Biochemical and High Throughput Microscopic Assessment of Fat Mass in Caenorhabditis Elegans

The nematode C. elegans has emerged as an important model for the study of conserved genetic pathways regulating fat metabolism as it relates to human obesity and its associated pathologies. Several previous methodologies developed for the visualization of C. elegans triglyceride-rich fat stores have proven to be erroneous, highlighting cellular compartments other than lipid droplets. Other methods require specialized equipment, are time-consuming, or yield inconsistent results. We introduce a rapid, reproducible, fixative-based Nile red staining method for the accurate and rapid detection of neutral lipid droplets in C. elegans. A short fixation step in 40% isopropanol makes animals completely permeable to Nile red, which is then used to stain animals. Spectral properties of this lipophilic dye allow it to strongly and selectively fluoresce in the yellow-green spectrum only when in a lipid-rich environment, but not in more polar environments. Thus, lipid droplets can be visualized on a fluorescent microscope equipped with simple GFP imaging capability after only a brief Nile red staining step in isopropanol. The speed, affordability, and reproducibility of this protocol make it ideally suited for high throughput screens. We also demonstrate a paired method for the biochemical determination of triglycerides and phospholipids using gas chromatography mass-spectrometry. This more rigorous protocol should be used as confirmation of results obtained from the Nile red microscopic lipid determination. We anticipate that these techniques will become new standards in the field of C. elegans metabolic research

Characterization of pandemic influenza immune memory signature after vaccination or infection

International audienceThe magnitude, quality, and maintenance of immunological memory after infection or vaccination must be considered for future design of effective influenza vaccines. In 2009, the influenza pandemic produced disease that ranged from mild to severe, even fatal, illness in infected healthy adults and led to vaccination of a portion of the population with the adjuvanted, inactivated influenza A(H1N1)pdm09 vaccine. Here, we have proposed a multi-parameter quantitative and qualitative approach to comparing adaptive immune memory to influenza 1 year after mild or severe infection or vaccination. One year after antigen encounter, severely ill subjects maintained high levels of humoral and polyfunctional effector/memory CD4$^+$ T cells responses, while mildly ill and vaccinated subjects retained strong cellular immunity, as indicated by high levels of mucosal homing and degranulation markers on IFN-$\gamma^+$ antigen-specific T cells. A principal component analysis distinguished 3 distinct clusters of individuals. The first group comprised vaccinated and mildly ill subjects, while clusters 2 and 3 included mainly infected individuals. Each cluster had immune memory profiles that differed in magnitude and quality. These data provide evidence that there are substantial similarities between the antiinfluenza response that mildly ill and vaccinated individuals develop and that this immune memory signature is different from that seen in severely ill individuals

Rictor/TORC2 Regulates Fat Metabolism, Feeding, Growth and Life Span in C. elegans

Rictor is a component of the target of rapamycin complex 2 (TORC2). While TORC2 has been implicated in insulin and other growth factor signaling pathways, the key inputs and outputs of this kinase complex remain unknown. We identified mutations in the Caenorhabditis elegans homolog of rictor in a forward genetic screen for increased body fat. Despite high body fat, rictor mutants are developmentally delayed, small in body size, lay an attenuated brood, and are short-lived, indicating that Rictor plays a critical role in appropriately partitioning calories between long-term energy stores and vital organismal processes. Rictor is also necessary to maintain normal feeding on nutrient-rich food sources. In contrast to wild-type animals, which grow more rapidly on nutrient-rich bacterial strains, rictor mutants display even slower growth, a further reduced body size, decreased energy expenditure, and a dramatically extended life span, apparently through inappropriate, decreased consumption of nutrient-rich food. Rictor acts directly in the intestine to regulate fat mass and whole-animal growth. Further, the high-fat phenotype of rictor mutants is genetically dependent on akt-1, akt-2, and serum and glucocorticoid-induced kinase-1 (sgk-1). Alternatively, the life span, growth, and reproductive phenotypes of rictor mutants are mediated predominantly by sgk-1. These data indicate that Rictor/TORC2 is a nutrient-sensitive complex with outputs to AKT and SGK to modulate the assessment of food quality and signal to fat metabolism, growth, feeding behavior, reproduction, and life span

Non-targeted metabolite profiling highlights the potential of strawberry leaves as a resource for specific bioactive compounds

Background: The non-edible parts of horticultural crops, such as leaves, contain substantial amounts of valuable bioactive compounds which are currently only little exploited. For example strawberry (Fragaria × ananassa) leaves may be a promising bioresource for diverse health-related applications. However, product standardization sets a real challenge, especially when the leaf material comes from varying cultivars. The first step towards better quality control of berry fruit leaf-based ingredients and supplements is to understand metabolites present and their stability in different plant cultivars, so we surveyed the distribution of potentially bioactive strawberry leaf metabolites in six different strawberry cultivars. Non-targeted metabolite profiling analysis using LC-qTOF-ESI-MS with data processing via principal component analysis and k-means clustering analysis were utilized to examine the differences and commonalities between the leaf metabolite profiles.Results: Quercetin and kaempferol derivatives were the dominant flavonol groups in strawberry leaves. Previously described and novel caffeic and chlorogenic acid derivatives were among the major phenolic acids. In addition, ellagitannins were one of the distinguishing compound classes in strawberry leaves. In general, strawberry leaves also contained high levels of octadecatrienoic acid derivatives, precursors of valuable odor compounds.Conclusions: The specific bioactive compounds found in the leaves of different strawberry cultivars offer the potential for the selection of optimized leaf materials for added-value food and for non-food applications.</p

Overview of the ATR power supplies

The AGS to RHIC transfer line (ATR) transports a variety of beams from the Alternating Gradient Synchrotron (AGS) which gets its input from the Booster Synchrotron. In turn, the Booster receives input beams from either a Tandem Van de Graaff (heavy ions) or a Linac (protons). The AGS extracts beam bunches, up to a rate of 30 Hertz, to the ATR which feeds the Relativistic Heavy Ion Collider (RHIC) starting with the sextant test in January of 1997. The ATR is made up of the upgraded U line and the new W, X, and Y lines. A test in 1995 transported beam to the end of the W line. During normal operation, a pulsed switching magnet at the end of the W line will bend the beam into the X line or the Y line so that the two storage rings in RHIC are filled with counter rotating beams. The ATR line is comprised of 80 power supplies (PS`s), 17 of which are upgraded AGS PS`s. The remaining 63 PS`s were newly purchased. These PS`s range from bipolar 600 watt linear type trim magnet PS`s to 1 Megawatt, thyristor, dipole PS`s. Results of the commissioning runs will be presented, as well as descriptions of regulation, filtering, and analog and digital controls

AGS slow extracted beam improvement

The Brookhaven AGS is a strong focusing accelerator which is used to accelerate protons and various heavy ion species to an equivalent proton energy of 29 GeV. Since the late 1960`s it has been serving high energy physics (HEP - proton beam) users of both slow and fast extracted beams. The AGS fixed target program presently uses primary proton and heavy ion beams (HIP) in slowly extracted fashion over spill lengths of 1.5 to 4.0 seconds. Extraction is accomplished by flattoping the main and extraction magnets and exciting a third integer resonance in the AGS. Over the long spill times, control of the subharmonic amplitude components up to a frequency of 1 kilohertz is very crucial. One of the most critical contributions to spill modulation is due to the AGS MMPS. An active filter was developed to reduce these frequencies and it`s operation is described in a previous paper. However there are still frequency components in the 60-720 Hz sub-harmonic ripple range, modulating the spill structure due to extraction power supplies and any remaining structures on the AGS MMPS. A recent scheme is being developed to use the existing tune-trim control horizontal quadrupole magnets and power supply to further reduce these troublesome noise sources. Feedback from an external beam sensor and overcoming the limitations of the quadrupole system by lead/lag compensation techniques will be described

mTOR: from growth signal integration to cancer, diabetes and ageing

In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.National Institutes of Health (U.S.)Howard Hughes Medical InstituteWhitehead Institute for Biomedical ResearchJane Coffin Childs Memorial Fund for Medical Research (Postdoctoral Fellowship)Human Frontier Science Program (Strasbourg, France

Test fast kicker pulser

In this paper, a test pulser of the Brookhaven AGS Booster extraction fast kicker is described. The pulser is projected for both proton and heavy ion operation. A load of total inductance 2.15 /mu/H is used for the test pulser. The PFN voltage is required to be below 40 kV for operation in air. Rise time of the pulse for proton extraction operation is about 120ns up to 97% of full current (1000A), and, for heavy ion extraction, 160ns up to 98% of full current (1615A). R-C compensation networks are used for pulse front edge sharpening. The flexibility of operation is obtained basically by switching an energy dumping resistor to match or mismatch the PFN impedance. Some comments on stray capacitance and stray inductance effects are included. 3 refs., 10 figs., 2 tabs