Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP

Specific Signal Enhancement on an RNA-Protein Interface by Dynamic Nuclear Polarization

Sensitivity and specificity are both crucial for the efficient solid-state NMR structure determination of large biomolecules. We present an approach that features both advantages by site-specific enhancement of NMR spectroscopic signals from the protein-RNA binding site within a ribonucleoprotein (RNP) by dynamic nuclear polarization (DNP). This approach uses modern biochemical techniques for sparse isotope labeling and exploits the molecular dynamics of 13C-labeled methyl groups exclusively present in the protein. These dynamics drive heteronuclear cross relaxation and thus allow specific hyperpolarization transfer across the biomolecular complex's interface. For the example of the L7Ae protein in complex with a 26mer guide RNA minimal construct from the box C/D complex in archaea, we demonstrate that a single methyl-nucleotide contact is responsible for most of the polarization transfer to the RNA, and that this specific transfer can be used to boost both NMR spectral sensitivity and specificity by DNP

Coagulation using organic carbonates opens up a sustainable route towards regenerated cellulose films

Due to their biodegradability, biocompatibility and sustainable nature, regenerated cellulose (RC) films are of enormous relevance for green applications including medicinal, environmental and separation technologies. However, the processes used so far are very hazardous to the environment and health. Here, we disclose a simple, fast, environmentally friendly, nontoxic and cost-effective processing method for preparing RC films. High quality non-transparent and transparent RC films and powders can be produced by dissolution with tetrabutylphosphonium hydroxide [TBPH]/[TBP]+[OH]− followed by coagulation with organic carbonates. Investigations on the coagulation mechanism revealed an extremely fast reaction between the carbonates and the hydroxide ions. The high-quality powders and films were fully characterized with respect to structure, surface morphology, permeation and selectivity. This method represents a future-oriented green alternative to known industrial processes. © 2020, The Author(s)

Exfoliated polymeric carbon nitride nanosheets for photocatalytic applications

Exfoliation into a 2D nanosheet structure can lead to enhanced surface activity and unique optical and electronic properties in polymeric carbon nitride (PCN). In this study, four common exfoliation strategies (liquid ultrasonication, thermal oxidation, hydrothermal oxidation, and chemical oxidation) were adopted, and their effects on the structural and electronic changes in PCN were analyzed in detail. This allows us to understand the relationship between the exfoliation mechanism and the structural/optical properties. Here, we demonstrate that the thermal and ultrasonic exfoliation methods can effectively reduce the thickness of PCN while preserving its original structure. In contrast, the chemical and hydrothermal treatments can strongly affect the morphology and structure of PCN, leading to a decreased performance in phenol photodegradation. Therefore, depending on the employed exfoliation method, the surface area, functionalization, band edge positions, charge carrier generation, and mobility are influenced differently up to the point where semiconducting behavior is entirely lost. Our results allow conclusions about the applicability of the different exfoliation methods to obtain distinct material properties for photocatalytic applications

Resolution and Polarization Distribution in Cryogenic DNP/MAS Experiments

This contribution addresses four potential misconceptions associated with high-resolution dynamic nuclear polarization/magic angle spinning (DNP/MAS) experiments. First, spectral resolution is not generally compromised at the cryogenic temperatures at which DNP experiments are performed. As we demonstrate at a modest field of 9 T (380 MHz [superscript 1]H), 1 ppm linewidths are observed in DNP/MAS spectra of a membrane protein in its native lipid bilayer, and <0.4 ppm linewidths are reported in a crystalline peptide at 85 K. Second, we address the concerns about paramagnetic broadening in DNP/MAS spectra of proteins by demonstrating that the exogenous radical polarizing agents utilized for DNP are distributed in the sample in such a manner as to avoid paramagnetic broadening and thus maintain full spectral resolution. Third, the enhanced polarization is not localized around the polarizing agent, but rather is effectively and uniformly dispersed throughout the sample, even in the case of membrane proteins. Fourth, the distribution of polarization from the electron spins mediated via spin diffusion between [superscript 1]H–[superscript 1]H strongly dipolar coupled spins is so rapid that shorter magnetization recovery periods between signal averaging transients can be utilized in DNP/MAS experiments than in typical experiments performed at ambient temperature.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026)National Institutes of Health (U.S.) (Grant EB001965)National Institutes of Health (U.S.) (Grant EB004866)National Science Foundation (U.S.). Graduate Research Fellowship Progra

EuReCa ONE—27 Nations, ONE Europe, ONE Registry A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe

AbstractIntroductionThe aim of the EuReCa ONE study was to determine the incidence, process, and outcome for out of hospital cardiac arrest (OHCA) throughout Europe.MethodsThis was an international, prospective, multi-centre one-month study. Patients who suffered an OHCA during October 2014 who were attended and/or treated by an Emergency Medical Service (EMS) were eligible for inclusion in the study. Data were extracted from national, regional or local registries.ResultsData on 10,682 confirmed OHCAs from 248 regions in 27 countries, covering an estimated population of 174 million. In 7146 (66%) cases, CPR was started by a bystander or by the EMS. The incidence of CPR attempts ranged from 19.0 to 104.0 per 100,000 population per year. 1735 had ROSC on arrival at hospital (25.2%), Overall, 662/6414 (10.3%) in all cases with CPR attempted survived for at least 30 days or to hospital discharge.ConclusionThe results of EuReCa ONE highlight that OHCA is still a major public health problem accounting for a substantial number of deaths in Europe.EuReCa ONE very clearly demonstrates marked differences in the processes for data collection and reported outcomes following OHCA all over Europe. Using these data and analyses, different countries, regions, systems, and concepts can benchmark themselves and may learn from each other to further improve survival following one of our major health care events

Electromagnetic properties of single-walled carbon nanotubes investigated by microwave absorption

Due to their unique properties, single-walled carbon nanotubes (SWNT) are very interesting candidates for the development of new electronic devices. Some of these properties, e.g., a possible transition to a superconducting phase or the existence of ordered magnetic states, are still under investigation and intensively discussed. Macroscopic amounts of SWNT can hitherto only be obtained as mixtures of tubes of different electronic properties. Therefore researchers have always been interested in a simple, fast, and reliable screeningmethod to detect the signatures of metallic or semiconducting SWNT. It is assumed quite generally that these “standard” electronic properties can be identified rather easily. In contrast to this, the above mentioned “unconventional” properties, i.e., superconductivity and magnetism, are anticipated to arise only in a small fraction of the nanotubes. Furthermore these features might be influenced by impurities, topological defects, or intertube interactions. Due to this fact, the sought-after screening method should be able to resolve the correlated signatures selectively, even if they are masked by other constituents in the sample. This study invokes microwave absorption, both in its resonant (electron paramagnetic resonance) and in its non-resonant variant (cavity perturbation). This method represents a versatile and selective tool to characterize magnetic and electronic phases and occurrent transitions. Whereas metallic SWNT are intrinsically paramagnetic due to Pauli paramagnetism, ideal semiconducting tubes are diamagnetic and therefore not accessible to electron paramagnetic resonance (EPR). Nevertheless extrinsic and intrinsic temperature-activated defects can introduce paramagnetic states observable by EPR. In additional experiments, nitrogen encapsulated in C60 has been incorporated inside SWNT as a paramagnetic probe, forming so called peapods. The synthesis of these N@C60 peapods allows the examination of the electromagnetic properties of the SWNT “from the inside” by EPR. In early studies, the EPR signal of SWNT grown by the electric arc-discharge method was masked by spurious signals of the catalyst remaining in the sample. By using nanotubes grown by a special chemical vapor deposition (CVD) technique, samples could be investigated which were almost catalyst-free. Thus it was possible to study the electronic properties of different types of SWNT over a wide temperature range by EPR. The high-temperature signals are dominated by itinerant spins. They result from the temperature activated delocalization of shallow defect states. At low temperatures, these charge carriers get trapped at specific sites. This trapping leads to a strong magnetic resonance of localized electron spins. Furthermore, no indication of the existence of elements different than carbon can be detected in the sample. This was proven by continuous wave (c.w.) EPR and also by modern techniques of pulsed EPR. Non-resonant microwave absorption is introduced as a powerful tool to study the electronic conductivity of bulk samples of SWNT. A custom microwave bridge was constructed therefore. By evoking this method, the temperature dependence of the complex resistivity at T > 20 K could be attributed to the existence of pseudo-metallic or small-band-gap semiconducting tubes. At T ≈ 12 K the transition from a non-linear dissipative state at low temperature to a conventional Ohmic loss behavior is observed. This transition is taken as an indication for the formation of superconducting domains in small parts of the sample. Furthermore, the existence of a weak ferromagnetic signal is detected via alternating current (AC) magnetization measurements. The features of this ferromagnetism, i.e., weak magnetization, low saturation field, and the absence of hysteresis effects, exclude remaining iron catalyst as source of this observation. Instead, the cooperative magnetism might arise from an intrinsic exchange interaction in SWNT

Bis-Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization

Kaushik M, Qi M, Godt A, Corzilius B. Bis-Gadolinium Complexes for Solid Effect and Cross Effect Dynamic Nuclear Polarization. Angewandte Chemie International Edition. 2017;56(15):4295–4299

Dynamic nuclear polarization on a hybridized hammerhead ribozyme: An explorative study of RNA folding and direct DNP with a paramagnetic metal ion cofactor.

While uniform isotope labeling of ribonucleic acids (RNA) can simply and efficiently be achieved by in-vitro transcription, the specific introduction of nucleotides in larger constructs is non-trivial and often ineffective. Here, we demonstrate how a medium-sized (67-mer), biocatalytically relevant RNA (hammerhead ribozyme, HHRz) can be formed by spontaneous hybridization of two differently isotope-labeled strands, each individually synthesized by in-vitro transcription. This allows on the one hand for a significant reduction in the number of isotope-labeled nucleotides and thus spectral overlap particularly under magic-angle spinning (MAS) dynamic nuclear polarization (DNP) NMR conditions, on the other hand for orthogonal C/N-labeling of complementary strands and thus for specific investigation of structurally or functionally relevant inter-strand and/or inter-stem contacts. By this method, we are able to confirm a non-canonical interaction due to single-site resolution and unique spectral assignments by two-dimensional C-C (PDSD) as well as N-C (TEDOR) correlation spectroscopy under "conventional" DNP enhancement. This contact is indicative of the ribozyme's functional conformation, and is present in frozen solution irrespective of the presence or absence of a Mg co-factor. Finally, we use different isotope-labeling schemes in order to investigate the distance dependence of paramagnetic interactions and direct metal-ion DNP if the diamagnetic Mg is substituted by paramagnetic Mn

Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR

The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, which underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields