In-Cell NMR Spectroscopy of Functional Riboswitch Aptamers in Eukaryotic Cells.

We report here the in-cell NMR-spectroscopic observation of the binding of the cognate ligand 2'-deoxyguanosine to the aptamer domain of the bacterial 2'-deoxyguanosine-sensing riboswitch in eukaryotic cells, namely Xenopus laevis oocytes and in human HeLa cells. The riboswitch is sufficiently stable in both cell types to allow for detection of binding of the ligand to the riboswitch. Most importantly, we show that the binding mode established by in vitro characterization of this prokaryotic riboswitch is maintained in eukaryotic cellular environment. Our data also bring important methodological insights: Thus far, in-cell NMR studies on RNA in mammalian cells have been limited to investigations of short (<15 nt) RNA fragments that were extensively modified by protecting groups to limit their degradation in the intracellular space. Here, we show that the in-cell NMR setup can be adjusted for characterization of much larger (≈70 nt) functional and chemically non-modified RNA.

Quantum Szilard Engine with Attractively Interacting Bosons.

We show that a quantum Szilard engine containing many bosons with attractive interactions enhances the conversion between information and work. Using an ab initio approach to the full quantum-mechanical many-body problem, we find that the average work output increases significantly for a larger number of bosons. The highest overshoot occurs at a finite temperature, demonstrating how thermal and quantum effects conspire to enhance the conversion between information and work. The predicted effects occur over a broad range of interaction strengths and temperatures.

Impact of spin label rigidity on extent and accuracy of distance information from PRE data.

Paramagnetic relaxation enhancement (PRE) is a versatile tool for NMR spectroscopic structural and kinetic studies in biological macromolecules. Here, we compare the quality of PRE data derived from two spin labels with markedly different dynamic properties for large RNAs using the I-A riboswitch aptamer domain (78 nt) from Mesoplamsa florum as model system. We designed two I-A aptamer constructs that were spin-labeled by noncovalent hybridization of short spin-labeled oligomer fragments. As an example of a flexible spin label, UreidoU-TEMPO was incorporated into the 3' terminal end of helix P1 while, the recently developed rigid spin-label Çm was incorporated in the 5' terminal end of helix P1. We determined PRE rates obtained from aromatic 13C bound proton intensities and compared these rates to PREs derived from imino proton intensities in this sizeable RNA (~78 nt). PRE restraints derived from both imino and aromatic protons yielded similar data quality, and hence can both be reliably used for PRE determination. For NMR, the data quality derived from the rigid spin label Çm is slightly better than the data quality for the flexible UreidoTEMPO as judged by comparison of the structural agreement with the I-A aptamer crystal structure (3SKI).

[Combination of Excessive Weight Gain and Interleukin-8: A Possible Predictor of Necrotising Enterocolitis in Neonates?]. / Kann ein Gewichtssprung in Kombination mit einem Anstieg des Interleukin-8 eine nekrotisierende Enterokolitis bei Neonaten vorhersagen?

BACKGROUND: Weight gain before the clinical diagnosis of necrotising enterocolitis (NEC) is described as a predictive factor. HYPOTHESIS: Weight gain of more than 5% one day prior to clinical suspicion plus increase of plasma Iinterleukin-8 (IL-8) are predictive for NEC. METHODS: 48 infants with diagnosis of NEC stage II and III were enrolled in a case-control study. Oral and parenteral nutrition, diuresis and kinetics of weight and of IL-8 were documented. RESULTS: 31 infants with NEC-II and 17 infants with NEC-III were enrolled. Weight gain>5% occurred in 35.3% of NEC-III, in 0% of NEC-II and in 4.2% of the control group. IL-8 increased significantly [NEC-III (6 561.4 pg/mL) vs. NEC-II: (326.7 pg/mL) vs. control group (38.9 pg/mL); p<0.05]. Sensitivity of IL-8 in NEC-II was 87.10% (70.15-96.25) and in NEC-III 100.00% (80.33-100.00). Sensitivity of weight gain was 0.00% (0.00-11.32) in NEC-II and 35.29% (14.30-61.65) in NEC-III. CONCLUSION: Weight gain>5% was found in only 35.3% of the cases with NEC-III. Combination of weight gain and IL-8 did not improve the diagnosis of NEC.

Total current blockade in an ultracold dipolar quantum wire.

Cold-atom systems offer a great potential for the future design of new mesoscopic quantum systems with properties that are fundamentally different from semiconductor nanostructures. Here, we investigate the quantum-gas analogue of a quantum wire and find a new scenario for the quantum transport: Attractive interactions may lead to a complete suppression of current in the low-bias range, a total current blockade. We demonstrate this effect for the example of ultracold quantum gases with dipolar interactions.

Field-resolved high-order sub-cycle nonlinearities in a terahertz semiconductor laser.

The exploitation of ultrafast electron dynamics in quantum cascade lasers (QCLs) holds enormous potential for intense, compact mode-locked terahertz (THz) sources, squeezed THz light, frequency mixers, and comb-based metrology systems. Yet the important sub-cycle dynamics have been notoriously difficult to access in operational THz QCLs. Here, we employ high-field THz pulses to perform the first ultrafast two-dimensional spectroscopy of a free-running THz QCL. Strong incoherent and coherent nonlinearities up to eight-wave mixing are detected below and above the laser threshold. These data not only reveal extremely short gain recovery times of 2 ps at the laser threshold, they also reflect the nonlinear polarization dynamics of the QCL laser transition for the first time, where we quantify the corresponding dephasing times between 0.9 and 1.5 ps with increasing bias currents. A density-matrix approach reproducing the emergence of all nonlinearities and their ultrafast evolution, simultaneously, allows us to map the coherently induced trajectory of the Bloch vector. The observed high-order multi-wave mixing nonlinearities benefit from resonant enhancement in the absence of absorption losses and bear potential for a number of future applications, ranging from efficient intracavity frequency conversion, mode proliferation to passive mode locking.

1H, 13C, and 15N backbone chemical shift assignments of coronavirus-2 non-structural protein Nsp10.

The international Covid19-NMR consortium aims at the comprehensive spectroscopic characterization of SARS-CoV-2 RNA elements and proteins and will provide NMR chemical shift assignments of the molecular components of this virus. The SARS-CoV-2 genome encodes approximately 30 different proteins. Four of these proteins are involved in forming the viral envelope or in the packaging of the RNA genome and are therefore called structural proteins. The other proteins fulfill a variety of functions during the viral life cycle and comprise the so-called non-structural proteins (nsps). Here, we report the near-complete NMR resonance assignment for the backbone chemical shifts of the non-structural protein 10 (nsp10). Nsp10 is part of the viral replication-transcription complex (RTC). It aids in synthesizing and modifying the genomic and subgenomic RNAs. Via its interaction with nsp14, it ensures transcriptional fidelity of the RNA-dependent RNA polymerase, and through its stimulation of the methyltransferase activity of nsp16, it aids in synthesizing the RNA cap structures which protect the viral RNAs from being recognized by the innate immune system. Both of these functions can be potentially targeted by drugs. Our data will aid in performing additional NMR-based characterizations, and provide a basis for the identification of possible small molecule ligands interfering with nsp10 exerting its essential role in viral replication.

Probing confined phonon modes by transport through a nanowire double quantum dot.

Strong radial confinement in semiconductor nanowires leads to modified electronic and phononic energy spectra. We analyze the current response to the interplay between quantum confinement effects of the electron and phonon systems in a gate-defined double quantum dot in a semiconductor nanowire. We show that current spectroscopy of inelastic transitions between the two quantum dots can be used as an experimental probe of the confined phonon environment. The resulting discrete peak structure in the measurements is explained by theoretical modeling of the confined phonon mode spectrum, where the piezoelectric coupling is of crucial importance.

Correlation-induced conductance suppression at level degeneracy in a quantum dot.

The large, level-dependent g factors in an InSb nanowire quantum dot allow for the occurrence of a variety of level crossings in the dot. While we observe the standard conductance enhancement in the Coulomb blockade region for aligned levels with different spins due to the Kondo effect, a vanishing of the conductance is found at the alignment of levels with equal spins. This conductance suppression appears as a canyon cutting through the web of direct tunneling lines and an enclosed Coulomb blockade region. In the center of the Coulomb blockade region, we observe the predicted correlation-induced resonance. Our findings are supported by numerical and analytical calculations.

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Aminoacylation of rat liver transfer RNA with L-penicillamine. On the specificity of the aminoacylation reaction.

L-]14C]Penicillamine is bound to RNA from rat liver in an in vitro reaction catalyzed by rat liver aminoacyl-tRNA synthetases. Addition of certain naturally occuring amino acids results in a significant decrease of L-penicillamine binding. The most potent inhibitor of this binding is L-valine, followed by L-isoleucine and L-threonine. Amino acids without structural relationship to L-penicillamine in the non-functional part of the molecule, such as L-phenylalanine, are ineffective. Studies on the competition of L-penicillamine and L-isoleucine, respectively, with L-valine demonstrate the high specificity of the aminoacylation reaction. They show that the change of L-penicillamine binding to tRNA Val is considerably lower than that of L-valine.

Unmethylated DNA in mouse L cells. I. DNA fibre autoradiography.

Enzymatic methylation of DNA in mouse L cells has been studied using DNA fibre autoradiography to analyse the distribution of 5-methylcytosine in chromosomal DNA. The autoradiographic pattern of DNA labelled in the 5-methylcytosine is in several respects similar to the pattern of DNA replication. Two mean features are apparent: (1) the silver grains appear in well defined sections, and (2) the labelled sections are arranged in tandem along each DNA double helix. After a short pulse of radioactivity in the rate of growth of labeled sections in the pattern of DNA replication and the enzymatic methylation of DNA are identical. Unlike the replication pattern, DNA labeled during the S phase with L-[Me-3H] methionine is not completely labeled. There are distinct, 8-20 mum intervals in the autoradiographic pattern of this DNA. The length of these intervals may correspond to unmethylated sections of chromosomal DNA of about 23 to 58 kilo base pairs. These unmethylated sections of chromosomal DNA represent about 10% of the total genome.

Enzymatic hypermethylation of DNA in mouse-mouse somatic cell hybrids.

Somatic cell hybrids between mouse L fibroblasts (A9 cells) and Ehrlich ascites tumour cells were constructed by use of poly(ethyleneglycol). The hybrids were selected in hypoxanthine/aminopterin/thymidine medium and morphologically different syncaryons were isolated by a micromanipulation. They were simultaneously analysed for their tumourogeneity, a release of certain proteins into the culture medium, the activity of pyrimidine salvage pathways, and the extent of enzymatic DNA methylation. None of these hybrids gave a rise of tumours if transplanted into DBA/2 mice. Two of the hybrids released a protein immunologically cross-reacting with antibody against C-peptide of human proinsulin. Activities of pyrimidine salvage pathways as measured by incorporation of [14C]deoxycytidine into DNA cytosine and thymine, respectively, are different in the analyzed cells. Enzymatic DNA methylation in somatic cell hybrids was significantly elevated as compared to the parental cell lines.

Dynamic scenarios of multistable switching in semiconductor superlattices.

We analyze the dynamics of charge distributions in weakly coupled, doped, dc voltage biased semiconductor superlattices subject to voltage steps of different sizes. Qualitatively different current responses to voltage switching processes have been observed experimentally. We explain them by invoking distinct scenarios for electric-field domain formation, validated by numerical simulations. Furthermore, we investigate the transient from an unstable to a stable point in the current-voltage characteristics after a steplike or ramplike increase of the external voltage.