Exercise increases the release of NAMPT in extracellular vesicles and alters NAD + activity in recipient cells

Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD⁺) declining over time. The decrease in NAD⁺ production has been linked to the age‐related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate‐limiting enzyme in the NAD⁺ biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV‐eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD⁺ and SIRT1 activity in recipient cells compared to pre‐exercise EVs, indicating a pathway for inter‐tissue signalling promoted through exercise. Our results suggest a mechanism to limit age‐related NAD⁺ decline, through the systemic delivery of eNAMPT via EVs released during exercise.     

Developing a vision for improved monitoring and reporting of riparian restoration projects

Representatives from agencies involved in natural resource management in the Murray‐Darling Basin gathered for a workshop in November 2010 to develop a vision for improved monitoring and reporting of riparian restoration projects. The resounding message from this workshop was that the effectiveness of riparian restoration depends on having sound, documented and agreed evidence on the ecological responses to restoration efforts. Improving our capacity to manage and restore riparian ecosystems is constrained by (i) a lack of ecological evidence on the effects of restoration efforts, and (ii) short‐termism in commitment to restoration efforts, in funding of monitoring and in expected time spans for ecosystem recovery. Restoration at the effective spatial scope will invariably require a long‐term commitment by researchers, funding agencies, management agencies and landholders. To address the knowledge gaps that constrain riparian restoration in the Basin, participants endorsed four major fields for future research: the importance of landscape context to restoration outcomes; spatio‐temporal scaling of restoration outcomes; functional effects of restoration efforts; and developing informative and effective indicators of restoration. To improve the monitoring and restoration of riparian zones throughout the Basin, participants advocated an integrated approach: a hierarchical adaptive management framework that incorporates long‐term ecological research.     

A Homogeneous,High-Throughput Assay for Phosphatidylinositol 5-Phosphate 4-Kinase with a Novel,Rapid Substrate Preparation

Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-³²P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC₅₀ of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.     

Distribution Survey and Threat Assessment of the Yellow-tailed Woolly Monkey (<Emphasis Type="BoldItalic">Oreonax flavicauda;</Emphasis> Humboldt 1812), Northeastern Peru

Peruvian yellow-tailed woolly monkeys (Oreonax flavicauda) are considered Critically Endangered (IUCN Categories A4c). The International Primatological Society also considers them one of the world’s 25 most endangered primate species and therefore a conservation priority. However, there is little concerted conservation action, and the existing protected area network may be inadequate to protect this species from extinction. Until recently this species has been the focus of few studies and its distributional limits remain unknown. I present results of a range-wide survey of Oreonax flavicauda in northeastern Peru. I conducted 53 presence/absence field surveys at 43 sites between March 2007 and March 2010, with data collected for an additional 7 sites from other researchers. I chose sites where the species was previously reported or following suggestions from predictive GIS modeling. Oreonax flavicauda was present at 35 sites, all presence records were in Ficus spp.–dominated cloud forests between 1500 and 2650 m above sea level. I give the geographical limits of this species distribution throughout the north, east, and west of its range; the exact extent of its range to the south requires further investigation. Oreonax flavicauda continues to be threatened throughout its range. The major threats I identified at the survey locations were the continued conversion of forests to cattle pasture, opening of new access routes into virgin areas, and both commercial and subsistence hunting. My results suggest that existing conservation measures may be inadequate at protecting this species but that substantial opportunities do exist. Further surveys need to be made in the southern distribution of this species to determine more accurately extant habitat.     

A C-terminal motif targets Hedgehog to axons, coordinating assembly of the Drosophila eye and brain

The developmental signal Hedgehog is distributed to two receptive fields by the photoreceptor neurons of the developing Drosophila retina. Delivery to the retina propagates ommatidial development across a precursor field. Transport along photoreceptor axons induces the development of postsynaptic neurons in the brain. Hedgehog is composed of N-terminal and C-terminal domains that dissociate in an autoproteolytic reaction that attaches cholesterol to the N-terminal cleavage product. Here, we show that the N-terminal domain is targeted to the retina when synthesized in the absence of the C-terminal domain. In contrast to studies that have focused on cholesterol as a determinant of subcellular localization, we find that the C-terminal domain harbors a conserved motif that overrides retinal localization, sending most of the autocleavage products into vesicles bound for growth cones or synapses. Competition between targeting signals at the opposite ends of Hedgehog apparently controls the match between eye and brain development.     

Thermal stability of turnip yellow mosaic virus RNA: effect of pH and multivalent cations on RNA deaggregation and degradation.

Light scattering studies of RNA isolated from turnip yellow mosaic virus (TYMV) revealed a molar mass of 1.9.10(6) g mol-1, which is close to the value of 2.0.10(6) g mol-1 published for intact genomic TYMV RNA (2M RNA). However, gel electrophoresis under denaturing conditions demonstrated that only 30-40% of this native RNA was 2M RNA. Sucrose gradient centrifugation revealed the occurrence of a series of smaller RNA size classes, the mass ratios of which were greatly influenced by the pH of the solution and the presence of EDTA. These results suggest that native TYMV RNA preparations originally contain a mixture of intact RNA particles and of aggregates of RNA fragments with the same molar mass of about 2.10(6) g mol-1, and that the size classes are intermediates in the deaggregation process of the degraded genomic TYMV RNA. The native RNA displayed pH-dependent deaggregation and degradation. The degradation process of 2M RNA followed (pseudo) first-order kinetics. Lower degradation rates were observed for RNA depleted of divalent cations and polyamines. For depleted 2M RNA an enthalpy of activation of about 100 kJ mol-1 and an almost zero entropy of activation was calculated. Similar values were also found for depleted E. coli ribosomal RNAs and depleted MS2 RNA, demonstrating that all RNAs are equally vulnerable to degradation. In the presence of multivalent cations the activation enthalpy for 2M TYMV RNA degradation increased to 150 kJ mol-1 and the entropy of activation to 150 J K-1 mol-1, indicative for a different degradation mechanism.     

Substrate and Target Sequence Length Influence RecTEPsy Recombineering Efficiency in Pseudomonas syringae

We are developing a new recombineering system to assist experimental manipulation of the Pseudomonas syringae genome. P. syringae is a globally dispersed plant pathogen and an important model species used to study the molecular biology of bacteria-plant interactions. We previously identified orthologs of the lambda Red bet/exo and Rac recET genes in P. syringae and confirmed that they function in recombineering using ssDNA and dsDNA substrates. Here we investigate the properties of dsDNA substrates more closely to determine how they influence recombineering efficiency. We find that the length of flanking homologies and length of the sequences being inserted or deleted have a large effect on RecTE_Psy mediated recombination efficiency. These results provide information about the design elements that should be considered when using recombineering.