Involvement of ligninolytic enzymes and Fenton-like reaction in humic acid degradation by Trametes sp

Trametes sp. M23, isolated from biosolids compost was found to decompose humic acids (HA). A low N (LN) medium (C/N, 53) provided suitable conditions for HA degradation, whereas in a high N (HN) medium (C/N, 10), HA was not degraded. In the absence of Mn²⁺, HA degradation was similar to that in Mn²⁺-containing medium. In contrast, MnP activity was significantly affected by Mn²⁺. Laccase activity exhibited a negative correlation to HA degradation, while LiP activity was not detected. Thus, ligninolytic enzymes activity could provide only a partial explanation for the HA-degradation mechanism. The decolorization of two dyes, Orange II and Brilliant Blue R250, was also determined. Similar to HA degradation, under LN conditions, decolorization occurred independently of the presence of Mn²⁺. We investigated the possible involvement of a Fenton-like reaction in HA degradation. The addition of DMSO, an OH-radical scavenger, to LN media resulted in a significant decrease in HA bleaching. The rate of extracellular Fe³⁺ reduction was much higher in the LN vs. HN medium. In addition, the rate of reduction was even higher in the presence of HA in the medium. In vitro HA bleaching in non-inoculated media was observed with H₂O₂ amendment to a final concentration of 200 mM (obtained by 50 mM amendments for 4 days) and Fe²⁺ (36 mM). After 4 days of incubation, HA decolorization was similar to the biological treatment. These results support our hypothesis that a Fenton-like reaction is involved in HA degradation by Trametes sp. M23.     

Cellular versus acellular matrix devices in treatment of diabetic foot ulcers: study protocol for a comparative efficacy randomized controlled trial

Background

The referral letter plays a key role both in the communication between primary and secondary care, and in the quality of the health care process. Many studies have attempted to evaluate and improve the quality of these referral letters, but few have assessed the impact of their quality on the health care delivered to each patient.

Methods

A cluster randomized trial, with the general practitioner office as the unit of randomization, has been designed to evaluate the effect of a referral intervention on the quality of health care delivered. Referral templates have been developed covering four diagnostic groups: dyspepsia, suspected colonic malignancy, chest pain, and chronic obstructive pulmonary disease. Of the 14 general practitioner offices primarily served by University Hospital of North Norway Harstad, seven were randomized to the intervention group. The primary outcome is a collated quality indicator score developed for each diagnostic group. Secondary outcomes include: quality of the referral, health process outcome such as waiting times, and adequacy of prioritization. In addition, information on patient satisfaction will be collected using self-report questionnaires. Outcome data will be collected on the individual level and analyzed by random effects linear regression.

Discussion

Poor communication between primary and secondary care can lead to inappropriate investigations and erroneous prioritization. This study’s primary hypothesis is that the use of a referral template in this communication will lead to a measurable increase in the quality of health care delivered.

Trial registration

This trial has been registered at ClinicalTrials.gov. The trial registration number is NCT01470963     

Whole-cell biochips for bio-sensing: integration of live cells and inanimate surfaces

Recent advances in the convergence of the biological, chemical, physical, and engineering sciences have opened new avenues of research into the interfacing of diverse biological moieties with inanimate platforms. A main aspect of this field, the integration of live cells with micro-machined platforms for high throughput and bio-sensing applications, is the subject of the present review. These unique hybrid systems are configured in a manner that ensures positioning of the cells in designated patterns, and enables cellular viability maintenance, and monitoring of cellular functionality. Here we review both animate and inanimate surface properties and how they affect cellular attachment, describe relevant modifications of both types of surfaces, list technologies for platform engineering and for cell deposition in the desired configurations, and discuss the influence of various deposition and immobilization methods on the viability and performance of the immobilized cells.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Initial Oxidation Products in the Metabolism of Pyrene, Anthracene, Fluorene, and Dibenzothiophene by the White Rot Fungus Pleurotus ostreatus

The initial metabolites in the degradation of pyrene, anthracene, fluorene, and dibenzothiophene by Pleurotus ostreatus were isolated by high-pressure liquid chromatography and characterized by UV-visible, gas-chromatographic, mass-spectrometric, and (sup1)H nuclear magnetic resonance spectral techniques. The metabolites from pyrene, dibenzothiophene, anthracene, and fluorene amounted to 45, 84, 64, and 96% of the total organic-solvent-extractable metabolites, respectively. Pyrene was metabolized predominantly to pyrene trans-4,5-dihydrodiol. Anthracene was metabolized predominantly to anthracene trans-1,2-dihydrodiol and 9,10-anthraquinone. In contrast, fluorene and dibenzothiophene were oxidized at the aliphatic bridges instead of the aromatic rings. Fluorene was oxidized to 9-fluorenol and 9-fluorenone; dibenzothiophene was oxidized to the sulfoxide and sulfone. Circular dichroism spectroscopy revealed that the major enantiomer of anthracene trans-1,2-dihydrodiol was predominantly in the S,S configuration and the major enantiomer of the pyrene trans-4,5-dihydrodiol was predominantly R,R. These results indicate that the white rot fungus P. ostreatus initially metabolizes polycyclic aromatic hydrocarbons by reactions similar to those previously reported for nonligninolytic fungi. However, P. ostreatus, in contrast to nonligninolytic fungi, can mineralize these polycyclic aromatic hydrocarbons. The identity of the dihydrodiol metabolites implicates a cytochrome P-450 monooxygenase mechanism.     

Metabolism of phenanthrene by the white rot fungus Pleurotus ostreatus.

The white rot fungus Pleurotus ostreatus, grown for 11 days in basidiomycetes rich medium containing [14C] phenanthrene, metabolized 94% of the phenanthrene added. Of the total radioactivity, 3% was oxidized to CO2. Approximately 52% of phenanthrene was metabolized to trans-9,10-dihydroxy-9,10-dihydrophenanthrene (phenanthrene trans-9,10-dihydrodiol) (28%), 2,2'-diphenic acid (17%), and unidentified metabolites (7%). Nonextractable metabolites accounted for 35% of the total radioactivity. The metabolites were extracted with ethyl acetate, separated by reversed-phase high-performance liquid chromatography, and characterized by 1H nuclear magnetic resonance, mass spectrometry, and UV spectroscopy analyses. 18O2-labeling experiments indicated that one atom of oxygen was incorporated into the phenanthrene trans-9,10-dihydrodiol. Circular dichroism spectra of the phenanthrene trans-9,10-dihydrodiol indicated that the absolute configuration of the predominant enantiomer was 9R,10R, which is different from that of the principal enantiomer produced by Phanerochaete chrysosporium. Significantly less phenanthrene trans-9,10-dihydrodiol was observed in incubations with the cytochrome P-450 inhibitor SKF 525-A (77% decrease), 1-aminobenzotriazole (83% decrease), or fluoxetine (63% decrease). These experiments with cytochrome P-450 inhibitors and 18O2 labeling and the formation of phenanthrene trans-9R,10R-dihydrodiol as the predominant metabolite suggest that P. ostreatus initially oxidizes phenanthrene stereoselectively by a cytochrome P-450 monoxygenase and that this is followed by epoxide hydrolase-catalyzed hydration reactions.     

Fungal activities involved in lignocellulose degradation by Pleurotus

Summary During growth of Pleurotus on cotton straw both the straw in general and the lignin in particular were degraded. After 4 days of fungal growth, activity of laccase, catechol oxidase, peroxidase, and cellulase were detected. This activity, however, declined rapidly after 8–10 days of growth.Lignin degradation began after 10 days and reached a maximum after 21 days. It would seem that the preliminary action of laccase is a prerequisite for lignin degradation.The Pleurotus ostreatus strain P₃ had no detectable laccase activity and showed very poor ability to degrade cotton straw and lignin.Water extract of cotton straw was found to be a potent inducer of laccase in liquid medium and had an effect much stronger than several small phenolic compounds. The degradation of washed cotton straw and lignin from this straw was lower than native straw, so was laccase activity on this medium. High carbon dioxide concentrations encouraged straw degradation by P. ostreatus florida but severly limited lignin degradation. Other fungi including the known lignin degrader Phanarochaete chrysosporium were able to degrade up to 40% of cotton straw dry weight within 21 days of fungal growth. The percentage degradation of lignin, however, was very low (only 10% in 21 days). Pleurotus ostreatus florida was able to degrade up to 56% of the lignin within this time.After treatment with P. ostreatus florida almost four times as much glucose was released when the straw was treated with commercial cellulases, showing increased availability of cellulose.It is suggested that treatment with P. ostreatus florida may be used to enrich low value food materials for ruminant animals.     

Inhibition of polyamine synthesis reduces the growth rate and delays the expression of differentiated phenotypes in primary cultures of embryonic mesoderm from chick

Summary Inhibition of polyamine synthesis in early chick embryos blocks their development at gastrulation. Analyses of arrested embryos show that mesodermal outgrowth and differentiation are drastically impaired. To study these effects in greater detail, we have used primary cultures of embryonic mesoderm from chick. The cultures were treated with -difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, the first and rate-limiting enzyme in polyamine synthesis. In control culture medium, mesodermal cells retained their in ovo outgrowth behavior and differentiation pattern. Addition of 10 mM DFMO to the culture medium, however, retarded attachment and outgrowth, and reduced the rate of proliferation of the mesodermal cells. Furthermore, the expression of differentiated phenotypes, such as beating heart tissue, erythroid cells, and adipocyte-like cells, was delayed. Simultaneous addition of 100 M putrescine prevented or reduced the effects of DFMO, showing that these were indeed caused by polyamine deficiency. In the DFMO-treated mesoderm, DNA synthesis was markedly suppressed by the first day. Similar effects on RNA and protein synthesis developed at a later time. Our data suggest that a reduction in the concentrations of the polyamines decreases the rate of mesodermal cell proliferation, and as a conseqence delays the expression of differentiated phenotypes.