Quantitative microbial indices in biogas and raw cattle slurries

Biogas slurry, the secondary product of the anaerobic digestion process, is increasingly being used as fertilizer. Information is available on its chemical and physical properties and their effects on plant growth. However, there is a demand to characterize the microbial quality of slurries, which may control further mineralization processes after application to soil. In this study, biogas and raw slurries obtained from six farms were analyzed for their ergosterol and amino sugar concentrations as indices for microbial biomass. A reliable, precise method for determining ergosterol in slurries is presented. Biogas slurries contained significantly less ergosterol (?34%), muramic acid (MurN; ?42%), galactosamine (GalN; ?32%), and fungal glucosamine (GlcN; ?40%) than raw slurries. The mean fungal GlcN to ergosterol ratio (50) and also the mean fungal carbon (C) to bacterial C ratio (0.29) did not significantly differ between the slurry types. The mean microbial C concentration in the biogas slurries was significantly lower than in the raw slurries. Consequently, the contribution of microbial C to slurry organic C was 3.6% in the biogas slurries and 5.7% in the raw slurries. Microbial C revealed significant nonlinear relationships with the fiber and ash concentration, pH, as well as the C/N ratio of the slurries.     

Exploring mild enzymatic sustainable routes for the synthesis of bio‐degradable aromatic‐aliphatic oligoesters

The application of Candida antarctica lipase B in enzyme‐catalyzed synthesis of aromatic‐aliphatic oligoesters is here reported. The aim of the present study is to systematically investigate the most favorable conditions for the enzyme catalyzed synthesis of aromatic‐aliphatic oligomers using commercially available monomers. Reaction conditions and enzyme selectivity for polymerization of various commercially available monomers were considered using different inactivated/activated aromatic monomers combined with linear polyols ranging from C₂ to C₁₂. The effect of various reaction solvents in enzymatic polymerization was assessed and toluene allowed to achieve the highest conversions for the reaction of dimethyl isophthalate with 1,4‐butanediol and with 1,10‐decanediol (88 and 87% monomer conversion respectively). M_w as high as 1512 Da was obtained from the reaction of dimethyl isophthalate with 1,10‐decanediol. The obtained oligomers have potential applications as raw materials in personal and home care formulations, for the production of aliphatic‐aromatic block co‐polymers or can be further functionalized with various moieties for a subsequent photo‐ or radical polymerization.     

Probing unnatural amino acid integration into enhanced green fluorescent protein by genetic code expansion with a high-throughput screening platform

Background

Genetic code expansion has developed into an elegant tool to incorporate unnatural amino acids (uAA) at predefined sites in the protein backbone in response to an amber codon. However, recombinant production and yield of uAA comprising proteins are challenged due to the additional translation machinery required for uAA incorporation.

Results

We developed a microtiter plate-based high-throughput monitoring system (HTMS) to study and optimize uAA integration in the model protein enhanced green fluorescence protein (eGFP). Two uAA, propargyl-L-lysine (Plk) and (S)-2-amino-6-((2-azidoethoxy) carbonylamino) hexanoic acid (Alk), were incorporated at the same site into eGFP co-expressing the native PylRS/tRNA^Pyl _CUA pair originating from Methanosarcina barkeri in E. coli. The site-specific uAA functionalization was confirmed by LC-MS/MS analysis. uAA-eGFP production and biomass growth in parallelized E. coli cultivations was correlated to (i) uAA concentration and the (ii) time of uAA addition to the expression medium as well as to induction parameters including the (iii) time and (iv) amount of IPTG supplementation. The online measurements of the HTMS were consolidated by end point-detection using standard enzyme-linked immunosorbent procedures.

Conclusion

The developed HTMS is powerful tool for parallelized and rapid screening. In light of uAA integration, future applications may include parallelized screening of different PylRS/tRNA^Pyl _CUA pairs as well as further optimization of culture conditions.

     

Ten Years after the International Committee of Medical Journal Editors’ Clinical Trial Registration Initiative,One Quarter of Phase 3 Pediatric Epilepsy Clinical Trials Still Remain Unpublished: A Cross Sectional Analysis

Introduction

Although selective reporting of clinical trial results introduces bias into evidence based clinical decision making, publication bias in pediatric epilepsy is unknown today. Since there is a considerable ambiguity in the treatment of an important and common clinical problem, pediatric seizures, we assessed the public availability of results of phase 3 clinical trials that evaluated treatments of seizures in children and adolescents as a surrogate for the extent of publication bias in pediatric epilepsy.

Methods

We determined the proportion of published and unpublished study results of phase 3 clinical trials that were registered as completed on ClinicalTrials.gov. We searched ClinicalTrials.gov, PubMed, and Google Scholar for publications and contacted principal investigators or sponsors. The analysis was performed according to STROBE criteria.

Results

Considering studies that were completed before 2014 (N = 99), 75 (76%) pediatric phase 3 clinical trials were published but 24 (24%) remained unpublished. The unpublished studies concealed evidence from 4,437 patients. Mean time-to-publication was 25 SD ± 15.6 months, more than twice as long as mandated.

Conclusion

Ten years after the ICMJE’s clinical trials registration initiative there is still a considerable amount of selective reporting and delay of publication that potentially distorts the body of evidence in the treatment of pediatric seizures.     

Construction of a new catabolic pathway for d-fructose in Escherichia coli K12 using an l-sorbose-specific enzyme from Klebsiella pneumoniae

Starting with a fruK (formerly fpk) mutant of Escherichia coli K12 lacking d-fructose-1-phosphate kinase (E.C. 2.7.1.3.), fructose positive derivatives were isolated after introduction of the cloned gene sorE from Klebsiella pneumoniae coding for an l-sorbose-1-phosphate reductase. The new pathway was shwon to proceed from d-fructose via d-fructose-1-phosphate and d-mannitol-1-phosphate to d-fructose 6-phosphate. It involves a transport system and enzymes encoded in the fru and the mtl operons from E. coli K12 as well as in the sor operon from K. pneumoniae respectively.     

Processing of continuous lines and edges by the visual system

Continuous lines and edges are described by chains of oriented filters. Combinatorical considerations leed to a complete set of differently shaped oriented filters, which show all the features of cortical receptive fields. Filters of this set may be linked to chains, enveloping arbitrary lines and edges. A hierarchical linking procedure avoids combinatorical explosion of the required neuronal network.