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IntroductionThe impact of water quality on the survival of human norovirus (NoV) was determined in irrigation water field run-off (tail water) and well water from a representative Central Coast vegetable production site in the Salinas Valley, California.MethodsTail water, well water, and ultrapure water samples were inoculated separately with two surrogate viruses for human NoV—Tulane virus (TV) and murine norovirus (MNV)—to achieve a titer of 1×105 plaque forming units (PFU)/ml. Samples were stored at 11, 19, and 24°C for 28 days. Additionally, inoculated water was applied to soil collected from a vegetable production site in the Salinas Valley or to the surface of growing romaine lettuce leaves, and virus infectivity was evaluated for 28 days in a growth chamber.ResultsVirus survival was similar for water stored at 11, 19, and 24°C and there was no difference in infectivity based on water quality. After 28 days, a maximum 1.5 log reduction was observed for both TV and MNV. TV decreased by 1.97-2.26 log and MNV decreased by 1.28- 1.48 logs after 28 days in soil; infectivity was not influenced by water type. Infectious TV and MNV were recovered from lettuce surfaces for up to 7 and 10 days after inoculation, respectively. Across the experiments there was no significant impact of water quality on the stability of the human NoV surrogates.DiscussionOverall, the human NoV surrogates were highly stable in water with a less than 1.5 log reduction over 28 days and no difference observed based on the water quality. In soil, the titer of TV declined by approximately 2 logs over 28 days, while MNV declined by 1 log during the same time interval, suggesting surrogate-specific inactivation dynamics in the soil tested in this study. A 5-log reduction in MNV (day 10 post inoculation) and TV (day 14 post inoculation) was observed on lettuce leaves, and the inactivation kinetics were not significantly impacted by the quality of water used. These results suggest that human NoV would be highly stable in water, and the quality of the water (e.g., nutrient content, salinity, and turbidity) does not significantly impact viral infectivity.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Additional file 15. List of primer sequences and amplicon sizes for TEF1α, TRI1, TRI8 and APS1.

Additional file 3 Summary of Illumina genome assembly statistics for 37 F. poae isolates profiled in this study.

Additional file 16. Detailed explanation of metabolomics data processing parameters, binary matrix conversion pipeline, qExactive instrument and data processing parameters and mass feature annotation pipeline details.

Additional file 9. Total ion current and extract ion current chromatographs illustrating butenolide-associated peak (red arrows) eluting during start of run, in region normally sent to waste (blue bracket). Accompanying text explains putative butenolide annotation process.

Additional file 6. Media conditions used. Formulations for CYA, MMK2, S1M, S2M, YES, YESIO are listed.

Additional file 8. Mirror plots of W-493 A (Top) and W-493 B (Bottom). Upper spectra in each mirror plot represent experimentally derived fragmentation patterns from F. poae extracts, bottom spectra are from GNPS libraries (spectral matches

Additional file 14. Duplex PCR screening for presence of APS1 in F. poae genomic DNA. Diagnostic bands for TEF1α and APS1 are indicated by arrows.

Additional file 11. Mirror plot of MS2 spectra from features annotated as apicidin (APS) and APS-G. Overlaid chemical structures are proposed ions associated with specific fragments. Top: spectra of m/z [M + H] + = 624.375 (APS structure top right). Spectra coloured purple are present in nearly all spectra associated with apicidins bearing O-methylated tryptophan moiety, including spectra annotated as APS, APS-B, APS-C, and APS-D2. Structures outlined in purple are those proposed to contain indole substructures. Notably, nominal masses 130 and 170 have been associated with tryptophan fragmentation elsewhere5,6. Bottom: spectra from parent m/z [M + H] + = 555.354 (annotated as APS-G, structure bottom right). Spectra coloured red are present uniquely from MS2 scans of this m/z, with corresponding proposed ion structures containing phenylalanine outlined in red. Spectra coloured green in bottom plot are m/z within 5 ppm match for spectra on top plot.

Additional file 5 LASTZ comparison of F. poae Fp157 with F. poae 2516 (assembly GCA_001675295.1) and F. graminearum PH1 (assembly GCA_000240135.3).

Additional file 2 Jukes-Cantor/Neighbor-joining consensus tree of Clustal Omega alignment of concatenated TEF1α – TRI1 – TRI8 genomic sequences of 19 representative Ontario and Quebec isolates and one Belgian F. poae isolate (genome assembly LYXU01; Vanheule et al. 2016). The isolate name is followed by the number of isolates represented in brackets of the 193 Ontario and Quebec isolates surveyed. The TEF1α, TRI1 and TRI8 genomic sequences of the 19 isolates have been deposited in Genbank as accession numbers MT571548-MT571566, MT578829-MT578829-MT578847, and MT571567-MT571585, respectively.

Additional file 10. Mirror plots of apicidin [M + Na] + (top), apicidin B [M + Na] + (middle), and apicidin C [M + Na] + (bottom). Upper spectra in each mirror plot represent experimentally derived fragmentation patterns from F. poae extracts, bottom spectra are from GNPS libraries (spectral matches

Additional file 13. Blastn comparison of FpAPS cluster to homologous clusters in F. sporotrichioides (PXOF00000000), F. langsethiae (JXCE00000000), and F. incarnatum (GQ331953).

Additional file 4 Genome assembly statistics for Fp157, assembly WOUF00000000.

A format for the structure-oriented analysis of immunoglobulin (Ig) variable region sequences is presented and applied to generate sequence profiles for comparison of heavy- and light-chain subgroups. The profile allows simultaneous evaluation of sequences and structural information and can be used for a number of different applications.

Enterococcus faecium NRRL B-2354 is a surrogate microorganism used in place of pathogens for validation of thermal processing technologies and systems. We evaluated the safety of strain NRRL B-2354 based on its genomic and functional characteristics. The genome of E. faecium NRRL B-2354 was sequenced and found to comprise a 2,635,572-bp chromosome and a 214,319-bp megaplasmid. A total of 2,639 coding sequences were identified, including 45 genes unique to this strain. Hierarchical clustering of the NRRL B-2354 genome with 126 other E. faecium genomes as well as pbp5 locus comparisons and multilocus sequence typing (MLST) showed that the genotype of this strain is most similar to commensal, or community-associated, strains of this species. E. faecium NRRL B-2354 lacks antibiotic resistance genes, and both NRRL B-2354 and its clonal relative ATCC 8459 are sensitive to clinically relevant antibiotics. This organism also lacks, or contains nonfunctional copies of, enterococcal virulence genes including acm, cyl, the ebp operon, esp, gelE, hyl, IS16, and associated phenotypes. It does contain scm, sagA, efaA, and pilA, although either these genes were not expressed or their roles in enterococcal virulence are not well understood. Compared with the clinical strains TX0082 and 1,231,502, E. faecium NRRL B-2354 was more resistant to acidic conditions (pH 2.4) and high temperatures (60°C) and was able to grow in 8% ethanol. These findings support the continued use of E. faecium NRRL B-2354 in thermal process validation of food products.

We have used a combinatorial mutagenesis strategy to humanize BR96, a monoclonal antibody that binds to the Lewis Y class of tumor antigens. This approach allows simultaneous assessment of hundreds of humanized variable regions to identify the molecules that best preserve affinity, thus overcoming the major drawback of current humanization procedures, the requirement to construct and analyze each humanized antibody separately. Murine residues of BR96 were mutated to human if they were solvent-exposed residues that did not participate in the formation of the antigen binding site and were not at the interface of the light and heavy chain. At positions that might be involved in binding to antigen, the choice between the murine and human residue was more difficult. Murine and human alternatives were incorporated into a combinatorial library at positions representing buried residues that might affect the structural integrity of the antigen binding site. By encoding this library of humanized BR96 Fabs in an M13 phage vector, we rapidly identified several candidates with nearly identical antigen binding, within 2-fold, of the chimeric Fab. Additional mutagenesis directed at sites suggested in the literature as potentially important for antigen binding in a similar anti-Lewis Y antibody yielded no further improvements.

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.

A human immunoglobulin G1 (IgG1) antibody oligomer was isolated from a transfected myeloma cell line that produced a monoclonal antibody to group B streptococci. Compared to the IgG1 monomer, the oligomer was significantly more effective at protecting neonatal rats from infection in vivo. The oligomer was also shown to cross the placenta and to be stable in neonatal rats. Immunochemical analysis and complementary DNA sequencing showed that the transfected cell line produced two distinct kappa light chains: a normal light chain (Ln) with a molecular mass of 25 kilodaltons and a 37-kilodalton species (L37), the domain composition of which was variable-variable-constant (V-V-C). Cotransfection of vectors encoding the heavy chain and L37 resulted in production of oligomeric IgG.

Comparison of seven high resolution x-ray structures shows that the conformations of canonical complementarity determining region (CDR) loops, which are shared by these antibodies, are very similar. However, large spatial displacements (up to 2.7 A) of the essentially identical CDR loops become evident when the antibody beta-sheet frameworks, to which the loops are attached, are least-squares superposed. The loop displacements follow, and amplify, small positional differences in framework/loop splice points. Intradomain structural variability and, to a lesser extent, domain-domain orientation appear to cause the observed loop divergences. The results suggest that the selection of framework regions for loop grafting procedures is more critical than previously thought.

We have characterized a human IgG1 monoclonal antibody composed of altered light chains. Each light chain consists of two identical variable domains and a kappa constant domain, in association with a normal gamma chain. This antibody assembled biosynthetically into a mixture of stable oligomers and monomers. Employing gel filtration, PAGE, and electron microscopy, we examined the antibody and the nature of the associations involved in oligomer formation. By engineering a protease factor Xa site between the duplicated light chain variable domains and examining the fragments produced following factor Xa cleavage, we demonstrated the association of the IgG monomers occurred through their duplicated VL domains. Electron microscopy showed the oligomeric antibody to be predominantly dimers and trimers in which the monomeric units were associated through the tips of the Fab portion of the antibody, presumably through the protuding N-terminal VL domains. Similar examination of monomers demonstrated several molecular forms, including individual molecules with self-crosslinked Fab arms and others displaying the open Y and T shapes typically observed for IgG antibodies. The monomers also displayed distally protruding domain-like structures. The oligomers produced by this cell line therefore occurred through the nonconvalent interaction between the extra light chain variable domains.

Monoclonal antibody 60.3 binds to the CD18 component of the beta 2 integrin family of adhesion molecules. 60.3 has potential clinical application in blocking the neutrophil-mediated organ damage which occurs following a myocardial infarct or hemorrhagic shock. Analysis of the nucleotide and deduced amino acid sequences of murine 60.3 shows that the light chain contains no amino acid substitutions relative to the closest germline sequence, while the heavy chain is heavily substituted. We report here the humanization of 60.3. The humanized antibody binds to CD18-bearing cells with approximately 4-fold less affinity than the murine or chimeric antibody. We have shown that modification of amino acid L50 in the L2 loop of the humanized antibody results in loss of binding, while modification of a structural determinant (H71) for the H2 loop has no effect.

The influence of valence and heavy chain on antibody activity was investigated using transfectoma-derived, class-switched IgG1 and IgM human monoclonal antibodies (MAbs) reactive with the bacterial pathogens Escherichia coli K1 and group B Streptococcus species. IgG-IgM pairs were compared in vitro for antigen binding and opsonic activities and in vivo for protective efficacy in neonatal rats. For the anti-E. coli pair, the IgM MAb was 1000-fold more potent in all assay formats. Importantly, the 50% protection dose (PD50) of the IgM MAb was 10-20 ng/rat, while 100 micrograms of the IgG MAb was only minimally protective. For the group B streptococcal MAbs, the IgM was 100- and 4500-fold more potent in binding and opsonization assays, respectively. However, while 20 micrograms of IgM protected neonatal rats, 100 micrograms of IgG MAb was partly protective. These experiments demonstrate the utility of recombinant DNA technology for creating a panel of antibodies that may aid in selecting potential immunotherapeutic candidates.