Bioorthogonal organic chemistry in living cells: novel strategies for labeling biomolecules

The chemical labeling of biomolecules continues to be an important tool for the study of their function and cellular fate. Attention is increasingly focused on labeling of biomolecules in living cells, since cell lysis introduces many artefacts. In addition, with the advances in biocompatible synthetic organic chemistry, a whole new field of opportunity has opened up, affording high diversity in the nature of the label as well as a choice of ligation reactions. In recent years, several different two-step labeling strategies have emerged. These rely on the introduction of a bioorthogonal attachment site into a biomolecule, then ligation of a reporter molecule to this site using bioorthogonal organic chemistry. This Perspective focuses on these techniques, their implications and future directions.     

Long range transport of biomass aerosol to Greenland: Multi-spectroscopic investigation of particles deposited in the snow

Summary Results are given from the NIST component of a pilot (“winter-over”) study of seasonal patterns of natural and anthropogenic species in air and snow transported to Summit, Greenland. Central to this research is the quantitative apportionment of fossil and biomass particulate carbon, based on advanced (micromolar) ¹⁴C accelerator mass spectrometry (AMS) applied to remote snow samples containing as little as 9 µg C/kg. The measurements were made practicable through stringent attention to the nature and sources of the isotopic-chemical blank, resulting in a blank reduction from »5 µg C to <0.5 µg C. An important result of this work is the first evidence of a seasonal pattern in biomass-C particles in Greenland snow. Although ¹⁴C AMS data serve to resolve fossil and biomass carbon quantitatively, a deeper understanding of the aerosol sources and character demands a multidisciplinary approach. This is illustrated with “multi-spectrometric” macro- and micro-analytical data for two cases involving substantial incursions of biomass aerosol to the Summit, Greenland snow.     

Molecular dynamics simulations of cyclosporin A: The crystal structure and dynamic modelling of a structure in apolar solution based on NMR data

Summary The conformation of the immunosuppressive drug cyclosporin A (CPA), both in apolar solution and in crystalline state, has been studied by computer simulation techniques. Three molecular dynamics (MD) simulations have been performed: one modelling the crystal structure and two modelling the structure in apolar solution, using a restrained MD approach in which data from nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy are taken into account. The simulation of the crystalline state (MDC) concerns a system of 4 unit cells containing 16 cyclosporin A molecules and 22 water molecules, which is simulated using crystalline periodic boundary conditions. The simulations modelling the apolar solvent conformation (MDS) concern one isolated cyclosporin A molecule. In these simulations an extra term in the interatomic potential function is used, which forces the molecule to satisfy a set of 57 atom-atom distance constraints originating from nuclear Overhauser effects (NOEs) obtained from NMR spectroscopy and one distance constraint deduced from IR spectroscopy.From a comparison of the results of the crystal simulation to those of the X-ray experiment in terms of structure, atomic fluctuations, hydrogen bond pattern, etc., it is concluded that the force field that is used yields an adequate representation of crystalline cyclosporin A. Secondly, it is shown that the dynamic modelling technique that is used to obtain a structure in a polar solution from NMR distance information works well. Starting from initial conformations which have a root mean square difference of 0.14 nm both distance restrained MD simulations converge to the same final solution structure. A comparison of the crystal structure of cyclosporin A and the one in apolar solution shows that there are significant differences. The overall difference in atomic positions is 0.09 nm for the C_x atoms and 0.17 nm for all atoms. In apolar solution, the molecule is slightly more bent and the side chains of 1 MeBmt and 10 MeLeu adopt a different conformation.Abbreviations MeBmt (4R)-4[(E)-2-butenyl]-4-methyl-l-Threonine - MD Molecular dynamics - EM Energy minimization - MDC Molecular dynamics simulation of the crystal - MDS1 Restrained molecular dynamics simulation to obtain the structure in solution starting from the crystal structure - MDS2 Like MDS1, but starting from the SMS structure - SMS Proposed structure in solution, obtained by model building - XRAY An X-ray structure - CPA Cyclosporin A - NMR Nuclear magnetic resonance spectroscopy - NOE Nuclear Overhauser enhancement - MDS1 Mean simulated structure obtained by averaging over the time period 20–40 ps of the MDS1 simulation - MDS2 Mean simulated structure obtained by averaging over the time period 10–30 ps of the MDS2 simulation - Mean simulated structure obtained by averaging over the time period 7–15 ps and over the 16 asymmetric units in the computational box of the MDC simulation.     

CCR5 antagonists: 3-(pyrrolidin-1-yl)propionic acid analogues with potent anti-HIV activity

[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.     

Comparison of (π− d→nn withdσ(π+ d→pp) to test charge symmetry in the Δ-region

First results on ^– d are reported. The measurements were made using 8 specially designed neutron counters, which were carefully calibratedin situ. The differential cross sections atT =142, 180, 217, and 254 MeV were obtained at four angles between 0° and 90°, they are compared to ⁺ d pp data measured at the same energies and angles with the same setup. At every beam energy, the shape of the angular distributions of ^– d nn and ⁺ d pp is the same to ±2%. The absolute cross sections differ by 1 to 10%. The error in this comparison is ±4% implying a small violation of charge symmetry.Dedicated to Prof. I. laus on the occasion of his 60th birthdayDeceased     

A model for making project funding decisions at the National Cancer Institute

This paper describes the development of a model for making project funding decisions at The National Cancer Institute (NCI). The American Stop Smoking Intervention Study (ASSIST) is a multiple-year, multiple-site demonstration project, aimed at reducing smoking prevalence. The initial request for ASSIST proposals was answered by about twice as many states as could be funded. Scientific peer review of the proposals was the primary criterion used for funding decisions. However, a modified Delphi process made explicit several criteria of secondary importance. A structured questionnaire identified the relative importance of these secondary criteria, some of which we incorporated into a composite preference function. We modeled the proposal funding decision as a zero-one program, and adjusted the preference function and available budget parametrically to generate many suitable outcomes. The actual funding decision, identified by our model, offers significant advantages over manually generated solutions found by experts at NCI.