The effect of prehydrolysis and improved mixing on high-solids batch simultaneous saccharification and fermentation of spruce to ethanol

In the production of ethanol from lignocellulosic material, it is necessary to reach a high ethanol concentration after fermentation. Simply increasing the substrate concentration leads to stirring problems and inhibition of the enzymes and yeast in the process.Batch simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce with 13.7% water-insoluble solids (WIS) (25% total solids (TS)) was run in a stirred-tank reactor as well as in two reactors designed to handle solid or semi-solid material. In all reactors, the overall ethanol yields were only between 5 and 6%. Fermentation of the liquid fraction of the steam-pretreated spruce slurry resulted in an overall ethanol yield of 85%.22 h of prehydrolysis at 48 °C prior to SSF at 32 °C significantly increased the overall ethanol yield to 72% (final ethanol concentration of 47.8 g/L), using the whole slurry of steam-pretreated spruce at a dry matter content of 13.7% WIS (25% TS).     

Plant survival in Iceland during periods of glaciation?

Aim The paper addresses the classical question of possible plant survival in Iceland during the last glacial period in the light of a palaeobotanical record from northern Iceland, spanning the period 11,300–9000 BP , including the Younger Dryas stadial. We review the Late Cenozoic fossil plant record, the past debate on glacial plant refugia in Iceland, and the evidence for ice-free areas during the Weichselian. Location The investigated lake sediment record comes from Lake Torfadalsvatn, which is situated in the northwestern part of the Skagi peninsula in northern Iceland. Methods The sediment chronology was constructed from the cccurrence of the Vedde Ash and the Saksunarvatn ash, two well-dated Icelandic tephras, together with the results from five AMS and conventional radiocarbon dates performed on bulk sediment samples. The vegetational reconstruction was based on detailed pollen analysis of the sediment sequence. Results The pollen analysis revealed that many of the taxa present in the area prior to the Younger Dryas stadial continued to produce pollen during that cold event. The more or less immediate reappearance of a few other pollen taxa at the Younger Dryas-Preboreal boundary suggests that these plants also survived, even if they did not produce sufficient pollen to be recorded during the Younger Dryas stadial. Main conclusions We conclude that the relatively high plant diversity found in high Arctic areas and present-day nunataks in Iceland and Greenland, together with the fact that many plant species were able to survive the Younger Dryas stadial on the Skagi peninsula, suggest that species with high tolerance for climate fluctuations also survived the whole Weichselian in Iceland. This conclusion is supported by recent palaeoclimatic data from ice-cores and deep-sea sediments, indicating that Icelandic climate during the last glacial was only occasionally slightly colder than during the Younger Dryas stadial.     

Matrilin-3 is dispensable for mouse skeletal growth and development

Matrilin-3 belongs to the matrilin family of extracellular matrix (ECM) proteins and is primarily expressed in cartilage. Mutations in the gene encoding human matrilin-3 (MATN-3) lead to autosomal dominant skeletal disorders, such as multiple epiphyseal dysplasia (MED), which is characterized by short stature and early-onset osteoarthritis, and bilateral hereditary microepiphyseal dysplasia, a variant form of MED characterized by pain in the hip and knee joints. To assess the function of matrilin-3 during skeletal development, we have generated Matn-3 null mice. Homozygous mutant mice appear normal, are fertile, and show no obvious skeletal malformations. Histological and ultrastructural analyses reveal endochondral bone formation indistinguishable from that of wild-type animals. Northern blot, immunohistochemical, and biochemical analyses indicated no compensatory upregulation of any other member of the matrilin family. Altogether, our findings suggest functional redundancy among matrilins and demonstrate that the phenotypes of MED disorders are not caused by the absence of matrilin-3 in cartilage ECM.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

A novel mammalian display system for the selection of protein-protein interactions by decoy receptor engagement

The emerging field of proteomics has created a need for new high-throughput methodologies for the analysis of gene products. An attractive approach is to develop systems that allow for clonal selection of interacting protein pairs from large molecular libraries. In this study, we have characterized a novel approach for identification and selection of protein-protein interactions, denoted SPIRE (selection of protein interactions by receptor engagement), which is based on a mammalian expression system. We have demonstrated proof of concept by creating a general plasma membrane bound decoy receptor, by displaying a protein or a peptide genetically fused to a trunctated version of the CD40 molecule. When this decoy receptor is engaged by a ligand to the displayed protein/peptide, the receptor expressing cell is rescued from apoptosis. To design a high-throughput system with a highly parallel capacity, we utilized the B cell line WEHI-231, as carrier of the decoy receptor. One specific peptide-displaying cell could be identified and amplified, based on a specific receptor engagement, in a background of 12 500 wild-type cells after four selections. This demonstrates that the approach may serve as a tool in post-genomic research for identifying protein-protein interactions, without prior knowledge of either component.     

Top-down approaches to the study of natural variation in complex physiological pathways using the white-footed mouse (Peromyscus leucopus) as a model

Variation in complex physiological pathways has important effects on human function and medical treatment. Complex pathways involve cells at multiple locations, which serve different functions regulated by many genes and include complex neuroendocrine pathways that regulate physiological function. One of two competing hypotheses regarding the effects of selection on complex pathways predicts that variability should be common within complex pathways. If this hypothesis is correct, then we should expect wide variation in neuroendocrine function to be typical within natural populations. To test this hypothesis, a complex neuroendocrine pathway that regulates photoperiod-dependent changes in fertility in a natural population of white-footed mice (Peromyscus leucopus) was used to test for natural genetic variability in multiple components of the pathway. After testing only six elements in the photoperiod pathway in P. leucopus, genetic variation in the following four of these elements was evident: the circadian clock, melatonin receptor abundance or affinity, sensitivity of the reproductive axis to steroid negative feedback, and gonadotropin-releasing hormone neuronal activity. If this result can be extended to humans, the prediction would be that significant variation at multiple loci in complex neuroendocrine pathways is common among humans, and that variation would exist even in human populations from a common genetic background. This finding could only be drawn from an "exotic" animal model derived from a natural source population, confirming the continuing importance of nontraditional models alongside the standard laboratory species.     

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.     

Comparison of family 12 glycoside hydrolases and recruited substitutions important for thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have studied the biochemical diversity of several GH 12 homologs. The H. schweinitzii Cel12A enzyme differs from the T. reesei Cel12A enzyme by only 14 amino acids (93% sequence identity), but is much less thermally stable. The bacterial Cel12A enzyme from S. sp. 11AG8 shares only 28% sequence identity to the T. reesei enzyme, and is much more thermally stable. Each of the 14 sequence differences from H. schweinitzii Cel12A were introduced in T. reesei Cel12A to determine the effect of these amino acid substitutions on enzyme stability. Several of the T. reesei Cel12A variants were found to have increased stability, and the differences in apparent midpoint of thermal denaturation (T(m)) ranged from a 2.5 degrees C increase to a 4.0 degrees C decrease. The least stable recruitment from H. schweinitzii Cel12A was A35S. Consequently, the A35V substitution was recruited from the more stable S. sp. 11AG8 Cel12A and this T. reesei Cel12A variant was found to have a T(m) 7.7 degrees C higher than wild type. Thus, the buried residue at position 35 was shown to be of critical importance for thermal stability in this structural family. There was a ninefold range in the specific activities of the Cel12 homologs on o-NPC. The most and least stable T. reesei Cel12A variants, A35V and A35S, respectively, were fully active. Because of their thermal tolerance, S. sp. 11AG8 Cel12A and T. reesei Cel12A variant A35V showed a continual increase in activity over the temperature range of 25 degrees C to 60 degrees C, whereas the less stable enzymes T. reesei Cel12A wild type and the destabilized A35S variant, and H. schweinitzii Cel12A showed a decrease in activity at the highest temperatures. The crystal structures of the H. schweinitzii, S. sp. 11AG8, and T. reesei A35V Cel12A enzymes have been determined and compared with the wild-type T. reesei Cel12A enzyme. All of the structures have similar Calpha traces, but provide detailed insight into the nature of the stability differences. These results are an example of the power of homolog recruitment as a method for identifying residues important for stability.