MOESM6 of A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Additional file 6: Fig. S6. Screen of FjoAcXEA activity towards selected pNP alkyl esters showing activity on short chain (< C4) substrates consistent with esterase rather than lipase activity. Reactions (200 µL) contained 0.5 µg of FjoAcXE, 50 mM HEPES (pH 8.0), and 2 mM of each substrate. Absorbance at 410 nm was measured after 2 h at 30 °C. pNP acetate (C2), pNP butyrate (C4), pNP hexanoate (C6), pNP octanoate (C8), pNP decanoate (C10), pNP dodecanoate (C12), pNP myristate (C14), and pNP palmitate (C16). n = 3; error bars correspond to standard deviation

MOESM5 of A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Additional file 5: Fig. S5. FjoAcXE activity screen against selected pNP substrates. Reactions (200 µL) contained 5 µg of FjoAcXE, 50 mM HEPES (pH 8.0), and 2 mM of each substrate. Absorbance was measured after 2 h at 30 °C

MOESM3 of A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Additional file 3: Fig. S3. Purified FjoAcXE is approximately 45.2 kDA

MOESM4 of A novel acetyl xylan esterase enabling complete deacetylation of substituted xylans

Additional file 4: Fig. S4. FjoAcXE activity screen against 0.5% (w/v) of selected polysaccharides. Reactions (50 µL) contained 5 µg of FjoAcXE, 50 mM HEPES (pH 8.0), and 0.5% w/v of each substrate, and were incubated for 16 h at 30 °C. Reducing sugars were measured using 1% final PAHBAH reagent [58]. BEX = beechwood xylan (Sigma, X4252); OSX = oat spelt xylan (Sigma, X0627); CMC = carboxymethylcellulose (Megazyme, P-CMC4 M); β-glucan (low viscosity; from barley; Megazyme, P-BGBL); starch (from corn; Sigma-Aldrich, S4126); pectin (from apple; Sigma, 76282); WAX = wheat arabinoxylan (high viscosity; Megazyme, P-WAXYH); arabinan (from sugarbeet; Megazyme, P-ARAB); glucomannan (low viscosity; from konjac; Megazyme, P-GLCML); galactomannan (from guar, GD28; Megazyme, enzyme modified); xyloglucan (amyloid, from tamarind seed; Megazyme, P-XYGLN); arabinogalactan (acacia gum, Sigma, G9752)

Additional file 1 of Functional characterization of fungal lytic polysaccharide monooxygenases for cellulose surface oxidation

Additional file 1: Figure S1: Product released by 5 μM C1 LPMOs after 16 h on PASC (0.1%), Avicel (1%), and SA-Avicel (1%)with 1 mM ascorbic acid as the electron donor. For each substrate, T. reesei cellulase cocktail was used to convert all C1-oxidized products into cellobionic acid, which was then quantified by HPAEC-PAD and reported as the total C1-oxidized ends generated (nanomoles per mg of starting fiber). Each bar is the average of three independent assays measured singly by HPAEC-PAD, with error bars indicating the standard error of the mean. Figure S2: Brightfield (A) and confocal (B) images of untreated SA-Avicel labelled using rhodamine chloride. 1% SA-Avicel with 1 mM of gallic acid was incubated at 50 °C for 24 h. Insoluble products were separated, labelled with fluorescent dye, and visualized using confocal microscopy. Figure S3: Process schematic for LPMO treatment of cellulosic substrates and subsequent soluble and insoluble product analysis. PASC (0.1%), Avicel (1%), and SA-Avicel (1%) were treated with LPMOs using either ascorbic acid, gallic acid, or cysteine as the electron donor. Insoluble products were separated from soluble products using centrifugation. For the soluble products analysis, HPAEC-PAD was used to annotate native and oxidized cello-oligosaccharide peaks. To quantify the C1-oxidized products, T. reesei cellulase cocktail was used to convert all C1-oxidized products into cellobionic acid, which was then quantified by HPAEC-PAD and reported as the total C1-oxidized ends generated (nanomoles per mg of starting fiber). For insoluble product analysis, separated insoluble products were labelled with either C1-specific or C4-specific fluorescent dye and subsequently visualized using confocal microscopy

Retesting BRCA1/BRCA2 mutation negative male breast cancer patients using next generation sequencing technologies

We read with great interest the study by Moran and colleagues, entitled ‘‘Revisiting breast cancer patients who previously tested negative for BRCA mutations using a 12- gene panel’’ recently published in this journal [1]. Using next generation sequencing (NGS) technologies, the authors re-assessed women affected by breast cancer who had previously tested negative for mutations in the large BRCA1 exon 11 and BRCA2 exons 10–11 by the protein truncation test (PTT). Specifically, they evaluated the prevalence of mutations in 12 breast cancer susceptibility genes, including BRCA1 and BRCA2, in 190 female breast cancer cases with a strong family history of breast cancer. Six mutations were detected in BRCA1 and BRCA2; in particular, one of these mutations (c.893_899delCAGTTGTinsTACTTCAG, p.Thr298fs) was detected in BRCA2 exon 10, previously screened by PTT. Overall, six women who had previously received a negative test result for BRCA1 and BRCA2 mutations using the PTT were found to carry pathogenic mutations in these two highpenetrance genes

Genes encoding LC-MS/MS detected proteins and exhibiting>2-fold regulation in comparisons of NELP and ELP cultures.¹

<p>Genes encoding LC-MS/MS detected proteins and exhibiting>2-fold regulation in comparisons of NELP and ELP cultures.^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#nt108" target="_blank">1</a>}</p

Transcripts accumulating>4-fold in non-extracted loblolly pine wood (NELP) relative to extracted loblolly pine wood (ELP).¹

<p>Transcripts accumulating>4-fold in non-extracted loblolly pine wood (NELP) relative to extracted loblolly pine wood (ELP).^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#nt105" target="_blank">1</a>}</p

Wood decay characteristics.

<p>Comparative weight loss of parental strain 11061 and single basidiospore derivatives on colonized loblolly pine wood (<i>Pinus taeda</i>) wood wafers were determined after 4, 8 and 12 weeks incubation (bottom left panel) as described in Methods. Single basidiospore strain 5–6 also aggressively decayed birch and spruce (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s057" target="_blank">Text S1</a>) and was selected for sequencing. Upper panels show scanning electron microscopy <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759-Blanchette2" target="_blank">[68]</a> of radial (left) and transverse (right) sections of pine wood tracheids that were substantially eroded or completely degraded by <i>P. gigantea</i> strain 5–6 by week twelve. Transverse section of sound wood (bottom photo) provides comparison. (Bar  = 40 µm).</p

Number and expression of genes likely involved in lignocellulose degradation.

<p>The number of genes encoding mass spectrometry-identified proteins was limited to those matching≥2 unique peptides after 5–9 days growth in media containing NELP or ELP. RPKM values>100 for RNA derived from these cultures were arbitrarily selected as the threshold for high transcript levels. Genes designated as ‘regulated’ showed significant accumulation (p<0.05;>2-fold) in NELP or ELP relative to glucose containing media. Methods and complete data are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s057" target="_blank">Text S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004759#pgen.1004759.s059" target="_blank">Dataset S2</a>.</p