "cancer", "cells", "(B16F10)", "in", "transwell", "system", "(Fig.", "1A", ").", "In", "addition,", "recombinant", "IL-1β", "increased", "the", "number", "of", "invading", "melanoma", "cancer", "cells", "(Fig.", "1B", ").", "These", "results", "demonstrate", "that", "IL-1β", "can", "enhance", "the", "metastatic", "potential", "of", "cancer", "cells.", "Figure", "1", "IL-1β", "increases", "the", "metastatic", "potential", "of", "melanoma", "cancer", "cells.", "(", "A", ",", "B", ")", "B16F10", "cells", "were", "plated", "in", "upper", "chamber", "of", "transwell.", "Serum-free", "medium", "containing", "recombinant", "IL-1β", "(0.5,", "1", "ng/ml)", "was", "placed", "in", "the", "bottom", "chamber.", "After", "24", "hr,", "the", "migration", "or", "invasion", "of", "B16F10", "cells", "was", "determined.", "Values", "are", "means", "±", "SEM", "(n", "=", "3).", "*p", "<", "0.05.", "We", "next", "investigated", "whether", "the", "activation", "of", "NLRP3", "inflammasome", "in", "immune", "cells", "in", "tumor", "microenvironment", "contributed", "to", "promoting", "metastatic", "potential", "of", "cancer", "cells.", "After", "primary", "mouse", "macrophages", "derived", "from", "wild-type", "(WT)", "or", "NLRP3-knockout", "(KO)", "mice", "were", "stimulated", "with", "ATP,", "an", "NLRP3", "inflammasome", "activator,", "conditioned", "media", "was", "collected.", "The", "media", "was", "added", "to", "the", "lower", "chamber", "of", "transwell", "and", "the", "migration", "of", "cancer", "cells", "plated", "on", "the", "upper", "chamber", "of", "transwell", "was", "measured.", "Conditioned", "media", "from", "ATP-stimulated", "WT", "macrophages", "increased", "the", "migration", "of", "melanoma", "cancer", "cells", "(B16F10)", "as", "compared", "with", "vehicle-treated", "WT", "macrophages", "(WT,", "vehicle", "vs", "WT,", "ATP:", "158.8", "±", "20.33", "vs", "553.8", "±", "130.09)", "(Fig.", "2A,B", ").", "In", "contrast,", "the", "ability", "to", "induce", "the", "cancer", "cell", "migration", "was", "dramatically", "reduced", "when", "conditioned", "media", "from", "ATP-stimulated", "NLRP3", "KO", "macrophages", "was", "added", "(WT,", "ATP", "vs", "KO,", "ATP:", "553.8", "±", "130.09", "vs", "214.6", "±", "59.07)", "(Fig.", "2A,B", ").", "Figure", "2", "NLRP3-deficient", "macrophages", "failed", "to", "increase", "the", "metastatic", "potential", "of", "melanoma", "cancer", "cells.", "Primary", "mouse", "macrophages", "derived", "from", "wild-type", "(WT)", "or", "NLRP3", "knockout", "(KO)", "mice", "were", "primed", "with", "LPS", "(100", "ng/ml)", "for", "4", "hr.", "The", "cells", "were", "stimulated", "with", "ATP", "(5", "mM)", "for", "1", "hr.", "Conditioned", "culture", "supernatants", "were", "collected", "and", "added", "to", "the", "bottom", "chambers", "of", "transwells", "which", "had", "B16F10", "cells", "plated", "in", "the", "upper", "chamber.", "After", "24", "hr,", "cell", "migration", "and", "invasion", "were", "determined.", "(", "A", ")", "Images", "of", "B16F10", "cell", "migration.", "(", "B", ")", "The", "number", "of", "migrating", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "(", "C", ")", "Images", "of", "B16F10", "cell", "invasion.", "(", "D", ")", "The", "number", "of", "invading", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "Values", "are", "means", "±", "SEM", "(n", "=", "5)", "for", "B", "and", "D.", "*p", "<", "0.05.", "We", "further", "examined", "whether", "the", "activation", "of", "NLRP3", "inflammasome", "in", "macrophages", "enhanced", "the", "invasive", "activity", "of", "cancer", "cells.", "Conditioned", "media", "obtained", "from", "ATP-stimulated", "WT", "macrophages", "increased", "the", "invasive", "potential", "of", "melanoma", "cancer", "cells", "(B16F10)", "(WT,", "vehicle", "vs", "WT,", "ATP:", "132", "±", "38.05", "vs", "564.6", "±", "133.52)", "(Fig.", "2C,D", ")", "while", "conditioned", "media", "collected", "from", "NLRP3", "KO", "macrophages", "stimulated", "with", "ATP", "failed", "to", "enhance", "invasion", "of", "cancer", "cells", "(WT,", "ATP", "vs", "KO,", "ATP:", "564.6", "±", "133.52", "vs", "170.6", "±", "55.48)", "(Fig.", "2C,D", ").", "We", "confirmed", "the", "involvement", "of", "NLRP3", "inflammasome", "using", "caspase-1", "deficient", "macrophages.", "Conditioned", "media", "from", "ATP-stimulated", "WT", "macrophages", "increased", "the", "migration", "of", "melanoma", "cancer", "cells", "(B16F10)", "(Fig.", "3", ")", "while", "the", "migration", "of", "melanoma", "cancer", "cells", "was", "decreased", "when", "conditioned", "media", "from", "caspase-1", "KO", "macrophages", "stimulated", "with", "ATP", "was", "added", "(Fig.", "3", ").", "Figure", "3", "Caspase-1-deficient", "macrophages", "have", "greatly", "reduced", "capacity", "to", "stimulate", "migration", "of", "melanoma", "cancer", "cells.", "Primary", "mouse", "macrophages", "derived", "from", "wild-type", "(WT)", "or", "caspase-1", "knockout", "(KO)", "mice", "were", "primed", "with", "LPS", "(100", "ng/ml)", "for", "4", "hr.", "The", "cells", "were", "stimulated", "with", "ATP", "(5", "mM)", "for", "1", "hr.", "Conditioned", "culture", "supernatants", "were", "collected", "and", "added", "to", "the", "bottom", "chambers", "of", "transwells", "which", "had", "B16F10", "cells", "plated", "in", "the", "upper", "chamber.", "After", "24", "hr,", "cell", "migration", "was", "determined.", "(", "A", ")", "Images", "of", "B16F10", "cell", "migration.", "(", "B", ")", "The", "number", "of", "migrating", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "Values", "are", "means", "±", "SEM", "(n", "=", "3).", "*p", "<", "0.05.", "To", "investigate", "whether", "tumor-promoting", "activity", "induced", "by", "cell", "supernatants", "of", "ATP-stimulated", "macrophages", "was", "mediated", "by", "IL-1β,", "the", "macrophages", "were", "treated", "with", "anti-IL-1β", "antibody", "after", "stimulation", "with", "ATP.", "Anti-IL-1β", "antibody", "prevented", "the", "migration", "and", "invasion", "of", "melanoma", "cancer", "cells", "(B16F10)", "induced", "by", "cell", "supernatants", "of", "ATP-stimulated", "macrophages", "(Fig.", "4", ").", "Figure", "4", "Tumor-promoting", "activity", "of", "macrophages", "is", "mediated", "through", "IL-1β.", "Primary", "mouse", "macrophages", "primed", "by", "LPS", "(100", "ng/ml)", "were", "stimulated", "with", "ATP", "(5", "mM)", "for", "1", "hr", "in", "the", "presence", "of", "isotype", "control", "IgG", "or", "anti-IL-1β", "antibody", "(10", "μg/ml).", "Conditioned", "culture", "supernatants", "were", "collected", "and", "added", "to", "the", "bottom", "chambers", "of", "transwells", "which", "had", "B16F10", "cells", "plated", "in", "the", "upper", "chamber.", "After", "24", "hr,", "cell", "migration", "and", "invasion", "were", "determined.", "(", "A", ")", "Images", "of", "B16F10", "cell", "migration.", "(", "B", ")", "The", "number", "of", "migrating", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "(", "C", ")", "Images", "of", "B16F10", "cell", "invasion.", "(", "D", ")", "The", "number", "of", "invading", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "Values", "are", "means", "±", "SEM", "(n", "=", "5).", "*p", "<", "0.05.", "The", "results", "show", "that", "the", "deficiency", "of", "NLRP3", "inflammasome", "components", "in", "macrophages", "leads", "to", "suppression", "of", "metastatic", "potential", "of", "cancer", "cells", "by", "reducing", "migration", "and", "invasion.", "These", "suggest", "that", "the", "activation", "of", "NLRP3", "inflammasome", "in", "macrophages", "by", "ATP", "of", "which", "level", "is", "high", "in", "tumor", "microenvironment,", "results", "in", "the", "increased", "production", "of", "IL-1β", "and", "the", "promotion", "of", "tumor", "cell", "metastasis.", "Celastrol,", "an", "inhibitor", "of", "NLRP3", "inflammasome,", "reduces", "macrophage", "ability", "to", "stimulate", "migration", "and", "invasion", "of", "cancer", "cells", "Next,", "we", "hypothesized", "that", "the", "pharmacological", "inhibitors", "of", "NLRP3", "inflammasome", "would", "suppress", "metastatic", "potential", "of", "cancer", "cells.", "We", "found", "that", "celastrol", "(Fig.", "5A", ")", "was", "effective", "to", "block", "activation", "of", "NLRP3", "inflammasome", "in", "macrophages", "(Fig.", "5", ").", "Celastrol", "blocked", "ATP-induced", "degradation", "of", "pro-caspase-1", "to", "caspase-1(p10)", "and", "cleavage", "of", "pro-IL-1β", "to", "mature", "IL-1β", "in", "macrophages", "as", "determined", "by", "immunoblotting", "assay", "(Fig.", "5B", ").", "Celastrol", "inhibited", "ATP-induced", "secretion", "of", "mature", "IL-1β", "as", "determined", "by", "ELISA", "of", "culture", "media", "(Fig.", "5C", ").", "Oligomerization", "of", "apoptosis-associated", "speck-like", "protein", "containing", "a", "CARD", "(ASC)", "is", "another", "hallmark", "of", "NLRP3", "inflammasome", "activation.", "Celastrol", "blocked", "ATP-induced", "ASC", "oligomerization", "in", "macrophages", "(Fig.", "5D", ").", "Confocal", "microscopy", "analysis", "showed", "that", "celastrol", "reduced", "ASC", "speckle", "formation", "induced", "by", "ATP", "(Fig.", "5E", ").", "These", "results", "show", "that", "celastrol", "is", "effective", "to", "suppress", "ATP-induced", "activation", "of", "NLRP3", "inflammasome", "in", "macrophages.", "In", "contrast,", "celastrol", "did", "not", "suppress", "the", "activation", "of", "other", "inflammasomes", "such", "as", "AIM2", "(absent", "in", "melanoma", "2)", "and", "NLRC4", "(also", "known", "as", "IPAF)", "since", "celastrol", "did", "not", "block", "the", "production", "of", "caspase-1(p10)", "and", "IL-1β", "induced", "by", "poly", "dA:dT", "(an", "AIM2", "activator)", "or", "flagellin", "(an", "NLRC4", "activator)", "in", "primary", "mouse", "macrophages", "(Supplemental", "Fig.", "1", ").", "Figure", "5", "Celastrol", "inhibits", "ATP-induced", "activation", "of", "NLRP3", "inflammasome", "in", "primary", "macrophages.", "(", "A", ")", "Chemical", "structure", "of", "celastrol.", "(", "B", "–", "E", ")", "Primary", "mouse", "macrophages", "were", "primed", "with", "LPS", "(100", "ng/ml)", "for", "4", "hr.", "The", "cells", "were", "treated", "with", "celastrol", "for", "1", "hr", "and", "then", "stimulated", "with", "ATP", "(5", "mM)", "for", "(", "B", ",", "D", ",", "E", ")", "1", "hr", "or", "(", "C", ")", "2", "hr.", "(", "B", ")", "The", "cell", "culture", "supernatants", "and", "cell", "lysates", "were", "immunoblotted", "for", "pro-caspase-1,", "caspase-1(p10),", "pro-IL-1β,", "and", "IL-1β.", "(", "C", ")", "The", "cell", "culture", "supernatants", "were", "analyzed", "for", "secreted", "IL-1β", "using", "ELISA.", "The", "values", "represent", "the", "means", "±", "SEM", "(n", "=", "3).", "*Significantly", "different", "from", "ATP", "alone,", "p", "<", "0.05.", "(", "D", ")", "Cell", "lysates", "were", "processed", "for", "immunoblotting", "of", "ASC.", "(", "E", ")", "The", "cells", "were", "stained", "for", "ASC", "(green).", "The", "nuclei", "were", "stained", "with", "4′,6-diamidino-2-phenylindole", "(DAPI;", "blue).", "The", "arrows", "indicate", "ASC", "speckles.", "The", "data", "are", "representative", "of", "three", "independent", "experiments.", "For", "immunoblotting", "results,", "the", "cropped", "blots", "from", "full", "length", "gels", "were", "presented.", "DIC,", "differential", "interference", "contrast.", "We", "investigated", "whether", "the", "suppression", "of", "NLRP3", "inflammasome", "activation", "by", "celastrol", "in", "macrophages", "contributed", "to", "the", "blockade", "of", "migration", "and", "invasion", "of", "cancer", "cells.", "Celastrol", "was", "treated", "to", "the", "macrophages", "which", "were", "stimulated", "with", "ATP", "and", "the", "conditioned", "media", "was", "collected", "to", "be", "added", "to", "lower", "chamber", "of", "transwell.", "The", "migration", "and", "invasion", "of", "melanoma", "cancer", "cells", "(B16F10)", "seeded", "on", "upper", "chamber", "were", "determined.", "Conditioned", "media", "derived", "from", "ATP-stimulated", "macrophages", "induced", "the", "migration", "of", "melanoma", "cancer", "cells", "(B16F10),", "while", "conditioned", "media", "obtained", "from", "ATP-stimulated", "macrophages", "in", "the", "presence", "of", "celastrol", "failed", "to", "induce", "cancer", "cell", "migration", "(ATP", "vs", "ATP", "±", "celastrol", "1", "μM:", "359", "±", "22.04", "vs", "63.4", "±", "16.82)", "(Fig.", "6A,B", ").", "Furthermore,", "celastrol", "treatment", "to", "ATP-stimulated", "macrophages", "resulted", "in", "the", "decrease", "of", "invasive", "properties", "of", "melanoma", "cancer", "cells", "(ATP", "vs", "ATP", "±", "celastrol", "1", "μM:", "301", "±", "58.37", "vs", "54.2", "±", "20.36)", "(Fig.", "6C,D", ").", "Figure", "6", "Celastrol", "treatment", "reduces", "the", "potency", "of", "macrophages", "to", "stimulate", "the", "metastatic", "potential", "of", "melanoma", "cancer", "cells.", "LPS-primed", "primary", "mouse", "macrophages", "were", "stimulated", "with", "ATP", "(5", "mM)", "for", "1", "hr", "in", "the", "presence", "or", "absence", "of", "celastrol.", "Conditioned", "culture", "supernatants", "were", "collected", "and", "added", "to", "the", "bottom", "chambers", "of", "transwells", "which", "had", "B16F10", "cells", "plated", "in", "the", "upper", "chamber.", "After", "24", "hr,", "cell", "migration", "and", "invasion", "were", "determined.", "(", "A", ")", "Images", "of", "B16F10", "cell", "migration.", "(", "B", ")", "The", "number", "of", "migrating", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "(", "C", ")", "Images", "of", "B16F10", "cell", "invasion.", "(", "D", ")", "The", "number", "of", "invading", "B16F10", "cells", "was", "counted", "in", "the", "microscopic", "field.", "Values", "are", "means", "±", "SEM", "(n", "=", "5)", "for", "B", "and", "D.", "*Significantly", "different", "from", "ATP", "alone,", "p", "<", "0.05.", "These", "results", "indicate", "that", "celastrol", "treatment", "to", "macrophages", "suppresses", "the", "stimulatory", "effects", "of", "macrophages", "on", "cancer", "migration", "and", "invasion,", "possibly", "mediated", "through", "the", "inhibition", "of", "NLRP3", "inflammasome", "activation", "and", "the", "consequent", "reduction", "of", "IL-1β", "secretion.", "Celastrol", "suppressed", "the", "potassium", "efflux", "induced", "by", "ATP", "and", "nigericin", "in", "macrophages", "We", "further", "investigated", "the", "mechanism", "as", "to", "how", "celastrol", "inhibited", "ATP-induced", "NLRP3", "inflammasome.", "Cellular", "mechanism", "by", "which", "ATP", "activates", "NLRP3", "inflammasome", "includes", "the", "activation", "of", "the", "purinergic", "P2X7", "receptor", "and", "the", "recruitment", "of", "pannexin-1", "to", "the", "receptor,", "resulting", "in", "the", "opening", "of", "a", "large", "pore", "and", "the", "following", "increase", "of", "potassium", "efflux", "11", ",", "12", ".", "Nigericin,", "another", "simulator", "of", "NLRP3", "inflammasome,", "a", "microbial", "toxin", "derived", "from", "Streptomyces", "hygroscopicus,", "induces", "the", "potassium", "efflux", "through", "pannexin-1", "13", ".", "Activation", "of", "NLRP3", "inflammasome", "by", "nigericin", "is", "independent", "of", "P2X7", "receptor,", "because", "nigericin", "itself", "acts", "as", "a", "potassium", "ionophore.", "Celastrol", "blocked", "nigericin-induced", "production", "of", "caspase-1(p10)", "and", "cleaved", "IL-1β", "in", "macrophages", "(Supplemental", "Fig.", "2A", ").", "Nigericin-induced", "IL-1β", "secretion", "was", "blocked", "by", "celastrol", "(Supplemental", "Fig.", "2B", ").", "Nigericin-induced", "ASC", "oligomerization", "was", "also", "suppressed", "by", "celastrol", "in", "macrophages", "(Supplemental", "Fig.", "2C,D", ").", "These", "suggest", "that", "the", "inhibitory", "effects", "of", "celastrol", "may", "not", "be", "dependent", "on", "P2X7", "receptor", "but", "related", "to", "the", "process", "of", "potassium", "efflux.", "To", "investigate", "the", "inhibitory", "effect", "of", "celastrol", "on", "the", "potassium", "efflux", "induced", "by", "ATP", "or", "nigericin,", "we", "determined", "the", "level", "of", "intracellular", "potassium", "ion", "after", "stimulation", "of", "macrophages", "with", "ATP", "and", "nigericin", "using", "a", "potassium", "sensitive", "dye,", "potassium", "binding", "benzofuran", "isophthalate", "(PBFI)-tetraammonium", "salt.", "The", "level", "of", "the", "potassium", "efflux", "was", "increased", "upon", "ATP", "stimulation", "while", "celastrol", "suppressed", "ATP-induced", "potassium", "efflux", "in", "macrophages", "(Fig.", "7A", ").", "Furthermore,", "nigericin-induced", "potassium", "efflux", "was", "also", "reduced", "by", "celastrol", "(Fig.", "7B", ").", "These", "results", "indicate", "that", "celastrol", "suppressed", "ATP-induced", "activation", "of", "NLRP3", "inflammasome", "by", "blocking", "the", "process", "of", "potassium", "efflux", "in", "macrophages.", "Figure", "7", "Celastrol", "suppresses", "the", "potassium", "efflux", "induced", "by", "ATP", "and", "nigericin", "in", "primary", "macrophages.", "Primary", "mouse", "macrophages", "were", "primed", "with", "LPS", "(100", "ng/ml)", "for", "4", "hr.", "The", "cells", "were", "treated", "with", "celastrol", "for", "1", "hr", "and", "then", "stimulated", "with", "(", "A", ")", "ATP", "(5", "mM)", "or", "(", "B", ")", "nigericin", "(10", "μM)", "together", "with", "potassium", "binding", "benzofuran", "isophthalate", "(PBFI)-tetraammonium", "salt", "for", "1", "hr.", "Values", "are", "means", "±", "SEM", "(n", "=", "3).", "*Significantly", "different", "from", "ATP", "or", "nigericin", "alone,", "p", "<", "0.05.", "Discussion", "Inflammation", "and", "immunity", "are", "considered", "important", "determinants", "of", "tumorigenesis", "from", "onset", "to", "cancer", "equilibrium", "during", "cancer", "development", "14", ",", "15", ".", "Once", "tumorigenesis", "is", "initiated", "by", "autonomous", "aberration", "of", "cells,", "the", "interaction", "between", "cancer", "cells", "and", "their", "microenvironment,", "particularly", "immune", "cells,", "is", "critical", "to", "govern", "tumor", "evasion.", "Much", "effort", "has", "been", "devoted", "to", "decoding", "the", "functions", "of", "the", "adaptive", "immune", "system", "in", "tumor", "immunity", "surveillance", "15", ",", "16", ",", "however,", "the", "contribution", "of", "innate", "immune", "pathways", "to", "this", "process", "is", "less", "understood.", "Among", "the", "effectors", "of", "the", "innate", "immune", "system,", "gonad-encoding", "pattern", "recognition", "receptors", "(PRRs)", "trigger", "inflammatory", "responses", "when", "detecting", "invading", "microorganisms", "or", "danger-associated", "molecules.", "Many", "PRRs,", "including", "members", "of", "the", "NOD-like", "receptor", "(NLR)", "and", "the", "HIN200", "families,", "work", "by", "combining", "macromolecular", "complexes", "called", "inflammasomes", "4", ".", "This", "signaling", "scaffold", "induces", "innate", "immunity", "by", "activating", "protease", "caspase-1,", "which", "cleaves", "the", "immune", "and", "metabolic", "matrix", "17", ",", "18", ".", "In", "particular,", "it", "induces", "the", "production", "of", "pro-inflammatory", "cytokines,", "IL-1β", "and", "IL-18,", "resulting", "in", "inflammation", "and", "pyroptosis", "19", ".", "Guo", "et", "al.", "showed", "that", "IL-1β", "enhanced", "the", "growth", "and", "metastasis", "of", "tumor", "cells", "and", "the", "recruitment", "of", "myeloid", "cells", "to", "tumor", "tissue", "sites", "in", "breast", "cancer", "mouse", "models,", "suggesting", "that", "tumor", "microenvironment", "produces", "IL-1β", "to", "promote", "the", "metastatic", "potential", "of", "tumor", "cells", "20", ".", "Late-stage", "human", "melanoma", "cells", "constitutively", "secrete", "IL-1β", "through", "spontaneous", "activation", "of", "the", "NLRP3", "inflammasome,", "promoting", "inflammatory", "environment,", "macrophage", "chemotaxis,", "and", "angiogenesis", "21", ".", "Our", "results", "demonstrate", "that", "IL-1β", "produced", "by", "macrophages", "stimulates", "the", "metastatic", "potential", "of", "melanoma", "cells,", "such", "as", "migration", "and", "invasion", "properties,", "showing", "the", "essential", "role", "of", "myeloid", "cells", "in", "the", "production", "of", "IL-1β", "in", "tumor", "microenvironment", "through", "activation", "of", "the", "NLRP3", "inflammasome.", "Furthermore,", "inhibition", "of", "the", "NLRP3", "inflammasome", "in", "myeloid", "cells", "by", "a", "pharmacological", "inhibitor", "such", "as", "celastrol", "resulted", "in", "the", "decreased", "metastatic", "potential", "of", "melanoma", "cells.", "These", "results", "suggest", "the", "importance", "of", "NLRP3", "inflammasome", "in", "the", "tumor", "microenvironment", "in", "the", "regulation", "of", "tumor", "metastasis.", "Therefore,", "the", "results", "further", "provide", "a", "beneficial", "strategy", "for", "preventing", "tumor", "metastasis", "by", "regulating", "NLRP3", "inflammasome", "in", "immune", "cells", "and", "thereby", "reducing", "IL-1β", "levels", "in", "tumor", "sites.", "Interestingly,", "the", "NLRP3", "inflammasome", "plays", "different", "roles", "in", "the", "tumorigenesis", "of", "melanoma", "in", "a", "stage-dependent", "manner", "22", ".", "Silencing", "ASC", "expression", "resulted", "in", "G1", "cell", "cycle", "arrest,", "reduced", "cell", "viability,", "and", "downregulation", "of", "tumorigenesis", "in", "metastatic", "melanoma", "while", "silencing", "ASC", "expression", "in", "primary", "melanoma", "led", "to", "increased", "cell", "viability", "with", "enhanced", "tumorigenesis", "22", ".", "In", "addition,", "Drexler", "et", "al.", "suggested", "that", "ASC", "exhibited", "different", "impacts", "on", "tumorigenesis", "depending", "on", "the", "different", "cell", "types", "23", ".", "Mice", "had", "more", "tumors", "than", "control", "group", "when", "ASC", "was", "specifically", "deficient", "in", "keratinocytes,", "suggesting", "that", "ASC", "may", "be", "a", "tumor-suppressor", "in", "keratinocytes", "23", ".", "The", "expression", "level", "of", "ASC", "protein", "in", "human", "metastatic", "melanoma", "was", "lower", "than", "in", "primary", "melanoma", "and", "ASC", "protein", "expression", "was", "lost", "in", "human", "cutaneous", "squamous", "cell", "carcinoma", "22", ",", "23", ".", "In", "contrast,", "similar", "to", "our", "results", "showing", "the", "role", "of", "NLRP3", "inflammasome", "in", "myeloid", "cells", "in", "enhancing", "the", "metastatic", "potential,", "mice", "with", "a", "specific", "deficiency", "of", "ASC", "in", "myeloid", "cells", "developed", "reduced", "skin", "cancer", "incidence,", "showing", "its", "pro-inflammatory", "role", "in", "myeloid", "cells", "23", ".", "The", "influence", "of", "the", "NLRP3", "inflammasome", "appears", "controversial", "in", "colitis-associated", "colon", "cancer.", "Unlike", "our", "results", "and", "those", "of", "Drexler", "et", "al.,", "the", "NLRP3", "inflammasome", "expressed", "in", "hematopoietic", "cells", "exerted", "a", "protective", "role", "against", "colitis-associated", "cancer", "as", "the", "increased", "tumor", "burden", "correlated", "with", "the", "downregulation", "of", "IL-1β", "expression", "at", "the", "tumor", "site", "24", ".", "Caspase-1", "deficiency", "resulted", "in", "enhanced", "tumor", "formation", "in", "the", "azoxymethane", "and", "dextran", "sodium", "sulfate", "colitis-associated", "colorectal", "cancer", "mouse", "model", "through", "the", "regulation", "of", "colonic", "epithelial", "cell", "proliferation", "and", "apoptosis,", "but", "not", "through", "the", "regulation", "of", "inflammatory", "cells", "25", ".", "The", "anti-inflammatory", "properties", "of", "celastrol", "have", "previously", "been", "reported.", "Celastrol", "inhibited", "lipopolysaccharides", "(LPS)-induced", "the", "activation", "of", "immune", "cells,", "mediated", "through", "the", "suppression", "of", "the", "transcription", "factors", "NF-κB", "and", "AP-1", "26", ".", "We", "previously", "reported", "that", "the", "inhibitory", "effects", "of", "celastrol", "on", "LPS-induced", "inflammation", "were", "mediated", "by", "blocking", "LPS", "binding", "to", "TLR4/MD2", "complex", "in", "macrophages", "27", ".", "In", "this", "study,", "we", "show", "that", "celastrol", "suppressed", "the", "activation", "of", "NLRP3", "inflammasome", "induced", "by", "ATP", "or", "nigericin.", "Celastrol", "inhibited", "the", "activation", "of", "NLRP3", "inflammasome", "induced", "by", "another", "activator,", "uric", "acid", "crystals", "(Supplemental", "Fig.", "3", ")", "although", "the", "inhibitory", "potency", "was", "weak.", "Interestingly,", "celastrol", "did", "not", "suppress", "the", "activation", "of", "other", "inflammasomes", "such", "as", "AIM2", "nor", "NLRC4", "inflammasome", "in", "primary", "mouse", "macrophages", "(Supplemental", "Fig.", "1", ").", "Decrease", "of", "cell", "viability", "was", "observed", "by", "ATP", "treatment", "possibly", "due", "to", "increase", "of", "pyroptosis", "accompanied", "by", "NLRP3", "inflammasome", "activation", "(Supplemental", "Fig.", "4", ").", "However,", "treatment", "of", "celastrol", "in", "addition", "to", "ATP", "did", "not", "significantly", "enhance", "cell", "death", "(Supplemental", "Fig.", "4", ").", "Furthermore,", "celastrol", "did", "not", "show", "any", "dose-dependent", "effect", "on", "cell", "viability", "(Supplemental", "Fig.", "4", ").", "It", "is", "reported", "that", "inhibition", "of", "NLRP3", "inflammasome", "by", "celastrol", "is", "possibly", "mediated", "through", "the", "down-regulation", "of", "reactive", "oxygen", "species", "production", "and", "NF-κB", "activation", "28", ".", "In", "addition,", "the", "induction", "of", "autophagy", "was", "suggested", "as", "the", "mechanism", "by", "which", "celastrol", "inhibits", "the", "NLRP3", "inflammasome", "29", ".", "Our", "results", "demonstrate", "that", "blocking", "potassium", "efflux", "is", "a", "new", "mechanism", "for", "celastrol", "inhibiting", "the", "NLRP3", "inflammasome", "in", "macrophages.", "Several", "anti-cancer", "targets", "and", "mechanisms", "for", "celastrol", "have", "been", "studied", "in", "cancer", "cells.", "Celastrol-induced", "autophagy", "blocked", "gastric", "cancer", "growth", "30", "and", "celastrol", "strongly", "downregulated", "the", "protein", "levels", "of", "Bcr-Abl", "in", "chronic", "myelogenous", "leukemia", "cells", "31", ".", "The", "anti-tumor", "effects", "of", "celastrol", "on", "MCF-7", "cells", "were", "mediated", "through", "AMPK-induced", "p53-polo-like", "kinase", "2", "(PLK-2)", "pathways", "32", ".", "Celastrol", "suppressed", "the", "activation", "of", "STAT3/JNK", "1/2/c-Src", "pathway", "in", "human", "hepatocellular", "carcinoma", "33", ".", "Celastrol", "is", "a", "proteasome", "inhibitor", "leading", "to", "enhanced", "apoptosis", "in", "prostate", "cancer", "models", "34", ".", "Our", "results", "propose", "the", "NLRP3", "inflammasome", "as", "a", "new", "anti-cancer", "target", "of", "celastrol", "for", "the", "regulation", "of", "myeloid", "cell", "activation", "in", "tumor", "microenvironment.", "The", "inhibition", "of", "NLRP3", "inflammasome", "by", "celastrol", "in", "macrophages", "results", "in", "the", "suppression", "of", "tumor", "metastatic", "potential.", "The", "results", "would", "provide", "a", "novel", "anti-cancer", "strategy", "using", "NLRP3", "inflammasome", "inhibitors,", "such", "as", "celastrol", "to", "modulate", "the", "myeloid", "cells", "in", "tumor", "microenvironment", "and", "tumor", "metastatic", "potential", "by", "reducing", "the", "IL-1β", "level", "at", "tumor", "sites.", "ATP", "abundant", "in", "tumor", "microenvironment", "stimulates", "the", "NLRP3", "inflammasome", "in", "macrophages,", "releasing", "mature", "IL-1β", "to", "the", "tumor", "microenvironment", "to", "enhance", "the", "metastatic", "potential", "of", "cancer", "cells.", "Our", "results", "demonstrate", "that", "suppression", "of", "ATP-induced", "NLRP3", "inflammasome", "activation", "and", "IL-1β", "secretion", "in", "tumor", "microenvironment,", "contributes", "to", "decreasing", "metastatic", "potential", "of", "cancer", "cells.", "Our", "results", "further", "suggest", "a", "possible", "therapeutic", "application", "of", "NLRP3", "inflammasome", "inhibitors", "that", "would", "prevent", "the", "onset", "of", "NLRP3", "inflammasome-mediated", "inflammatory", "and", "immune", "diseases.", "Materials", "and", "Methods", "Animals", "and", "cell", "culture", "Animal", "care", "and", "the", "study", "protocols", "were", "carried", "out", "in", "accordance", "with", "the", "guidelines", "of", "the", "Institutional", "Animal", "Care", "and", "Use", "Committee", "(IACUC)", "of", "the", "Catholic", "University", "of", "Korea.", "The", "experimental", "protocols", "were", "approved", "by", "the", "IACUC", "of", "the", "Catholic", "University", "of", "Korea", "with", "a", "permission", "#", "2012-5-001.", "C57BL/6", "mice", "were", "purchased", "from", "Orient", "Bio", "(Seoul,", "Korea).", "NLRP3-knockout", "mice", "(B6N.129-Nlrp3tm3Hhf/J)", "and", "caspase-1", "knockout", "mice", "(B6N.129S2-Casp1tm1Flv/J)", "were", "purchased", "from", "The", "Jackson", "Laboratory", "(Bar", "Harbor,", "ME,", "USA).", "The", "mice", "were", "acclimated", "under", "specific", "pathogen-free", "conditions", "in", "an", "animal", "facility", "for", "at", "least", "a", "week", "before", "the", "experiments.", "They", "were", "housed", "in", "a", "room", "controlled", "for", "temperature", "(23", "±", "3", "°C)", "and", "relative", "humidity", "(40–60%)", "and", "acclimated", "under", "specific", "pathogen-free", "conditions", "in", "the", "animal", "facility", "for", "at", "least", "a", "week", "before", "the", "experiments.", "Bone", "marrow", "cells", "were", "isolated", "from", "C57BL/6", "mice", "and", "differentiated", "to", "macrophage", "as", "described", "previously", "35", ".", "Bone", "marrow", "cells", "were", "cultured", "in", "in", "Dulbecco", "Modified", "Eagle", "Medium", "(DMEM)", "(Gibco,", "Hampton,", "NH,", "USA)", "containing", "10%", "(v/v)", "heat-inactivated", "fetal", "bovine", "serum", "(Corning,", "NY,", "USA),", "2", "mM", "L-glutamine,", "1", "mM", "sodium", "pyruvate,", "10", "mM", "Hepes", "buffer", "and", "20%", "L929", "cell-conditioned", "medium", "for", "6", "days,", "and", "adherent", "cells", "were", "used", "as", "macrophages.", "Mouse", "melanoma", "cell", "line", "(B16F10,", "CRL-6475,", "American", "Type", "Culture", "Collection,", "Manassas,", "VA,", "USA)", "were", "cultured", "in", "DMEM", "containing", "10%", "(v/v)", "heat-inactivated", "fetal", "bovine", "serum,", "100", "units/ml", "of", "penicillin", "and", "100", "μg/ml", "of", "streptomycin", "(Gibco).", "The", "cells", "were", "maintained", "at", "37", "°C", "in", "a", "5%", "CO", "2", "/air", "environment.", "For", "inflammasome", "activation", "study,", "primary", "mouse", "macrophages", "were", "primed", "with", "lipopolysaccharides", "(LPS)", "for", "4", "h.", "To", "examine", "the", "effect", "of", "celastrol", "on", "inflammasome", "with", "excluding", "the", "effect", "on", "LPS,", "LPS", "was", "removed", "by", "washing", "with", "phosphate-buffered", "saline", "(PBS)", "before", "treatment", "of", "celastrol.", "After", "cells", "were", "treated", "with", "celastrol", "for", "1", "h,", "cells", "were", "stimulated", "with", "inflammasome", "activators", "such", "as", "adenosine", "triphosphate", "(ATP),", "nigericin,", "monosodium", "uric", "acid", "crystals,", "poly", "dA:dT,", "or", "flagellin.", "Reagents", "Purified", "LPS", "from", "Escherichia", "coli", "was", "obtained", "from", "List", "Biological", "Laboratory", "Inc.", "(Campbell,", "CA,", "USA)", "and", "dissolved", "in", "endotoxin-free", "water.", "Celastrol", "was", "purchased", "from", "Sigma-Aldrich", "(St.", "Louis,", "MO,", "USA)", "and", "dissolved", "in", "dimethyl", "sulfoxide", "(DMSO).", "ATP", "and", "flagellin", "were", "purchased", "from", "Invivogen", "(San", "Diego,", "CA,", "USA).", "Nigericin", "was", "purchased", "from", "Sigma-Aldrich.", "In", "vitro", "migration", "assay", "Cellular", "migration", "assay", "was", "performed", "using", "Boyden", "transwell", "chamber", "(8-μm", "pore", "size,", "Becton", "Dickinson,", "Bedford,", "MA).", "Tumor", "cells", "(2", "×", "10", "5", "cells/100", "μl)", "were", "placed", "in", "the", "filter", "membrane.", "Conditioned", "medium", "(300", "μl)", "obtained", "from", "primary", "mouse", "macrophages", "were", "placed", "in", "the", "lower", "chamber.", "After", "transwell", "chambers", "with", "cells", "were", "maintained", "at", "37", "°C", "for", "24", "h,", "cells", "on", "the", "upper", "membrane", "surface", "were", "completely", "removed", "by", "wiping", "with", "cotton", "swab", "and", "the", "migrant", "cells", "on", "the", "lower", "membrane", "surface", "were", "fixed", "with", "4%", "paraformaldehyde", "and", "then", "stained", "with", "0.5%", "crystal", "violet", "(Sigma-Aldrich).", "The", "membranes", "were", "examined", "microscopically", "and", "stained", "migrant", "cells", "were", "counted", "in", "at", "least", "5", "randomly", "selected", "fields.", "In", "vitro", "invasion", "assay", "Cell", "invasion", "assay", "were", "performed", "using", "Boyden", "transwell", "chamber", "(8-μm", "pore", "size,", "Becton", "Dickinson).", "Matrigel", "(Becton", "Dickinson)", "diluted", "with", "serum-free", "DMEM", "was", "placed", "in", "the", "upper", "chamber", "of", "filter", "membrane,", "and", "incubated", "at", "37", "°C", "for", "4", "h.", "A", "total", "of", "2", "×", "10", "5", "tumor", "cells", "were", "seeded", "with", "serum-free", "DMEM", "in", "the", "upper", "chamber.", "Conditioned", "medium", "(300", "μl)", "obtained", "from", "bone-marrow", "derived", "macrophages", "were", "placed", "in", "the", "lower", "chamber.", "After", "transwell", "chambers", "with", "cells", "were", "maintained", "at", "37", "°C", "for", "24", "h,", "cells", "on", "the", "upper", "membrane", "surface", "were", "completely", "removed", "by", "wiping", "with", "cotton", "swab", "and", "the", "migrant", "cells", "on", "the", "lower", "membrane", "surface", "were", "fixed", "with", "4%", "paraformaldehyde", "and", "then", "stained", "with", "0.5%", "crystal", "violet", "(Sigma-Aldrich).", "The", "membranes", "were", "examined", "microscopically", "and", "stained", "migrant", "cells", "were", "counted", "in", "at", "least", "5", "randomly", "selected", "fields.", "Immunoblotting", "analysis", "This", "was", "performed", "as", "described", "previously", "36", ".", "The", "antibody", "to", "detect", "pro-caspase-1", "(45", "kDa)", "and", "caspase-1(p10)", "(10", "kDa)", "was", "obtained", "from", "Santa", "Cruz", "Biotechnology", "(sc-514,", "Dallas,", "TX,", "USA).", "An", "antibody", "to", "detect", "pro-IL-1β", "(31", "kDa)", "and", "mature", "IL-1β", "(17", "kDa)", "was", "from", "R&D", "systems", "(Minneapolis,", "MN,", "USA).", "ELISA", "The", "levels", "of", "IL-1β", "in", "culture", "media", "were", "determined", "using", "enzyme-linked", "immunosorbent", "assay", "(ELISA)", "kits", "(R&D", "Systems,", "Minneapolis,", "MN,", "USA).", "The", "concentration", "ranges", "for", "the", "standard", "curves", "is", "from", "12.5", "to", "1000", "pg/mL", "and", "the", "minimum", "detectable", "dose", "ranged", "from", "0.46", "to", "4.8", "pg/mL", "concentration", "ranges", "for", "the", "standard", "curves.", "Determination", "of", "ASC", "oligomerization", "This", "was", "performed", "as", "described", "previously", "37", ".", "Cell", "pellet", "fractions", "were", "immunoblotted", "with", "anti-ASC", "antibody", "(sc-22514-12,", "Santa", "Cruz", "Biotechnology).", "Confocal", "imaging", "analysis", "This", "was", "performed", "as", "described", "previously", "38", ".", "Briefly,", "after", "LPS-primed", "primary", "macrophages", "were", "treated", "with", "celastrol", "for", "1", "h,", "cells", "were", "stimulated", "with", "ATP", "and", "nigericin.", "Cells", "were", "fixed", "and", "incubated", "with", "anti-ASC", "antibody", "(sc-22514-12,", "Santa", "Cruz", "Biotechnology)", "and", "DAPI", "(4′,", "6-diamidino-2-phenylindole).", "Cells", "were", "further", "incubated", "with", "FITC-conjugated", "anti-rabbit", "IgG", "antibody", "(Sigma-Aldrich).", "The", "samples", "were", "examined", "with", "an", "LSM710", "confocal", "laser", "scanning", "microscope", "(Carl", "Zeiss,", "Oberkochen,", "Germany)", "equipped", "with", "40x", "objectives.", "Images", "were", "obtained", "with", "ZEN2011", "software", "(Carl", "Zeiss).", "Determination", "of", "extracelluar", "potassium", "concentration", "Primary", "mouse", "macrophages", "were", "grown", "in", "96-well", "plates", "until", "80–90%", "confluency.", "After", "cells", "were", "pre-treated", "with", "celastrol", "for", "1", "h,", "cells", "were", "stimulated", "with", "ATP", "and", "nigericin", "together", "with", "potassium", "binding", "benzofuran", "isophthalate", "(PBFI)-tetraammonium", "salt", "(Molecular", "Probes,", "Inc.,", "Eugene,", "OR,", "USA)", "for", "1", "h.", "Samples", "were", "read", "with", "fluorescence", "plate", "reader", "(SpectraMax", "M5,", "Molecular", "Devices,", "Sunnyvale,", "CA,", "USA)", "at", "excitation", "340", "nm", "and", "emission", "515", "nm.", "Statistical", "analysis", "Data", "are", "expressed", "as", "means", "±", "SEM", "(n", "=", "3–5).", "Comparisons", "of", "data", "between", "groups", "were", "performed", "by", "one-way", "analysis", "of", "variance", "(ANOVA)", "followed", "by", "Duncan’s", "multiple", "range", "test.", "Values", "of", "p", "<", "0.05", "were", "considered", "significant." ]
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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, 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, 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{"negatives/white": ["200+1", "493+1", "954+1", "1422+1", "1783+1", "2297+1", "2804+1", "3260+1", "3645+1", "4158+1", "2458+1", "3163+1"]}
CC-BY
4.0
http://creativecommons.org/licenses/by/4.0/
Original work titled: 'Inhibition of NLRP3 inflammasome in tumor microenvironment leads to suppression of metastatic potential of cancer cells' (Scientific Reports/Nature Publishing Group UK) by the following authors: Hye Eun Lee; Jin Young Lee; Gabsik Yang; Han Chang Kang; Yong-Yeon Cho; Hye Suk Lee; Joo Young Lee DOI: 10.1038/s41598-019-48794-x
925f953a678d2142afaa0aa423712046
[ "Introduction", "Invasive", "species", "are", "a", "major", "threat", "to", "food", "security", "and", "conservation", "of", "natural", "biodiversity", "1", ",", "2", ".", "Typically,", "such", "biological", "invasions", "follow", "a", "jump", "dispersal", "pattern", "reflecting", "human", "assisted", "transmission", "often", "across", "the", "entire", "globe", "3", ",", "4", ".", "In", "particular,", "there", "is", "growing", "evidence", "that", "international", "trade", "can", "play", "a", "major", "role", "in", "spreading", "alien", "species", "5", ",", "6", ",", "but", "in", "many", "cases", "the", "actual", "trading", "routes", "and", "goods", "involved", "remain", "poorly", "understood.", "Aiming", "at", "mitigating", "the", "global", "impact", "of", "invasive", "species,", "a", "better", "understanding", "of", "the", "role", "of", "specific", "international", "trading", "routes", "as", "transmission", "pathways", "appears", "therefore", "crucial.", "Since", "genetic", "tools", "are", "routinely", "used", "to", "reconstruct", "routes", "of", "invasion", "7", ",", "one", "feasible", "approach", "seems", "to", "be", "a", "combination", "of", "genetics", "data", "with", "public", "data", "on", "international", "trade", "8", "for", "specific", "cases", "of", "invasive", "species.", "This", "also", "holds", "true", "for", "invasive", "pests", "and", "pathogens", "associated", "with", "managed", "honey", "bees,", "Apis", "mellifera", "Linnaeus", "(Hymenoptera:", "Apidae)", "9", ".", "One", "invasive", "species", "associated", "with", "honey", "bees", "is", "the", "small", "hive", "beetle", "(=SHBs),", "Aethina", "tumida", "Murray", "(Coleoptera:", "Nitidulidae)", "10", ".", "SHBs", "are", "parasites", "of", "honey", "bee", "colonies", "native", "to", "sub-Saharan", "Africa", "11", ",", "12", ".", "Larval", "and", "adult", "SHBs", "feed", "on", "honey,", "pollen,", "bee", "larvae,", "as", "well", "as", "dead", "or", "live", "adult", "bees", "13", "–", "15", ".", "SHB", "larvae", "can", "cause", "severe", "damage", "to", "honey", "bee", "colonies", "14", ",", "often", "resulting", "in", "the", "full", "structural", "collapse", "of", "the", "entire", "nest", "12", ".", "Since", "1996,", "SHBs", "have", "emerged", "as", "an", "invasive", "species", "and", "have", "now", "established", "on", "all", "continents", "except", "Antarctica", "10", ",", "16", "–", "18", ".", "At", "present,", "it", "is", "still", "unclear", "whether", "a", "single", "or", "multiple", "introductions", "into", "the", "USA", "have", "occurred", "19", "–", "21", ".", "SHBs", "in", "Australia", "appear", "to", "have", "a", "different", "origin", "than", "beetles", "in", "North", "America", "and", "the", "initial", "North", "American", "beetles", "shared", "the", "same", "source", "21", ".", "The", "outbreaks", "in", "Quebec", "(Canada)", "22", "appear", "to", "have", "originated", "from", "the", "USA", "10", ",", "21", ",", "22", "and", "the", "SHBs", "confirmed", "in", "Alberta", "(Canada)", "in", "2006", "22", "were", "probably", "a", "novel", "introduction", "via", "Australian", "package", "bees", "21", ".", "SHBs", "confirmed", "in", "the", "Calabria", "region", "(Italy)", "in", "2014", "23", "appear", "to", "have", "been", "introduced", "from", "Cameroon", "24", ".", "This", "introduction", "into", "Calabria", "was", "followed", "by", "man-mediated", "migration", "to", "Sicily", "24", ".", "SHBs", "detected", "and", "originally", "reported", "to", "be", "widespread", "in", "Egypt", "25", "were", "not", "confirmed", "by", "a", "latter", "extensive", "survey", "26", ",", "thereby", "suggesting", "a", "possible", "false", "positive", "diagnosis", "in", "the", "first", "place.", "In", "2004,", "SHB", "larvae", "were", "intercepted", "in", "a", "shipment", "of", "queens", "from", "the", "USA", "to", "Portugal,", "but", "pest", "establishment", "was", "successfully", "prevented", "by", "rigorous", "sanitation", "27", ".", "Recently,", "introductions", "into", "Hawaii", "(2010)", "10", "and", "Brazil", "(2015)", "16", ",", "were", "traced", "back", "to", "South", "Africa", "28", "and", "the", "USA", "29", ",", "respectively.", "None", "of", "the", "confirmed", "SHB", "introductions", "reported", "in", "Portugal", "(2004)", "27", ",", "Jamaica", "(2005)", "10", ",", "Mexico", "(2007)", "10", ",", "Cuba", "(2012)", "10", ",", "El", "Salvador", "(2013)", "10", ",", "Nicaragua", "(2014)", "10", ",", "Philippines", "(2014)", "10", ",", "South", "Korea", "(2016)", "17", "and", "Mauritius", "(2016)", "18", "have", "so", "far", "been", "studied", "genetically.", "In", "its", "new", "ranges,", "SHBs", "can", "have", "a", "strong", "impact", "on", "local", "honey", "bees", "and", "can", "also", "infest", "colonies", "of", "other", "social", "bees", "10", "as", "well", "as", "nests", "of", "solitary", "bees", "30", ".", "It", "appears", "therefore", "high", "time", "to", "slow", "down", "the", "ongoing", "spread", "of", "SHBs", "until", "better", "mitigating", "options", "will", "become", "available", "31", ".", "Central", "to", "that", "is", "clearly", "the", "identification", "of", "the", "actual", "transmission", "means.", "So", "far,", "a", "whole", "range", "of", "actual", "and", "possible", "transmission", "pathways", "has", "been", "identified,", "with", "import", "of", "bees", "and", "bee", "products", "being", "the", "major", "confirmed", "contributors", "so", "far", "10", ".", "Accordingly,", "countries", "have", "put", "in", "place", "legislation", "and", "quarantine", "measures", "to", "reduced", "chances", "for", "entry", "of", "SHBs", "as", "well", "as", "other", "bee", "pests", "and", "diseases", "9", ".", "While", "most", "of", "these", "efforts", "have", "focused", "on", "the", "control", "of", "living", "bee", "imports,", "hive", "products", "have", "so", "far", "been", "rather", "neglected,", "even", "though", "one", "introduction", "of", "SHBs", "into", "Canada", "could", "be", "traced", "back", "to", "beeswax", "imports", "22", ".", "Therefore,", "it", "seems", "obvious", "that", "at", "least", "some", "additional", "SHB", "introductions", "may", "have", "occurred", "in", "association", "with", "international", "trade", "of", "beeswax.", "Beeswax", "is", "a", "creamy", "coloured", "substance", "used", "by", "worker", "bees", "to", "build", "the", "comb", "that", "forms", "the", "structure", "of", "their", "nest", "32", ".", "It", "is", "widely", "used", "by", "humans,", "e.g.", "in", "cosmetics,", "pharmaceutical", "preparations", "and", "food", "production", "33", ".", "In", "particular,", "beekeepers", "use", "large", "quantities", "of", "beeswax", "for", "making", "beeswax", "comb", "foundation", "32", ",", "33", ".", "Beeswax", "can", "significantly", "range", "in", "quality", "34", "and", "respective", "international", "trade", "can", "occur", "in", "various", "forms,", "ranging", "from", "non-processed", "“crude”", "wax", "over", "wax", "foundations", "to", "highly", "processed", "ones", "(i.e.", "melted", "and", "purified)", "9", ",", "32", "–", "35", ".", "For", "beeswax", "in", "the", "form", "of", "“honeycomb”,", "there", "are", "trade", "regulations", "with", "respect", "to", "SHBs", "available", "36", ".", "Either", "the", "beeswax", "originates", "from", "a", "country", "free", "of", "A.", "tumida", "infestation", "or", "precautions,", "such", "as", "“thoroughly”", "cleaning,", "have", "been", "taken", "to", "prevent", "infestation/contamination", "with", "SHB", "life", "stages", "9", ",", "36", ".", "However,", "for", "processed", "beeswax", "there", "are", "currently", "no", "trade", "restrictions", "with", "respect", "to", "SHBs", "9", ",", "36", ",", "probably", "because", "wax", "processing", "is", "assumed", "to", "kill", "all", "SHB", "life", "stages", "(e.g.", "melting).", "Interestingly,", "countries", "in", "sub-Saharan", "Africa,", "the", "endemic", "range", "of", "SHBs", "10", ",", "12", ",", "only", "participate", "in", "international", "trade", "with", "the", "sale", "of", "beeswax", "and", "honey", "8", ",", "the", "latter", "not", "being", "confirmed", "as", "a", "SHB", "transport", "means", "so", "far", "10", ".", "Nevertheless,", "correlations", "between", "global", "beeswax", "trade", "routes", "8", "and", "confirmed", "SHB", "introductions", "with", "respective", "areas", "of", "origin", "have", "yet", "to", "be", "taken", "into", "consideration.", "This", "is", "mainly", "due", "to", "few", "data", "sets", "existing", "on", "molecular", "markers", "tracing", "back", "the", "origin", "and", "possible", "transmission", "pathways", "of", "SHBs", "19", "–", "21", ",", "24", ",", "28", ",", "29", ".", "These", "previous", "studies", "could", "obviously", "not", "have", "covered", "all", "confirmed", "introductions", "so", "far", "and/or", "only", "considered", "comparatively", "few", "native", "locations", "of", "SHBs.", "The", "aim", "of", "this", "study", "is", "to", "shed", "further", "light", "on", "possible", "SHB", "invasion", "pathways", "to", "mitigate", "its", "further", "spread.", "For", "that", "purpose,", "we", "here", "combine", "for", "the", "first", "time", "genetic", "data", "of", "12", "confirmed", "SHB", "invasions", "with", "FAO", "data", "on", "international", "trade", "of", "beeswax", "8", ".", "If", "the", "latter", "constitutes", "a", "major", "means", "of", "SHB", "transport", "globally,", "we", "suspect", "that", "in", "the", "majority", "of", "cases", "genetic", "traces", "of", "origin", "match", "with", "respective", "data", "on", "the", "imports", "of", "beeswax.", "Results", "Mitochondrial", "COI", "haplotypes", "Analysis", "of", "mitochondrial", "COI", "DNA", "sequence", "data", "from", "296", "individuals", "revealed", "90", "unique", "haplotypes", "representing", "0.857", "±", "0.018", "and", "0.02280", "±", "0.00103", "(mean", "±", "SD)", "haplotype", "and", "nucleotide", "diversity,", "respectively.", "Among", "the", "sequences", "analysed,", "118", "segregating", "(polymorphic)", "sites", "were", "detected", "(Table", "1", "),", "89", "of", "which", "were", "parsimony-informative", "and", "29", "were", "singleton", "sites.", "Table", "1", "Number", "of", "analysed", "individuals", "(N),", "polymorphic", "sites", "(S),", "parsimony", "informative", "sites", "(PIS),", "number", "of", "haplotypes", "(h),", "haplotype", "diversity", "(Hd),", "nucleotide", "diversity", "(pi)", "and", "average", "number", "of", "differences", "(K)", "in", "mt-DNA", "sequences", "computed", "for", "Aethina", "tumida", "collected", "from", "five", "geographical", "regions.", "Regions", "Sample", "size", "(N)", "Polymorphic", "sites", "(S)", "Parsimony", "informative", "sites", "(PIS)", "Number", "of", "haplotypes", "(h)", "Haplotype", "diversity", "(Hd)", "Nucleotide", "diversity", "(pi)", "Average", "number", "of", "nucleotide", "differences", "(K)", "Australia", "30", "19", "10", "4", "0.510", "±", "0.109", "0.00285", "±", "0.00076", "2.522", "Americas", "115", "16", "13", "5", "0.301", "±", "0.056", "0.00183", "±", "0.00039", "1.518", "Europe", "10", "45", "43", "4", "0.889", "±", "0.075", "0.02070", "±", "0.00256", "20.289", "Asia", "21", "36", "36", "2", "0.381", "±", "0.101", "0.01404", "±", "0.00370", "13.714", "Africa", "120", "105", "73", "80", "0.983", "±", "0.005", "0.02490", "±", "0.00148", "13.646", "All", "296", "118", "89", "90", "0.857", "±", "0.018", "0.02280", "±", "0.00103", "12.450", "81", "haplotypes", "were", "found", "in", "a", "single", "country", "(Australia,", "Benin,", "Burkina-Faso,", "Burundi,", "DR", "Congo,", "Ethiopia,", "Italy,", "Kenya,", "Madagascar,", "Malawi,", "Nigeria,", "Central", "African", "Republic,", "South-Sudan,", "Sudan,", "Tanzania,", "Togo,", "Uganda,", "and", "the", "USA)", "while", "the", "nine", "others", "were", "present", "in", "at", "least", "two", "different", "countries.", "The", "most", "widely", "distributed", "haplotype", "(Hap_11)", "was", "found", "with", "a", "global", "frequency", "of", "35.1%", "and", "was", "detected", "in", "Africa", "(Tanzania)", "and", "the", "Americas", "(Brazil,", "Canada,", "Cuba,", "Jamaica,", "Mexico,", "and", "USA)", "(Fig.", "1", ").", "The", "two", "other", "most", "frequent", "haplotypes", "were", "Hap_1", "and", "Hap_23", "with", "global", "frequencies", "of", "11.5", "and", "4.7%,", "respectively.", "Whereas", "Hap_1", "occurred", "in", "three", "countries", "(South", "Africa,", "Hawaii", "and", "Australia),", "Hap_23", "was", "found", "in", "four", "countries", "(Canada,", "Costa", "Rica,", "USA,", "and", "South", "Korea)", "(Fig.", "1", ").", "Figure", "1", "Mitochondrial", "haplotype", "network", "constructed", "from", "samples", "of", "Aethina", "tumida", "(N", "=", "296)", "using", "the", "TCS", "method", "as", "implemented", "in", "PopArt.", "The", "areas", "of", "the", "circles", "are", "proportional", "to", "the", "number", "of", "samples", "sharing", "each", "haplotype.", "Small", "ticks", "on", "branches", "indicate", "the", "number", "of", "mutations", "separating", "haplotypes.", "Each", "colour", "represents", "haplotypes", "found", "in", "a", "region.", "The", "highest", "COI", "diversity", "was", "found", "in", "Africa", "with", "80", "out", "of", "90", "haplotypes", "present", "(Hd", "=", "0.983", "±", "0.005,", "pi", "=", "0.02490", "±", "0.00148,", "K", "=", "13.6)", "(Table", "1", ").", "Among", "the", "invaded", "areas,", "Europe", "(Portugal", "and", "Italy),", "where", "four", "haplotypes", "were", "detected,", "displayed", "the", "highest", "genetic", "diversity", "(Hd", "=", "0.889", "±", "0.075,", "pi", "=", "0.02070", "±", "0.00256,", "K", "=", "20.3)", "(Table", "1", ").", "The", "Americas,", "with", "five", "identified", "haplotypes,", "showed", "the", "lowest", "genetic", "diversity", "(Hd", "=", "0.301", "±", "0.056,", "pi", "=", "0.00183", "±", "0.00039,", "K", "=", "1.518).", "From", "the", "five", "detected", "haplotypes", "in", "the", "Americas,", "four", "were", "found", "in", "the", "USA.", "Phylogenetic", "reconstruction", "The", "Bayesian", "phylogenetic", "reconstruction", "based", "on", "COI", "sequences", "separates", "the", "samples", "into", "six", "different", "clades,", "labelled", "A,", "A1,", "A2,", "B,", "B1,", "and", "B2", "(Fig.", "2", ").", "These", "different", "clades", "can", "be", "grouped", "in", "two", "main", "African", "populations", "labelled", "Pop1", "and", "Pop2", "(Fig.", "2", ").", "Pop1", "is", "consisted", "of", "Western", "(Benin,", "Nigeria,", "Togo,", "and", "Burkina", "Faso)", "and", "Central", "African", "(Cameroon,", "Central", "African", "Republic,", "and", "Democratic", "Republic", "of", "Congo)", "populations,", "but", "also", "some", "Eastern", "regions", "(Uganda,", "South", "Sudan,", "Sudan,", "Burundi,", "and", "Ethiopia).", "Pop2", "includes", "some", "Eastern", "(Tanzania,", "Kenya,", "South", "Sudan,", "and", "Madagascar),", "Southern", "(South", "Africa,", "Malawi,", "and", "Zimbabwe)", "and", "Western", "African", "(Burkina", "Faso)", "populations.", "Pop2", "shows", "93%", "similarity", "with", "Burkina", "Faso", "samples", "from", "Fada-Ngourma.", "In", "Pop1", "and", "Pop2", "different", "clades", "are", "well-supported", "by", "posterior", "probabilities", "(>90%).", "Figure", "2", "Bayesian", "phylogenetic", "tree", "of", "the", "COI", "gene", "showing", "relationships", "among", "Aethina", "tumida", "samples", "from", "Africa,", "the", "Americas,", "Asia,", "Australia", "and", "Europe.", "Posterior", "probabilities", "are", "shown", "above", "nodes", "with", "over", "70%", "support.", "The", "different", "letters", "represent", "main", "clades", "(A,", "A1,", "A2,", "B,", "B1,", "B2).", "Each", "branch", "and", "label", "colour", "shows", "A.", "tumida", "samples", "collected", "from", "the", "same", "country", "and/or", "continent.", "Each", "colour", "range", "shows", "the", "main", "African", "population", "Pop1", "(=beige)", "or", "Pop2", "(=frost", "blue).", "A.", "concolor", "was", "used", "as", "outgroup.", "Most", "of", "the", "Italian", "specimens", "belong", "to", "clade", "A,", "mainly", "A1", "and", "A2.", "The", "other", "Italian", "specimens,", "which", "are", "not", "included", "in", "clade", "A,", "show", "similarity", "with", "each", "other.", "In", "clade", "A1,", "the", "Italian", "samples", "(ItalyCal8,", "and", "ItalyCal9)", "cluster", "with", "one", "Ugandan", "sample", "(UgandaAl2)", "with", "a", "Bayesian", "posterior", "probability", "of", "86%.", "In", "the", "same", "clade,", "other", "Italian", "samples", "(ItalyCal2,", "Italy12,", "Italy3,", "Italy4", "2015)", "show", "a", "high", "similarity", "with", "the", "Portuguese", "sample", "(96%).", "In", "clade", "A2,", "Italian", "samples,", "which", "contains", "the", "first", "detected", "specimen", "in", "Calabria", "(ItalyCal1_KT380624),", "cluster", "with", "Ethiopian", "samples", "with", "a", "Bayesian", "posterior", "probability", "of", "97%.", "Clade", "B1", "includes", "SHB", "from", "the", "USA", "(USA2,", "US", "NthAm2),", "Mexico,", "Jamaica,", "Cuba,", "Canada", "(British", "Columbia,", "Quebec)", "and", "Brazil.", "Interestingly,", "within", "this", "clade", "all", "samples", "cluster", "with", "Kenyan,", "Zimbabwean,", "and", "Tanzanian", "samples", "with", "a", "Bayesian", "posterior", "probability", "of", "95%.", "In", "clade", "B,", "all", "samples", "from", "Australia,", "USA", "(USA1,", "USNthAm1),", "Canada", "(Alberta,", "Ashville,", "and", "Concordia),", "Costa", "Rica,", "Hawaii", "and", "South", "Korea", "show", "similarity", "with", "Tanzanian", "and", "South", "African", "samples.", "FAO", "data", "We", "considered", "beeswax", "imports", "8", "from", "countries", "with", "known", "native", "or", "introduced", "SHB", "populations", "up", "to", "two", "years", "before", "the", "introduction", "into", "new", "ranges", "(Table", "2", ").", "Based", "on", "prior", "invasion", "history", "(USA,", "Australia)", "10", ",", "15", ",", "22", ",", "this", "appears", "to", "be", "a", "suitable", "time", "window.", "SHB", "populations", "probably", "have", "to", "increase", "first", "to", "cause", "clinical", "symptoms", "of", "infestation", "37", ",", "thereby", "fostering", "their", "detection", "by", "beekeepers.", "Table", "2", "Combined", "genetics", "data", "(this", "study)", "and", "beeswax", "trade", "data", "(FAO)", "8", "for", "12", "confirmed", "small", "hive", "beetle", "(SHB)", "invasions.", "Invasion", "Genetics", "Beeswax", "Origin", "USA", "(mainland)", "Kenya,", "Tanzania,", "Zimbabwe,", "South", "Africa", "Tanzania", "Tanzania", "Mexico", "Kenya,", "Tanzania,", "Zimbabwe", "USA,", "Germany", "(no", "SHBs)", "USA", "Jamaica", "Kenya,", "Tanzania,", "Zimbabwe", "USA", "USA", "Cuba", "Kenya,", "Tanzania,", "Zimbabwe", "—", "USA", "Canada", "Kenya,", "Tanzania,", "Zimbabwe,", "South", "Africa", "USA", "USA", "Brazil", "Kenya,", "Tanzania,", "Zimbabwe", "USA,", "India", "(no", "SHBs)", "USA", "Costa", "Rica", "Tanzania,", "South", "Africa", "USA", "USA", "USA", "(Hawaii)", "Tanzania,", "South", "Africa", "—", "USA,", "South", "Africa", "South", "Korea", "South", "Africa,", "Tanzania", "USA", "USA", "Australia", "South", "Africa,", "Tanzania", "South", "Africa,", "China", "(no", "SHBs)", "South", "Africa", "Portugal", "Unknown", "(same", "as", "Italy)", "Spain,", "Germany,", "UK,", "France,", "Netherlands", "(all", "no", "SHBs)", "USA", "Italy", "Unknown,", "Ethiopia,", "Uganda", "Various", "countries,", "incl.", "Ethiopia", "Ethiopia", "The", "country", "of", "SHB", "Invasion,", "the", "possible", "country", "of", "origin", "in", "the", "endemic", "range", "in", "Africa", "(=Genetics),", "the", "country", "of", "origin", "for", "imported", "beeswax", "(=Beeswax)", "and", "the", "most", "parsimonious", "country", "of", "SHB", "origin", "(=Origin)", "are", "shown", "(−=", "no", "data", "available).", "In", "nine", "out", "of", "12", "invasion", "cases,", "the", "genetics", "and", "beeswax", "data", "match", "as", "indicated", "in", "bold", ".", "For", "the", "Portugal", "case,", "import", "of", "queen", "bees", "was", "shown", "35", "and", "for", "two", "further", "cases,", "beeswax", "trade", "data", "are", "not", "available", "(−=", "lack", "of", "FAO", "data", "8", ").", "Beeswax", "imports", "into", "the", "Americas", "From", "1994", "to", "1996,", "Tanzania", "was", "the", "first", "African", "beeswax", "trade", "partner", "to", "the", "USA.", "The", "USA", "imported", "39,", "29", "and", "70", "tons", "(=T)", "in", "1994,", "1995", "and", "1996", "respectively.", "From", "2000", "to", "2002,", "Canada", "imported", "363.84", "T", "of", "beeswax", "per", "year,", "of", "which", "∼87%", "was", "from", "the", "USA.", "During", "this", "period,", "Canada", "did", "not", "import", "any", "beeswax", "from", "Africa.", "In", "the", "suspected", "period", "of", "introduction", "of", "SHBs", "into", "Jamaica", "(i.e.", "2003–2005),", "FAO", "data", "clearly", "show", "that", "this", "country", "imported", "3", "T", "of", "beeswax", "only", "in", "2003", "and", "exclusively", "from", "the", "USA.", "From", "2005", "to", "2007,", "the", "USA", "and", "Germany", "(no", "confirmed", "SHB", "cases)", "were", "the", "main", "beeswax", "suppliers", "of", "Mexico", "with", "an", "average", "of", "81.33", "and", "75.33", "T", "per", "year", "respectively", "(with", "a", "maximum", "import", "of", "108", "and", "78", "T", "in", "2007).", "Costa", "Rica", "is", "a", "small", "beeswax", "importer", "with", "an", "average", "of", "3.33", "T", "per", "year", "from", "2012", "to", "2014.", "During", "this", "period,", "Costa", "Rica", "imported", "only", "from", "two", "countries:", "Argentina", "(2", "T;", "no", "confirmed", "SHB", "cases)", "and", "USA", "(1.33", "T).", "In", "2012", "and", "2013,", "Costa", "Rica", "imported", "beeswax", "exclusively", "from", "the", "USA", "with", "an", "average", "of", "1", "T", "per", "year.", "The", "USA,", "after", "India", "(no", "confirmed", "SHB", "cases),", "was", "the", "second", "largest", "beeswax", "exporter", "to", "Brazil", "with", "an", "average", "of", "5.67", "T", "per", "year", "from", "2013", "to", "2015.", "There", "are", "no", "data", "available", "on", "the", "quantity", "of", "beeswax", "imported", "to", "Cuba.", "Beeswax", "imports", "into", "Australia", "For", "the", "period", "1999–2001,", "data", "are", "not", "available", "in", "the", "FAO", "database.", "However,", "from", "2005", "to", "2016", "South", "Africa", "was", "the", "second", "largest", "beeswax", "supplier", "(after", "China)", "to", "Australia", "with", "an", "average", "of", "80.6", "T", "per", "year.", "Beeswax", "imports", "into", "Italy,", "Portugal", "and", "South", "Korea", "From", "2012", "to", "2014,", "Italy", "imported", "from", "Asia", "(283.33", "T),", "Europe", "(242.01", "T),", "Africa", "(20", "T)", "and", "the", "Americas", "(2", "T).", "In", "2012,", "Italy", "imported", "beeswax", "from", "Africa", "exclusively", "from", "Ethiopia", "(18", "T).", "During", "this", "period,", "Italy", "did", "not", "import", "any", "beeswax", "from", "Cameroon.", "From", "2002", "to", "2004,", "Portugal", "exclusively", "imported", "from", "Europe:", "Spain", "(20", "T),", "Germany", "(5.67", "T),", "UK", "(3.67", "T),", "France", "(0.33", "T)", "and", "Netherlands", "(0.33", "T).", "None", "of", "these", "countries", "had", "any", "confirmed", "SHB", "cases.", "Portugal", "did", "not", "import", "any", "beeswax", "from", "the", "USA.", "South", "Korea", "imported", "beeswax", "from", "Americas", "exclusively", "from", "the", "USA", "with", "11", "and", "16", "T", "in", "2015", "and", "2016", "respectively.", "Combination", "of", "genetics", "and", "beeswax", "trade", "data", "To", "shed", "light", "on", "the", "role", "of", "beeswax", "imports", "for", "the", "spread", "of", "SHBs,", "we", "combined", "our", "genetic", "data", "with", "beeswax", "trade", "data", "(FAO)", "8", "for", "12", "confirmed", "SHB", "invasions.", "For", "that", "purpose,", "we", "considered", "imports", "from", "countries", "in", "the", "endemic", "range", "of", "SHBs", "and", "with", "prior", "confirmed", "cases", "10", ",", "15", "–", "18", ",", "22", ".", "In", "nine", "out", "of", "12", "invasion", "cases,", "the", "genetics", "and", "beeswax", "data", "matched,", "i.e.", "countries", "of", "origin", "suggested", "by", "genetics", "data", "matched", "with", "beeswax", "trade", "records", "(Table", "2", ",", "Fig.", "3", ").", "Figure", "3", "Small", "hive", "beetle", "native", "range,", "confirmed", "global", "introductions", "(up", "to", "May", "2019)", "and", "invasion", "pathways.", "Please", "refer", "to", "Neumann", "et", "al.", "10", "for", "further", "references.", "Endemic", "distribution", "range", "in", "sub-Saharan", "Africa", "(=dark", "grey", "areas),", "introductions", "(=white", "circles)", "and", "most", "likely", "invasion", "routes", "(arrows)", "are", "shown:", "(1)", "1996,", "Charleston,", "South", "Carolina,", "USA,", "(2)", "2000,", "Itay-Al-Baroud,", "Egypt,", "(3)", "2001,", "Richmond,", "NSW,", "Australia,", "(4)", "2002,", "Manitoba,", "Canada,", "(5)", "2004,", "Lisbon,", "Portugal,", "(6)", "2005,", "Jamaica", "(2010),", "(7)", "2006,", "Alberta", "and", "Manitoba,", "Canada,", "(8)", "2007,", "Coahuila,", "Mexico,", "(9)", "2007,", "Kununurra,", "North", "Australia,", "(10)", "2008,", "Perth", "Australia,", "(11)", "2008,", "2009,", "Quebec,", "Canada,", "(12)", "2008,", "2013", "Ontario,", "Canada,", "(13)", "2010,", "Pana’ewa,", "Big", "Island,", "Hawaii,", "(14)", "2012,", "Cuba,", "(15)", "2012,", "Naracoorte", "in", "Eastern", "South", "Australia;", "(16)", "2013,", "El", "Salvador,", "(17)", "2014,", "Nicaragua),", "(18)", "Sovereto,", "Calabria,", "Italy,", "(19)", "2014,", "Renmark,", "Australia,", "(20)", "2014,", "Lupon,", "Philippines,", "(21)", "2015,", "Piracicaba,", "São", "Paulo", "State,", "Brazil", "16", ",", "(22)", "2016,", "Miryang-si,", "GN,", "Korea", "17", ",", "(23)", "2016,", "Mauritius", "island", "18", ".", "(24)", "2016,", "Guanacoste,", "Costa-Rica", "(this", "study);", "(red", "line", "=", "our", "genetic", "data;", "solid", "red", "lines", "=", "invasion", "pathways", "suggested", "by", "genetics;", "dashed", "red", "lines", "=", "origin", "from", "Australia", "and/or", "South", "Africa;", "dotted", "red", "line", "=", "confirmed", "introduction", "from", "the", "USA", "via", "bee", "import", "7", ",", "but", "our", "data", "suggest", "the", "same", "African", "origin", "as", "for", "Italy;", "yellow", "line", "=", "matching", "beeswax", "trade", "data", "8", "with", "our", "genetic", "data).", "Discussion", "Our", "genetic", "data", "combined", "with", "FAO", "data", "strongly", "suggest", "that", "imports", "of", "beeswax", "play", "a", "previously", "underestimated", "key", "role", "in", "propagating", "SHBs", "globally.", "Indeed,", "in", "nine", "out", "of", "12", "cases,", "beeswax", "trade", "data", "and", "our", "genetic", "data", "match.", "When", "excluding", "one", "confirmed", "introduction", "pathway", "(bee", "imports,", "Portugal", "27", ")", "and", "the", "two", "cases,", "for", "which", "no", "FAO", "data", "were", "available,", "the", "genetics", "and", "FAO", "trade", "data", "consistently", "predict", "the", "same", "source", "of", "SHBs.", "This", "creates", "an", "urgent", "demand", "to", "better", "consider", "the", "role", "of", "international", "beeswax", "trade", "for", "the", "spread", "of", "this", "invasive", "species.", "The", "results", "confirm", "the", "African", "origin", "of", "this", "invasive", "species", "10", ",", "12", ",", "15", "and", "that", "SHBs", "found", "in", "Australia", "and", "Canada", "are", "most", "likely", "originating", "from", "South", "Africa", "and", "USA", "respectively", "21", "(Fig.", "3", ").", "Similarly,", "our", "genetic", "data", "are", "also", "in", "line", "with", "recent", "reports", "on", "the", "origin", "of", "SHBs", "in", "Hawaii", "28", "and", "Brazil", "29", ",", "suggesting", "that", "SHBs", "discovered", "in", "Hawaii", "originated", "from", "South", "Africa", "and", "those", "found", "in", "Brazil", "came", "from", "the", "USA.", "Tanzania,", "South", "Africa,", "Ethiopia", "and", "Uganda", "have", "been", "identified", "as", "sources", "of", "invasive", "populations", "respectively", "found", "in", "the", "USA,", "Australia", "and", "Italy.", "The", "data", "further", "suggest", "that", "the", "USA", "is", "the", "most", "probably", "source", "of", "spread", "of", "SHBs", "in", "Cuba,", "Jamaica,", "Costa", "Rica,", "Mexico", "and", "South", "Korea.", "The", "samples", "from", "Costa", "Rica", "were", "here", "confirmed", "to", "be", "A.", "tumida.", "The", "highest", "COI", "diversity", "was", "found", "in", "Africa", "with", "80", "out", "of", "90", "identified", "mitochondrial", "haplotypes.", "This", "high", "genetic", "diversity", "confirms", "previous", "findings", "21", "as", "well", "as", "the", "African", "origin", "of", "SHBs.", "Furthermore,", "the", "phylogenetic", "reconstruction", "suggests", "the", "existence", "of", "at", "least", "two", "large", "African", "populations,", "in", "which", "several", "haplotypes", "are", "present.", "The", "population", "Pop1", "is", "more", "heterogeneous", "than", "population", "Pop2,", "thereby", "suggesting", "that", "the", "large", "region", "(West,", "Central", "and", "Eastern", "Africa)", "is", "probably", "the", "SHBs", "nucleus", "of", "origin.", "The", "comparatively", "high", "mobility", "of", "adult", "SHBs", "10", "and", "resulting", "panmixis", "21", "could", "explain", "the", "non-grouping", "of", "the", "different", "sub-populations", "by", "country", "in", "the", "phylogenetic", "tree.", "In", "addition,", "Pop2", "showed", "a", "considerable", "similarity", "with", "samples", "from", "Burkina", "Faso", "(Fada-Ngourma)", "belonging", "to", "Pop1.", "This", "population", "may", "have", "invaded", "the", "southern", "and", "eastern", "regions.", "This", "could", "explain", "the", "homogeneity", "and", "low", "diversity", "observed", "within", "population", "Pop2.", "Based", "on", "our", "data,", "it", "is", "not", "clear", "if", "these", "two", "SHB", "populations", "actually", "overlap", "or", "not.", "A", "more", "exhaustive", "sampling", "in", "the", "neighbouring", "geographic", "areas", "between", "the", "two", "populations", "and", "more", "genetic", "markers", "will", "be", "required", "to", "shed", "light", "on", "the", "causes", "for", "this", "observed", "divergence.", "In", "the", "invaded", "areas", "(Americas,", "Australia", "and", "Asia)", "the", "COI", "diversity", "was", "low", "when", "compared", "to", "Africa,", "which", "is", "a", "logic", "consequence", "of", "genetic", "bottlenecks", "due", "to", "invasion", "events", "38", ".", "Among", "these", "areas,", "the", "Americas", "showed", "the", "lowest", "genetic", "diversity", "probably", "reflecting", "comparatively", "few", "introduction", "events", "21", ".", "Alternatively,", "but", "not", "mutually", "exclusive,", "an", "original", "genetic", "diversity", "may", "have", "been", "lost", "secondarily.", "Our", "results", "clearly", "show", "that", "one", "of", "the", "haplotypes", "(Hap_11)", "present", "in", "the", "Americas", "(USA,", "Brazil,", "Canada,", "Cuba,", "Jamaica", "and", "Mexico)", "is", "the", "same", "as", "that", "found", "in", "Tanzania.", "Combining", "our", "genetics", "data", "with", "the", "FAO", "data", "base", "8", ",", "the", "most", "likely", "invasion", "scenario", "is", "that", "the", "first", "specimens", "found", "in", "South", "Carolina", "in", "November", "1996", "15", "were", "introduced", "via", "infested", "beeswax", "containers", "from", "Tanzania.", "Since", "it", "can", "take", "years", "for", "mass", "reproduction", "with", "obvious", "clinical", "symptoms", "to", "occur", "15", ",", "SHBs", "could", "have", "easily", "been", "introduced", "one", "to", "two", "years", "before", "their", "confirmation.", "Indeed,", "the", "USA", "imported", "39", "and", "29", "T", "of", "beeswax", "respectively", "in", "1994", "and", "1996", "from", "Tanzania", "8", ".", "These", "SHBs", "almost", "certainly", "crossed", "the", "USA", "border", "to", "invade", "Quebec", "in", "2008", "(<25", "km)", "22", ".", "Our", "data", "do", "not", "confirm", "the", "previous", "finding", "of", "an", "Australian", "origin", "of", "Canadian", "SHBs", "21", ".", "Most", "likely,", "the", "SHBs", "introduced", "from", "Australia", "back", "then", "were", "not", "able", "to", "establish", "a", "local", "population", "in", "their", "northern", "distribution", "limit", "in", "Canada", "(but", "see", "Essex", "county,", "Ontario", "for", "an", "exception)", "10", ".", "It", "appears", "therefore", "most", "likely", "that", "novel", "introductions", "from", "the", "USA", "have", "occurred", "in", "the", "meantime.", "Similarly,", "according", "to", "our", "data,", "the", "USA", "is", "the", "most", "probable", "source", "of", "SHBs", "in", "other", "countries", "of", "the", "Americas", "(Brazil,", "Cuba,", "Jamaica", "and", "Mexico),", "which", "is", "also", "reflected", "in", "respective", "beeswax", "trade", "activities", "8", ".", "The", "results", "of", "the", "haplotype", "analysis", "show", "that", "the", "second", "haplotype", "(Hap_23)", "found", "in", "the", "USA", "is", "also", "present", "in", "Canada,", "Costa", "Rica", "and", "South", "Korea.", "According", "to", "FAO", "statistics,", "Costa", "Rica", "and", "South", "Korea", "have", "not", "imported", "beeswax", "from", "Africa", "8", ".", "However,", "both", "countries", "have", "mostly", "imported", "beeswax", "from", "the", "USA", "8", ",", "thereby", "supporting", "that", "the", "SHBs", "detected", "in", "Costa", "Rica", "and", "more", "recently", "in", "South", "Korea", "originated", "from", "the", "USA.", "Alternatively,", "but", "not", "mutually", "exclusive,", "the", "SHBs", "in", "Costa", "Rica", "simply", "reflect", "the", "ongoing", "SHB", "invasion", "front", "in", "Central", "America", "10", ".", "Our", "results", "confirm", "previous", "findings", "that", "SHBs", "found", "in", "Australia", "are", "most", "likely", "originating", "from", "South", "Africa", "21", ".", "Interestingly,", "the", "SHBs", "found", "in", "Hawaii", "are", "also", "similar", "to", "South", "African", "haplotypes", "25", ",", "thereby", "implying", "that", "Australian", "and", "Hawaiian", "beetles", "both", "originated", "from", "the", "same", "African", "source", "population.", "Alternatively,", "but", "not", "mutually", "exclusive,", "the", "Hawaiian", "SHBs", "may", "have", "originated", "from", "the", "Australian", "populations.", "Unfortunately,", "there", "are", "no", "FAO", "data", "available", "concerning", "beeswax", "imports", "from", "South", "Africa", "to", "Australia", "and", "to", "Hawaii,", "respectively.", "When", "compared", "to", "the", "Americas,", "SHB", "populations", "found", "in", "Europe", "show", "a", "higher", "haplotype", "diversity,", "thereby", "supporting", "multiple", "introduction", "events", "24", ".", "Phylogenetic", "analysis", "shows", "that", "specimens", "from", "Italy", "belong", "to", "a", "different", "clade", "than", "the", "one", "in", "the", "USA", "and", "Australia.", "Our", "data", "therefore", "support", "earlier", "results", "that", "the", "occurrence", "of", "SHB", "in", "Italy", "is", "due", "to", "an", "independent", "introduction", "from", "Africa", "and", "not", "from", "the", "USA", "or", "Australia", "24", ".", "The", "phylogenetic", "tree", "clearly", "shows", "two", "clades,", "A1", "and", "A2,", "to", "which", "most", "SHB", "specimen", "from", "Italy", "belong,", "therefore,", "suggesting", "two", "separate", "introductions", "into", "the", "Calabria", "region.", "The", "first", "specimen", "detected", "in", "September", "2014", "in", "Gioia", "Tauro", "in", "the", "Calabria", "region", "23", "most", "likely", "originated", "from", "Ethiopia", "(clade", "A2).", "A", "second", "introduction", "into", "the", "same", "region", "most", "likely", "originated", "from", "Uganda", "(clade", "A1).", "Ethiopia", "and", "Uganda", "are", "among", "the", "largest", "producers", "of", "beeswax", "with", "respective", "productions", "of", "5,542", "and", "1,308", "T", "in", "2016", "8", ".", "Therefore,", "SHBs", "detected", "in", "the", "region", "of", "Calabria", "(only", "a", "few", "kilometres", "from", "the", "port", "of", "Gioia", "Tauro)", "most", "probably", "came", "from", "infested", "wax", "containers", "from", "Ethiopia", "and/or", "Uganda.", "Interestingly,", "Italy", "however", "only", "imported", "beeswax", "from", "Ethiopia", "until", "2012", "8", ".", "This", "suggests", "that", "SHBs", "were", "probably", "present", "in", "Calabria", "at", "least", "two", "years", "before", "their", "official", "confirmation", "similar", "to", "the", "USA", "and", "Australia", "15", ".", "Moreover,", "it", "also", "shows", "that", "those", "SHBs", "in", "Calabria", "most", "likely", "originating", "from", "Uganda", "must", "have", "arrived", "via", "another", "yet", "unidentified", "pathway,", "e.g.", "bee", "imports", "as", "in", "the", "case", "of", "Portugal", "2004", "27", ".", "Fascinatingly,", "the", "Portuguese", "sample", "shows", "a", "very", "high", "similarity", "(96%)", "with", "some", "Italian", "samples", "from", "Calabria,", "thereby", "strongly", "suggesting", "the", "same", "yet", "unidentified", "African", "origin", "(assuming", "that", "SHBs", "were", "not", "present", "in", "Italy", "back", "in", "2004).", "The", "FAO", "data", "show", "that", "beeswax", "was", "not", "imported", "into", "Portugal", "from", "any", "country", "with", "known", "SHB", "populations", "8", ".", "Indeed,", "the", "A.", "tumida", "larvae", "were", "detected", "in", "a", "shipment", "of", "queens", "from", "Texas", "(USA)", "27", ".", "Since", "the", "Portuguese", "sample", "therefore", "unequivocally", "originated", "from", "the", "USA,", "this", "strongly", "implies", "that", "at", "least", "a", "second,", "previously", "not", "detected,", "introduction", "into", "the", "USA", "must", "have", "occurred", "independently", "from", "the", "Tanzanian", "one", "(see", "above).", "Therefore,", "we", "can", "here", "clarify", "the", "question", "whether", "a", "single", "or", "multiple", "introductions", "into", "the", "USA", "have", "occurred", "21", ".", "Based", "on", "our", "genetic", "data,", "it", "seems", "safe", "to", "assume", "that", "at", "least", "two", "independent", "introductions", "into", "the", "USA", "must", "have", "occurred.", "The", "combination", "of", "our", "genetic", "and", "beeswax", "trade", "data", "8", "enabled", "light", "to", "be", "shed", "on", "a", "previously", "overlooked", "key", "factor", "for", "SHB", "spread.", "Since", "the", "origin", "of", "the", "Portugal", "SHB", "introduction", "in", "2004", "can", "unambiguously", "be", "traced", "back", "to", "shipment", "of", "queen", "bees", "from", "the", "USA", "36", ",", "only", "for", "11", "out", "of", "the", "12", "confirmed", "introductions,", "which", "were", "investigated", "here,", "sources", "could", "not", "certainly", "be", "traced", "back", "so", "far.", "Correlations", "are", "not", "causations,", "however,", "given", "that", "two", "independent", "data", "sets", "match", "in", "their", "correlations", "(genetics", "and", "FAO),", "this", "further", "supports", "a", "causal", "relationship.", "Indeed,", "in", "the", "nine", "remaining", "cases,", "for", "which", "both", "genetic", "and", "FAO", "data", "were", "available,", "both", "data", "sets", "consistently", "suggest", "the", "same", "country", "of", "origin.", "This", "strongly", "suggests", "that", "international", "beeswax", "trade", "is", "the", "major", "factor", "explaining", "the", "past", "invasion", "history", "of", "small", "hive", "beetles.", "It", "seems", "therefore", "most", "unfortunate", "that", "nobody", "paid", "really", "attention", "up", "to", "now", "on", "the", "role", "of", "beeswax", "for", "SHB", "spread.", "Interestingly,", "the", "international", "transport", "of", "beeswax", "usually", "occurs", "via", "ships", "39", "and", "some", "SHB", "introductions", "so", "far", "occurred", "near", "major", "harbors", "(Charleston,", "USA", "15", ";", "Gioia", "Tauro,", "Italy", "23", ")", "or", "on", "islands", "(Jamaica,", "Hawaii,", "Cuba,", "Philippines)", "10", ".", "Since", "the", "international", "shipping", "industry", "is", "responsible", "for", "the", "carriage", "of", "~90%", "of", "world", "trade", "39", ",", "it", "appears", "unsurprising", "that", "the", "surroundings", "of", "harbours", "were", "starting", "points", "of", "several", "SHB", "invasions.", "It", "seems", "therefore", "prudent", "to", "take", "into", "account", "beeswax", "trade", "for", "adequate", "mitigation", "measures", "to", "further", "limit", "the", "spread", "of", "this", "pest", "species.", "Ideally,", "beeswax", "shipments", "are", "not", "contaminated", "prior", "to", "sending.", "However,", "this", "appears", "notoriously", "difficult", "in", "regions", "with", "SHB", "populations", "due", "to", "the", "highly", "mobile", "and", "cryptic", "behaviour", "of", "the", "adult", "beetles", "10", ",", "40", ".", "Regardless", "of", "the", "actual", "beeswax", "processing", "stage", "33", ",", "34", ",", "adult", "SHBs", "are", "therefore", "very", "likely", "to", "invade", "and", "successfully", "hide", "in", "any", "beeswax", "trade", "package.", "Indeed,", "even", "small", "commercial", "honey", "bee", "queen", "cages,", "which", "are", "obviously", "much", "easier", "to", "visually", "screen", "by", "the", "beekeepers", "compared", "to", "containers", "and", "large", "boxes,", "were", "already", "responsible", "for", "two", "independent", "SHB", "invasions", "(Portugal", "27", ";", "Northern", "Territory,", "Australia", "10", ").", "It", "therefore", "appears", "inevitable", "that", "adult", "SHBs", "can", "and", "eventually", "will", "invade", "any", "beeswax", "trade", "containers", "during", "packaging.", "Considering", "adult", "SHB", "survival", "under", "starvation", "(at", "least", "12", "days", "without", "any", "food", "and", "water;", "Peter", "Neumann,", "unpublished", "data),", "chances", "for", "adult", "SHB", "invasion", "of", "and", "hiding", "39", "in", "beeswax", "trade", "containers", "as", "well", "as", "the", "impact", "of", "this", "invasion", "on", "bees", "in", "the", "new", "ranges,", "it", "seems", "as", "if", "beeswax", "imports", "from", "countries", "with", "known", "SHB", "populations", "should", "be", "banned.", "Alternatively,", "rigorous", "quarantine", "measures", "in", "the", "importing", "countries", "might", "help,", "but", "in", "light", "of", "our", "rather", "fragmentary", "knowledge", "of", "SHB", "physiology,", "this", "appears", "risky.", "In", "conclusion,", "our", "genetic", "results", "(1)", "confirm", "the", "African", "origin", "of", "SHBs,", "(2)", "show", "a", "high", "genetic", "diversity", "in", "Africa,", "(3)", "suggest", "two", "main", "native", "populations,", "(4)", "confirm", "previous", "findings", "and", "(5)", "suggest", "novel", "origins.", "Since", "our", "genetic", "data", "match", "well", "with", "data", "on", "international", "trade", "of", "beeswax", "8", ",", "a", "closer", "investigation", "of", "international", "beeswax", "trade", "may", "enable", "to", "slow", "down", "the", "further", "spread", "of", "this", "invasive", "species.", "In", "general,", "it", "appears", "worthwhile", "to", "use", "this", "combination", "of", "genetics", "and", "public", "trade", "data", "in", "more", "cases", "of", "invasive", "species.", "Methods", "Sample", "collection", "Adult", "SHBs", "(N", "=", "1542)", "were", "manually", "sampled", "41", "from", "101", "naturally", "infested", "A.", "mellifera", "colonies", "from", "98", "locations", "both", "in", "the", "native", "range", "of", "SHBs", "in", "sub-Saharan", "Africa", "(Benin,", "Togo,", "Nigeria,", "Burkina-Faso,", "Central", "African", "Republic,", "Democratic", "Republic", "of", "Congo,", "Uganda,", "Sudan,", "South", "Sudan,", "Ethiopia,", "Kenya,", "Tanzania,", "Malawi,", "Burundi,", "Madagascar", "and", "South", "Africa)", "as", "well", "as", "from", "11", "confirmed", "SHB", "introductions", "(USA,", "Australia,", "Canada,", "Philipines,", "Mexico,", "Jamaica,", "Hawaii,", "Cuba,", "Italy,", "Brazil", "and", "South", "Korea).", "Samples", "from", "Guanacoste,", "Costa", "Rica", "taken", "in", "2016", "by", "J.", "Pettis", "were", "also", "included", "because", "detailed", "morphometric", "analysis", "41", "confirmed", "that", "the", "samples", "do", "belong", "to", "A.", "tumida.", "The", "last", "remaining", "SHB", "larvae", "from", "the", "Portugal", "introduction", "in", "2004", "27", "was", "also", "included.", "Aethina", "concolor", "specimen", "collected", "in", "Australia", "were", "used", "as", "outgroup.", "Please", "refer", "to", "Table", "3", "for", "details", "of", "the", "sampling.", "All", "samples", "were", "preserved", "in", "70%", "Ethanol,", "transported", "at", "room", "temperature", "and", "stored", "at", "−20", "°C", "in", "a", "laboratory", "until", "further", "analyses.", "Table", "3", "Sampling", "overview.", "Country", "Site", "(N", "of", "sequenced", "individuals)", "Year", "Australia", "Townsville", "(5),", "Cairns", "(6),", "Nambour", "(5),", "Victoria-Melbourne", "(4),", "Bathurst", "(3),", "S.E.", "Queensland", "(2)", "2016", "Benin", "Ketu", "(3),", "Abomey", "(3)", "2015", "Brazil", "Piracicaba", "(30),", "Sao", "Pedro", "(6)", "2016", "Burkina-Faso", "Fada", "N’gourma", "(2),", "Tenkodogo", "(3),", "Ziniaré", "(2),", "Bobo", "Dioulasso", "(3)", "2015", "Burundi", "Rusiga", "(1)", "2016", "Canada", "Canary", "(6),", "Mckenzie", "Road", "(1),", "LeFeuvre", "Road", "(1),", "Eco-dairy", "(1)", "2015", "Central", "African", "Republic", "Kelengô", "(2),", "Ndara", "(3),", "Sibut", "(2),", "Yeremon", "(3)", "2015", "Costa", "Rica", "Guanacoste", "(3)", "2016", "Cuba", "Jovenallos", "(5),", "San", "Nicolas", "de", "Bari", "(5),", "Jaruco", "(5)", "2016", "Democratic", "Republic", "of", "Congo", "Maboya", "1", "(2),", "Maboya", "2", "(1),", "Maboya", "3", "(1),", "Maboya", "4", "(1),", "Mulo", "1", "(1),", "Mulo", "2", "(2),", "Mulo", "3", "(1),", "Mulo", "4", "(1),", "Kambo", "1", "(1),", "Kambo", "2", "(1),", "Kambo", "3", "(2),", "Kambo", "4", "(2)", "2016", "Ethiopia", "Gedeo", "zone", "(5),", "Sidama", "zone", "(5)", "2016", "Italy", "Candinoni", "(1),", "Rizziconi", "(1),", "Melicucco", "(1),", "Polistena", "(1),", "Feroleto", "Della", "Chiesa", "(1),", "Taurianova", "(1),", "Altilia", "(3)", "2016", "Jamaica", "Old", "Habour", "(5)", "2016", "Kenya", "Meru", "(2)", "2016", "South", "Korea", "Miryang", "(5)", "2017", "Madagascar", "Ambat", "(3),", "Anjozorobe", "(1),", "Beomby", "(2),", "Isatra", "(3),", "Mantasoa", "(3)", "2016", "Malawi", "Chawala", "(1),", "Chakhuntha", "(1)", "2015", "Mexico", "San", "Ignacio", "(4),", "Llera", "(4),", "Linares", "(4),", "Tancitaro", "(4),", "Carretera", "(4),", "Candelaria", "(4),", "Benito", "Juarez", "(4),", "Tepich", "(4),", "Yucatan", "(4)", "2016", "Nigeria", "Osogbo", "(2),", "Ayangba", "(2),", "Otan", "(2),", "Oyan", "(2),", "Taraba", "(1)", "2015", "Philipines", "Davao", "2", "(6),", "Davao", "3", "(5),", "Davao", "4", "(5)", "2015", "Portugal", "Lisboa", "region", "(1", "Larvae)", "2004", "Republic", "of", "South", "Africa", "Northern", "Cape", "(9)", "2016", "South", "Sudan", "Yei", "City", "(2)", "2015", "Sudan", "Khartoum", "(1)", "2015", "Tanzania", "Kilimanjaro", "(8),", "Arusha", "(6)", "2016", "Togo", "Seva", "(2),", "Notsie", "(3)", "2015", "Uganda", "Jjangano", "(2),", "Keirere", "(2),", "Rubona", "(1),", "Kyanyamutale", "(1),", "Oryang-Ojuma", "(1),", "Busiwu", "(2),", "Alik", "(2),", "Wasswa/Kavunza", "(2),", "Kanyonza", "(2)", "2015", "USA", "Baton", "Rouge", "1", "(2),", "Baton", "Rouge", "2", "(3),", "Baton", "Rouge", "3", "(3),", "Oahu", "kunia", "(2)", "2016", "The", "country,", "site", "and", "year", "of", "sampling", "are", "shown", "as", "well", "as", "the", "number", "of", "sequenced", "individuals", "per", "site.", "Genomic", "DNA", "extraction", "A", "modified", "protocol", "from", "Neumann", "et", "al.", "41", "was", "used.", "In", "brief,", "all", "samples", "were", "crushed", "individually", "in", "2", "mL", "microcentrifuge", "tubes", "containing", "5", "mm", "metal", "beads", "and", "100", "µL", "TN", "buffer", "(10", "mmol/L", "Tris,", "10", "mmol/L", "NaCl;", "pH", "7.6).", "Crushed", "samples", "were", "homogenized", "with", "a", "TissueLyser", "(QIAGEN,", "Hombrechtikon,", "Switzerland)", "for", "25", "sec", "at", "20", "1/sec", "frequency", "using", "a", "Qiagen", "Retsch", "®", "MM", "300", "mixer", "mill", "(Thermo", "Fisher", "Scientific,", "Zurich,", "Switzerland)", "and", "centrifuged", "for", "60", "sec", "at", "2000", "rpm.", "50", "µL", "of", "supernatant", "was", "taken", "from", "the", "homogenate", "and", "used", "for", "DNA", "extraction", "using", "innuPrep", "DNA", "Mini", "Kit", "(Analytik", "Jena,", "Jena,", "Germany)", "by", "following", "the", "manufacturer’s", "recommendations.", "The", "DNA", "yield", "and", "purity", "of", "each", "sample", "were", "checked", "using", "a", "Spectrophotometer", "Thermo", "Scientific", "TM", "Nanodrop", "2000", "(Axon", "lab,", "Baden-Dättwill,", "Switzerland).", "PCR", "amplification", "and", "COI", "gene", "sequencing", "A", "fragment", "of", "Cytochrome", "C", "Oxidase", "subunit", "I", "(COI)", "gene", "was", "amplified", "using", "primers", "AT1904S", "(5′-GGTGGATCTTCAGTTGATTTAGC-3′)", "and", "AT2953A", "(5′-TCAGCTGGGGGATAAAATTG-3′)", "for", "SHB", "specimens", "19", ".", "Additionally,", "another", "COI", "fragment", "was", "also", "amplified", "with", "LCO1490", "(5′-GGTCAACAAATCATAAAGATATTGG-3′)", "and", "HCO2198", "(5′-TAAACTTCAGGGTGACCAAAAAATCA-3′)", "for", "A.", "concolor", "specimen", "41", ".", "PCR", "tests", "were", "carried", "out", "in", "25", "μL", "(15.88", "μL", "millipore", "water,", "5", "μL", "5x", "reaction", "buffer,", "1", "μL", "(0.4", "μmol/L)", "each", "primer", "(reverse", "and", "forward),", "0.125", "μL", "(0.63", "units)", "Taq", "DNA", "polymerase,", "2", "μL", "tenfold-diluted", "DNA)", "in", "a", "Biometra", "®", "Thermal", "Cycler", "(Thermo", "Fisher", "Scientific,", "Switzerland)", "as", "follows:", "95", "°C", "for", "2", "min,", "35", "cycles", "of", "95", "°C", "for", "20", "sec,", "56", "°C", "for", "30", "sec,", "72", "°C", "for", "30", "sec,", "and", "72", "°C", "for", "2", "min.", "Positive", "and", "negative", "controls", "were", "included", "in", "each", "PCR", "(DNA", "of", "previously", "identified", "SHB", "specimen", "from", "Nigeria", "(based", "on", "both", "morphometrics", "and", "DNA", "sequence", "data)", "or", "millipore", "water).", "Aliquots", "of", "PCR", "products", "were", "run", "by", "electrophoresis", "on", "a", "2%", "(w/v)", "agarose", "gel.", "The", "amplicons", "were", "purified", "with", "ExoSAP-IT", "TM", "(Thermo", "Fisher", "Scientific,", "Zürich,", "Switzerland)", "and", "sequenced", "in", "both", "directions", "(forward", "and", "reverse).", "For", "a", "better", "coverage", "of", "the", "1091", "bp", "fragment", "to", "be", "sequenced,", "two", "internal", "primers,", "Aet-int-F", "(5′-CTTCTGCTACAATAATTATTGC-3′)", "and", "Aet-int-R", "(5′-TTGTGTACCATGAAGAGTAGC-3′)", "were", "added", "to", "the", "sequencing", "reactions", "42", ".", "All", "samples", "were", "analyzed", "using", "the", "Big", "Dye", "®", "Terminator", "v3.1", "cycle", "sequencing", "kit", "(Thermo", "Fisher", "Scientific,", "Zurich,", "Switzerland).", "Sequencing", "was", "performed", "using", "a", "96-capillary", "ABI", "PRISM", "®", "3730XL", "Genetic", "Analyzer", "(Thermo", "Fisher", "Scientific,", "Zurich,", "Switzerland).", "The", "nucleotide", "sequences", "were", "deposited", "in", "the", "GenBank", "database", "under", "accessions", "MK024945-MK025231.", "DNA", "sequences", "alignment", "and", "analyses", "The", "sequencing", "data", "were", "assembled", "and", "edited", "with", "Sequencher", "®", "software", "v5.4.1", "(Gene", "codes", "corporation,", "Michigan,", "USA).", "The", "consensus", "sequences", "obtained", "were", "aligned", "using", "the", "MEGA", "7", "package", "and", "the", "MUSCLE", "alignment", "algorithm", "43", ",", "and", "compared", "to", "reference", "nucleotide", "sequence", "available", "in", "the", "GenBank", "database", "(", "http://www.ncbi.nlm.nih.gov/genbank/", ").", "All", "sequences", "with", "bad", "quality", "or", "ambiguous", "electropherograms", "were", "sequenced", "one", "in", "each", "direction", "twice", "and", "completely", 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CC-BY
4.0
http://creativecommons.org/licenses/by/4.0/
Original work titled: 'International beeswax trade facilitates small hive beetle invasions' (Scientific Reports/Nature Publishing Group UK) by the following authors: Franck Ouessou Idrissou; Qiang Huang; Orlando Yañez; Peter Neumann DOI: 10.1038/s41598-019-47107-6
060f177a337317f5f1f4f885c0834fb0
[ "Background", "Acute", "uncomplicated", "lower", "urinary", "tract", "infection", "(UTI)", "is", "one", "of", "the", "most", "common", "bacterial", "infections.", "Patients", "usually", "present", "with", "dysuria,", "urinary", "urgency,", "urinary", "frequency,", "and", "suprapubic", "pain/tenderness", "[", "1", "].", "In", "the", "United", "States,", "there", "are", ">", "7", "million", "outpatients", "with", "UTIs", "and", "about", "1", "million", "inpatients", "each", "year", "[", "2", "].", "Approximately", "150", "million", "people", "are", "diagnosed", "with", "UTI", "each", "year", "worldwide", "[", "3", "].", "UTI,", "which", "can", "cause", "shock", "and", "death,", "ranks", "third", "among", "all", "diseases", "that", "die", "from", "infection", "[", "4", "].", "Gram-negative", "bacteria,", "especially", "Enterobacteriaceae,", "is", "the", "common", "cause", "of", "both", "community-acquired", "and", "hospital-acquired", "UTIs", "[", "3", ",", "5", "].", "Many", "Chinese", "medicines", "that", "have", "a", "heat-clearing", "and", "detoxicating", "function", "have", "been", "proven", "to", "have", "the", "bacteriostasis", "on", "pathogenic", "microorganisms,", "which", "can", "inhibit", "or", "destroy", "the", "formation", "of", "toxic", "substances", "[", "6", ",", "7", "].", "In", "the", "antibacterial", "test", "against", "mice,", "it", "was", "found", "that", "Sanjin", "tablets", "(SJT)", "have", "strong", "bacteriostasis", "activity", "[", "8", "].", "SJT", "have", "the", "effects", "of", "clearing", "heat,", "detoxicating,", "eliminating", "dampness,", "and", "treating", "stranguria", "[", "9", "].", "It", "is", "used", "to", "treat", "urinal", "urgency,", "frequency,", "and", "burning", "caused", "by", "syndromes", "of", "dampness-heat", "in", "the", "lower", "jiao,", "which", "coincides", "with", "the", "symptoms", "of", "acute", "uncomplicated", "lower", "UTI.", "SJT", "are", "composed", "of", "five", "kinds", "of", "Chinese", "herbal", "medicines:", "Jinyinggen", "(Root", "of", "Cherokee", "Rose),", "Baqia", "(Chinaroot", "Greenbrier", "Rhizome),", "Yangkaikou", "(Fruit", "of", "Fiverleaf", "Akebia),", "Jinshateng", "(Lygodii", "Herba)", "and", "Jixuecao", "(Asiatic", "Pennywort", "Herb),", "all", "of", "which", "are", "recorded", "in", "the", "Chinese", "Pharmacopoeia", "(V.2015).", "Jinyinggen", "(Root", "of", "Cherokee", "Rose)", "is", "acerb", "and", "neutral", "in", "nature;", "it", "can", "control", "nocturnal", "emission", "and", "astringent", "intestine.", "Baqia", "(Chinaroot", "Greenbrier", "Rhizome)", "is", "cosin", "and", "neutral", "in", "nature;", "it", "can", "remove", "rheumatism,", "promote", "blood", "circulation,", "detoxicate,", "relieve", "convulsion,", "and", "calm", "endogenous", "wind.", "Jinshateng", "(Lygodii", "Herba)", "is", "slightly", "sweet", "and", "cold-natured;", "it", "can", "clear", "heat,", "detoxicate,", "and", "remove", "dampness.", "The", "above", "three", "herbs", "are", "monarch", "drugs", "in", "a", "prescription,", "which", "have", "a", "mutual", "promotion", "effect", "on", "the", "function", "of", "clearing", "heat,", "removing", "dampness,", "and", "inducing", "diuresis", "for", "treating", "stranguria.", "The", "above", "three", "medicines,", "supplemented", "with", "Yangkaikou", "(Fruit", "of", "Fiverleaf", "Akebia)", "and", "Jixuecao", "(Asiatic", "Pennywort", "Herb),", "can", "enhance", "the", "effects", "of", "removing", "dampness", "and", "treating", "stranguria", "[", "10", ",", "11", "].", "SJT", "are", "a", "completely", "natural", "preparation", "with", "significant", "efficacy", "in", "treating", "UTI", "[", "12", "].", "Previous", "clinical", "studies", "have", "indicated", "that", "SJT", "can", "reduce", "the", "symptoms", "of", "chronic", "UTI", "and", "number", "of", "acute", "attacks", "and", "can", "reduce", "the", "secretory", "level", "of", "urinary", "sIL-2R,", "IL-6,", "IL-8", "in", "patients", "with", "chronic", "nephropyelitis", "[", "8", "].", "Observation", "by", "electron", "microscope", "found", "that", "SJT", "can", "make", "Escherichia", "coli", "flagella", "drop", "without", "any", "danger.", "A", "meta-analysis", "has", "demonstrated", "that", "the", "current", "evidence", "is", "insufficient", "to", "support", "the", "efficacy", "and", "safety", "of", "SJT", "for", "acute", "uncomplicated", "lower", "UTI", "due", "to", "lack", "of", "high-quality", "randomized", "controlled", "trials", "(RCT)", "[", "13", "].", "The", "high", "recurrence", "rate", "of", "lower", "UTI", "is", "a", "common", "problem", "of", "clinical", "treatment", "[", "14", "].", "The", "misuse", "of", "antibiotics", "has", "led", "to", "the", "emergence", "of", "a", "number", "of", "resistant", "bacterial", "strains", "[", "15", "].", "Unfortunately,", "the", "efforts", "to", "produce", "new", "antibiotics", "have", "not", "been", "sufficient", "to", "cope", "with", "the", "emergence", "of", "these", "new", "antibiotic-resistant", "(AR)", "strains", "[", "16", "].", "Therefore,", "the", "objective", "of", "our", "study", "is", "to", "evaluate", "effectiveness,", "safety,", "and", "recurrence", "rate", "of", "SJT", "and", "explore", "whether", "SJT", "can", "reduce", "or", "substitute", "the", "use", "of", "antibiotics", "for", "acute", "uncomplicated", "lower", "UTI", "by", "a", "rigorous", "RCT.", "Method/design", "Objective", "and", "design", "This", "is", "a", "randomized,", "double-blind,", "double-dummy,", "parallel", "control", "of", "positive", "drug,", "multicentre", "clinical", "study.", "The", "study", "will", "be", "conducted", "at:", "the", "XiYuan", "Hospital,", "China", "Academy", "of", "Chinese", "Medical", "Sciences;", "the", "Longhua", "Hospital", "Shanghai", "University", "of", "Traditional", "Chinese", "Medicine;", "the", "Guangdong", "Provincial", "Hospital", "of", "Traditional", "Chinese", "Medicine;", "the", "Hubei", "Hospital", "of", "Traditional", "Chinese", "Medicine;", "the", "Affiliated", "hospital", "of", "Chengdu", "University", "of", "Traditional", "Chinese", "Medicine;", "and", "the", "Yunnan", "Provincial", "Hospital", "of", "Traditional", "Chinese", "Medicine.", "Ethical", "clearance", "for", "the", "trial", "was", "obtained", "from", "ethics", "committee", "of", "the", "six", "hospitals.", "Patients", "with", "acute", "uncomplicated", "lower", "UTI", "will", "undergo", "a", "standardized", "baseline", "evaluation", "before", "treatment,", "comprising", "detailed", "history", "taking,", "physical", "examination,", "and", "laboratory", "testing.", "All", "included", "patients", "are", "randomly", "divided", "into", "three", "groups:", "experimental", "group;", "control", "group", "1;", "and", "control", "group", "2.", "The", "experimental", "group", "receives", "SJT", "plus", "levofloxacin", "tablets", "(LT)", "placebo;", "control", "group", "1", "receives", "LT", "plus", "SJT", "placebo;", "and", "control", "group", "2", "receives", "SJT", "plus", "LT", "on", "the", "first", "five", "days,", "SJT", "plus", "LT", "placebo", "on", "the", "last", "two", "days.", "After", "seven", "days", "of", "treatment,", "the", "efficacy", "and", "safety", "of", "three", "groups", "will", "be", "evaluated.", "The", "first", "aim", "is", "to", "determine", "whether", "SJT", "can", "reduce", "the", "use", "of", "antibiotics", "(control", "group", "2", "versus", "control", "group", "1,", "non-inferiority", "test);", "the", "second", "aim", "is", "to", "determine", "whether", "SJT", "can", "substitute", "the", "use", "of", "antibiotics", "(experimental", "group", "versus", "control", "group", "1,", "non-inferiority", "test).", "The", "recurrence", "rate", "will", "be", "assessed", "at", "the", "28-day", "follow-up", "after", "being", "cured", "to", "determine", "whether", "SJT", "can", "reduce", "the", "recurrence", "rate.", "The", "trial", "is", "conducted", "in", "accordance", "with", "the", "World", "Medical", "Association", "Declaration", "of", "Helsinki", "and", "Good", "Clinical", "Practice", "of", "Pharmaceutical", "Products", "[", "17", ",", "18", "].", "After", "a", "full", "explanation", "by", "the", "clinicians,", "written", "informed", "consent", "will", "be", "obtained", "from", "the", "participants", "before", "intervention", "[", "19", ",", "20", "].", "Strict", "data", "management", "and", "quality", "control", "will", "be", "conducted", "in", "this", "trial", "[", "21", ",", "22", "].", "This", "trial", "was", "registered", "in", "the", "Clinical", "Trials", "USA", "registry", "(registration", "No.", "NCT03658291)", "on", "4", "September", "2018", "and", "will", "be", "carried", "out", "from", "January", "2019", "to", "December", "2019.", "The", "study", "design", "is", "shown", "in", "Fig.", "1", ".", "The", "protocol", "follows", "the", "recommendations", "of", "the", "SPIRIT", "initiative", "(see", "Additional", "file", "1", ")", "[", "23", "]", "and", "the", "trial", "results", "will", "be", "reported", "according", "to", "the", "latest", "version", "of", "the", "CONSORT", "statement", "[", "24", "].", "Fig.", "1", "Flow", "diagram.", "SJT", "Sanjin", "tablets,", "LT", "levofloxacin", "tablets", "Patient", "and", "public", "involvement", "Patients", "were", "not", "involved", "in", "the", "research", "question,", "design,", "conduct,", "outcome", "measures,", "and", "data", "analysis", "of", "the", "study.", "Only", "medically", "trained", "clinicians", "carry", "out", "the", "patient", "recruitment", "and", "management", "in", "the", "study.", "The", "clinicians", "will", "describe", "the", "purpose,", "burden", "of", "the", "intervention,", "procedure,", "and", "potential", "risks", "of", "this", "trial", "to", "the", "participants", "themselves", "or", "their", "designated", "representative", "before", "the", "recruitment.", "We", "will", "disseminate", "the", "results", "of", "this", "study", "to", "participants", "through", "patient", "organizations", "and", "open", "lectures.", "Sample", "size", "The", "effective", "rate", "will", "be", "taken", "as", "the", "main", "outcome", "measure", "of", "the", "clinical", "trial.", "According", "to", "a", "previous", "study,", "it", "showed", "that", "the", "effective", "rate", "of", "SJT", "was", "77.14%", "in", "the", "treatment", "of", "simple", "bacterial", "lower", "urinary", "tract", "infection", "[", "25", "].", "On", "the", "basis", "of", "a", "0.9", "power", "to", "detect", "a", "significant", "difference", "(α", "=", "0.01,", "two-sided),", "76", "participants", "will", "be", "required", "for", "each", "group.", "228", "participants", "will", "be", "required", "for", "the", "three", "groups", "in", "a", "1:1:1", "ratio.", "Considering", "a", "20%", "drop-out", "rate,", "we", "plan", "to", "include", "252", "patients", "in", "the", "whole", "trial.", "Participants", "and", "recruitment", "Diagnostic", "criteria", "of", "western", "medicine", "and", "traditional", "Chinese", "medicine", "The", "western", "medicine", "diagnostic", "criteria", "of", "acute", "uncomplicated", "lower", "UTI", "will", "refer", "to", "the", "Guidelines", "for", "the", "diagnosis", "and", "treatment", "of", "urological", "diseases", "in", "China", "(2014", "ed)", "[", "26", "].", "The", "traditional", "Chinese", "medicine", "(TCM)", "diagnostic", "criteria", "of", "acute", "uncomplicated", "lower", "UTI", "will", "refer", "to", "the", "Clinic", "terminology", "of", "traditional", "Chinese", "medical", "diagnosis", "and", "treatment-syndromes,", "Internal", "Medicine", "of", "TCM", "[", "27", "–", "29", "].", "According", "to", "the", "characteristics", "and", "functions", "of", "SJT,", "it", "is", "proposed", "to", "select", "the", "syndrome", "of", "dampness-heat", "in", "the", "lower", "jiao", "as", "the", "syndrome", "of", "TCM", "in", "this", "trial.", "Inclusion", "criteria", "Inclusion", "criteria", "are", "as", "follows:", "(i)", "age", ">", "18", "and", "<", "50", "years;", "(ii)", "meet", "the", "western", "diagnostic", "criterion", "of", "acute", "uncomplicated", "lower", "UTI", "[", "26", "]", "that", "the", "disease", "duration", "does", "not", "exceed", "72", "h;", "(iii)", "bacterial", "culture", "result", "will", "be", "sensitive", "to", "levofloxacin", "(bacterial", "culture", "and", "clinical", "treatment", "will", "be", "conducted", "at", "the", "same", "time);", "(iv)", "the", "syndrome", "differentiation", "of", "TCM", "is", "syndrome", "of", "dampness-heat", "in", "the", "lower", "jiao;", "(v)", "did", "not", "receive", "antibiotic", "treatment", "within", "48", "h", "before", "being", "selected;", "(vi)", "not", "pregnant", "or", "lactating;", "and", "(vii)", "sign", "the", "written", "informed", "consent", "form", "for", "the", "clinical", "trial.", "Exclusion", "criteria", "Exclusion", "criteria", "are", "as", "follows:", "(i)", "allergic", "to", "the", "test", "drug", "or", "quinolones;", "(ii)", "there", "was", "a", "history", "of", "bacterial", "culture", "that", "was", "not", "sensitive", "to", "levofloxacin;", "(iii)", "diagnosed", "as", "complicated", "UTI;", "(iv)", "patients", "with", "urinary", "calculi", "or", "obstruction,", "urinary", "tuberculosis,", "renal", "papillary", "necrosis,", "perinephric", "abscess,", "or", "neurogenic", "bladder;", "(v)", "combined", "with", "vaginitis", "symptoms,", "genital", "ulcers,", "or", "gonorrhea;", "(vi)", "combined", "with", "severe", "cardiopulmonary", "disease,", "liver", "and", "kidney", "disease,", "advanced", "tumor,", "and", "other", "serious", "or", "progressive", "disease;", "(vii)", "have", "a", "neurological", "or", "mental", "illness", "and", "not", "able", "to", "cooperate;", "(viii)", "use", "of", "other", "antibacterial", "drugs", "in", "combination;", "(ix)", "pregnant,", "lactating,", "planning", "to", "become", "pregnant", "women;", "(x)", "taking", "part", "in", "another", "clinical", "trial", "within", "three", "months", "before", "being", "selected.", "Exit", "criteria", "Patients", "will", "leave", "the", "trial", "when", "one", "of", "the", "following", "criteria", "is", "met:", "(i)", "incorrectly", "included;", "(ii)", "are", "poorly", "compliant", "or", "pregnant;", "(iii)", "no", "medication", "or", "any", "follow-up", "records;", "(iv)", "occurrence", "of", "allergic", "reactions", "or", "serious", "adverse", "events", "(AE);", "(v)", "participants", "have", "other", "complications", "or", "special", "physiological", "changes", "during", "the", "trial;", "(vi)", "patients", "have", "been", "treated", "with", "other", "medicines", "during", "the", "trial;", "and", "(vii)", "not", "alleviated", "or", "the", "symptoms", "are", "aggravated.", "Participants", "may", "withdraw", "from", "the", "study", "at", "any", "time", "for", "any", "reason.", "Blinding", "This", "is", "a", "double-blind", "(patients", "and", "clinicians", "are", "blinded)", "and", "double-dummy", "study", "[", "30", "].", "A", "two-stage", "blind", "design", "was", "used.", "The", "first", "stage", "was", "group", "A,", "group", "B,", "and", "group", "C;", "the", "second", "stage", "was", "randomization", "to", "the", "experimental", "group,", "control", "group", "1,", "and", "control", "group", "2", "according", "to", "SAS", "software,", "without", "correspondence", "to", "group", "A,", "group", "B,", "and", "group", "C.", "Treatment", "assignments", "will", "not", "be", "revealed", "until", "the", "whole", "process", "is", "complete.", "If", "patients", "have", "severe", "AEs,", "clinicians", "will", "log", "on", "to", "the", "central", "platform", "to", "unblind", "the", "patients", "as", "an", "emergency", "and", "provide", "relevant", "treatment.", "Once", "the", "blinding", "is", "broken,", "the", "patient", "with", "this", "number", "will", "be", "withdrawn", "from", "the", "trial", "and", "the", "clinicians", "will", "record", "the", "reasons", "in", "the", "case", "report", "form", "(CRF).", "To", "achieve", "blinding,", "all", "three", "groups", "will", "use", "the", "same", "kind", "of", "packaging", "to", "encase", "the", "drug", "or", "placebo.", "Size,", "color,", "shape,", "taste,", "smell,", "and", "packaging", "of", "the", "placebo", "are", "made", "identical", "to", "that", "of", "the", "corresponding", "medicine", "by", "adding", "artificial", "pigment.", "Randomization", "and", "allocation", "concealment", "Randomization", "will", "be", "used", "for", "patient", "allocation", "[", "31", "].", "The", "randomization", "will", "be", "performed", "by", "an", "independent", "statistician.", "The", "random", "numbers", "are", "divided", "into", "three", "groups", "sequentially:", "experimental", "group;", "control", "group", "1;", "and", "control", "group", "2.", "SAS", "9.1.3", "statistical", "software", "PROC", "PLAN", "procedure", "statements", "will", "be", "used,", "the", "seed", "number", "given,", "and", "randomized", "grouping", "tables", "will", "be", "generated", "for", "the", "252", "patients", "receiving", "treatment.", "On-site", "drug", "blinding", "will", "be", "carried", "out,", "and", "emergency", "unblinding", "procedures", "will", "be", "created.", "Blinded", "materials", "will", "be", "kept", "by", "a", "full-time", "investigator", "who", "will", "not", "participate", "in", "any", "part", "of", "the", "trial.", "During", "the", "trial,", "the", "investigator", "will", "be", "able", "to", "obtain", "the", "randomized", "number", "and", "drug", "number", "of", "each", "patient", "from", "the", "designated", "central", "randomized", "platform.", "Interventions", "All", "researchers", "are", "clinical", "doctors", "and", "receive", "standardized", "training", "for", "the", "diagnostic", "interview", "before", "the", "start", "of", "the", "trial.", "LT", "are", "licensed", "for", "UTI", "with", "proven", "curative", "effect", "and", "safety", "[", "32", "].", "Therefore,", "levofloxacin", "had", "been", "selected", "as", "the", "active", "control", "medicine.", "Participants", "in", "the", "experimental", "group", "will", "receive", "SJT", "plus", "LT", "placebo", "orally", "four", "times", "a", "day", "for", "seven", "days.", "Participants", "in", "control", "group", "1", "will", "take", "LT", "plus", "SJT", "placebo", "four", "times", "a", "day", "for", "seven", "days.", "Participants", "in", "control", "group", "2", "will", "take", "SJT", "and", "LT", "four", "times", "a", "day", "for", "the", "first", "five", "days", "and", "SJT", "plus", "LT", "placebo", "four", "times", "a", "day", "for", "the", "last", "two", "days.", "Patient", "visits", "are", "required", "after", "seven", "days", "of", "treatment.", "This", "is", "a", "placebo-controlled", "study;", "all", "participants", "receive", "the", "same", "number", "of", "pills", "with", "varying", "proportions", "of", "active", "drug", "and", "placebo", "to", "ensure", "that", "patients", "and", "clinicians", "are", "not", "aware", "of", "the", "allocated", "treatment", "aim.", "The", "SJT", "placebo", "is", "composed", "of", "55%", "microcrystalline", "cellulose,", "41%", "starch,", "2%", "caramel,", "1.5%", "silicon", "dioxide,", "and", "0.5%", "talcum", "powder.", "The", "LT", "placebo", "is", "made", "of", "56%", "microcrystalline", "cellulose,", "42%", "starch,", "1.5%", "silicon", "dioxide,", "and", "0.5%", "talcum", "powder.", "The", "SJT", "and", "LT", "placebos", "are", "similar", "to", "the", "SJT", "and", "LT", "in", "size,", "color,", "shape,", "taste,", "smell,", "and", "packaging.", "SJT", "and", "its", "placebo", "and", "the", "LT", "placebo", "are", "produced", "by", "Guilin", "Sanjin", "Pharmaceutical", "Co.,", "Ltd.", "and", "can", "be", "preserved", "for", "two", "years.", "LT", "is", "provided", "by", "First", "Sankyo", "Pharmaceutical", "(Beijing)", "Co.,", "Ltd.", "at", "a", "dosage", "of", "100", "mg", "and", "can", "be", "preserved", "for", "two", "years.", "The", "recommended", "dosage", "of", "LT", "is", "2–3", "times", "a", "day,", "one", "pill", "at", "a", "time;", "hence,", "in", "order", "to", "meet", "the", "requirements", "of", "the", "blinding,", "the", "administration", "method", "of", "this", "medicine", "has", "been", "designed", "as", "described", "in", "Table", "1", ".", "Table", "1", "Administration", "methods", "in", "the", "experimental", "and", "control", "groups", "Time", "Experimental", "group", "Control", "group", "1", "Control", "group", "2", "First", "5", "days", "Last", "2", "days", "08:00", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "placebo", "3", "pills", "of", "SJT", "placebo,", "1", "pill", "of", "LT", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "placebo", "12:00", "3", "pills", "of", "SJT", "3", "pills", "of", "SJT", "placebo", "3", "pills", "of", "SJT", "3", "pills", "of", "SJT", "17:00", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "placebo", "3", "pills", "of", "SJT", "placebo,", "1", "pill", "of", "LT", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "3", "pills", "of", "SJT,", "1", "pill", "of", "LT", "placebo", "22:00", "3", "pills", "of", "SJT", "3", "pills", "of", "SJT", "placebo", "3", "pills", "of", "SJT", "3", "pills", "of", "SJT", "Follow-up", "All", "included", "patients", "will", "be", "re-evaluated", "after", "seven", "days", "of", "treatment", "follow-ups", "as", "an", "outpatient", "to", "assess", "the", "efficacy", "and", "safety.", "The", "patients", "who", "were", "cured", "will", "receive", "the", "second", "follow-up", "after", "28", "days", "to", "assess", "the", "recurrence", "rate.", "Patients", "whose", "symptoms", "worsened", "will", "receive", "a", "supply", "of", "relevant", "medicine", "and", "a", "written", "withdrawal", "schedule.", "Assessment", "of", "study", "endpoints", "and", "duration", "of", "follow-up", "is", "shown", "in", "Fig.", "2", ".", "Fig.", "2", "Standard", "Protocol", "Items:", "Recommendation", "for", "Interventional", "Trials", "(SPIRIT)", "figure", "Outcome", "measures", "Effective", "rate", "The", "effective", "rate", "will", "be", "evaluated", "from", "three", "dimensions:", "symptoms;", "urine", "leukocytes;", "and", "bacteriological", "examination.", "Primary", "outcome", "measure", "The", "symptoms", "of", "acute", "uncomplicated", "lower", "UTI", "will", "be", "assessed.", "The", "assessment", "tools", "for", "symptoms", "of", "acute", "uncomplicated", "lower", "UTI", "are", "three", "participant", "self-rating", "scales", "(see", "Additional", "file", "2", "):", "criteria", "sheet", "for", "evaluating", "the", "severity", "of", "urinary", "system", "infection", "(CSESUSI;", "mild:", "total", "score", "≤", "8,", "moderate:", "total", "score", "9–15,", "severe:", "total", "score", "≥", "16),", "if", "the", "severity", "of", "the", "disease", "decreases", "from", "severe", "to", "moderate,", "or", "from", "moderate", "to", "mild,", "the", "symptoms", "are", "improved;", "symptoms", "score", "sheet", "of", "TCM", "(SSST;", "cure:", "total", "score", "=", "0,", "effective:", "total", "score", "0–6,", "invalid:", "total", "score", "≥", "6);", "overactive", "bladder", "symptom", "score", "(OABSS;", "if", "the", "urgent", "urination", "score", ">", "2", "and", "total", "score", ">", "3,", "OAB", "will", "be", "diagnosed;", "mild:", "total", "score", "≤", "5,", "moderate:", "total", "score", "6–11,", "severe:", "total", "score", "≥", "12).", "The", "CSESUSI", "will", "use", "a", "scoring", "method", "to", "record", "the", "changes", "of", "symptoms", "and", "signs", "before", "and", "after", "treatment,", "such", "as", "shivering,", "fever,", "dysuria,", "urinal", "pain,", "urinary", "urgency,", "urinary", "frequency,", "lower", "abdomen", "distending", "pain,", "lumbago,", "and", "percussion", "tenderness", "over", "kidney", "region.", "The", "SSST", "uses", "a", "scoring", "method", "to", "record", "the", "changes", "of", "symptoms,", "tongue,", "and", "pulse", "in", "TCM", "before", "and", "after", "treatment.", "The", "OABSS", "has", "been", "found", "to", "be", "reliable", "and", "valid", "and", "highly", "responsive", "to", "treatment-related", "fluctuations", "in", "overactive", "bladder", "symptoms.", "Repeating", "the", "test", "after", "an", "acceptable", "treatment", "interval", "is", "an", "effective", "way", "to", "assess", "the", "efficacy", "of", "a", "particular", "drug", "regimen", "on", "OAB", "symptoms", "[", "33", ",", "34", "].", "If", "the", "symptoms", "disappeared", "after", "seven", "days", "of", "treatment,", "it", "indicates", "that", "the", "patient", "is", "cured.", "If", "these", "symptoms", "partially", "disappeared", "after", "seven", "days", "of", "treatment,", "it", "indicates", "that", "the", "treatment", "is", "effective", "but", "not", "cured.", "If", "there", "was", "no", "obvious", "remission", "and", "the", "symptoms", "of", "infection", "persisted", "after", "seven", "days", "of", "treatment,", "it", "indicates", "that", "the", "treatment", "is", "invalid.", "Secondary", "outcome", "measure", "The", "urine", "leukocytes", "of", "252", "participants", "will", "be", "assessed.", "If", "the", "value", "of", "urine", "leukocytes", "is", "returned", "to", "normal", "after", "seven", "days", "of", "treatment,", "it", "indicates", "the", "patient", "is", "cured.", "If", "the", "value", "of", "urine", "leukocytes", "is", "reduced", "but", "not", "returned", "to", "normal", "after", "seven", "days", "of", "treatment,", "it", "indicates", "that", "the", "treatment", "is", "effective.", "If", "the", "value", "of", "urine", "leukocytes", "is", "still", "higher", "than", "the", "normal", "value", "after", "seven", "days,", "it", "indicates", "the", "treatment", "is", "invalid.", "The", "bacteriological", "examination", "of", "252", "participants", "will", "be", "assessed.", "If", "the", "original", "infected", "part", "of", "the", "specimen", "did", "not", "regenerate", "the", "original", "infected", "pathogen", "after", "seven", "days", "of", "treatment,", "it", "indicates", "the", "original", "bacteria", "is", "cleared.", "If", "the", "original", "pathogen", "was", "still", "cultured", "in", "the", "culture", "of", "the", "specimen", "from", "the", "original", "infection", "position,", "it", "indicates", "the", "original", "bacteria", "is", "not", "cleared.", "Criteria", "of", "comprehensive", "efficacy", "Criteria", "of", "cure:", "the", "evaluation", "of", "clinical", "symptoms", "reached", "the", "level", "of", "cure", "after", "seven", "days", "of", "treatment;", "the", "value", "of", "urine", "leukocytes", "returned", "to", "the", "normal", "value;", "the", "original", "infected", "bacteria", "was", "cleared", "after", "seven", "days", "of", "treatment.", "All", "three", "criteria", "must", "be", "met.", "Criteria", "of", "effective", "treatment:", "the", "clinical", "symptoms", "were", "alleviated", "but", "did", "not", "reach", "the", "level", "of", "cure", "after", "seven", "days", "of", "treatment;", "the", "value", "of", "urine", "leukocytes", "was", "reduced", "but", "not", "returned", "to", "normal", "after", "seven", "days", "of", "treatment.", "Either", "of", "these", "criteria", "must", "be", "met.", "Criteria", "of", "invalid", "treatment:", "the", "clinical", "symptoms", "were", "not", "alleviated", "after", "seven", "days", "of", "treatment;", "the", "value", "of", "urine", "leukocytes", "was", "still", "higher", "than", "normal", "after", "seven", "days", "of", "treatment;", "the", "original", "infected", "bacteria", "were", "still", "cultured", "from", "the", "original", "infection", "position", "after", "seven", "days", "of", "treatment.", "All", "three", "criteria", "must", "be", "met.", "Recurrence", "rate", "Patients", "who", "were", "cured", "will", "attend", "the", "second", "follow-up", "after", "28", "days", "to", "assess", "the", "recurrence", "rate.", "Recurrence", "criteria", "must", "include", "item", "(i)", "and", "either", "(ii)", "or", "(iii)", "at", "the", "same", "time,", "or", "item", "(i)", "alone.", "Primary", "outcome", "measure", "The", "recurrence", "rate", "of", "cured", "participants", "will", "be", "assessed", "by", "the", "symptoms", "of", "acute", "uncomplicated", "lower", "UTI.", "The", "assessment", "tools", "of", "renewed", "symptoms", "will", "also", "use", "the", "three", "participant", "self-rating", "scales", "(see", "Additional", "file", "2", "):", "CSESUSI;", "SSST;", "and", "OABSS.", "If", "the", "symptoms", "of", "cured", "patients", "reappeared", "in", "the", "fourth", "week", "after", "the", "end", "of", "medication,", "it", "indicates", "the", "participant", "has", "relapsed.", "Secondary", "outcome", "measures", "The", "recurrence", "rate", "of", "cured", "participants", "will", "be", "assessed", "by", "urine", "leukocytes.", "If", "the", "urine", "leukocyte", "value", "of", "cured", "patients", "increased", "again", "in", "the", "fourth", "week", "after", "the", "end", "of", "medication,", "it", "indicates", "the", "participant", "has", "relapsed.", "The", "recurrence", "rate", "of", "cured", "participants", "will", "be", "assessed", "by", "bacteriological", "examination.", "If", "the", "urine", "culture", "of", "cured", "patients", "indicated", "that", "the", "original", "urinary", "tract", "pathogen", "is", "positive", "again", "in", "the", "fourth", "week", "after", "the", "end", "of", "medication,", "it", "indicates", "the", "participant", "has", "relapsed.", "Safety", "assessments", "Safety", "measurements", "included", "laboratory", "indices", "and", "AEs.", "All", "patients", "will", "undergo", "the", "following", "laboratory", "examination", "before", "enrollment", "and", "at", "the", "end", "of", "the", "clinical", "trial.", "Changes", "in", "laboratory", "indices", "before", "and", "after", "the", "clinical", "trial", "will", "be", "compared", "to", "conducting", "a", "safety", "analysis.", "Laboratory", "indices", "are:", "(i)", "routine", "blood", "and", "urine", "testing;", "(ii)", "liver", "function", "(AST,", "ALT,", "TBIL,", "DBIL,", "γ-GT,", "ALP)", "and", "kidney", "function", "(Scr,", "BUN);", "and", "(iii)", "ECG.", "The", "occurrence", "of", "any", "AEs", "in", "trial", "participants,", "such", "as", "subjective", "discomfort", "of", "patients", "and", "abnormal", "laboratory", "results,", "will", "be", "recorded", "in", "the", "CRF", "during", "the", "whole", "trial", "process.", "We", "will", "withdraw", "patients", "who", "have", "severe", "AEs,", "as", "it", "will", "be", "unsafe", "for", "them", "to", "continue", "the", "trial.", "Meanwhile,", "we", "will", "give", "them", "relevant", "medical", "care", "and", "follow", "them", "up", "until", "the", "reaction", "has", "terminated.", "Informed", "consent", "Before", "enrolling", "patients", "in", "the", "trial,", "the", "investigating", "doctor", "will", "completely", "and", "comprehensively", "describe", "the", "purpose,", "procedure,", "and", "potential", "risks", "of", "this", "trial", "in", "writing", "to", "the", "patient", "themselves", "or", "their", "designated", "representative.", "Patients", "will", "be", "informed", "that", "they", "have", "the", "right", "to", "withdraw", "from", "the", "trial", "at", "any", "time.", "Each", "patient", "must", "provide", "written", "informed", "consent", "before", "participating", "in", "this", "study;", "this", "consent", "will", "be", "kept", "in", "the", "study", "file.", "Quality", "control", "Quality", "assessment", "will", "be", "conducted", "in", "the", "following", "aspects", "in", "this", "trial:", "the", "progress", "of", "the", "trial;", "the", "qualifications", "of", "the", "investigators;", "the", "mastery", "of", "the", "program;", "the", "authenticity,", "accuracy,", "and", "completeness", "of", "the", "CRF;", "archival", "preservation;", "program", "implementation;", "AEs;", "drug", "preservation", "and", "storage;", "written", "informed", "consent;", "participant", "compliance;", "and", "laboratory", "examination", "data.", "In", "particular,", "the", "authenticity", "and", "accuracy", "of", "the", "CRF,", "program", "implementation,", "and", "determination", "of", "AEs", "will", "be", "strictly", "inspected.", "Data", "management", "The", "patients", "in", "this", "trial", "will", "be", "recruited", "patients.", "Therefore,", "the", "original", "data", "will", "include", "the", "CRF,", "patient", "log", "card,", "original", "laboratory", "examination,", "and", "written", "informed", "consent.", "The", "inspector", "will", "regularly", "visit", "all", "centers", "to", "conduct", "a", "data", "quality", "inspection.", "The", "authenticity", "and", "accuracy", "of", "data", "will", "be", "checked", "by", "original", "laboratory", "comparison,", "telephone", "follow-up", "with", "patients,", "and", "examination", "of", "the", "integrity,", "timeliness,", "and", "normalization", "of", "data.", "The", "paper", "form", "of", "the", "data", "will", "be", "collected", "after", "approval", "inspection.", "The", "researcher", "who", "is", "responsible", "for", "data", "entry", "will", "build", "an", "EpiData-based", "database", "with", "double-recorded", "data", "entry", "by", "two", "people", "and", "consistency", "testing", "will", "be", "carried", "out", "to", "ensure", "the", "accuracy", "of", "data.", "Data", "analysis", "Data", "analysis", "will", "be", "performed", "by", "professional", "statisticians", "using", "SPSS", "22.0.", "Three", "datasets", "will", "be", "conducted:", "intention-to-treat;", "per-protocol", "set;", "and", "safety", "dataset.", "The", "intention-to-treat", "refers", "to", "the", "patients", "who", "have", "been", "randomized;", "an", "intentional", "analysis", "will", "be", "conducted", "for", "curative", "effect.", "The", "per-protocol", "set", "refers", "to", "all", "cases", "that", "do", "not", "violate", "the", "protocol", "and", "complete", "the", "trial;", "per-protocol", "set", "analysis", "will", "be", "conducted", "for", "curative", "effect.", "The", "safety", "dataset", "refers", "to", "the", "randomized", "cases", "that", "have", "taken", "a", "tested", "drug", "at", "least", "once", "with", "safety", "evaluation", "data", "after", "treatment.", "We", "use", "mean", "±", "standard", "deviation", "(SD)", "for", "continuous", "variables", "and", "percentages", "for", "categorical", "variables.", "In", "measured", "indices,", "the", "independent", "t-test", "will", "be", "used", "for", "hypothesis", "testing", "of", "the", "normal", "variables,", "while", "the", "Wilcoxon", "rank", "sum", "test", "will", "be", "used", "for", "hypothesis", "testing", "of", "the", "skewed", "variables.", "The", "χ", "2", "test", "will", "be", "used", "for", "hypothesis", "testing", "of", "the", "counted", "indices.", "The", "statistical", "analyses", "will", "use", "the", "two-sided", "hypothesis", "test.", "P", "≤", "0.05", "will", "be", "set", "as", "the", "significant", "level.", "Ethics", "and", "dissemination", "The", "protocol", "has", "been", "approved", "by:", "the", "Ethics", "Committee", "of", "XiYuan", "Hospital,", "China", "Academy", "of", "Chinese", "Medical", "Sciences", "(identifier", "2018XLA03l-3);", "Ethics", "Committee", "of", "Longhua", "Hospital", "Shanghai", "University", "of", "Traditional", "Chinese", "Medicine", "(identifier", "2018LCSY034);", "the", "Ethics", "Committee", "of", "Guangdong", "Provincial", "Hospital", "of", "Traditional", "Chinese", "Medicine", "(identifier", "BF2018–083-01);", "the", "Ethics", "Committee", "of", "Hubei", "Hospital", "of", "Traditional", "Chinese", "Medicine", "(identifier", "SL2018-C19–01);", "the", "Ethics", "Committee", "of", "the", "Affiliated", "hospital", "of", "Chengdu", "University", "of", "Traditional", "Chinese", "Medicine", "(identifier", "2018KL-053);", "and", "the", "Ethics", "Committee", "of", "Yunnan", "Provincial", "Hospital", "of", "Traditional", "Chinese", "Medicine", "(identifier", "2018LLZ-014-NO.01).", "The", "protocol", "has", "been", "registered", "in", "the", "Clinical", "Trials", "USA", "registry", "(registration", "no.", "NCT03658291)", "on", "4", "September", "2018", "(see", "Additional", "file", "3", ").", "The", "trial", "will", "help", "to", "demonstrate", "if", "SJT", "is", "effective", "and", "safe", "for", "patients", "with", "acute", "uncomplicated", "lower", "UTI.", "We", "will", "publish", "the", "results", "of", "this", "study", "in", "peer-reviewed", "journals", "to", "ensure", "widespread", "dissemination.", "Discussion", "This", "study", "was", "designed", "to", "evaluate", "the", "efficacy,", "safety,", "and", "recurrence", "rate", "of", "SJT", "for", "the", "patients", "with", "acute", "uncomplicated", "lower", "UTI.", "A", "total", "of", "252", "patients", "were", "divided", "into", "three", "groups", "in", "a", "randomized,", "double-blind,", "double-dummy,", "parallel", "control,", "multicenter", "clinical", "trial.", "To", "our", "knowledge,", "this", "is", "the", "first", "study", "protocol", "to", "evaluate", "the", "recurrence", "rate", "and", "the", "effects", "of", "reducing", "or", "substituting", "antibiotics", "of", "SJT", "for", "the", "treatment", "of", "acute", "uncomplicated", "lower", "UTI.", "The", "first", "aim", "of", "this", "trial", "is", "to", "determine", "whether", "SJT", "can", "reduce", "the", "use", "of", "antibiotics", "(control", "group", "2", "versus", "control", "group", "1,", "non-inferiority", "test);", "the", "second", "aim", "is", "to", "determine", "whether", "SJT", "can", "substitute", "the", "use", "of", "antibiotics", "(experimental", "group", "versus", "control", "group", "1,", "non-inferiority", "test).", "The", "recurrence", "rate", "will", "be", "assessed", "at", "28-day", "follow-up", "after", "being", "cured", "to", "determine", "whether", "SJT", "can", "reduce", "the", "recurrence", "rate.", "To", "facilitate", "high", "validity", "and", "reliability,", "a", "strict", "quality", "control", "and", "high-quality", "methodology", "is", "indispensable.", "We", "describe", "in", "detail", "the", "method", "of", "randomization,", "allocation", "concealment,", "blinding,", "interventions,", "recruitment,", "and", "data", "collection.", "The", "results", "from", "this", "trial", "may", "provide", "evidence", "on", "the", "effectiveness,", "safety,", "recurrence", "rate,", "and", "reduce", "or", "substitute", "antibiotics", "of", "SJT.", "Strengths", "and", "limitations", "of", "this", "study", "This", "is", "a", "well-designed", "study", "to", "assess", "the", "efficacy,", "safety,", "and", "recurrence", "rate", "of", "SJT", "for", "acute", "uncomplicated", "lower", "UTI", "in", "adults.", "The", "results", "from", "this", "randomized,", "double-blind,", "double-dummy,", "parallel", "control", "of", "positive", "drug,", "multicenter", "clinical", "trial", "will", "provide", "new", "evidence", "of", "the", "efficacy", "and", "recurrence", "rate", "of", "SJT", "for", "acute", "uncomplicated", "lower", "UTT.", "This", "study", "will", "provide", "evidence", "of", "SJT", "for", "reducing", "or", "substituting", "the", "use", "of", "antibiotics", "for", "acute", "uncomplicated", "lower", "UTI,", "which", "can", "guide", "rational", "application", "of", "antibiotics.", "This", "trial", "will", "be", "implemented", "in", "six", "hospitals", "in", "Chinese", "patients;", "this", "can", "strengthen", "its", "generalizability.", "One", "limitation", "is", "the", "results", "of", "participant", "symptoms", "evaluate", "scales", "might", "have", "subjective", "factors.", "Another", "limitation", "is", "the", "participant", "symptoms", "evaluate", "scales,", "which", "might", "exaggerate", "the", "severity", "of", "the", "symptoms", "of", "acute", "uncomplicated", "lower", "UTI.", "Trial", "status", "This", "trial", "was", "registered", "in", "the", "Clinical", "Trials", "USA", "registry", "(registration", "no.", "NCT03658291)", "on", "4", "September", "2018.", "Recruitment", "will", "begin", "in", "January", "2019", "and", "it", "is", "anticipated", "that", "enrollment", "will", "be", "completed", "in", "December", "2019." ]
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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, 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 ]
{"negatives/white": ["2756+1", "3098+1", "123+1", "2991+1", "2352+1", "1904+1", "422+1", "1377+1", "3493+1", "4127+1"]}
CC-BY
4.0
http://creativecommons.org/licenses/by/4.0/
Original work titled: 'Sanjin tablets for acute uncomplicated lower urinary tract infection (syndrome of dampness-heat in the lower jiao): protocol for randomized, double-blind, double-dummy, parallel control of positive drug, multicenter clinical trial' (Trials/BioMed Central) by the following authors: Jian Lyu; Yan-ming Xie; Zhan Gao; Jian-wu Shen; Yue-yi Deng; Song-tao Xiang; Wen-xi Gao; Wen-tong Zeng; Chun-he Zhang; Dan-hui Yi; Lian-xin Wang; Zhi-fei Wang DOI: 10.1186/s13063-019-3539-5
298f0b4ad99836dbc04299d84f54f8c8
[ "Introduction", "Constipation", "is", "a", "bowel", "disease", "characterized", "by", "symptoms", "such", "as", "infrequent", "bowel", "movements,", "difficulty", "during", "defecation,", "and", "sensation", "of", "incomplete", "bowel", "evacuation", "[", "1", "–", "4", "].", "This", "disease", "is", "often", "the", "outcome", "of", "numerous", "reasons", "including", "insufficient", "dietary", "fiber", "intake,", "inadequate", "fluid", "intake,", "decreased", "physical", "activity,", "side", "effects", "of", "medication,", "hypothyroidism,", "obstruction", "by", "colorectal", "cancer,", "and", "side", "effects", "due", "to", "drug", "administrations", "[", "5", "–", "7", "].", "Several", "chemical", "drugs", "and", "natural", "products", "have", "been", "investigated", "as", "laxatives", "to", "treat", "constipation.", "Chemical", "drugs", "(laxatives)", "such", "as", "senna,", "correctol,", "exlax,", "senokot", "and", "gaviscon", "are", "widely", "prescribed", "to", "increase", "bulkiness", "and", "soften", "stool,", "or", "as", "osmotic", "agents", "that", "enhance", "water", "flow", "into", "the", "colon", "to", "promote", "elimination", "and", "trigger", "bowel", "movements,", "although", "they", "have", "undesirable", "side", "effects", "[", "8", ",", "9", "].", "Furthermore,", "several", "herbal", "medicines", "such", "as", "Aloe", "ferox", "Mill.,", "Liriope", "platyphylla,", "Fumaria", "parviflora", "and", "Mareya", "micrantha", "Mull", "are", "known", "to", "dramatically", "improve", "the", "symptoms", "of", "constipation", "in", "animal", "models,", "without", "any", "significant", "adverse", "side", "effects", "[", "6", ",", "10", ",", "11", "].", "In", "an", "effort", "to", "identify", "novel", "drugs", "for", "the", "treatment", "of", "constipation,", "a", "recent", "study", "has", "investigated", "the", "therapeutic", "effects", "of", "Red", "Liriope", "platyphylla", "extract", "(EtRLP)", "in", "Lop-induced", "constipated", "SD", "rats", "[", "12", "].", "EtRLP", "was", "manufactured", "from", "L.", "platyphylla", "roots", "using", "a", "specific", "process", "consisting", "of", "two", "steps", "(steaming", "of", "L.", "platyphylla", "roots", "at", "99", "°C", "for", "3", "h,", "followed", "by", "air-drying", "at", "70", "°C", "for", "24", "h)", "for", "different", "number", "of", "repetitions", "[", "13", "].", "The", "extract", "contains", "a", "large", "amount", "of", "total", "phenolic", "compounds,", "total", "flavonoids", "and", "5-hydroxmethyl-2-furfural,", "and", "is", "mainly", "composed", "of", "carbohydrates,", "moisture,", "crude", "protein,", "crude", "ash,", "and", "crude", "fat", "[", "5", "].", "Furthermore,", "EtRLP", "treatment", "induces", "the", "enhancement", "of", "insulin", "secretion", "and", "decrease", "in", "the", "glucose", "concentration", "in", "INS", "cells,", "STZ-induced", "diabetic", "ICR", "mice,", "and", "OLETF", "rats", "[", "5", ",", "13", ",", "14", "].", "Furthermore,", "the", "symptoms", "of", "constipation,", "including", "stool", "excretions,", "histological", "structure,", "mucin", "section,", "muscarinic", "acetylcholine", "receptors", "(mAChR)", "signaling", "pathway,", "endoplasmic", "reticulum", "(ER)", "stress", "response,", "and", "mucin", "secretion", "are", "markedly", "improved", "after", "the", "RLP", "administration", "[", "12", "].", "However,", "to", "date,", "there", "has", "been", "no", "research", "in", "screening", "of", "metabolomics", "biomarkers", "that", "anticipate", "the", "laxative", "effects", "induced", "by", "RLP", "treatment.", "As", "part", "of", "the", "search", "for", "sensitive", "and", "reliable", "biomarkers", "of", "laxative", "effects,", "this", "study", "was", "designed", "to", "compare", "the", "serum", "biomarkers", "obtained", "from", "Lop", "+", "EtRLP", "treated", "SD", "rats", "using", "the", "metabolomics-based", "proton", "(NMR)", "platform.", "The", "present", "results", "indicate", "that", "the", "metabolomics", "profile", "of", "serum", "collected", "from", "EtRLP", "treated", "constipation", "SD", "rats", "may", "provide", "useful", "information", "in", "the", "future", "development", "of", "novel", "sensitive", "and", "reliable", "biomarkers", "for", "laxative", "drugs.", "Results", "Effect", "of", "EtRLP", "on", "the", "feeding", "behavior", "and", "excretion", "parameters", "of", "the", "constipation", "rats", "To", "investigate", "whether", "EtRLP", "treatment", "affects", "the", "feeding", "behavior", "and", "excretion", "parameters", "of", "constipated", "rats,", "we", "measured", "the", "food", "intake,", "water", "consumption", "and", "excretion", "parameters", "in", "SD", "rats", "of", "subset", "groups.", "As", "shown", "in", "Fig.", "1", ",", "water", "consumption", "was", "slightly", "recovered", "in", "the", "Lop", "+", "EtRLP", "treated", "group,", "whereas", "no", "significant", "difference", "was", "observed", "in", "the", "body", "weight", "and", "food", "intake", "of", "the", "LOP", "treated", "and", "Lop", "+", "EtRLP", "treated", "groups.", "Also,", "a", "decrease", "in", "stool", "number,", "weight", "and", "water", "contents", "in", "the", "LOP", "treated", "group", "was", "almost", "recovered", "in", "the", "Lop", "+", "EtRLP", "and", "LOP", "+", "BisaC", "treated", "groups.", "These", "results", "indicate", "that", "EtRLP", "treatment", "improves", "the", "excretion", "parameters", "of", "Lop-induced", "constipation", "in", "SD", "rats", "without", "any", "changes", "in", "the", "feeding", "behavior.", "Fig.", "1", "Feeding", "behavior", "and", "stool", "parameters", "after", "EtRLP", "treatment.", "a", "-", "c", "Three", "parameters", "related", "with", "the", "feeding", "behavior", "were", "measured", "in", "SD", "rats", "of", "subset", "groups", "in", "duplicate.", "d", "-", "e", "Three", "stool", "parameters", "were", "measured", "in", "SD", "rats", "of", "subset", "groups", "in", "duplicate.", "Five", "to", "six", "rats", "per", "group", "were", "assayed", "in", "duplicate", "by", "parameter", "counting.", "The", "data", "are", "reported", "as", "the", "mean", "±", "SD.", "*", "indicates", "p", "<", "0.05", "compared", "to", "the", "NO", "group.", "#", "indicates", "p", "<", "0.05", "compared", "to", "the", "Lop", "+", "Veh", "treated", "group", "Recovery", "effect", "of", "EtRLP", "on", "the", "histological", "alteration", "of", "colon", "To", "investigate", "the", "effects", "of", "EtRLP", "treatment", "on", "the", "recovery", "of", "the", "histological", "structure", "of", "the", "colon,", "we", "evaluated", "alterations", "in", "mucosa", "and", "muscle", "thickness", "in", "colon", "sections", "of", "subset", "groups", "stained", "with", "H&E.", "The", "average", "thickness", "of", "mucosa", "and", "muscle", "was", "observed", "to", "be", "significantly", "shorter", "in", "the", "Lop", "+", "Veh", "treated", "group", "as", "compared", "to", "the", "No", "treated", "group.", "Following", "Lop", "+", "EtRLP", "or", "LOP", "+", "BisaC", "cotreatment,", "this", "level", "greatly", "increased", "when", "compared", "with", "the", "Lop", "+", "Vehicle", "treated", "group", "(Fig.", "2", ").", "These", "results", "suggest", "that", "EtRLP", "induces", "in", "recovering", "the", "abnormal", "structure", "of", "the", "colon", "in", "constipated", "SD", "rats", "after", "Lop", "treatment.", "Fig.", "2", "Histological", "structure", "of", "EtRLP", "treated", "colon.", "H&E-stained", "sections", "of", "transverse", "colons", "collected", "from", "No,", "EtRLP,", "Lop", "+", "Vehicle,", "Lop", "+", "EtRLP", "and", "Lop", "+", "BisaC", "treated", "rats", "were", "observed", "at", "two", "different", "magnifications", "(100x", "and", "400x)", "using", "a", "light", "microscope.", "Five", "to", "six", "rats", "per", "group", "were", "assayed", "in", "triplicate,", "after", "H&E", "staining.", "The", "data", "are", "reported", "as", "the", "mean", "±", "SD.", "*", "indicates", "p", "<", "0.05", "compared", "to", "the", "NO", "group.", "#", "indicates", "p", "<", "0.05", "compared", "to", "the", "Lop", "+", "Veh", "treated", "group", "Effects", "of", "EtRLP", "treatment", "on", "endogenous", "metabolites", "in", "serum", "To", "observe", "the", "effects", "of", "EtRLP", "treatment", "on", "the", "endogenous", "metabolites", "of", "subset", "groups,", "pattern", "recognitions", "were", "analyzed", "in", "the", "serum", "of", "subset", "groups", "using", "PCA", "and", "OPLS-DA.", "NMR", "spectra", "in", "global", "profiling", "show", "clustering", "between", "the", "No", "group", "and", "Lop", "+", "Veh", "treated", "group,", "or", "between", "Lop", "+", "Veh", "treated", "group", "and", "Lop", "+", "EtRLP", "treated", "group", "(Fig.", "3", "a).", "Also,", "the", "Chenomx", "NMR", "Suite", "(ver.", "4.6,", "Chenomx", "Inc.,", "Edmonton,", "Alberta,", "Canada),", "applied", "for", "targeted", "NMR", "spectral", "analysis,", "detected", "a", "total", "of", "33", "metabolites", "(Additional", "file", "1", ":", "Table", "S1);", "the", "PCA", "and", "OPLS-DA", "score", "plots", "differed", "between", "the", "No-", "and", "Lop", "+", "Veh", "treated", "groups", "(Fig.", "3", "b).", "Of", "these,", "some", "endogenous", "metabolites", "were", "selected", "to", "investigate", "the", "effects", "of", "EtRLP", "treatment", "using", "VIP.", "Especially,", "15", "metabolites", "were", "detected", "in", "response", "to", "EtRLP", "treatment.", "These", "metabolites", "were", "categorized", "into", "two", "major", "groups:", "an", "amino", "acid", "group", "containing", "alanine,", "arginine,", "glutamine,", "glutamate,", "glycine,", "threonine", "and", "valine,", "and", "a", "metabolite", "group", "containing", "betaine,", "creatine,", "glycerol,", "glucose,", "ethanol,", "lactate,", "succinate", "and", "taurine.", "In", "serum", "samples", "of", "the", "Lop", "+", "Veh", "treated", "group,", "these", "metabolite", "concentrations", "were", "significantly", "decreased", "relative", "to", "those", "of", "the", "No", "treated", "group.", "However,", "significant", "recovery", "was", "observed", "after", "Lop", "+", "EtRLP", "cotreatment.", "Furthermore,", "the", "level", "of", "most", "metabolites", "in", "the", "Lop", "+", "EtRLP", "treated", "group", "was", "very", "similar", "with", "those", "of", "the", "Lop", "+", "BisaC", "treated", "group", "(Fig.", "4", ").", "Therefore,", "these", "results", "suggest", "that", "EtRLP", "treatment", "causes", "a", "recovery", "of", "15", "endogenous", "metabolites", "relative", "to", "the", "constipations.", "Fig.", "3", "Spectral", "analysis", "of", "metabolomic", "pattern", "using", "PCA", "and", "OPLS-DA.", "a", "Global", "profiling", "of", "loperamide", "treatment", "in", "serum", "samples.", "Targeted", "profiling", "of", "loperamide", "treatment", "in", "serum", "samples.", "b", "The", "VIP", "shows", "the", "major", "metabolites", "contributing", "to", "cluster", "separation.", "The", "data", "are", "reported", "as", "the", "mean", "±", "SD", "Fig.", "4", "Concentration", "of", "7", "amino", "acids", "and", "8", "metabolites", "after", "EtRLP", "administration", "in", "SD", "rats.", "The", "pathway", "relates", "to", "the", "endogenous", "metabolites", "that", "are", "altered", "after", "Lop", "+", "EtRLP", "treatment,", "while", "the", "box", "shows", "the", "altered", "metabolites.", "The", "data", "are", "reported", "as", "the", "mean", "±", "SD", "Confirmation", "of", "glucose", "and", "creatine", "concentration", "Serum", "biochemical", "analyses", "were", "conducted", "to", "validate", "the", "changes", "in", "the", "concentration", "of", "the", "two", "metabolites", "identified", "by", "metabolomics", "analysis.", "As", "shown", "in", "Fig.", "5", ",", "the", "regulation", "pattern", "of", "glucose", "and", "creatine", "observed", "upon", "serum", "biochemical", "analyses", "was", "very", "similar", "to", "those", "in", "NMR", "spectrometry.", "They", "were", "significantly", "enhanced", "in", "Lop", "+", "EtRLP", "or", "Lop", "+", "BisaC", "treated", "group,", "although", "their", "levels", "were", "significantly", "lower", "in", "the", "Lop", "+", "Veh", "treated", "group", "than", "No", "treated", "group.", "Therefore,", "these", "results", "indicate", "that", "the", "alteration", "of", "metabolites", "detected", "by", "NMR", "spectrometry", "reflects", "change", "in", "metabolite", "concentration", "in", "the", "serum", "of", "the", "Lop", "+", "EtRLP", "and", "Lop", "+", "Veh", "treated", "group.", "Fig.", "5", "Serum", "biochemical", "analysis", "for", "glucose", "a", "and", "creatine", "b", "concentration.", "After", "the", "collection", "of", "serum,", "their", "levels", "were", "measured", "using", "an", "automatic", "biochemical", "analyzer.", "Five", "to", "six", "rats", "per", "group", "were", "assayed", "in", "duplicate", "by", "parameter", "counting.", "The", "data", "are", "reported", "as", "the", "mean", "±", "SD.", "*", "indicates", "p", "<", "0.05", "compared", "to", "the", "NO", "group.", "#", "indicates", "p", "<", "0.05", "compared", "to", "the", "Lop", "+", "Veh", "treated", "group", "Discussion", "Some", "extracts", "derived", "from", "natural", "products", "are", "considered", "as", "key", "candidates", "for", "treating", "chronic", "constipation,", "since", "they", "are", "able", "to", "increase", "the", "intestinal", "motility,", "frequency", "and", "weight", "of", "stools,", "and", "the", "ileum", "tension.", "Especially,", "the", "extract", "of", "Aloe", "ferox", "Mill.,", "agarwood", "(Aquilaria", "sinensis", "and", "Aquilaria", "crasna)", "leaves", "and", "Ficus", "carica", "paste", "induces", "an", "improvement", "in", "the", "intestinal", "motility,", "increases", "fecal", "volume,", "and", "normalizes", "the", "body", "weight", "in", "Lop-induced", "constipated", "rats", "[", "5", ",", "11", ",", "15", "].", "Also,", "extracts", "of", "LP", "are", "reported", "to", "increase", "the", "frequency", "and", "weight", "of", "stools,", "villus", "length,", "crypt", "layer,", "muscle", "thickness,", "mucin", "secretion,", "and", "accumulation", "of", "lipid", "droplets,", "and", "dramatically", "reduce", "the", "mAChRs", "signal", "pathway,", "in", "enterocytes", "of", "the", "crypt", "in", "Lop-induced", "SD", "rats", "[", "10", "].", "However,", "no", "studies", "have", "investigated", "alterations", "in", "the", "metabolomics", "profiles", "during", "the", "process", "of", "EtRLP", "therapeutic", "outcome", "against", "constipation.", "We", "believe", "that", "our", "study", "therefore", "provides", "the", "first", "evidence", "for", "metabolomics", "profiles", "obtained", "from", "the", "laxative", "effects", "of", "EtRLP", "in", "Lop-induced", "constipation", "SD", "rats.", "Twenty", "amino", "acids", "are", "used", "as", "the", "major", "units", "to", "structure", "proteins", "and", "other", "biomolecules,", "and", "are", "known", "to", "oxidize", "to", "urea", "and", "carbon", "dioxide", "as", "key", "sources", "of", "energy", "[", "16", "].", "Of", "these,", "9", "amino", "acid", "(histidine,", "isoleucine,", "leucine,", "lysine,", "methionine,", "phenylalanine,", "threonine,", "tryptophan", "and", "valine)", "are", "assigned", "as", "essential", "amino", "acids", "since", "they", "cannot", "be", "synthesized", "from", "other", "compounds", "at", "the", "concentration", "required", "for", "normal", "growth", "in", "the", "human", "body", "[", "17", "].", "Especially,", "dietary", "amino", "acids", "are", "used", "as", "key", "substrates", "for", "the", "synthesis", "of", "intestinal", "proteins,", "NO", "and", "polyamines,", "as", "well", "as", "consumed", "as", "the", "major", "fuel", "in", "the", "small", "intestine", "[", "18", "].", "Some", "specific", "amino", "acids,", "including", "glutamine,", "glutamate,", "arginine,", "glycine,", "lysine,", "threonine,", "and", "sulfur-containing", "amino", "acids,", "have", "a", "potentially", "therapeutic", "role", "against", "gut-related", "diseases", "[", "19", ",", "20", "].", "However,", "scientific", "evidence", "correlating", "the", "laxative", "effect", "of", "EtRLP", "and", "amino", "acids", "has", "not", "been", "presented", "to", "date.", "In", "the", "present", "study,", "the", "results", "of", "metabolomics", "profile", "analysis", "indicate", "that", "the", "serum", "level", "of", "7", "amino", "acids", "can", "be", "recovered", "by", "EtRLP", "treatment.", "Our", "findings", "also", "show", "that", "these", "amino", "acids", "may", "be", "key", "markers", "indicating", "the", "laxative", "effects", "in", "Lop", "induced", "constipation", "rats.", "Meanwhile,", "serum", "has", "been", "used", "as", "an", "important", "specimen", "for", "studying", "the", "action", "mechanism", "and", "evaluating", "therapeutic", "efficacy", "in", "constipation", "and", "irritable", "bowel", "syndrome", "(IBS).", "The", "production", "levels", "of", "gastrointestinal", "metabolic", "components", "including", "motilin", "(MTL),", "gastrin", "(Gas),", "endothelin", "(ET),", "substance", "P", "(SP),", "acetylcholinesterase", "(AChE)", "and", "vasoactive", "intestinal", "peptide", "(VIP)", "were", "measured", "in", "serum", "to", "investigate", "the", "laxative", "effects", "of", "Lactobacillus", "fermentum", "Suo", "and", "naringenin", "[", "21", ",", "22", "].", "Serum", "serotonin", "and", "glucose-like", "peptide", "(GLP-1)", "concentration", "were", "measured", "by", "ELISA", "in", "patient", "with", "IBS", "[", "23", ",", "24", "].", "Serum", "proteomic", "analysis", "was", "performed", "upon", "patients", "with", "constipation", "predominant", "IBS", "symptoms.", "Among", "1,317", "proteins,", "12", "proteins,", "such", "as", "TGFβ1,", "PF4V1,", "PF4,", "APP", "and", "MMP", "showed", "significance", "different", "in", "level", "of", "expression", "[", "25", "].", "Based", "on", "above", "previous", "results,", "we", "selected", "serum", "as", "a", "sample", "for", "analyzing", "metabolites.", "The", "metabolic", "profiles", "associated", "with", "constipation", "were", "investigated", "in", "few", "studies", "using", "samples", "of", "animals", "and", "human.", "The", "metabolic", "profile", "before", "and", "after", "a", "meal", "challenge", "in", "a", "cohort", "of", "children", "with", "constipation", "was", "investigated", "to", "determine", "its", "relationship", "with", "postprandial", "colon", "motility", "patterns", "and", "identify", "metabolic", "targets", "for", "treatment", "of", "constipation.", "Some", "significant", "alterations", "were", "measured", "on", "the", "16", "amino", "acid", "and", "22", "lipid", "metabolites", "of", "the", "postprandial", "group", "among", "187", "metabolites.", "Some", "correlation", "between", "normal", "and", "abnormal", "postprandial", "motility", "pattern", "was", "detected", "in", "the", "concentration", "of", "specific", "metabolites", "including", "glycerol,", "carnosine,", "alanine,", "asparagine,", "cytosine,", "choline,", "phosphocholine,", "thyroxine", "and", "triiodothyronine", "[", "26", "].", "Also,", "metabolomics", "approach", "was", "performed", "in", "the", "serum", "of", "Lop-induced", "constipation", "in", "SD", "rats.", "Among", "35", "endogenous", "metabolites,", "four", "amino", "acids", "(alanine,", "glutamate,", "glutamine", "and", "glycine)", "and", "six", "endogenous", "metabolites", "(acetate,", "glucose,", "glycerol,", "lactate,", "succinate", "and", "taurine)", "were", "significantly", "decreased", "in", "Lop-treated", "SD", "rats", "[", "27", "].", "In", "this", "study,", "the", "Lop", "+", "EtRLP", "treated", "SD", "rats", "showed", "a", "dramatic", "alteration", "on", "the", "7", "amino", "acids", "(alanine,", "arginine,", "glutamate,", "glutamine,", "glycine,", "threonine", "and", "valine)", "and", "8", "endogenous", "metabolites", "(betaine,", "creatine,", "glucose,", "taurine,", "ethanol,", "lactate,", "glycerol", "and", "succinate).", "These", "patterns", "were", "very", "similar", "with", "metabolomics", "profile", "of", "Lop-induced", "constipation", "rats", "although", "they", "were", "different", "from", "the", "results", "analyzed", "from", "children", "with", "constipation.", "Especially,", "glycerol", "was", "commonly", "detected", "as", "an", "important", "key", "marker", "for", "constipation", "among", "various", "metabolites", "in", "above", "three", "studies.", "Meanwhile,", "recent", "studies", "support", "the", "correlation", "between", "L.", "platyphylla", "and", "glucose", "regulation,", "although", "there", "is", "no", "evidence", "for", "the", "role", "of", "glucose", "in", "the", "laxative", "effects", "of", "L.", "platyphylla.", "The", "homoisoflavone-enriched", "fraction", "and", "LP9M80-H", "from", "L.", "platyphylla", "increases", "the", "insulin-stimulated", "glucose", "uptake", "in", "3", "T3-L1", "adipocytes", "and", "enhances", "the", "secretion", "of", "insulin", "in", "the", "HIT-T15", "pancreatic", "ß-cell", "line", "[", "28", ",", "29", "].", "Also,", "LP9M80-H", "and", "aqueous", "extracts", "induce", "a", "significant", "increase", "of", "glucose", "levels", "and", "decrease", "in", "insulin", "concentrations", "in", "the", "blood", "of", "ICR", "mice", "and", "OLETF", "rats", "[", "29", ",", "30", "].", "Furthermore,", "similar", "effects", "on", "the", "insulin", "secretion", "by", "L.", "platyphylla", "have", "also", "been", "observed", "after", "EtRLP", "treatment;", "insulin", "secretion", "significantly", "increases", "after", "EtRLP", "treatment", "in", "INS", "cells,", "streptozotocin-induced", "diabetic", "ICR", "mice,", "and", "OLETF", "rats", "[", "13", ",", "14", ",", "31", "].", "In", "the", "current", "study,", "we", "investigated", "the", "profile", "of", "endogenous", "metabolites", "in", "serum", "of", "constipation", "SD", "rats", "treated", "with", "EtRLP,", "to", "characterize", "putative", "biomarkers", "for", "laxative", "effects.", "We", "observed", "that", "the", "concentration", "of", "D-glucose", "dramatically", "recovers", "in", "the", "Lop", "+", "EtRLP", "treated", "group,", "as", "compared", "to", "the", "Lop", "+", "Veh", "treated", "group", "(Fig.", "4", ").", "Therefore,", "we", "construe", "that", "the", "increased", "glucose", "concentration", "in", "the", "profile", "of", "endogenous", "metabolites", "may", "be", "caused", "by", "the", "ability", "of", "EtRLP", "for", "insulin", "secretion,", "although", "additional", "research", "is", "required", "to", "confirm", "the", "same.", "Only", "a", "few", "studies", "have", "provided", "some", "evidences", "for", "the", "correlation", "between", "some", "metabolites", "and", "laxative", "effects.", "L-arginine", "effectively", "regulated", "the", "opioid-induced", "constipation", "via", "a", "stereospecific", "and", "peripheral", "action", "[", "32", "].", "Average", "intake", "of", "taurine", "was", "significantly", "higher", "in", "functional", "constipation", "patients", "than", "control", "group", "[", "33", "].", "Also,", "prucalopride", "succinate", "improved", "constipation", "through", "the", "stimulation", "of", "colonic", "motility", "[", "34", "].", "Therefore,", "above", "results", "from", "previous", "studies", "support", "the", "reliability", "of", "metabolites", "identified", "by", "present", "study", "although", "additional", "studies", "will", "be", "need.", "Conclusions", "Overall,", "we", "believe", "that", "the", "present", "research", "is", "the", "first", "study", "to", "examine", "metabolic", "changes", "in", "SD", "rats", "treated", "with", "Lop", "+", "EtRLP,", "and", "to", "correlate", "changes", "in", "specific", "metabolites", "consistent", "with", "laxative", "effects", "of", "EtRLP.", "In", "addition,", "the", "results", "presented", "herein", "provide", "evidence", "that", "7", "amino", "acids", "and", "8", "metabolites", "are", "potential", "biomarkers", "for", "predicting", "the", "laxative", "effects", "induced", "by", "natural", "product", "remedies.", "Materials", "and", "methods", "Preparation", "and", "analysis", "of", "EtRLP", "EtRLP", "was", "prepared", "using", "the", "methods", "described", "in", "previous", "studies", "[", "31", "].", "Briefly,", "to", "manufacture", "RLP", "from", "L.", "platyphylla", "(LP)", "at", "nine", "different", "steaming", "frequencies,", "the", "specific", "process", "comprising", "two", "steps", "(200", "g", "of", "dry", "roots", "steamed", "at", "99", "°C", "for", "3", "h", "and", "air-dried", "at", "70", "°C", "for", "24", "h)", "was", "carried", "out", "for", "different", "numbers", "of", "repetitions", "a", "total", "of", "9", "times.", "The", "RLP", "was", "then", "subjected", "to", "the", "appropriate", "extraction", "process,", "and", "the", "resultant", "EtRLP", "was", "collected.", "Furthermore,", "the", "composition", "and", "the", "concentration", "of", "active", "compounds", "in", "the", "EtRLP", "were", "analyzed", "by", "the", "methods", "suggested", "in", "previous", "studies", "[", "12", ",", "31", "].", "Experimental", "design", "for", "animals", "The", "animal", "protocol", "used", "in", "this", "study", "was", "reviewed", "and", "approved", "based", "on", "ethical", "procedures", "and", "scientific", "care", "by", "the", "Pusan", "National", "University-Institutional", "Animal", "Care", "and", "Use", "Committee", "(PNU-IACUC;", "Approval", "Number", "PNU-2012-0010).", "Adult", "SD", "rats", "were", "purchased", "from", "Samtacho", "BioKorea", "Co.", "(Osan,", "Korea)", "and", "handled", "in", "a", "accredited", "by", "the", "Korea", "Food", "and", "Drug", "Administration", "(FDA)", "(Accredited", "Unit", "Number-000231)", "and", "AAALAC", "International", "(Accredited", "Unit", "Number;", "001525).", "All", "rats", "were", "provided", "with", "standard", "irradiated", "chow", "diet", "(Purina", "Mills,", "Seoungnam,", "Korea)", "ad", "libitum", "and", "were", "maintained", "in", "a", "specific", "pathogen-free", "state", "under", "a", "strict", "light", "cycle", "(lights", "on", "at", "06:00", "h", "and", "off", "at", "18:00", "h)", "at", "a", "temperature", "of", "22", "±", "2", "°C", "and", "a", "relative", "humidity", "of", "50", "±", "10%.", "Constipation", "was", "induced", "in", "SD", "rats", "by", "subcutaneous", "injection", "of", "Lop", "(4", "mg/kg", "weight)", "in", "0.9%", "sodium", "chloride", "twice", "a", "day", "for", "3", "days,", "whereas", "the", "non-constipation", "group", "was", "injected", "with", "0.9%", "sodium", "chloride", "alone", "[", "11", "].", "For", "the", "experiment,", "8-week-old", "SD", "rats", "(n", "=", "30)", "were", "assigned", "to", "either", "a", "non-constipation", "group", "(n", "=", "12)", "or", "a", "constipation", "group", "(n", "=", "18).", "The", "non-constipation", "group", "was", "further", "divided", "into", "a", "No", "treated", "group", "(n", "=", "6)", "and", "an", "EtRLP", "treated", "group", "(n", "=", "6).", "The", "No", "treated", "group", "was", "untreated", "during", "the", "experimental", "period,", "whereas", "the", "EtRLP", "treated", "group", "received", "15", "μl/g", "body", "weight", "of", "EtRLP", "(1,000", "mg/kg", "weight)", "one", "time.", "The", "constipation", "group", "was", "further", "divided", "into", "a", "Lop", "+", "Veh", "treated", "group", "(n", "=", "6),", "Lop", "+", "EtRLP", "treated", "group", "(n", "=", "6)", "and", "Lop", "+", "BisaC", "treated", "group", "(n", "=", "6).", "The", "Lop", "+", "Veh", "treated", "group", "received", "a", "consistent", "volume", "of", "water", "via", "gavage,", "whereas", "the", "other", "cotreatment", "groups", "were", "administered", "once,", "either", "1,000", "mg/kg", "body", "weight", "of", "EtRLP", "(Lop", "+", "EtRLP", "treated", "group)", "or", "3.3", "mg/kg", "body", "weight", "of", "BisaC", "(Lop", "+", "BisaC", "treated", "group)", "after", "the", "induction", "of", "constipation.", "BisaC", "majorly", "included", "bisacodyl,", "docusate", "sodium", "and", "sennoside", "calcium,", "and", "was", "purchased", "from", "Kolon", "Pharmaceuticals", "INC.", "(Gyenggido,", "Korea).", "At", "24", "h", "after", "the", "EtRLP,", "BisaC", "and", "Veh", "treatment,", "all", "animals", "were", "euthanized", "using", "CO", "2", "gas,", "and", "tissue", "samples", "were", "acquired", "and", "stored", "in", "Eppendorf", "tubes", "at", "−", "70", "°C", "until", "assay.", "Measurement", "of", "stool", "parameters", "and", "urinary", "volume", "All", "SD", "rats", "were", "bred", "in", "metabolic", "cages", "during", "the", "entire", "experimental", "period", "to", "avoid", "contamination.", "Stools", "and", "urine", "excreted", "from", "each", "SD", "rat", "were", "collected", "daily", "at", "10:00", "a.m.", "Stool", "weight", "and", "number", "was", "measured", "three", "times", "per", "sample", "using", "an", "electric", "balance", "and", "hand", "counter,", "whereas", "the", "water", "content", "was", "determined", "as", "the", "difference", "between", "the", "wet", "and", "dry", "weights", "of", "the", "stool,", "as", "described", "previously", "[", "11", ",", "31", "].", "Histological", "analysis", "Colons", "collected", "from", "SD", "rats", "of", "subset", "group", "were", "fixed", "with", "10%", "formalin", "for", "48", "h,", "embedded", "in", "paraffin", "wax,", "and", "then", "sectioned", "into", "4", "μm", "thick", "slices", "that", "were", "subsequently", "subjected", "to", "hematoxylin", "and", "eosin", "staining", "(H&E,", "Sigma-Aldrich", "Co.,", "St.", "Louis,", "MO,", "USA).", "Morphological", "features,", "including", "thickness", "of", "mucosa", "and", "muscle,", "were", "observed", "under", "light", "microscopy", "at", "100x", "and", "400x", "magnification", "(Leica", "Microsystems,", "Switzerland).", "Metabolomics", "analysis", "Appropriate", "serum", "samples", "(300", "μl)", "obtained", "from", "animals", "were", "placed", "in", "micro", "centrifuge", "tubes", "containing", "300", "μl", "D", "2", "O", "solutions", "with", "4", "mM", "TSP", "as", "a", "qualitative", "standard", "for", "the", "chemical", "shift", "scale.", "After", "vortexing,", "serum", "samples", "were", "analyzed", "by", "NMR", "spectrometry", "within", "48", "h.", "All", "spectra", "were", "determined", "using", "a", "Varian", "Unity", "Inova", "600", "MHz", "spectrometer", "operating", "at", "a", "temperature", "of", "26", "°C", "supported", "by", "Pusan", "National", "University", "(Busan,", "Korea).", "The", "one-dimensional", "NMR", "spectra", "were", "acquired", "with", "the", "following", "acquisition", "parameters:", "spectral", "width", "24,038.5", "Hz,", "7.55", "min", "acquisition", "time,", "and", "128", "nt.", "Additional", "conditions", "of", "a", "relaxation", "delay", "time", "of", "1", "s", "and", "saturation", "power", "of", "4", "were", "set", "to", "suppress", "a", "massive", "water", "peak.", "NMR", "spectra", "were", "reduced", "to", "data", "using", "the", "program", "Chenomx", "NMR", "Suite", "(ver", "4.6,", "Chenomx", "Inc.,", "Edmonton,", "Alberta,", "Canada).", "The", "spectral", "region", "of", "δ0.0–10.0,", "excluding", "the", "water", "peak", "(δ4.5–5.0),", "was", "segmented", "into", "regions", "of", "0.04", "ppm,", "which", "provided", "250", "integrated", "regions", "in", "each", "NMR", "spectrum.", "This", "binning", "process", "endowed", "each", "segment", "with", "integral", "values,", "giving", "an", "intensity", "distribution", "of", "the", "whole", "spectrum", "with", "250", "variables", "prior", "to", "pattern", "recognition", "analysis.", "All", "data", "were", "converted", "from", "the", "NMR", "suite", "Professional", "software", "format", "into", "Microsoft", "Excel", "format", "(*.xls).", "One-dimensional", "NMR", "spectra", "data", "were", "imported", "into", "the", "SIMCA-P", "(version", "12.0,", "Umetrics", "Inc.,", "Kinnelon,", "NJ,", "USA)", "for", "multivariate", "statistical", "analysis", "to", "examine", "the", "intrinsic", "variation", "in", "the", "data", "set.", "These", "data", "were", "scaled", "using", "centered", "scaling", "prior", "to", "principal", "component", "analysis", "(PCA)", "and", "partial", "least", "square-discriminant", "analysis", "(PLS-DA).", "With", "the", "scaling", "process,", "we", "calculated", "the", "average", "value", "of", "each", "variable,", "and", "then", "subtracted", "it", "from", "the", "data.", "Score", "plots", "of", "PCA", "and", "PLS-DA", "were", "used", "to", "interpret", "the", "intrinsic", "variation", "of", "the", "data.", "Variable", "importance", "plots", "(VIP)", "were", "also", "utilized", "to", "select", "putative", "metabolites", "related", "to", "loperamide.", "Serum", "biochemical", "analysis", "After", "fasting", "for", "8", "h,", "whole", "blood", "from", "each", "rats", "in", "all", "groups", "was", "collected", "from", "their", "abdominal", "veins", "and", "incubated", "for", "30", "min", "at", "room", "temperature", "in", "a", "serum", "separating", "tube", "(BD", "Container,", "Franklin", "Lakes,", "NJ,", "USA).", "Serum", "was", "then", "obtained", "by", "centrifugation", "at", "1,500×g", "and", "analyzed", "for", "D-glucose", "and", "creatinine", "using", "an", "automatic", "biochemical", "analyzer", "(BS-120,", "Mindray,", "China).", "All", "assays", "were", "conducted", "in", "duplicate", "using", "fresh", "serum.", "Statistical", "analysis", "Statistical", "significance", "was", "evaluated", "using", "the", "One-way", "Analysis", "of", "Variance", "(ANOVA)", "(SPSS", "for", "Windows,", "Release", "10.10,", "Standard", "Version,", "Chicago,", "IL,", "USA),", "followed", "by", "Tukey’s", "post", "hoc", "t-test", "for", "multiple", "comparison.", "All", "values", "are", "expressed", "as", "the", "means", "±", "SD,", "and", "a", "p", "value", "<", "0.05", "is", "considered", "statistically", "significant." ]
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CC-BY
4.0
http://creativecommons.org/licenses/by/4.0/
Original work titled: 'Metabolomics approach to serum biomarker for laxative effects of red Liriope platyphylla in loperamide-induced constipation of SD rats' (Laboratory Animal Research/BioMed Central) by the following authors: Ji Eun Kim; Young Ju Lee; Sung Ha Ryu; Ji Won Park; Mi Ju Kang; Hyeon Jun Choi; Su Ji Bae; Yusang Choi; Hyun Gu Kang; Kyu-Bong Kim; Suhkmann Kim; Yong Lim; Dae Youn Hwang DOI: 10.1186/s42826-019-0009-x