Method for Comparative Analysis of Ribonucleic Acids Using Isotope Labeling and Mass Spectrometry

Here, we describe a method for the comparative analysis of ribonucleic acids (RNAs). This method allows sequence or modification information from a previously uncharacterized RNA to be obtained by direct comparison with a reference RNA, whose sequence or modification information is known. This simple and rapid method is enabled by the differential labeling of two RNA samples. One sample, the reference RNA, is labeled with 16O during enzymatic digestion. The second sample, the candidate or unknown RNA, is labeled with 18O. By combining the two digests, digestion products that share the same sequence or post-transcriptional modification(s) between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. We illustrate the application of this approach for sequencing individual RNAs and demonstrate how this method can be used to identify sequence-specific differences in RNA modification. This comparative analysis of RNA digests (CARD) approach is scalable to multiple candidate RNAs using one or multiple reference RNAs and is compatible with existing methods for quantitative analysis of RNAs

Crystal structure of CARMA1-CARD and comparison with other CARD family members.

<p>(<b>A</b>) Cartoon representation of CARMA1-CARD. The six helices are colored magenta, and the loops are colored yellow. (<b>B</b>) The conserved central hydrophobic core. The conserved hydrophobic residues are essential for the stabilization of the structure. The side chains are shown as sticks and are colored green. (<b>C</b>) Sequence alignment of CARMA1-CARD and seven representative CARD domains. Conserved residues are labeled and colored red. Secondary structures (helices α1 to α6) are shown above the sequence. (<b>D</b>) A stereo view of the structural superposition of CARMA1-CARD and Apaf-1-CARD is shown in magenta and cyan, respectively.</p

Structural Insights into the Assembly of CARMA1 and BCL10

<div><p>The CBM complex (CARMA1, BCL10 and MALT1) plays a crucial role in B and T lymphocyte activation. CARMA1 serves as a scaffold for BCL10, MALT1 and other effector proteins and regulates various signaling pathways related to the immune response. The assembly of CARMA1 and BCL10 is mediated through a CARD-CARD interaction. Here, we report the crystal structure of the CARD domain of CARMA1 at a resolution of 1.75 Å. The structure consists of six helices, as previously determined for CARD domains. Structural and computational analysis identified the binding interface between CARMA1-CARD and BCL10-CARD, which consists of a basic patch in CARMA1 and an acidic patch in BCL10. Site-directed mutagenesis, co-immunoprecipitation and an NF-κB activation assay confirmed that the interface is necessary for association and downstream signaling. Our studies provide molecular insight into the assembly of CARMA1 and BCL10.</p> </div

EDTA-Assisted Self-Assembly of Fluoride-Substituted Hydroxyapatite Coating on Enamel Substrate

Reconstructed layers containing ordered enamel-like structures of fluoride-substituted hydroxyapatite (FHAp) microcrystals were constructed on a human enamel surface using ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) as the mediating agent under near-physiological conditions (pH 6.00, 37 °C, 1 atm). The effects of initial pH value, fluoride concentration, as well as reaction time on the formation of the FHAp microcrystals, including their microarchitectural structure, crystalline phase, chemical components, and hardness properties, were investigated using transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and Vickers microhardness measurements. The results of these in vitro experiments indicated that EDTA induced the assembly of hexagonal prismlike FHAp microcrystals along their c-axis direction, and the microcrystals further amalgamated with the extended reaction time. In addition, fluoride ions were found to play a critical role in the formation of hexagonal FHAp microcrystals. Interestingly, after reaction for 5 days, the Vickers microhardness of the new layer (347–370 VHN) was harder than that of natural tooth. On the basis of the experimental evidence, a mesoscale self-assembly mechanism was proposed to explain the growth of FHAp microcrystals

Data collection and refinement statistics.

a<p>the highest resolution shell.</p>b<p>.</p>c<p><b><i>R</i></b>_crystal = .</p>d<p><b><i>R</i></b>_free, calculated the same as <b><i>R</i></b>_crystal, but from a test set containing 5% of data excluded from the refinement calculation.</p

The binding surface of BCL10-CARD.

<p>(<b>A</b>) Superposition of homology models of BCL10-CARD. The computational results from the programs MODELLER and SWISS-MODEL are represented with green and blue cartoon models, respectively. (<b>B</b>) The electrostatic surface of BCL10-CARD. Red: negative; blue: positive; and white: neutral. Residues E50, E53 and E54 are labeled. (<b>C</b>) Sequence alignment of BCL10-CARD proteins from different species. The conserved amino acids are highlighted with red, and the conserved acidic residues that make up the acidic patch are denoted with asterisks. (<b>D</b>) Protein docking models of the CARMA1-CARD and BCL10-CARD calculated using the ZDOCK server. CARMA1-CARD and BCL10-CARD are colored magenta and gray, respectively. Three out of ten best scoring complexes place the BCL10-CARD domain approaching the interface containing residues R35, K41, K69 and R72 of CARMA1-CARD. The side chains of residues R35, K41, K69 and R72 are shown as sticks. (<b>E</b>) The interactions between CARD domains in the best docking complex model. CARMA1-CARD and BCL10-CARD are colored magenta and gray, respectively. Side chains of E50, E53 and E54 of BCL10 as well as R53, K41, K69 and R72 of CARMA1 are shown as stick.</p

The binding surface of CARMA1-CARD.

<p>(<b>A</b>) The representative basic residues (R35, K41, K69 and R72) on the positive surface of the CARMA1-CARD are colored blue. (<b>B</b>) Interactions between the basic residues and surrounding sulfate ions. The side chains of basic residues R35, K41, K69 and R72 and sulfate ions are shown as sticks. The oxygen atoms and sulfur atoms are colored red and yellow, respectively. Hydrogen bonds are shown as red dashed lines. (<b>C</b>) Sequence alignment of CARMA1-CARD proteins from different species. The conserved amino acids are highlighted in red. Conserved residues in the basic patch are denoted with asterisks.</p

Association of CARMA1 and BCL10.

<p>(<b>A</b>) Co-IP analysis of interactions between BCL10-CARD and variants of CARMA1-CARD. HEK293T cells were transiently co-transfected with GFP-tagged BCL10-CARD and wild type or mutants of Myc-tagged CARMA1-CARD constructs. Cell extracts were immunoprecipitated using an anti-Myc antibody and blotted using anti-GFP. (<b>B</b>) Co-IP analysis of interactions between Myc-tagged CARMA1-CARD and wild type or mutants of GFP-tagged BCL10-CARD constructs. (<b>C,D</b>) Bar graph displaying interactions between CARMA1-CARD and BCL10-CARD. (<b>E</b>) The effect of wild type and mutants of CARMA1 on the NF-κB reporter assay. (<b>F</b>) The effect of wild type and mutants of BCL10 on NF-κB activity. RLU: relative luciferase unit; Luc: firefly luciferase activity; and Ren: Renilla luciferase activity. The error bars indicate the standard error of the mean (n = 3 separate experiments). * indicates a P value<0.05, ** indicates a P value<0.001. (<b>G, H</b>) The expression levels of CARMA1 and BCL10 in NF-κB assays were checked by immunoblotting with anti-GFP, anti-MYC and anti-GAPDH antibodys, respectively.</p

Zn-Doped CoS₂ Nanoarrays for an Efficient Oxygen Evolution Reaction: Understanding the Doping Effect for a Precatalyst

Development of low-cost, efficient, and durable electrocatalysts for the oxygen evolution reaction (OER) is crucial for multiple energy conversions and storage devices. Herein, Zn-doped CoS2 nanoarrays supported on carbon cloth, Co­(Zn)­S2/CC, are fabricated through a facile sulfidization of CoZn metal–organic frameworks. This precatalyst, Co­(Zn)­S2/CC, with a well-defined nanoarray structure affords excellent OER catalytic activity (η = 248 mV at 10 mA/cm2) and long-term durability in 1 M KOH. X-ray photoelectron and in situ Raman spectroscopic studies indicate that Co­(Zn)­S2 undergoes surface reconstruction with the generation of Co­(Zn)­OOH adsorbed with SO42– at the surface during the OER process. The Zn dopant is calculated to impact on the electronic structure of Co species and further the adsorption of intermediates. This work not only provides a novel method for the synthesis of bimetallic sulfides but also gives insights into the doping effect on the OER performance of transition metal sulfides

Additional file 1: of Baduanjin exercise for patients with ischemic heart failure on phase-II cardiac rehabilitation (BEAR trial): study protocol for a prospective randomized controlled trial

Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) Checklist. (PDF 131 kb