A high-fat diet suppresses de novo lipogenesis and desaturation but not elongation and triglyceride synthesis in mice

Intracellular lipids and their synthesis contribute to the mechanisms and complications of obesity-associated diseases. We describe an NMR approach that provides an abbreviated lipidomic analysis with concurrent lipid biosynthetic fluxes. Following deuterated water administration, positional isotopomer analysis by deuterium NMR of specific lipid species was used to examine flux through de novo lipogenesis (DNL), FA elongation, desaturation, and TG-glycerol synthesis. The NMR method obviated certain assumptions regarding sites of enrichment and exchangeable hydrogens required by mass isotope methods. The approach was responsive to genetic and pharmacological gain or loss of function of DNL, elongation, desaturation, and glyceride synthesis. BDF1 mice consuming a high-fat diet (HFD) or matched low-fat diet for 35 weeks were examined across feeding periods to determine how flux through these pathways contributes to diet induced fatty liver and obesity. HFD mice had increased rates of FA elongation and glyceride synthesis. However DNL was markedly suppressed despite insulin resistance and obesity. We conclude that most hepatic TGs in the liver of HFD mice were formed from the reesterification of existing or ingested lipids, not DNL.     

Task-Specific Response Strategy Selection on the Basis of Recent Training Experience

The goal of training is to produce learning for a range of activities that are typically more general than the training task itself. Despite a century of research, predicting the scope of learning from the content of training has proven extremely difficult, with the same task producing narrowly focused learning strategies in some cases and broadly scoped learning strategies in others. Here we test the hypothesis that human subjects will prefer a decision strategy that maximizes performance and reduces uncertainty given the demands of the training task and that the strategy chosen will then predict the extent to which learning is transferable. To test this hypothesis, we trained subjects on a moving dot extrapolation task that makes distinct predictions for two types of learning strategy: a narrow model-free strategy that learns an input-output mapping for training stimuli, and a general model-based strategy that utilizes humans'' default predictive model for a class of trajectories. When the number of distinct training trajectories is low, we predict better performance for the mapping strategy, but as the number increases, a predictive model is increasingly favored. Consonant with predictions, subject extrapolations for test trajectories were consistent with using a mapping strategy when trained on a small number of training trajectories and a predictive model when trained on a larger number. The general framework developed here can thus be useful both in interpreting previous patterns of task-specific versus task-general learning, as well as in building future training paradigms with certain desired outcomes.     

Quadriceps tendon allografts as an alternative to Achilles tendon allografts: a biomechanical comparison

Quadriceps tendon with a patellar bone block may be a viable alternative to Achilles tendon for anterior cruciate ligament reconstruction (ACL-R) if it is, at a minimum, a biomechanically equivalent graft. The objective of this study was to directly compare the biomechanical properties of quadriceps tendon and Achilles tendon allografts. Quadriceps and Achilles tendon pairs from nine research-consented donors were tested. All specimens were processed to reduce bioburden and terminally sterilized by gamma irradiation. Specimens were subjected to a three phase uniaxial tension test performed in a custom environmental chamber to maintain the specimens at a physiologic temperature (37 ± 2 °C) and misted with a 0.9 % NaCl solution. There were no statistical differences in seven of eight structural and mechanical between the two tendon types. Quadriceps tendons exhibited a significantly higher displacement at maximum load and significantly lower stiffness than Achilles tendons. The results of this study indicated a biomechanical equivalence of aseptically processed, terminally sterilized quadriceps tendon grafts with bone block to Achilles tendon grafts with bone block. The significantly higher displacement at maximum load, and lower stiffness observed for quadriceps tendons may be related to the failure mode. Achilles tendons had a higher bone avulsion rate than quadriceps tendons (86 % compared to 12 %, respectively). This was likely due to observed differences in bone block density between the two tendon types. This research supports the use of quadriceps tendon allografts in lieu of Achilles tendon allografts for ACL-R.     

Toxicity and localization studies of a potential photodynamic therapy agent in Drosophila

Photodynamic therapy utilizes light, a photosensitizer, and molecular oxygen as a treatment modality for a variety of cancers. We have recently combined ruthenium(II) polypyridyl groups with a zinc(II) centered porphyrin as a new photosensitizer for the treatment of melanoma. In‐vitro studies have indicated that this photosensitizer is toxic to melanoma cells when irradiated with low energy light; however, it is nontoxic to normal cells under similar conditions. To determine the toxicity and cell viability of this compound in‐vivo we present, herein, a study using Drosophila melanogaster. In the absence of light, the new photosensitizer shows no discernible effects to fly larvae at various concentrations of compound and stages of larval development. When the larvae were fed the photosensitizer it was observed, by fluorescence microscopy, that the compound passes through the cell membrane and localizes in the cytosol at lower concentrations and the nucleus at slightly higher concentrations indicating that the compound is not immediately metabolized. genesis 52:309–314, 2014. © 2014 Wiley Periodicals, Inc.     

Novel Src homology 3 domain-binding motifs identified from proteomic screen of a Pro-rich region

The Src homology (SH) 3 domain has been shown recently to bind peptide sequences that lack the canonical PXXP motif. The diverse specificity in ligand recognition for a group of 15 SH3 domains has now been investigated using arrays of peptides derived from the proline-rich region of the SH2 domain-containing leukocyte protein of 76 kDa (SLP-76). A screen of the peptide arrays using individual or mixed SH3 domains has allowed the identification of a number of candidate SH3-binding peptides. Although some peptides contain the conventional PXXP motif, most are devoid of such a motif and are instead enriched in basic residues. Fluorescent polarization measurements using soluble peptides and purified SH3 domains demonstrated that several SH3 domains, including those from growth factor receptor-bound protein 2 (Grb2), NCK, and phospholipase C (PLC)-gamma1, bound with moderate affinities (10-100 microm) to a group of non-conventional peptides. Of particular interest, the PLC-gamma1 SH3 domain was found to associate with SLP-76 through at least three distinct sites, two of which bore a novel KKPP motif and the other contained the classic PXXP sequence. Intriguingly mutation of critical residues for the three sites not only affected binding of SLP-76 to the PLC-gamma1 SH3 domain but also to the Grb2 C-terminal SH3 domain, indicating that the binding sites in SLP-76 for the two SH3 domains are overlapped. Our studies suggest that the SH3 domain is an inherently promiscuous interaction module capable of binding to peptides that may or may not contain a PXXP motif. Furthermore the identification of numerous non-conventional SH3-binding peptides in SLP-76 implies that the global ligand pool for SH3 domains in a mammalian proteome may be significantly greater than previously acknowledged.     

Optimization of the multiple enzymatic activities of the hepatitis C virus NS3 protein

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is known to possess multiple enzymatic activities. In addition to its well-characterized protease activity, HCV NS3 also has ATP hydrolase (ATPase) and nucleic acid unwinding (helicase) activities. We systematically studied the effect of common reagents on all three enzymatic activities with a view to improving assay sensitivity for compound screening and profiling. Inclusion of the detergent lauryl dimethylamine oxide (LDAO) improves protease and helicase activities significantly, allowing robust assays at much lower NS3 concentrations. These conditions enable a particularly sensitive protease assay that uses picomolar concentrations of NS3.     

Characterization and Identification of Hepatic mRNA Related to Copper Metabolism and Homeostasis in Cattle

Copper is an essential trace mineral required for growth and development. Copper homeostasis within the cell is mediated by the expression of the Cu transporter protein (CTR1), ATPase7A (ATP7A), ATPase7B (ATP7B), Cox17, and Cu chaperone for Cu–Zn superoxide dismutase (CCS) which helps to regulate Cu uptake, export, and intracellular compartmentalization in non-ruminants. Copper also serves as a cofactor of antioxidant, superoxide dismutase1 (SOD1). Liver tissue from eighteen Holstein bull calves (average BW 201?±?58.5 kg, 7.3?±?1.9 months) from a previous experiment were utilized to characterize and identify hepatic mRNA related to Cu metabolism and homeostasis in cattle. Hepatic Cu concentration was determined via flame atomic absorption, and total RNA was extracted using TRI reagent and purified using RNeasy. Hepatic Cu concentrations ranged from 86 to 801 mg of Cu/kg DM. Real-time polymerase chain reaction analysis revealed that CTR1, ATP7A, and ATP7B mRNA expressions were negatively correlated with hepatic Cu concentration, while CCS (P?=?0.0887) and SOD1 had a tendency (P?=?0.0733) to be negatively correlated to hepatic Cu concentration. These data indicate that higher than normal hepatic Cu concentration downregulates gene expression of CTR1, ATP7A, ATP7B, and Cox17, which are involved in bovine liver copper homeostasis.