Hot dense capsule implosion cores produced by z-pinch dynamic hohlraum radiation

Hot dense capsule implosions driven by z-pinch x-rays have been measured for the first time. A ~220 eV dynamic hohlraum imploded 1.7-2.1 mm diameter gas-filled CH capsules which absorbed up to ~20 kJ of x-rays. Argon tracer atom spectra were used to measure the Te~ 1keV electron temperature and the ne ~ 1-4 x10^23 cm-3 electron density. Spectra from multiple directions provide core symmetry estimates. Computer simulations agree well with the peak compression values of Te, ne, and symmetry, indicating reasonable understanding of the hohlraum and implosion physics.Comment: submitted to Phys. Rev. Let

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells.

A network model for the determination of tumor metabolic fluxes from (13)C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-(13)C2]glucose under normoxic conditions at 37 °C and monitored by (13)C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism

The LARP1 La-Module recognizes both ends of TOP mRNAs

La-Related Protein 1 (LARP1) is an RNA-binding protein that regulates the stability and translation of mRNAs encoding the translation machinery, including ribosomal proteins and translation factors. These mRNAs are characterized by a 5ʹ-terminal oligopyrimidine (TOP) motif that coordinates their temporal and stoichiometric expression. While LARP1 represses TOP mRNA translation via the C-terminal DM15 region, the role of the N-terminal La-Module in the recognition and translational regulation of TOP mRNAs remains elusive. Herein we show that the LARP1 La-Module also binds TOP motifs, although in a cap-independent manner. We also demonstrate that it recognizes poly(A) RNA. Further, our data reveal that the LARP1 La-Module can simultaneously engage TOP motifs and poly(A) RNA. These results evoke an intriguing molecular mechanism whereby LARP1 could regulate translation and stabilization of TOP transcripts

In vitro transactivation of Bacillus subtilis RNase P RNA

AbstractDeletion of the ‘signature’ PL5.1 stem-loop structure of a Type II RNase P RNA diminished its catalytic activity. Addition of PL5.1 in trans increased catalytic efficiency (kcat/KM) rather than kcat. Transactivation was due to the binding of a single PL5.1 species per ribozyme with an apparent Kd near 600 nM. The results are consistent with the role of PL5.1 being to position the substrate near the active site of the ribozyme, and with the hypothesis that ribozymes can evolve by accretion of preformed smaller structures

Hsp27 anti-sense oligonucleotides sensitize the microtubular cytoskeleton of Chinese hamster ovary cells grown at low pH to 42 degrees C-induced reorganization

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH

Imaging cell surface glycosylation in vivo using "double click" chemistry.

Dynamic alterations in cell surface glycosylation occur in numerous biological processes that involve cell-cell communication and cell migration. We report here imaging of cell surface glycosylation in live mice using double click chemistry. Cell surface glycans were metabolically labeled using peracetylated azido-labeled N-acetylgalactosamine and then reacted, in the first click reaction, with either a cyclooctyne, in a Huisgen [3 + 2] cycloaddition, or with a Staudinger phosphine, via Staudinger ligation. The second click reaction was a [4 + 2] inverse electron demand Diels-Alder reaction between a trans-cyclooctene and a tetrazine, where the latter reagent had been fluorescently labeled with a far-red fluorophore. After administration of the fluorescent tetrazine, the bifunctional cyclooctyne-cyclooctene produced significant azido sugar-dependent fluorescence labeling of tumor, kidney, liver, spleen, and small intestine in vivo, where the kidney and tumor could be imaged noninvasively in the live mouse

(13)C MRS and LC-MS Flux Analysis of Tumor Intermediary Metabolism.

We present the first validated metabolic network model for analysis of flux through key pathways of tumor intermediary metabolism, including glycolysis, the oxidative and non-oxidative arms of the pentose pyrophosphate shunt, the TCA cycle as well as its anaplerotic pathways, pyruvate-malate shuttling, glutaminolysis, and fatty acid biosynthesis and oxidation. The model that is called Bonded Cumomer Analysis for application to (13)C magnetic resonance spectroscopy ((13)C MRS) data and Fragmented Cumomer Analysis for mass spectrometric data is a refined and efficient form of isotopomer analysis that can readily be expanded to incorporate glycogen, phospholipid, and other pathways thereby encompassing all the key pathways of tumor intermediary metabolism. Validation was achieved by demonstrating agreement of experimental measurements of the metabolic rates of oxygen consumption, glucose consumption, lactate production, and glutamate pool size with independent measurements of these parameters in cultured human DB-1 melanoma cells. These cumomer models have been applied to studies of DB-1 melanoma and DLCL2 human diffuse large B-cell lymphoma cells in culture and as xenografts in nude mice at 9.4 T. The latter studies demonstrate the potential translation of these methods to in situ studies of human tumor metabolism by MRS with stable (13)C isotopically labeled substrates on instruments operating at high magnetic fields (≥7 T). The melanoma studies indicate that this tumor line obtains 51% of its ATP by mitochondrial metabolism and 49% by glycolytic metabolism under both euglycemic (5 mM glucose) and hyperglycemic conditions (26 mM glucose). While a high level of glutamine uptake is detected corresponding to ~50% of TCA cycle flux under hyperglycemic conditions, and ~100% of TCA cycle flux under euglycemic conditions, glutaminolysis flux and its contributions to ATP synthesis were very small. Studies of human lymphoma cells demonstrated that inhibition of mammalian target of rapamycin (mTOR) signaling produced changes in flux through the glycolytic, pentose shunt, and TCA cycle pathways that were evident within 8 h of treatment and increased at 24 and 48 h. Lactate was demonstrated to be a suitable biomarker of mTOR inhibition that could readily be monitored by (1)H MRS and perhaps also by FDG-PET and hyperpolarized (13)C MRS methods

An NMR-Guided Screening Method for Selective Fragment Docking and Synthesis of a Warhead Inhibitor

Selective hits for the glutaredoxin ortholog of Brucella melitensis are determined using STD NMR and verified by trNOE and (15)N-HSQC titration. The most promising hit, RK207, was docked into the target molecule using a scoring function to compare simulated poses to experimental data. After elucidating possible poses, the hit was further optimized into the lead compound by extension with an electrophilic acrylamide warhead. We believe that focusing on selectivity in this early stage of drug discovery will limit cross-reactivity that might occur with the human ortholog as the lead compound is optimized. Kinetics studies revealed that lead compound 5 modified with an ester group results in higher reactivity than an acrylamide control; however, after modification this compound shows little selectivity for bacterial protein versus the human ortholog. In contrast, hydrolysis of compound 5 to the acid form results in a decrease in the activity of the compound. Together these results suggest that more optimization is warranted for this simple chemical scaffold, and opens the door for discovery of drugs targeted against glutaredoxin proteins-a heretofore untapped reservoir for antibiotic agents

Degeneracy of consistency equations in braneworld inflation

In a Randall-Sundrum type II inflationary scenario we compute perturbation amplitudes and spectral indices up to next-to-lowest order in the slow-roll parameters, starting from the well-known lowest-order result for a de Sitter brane. Using two different prescriptions for the tensor amplitude, we show that the braneworld consistency equations are not degenerate with respect to the standard relations and we explore their observational consequences. It is then shown that, while the degeneracy between high- and low-energy regimes can come from suitable values of the cosmological observables, exact functional matching between consistency expressions is plausibly discarded. This result is then extended to the Gauss-Bonnet case.Comment: 16 pages, 3 figures. v3: major revision. Changed title, updated references, rearranged material, new prescription for the tensor spectrum, new figures, extended and more robust conclusion