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The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Portable emission measurement systems (PEMS) [1] are used by the US Environmental Protection Agency (EPA) to measure gaseous and particulate matter mass emissions from vehicles in normal, in-use, on-the-road, and “real-world” operations to support many of its programs. These programs include vehicle modeling, emissions compliance, regulatory development, emissions inventory development, and investigations of the effects of real, in-use driving conditions on NOx, CO2, and other regulated pollutants. This article discusses EPA’s analytical methodology for evaluating light-duty vehicle energy and EU Real Driving Emissions (RDE). A simple, data-driven model was developed and validated using measured PEMS emissions test data. The work also included application of the EU RDE procedures and comparison to the PEMS test methodologies and FTP and other chassis dynamometer test data used by EPA for characterizing in-use light- and heavy-duty vehicle emissions. This work was conducted as part of EPA’s participation in the development of UNECE Global Technical Regulations and also supports EPA mobile source emission inventory development. This article discusses the real-world emissions of light-duty vehicles with 12V Start-Stop technology and light-duty vehicles using both gasoline and diesel fuels.

The quest for the discovery and validation of radiosensitivity biomarkers is ongoing and while conventional bioassays are well established as biomarkers, molecular advances have unveiled new emerging biomarkers. Herein, we present the validation of a new 4-gene signature panel of CDKN1, FDXR, SESN1 and PCNA previously reported to be radiation-responsive genes, using the conventional G2 chromosomal radiosensitivity assay. Radiation-induced G2 chromosomal radiosensitivity at 0.05 Gy and 0.5 Gy IR is presented for a healthy control (n = 45) and a prostate cancer (n = 14) donor cohort. For the prostate cancer cohort, data from two sampling time points (baseline and Androgen Deprivation Therapy (ADT)) is provided, and a significant difference (p > 0.001) between 0.05 Gy and 0.5 Gy was evident for all donor cohorts. Selected donor samples from each cohort also exposed to 0.05 Gy and 0.5 Gy IR were analysed for relative gene expression of the 4-gene signature. In the healthy donor cohort, there was a significant difference in gene expression between IR dose for CDKN1, FXDR and SESN1 but not PCNA and no significant difference found between all prostate cancer donors, unless they were classified as radiation-induced G2 chromosomal radiosensitive. Interestingly, ADT had an effect on radiation response for some donors highlighting intra-individual heterogeneity of prostate cancer donors.

14 páginas.- 6 figuras. 1 tabla.- 50 referencias.- Supplementary Information Te online version contains supplementary material available at https://doi.org/10.1038/s41598-021-91756-5, Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi. © 2021, The Author(s)., The work partly conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Research was also supported by NSF Grants 1457695 and 1457721 to J.M.B. and D.C., respectively, by CSIC project 201740E071 to A.G. and by JSPS Grant-in-Aid for Scientific Research 19K15881, JST-ACTX PJ2519A059 and 2017 Feasibility Study Program of the Frontier Chemistry Center, Faculty of Engineering, Hokkaido University to C.H.

Fungi that decay wood have characteristic associations with certain tree species, but the mechanistic bases for these associations are poorly understood. We studied substrate-specific gene expression and RNA editing in six species of wood-decaying fungi from the 'Antrodia clade' (Polyporales, Agaricomycetes) on three different wood substrates (pine, spruce, and aspen) in submerged cultures. We identified dozens to hundreds of substrate-biased genes (i.e., genes that are significantly upregulated in one substrate relative to the other two substrates) in each species, and these biased genes are correlated with their host ranges. Evolution of substrate-biased genes is associated with gene family expansion, gain and loss of genes, and variation in cis- and trans- regulatory elements, rather than changes in protein coding sequences. We also demonstrated widespread RNA editing events in the Antrodia clade, which differ from those observed in the Ascomycota in their distribution, substitution types, and the genomic environment. Moreover, we found that substrates could affect editing positions and frequency, including editing events occurring in mRNA transcribed from wood-decay-related genes. This work shows the extent to which gene expression and RNA editing differ among species and substrates, and provides clues into mechanisms by which wood-decaying fungi may adapt to different hosts.

19 p.-6 fig., As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates -namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases- we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf-litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes, but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways, and mapped the appearance of the different enzyme types in a time-calibrated species tree., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (BIO2017-86559-R to F.J.R.-D., S.C. and A.T.M., BIO2015-7369-JIN to J.B, and AGL2014-55971-R to A.G.P and L.R., projects cofinanced by FEDER funds); National Science Foundation (grant 1457721 to D.C.); Bundesministerium für Bildung und Forschung (CEFOX 031B0831B to H.K.); Deutsche Forschungsgemeinschaft (Biodiversity-Exploratories BLD-MFD-HZG III, KE 1742/2-1 to H.K.); Consejo Superior de Investigaciones Científicas (PIE-201620E081 to A.T.M.); and the Laboratory of Excellence ARBRE (ANR-11-LABX-0002-01), the Region Lorraine, the European Regional Development Fund, and the Plant–Microbe Interfaces Scientific Focus Area in the Genomic Science Program, U.S. DOE Office of Science to F.M. The work conducted by the JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE under contract DE-AC02-05CH11231.

The success of radiotherapy in tumour control depends on the total dose given. However, the tolerance of the normal tissues surrounding the tumour limits this dose. It is not known why some patients develop radiation toxicity and, currently, it is not possible to predict before treatment which patients will experience adverse effects. Thus, there is an unmet clinical need for a new test to identify patients at risk of radiation toxicity. Here, we report a new approach based on Raman spectroscopy.Blood samples were collected from 42 patients who had undergone radiotherapy for prostate cancer and had shown either severe or no/minimal late radiation toxicity in follow up. Radiation response was assessed following in vitro irradiation using Raman spectroscopy in addition to the G2 chromosomal radiosensitivity assay and the H2AX DNA damage assay.A Partial Least Squares Discriminant Analysis model was developed to classify patients using known radiation toxicity scores. A sensitivity of 95%, specificity of 92% and overall accuracy of 93% was achieved. In the future, this technology may have potential to lead to individualised patient radiotherapy by identifying which patients are at risk of radiation toxicity.

The potato aphid, Macrosiphum euphorbiae Thomas (Hemiptera: Aphididae: Macrosiphini), is a common polyphagous aphid in Europe and North America. However, the factors influencing potato aphid dynamics and susceptibility to natural enemies are largely undescribed, particularly in relation to facultative endosymbiotic bacteria, which can provide protection against parasitism and disease in some aphid species. This study investigated whether potato aphid susceptibility to one of its principal natural enemies, the parasitoid Aphidius ervi Haliday (Hymenoptera: Braconidae: Aphidiinae), varied in relation to aphid genotype and/or endosymbiont presence. Parasitism and aphid fitness assays were conducted on clonal lineages of aphids, harbouring their natural endosymbiont infections, collected over 3 years from separate geographic locations. Parasitized aphids were dissected to quantify parasitoid oviposition, larval development, and mummification. Amongst the 19 clonal lines of M. euphorbiae tested, seven aphid genotypes were identified, and 11 lines harboured one or both of the facultative endosymbionts Hamiltonella defensa Moran et al. and Regiella insecticola Moran et al.; H. defensa infections were associated exclusively with two of the seven M. euphorbiae genotypes. Parasitism resistance was detected in clonal lines belonging to a single aphid genotype and resulted from failure of parasitoid eggs to develop into larvae rather than failure of the parasitoid to oviposit. Contrary with studies of several other aphid species, there was little evidence that H. defensa provided strong protection to M. euphorbiae from parasitism by A. ervi. Furthermore, there were no clear fitness costs to the aphid associated with parasitism resistance or with H. defensa infection. The two M. euphorbiae genotypes in which H. defensa occurred, which included the resistant genotype, exhibited faster development, higher survival, and greater fecundity than the other five aphid genotypes. These findings suggest that biological control of M. euphorbiae using A. ervi alone could exacerbate pest problems by selecting for the fittest parasitism‐resistant genotypes.

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)-FTIR and HT-Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.

Radiation therapy (RT) is used to treat approximately 50% of all cancer patients. However, RT causes a wide range of adverse late effects that can affect a patient&rsquo, s quality of life. There are currently no predictive assays in clinical use to identify patients at risk of normal tissue radiation toxicity. This study aimed to investigate the potential of Fourier transform infrared (FTIR) spectroscopy for monitoring radiotherapeutic response. Blood plasma was acquired from 53 prostate cancer patients at five different time points: prior to treatment, after hormone treatment, at the end of radiotherapy, two months post radiotherapy and eight months post radiotherapy. FTIR spectra were recorded from plasma samples at all time points and the data was analysed using MATLAB software. Discrimination was observed between spectra recorded at baseline versus follow up time points, as well as between spectra from patients showing minimal and severe acute and late toxicity using principal component analysis. A partial least squares discriminant analysis model achieved sensitivity and specificity rates ranging from 80% to 99%. This technology may have potential to monitor radiotherapeutic response in prostate cancer patients using non-invasive blood plasma samples and could lead to individualised patient radiotherapy.

The conifer needle endophyte Phialocephala scopiformis DAOMC 229536 was cultivated in medium containing ground Pinus contorta wood as the sole carbon source. Mass spectrometry analyses identified 590 proteins., The conifer needle endophyte Phialocephala scopiformis DAOMC 229536 was cultivated in medium containing ground Pinus contorta wood as the sole carbon source. Mass spectrometry analyses identified 590 proteins. The expression of extracellular hydrolases and oxidoreductases indicates a capacity to degrade wood. The results clearly demonstrate the latent saprophytic potential of P. scopiformis.

e18596 Background: Several oncology guidelines recommend using oral drugs vs. IV to minimize COVID-19 risk for patients with cancer. We examined the association between prescribing patterns of oral capecitabine vs. IV 5FU for GI cancers and social distancing, measured by the change in population mobility patterns in response to shelter-in place policies, during the pandemic. Methods: Using claims data for commercially insured members, we included patients 18 years of age or older with colorectal, gastroesophageal, or pancreatic cancer, who had continuous health plan coverage for at least 2 months before and 1 month after initiating chemotherapy with capecitabine or 5-FU from January 2017 to August 2020. We analyzed unadjusted trends in proportion of chemotherapy that was oral during pandemic (March 1st to August 31st, 2020) compared to previous years. Then, we conducted difference-in-differences analysis using COVID-19 Community Mobility Reports, by Google, and utilizing different levels of changes in mobility trends across states over time. In our main model, we used a 20% decrease in retail and recreation visits as our threshold and compared the prescribing rates in states below and above the threshold as well as before and after the pandemic began. We also used different thresholds and categories of places to check the sensitivity of our findings. Models are adjusted for age, gender, month of year, urban status, comorbidities, and state of residence at chemotherapy start date. Results: A total of 17,414 nationally distributed patients (69% colorectal, 13% gastroesophageal, 18% pancreatic) were included (mean age, 58.8 years; 41% female). During the pandemic, 1,875 patients (65% colorectal, 15% gastroesophageal, 20% pancreatic) were identified. The proportion of oral regimens did not change significantly for colorectal and gastroesophageal patients and decreased by 7.4 percentage points (pp) (p < 0.01) for pancreatic patients. In regression modelling with mobility data, oral prescribing rates for colorectal patients increased by 3.1 pp (p < 0.01), largely driven by increases for female patients (9.2 pp, p = 0.02). We observed a decrease in oral prescribing rates among pancreatic patients (-1.20 pp, p = 0.04) and did not observe a significant change for gastroesophageal patients. Our results are not sensitive to different social distancing specifications. Conclusions: We observed differential impact of the pandemic on oral prescribing rates by GI cancer type and gender. Oral prescribing increased among colorectal cancer patients driven mostly by higher oral prescribing in females. For pancreatic and gastroesophageal patients, oral prescriptions either remained unchanged or decreased. This observation may reflect a variable impact of the pandemic on women as compared to men and might involve heightened caregiving responsibilities for women.

Activation and proliferation of immune cells such as lymphocytes and monocytes are appropriate inflammatory responses to invading pathogens and are key to overcoming an infection. In contrast, uncontrolled and prolonged activation of these cellular signalling pathways can be deleterious to the body and result in the development of autoimmune conditions. The understanding of cellular activatory status therefore plays a significant role in disease diagnosis and progression. Conventional automated approaches such as enzyme linked immunosorbent assays (ELISA) and immune-labelling techniques are time-consuming and expensive, relying on a commercially available and specific antibody to identify cell activation. Developing a label-free method for assessing molecular changes would therefore offer a quick and cost-efficient alternative in biomedical research. Here Raman spectroscopy is presented as an effective spectroscopic method for the identification of activated immune cells using both cell lines and primary cells (including purified monocyte and lymphocyte subgroups and mixed peripheral blood mononuclear cell (PBMC) populations) obtained from healthy donors. All cell lines and primary cells were exposed to different stimulants and cellular responses confirmed by flow cytometry or ELISA. Machine learning models of cell discrimination using Raman spectra were developed and compared to reference flow-cytometry, with spectral discrimination levels comparing favourably with the reference method. Spectral signatures of molecular expression after activation were also extracted with results demonstrating alignment with expected profiles. High performance classification models constructed in these in-vitro and ex-vivo studies enabled identification of the spectroscopic discrimination of immune cell subtypes in their resting and activated state. Further spectral fitting analysis identified a number of potential spectral biomarkers that elucidate the spectral classification.

Roger Scruton's philosophical enterprise is an effort to “save the appearances” of value in the human world that elude scientific explanation. The meaning of the human things appears only to a first-person perspective, which is incommensurable with the objective perspective of cause and effect. To make sense of our experience requires a “cognitive dualism” that can account for the human or “lifeworld” as well as physical reality. Ranging rather indiscriminately over Scruton's diverse writings, I expound the unifying conception of personhood that makes them a coherent whole and also serves as the touchstone for Scruton's conservative critique of liberal theory and practice. Some questions are raised about that two-dimensional critique, about its separation into “metaphysical” and “empirical” components, and about whether the latter does or doesn't “operationalize” the former.

The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium . The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H 2 O 2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus , GLOX1 ( Pci GLOX1) and GLOX2 ( Pci GLOX2), were heterologously produced in Aspergillus niger strain D15#26 ( pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for Pci GLOX1 and Pci GLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but Pci GLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H 2 O 2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.

The conifer needle endophyte,Phialocephala scopiformis, was cultivated in media containing groundPinus contortawood as sole carbon source. After five and seven days growth, concentrated extracellular fluids were subjected to LC-MS/MS analyses. A total of 590 proteins were identified of which 99 were assigned to glycoside hydrolase families within the Carbohydrate Active Enzyme (CAzyme) system. Multiple isozymes of exo-and endo-acting cellulases were among the most abundant proteins, and oxidative degradation of cellulose was supported by the presence of lytic polysaccharide monooxygenases, glucooligosaccharide oxidase and cellobiose dehydrogenase. Oxidoreductases were also plentiful and included GMC oxidoreductases, alcohol dehydrogenases, laccases, copper radical oxidases, tyrosinases and catalase. The expression and diversity of extracellular oxidoreductases indicates a capacity to metabolize alcohols and aromatic compounds.

Liquid biopsies are a potentially rich store of biochemical information that can be linked to an individual's response to therapeutic treatments, including radiotherapy, and which may ultimately play a role in the individualization of treatment regimens. Peripheral blood mononuclear cells (PBMCs) can be used not only for the biochemical profiling of the individual, but also, being living cells, can provide insights into the individuals response to ionizing radiation exposure.The present study attempts to link the biochemical profile of lymphocytes within PBMCs obtained through Raman spectroscopy to in vitro measures of low-dose (0.5Gy) DNA damage response and cytogenetic metrics of radiosensitivity in a cohort of healthy controls and prostate cancer patients (from CTRIAL-IE(ICORG) 08-17, NCT00951535). All parallel metrics to the Raman spectra of the cells were obtained ex vivo in cycling peripheral blood lymphocytes, with radiosensitivity estimated using the G2 chromosomal assay and DNA damage assessed using γH2AX fluorescence. Spectra from a total of 26 healthy volunteers and 22 prostate cancer patients were obtained.The links between both measures of cellular response to ionizing radiation and the Raman spectra were modeled using partial least squares regression (PLSR) and support-vector regression (SVR). It was found that neither regression approach could predict radiation-induced G2 score well, but could predict γH2AX MFI with the SVR outperforming PLSR, implying a non-linear relationship between spectral measurements and measures of DNA damage.Raman spectroscopy of PBMCs represents a label-free approach for prediction of DNA damage levels for either prospective or retrospective analysis.

The ability to extract and purify messenger RNA directly from plants, decomposing organic matter and soil, followed by high-throughput sequencing of the pool of expressed genes, has spawned the emerging research area of metatranscriptomics. Each metatranscriptome provides a snapshot of the composition and relative abundance of actively transcribed genes, and thus provides an assessment of the interactions between soil microorganisms and plants, and collective microbial metabolic processes in many environments. We highlight current approaches for analysis of fungal transcriptome and metatranscriptome datasets across a gradient of community complexity, and note benefits and pitfalls associated with those approaches. We discuss knowledge gaps that limit our current ability to interpret metatranscriptome datasets and suggest future research directions that will require concerted efforts within the scientific community.

Glyoxal Oxidases from Pycnoporus Cinnabarinus for Green Chemistry Applications . International Symposium on Green Chemistry (2017)

Furylglycolic acid (FA), a pseudoaromatic hydroxy-acid suitable for copolymerization with lactic acid, can be produced from glucose via enzymatically derived cortalcerone using a combination of Bronsted and Lewis acid catalysts. Cortalcerone is first converted to furylglyoxal hydrate (FH) over a Bronsted acid site (HCl or Al-containing beta-zeolite), and FH is subsequently converted to FA over a Lewis acid site (Sn-beta zeolite). Selectivity for conversion of FH to FA is as high as 80% at 12% conversion using tetrahydrofuran (THF) as a solvent at 358 K. Higher conversion of FH leads to FA-catalyzed degradation of FH and subsequent deactivation of the catalyst by the deposition of carbonaceous residues. The deactivated catalyst can be regenerated by calcination. Cortalcerone can be produced from 10% glucose solution using recombinant Escherichia coli strains expressing pyranose 2-oxidase and aldos-2-ulose dehydratase from the wood-decay fungus Phanerochaete chrysosporium BKM-F-1767. This enzymatically deri...

Certain wood decay basidiomycetes, collectively referred to as brown rot fungi, rapidly depolymerize cellulose while leaving behind the bulk of cell wall lignin as a modified residue. The mechanism(s) employed is unclear, but considerable evidence implicates the involvement of diffusible oxidants generated via Fenton-like chemistry. Toward a better understanding of this process, we have examined the transcriptome and secretome of Wolfiporia cocos when cultivated on media containing glucose, purified crystalline cellulose, aspen ( Populus grandidentata ), or lodgepole pine ( Pinus contorta ) as the sole carbon source. Compared to the results obtained with glucose, 30, 183, and 207 genes exhibited 4-fold increases in transcript levels in cellulose, aspen, and lodgepole pine, respectively. Mass spectrometry identified peptides corresponding to 64 glycoside hydrolase (GH) proteins, and of these, 17 corresponded to transcripts upregulated on one or both woody substrates. Most of these genes were broadly categorized as hemicellulases or chitinases. Consistent with an important role for hydroxyl radical in cellulose depolymerization, high transcript levels and upregulation were observed for genes involved in iron homeostasis, iron reduction, and extracellular peroxide generation. These patterns of regulation differ markedly from those of the closely related brown rot fungus Postia placenta and expand the number of enzymes potentially involved in the oxidative depolymerization of cellulose. IMPORTANCE The decomposition of wood is an essential component of nutrient cycling in forest ecosystems. Few microbes have the capacity to efficiently degrade woody substrates, and the mechanism(s) is poorly understood. Toward a better understanding of these processes, we show that when grown on wood as a sole carbon source the brown rot fungus W. cocos expresses a unique repertoire of genes involved in oxidative and hydrolytic conversions of cell walls.

The unique degradative activities of wood decay fungi, together with the rapid increase in genome data, offer unparalleled opportunities for biotechnological exploitation. Enzymatic conversion of pretreated woody material to high value products is of particular interest for biofuels production. A diverse array of extracellular peroxidases have been identified and these have considerable promise for the oxidation of the recalcitrant cell wall polymer, lignin. These enzymes, together with an array of intracellular oxidoreductases, are also able to transform various xenobiotics. These ligninolytic fungi also feature large numbers of genes encoding structurally diverse hydrolases and these too have considerable potential for the conversion of cellulose and hemicellulose to high value products. Certain wood decay fungi, collectively referred to as brown rot fungi, do not appreciably remove lignin and employ small molecular weight oxidants to rapidly depolymerize cellulose in the absence of hydrolases. Although rapid and efficient, it remains to be seen whether such non-enzymatic processes could be effectively harnessed for conversion of ‘cellulosics’. A complete understanding of lignocellulose degradation has been hampered by limited experimental tools and by the enormous number of genes encoding proteins of unknown function. Based on transcriptome and proteome studies, many of these ‘hypothetical’ proteins likely play an important, but poorly understood, role in lignocellulose degradation, Addressing this longstanding problem, new approaches for genetic transformation offer opportunities to establish the function of genes. Together with a growing number of metagenome investigations, current and future research will surely identify novel and potentially useful enzymes for bioprocess development.

Identification of specific genes and enzymes involved in conversion of lignocellulosics from an expanding number of potential feedstocks is of growing interest to bioenergy process development. The basidiomycetous wood decay fungi Phanerochaete chrysosporium and Postia placenta are promising in this regard because they are able to utilize a wide range of simple and complex carbon compounds. However, systematic comparative studies with different woody substrates have not been reported. To address this issue, we examined gene expression of these fungi colonizing aspen ( Populus grandidentata ) and pine ( Pinus strobus ). Transcript levels of genes encoding extracellular glycoside hydrolases, thought to be important for hydrolytic cleavage of hemicelluloses and cellulose, showed little difference for P. placenta colonizing pine versus aspen as the sole carbon source. However, 164 genes exhibited significant differences in transcript accumulation for these substrates. Among these, 15 cytochrome P450s were upregulated in pine relative to aspen. Of 72 P. placenta extracellular proteins identified unambiguously by mass spectrometry, 52 were detected while colonizing both substrates and 10 were identified in pine but not aspen cultures. Most of the 178 P. chrysosporium glycoside hydrolase genes showed similar transcript levels on both substrates, but 13 accumulated >2-fold higher levels on aspen than on pine. Of 118 confidently identified proteins, 31 were identified in both substrates and 57 were identified in pine but not aspen cultures. Thus, P. placenta and P. chrysosporium gene expression patterns are influenced substantially by wood species. Such adaptations to the carbon source may also reflect fundamental differences in the mechanisms by which these fungi attack plant cell walls.

Sequencing the Fungal Tree of Life F. Martin 1 , D. Cullen 2 , D. Hibbett 3 , A. Pisabarro 4 , J. W. Spatafora 5 , S. E. Baker 6,7, I. V. Grigoriev 7 UMR INRA/UHP 1136, Interactions Arbres/Micro-Organismes, INRA-Nancy, 54280 Champenoux, France University of Wisconsin-Madison, Madison, WI, USA Clark University, Worcester, MA, USA Department of Agrarian Production, Public University of Navarre, 31006 Pamplona, Spain Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331 Chemical and Biological Process Development Group, Pacific Northwest National Laboratory, Richland, Washington, USA, 99352 US DOE Joint Genome Institute, Walnut Creek, California, USA March 2011 The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02- 05CH11231 DISCLAIMER This document was prepared as an account of work sponsored by the United States Government. While this document is believed to contain correct information, neither the United States Government nor any agency thereof, nor The Regents of the University of California, nor any of their employees, makes any warranty, express or implied, or assumes any legal responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by its trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the United States Government or any agency thereof, or The Regents of the University of California. The views and opinions of authors expressed herein do not necessarily state or reflect those of the United States Government or any agency thereof or The Regents of the University of California.

Plasma electrochemistry consists of an electrolytic cell where the cathode or the anode is replaced by a direct current (DC) plasma. Unlike conventional electrolysis, highly reactive species such as the solvated electron (e- aq), and the hydroxyl (OH) radical dominate the surface reactions, instead of the properties of the solid electrode. Using this configuration, plasmas have been used to process chemicals, create long-lived chemical species, and synthesize nanoparticles in electrolytic cells. However, which chemical species are introduced to the electrolyte, in what amounts, and how their respective reactions compete, are lingering questions in the field. Here, a non-thermal, atmospheric argon DC plasma was used as a cathode coupled with a platinum anode and aqueous sodium perchlorate (NaClO4) or sodium chloride (NaCl) solutions as background electrolytes. Two chemical compounds, ferricyanide (Fe(CN)6 3-) or methylene blue (MB), were used as chemical indicators to study the competitive reactions driven by e- aq and OH, and various scavengers were used to help identify plausible reaction pathways. A simple kinetic model was used to analyze and interpret the data, revealing that the Faradaic efficiency in these systems is severely limited by the ubiquitous presence of OH.

Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe 3+ , thus generating perhydroxyl radicals and Fe 2+ , which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe 3+ tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer Postia placenta contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute P. placenta protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by P. placenta laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 10 4 M −1 s −1 , and measurements of the H 2 O 2 produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by P. placenta laccase in wood is large enough that it likely contributes to incipient decay.