Molecular engineering of a thermostable carbohydrate-binding module

Structure–function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel?, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

Estimating and Analyzing Savannah Phenology with a Lagged Time Series Model

Savannah regions are predicted to undergo changes in precipitation patterns according to current climate change projections. This change will affect leaf phenology, which controls net primary productivity. It is of importance to study this since savannahs play an important role in the global carbon cycle due to their areal coverage and can have an effect on the food security in regions that depend on subsistence farming. In this study we investigate how soil moisture, mean annual precipitation, and day length control savannah phenology by developing a lagged time series model. The model uses climate data for 15 flux tower sites across four continents, and normalized difference vegetation index from satellite to optimize a statistical phenological model. We show that all three variables can be used to estimate savannah phenology on a global scale. However, it was not possible to create a simplified savannah model that works equally well for all sites on the global scale without inclusion of more site specific parameters. The simplified model showed no bias towards tree cover or between continents and resulted in a cross-validated r² of 0.6 and root mean squared error of 0.1. We therefore expect similar average results when applying the model to other savannah areas and further expect that it could be used to estimate the productivity of savannah regions.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Effects of food abundance and early clutch predation on reproductive timing in a high Arctic shorebird exposed to advancements in arthropod abundance

Climate change may influence the phenology of organisms unequally across trophic levels and thus lead to phenological mismatches between predators and prey. In cases where prey availability peaks before reproducing predators reach maximal prey demand, any negative fitness consequences would selectively favor resynchronization by earlier starts of the reproductive activities of the predators. At a study site in northeast Greenland, over a period of 17 years, the median emergence of the invertebrate prey of Sanderling Calidris alba advanced with 1.27 days per year. Yet, over the same period Sanderling did not advance hatching date. Thus, Sanderlings increasingly hatched after their prey was maximally abundant. Surprisingly, the phenological mismatches did not affect chick growth, but the interaction of the annual width and height of the peak in food abundance did. Chicks grew especially better in years when the food peak was broad. Sanderling clutches were most likely to be depredated early in the season, which should delay reproduction. We propose that high early clutch predation may favor a later reproductive timing. Additionally, our data suggest that in most years food was still abundant after the median date of emergence, which may explain why Sanderlings did not advance breeding along with the advances in arthropod phenology.     

Postnatal remains of the foetal membranes affect growth and survival of wild European rabbit pups

During a long-term study on reproduction of the European rabbit (Oryctolagus cuniculus L.) that was carried out on a population living under semi-natural conditions, occasionally single pups per litter were discovered being stuck in remnants of the foetal membranes. Here we describe this so far unknown phenomenon, quantify the occurrence, and investigate the consequences for the individual growth rate and for survival. Furthermore, we tested if maternal social rank and age were correlated with the occurrence of this phenomenon. Pups comprising the described adherence of the foetal membranes had lower growth rates than their unaffected litter siblings, and usually died during the nestling period or short after emergence above ground. Our findings point out that there was an individual predisposition of the mother for having affected litters. However, an impact of maternal social rank or age on the occurrence of this feature in their offspring was not supported by our data. Nevertheless, a pathological background also has to be considered as a possible cause.     

Fractionation and characterization of rapidly phosphorylated nuclear proteins in salivary gland cells of Chironomus tentans

Rapidly phosphorylated nuclear proteins were investigated in explanted salivary gland cells of Chironomus tentans after labeling with ³²P_i. After sonication nuclei were fractionated by centrifugation at 18,000 g into sedimentable (80% of ³²P) and not sedimentable (supernatant) material. About 90% of ³²P in the supernatant fraction was sedimentable at 100,000 g (disperse chromatin). The disperse chromatin contained 20%–40% of the total nuclear DNA but only 5%–20% of ³²P. The ³²P-labeled phosphoproteins in the material pelleted at 20,000 g were further fractionated by differential solubility in lysis buffer. Electrophoretic analyses on SDS polyacrylamide gels resolved the ³²P-labeled nuclear proteins into 12 major bands in the M_r range of 12,000–120,000. The incorporation of ³²P into most bands reached a steady-state within 5–10 min of incubation with ³²P_i and was not measurably influenced by cycloheximide, an inhibitor of protein synthesis. The phosphate groups are linked to polypeptide chains by bonds vulnerable to pronase and alkaline phosphatase. All major bands in the pelleted chromatin were also present in the disperse chromatin except for an M_r 95,000 phosphoprotein. Two of the fastest moving ³²P-bands comigrated with the core histones H2A and H4. Both possessed a high pI value and were insoluble in 0.35 M NaCl. The H2A-like protein was partially soluble in lysis buffer while the H4-like one was not. The two fast moving ³²P-labeled bands with rapidly turned over phosphates may be fractions or variants of the core histones H2A and H4.     

The intercellular adhesion molecule-1 K469E polymorphism in type 1 diabetes

Type 1 (insulin-dependent) diabetes is a complex trait. The region harboring the ICAM1 gene on 19p13 links to type 1 diabetes, and a growing body of evidence indicates that intercellular adhesion molecule-1 (ICAM-1) could play a role in type 1 diabetes development. Recently, association studies of an ICAM-1 K469E polymorphism in type 1 diabetes populations have reported conflicting results. Hence, we performed a transmission disequilibrium test analysis of the ICAM-1 K469E variations in 253 Danish type 1 diabetes families. Linkage and association was not found between the ICAM-1 K469E variation and type 1 diabetes in Danish patients (P(tdt)> or =0.48), and our data did not indicate an interaction between ICAM1 and IDDM1 in predisposition to type 1 diabetes in Danes (P=0.78). We did not observe significant association with late-onset type 1 diabetes (P(tdt)> or =0.12) or differences in transmission patterns between groups of affected offspring stratified for age at onset (P> or =0.19), as suggested in Japanese patients. Combined analysis of the present and previously reported transmission data comprising 728 affected offspring of Romanian, Finnish, and Danish ancestry suggested association between the ICAM-1 E469 allele and type 1 diabetes (P(tdt)=0.013), but association was not found in the combined Scandinavian material. In conclusion, we found no association of the ICAM-1 K469E polymorphism with type 1 diabetes or its subsets stratified for age at onset and HLA risk in Danish patients. Analysis of ICAM-1 K469E transmissions reported in three populations suggested association to type 1 diabetes, but also demonstrated heterogeneity between populations.