Cell-free production of the bifunctional glycoside hydrolase GH78 from Xylaria polymorpha

The ability to catalyze diverse reactions with relevance for chemical and pharmaceutical research and industry has led to an increasing interest in fungal enzymes. There is still an enormous potential considering the sheer amount of new enzymes from the huge diversity of fungi. Most of these fungal enzymes have not been characterized yet due to the lack of high throughput synthesis and analysis methods. This bottleneck could be overcome by means of cell-free protein synthesis. In this study, cell-free protein synthesis based on eukaryotic cell lysates was utilized to produce a functional glycoside hydrolase (GH78) from the soft-rot fungus Xylaria polymorpha (Ascomycota). The enzyme was successfully synthesized under different reaction conditions. We characterized its enzymatic activities and immobilized the protein via FLAG-Tag interaction. Alteration of several conditions including reaction temperature, template design and lysate supplementation had an influence on the activity of cell-free synthesized GH78. Consequently this led to a production of purified GH78 with a specific activity of 15.4 U mg− 1. The results of this study may be foundational for future high throughput fungal enzyme screenings, including substrate spectra analysis and mutant screenings

Genome and secretome of Chondrostereum purpureum correspond to saprotrophic and phytopathogenic life styles

We thank R. Ullrich for his mycological expertise and K. Eismann for useful technical assistance.The basidiomycete Chondrostereum purpureum (Silverleaf fungus) is a saprotroph and plant pathogen commercially used for combatting forest “weed” trees in vegetation management. However, little is known about its lignocellulose-degrading capabilities and the enzymatic machinery that is responsible for the degradative potential, and it is not yet clear to which group of wood-rot fungi it actually belongs. Here, we sequenced and analyzed the draft genome of C. purpureum (41.2 Mbp) and performed a quantitative proteomic approach during growth in submerged and solid-state cultures based on soybean meal suspension or containing beech wood supplemented with phenol-rich olive mill residues, respectively. The fungus harbors characteristic lignocellulolytic hydrolases (GH6 and GH7) and oxidoreductases (e.g. laccase, heme peroxidases). High abundance of some of these genes (e.g. 45 laccases, nine GH7) can be explained by gene expansion, e.g. identified for the laccase orthogroup ORTHOMCL11 that exhibits a total of 18 lineage-specific duplications. Other expanded genes families encode for proteins more related to a pathogenic lifestyle (e.g. protease and cytochrome P450s). The fungus responds to the presence of complex growth substrates (lignocellulose, phenolic residues) by the secretion of most of these lignocellulolytic and lignin-modifying enzymes (e.g. alcohol and aryl alcohol oxidases, laccases, GH6, GH7). Based on the genetic and enzymatic constitution, we consider the ‘marasmioid’ fungus C. purpureum as a ‘phytopathogenic’ white-rot fungus (WRF) that possesses a complex extracellular enzyme machinery to accomplish efficient lignocellulose degradation during both saprotrophic and phytopathogenic life phases.The work was financially and scientifically supported by the European Union [integrated projects INDOX – KBBE-7-2013-613549; ENZOX2 – 720297], by the DFG project PeroxiDiv HO 1961/8-1 and the AiF project PeroxyMEER IGF 19636 BG/3. The work has been partly funded by the DFG Priority Program 1374 "Infrastructure-Biodiversity-Exploratories" with the projects HO 1961/6-1, KE 1742/2-1 and JE 724/7-4 (AOBJ: 635952) and the Spanish Ministry of Economy and Competitiveness [project AGL2012-32873]. RR thanks the JAE-Program of the Spanish National Research Council [CSIC] and EA thanks MINECO and FEDER Co-Funds [RyC-2013-12481]

Simulated Atmospheric N Deposition Alters Fungal Community Composition and Suppresses Ligninolytic Gene Expression in a Northern Hardwood Forest

High levels of atmospheric nitrogen (N) deposition may result in greater terrestrial carbon (C) storage. In a northern hardwood ecosystem, exposure to over a decade of simulated N deposition increased C storage in soil by slowing litter decay rates, rather than increasing detrital inputs. To understand the mechanisms underlying this response, we focused on the saprotrophic fungal community residing in the forest floor and employed molecular genetic approaches to determine if the slower decomposition rates resulted from down-regulation of the transcription of key lignocellulolytic genes, by a change in fungal community composition, or by a combination of the two mechanisms. Our results indicate that across four Acer-dominated forest stands spanning a 500-km transect, community-scale expression of the cellulolytic gene cbhI under elevated N deposition did not differ significantly from that under ambient levels of N deposition. In contrast, expression of the ligninolytic gene lcc was significantly down-regulated by a factor of 2–4 fold relative to its expression under ambient N deposition. Fungal community composition was examined at the most southerly of the four sites, in which consistently lower levels of cbhI and lcc gene expression were observed over a two-year period. We recovered 19 basidiomycete and 28 ascomycete rDNA 28S operational taxonomic units; Athelia, Sistotrema, Ceratobasidium and Ceratosebacina taxa dominated the basidiomycete assemblage, and Leotiomycetes dominated the ascomycetes. Simulated N deposition increased the proportion of basidiomycete sequences recovered from forest floor, whereas the proportion of ascomycetes in the community was significantly lower under elevated N deposition. Our results suggest that chronic atmospheric N deposition may lower decomposition rates through a combination of reduced expression of ligninolytic genes such as lcc, and compositional changes in the fungal community

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

Determinants of Deadwood-Inhabiting Fungal Communities in Temperate Forests: Molecular Evidence From a Large Scale Deadwood Decomposition Experiment

Despite the important role of wood-inhabiting fungi (WIF) in deadwood decomposition, our knowledge of the factors shaping the dynamics of their species richness and community composition is scarce. This is due to limitations regarding the resolution of classical methods used for characterizing WIF communities and to a lack of well-replicated long-term experiments with sufficient numbers of tree species. Here, we used a large scale experiment with logs of 11 tree species at an early stage of decomposition, distributed across three regions of Germany, to identify the factors shaping WIF community composition and Operational Taxonomic Unit (OTU) richness using next generation sequencing. We found that tree species identity was the most significant factor, corresponding to (P < 0.001) and explaining 10% (representing 48% of the explainable variance) of the overall WIF community composition. The next important group of variables were wood-physicochemical properties, of which wood pH was the only factor that consistently corresponded to WIF community composition. For overall WIF richness patterns, we found that approximately 20% of the total variance was explained by wood N content, location, tree species identity and wood density. It is noteworthy that the importance of determinants of WIF community composition and richness appeared to depend greatly on tree species group (broadleaved vs. coniferous) and it differed between the fungal phyla Ascomycota and Basidiomycota

Rheumatoid arthritis - treatment: 180. Utility of Body Weight Classified Low-Dose Leflunomide in Japanese Rheumatoid Arthritis

Background: In Japan, more than 20 rheumatoid arthritis (RA) patients died of interstitial pneumonia (IP) caused by leflunomide (LEF) were reported, but many of them were considered as the victims of opportunistic infection currently. In this paper, efficacy and safety of low-dose LEF classified by body weight (BW) were studied. Methods: Fifty-nine RA patients were started to administrate LEF from July 2007 to July 2009. Among them, 25 patients were excluded because of the combination with tacrolimus, and medication modification within 3 months before LEF. Remaining 34 RA patients administered 20 to 50 mg/week of LEF were followed up for 1 year and enrolled in this study. Dose of LEF was classified by BW (50 mg/week for over 50 kg, 40 mg/week for 40 to 50 kg and 20 to 30 mg/week for under 40 kg). The average age and RA duration of enrolled patients were 55.5 years old and 10.2 years. Prednisolone (PSL), methotrexate (MTX) and etanercept were used in 23, 28 and 2 patients, respectively. In case of insufficient response or adverse effect, dosage change or discontinuance of LEF were considered. Failure was defined as dosages up of PSL and MTX, or dosages down or discontinuance of LEF. Last observation carried forward method was used for the evaluation of failed patients at 1 year. Results: At 1 year after LEF start, good/ moderate/ no response assessed by the European League Against Rheumatism (EULAR) response criteria using Disease Activity Score, including a 28-joint count (DAS28)-C reactive protein (CRP) were showed in 14/ 10/ 10 patients, respectively. The dosage changes of LEF at 1 year were dosage up: 10, same dosage: 5, dosage down: 8 and discontinuance: 11 patients. The survival rate of patients in this study was 23.5% (24 patients failed) but actual LEF continuous rate was 67.6% (11 patients discontinued) at 1 year. The major reason of failure was liver dysfunction, and pneumocystis pneumonia was occurred in 1 patient resulted in full recovery. One patient died of sepsis caused by decubitus ulcer infection. DAS28-CRP score was decreased from 3.9 to 2.7 significantly. Although CRP was decreased from 1.50 to 0.93 mg/dl, it wasn't significant. Matrix metalloproteinase (MMP)-3 was decreased from 220.0 to 174.2 ng/ml significantly. Glutamate pyruvate transaminase (GPT) was increased from 19 to 35 U/l and number of leukocyte was decreased from 7832 to 6271 significantly. DAS28-CRP, CRP, and MMP-3 were improved significantly with MTX, although they weren't without MTX. Increase of GPT and leukopenia were seen significantly with MTX, although they weren't without MTX. Conclusions: It was reported that the risks of IP caused by LEF in Japanese RA patients were past IP history, loading dose administration and low BW. Addition of low-dose LEF is a potent safe alternative for the patients showing unsatisfactory response to current medicines, but need to pay attention for liver function and infection caused by leukopenia, especially with MTX. Disclosure statement: The authors have declared no conflicts of interes

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Risk profiles and one-year outcomes of patients with newly diagnosed atrial fibrillation in India: Insights from the GARFIELD-AF Registry.

BACKGROUND: The Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF) is an ongoing prospective noninterventional registry, which is providing important information on the baseline characteristics, treatment patterns, and 1-year outcomes in patients with newly diagnosed non-valvular atrial fibrillation (NVAF). This report describes data from Indian patients recruited in this registry. METHODS AND RESULTS: A total of 52,014 patients with newly diagnosed AF were enrolled globally; of these, 1388 patients were recruited from 26 sites within India (2012-2016). In India, the mean age was 65.8 years at diagnosis of NVAF. Hypertension was the most prevalent risk factor for AF, present in 68.5% of patients from India and in 76.3% of patients globally (P < 0.001). Diabetes and coronary artery disease (CAD) were prevalent in 36.2% and 28.1% of patients as compared with global prevalence of 22.2% and 21.6%, respectively (P < 0.001 for both). Antiplatelet therapy was the most common antithrombotic treatment in India. With increasing stroke risk, however, patients were more likely to receive oral anticoagulant therapy [mainly vitamin K antagonist (VKA)], but average international normalized ratio (INR) was lower among Indian patients [median INR value 1.6 (interquartile range {IQR}: 1.3-2.3) versus 2.3 (IQR 1.8-2.8) (P < 0.001)]. Compared with other countries, patients from India had markedly higher rates of all-cause mortality [7.68 per 100 person-years (95% confidence interval 6.32-9.35) vs 4.34 (4.16-4.53), P < 0.0001], while rates of stroke/systemic embolism and major bleeding were lower after 1 year of follow-up. CONCLUSION: Compared to previously published registries from India, the GARFIELD-AF registry describes clinical profiles and outcomes in Indian patients with AF of a different etiology. The registry data show that compared to the rest of the world, Indian AF patients are younger in age and have more diabetes and CAD. Patients with a higher stroke risk are more likely to receive anticoagulation therapy with VKA but are underdosed compared with the global average in the GARFIELD-AF. CLINICAL TRIAL REGISTRATION-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01090362