A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Safety,Immunogenicity and Duration of Immunity Elicited by an Inactivated Bovine Ephemeral Fever Vaccine

Bovine ephemeral fever (BEF) is an economically important viral vector-borne cattle disease. Several live-attenuated, inactivated and recombinant vaccines have been tested, demonstrating varying efficacy. However, to the best of our knowledge, duration of immunity conferred by an inactivated vaccine has never been reported. In the last decade, Israel has faced an increasing number of BEF outbreaks. The need for an effective vaccine compatible with strains circulating in the Middle East region led to the development of a MONTANIDE™ ISA 206 VG (water-in-oil-in-water), inactivated vaccine based on a local strain. We tested the safety, immunogenicity and duration of immunity conferred by this vaccine. The induced neutralizing antibody (NA) response was followed for 493 days in 40 cows vaccinated by different protocols. The vaccine did not cause adverse reactions or a decrease in milk production. All cows [except 2 (6.7%) which did not respond to vaccination] showed a significant rise in NA titer of up to 1:256 following the second, third or fourth booster vaccination. Neutralizing antibody levels declined gradually to 1:16 up to 120 days post vaccination. This decline continued in cows vaccinated only twice, whereas cows vaccinated 3 or 4 times showed stable titers of approximately 1:16 for up to 267 days post vaccination. At least three vaccinations with the inactivated BEF vaccine were needed to confer long-lasting immunity. These results may have significant implications for the choice of vaccination protocol with inactivated BEF vaccines. Complementary challenge data should however be added to the above results in order to determine what is the minimal NA response conferring protection from clinical disease.     

Significance of the Natural Occurrence of L‐ Versus D‐Pipecolic Acid: A Review

Pipecolic acid naturally occurs in microorganisms, plants, and animals, where it plays many roles, including the interactions between these organisms, and is a key constituent of many natural and synthetic bioactive molecules. This article provides a review of current knowledge on the natural occurrence of pipecolic acid and the known and potential significance of its L‐ and D‐enantiomers in different scientific disciplines. Knowledge gaps with perspectives for future research identified within this article include the roles of the L‐ versus the D‐enantiomer of pipecolic acid in plant resistance, nutrient acquisition, and decontamination of polluted soils, as well as rhizosphere ecology and medical issues. Chirality 25:823–831, 2013. © 2013 Wiley Periodicals, Inc.     

Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Human dUTPase, essential for DNA integrity, is an important survival factor for cancer cells. We determined the crystal structure of the enzyme:alpha,beta-imino-dUTP:Mg complex and performed equilibrium binding experiments in solution. Ordering of the C-terminus upon the active site induces close juxtaposition of the incoming nucleophile attacker water oxygen and the alpha-phosphorus of the substrate, decreasing their distance below the van der Waals limit. Complex interactions of the C-terminus with both substrate and product were observed via a specifically designed tryptophan sensor, suitable for further detailed kinetic and ligand binding studies. Results explain the key functional role of the C-terminus.     

The heterotrimeric G protein G(i3) regulates hepatic autophagy downstream of the insulin receptor

Compelling evidence suggests that the heterotrimeric G protein G(i3) specifically transmits the antiautophagic effects of insulin and amino acids in the liver. This points to a previously unrecognized cross talk between the insulin receptor tyrosine kinase and G(i3). Interestingly, G(i3) is localized not only to plasma membranes but also to membranes of the autophagosomal compartment. Furthermore, as part of insulin's or phenylalanine's actions to inhibit autophagy, G(i3) is redistributed away from autophagosomes. Therefore, endomembrane-associated rather than plasma membrane-localized G(i3) may serve as the target of insulin's endocrine and metabolic actions. We therefore propose that the function and regulation of organelle-associated heterotrimeric G proteins may be different from their roles at the plasma membrane where they act as signal transducers of seven-transmembrane receptors. Here, we discuss recent findings and propose a function for G(i3) in mTOR-dependent signaling pathways. We hypothesize that G(i) family members may have tissue-specific roles in the regulation of autophagy under different physiological and pathological conditions.     

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein

Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.     

Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor

Systemic bile acid (BA) homeostasis is a critical determinant of dietary fat digestion, enterohepatic function, and postprandial thermogenesis. However, major checkpoints for the dynamics and the molecular regulation of BA homeostasis remain unknown. Here we show that hypothalamic-pituitary-adrenal (HPA) axis impairment in humans and liver-specific deficiency of the glucocorticoid receptor (GR) in mice disrupts the normal changes in systemic BA distribution during the fasted-to-fed transition. Fasted mice with hepatocyte-specific GR knockdown had smaller gallbladder BA content and were more susceptible to developing cholesterol gallstones when fed a cholesterol-rich diet. Hepatic GR deficiency impaired liver BA uptake/transport via lower expression of the major hepatocyte basolateral BA transporter, Na(+)-taurocholate transport protein (Ntcp/Slc10a1), which affected dietary fat absorption and brown adipose tissue activation. Our results demonstrate a role of the HPA axis in the endocrine regulation of BA homeostasis through the liver GR control of enterohepatic BA recycling.     

Application of Bayesian evidence synthesis to modelling the effect of ketogenic therapy on survival of high grade glioma patients

Background

Ketogenic therapy in the form of ketogenic diets or calorie restriction has been proposed as a metabolic treatment of high grade glioma (HGG) brain tumors based on mechanistic reasoning obtained mainly from animal experiments. Given the paucity of clinical studies of this relatively new approach, our goal is to extrapolate evidence from the greater number of animal studies and synthesize it with the available human data in order to estimate the expected effects of ketogenic therapy on survival in HGG patients. At the same time we are using this analysis as an example for demonstrating how Bayesianism can be applied in the spirit of a circular view of evidence.

Results

A Bayesian hierarchical model was developed. Data from three human cohort studies and 17 animal experiments were included to estimate the effects of four ketogenic interventions (calorie restriction/ketogenic diets as monotherapy/combination therapy) on the restricted mean survival time ratio in humans using various assumptions for the relationships between humans, rats and mice. The impact of different biological assumptions about the relevance of animal data for humans as well as external information based on mechanistic reasoning or case studies was evaluated by specifying appropriate priors. We provide statistical and philosophical arguments for why our approach is an improvement over existing (frequentist) methods for evidence synthesis as it is able to utilize evidence from a variety of sources. Depending on the prior assumptions, a 30–70% restricted mean survival time prolongation in HGG patients was predicted by the models. The highest probability of a benefit (>?90%) for all four ketogenic interventions was obtained when adopting an enthusiastic prior based on previous case reports together with assuming synergism between ketogenic therapies with other forms of treatment. Combinations with other treatments were generally found more effective than ketogenic monotherapy.

Conclusions

Combining evidence from both human and animal studies is statistically possible using a Bayesian approach. We found an overall survival-prolonging effect of ketogenic therapy in HGG patients. Our approach is best compatible with a circular instead of hierarchical view of evidence and easy to update once more data become available.

     

Immobilized metal affinity chromatography optimized for the analysis of extracellular phosphorylation

Phosphorylation is the most widely studied posttranslational modification. Its role within the cell has been the focus of numerous large‐scale studies. Recently there is growing evidence on the biological significance of extracellular phosphorylation. The analysis of these phosphopeptides is complicated by the abundance of glycosylation in the extracellular space, since glycopeptides are also enriched by the methods used for phosphopeptide isolation. Thus, we optimized IMAC for phosphorylation analysis of secreted proteins, specifically in human serum. Selectivity and efficiency of different enrichment conditions used in earlier large‐scale phosphoproteomic studies were evaluated. We found that minimizing hydrophilic interactions in the enrichment allowed selective phosphopeptide isolation. Using a two‐step IMAC enrichment protocol under these conditions led to the identification of ～100 phosphorylation sites from the tryptic digest of as little as 40 μL human serum.