High-resolution mucociliary transport measurement in live excised large animal trachea using synchrotron X-ray imaging

Background: The Australian Synchrotron Imaging and Medical Beamline (IMBL) was designed as the world's widest synchrotron X-ray beam, enabling both clinical imaging and therapeutic applications for humans as well as the imaging of large animal models. Our group is developing methods for imaging the airways of newly developed CF animal models that display human-like lung disease, such as the CF pig, and we expect that the IMBL can be utilised to image airways in animals of this size. Methods: This study utilised samples of excised tracheal tissue to assess the feasibility, logistics and protocols required for airway imaging in large animal models such as pigs and sheep at the IMBL. We designed an image processing algorithm to automatically track and quantify the tracheal mucociliary transport (MCT) behaviour of 103 μm diameter high refractive index (HRI) glass bead marker particles deposited onto the surface of freshly-excised normal sheep and pig tracheae, and assessed the effects of airway rehydrating aerosols. Results: We successfully accessed and used scavenged tracheal tissue, identified the minimum bead size that is visible using our chosen imaging setup, verified that MCT could be visualised, and that our automated tracking algorithm could quantify particle motion. The imaging sequences show particles propelled by cilia, against gravity, up the airway surface, within a well-defined range of clearance speeds and with examples of 'clumping' behaviour that is consistent with the in vivo capture and mucus-driven transport of particles. Conclusion: This study demonstrated that the wide beam at the IMBL is suitable for imaging MCT in ex vivo tissue samples. We are now transitioning to in vivo imaging of MCT in live pigs, utilising higher X-ray energies and shorter exposures to minimise motion blur.Martin Donnelley, Kaye S. Morgan, Maged Awadalla, Nigel R. Farrow, Chris Hall and David W. Parson

The Vehicle, 1967, Vol. 9 no. 1

Vol. 9, No. 1 Table of Contents Commentarypage 3 PoofMolly J. Evanspage 4 PreludeMike Baldwinpage 5 UntitledMike Baldwinpage 5 Where is Tomorrow?Paula Bresnanpage 6 Could It Be Or NotMary Hoeggerpage 7 PsalmAnthony Griggspage 7 Where Am I Going?William A. Framepage 8 Out of DarknessMarilyn Henry Hoodpage 9 She CriedMolly J. Evanspage 12 When I MoveAnthony Griggspage 13 Hi Ya, MorningWilliam A. Framepage 13 Summer Twilight ThoughtsSteve Allenpage 14 Too MuchBill Moserpage 16 Ink SketchWilliam A. Framepage 17 No. 1Molly J. Evanspage 18 Youth, So Hated and DamnedJeff Hendrickspage 18 GoneJackie Jaquespage 19 The JesterWilliam A. Framepage 20 ReflectionMike Baldwinpage 20 No. 3Molly J. Evanspage 21 EpitaphBill Moserpage 22 I Take A Long-Out-of-Use BookAnthony Griggspage 23https://thekeep.eiu.edu/vehicle/1016/thumbnail.jp

The Vehicle, 1967, Vol. 9 no. 1

Vol. 9, No. 1 Table of Contents Commentarypage 3 PoofMolly J. Evanspage 4 PreludeMike Baldwinpage 5 UntitledMike Baldwinpage 5 Where is Tomorrow?Paula Bresnanpage 6 Could It Be Or NotMary Hoeggerpage 7 PsalmAnthony Griggspage 7 Where Am I Going?William A. Framepage 8 Out of DarknessMarilyn Henry Hoodpage 9 She CriedMolly J. Evanspage 12 When I MoveAnthony Griggspage 13 Hi Ya, MorningWilliam A. Framepage 13 Summer Twilight ThoughtsSteve Allenpage 14 Too MuchBill Moserpage 16 Ink SketchWilliam A. Framepage 17 No. 1Molly J. Evanspage 18 Youth, So Hated and DamnedJeff Hendrickspage 18 GoneJackie Jaquespage 19 The JesterWilliam A. Framepage 20 ReflectionMike Baldwinpage 20 No. 3Molly J. Evanspage 21 EpitaphBill Moserpage 22 I Take A Long-Out-of-Use BookAnthony Griggspage 23https://thekeep.eiu.edu/vehicle/1016/thumbnail.jp

The Laccase Engineering Database: a classification and analysis system for laccases and related multicopper oxidases

Laccases and their homologues form the protein superfamily of multicopper oxidases (MCO). They catalyze the oxidation of many, particularly phenolic substances, and, besides playing an important role in many cellular activities, are of interest in biotechnological applications. The Laccase Engineering Database (LccED, http://www.lcced.uni-stuttgart.de) was designed to serve as a tool for a systematic sequence-based classification and analysis of the diverse multicopper oxidase protein family. More than 2200 proteins were classified into 11 superfamilies and 56 homologous families. For each family, the LccED provides multiple sequence alignments, phylogenetic trees and family-specific HMM profiles. The integration of structures for 14 different proteins allows a comprehensive comparison of sequences and structures to derive biochemical properties. Among the families, the distribution of the proteins regarding different kingdoms was investigated. The database was applied to perform a comprehensive analysis by MCO- and laccase-specific patterns

Induction, expression and characterisation of laccase genes from the marine-derived fungal strains Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330

The capability of the fungi Nigrospora sp. CBMAI 1328 and Arthopyrenia sp. CBMAI 1330 isolated from marine sponge to synthesise laccases (Lcc) in the presence of the inducer copper (110 M) was assessed. In a liquid culture medium supplemented with 5 M of copper sulphate after 5 days of incubation, Nigrospora sp. presented the highest Lcc activity (25.2 UL1). The effect of copper on Lcc gene expression was evaluated by reverse transcriptase polymerase chain reaction. Nigrospora sp. showed the highest gene expression of Lcc under the same conditions of Lcc synthesis. The highest Lcc expression by the Arthopyrenia sp. was detected at 96 h of incubation in absence of copper. Molecular approaches allowed the detection of Lcc isozymes and suggest the presence of at least two undescribed putative genes. Additionally, Lcc sequences from the both fungal strains clustered with other Lcc sequences from other fungi that inhabit marine environments.M. Passarini was supported by Ph.D. grant from FAPESP (2008/06720-7), Sao Paulo, Brazil. The authors thank FAPESP for financial support (BIOTA-FAPESP grant 2010/50190-2 and FAPESP grant 2013/19486-0) and Roberto G.S. Berlinck and CEBIMAR for the support related to samples collecting. L.D. Sette thanks CNPq for Productivity Fellowships 304103/2013-6

Bioinformatic Analysis Reveals High Diversity of Bacterial Genes for Laccase-Like Enzymes

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three- domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications

Fungi Unearthed: Transcripts Encoding Lignocellulolytic and Chitinolytic Enzymes in Forest Soil

BACKGROUND: Fungi are the main organisms responsible for the degradation of biopolymers such as lignin, cellulose, hemicellulose, and chitin in forest ecosystems. Soil surveys largely target fungal diversity, paying less attention to fungal activity. METHODOLOGY/PRINCIPAL FINDINGS: Here we have focused on the organic horizon of a hardwood forest dominated by sugar maple that spreads widely across Eastern North America. The sampling site included three plots receiving normal atmospheric nitrogen deposition and three that received an extra 3 g nitrogen m(2) y(1) in form of sodium nitrate pellets since 1994, which led to increased accumulation of organic matter in the soil. Our aim was to assess, in samples taken from all six plots, transcript-level expression of fungal genes encoding lignocellulolytic and chitinolytic enzymes. For this we collected RNA from the forest soil, reverse-transcribed it, and amplified cDNAs of interest, using both published primer pairs as well as 23 newly developed ones. We thus detected transcript-level expression of 234 genes putatively encoding 26 different groups of fungal enzymes, notably major ligninolytic and diverse aromatic-oxidizing enzymes, various cellulose- and hemicellulose-degrading glycoside hydrolases and carbohydrate esterases, enzymes involved in chitin breakdown, N-acetylglucosamine metabolism, and cell wall degradation. Among the genes identified, 125 are homologous to known ascomycete genes and 105 to basidiomycete genes. Transcripts corresponding to all 26 enzyme groups were detected in both control and nitrogen-supplemented plots. CONCLUSIONS/SIGNIFICANCE: Many of these enzyme groups are known to be important in soil turnover processes, but the contribution of some is probably underestimated. Our data highlight the importance of ascomycetes, as well as basidiomycetes, in important biogeochemical cycles. In the nitrogen-supplemented plots, we have detected no transcript-level gap likely to explain the observed increased carbon storage, which is more likely due to community changes and perhaps transcriptional and/or post-transcriptional down-regulation of relevant genes

Measuring phenol oxidase and peroxidase activities with pyrogallol, l-DOPA, and ABTS: Effect of assay conditions and soil type

Microbial phenol oxidases and peroxidases mediate biogeochemical processes in soils, including microbial acquisition of carbon and nitrogen, lignin degradation, carbon mineralization and sequestration, and dissolved organic carbon export. Measuring oxidative enzyme activities in soils is more problematic than assaying hydrolytic enzyme activities because of the non-specific, free radical nature of the reactions and complex interactions between enzymes, assay substrates, and the soil matrix. We compared three substrates commonly used to assay phenol oxidase and peroxidase in soil: pyrogallol (PYGL, 1,2,3-trihydroxybenzene), l-DOPA (l-3,4-dihydroxyphenylalanine), and ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid). We measured substrate oxidation in three soils across a pH gradient from 3.0 to 10.0 to determine the pH optimum for each substrate. In addition, we compared activities across 17 soils using the three substrates. In general, activities on the substrates followed the trend PYGL>. l-DOPA>ABTS and were inversely related to substrate redox potential. PYGL and ABTS were not suitable substrates at pH>5, and ABTS oxidation often declined with addition of peroxide to the assay. Absolute and relative oxidation rates varied widely among substrates in relation to soil type and assay pH. We also tested whether autoclaved or combusted soils could be used as negative controls for the influence of abiotic factors (e.g., soil mineralogy) on oxidative activity. However, neither autoclaving nor combustion produced reliable negative controls because substrate oxidation still occurred; in some cases, these treatments enhanced substrate oxidation rates. For broad scale studies, we recommend that investigators use all three substrates to assess soil oxidation potentials. For focused studies, we recommend evaluating substrates before choosing a single option, and we recommend assays at both the soil pH and a reference pH (e.g., pH 5.0) to determine the effect of assay pH on oxidase activity. These recommendations should contribute to greater comparability of oxidase potential activities across studies. © 2013 Elsevier Ltd

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus)

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 108 synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi