Clinical outcomes from the Assessing Donor-derived cell-free DNA Monitoring Insights of kidney Allografts with Longitudinal surveillance (ADMIRAL) study.

The use of routine monitoring of donor-derived cell-free DNA (dd-cfDNA) after kidney transplant may allow clinicians to identify subclinical allograft injury and intervene prior to development of clinically evident graft injury. To evaluate this, data from 1092 kidney transplant recipients monitored for dd-cfDNA over a three-year period was analyzed to assess the association of dd-cfDNA with histologic evidence of allograft rejection. Elevation of dd-cfDNA (0.5% or more) was significantly correlated with clinical and subclinical allograft rejection. dd-cfDNA values of 0.5% or more were associated with a nearly three-fold increase in risk development of de novo donor-specific antibodies (hazard ratio 2.71) and were determined to be elevated a median of 91 days (interquartile range of 30-125 days) ahead of donor specific antibody identification. Persistently elevated dd-cfDNA (more than one result above the 0.5% threshold) predicted over a 25% decline in the estimated glomerular filtration rate over three years (hazard ratio 1.97). Therefore, routine monitoring of dd-cfDNA allowed early identification of clinically important graft injury. Biomarker monitoring complemented histology and traditional laboratory surveillance strategies as a prognostic marker and risk-stratification tool post-transplant. Thus, persistently low dd-cfDNA levels may accurately identify allograft quiescence or absence of injury, paving the way for personalization of immunosuppression trials.

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS).

BACKGROUND: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. RESULTS: Two ß-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the ß-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 µL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. CONCLUSIONS: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic ß-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol ß-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol ß-O-4 type of molecules with laccase and MnP.

Hydrogenolytic depolymerization of procyanidin polymers from hi-tannin sorghum bran.

Depolymerization of procyanidin polymers into oligomers enhances their bioavailability and bioactivity because oligomers are bioavailable. Hydrogenolysis was applied in this study to depolymerize hi-tannin sorghum bran procyanidin polymers into oligomers. The yield and composition of oligomers under different hydrogenolysis conditions was investigated. Results showed that raising temperature from 50 to 100 °C significantly increased total yield of oligomers. Higher temperatures (150 and 200 °C) produced monomers with lower yield. The highest yield of oligomers (38.8%) was obtained using 1 MPa hydrogen whereas 3 MPa hydrogen in reaction vessel decreased yield. Total yield of oligomers reached the highest at 1-3 h and then decreased with prolonged reaction time. Yield increased with palladium-on-carbon (Pd/C, a catalyst) amount from 0.5 to 3 mg and plateaued with Pd/C amount from 3 to 10 mg. The maximum yield of produced oligomers was achieved under 100 °C, 1 MPa hydrogen pressure, 1-3 h, and 3-10 mg Pd/C.

Biomimetic Fenton-catalyzed lignin depolymerization to high-value aromatics and dicarboxylic acids.

By mimicking natural lignin degradation systems, the Fenton catalyst (Fe(3+), H2O2) can effectively facilitate lignin depolymerization in supercritical ethanol (7 MPa, 250 °C) to give organic oils that consist of mono- and oligomeric aromatics, phenols, dicarboxylic acids, and their derivatives in yields up to (66.0±8.5) %. The thermal properties, functional groups, and surface chemistry of lignin before and after Fenton treatment were examined by thermogravimetric analysis, pyrolysis-gas chromatography-mass spectrometry, (31)P NMR spectroscopy, and X-ray photoelectron spectroscopy. The results suggest that the Fenton catalyst facilitates lignin depolymerization through cleavage of ß-ether bonds between lignin residues. The formation of a lignin-iron chelating complex effectively depresses lignin recondensation; thus minimizing charcoal formation and enhancing the yield of liquid products.

Effects of lignin modification on wheat straw cell wall deconstruction by Phanerochaete chrysosporium.

BACKGROUND: A key focus in sustainable biofuel research is to develop cost-effective and energy-saving approaches to increase saccharification of lignocellulosic biomass. Numerous efforts have been made to identify critical issues in cellulose hydrolysis. Aerobic fungal species are an integral part of the carbon cycle, equip the hydrolytic enzyme consortium, and provide a gateway for understanding the systematic degradation of lignin, hemicelluloses, and cellulose. This study attempts to reveal the complex biological degradation process of lignocellulosic biomass by Phanerochaete chrysosporium in order to provide new knowledge for the development of energy-efficient biorefineries. RESULTS: In this study, we evaluated the performance of a fungal biodegradation model, Phanerochaete chrysosporium, in wheat straw through comprehensive analysis. We isolated milled straw lignin and cellulase enzyme-treated lignin from fungal-spent wheat straw to determine structural integrity and cellulase absorption isotherms. The results indicated that P. chrysosporium increased the total lignin content in residual biomass and also increased the cellulase adsorption kinetics in the resulting lignin. The binding strength increased from 117.4 mL/g to 208.7 mL/g in milled wood lignin and from 65.3 mL/g to 102.4 mL/g in cellulase enzyme lignin. A detailed structural dissection showed a reduction in the syringyl lignin/guaiacyl lignin ratio and the hydroxycinnamate/lignin ratio as predominant changes in fungi-spent lignin by heteronuclear single quantum coherence spectroscopy. CONCLUSION: P. chrysosporium shows a preference for degradation of phenolic terminals without significantly destroying other lignin components to unzip carbohydrate polymers. This is an important step in fungal growth on wheat straw. The phenolics presumably locate at the terminal region of the lignin moiety and/or link with hemicellulose to form the lignin-carbohydrate complex. Findings may inform the development of a biomass hydrolytic enzyme combination to enhance lignocellulosic biomass hydrolysis and modify the targets in plant cell walls.

Improved lipid accumulation by morphology engineering of oleaginous fungus Mortierella isabellina.

Oleaginous fungi capable of accumulating a considerable amount of lipids are promising sources for lipid-based biofuel production. The specific productivities of filamentous fungi in submerged fermentation are often correlated with morphological forms. However, the relationship between morphological development and lipid accumulation is not known. In this study, distinct morphological forms of oleaginous fungus Mortierella isabellina including pellets of different sizes, free dispersed mycelia, and broken hyphal fragments were developed by additions of different concentrations of magnesium silicate microparticles. Different morphological forms led to different levels of lipid accumulation as well as different spatial patterns of lipid distribution within pellets/mycelial aggregates. Significant higher lipid content (0.75 g lipid/g cell biomass) and lipid yield (0.18 g lipid/g glucose consumed) were achieved in free dispersed mycelia than in pellets. Moreover, extracellular metabolite analysis showed that production of undesirable by-product malate was repressed in free dispersed mycelium form. Unveiling the desired morphological form of M. isabellina for lipid accumulation provided insights into molecular mechanism of lipid biosynthesis linked with morphological development, as well as design and optimization of bioprocess to produce lipid-based biofuels.

Isolation and structural characterization of sugarcane bagasse lignin after dilute phosphoric acid plus steam explosion pretreatment and its effect on cellulose hydrolysis.

The structure of lignin after dilute phosphoric acid plus steam explosion pretreatment process of sugarcane bagasse in a pilot scale and the effect of the lignin extracted by ethanol on subsequent cellulose hydrolysis were investigated. The lignin structural changes caused by pretreatment were identified using advanced nondestructive techniques such as gel permeation chromatography (GPC), quantitative (13)C, and 2-D nuclear magnetic resonance (NMR). The structural analysis revealed that ethanol extractable lignin preserved basic lignin structure, but had relatively lower amount of ß-O-4 linkages, syringyl/guaiacyl units ratio (S/G), p-coumarate/ferulate ratio, and other ending structures. The results also indicated that approximately 8% of mass weight was extracted by pure ethanol. The bagasse after ethanol extraction had an approximate 22% higher glucose yield after enzyme hydrolysis compared to pretreated bagasse without extraction.

Quantification of wheat straw lignin structure by comprehensive NMR analysis.

A further understanding of the structure of lignin from herbaceous crops is needed for advancing technologies of lignocellulosic biomass processing and utilization. A method was established in this study for analyzing structural motifs found in milled straw lignin (MSL) and cellulase-digested lignin (CEL) isolated from wheat straw by combining quantitative (13)C and HSQC NMR spectral analyses. The results showed that guaiacyl (G) was the predominant unit in wheat straw cell wall lignin over syringyl (S) and hydroxyphenyl (H) units. Up to 8.0 units of tricin were also detected in wheat straw lignin per 100 aromatic rings. Various interunit linkages, including ß-O-4, ß-5, ß-ß', ß-1, α, ß-diaryl ether, and 5-5'/4-O-ß' as well as potential lignin-carbohydrate complex (LCC) bonds, were identified and quantified. These findings provide useful information for the development of biofuels and lignin-based materials.

Microbial lipid production from xylose by Mortierella isabellina.

Culture conditions including nitrogen source and concentration, xylose concentration, and inoculum level were evaluated for the effect on cell growth and lipid production of an oleaginous fungus, Mortierella isabellina, grown on xylose. Yeast extract and ammonium sulfate were found to be the best amongst the organic and inorganic nitrogen sources tested, respectively. Subsequent combination of these two nitrogen sources at a nitrogen ratio of 1:1 further enhanced lipid production. The highest cell biomass 28.8 g L(-1) and lipid 18.5 g L(-1) were obtained on a medium containing 100 g L(-1) xylose and 50.4 mM nitrogen with a spore concentration of 10(8) mL(-1). Specifically, nitrogen concentration and inoculum level were demonstrated to be important for obtaining a high lipid yield on xylose consumed of 0.182 g g(-1). The results suggest that M. isabellina holds great potential to be a candidate for biofuel production from xylose, the second most abundant sugar from lignocellulose.

Lignocellulosic biomass as a carbohydrate source for lipid production by Mortierella isabellina.

Various carbon sources including monosugars, disaccharides and carboxymethyl-cellulose (CMC) were used for single-cell oil production by the filamentous fungus Mortierella isabellina. In addition, the inhibitory effects of lignocellulose-derived compounds (lignin aldehydes, furan aldehydes and weak acid) were investigated. C6 sugars were preferably used for growth compared to C5 sugars. CMC was not an usable substrate, implying the absence of a cellulase system in this fungus. Lignin derivatives showed the most inhibitory effects, but acetic and formic acids at concentrations of 4 g/L improved lipid production, achieving 6.81 ± 0.07 g/L and 6.66 ± 0.33 g/L respectively, which was twice as high as that of the control. A 16.8% lipid yield from hydrolysate suggested that this fungus could be useful for microbial lipid production.

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw.

BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.

Biological pretreatment of wheat straw by Phanerochaete chrysosporium supplemented with inorganic salts.

Inorganic salts and tween 80 are known to induce the lignin degrading peroxidase expression of Phanerochaete chrysosporium in submerged culture. In this study, the wheat straw pretreatment supplemented with inorganic salts (salts group), tween 80 (plus) and no supplementation to the biomass (minus) were examined. Among the solid state fermentation groups, salts group resulted in a substantial degradation of wheat straw within one week, along with the highest lignin loss (25%) and ∼250% higher efficiency for the total sugar release through enzymatic hydrolysis. The results were correlated with pyrolysis GC-MS (Py-GC-MS), thermogravimetric (TG)/differential thermogravimetric (DTG) and X-ray diffraction (XRD). The results suggested that the supplementation of inorganic salts in the solid state fermentation of wheat straw significantly enhances the degradation rate of the biomass by P. chrysosporium which can be exploited as an alternative means to existing pretreatment technologies.