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Protein in-cell NMR spectroscopy at 1.2 GHz
Full text linkIn-cell NMR spectroscopy provides precious structural and functional information on biological macromolecules in their native cellular environment at atomic resolution. However, the intrinsic low sensitivity of NMR imposes a big limitation in the applicability of the methodology. In this respect, the recently developed commercial 1.2GHz NMR spectrometer is expected to introduce significant benefits. However, cell samples may suffer from detrimental effects at ultrahigh fields, that must be carefully evaluated. Here we show the first in-cell NMR spectra recorded at 1.2GHz on human cells, and we compare resolution and sensitivity against those obtained at 900 and 950MHz. To evaluate the effects of different spin relaxation rates, SOFAST-HMQC and BEST-TROSY spectra were recorded on intracellular alpha-synuclein and carbonic anhydrase. Major improvements are observed at 1.2GHz when analyzing unfolded proteins, such as alpha-synuclein, while the TROSY scheme improves the resolution for both globular and unfolded proteins
Radio Signals from Live Cells: The Coming of Age of In-Cell Solution NMR
Full text linkA detailed knowledge of the complex processes that make cells and organisms alive is fundamental in order to understand diseases and to develop novel drugs and therapeutic treatments. To this aim, biological macromolecules should ideally be characterized at atomic resolution directly within the cellular environment. Among the existing structural techniques, solution NMR stands out as the only one able to investigate at high resolution the structure and dynamic behavior of macromolecules directly in living cells. With the advent of more sensitive NMR hardware and new biotechnological tools, modern in-cell NMR approaches have been established since the early 2000s. At the coming of age of in-cell NMR, we provide a detailed overview of its developments and applications in the 20 years that followed its inception. We review the existing approaches for cell sample preparation and isotopic labeling, the application of in-cell NMR to important biological questions, and the development of NMR bioreactor devices, which greatly increase the lifetime of the cells allowing real-time monitoring of intracellular metabolites and proteins. Finally, we share our thoughts on the future perspectives of the in-cell NMR methodology
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Determination of intracellular protein-ligand binding affinity by competition binding in-cell NMR
Full text linkStructure-based drug development suffers from high attrition rates due to the poor activity of lead compounds in cellular and animal models caused by low cell penetrance, off-target binding or changes in the conformation of the target protein in the cellular environment. The latter two effects cause a change in the apparent binding affinity of the compound, which is indirectly assessed by cellular activity assays. To date, direct measurement of the intracellular binding affinity remains a challenging task. In this work, in-cell NMR spectroscopy was applied to measure intracellular dissociation constants in the nanomolar range by means of protein-observed competition binding experiments. Competition binding curves relative to a reference compound could be retrieved either from a series of independent cell samples or from a single real-time NMR bioreactor run. The method was validated using a set of sulfonamide-based inhibitors of human carbonic anhydrase II with known activity in the subnanomolar to submicromolar range. The intracellular affinities were similar to those obtained in vitro, indicating that these compounds selectively bind to the intracellular target. In principle, the approach can be applied to any soluble intracellular target that gives rise to measurable chemical shift changes upon ligand binding
Intracellular Binding/Unbinding Kinetics of Approved Drugs to Carbonic Anhydrase II Observed by in-Cell NMR
Full text linkCandidate drugs rationally designed in vitro often fail due to low efficacy in vivo caused by low tissue availability or because of unwanted side effects. To overcome the limitations of in vitro rational drug design, the binding of candidate drugs to their target needs to be evaluated in the cellular context. Here, we applied in-cell NMR to investigate the binding of a set of approved drugs to the isoform II of carbonic anhydrase (CA) in living human cells. Some compounds were originally developed toward other targets and were later found to inhibit CAs. We observed strikingly different dose- and time-dependent binding, wherein some drugs exhibited a more complex behavior than others. Specifically, some compounds were shown to gradually unbind from intracellular CA II, even in the presence of free compound in the external medium, therefore preventing the quantitative formation of a stable protein-ligand complex. Such observations could be correlated to the known off-target binding activity of these compounds, suggesting that this approach could provide information on the pharmacokinetic profiles of lead candidates at the early stages of multitarget drug design
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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