Titration-based screening for evaluation of natural product extracts: identification of an aspulvinone family of luciferase inhibitors

The chemical diversity of nature has tremendous potential for the discovery of molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, and macro- and microorganisms has curtailed their use in lead discovery. Here, we describe a process for leveraging the concentration-response curves obtained from quantitative HTS to improve the initial selection of "actives" from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm improves the probability that labor-intensive subsequent steps of reculturing, extraction, and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by X-ray crystallography.     

Phase diagram and thermodynamic modeling of PEO + organic salts + H2O and PPO + organic salts + H2O aqueous two-phase systems

The phase diagrams of PEO1500 + sodium tartrate + water, PPO400 + sodium tartrate + water, PEO1500 + sodium succinate + water, PPO400 + sodium succinate + water, PEO1500 + sodium citrate + water, PPO400 + sodium citrate + water and PPO400 + sodium acetate + water aqueous two-phase systems were determined at (283.15, 298.15, and 313.15) K. Both equilibrium phases composition were analyzed by conductimetry and refractive index. In this paper, the influences of polymer hydrophobicity, salt nature and temperature on the phase diagram were analyzed. The phase separation processes was endothermic and the hydrophobic increase make easier the phase splitting, while the electrolyte capacity to induce phase separation follow the order: citrate > tartrate > succinate. The consistency of the tie-line data was ascertained by applying the Othmer-Tobias correlation. The experimental data were correlated with the NRTL model for the activity coefficient, with estimation of new interaction energy parameters. The results, analyzed in terms of root mean square deviations between experimental and calculated compositions, were considered satisfactory.     

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

Recombinant human erythropoietin (rhEPO) analogues are known to have been used in horse sports for their assumed performance enhancing properties. Recently, several authors have published liquid chromatographic-tandem mass spectrometric (LC-MS/MS) methods for confirming the presence of rhEPO analogues in horse plasma. In the current study, an improved LC-MS/MS confirmatory procedure for rhEPO, darbepoetin (DPO) and continuous erythropoietin receptor activator (CERA) in horse plasma was developed and validated. The method was also adapted for and applied to urine samples for the first time. Similar to previously published plasma assays, the methods utilise size exclusion and immunoaffinity extraction prior to tryptic cleavage, enzymatic deglycosylation and LC-MS/MS analysis of the resulting signature tryptic peptides (rhEPO/CERA T5, rhEPO/CERA/DPO T6 and DPO T9). However, the novel application of UPLC chromatography significantly improves the run time of the method compared to nano- or micro-LC and its robustness compared to nano-LC. Furthermore, recombinant canine EPO was found to serve as an effective internal standard, thus allowing confidence in interpretation of the success/failure of every step in the procedure. Limits of detection for confirming the presence of rhEPO, CERA and DPO in plasma were 0.1, 0.25 and 0.05 ng mL^?1, respectively, which were equal to or lower than limits achieved using previously published LC-MS/MS based methods. Limits of detection for confirming the presence of rhEPO, CERA and DPO in urine were 0.05, 0.15 and 0.025 ng mL^?1 and the analysis of urine samples collected from horses administered rhEPO (Eprex?) or DPO (Aranesp?) demonstrated the use of this matrix as a suitable alternative in situations where plasma is not available.     

Chromium-mediated stereoselective synthesis of carbohydrate-derived (E)-α,β-unsaturated esters or amides

A chromium-mediated novel synthesis of carbohydrate-derived di- and trisubstituted (E)-α,β-unsaturated esters or amides from a range of dichloroesters or amides and a variety of sugar aldehydes is reported. The process took place with total stereoselectivity and in high yields. A mechanism based on a sequential chromium-promoted aldol-type reaction and a completely stereoselective β-elimination reaction is proposed to explain these results.     

In Vitro and In Vivo Enzyme‐Mediated Biomineralization of Oligo(poly(ethylene glycol) Fumarate Hydrogels

The enzyme alkaline phosphatase (ALP) is added at different concentrations (i.e., 0, 2.5, and 10 mg · ml^?1) to oligo(poly(ethylene glycol)fumarate) (OPF) hydrogels. The scaffolds are either incubated in 10 mM calcium glycerophosphate (Ca–GP) solution for 2 weeks or implanted in a rat subcutaneous model for 4 weeks. Fourier transform infrared (FTIR) spectroscopy, X‐ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and alizarin red staining show a strong ability to form minerals exclusively in ALP‐containing hydrogels in vitro. Additionally, the calcium content increases with increasing ALP concentration. Similarly, only ALP‐containing hydrogels induce mineralization in vivo. Specifically, small (≈5–20 µm) mineral deposits are observed at the periphery of the hydrogels near the dermis/scaffold interface using Von Kossa and alizarin red staining.

     

Thin sequences and the Gram matrix

We provide a new proof of Volberg’s Theorem characterizing thin interpolating sequences as those for which the Gram matrix associated to the normalized reproducing kernels is a compact perturbation of the identity. In the same paper, Volberg characterized sequences for which the Gram matrix is a compact perturbation of a unitary as well as those for which the Gram matrix is a Schatten-2 class perturbation of a unitary operator. We extend this characterization from 2 to p, where 2 ≤ p ≤ ∞.     

Structure-preserving Runge-Kutta methods for stochastic Hamiltonian equations with additive noise

There has been considerable recent work on the development of energy conserving one-step methods that are not symplectic. Here we extend these ideas to stochastic Hamiltonian problems with additive noise and show that there are classes of Runge-Kutta methods that are very effective in preserving the expectation of the Hamiltonian, but care has to be taken in how the Wiener increments are sampled at each timestep. Some numerical simulations illustrate the performance of these methods.     

Enhanced Light‐Harvesting Capacity by Micellar Assembly of Free Accessory Chromophores and LH1‐like Antennas

Biohybrid light‐harvesting antennas are an emerging platform technology with versatile tailorability for solar‐energy conversion. These systems combine the proven peptide scaffold unit utilized for light harvesting by purple photosynthetic bacteria with attached synthetic chromophores to extend solar coverage beyond that of the natural systems. Herein, synthetic unattached chromophores are employed that partition into the organized milieu (e.g. detergent micelles) that house the LH1‐like biohybrid architectures. The synthetic chromophores include a hydrophobic boron‐dipyrrin dye (A1) and an amphiphilic bacteriochlorin (A2), which transfer energy with reasonable efficiency to the bacteriochlorophyll acceptor array (B875) of the LH1‐like cyclic oligomers. The energy‐transfer efficiencies are markedly increased upon covalent attachment of a bacteriochlorin (B1 or B2) to the peptide scaffold, where the latter likely acts as an energy‐transfer relay site for the (potentially diffusing) free chromophores. The efficiencies are consistent with a Förster (through‐space) mechanism for energy transfer. The overall energy‐transfer efficiency from the free chromophores via the relay to the target site can approach those obtained previously by relay‐assisted energy transfer from chromophores attached at distant sites on the peptides. Thus, the use of free accessory chromophores affords a simple design to enhance the overall light‐harvesting capacity of biohybrid LH1‐like architectures.     

Cold-Active Xylanase Produced by Fungi Associated with Antarctic Marine Sponges

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5–7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.     

Palladium-catalyzed cross-coupling reaction of tricyclopropylbismuth with aryl halides and triflates

The palladium-catalyzed cross-coupling reaction of tricyclopropylbismuth with aryl and heterocyclic halides and triflates is reported. The reaction tolerates numerous functional groups and does not require anhydrous conditions. The method was successfully extended to the cross-coupling of triethylbismuth.