QTL Mapping of Enzymatic Saccharification in Short Rotation Coppice Willow and Its Independence from Biomass Yield

Short rotation coppice (SRC) willows (Salix spp.) are fast-growing woody plants which can achieve high biomass yields over short growth cycles with low agrochemical inputs. Biomass from SRC willow is already used for heat and power, but its potential as a source of lignocellulose for liquid transport biofuels has still to be assessed. In bioethanol production from lignocellulose, enzymatic saccharification is used as an approach to release glucose from cellulose in the plant cell walls. In this study, 138 genotypes of a willow mapping population were used to examine variation in enzymatic glucose release from stem biomass to study relationships between this trait and biomass yield traits and to identify quantitative trait loci (QTL) associated with enzymatic saccharification yield. Significant natural variation was found in glucose yields from willow stem biomass. This trait was independent of biomass yield traits. Four enzyme-derived glucose QTL were mapped onto chromosomes V, X, XI, and XVI, indicating that enzymatic saccharification yields are under significant genetic influence. Our results show that SRC willow has strong potential as a source of bioethanol and that there may be opportunities to improve the breeding programs for willows for increasing enzymatic saccharification yields and biofuel production.     

Light controllable siRNAs regulate gene suppression and phenotypes in cells

Small interfering RNA (siRNA) is widely recognized as a powerful tool for targeted gene silencing. However, siRNA gene silencing occurs during transfection, limiting its use is in kinetic studies, deciphering toxic and off-target effects and phenotypic assays requiring temporal, and/or spatial regulation. We developed a novel controllable siRNA (csiRNA) that is activated by light. A single photo removable group is coupled during oligonucleotide synthesis to the 5' end of the antisense strand of the siRNA, which blocks the siRNA's activity. A low dose of light activates the siRNA, independent of transfection resulting in knock down of specific target mRNAs and proteins (GAPDH, p53, survivin, hNuf2) without stimulating non-specific effects such as regulated protein kinase PKR and induction of the interferon response. We demonstrate survivin and hNuf2 csiRNAs temporally knockdown their mRNAs causing multinucleation and cell death by mitotic arrest, respectively. Furthermore, we demonstrate a dose-dependent light regulation of hNuf2 csiRNA activity and resulting phenotype. The light controllable siRNAs are introduced into cells using commercially available reagents including the MPG peptide based delivery system. The csiRNAs are comparable to standard siRNAs in their transfection efficiency and potency of gene silencing. This technology should be of interest for phenotypic assays such as cell survival, cell cycle regulation, and cell development.     

Tyrosine plays a dominant functional role in the paratope of a synthetic antibody derived from a four amino acid code

The antigen-binding fragment Fab-YADS2 recognizes vascular endothelial growth factor (VEGF) and was derived from a library with chemical diversity restricted to only four amino acids (Tyr, Ser, Ala and Asp). The structure of the Fab:antigen complex revealed that the structural paratope is dominated by Tyr side-chains. Isothermal titration calorimetry and cell-based assays show that restricted chemical diversity does not limit the affinity or specificity of Fab-YADS2, which behaves in a manner comparable to natural antibodies. Mutagenesis experiments reveal that the functional paratope is dominated by Tyr, which represents 11 of the 15 functionally important residues. However, mutagenesis experiments also indicate that substitution of any of these tyrosine residues by Phe does not significantly affect binding to VEGF. Furthermore, saturation mutagenesis shows that replacement of three functionally important tyrosine residues by combinations of other hydrophobic residues is not only tolerated, but can actually improve affinity. The results support a model for na?ve antigen recognition in which large Tyr side-chains establish binding contacts with antigen, and small Ser and Ala side-chains serve as auxiliaries that help to position Tyr in favorable binding conformations. While Tyr may not be optimal for any particular antigen contact, it is nonetheless capable of mediating favorable interactions with a diverse array of surfaces. Furthermore, the side-chain hydroxyl group makes Tyr significantly more hydrophilic than Phe and other hydrophobic amino acids. Increased hydrophilicity may reduce non-specific binding in the unbound state, and this may be critical for a na?ve repertoire that is exposed to a diverse range of potential antigenic surfaces. The results show that the chemical nature of Tyr endows the amino acid with a privileged role in antigen recognition, and this likely explains the high abundance of Tyr in natural antigen-binding sites.     

Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy balance and of the effects of leptin on food intake and fatty acid oxidation. Obesity is usually associated with resistance to the effects of leptin on food intake and body weight. To determine whether diet-induced obesity (DIO) impairs the AMPK response to leptin in muscle and/or hypothalamus, we fed FVB mice a high fat (55%) diet for 10-12 weeks. Leptin acutely decreased food intake by approximately 30% in chow-fed mice. DIO mice tended to eat less, and leptin had no effect on food intake. Leptin decreased respiratory exchange ratio in chow-fed mice indicating increased fatty acid oxidation. Respiratory exchange ratio was low basally in high fat-fed mice, and leptin had no further effect. Leptin (3 mg/kg intraperitoneally) increased alpha2-AMPK activity 2-fold in muscle in chow-fed mice but not in DIO mice. Leptin decreased acetyl-CoA carboxylase activity 40% in muscle from chow-fed mice. In muscle from DIO mice, acetyl-CoA carboxylase activity was basally low, and leptin had no further effect. In paraventricular, arcuate, and medial hypothalamus of chow-fed mice, leptin inhibited alpha2-AMPK activity but not in DIO mice. In addition, leptin increased STAT3 phosphorylation 2-fold in arcuate of chow-fed mice, but this effect was attenuated because of elevated basal STAT3 phosphorylation in DIO mice. Thus, DIO in FVB mice alters alpha2-AMPK in muscle and hypothalamus and STAT3 in hypothalamus and impairs further effects of leptin on these signaling pathways. Defective responses of AMPK to leptin may contribute to resistance to leptin action on food intake and energy expenditure in obese states.     

Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha

Diminished mitochondrial oxidative phosphorylation and aerobic capacity are associated with reduced longevity. We tested whether resveratrol (RSV), which is known to extend lifespan, impacts mitochondrial function and metabolic homeostasis. Treatment of mice with RSV significantly increased their aerobic capacity, as evidenced by their increased running time and consumption of oxygen in muscle fibers. RSV's effects were associated with an induction of genes for oxidative phosphorylation and mitochondrial biogenesis and were largely explained by an RSV-mediated decrease in PGC-1alpha acetylation and an increase in PGC-1alpha activity. This mechanism is consistent with RSV being a known activator of the protein deacetylase, SIRT1, and by the lack of effect of RSV in SIRT1(-/-) MEFs. Importantly, RSV treatment protected mice against diet-induced-obesity and insulin resistance. These pharmacological effects of RSV combined with the association of three Sirt1 SNPs and energy homeostasis in Finnish subjects implicates SIRT1 as a key regulator of energy and metabolic homeostasis.     

Anisotropic lattice distortions in biogenic calcite induced by intra-crystalline organic molecules

We have performed precise structural measurements on five different calcitic seashells by high-resolution X-ray powder diffraction on a synchrotron beam line and by laboratory single crystal X-ray diffraction. The unit cell parameters a and c of biogenic calcite were found to be systematically larger than those measured in the non-biogenic calcite. The maximum lattice distortion (about 2.10(-3)) was detected along the c-axis. Under heat treatment above 200 degrees C, a pronounced lattice relaxation was observed, which allowed us to conclude that anisotropic lattice swelling in biogenic calcite is induced by organic macromolecules incorporated within the single crystal calcitic prisms during biomineralization. This conclusion is supported by the results of crystallization experiments in the presence of specific protein extracted from one of the shells.