NUScon: a community-driven platform for quantitative evaluation of nonuniform sampling in NMR

Although the concepts of nonuniform sampling (NUS​​​​​​​) and non-Fourier spectral reconstruction in multidimensional NMR began to emerge 4 decades ago (Bodenhausen and Ernst, 1981; Barna and Laue, 1987), it is only relatively recently that NUS has become more commonplace. Advantages of NUS include the ability to tailor experiments to reduce data collection time and to improve spectral quality, whether through detection of closely spaced peaks (i.e., “resolution”) or peaks of weak intensity (i.e., “sensitivity”). Wider adoption of these methods is the result of improvements in computational performance, a growing abundance and flexibility of software, support from NMR spectrometer vendors, and the increased data sampling demands imposed by higher magnetic fields. However, the identification of best practices still remains a significant and unmet challenge. Unlike the discrete Fourier transform, non-Fourier methods used to reconstruct spectra from NUS data are nonlinear, depend on the complexity and nature of the signals, and lack quantitative or formal theory describing their performance. Seemingly subtle algorithmic differences may lead to significant variabilities in spectral qualities and artifacts. A community-based critical assessment of NUS challenge problems has been initiated, called the “Nonuniform Sampling Contest” (NUScon), with the objective of determining best practices for processing and analyzing NUS experiments. We address this objective by constructing challenges from NMR experiments that we inject with synthetic signals, and we process these challenges using workflows submitted by the community. In the initial rounds of NUScon our aim is to establish objective criteria for evaluating the quality of spectral reconstructions. We present here a software package for performing the quantitative analyses, and we present the results from the first two rounds of NUScon. We discuss the challenges that remain and present a roadmap for continued community-driven development with the ultimate aim of providing best practices in this rapidly evolving field. The NUScon software package and all data from evaluating the challenge problems are hosted on the NMRbox platform

CONFOCAL MICROSCOPY STUDY OF BIOLOGICAL PECULIARITIES OF SCAFFOLD MADE FROM RECOMBINANT SPIDER SILK

We studied the viability and dynamic of cell distribution during long-term cultivation of broblasts 3T3 in spider silk spidroin 1-based scaffold. Laser scanning confocal microscopy is shown to have advantages for visualization of cells situated on the external and internal surfaces of scaffold. Fibroblasts maintain high proliferative ability and viability during long term cultivation. Spidroin 1-based scaffold are the perspective materials for bioengineering

ИСПОЛЬЗОВАНИЕ МЕТОДА КОНФОКАЛЬНОЙ МИКРОСКОПИИ ДЛЯ ИЗУЧЕНИЯ БИОЛОГИЧЕСКИХ СВОЙСТВ МАТРИКСА ИЗ РЕКОМБИНАНТНОЙ ПАУТИНЫ

We studied the viability and dynamic of cell distribution during long-term cultivation of broblasts 3T3 in spider silk spidroin 1-based scaffold. Laser scanning confocal microscopy is shown to have advantages for visualization of cells situated on the external and internal surfaces of scaffold. Fibroblasts maintain high proliferative ability and viability during long term cultivation. Spidroin 1-based scaffold are the perspective materials for bioengineering. Изучена жизнеспособность и динамика распределения мышиных фибробластов 3T3 в матрице на ос- нове рекомбинантного белка каркасной нити паутины спидроина 1 при длительном культивировании. Показана возможность и преимущества использования конфокальной микроскопии как для визуализа- ции клеток, находящихся в глубоких слоях трехмерной матрицы, так и для наблюдения за изменением состояния культивируемых клеток. Популяция фибробластов сохраняла высокие показатели жизнеспо- собности и пролиферативной активности, а инфильтрация матрицы клеточной популяцией становилась однородной в течение длительного времени культивирования. Это позволяет рассматривать матрицы на основе спидроина 1 в качестве перспективного материала для создания биоинженерных конструкций.

NMR Structure and Dynamics of the C-Terminal Domain from Human Rev1 and Its Complex with Rev1 Interacting Region of DNA Polymerase η

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer–template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157–1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524–539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.National Institute of Environmental Health Sciences (ES015818)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant P30 ES002109