Structure of Essential RNA Regulatory Elements in the West Nile Virus 3'-Terminal Stem Loop.

Flaviviruses, such as West Nile and Dengue Virus, pose a significant and growing threat to global health. Central to the flavivirus life cycle are highly structured 5'- and 3'-untranslated regions (UTRs), which harbor conserved cis-acting RNA elements critical for viral replication and host adaptation. Despite their essential roles, detailed molecular insights into these RNA elements have been limited.

Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors.

Therapeutic plasma exchange for peripheral neuropathy associated with trisulfated heparan disaccharide IgM antibodies: A case series of 17 patients.

Background: Small fiber neuropathy (SFN) can be associated with autoantibodies, including those of IgM class with specificity for the trisulfated heparan disaccharide (TS-HDS) antigen. We hypothesized that, as an IgM autoantibody-mediated disorder, TS-HDS-associated SFN symptoms may be reduced with therapeutic plasma exchange (TPE).

Study Methods: This was an observational analysis of all patients referred for TPE from 2018 to 2020 following laboratory confirmation of SFN with TS-HDS autoantibodies; a loading course of 3 to 5 procedures over 2 weeks was completed, with some patients returning for monthly procedures.

A novel sample handling system for dissolution dynamic nuclear polarization experiments.

We present a system for facilitated sample vitrification, melting, and transfer in dissolution dynamic nuclear polarization (DDNP) experiments. In DDNP, a sample is typically hyperpolarized at cryogenic temperatures before dissolution with hot solvent and transfer to a nuclear magnetic resonance (NMR) spectrometer for detection in the liquid state. The resulting signal enhancements can exceed 4 orders of magnitude.

Evaluation of A plasma for incompatible patients.

Background: Plasma transfusion is a critical treatment in managing bleeding patients. In an effort to make plasma immediately available in spite of the limited amount of AB plasma, providers have begun using A plasma in life-threatening emergencies. As this practice becomes widely adopted it is important to evaluate safety.

Assessing Site-Specific Enhancements Imparted by Hyperpolarized Water in Folded and Unfolded Proteins by 2D HMQC NMR.

Hyperpolarized water can be a valuable aid in protein NMR, leading to amide group H polarizations that are orders of magnitude larger than their thermal counterparts. Suitable procedures can exploit this to deliver 2D H-N correlations with good resolution and enhanced sensitivity. These enhancements depend on the exchange rates between the amides and the water, thereby yielding diagnostic information about solvent accessibility.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water.

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and H-N 2D correlation experiments.

A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR.

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW's enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened.

Natural Abundance, Single-Scan C-C-Based Structural Elucidations by Dissolution DNP NMR.

While C-based Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) experiments offer an attractive alternative for establishing molecular structures, they suffer from low sensitivities arising from the scarcity of spin pairs present at natural abundance. Herein we demonstrate that dissolution dynamic nuclear polarization (dDNP) provides sufficient sensitivity to acquire 1D C INADEQUATE spectra in a single scan and at natural abundance. Moreover, if steps are adopted to endow sub-Hertz precision to these measurements, they allow one to measure carbon-carbon J couplings over both one and multiple bonds for each chemical site.

Identification and Rationalization of Kinetic Folding Intermediates for a Low-Density Lipoprotein Receptor Ligand-Binding Module.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time.

High-Resolution 2D NMR of Disordered Proteins Enhanced by Hyperpolarized Water.

This study demonstrates the usefulness derived from relying on hyperpolarized water obtained by dissolution DNP, for site-resolved biophysical NMR studies of intrinsically disordered proteins. Thanks to the facile amide-solvent exchange experienced by protons in these proteins, 2D NMR experiments that like HMQC rely on the polarization of the amide protons, can be enhanced using hyperpolarized water by several orders of magnitude over their conventional counterparts. Optimizations of the DNP procedure and of the subsequent injection into the protein sample are necessary to achieve these gains while preserving state-of-the-art resolution; procedures enabling this transfer of the hyperpolarized water and the achievement of foamless hyperpolarized protein solutions are demonstrated.

Sensitivity-enhanced detection of non-labile proton and carbon NMR spectra on water resonances.

Chemical exchange saturation transfer (CEST) experiments enhance the NMR signals of labile protons by continuously transferring these protons' saturation to an abundant solvent pool like water. The present study expands these principles by fusing into these experiments homonuclear isotropic mixing sequences, enabling the water-enhanced detection of non-exchangeable species. Further opportunities are opened by the addition of coupling-mediated heteronuclear polarization transfers, which then impose on the water resonance a saturation stemming from non-labile heteronuclear species like C.

Heteronuclear 1D and 2D NMR Resonances Detected by Chemical Exchange Saturation Transfer to Water.

A method to detect NMR spectra from heteronuclei through the modulation that they impose on a water resonance is exemplified. The approach exploits chemical exchange saturation transfers, which can magnify the signal of labile protons through their influence on a water peak. To impose a heteronuclear modulation on water, an HMQC-type sequence was combined with the FLEX approach.

Acquiring and processing ultrafast biomolecular 2D NMR experiments using a referenced-based correction.

Thanks to their special spatiotemporal encoding/decoding scheme, ultrafast (UF) NMR sequences can deliver arbitrary 2D spectra following a single excitation. Regardless of their nature, these sequences have in common their tracing of a path in the [Formula: see text]-[Formula: see text] plane, that will deliver the spectrum being sought after a 1D Fourier transformation versus [Formula: see text]. This need to simultaneously digitize two domains, tends to impose bandwidth limitations along all spectral axes.

The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors.

The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts.

Toward single-shot pure-shift solution 1H NMR by trains of BIRD-based homonuclear decoupling.

Achieving homonuclear 1H decoupling remains one of the key challenges in liquid-state NMR. Such spectra would endow a variety of organic and analytical applications with an increased resolution, and would ideally do so even in a one-dimensional format. A number of parallel efforts aimed at achieving this goal using two-dimensional acquisitions have been proposed; approaches demonstrated over recent years include, among others, new modes for achieving purely-absorptive J spectroscopy, the use of spatially-selective manipulations, and exploiting the natural spin dilution afforded by heteronuclei.

Monitoring mechanistic details in the synthesis of pyrimidines via real-time, ultrafast multidimensional NMR spectroscopy.

Recent years have witnessed unprecedented advances in the development of fast multidimensional NMR acquisition techniques. This progress could open valuable new opportunities for the elucidation of chemical and biochemical processes. This study demonstrates one such capability, with the first real-time Two-dimensional (2D) dynamic analysis of a complex organic reaction relying on unlabeled substrates.

Theory of nonrigid rotational motion applied to NMR relaxation in RNA.

Solution NMR spectroscopy can elucidate many features of the structure and dynamics of macromolecules, yet relaxation measurements, the most common source of experimental information on dynamics, can sample only certain ranges of dynamic rates. A complete characterization of motion of a macromolecule thus requires the introduction of complementary experimental approaches. Solid-state NMR spectroscopy successfully probes the time scale of nanoseconds to microseconds, a dynamic window where solution NMR results have been deficient, and probes conditions where the averaging effects of rotational diffusion of the molecule are absent.

Keratoameloblastoma: complex histologic variant of ameloblastoma.

Keratoameloblastoma is a rare subtype of ameloblastoma that has been reported only 12 times previously in the literature. Ameloblastomas are benign, locally aggressive neoplasms that constitute approximately 1% of odontogenic neoplasms. The mandible is involved in 80% of cases and the maxilla 20%.

Deuterium magic angle spinning NMR used to study the dynamics of peptides adsorbed onto polystyrene and functionalized polystyrene surfaces.

LKα14 is a 14 amino acid peptide with a periodic sequence of leucine and lysine residues consistent with an amphipathic α-helix. This "hydrophobic periodicity" has been found to result in an α-helical secondary structure at air-water interfaces and on both polar and nonpolar solid polymer surfaces. In this paper, the dynamics of LKα14 peptides, selectively deuterated at a single leucine and adsorbed onto polystyrene and carboxylated polystyrene beads, are studied using (2)H magic angle spinning (MAS) solid state NMR over a 100 °C temperature range.

Slow exchange model of nonrigid rotational motion in RNA for combined solid-state and solution NMR studies.

Functional RNA molecules are conformationally dynamic and sample a multitude of dynamic modes over a wide range of frequencies. Thus, a comprehensive description of RNA dynamics requires the inclusion of a broad range of motions across multiple dynamic rates which must be derived from multiple spectroscopies. Here we describe a slow conformational exchange theoretical approach to combining the description of local motions in RNA that occur in the nanosecond to microsecond window and are detected by solid-state NMR with nonrigid rotational motion of the HIV-1 transactivation response element (TAR) RNA in solution as observed by solution NMR.

Platelet-derived growth factor receptor beta signaling is required for efficient epicardial cell migration and development of two distinct coronary vascular smooth muscle cell populations.

The epicardium plays an essential role in coronary artery formation and myocardial development, but signals controlling the development and differentiation of this tissue are not well understood. To investigate the role of platelet-derived growth factor receptor (PDGFR)beta in development of epicardial-derived vascular smooth muscle cells (VSMCs), we examined PDGFRbeta(-/-) and PDGFRbeta epicardial mutant hearts. We found that PDGFRbeta(-/-) hearts failed to form dominant coronary vessels on the ventral heart surface, had a thinned myocardium, and completely lacked coronary VSMCs (cVSMCs).