Ca²⁺ stress induces tRNA met-misacylation and identification of Met-mistranslated CaMKII proteins.

<p>(<b>A</b>) Flow diagram summarizing the experiments and goals of this work. (<b>B</b>) tRNA misacylation with methionine measured by tRNA microarrays in the absence or presence of Ca²⁺ stress. Like many chemical inducers demonstrated earlier [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005745#pgen.1005745.ref001" target="_blank">1</a>], Ca²⁺ stress also increased tRNA misacylation. The array layout on the right shows the location of the Met-tRNA probes. (<b>C</b>) Cellular ROS measurement. HEK293T cells were suddenly exposed to Ca²⁺ at different concentrations (0, 0.1 or 1 mM) for 16 h, and cellular ROS level was measured. Tert-butyl hydrogen peroxide (TBHP) was used as positive control; n = 8 biological replicates. (<b>D</b>) Caspase-3 activity upon addition of 0.1 or 1 mM Ca²⁺. Z-DEVD-AMC was used as the substrate and Ac-DEVD-CHO was used as the inhibitor to confirm caspase-3 activity; n = 6 biological replicates. In panels (C) and (D), data are expressed as Mean ± S.D. of independent replicates, data between groups are compared by Student's t test and the P values are shown. (<b>E</b>) A Met-mistranslated CaMKII peptide identified by mass spectrometry. The Tyr-to-Met conversion is indicated by an arrow, and the peptide sequence is shown in the left side of the spectrum.</p

Characterization of CaMKII proteins isolated under no-stress and Ca²⁺-stress conditions.

<p>(<b>A</b>) C-terminal Flag-tagged proteins were purified from HEK293T cells and its purity examined by SDS-PAGE. Each sample is loaded in different amounts in two lanes. (<b>B</b>) Kinase activity assay of the proteins isolated from both conditions. Using the same amount of protein, CaMKII isolated under Ca²⁺ stress shows consistently higher activity than the protein isolated from no-stress condition. (<b>C</b>) Kinase activities of stressed and non-stressed samples at different substrate concentrations upon treatment with H₂O₂, or ATP (<b>D</b>).</p

Supplemental material for Comparison of the efficacy and safety of non-steroidal anti-inflammatory drugs for patients with primary dysmenorrhea: A network meta-analysis

<p>Supplemental material for Comparison of the efficacy and safety of non-steroidal anti-inflammatory drugs for patients with primary dysmenorrhea: A network meta-analysis by Xuan Feng and Xiaoyun Wang in Molecular Pain</p

Characterization of Met-mistranslated CaMKII <i>in vitro</i>.

<p>(<b>A</b>) Comparison of kinase activity between Flag-tagged WT and four Met-mistranslated CaMKII proteins purified from HEK293T cells after treatment with 10 μM H₂O₂. Activities are normalized to the WT protein. (<b>B</b>) Kinase activity of the WT and V208M mutant after pre-incubation with varying H₂O₂ concentration (0 to 1000 μM). Activities are normalized to the WT protein at 0 H₂O₂ (shown at 0.01 μM in this log-plot for better visualization). (<b>C</b>) Comparison of ATP dependent kinase activity between WT and four mutant CaMKII proteins after pre-incubation with 0.1 mM ATP. Activities are normalized to the WT protein. (<b>D</b>) Kinase activity of the WT and E359M mutant after ATP pre-incubation at varying ATP concentrations (0 to 2 mM). Activities are normalized to the WT protein at 0 ATP (shown at 0.004 mM in this log-plot for better visualization). (<b>E</b>) Autophosphorylation level of WT and four mutant CaMKII proteins. Levels are normalized to the WT protein. CaMKII kinase activity was measured as a function of γ³²P-ATP incorporation into the peptide substrate autocamtide. Autophosphorylation of CaMKII was measured as a function of γ³²P-ATP incorporation into CaMKII protein in the absence of substrate.</p

Subcellular levels of WT and two Met-mistranslated CaMKIIs.

<p>Subcelluar fractions isolated separately were subjected to SDS-PAGE followed by Western blotting to track the level of the C-terminal Flag-tagged WT and V208M, E359M CaMKII proteins. Respective subcellular markers were also shown, and control markers were used to exclude cross-contaminations. (<b>A</b>) Total lysate. (<b>B</b>) Cytoplasm. (<b>C</b>) Nucleus. (<b>D</b>) Mitochondria. (<b>E</b>) Endoplasmic reticulum. In all graphs, band signals from the CaMKII are divided by the signals from the subcellular markers, and then normalized to the WT levels at 0 mM Ca²⁺.</p

Ca²⁺ stress-induced elevation of caspase-3 activity by the WT and two Met-mistranslated CaMKIIs.

<p><b>(A)</b> CaMKII proteins isolated under 0 and 0.1 mM Ca²⁺ conditions analyzed by SDS-PAGE. (<b>B</b>) Caspase-3 activity in cells upon transfection with CaMKII protein isolated from stressed (0.1 mM Ca²⁺) or non-stressed (0 mM Ca²⁺) conditions; n = 12 biological replicates. (<b>C</b>) Caspase-3 activity in cells upon transfection with WT or mutant CaMKIIs (V208M and E359M) plasmids, n = 6 biological replicates. In panels (B) and (C), data are expressed as Mean ± S.D. of independent replicates, data between groups are compared by Student's t test and the P values are shown.</p

Met-mistranslation as nature’s way to expand the activity profile of proteomes.

<p>(<b>A</b>) The location of the 10 Met-mistranslated CaMKII residues detected in this work in the monomeric structure of CaMKII (PDB file 3SOA). Met-mistranslated substitutions are generally located on the surface of the CaMKII monomer. (<b>B</b>) The location of the V208M (red) and E359M (cyan) mutants in the hexamer ring of CaMKII (3SOA). The Ca²⁺/calmodulin regulatory segment is colored in dark yellow. V208 is at the base of the regulatory segment, and E359 is at the interface between subunits. (<b>C</b>) Limited proteolysis of WT and V208M/E359M mutant CaMKII proteins visualized using Coomassie staining. (<b>D</b>) A functional model of Met mistranslation on CaMKII activity. Known post-translational modifications that activate CaMKII include Met281/282 oxidation and Thr287/Thr306/Thr307 phosphorylation (above the domain structure of CaMKII). Mistranslation (below the domain structure) generates many mutant proteins, individually at low frequency, but collectively affecting CaMKII activity.</p

Supplementary Material, onlineAppendix_NVSQ – Generational Succession in American Giving: Donors Down, Dollars Per Donor Holding Steady But Signs That It Is Starting to Slip

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Table_1_Mendelian randomization analysis reveals an independent causal relationship between four gut microbes and acne vulgaris.xlsx

BackgroundNumerous studies have suggested a correlation between gut microbiota and acne vulgaris; however, no specific causal link has been explored.Materials and methodsTo investigate the possible causal relationship between gut microbiota and acne vulgaris, this study employed a large-scale genome-wide association study (GWAS) summary statistic. Initially, a two-sample Mendelian randomization (MR) analysis was utilized to identify the specific gut microflora responsible for acne vulgaris. We used the Inverse Variance Weighted (IVW) method as the main MR analysis method. Additionally, we assessed heterogeneity and horizontal pleiotropy, while also examining the potential influence of individual single-nucleotide polymorphisms (SNPs) on the analysis results. In order to eliminate gut microbiota with reverse causal associations, we conducted reverse MR analysis. Multivariate Mendelian randomization analysis (MVMR) was then employed to verify the independence of the causal associations. Finally, we performed SNP annotation on the instrumental variables of independent gut microbiota and acne vulgaris to determine the genes where these genetic variations are located. We also explored the biological functions of these genes through enrichment analysis.ResultThe IVW method of forward MR identified nine gut microbes with a causal relationship with acne vulgaris (p ConclusionOur study found independent causal relationships between four gut microbes and acne vulgaris, and revealed a genetic association between acne vulgaris patients and gut microbiota. Consider preventing and treating acne vulgaris by interfering with the relative content of these four gut microbes.</p

Data_Sheet_1_Mendelian randomization analysis reveals an independent causal relationship between four gut microbes and acne vulgaris.docx

BackgroundNumerous studies have suggested a correlation between gut microbiota and acne vulgaris; however, no specific causal link has been explored.Materials and methodsTo investigate the possible causal relationship between gut microbiota and acne vulgaris, this study employed a large-scale genome-wide association study (GWAS) summary statistic. Initially, a two-sample Mendelian randomization (MR) analysis was utilized to identify the specific gut microflora responsible for acne vulgaris. We used the Inverse Variance Weighted (IVW) method as the main MR analysis method. Additionally, we assessed heterogeneity and horizontal pleiotropy, while also examining the potential influence of individual single-nucleotide polymorphisms (SNPs) on the analysis results. In order to eliminate gut microbiota with reverse causal associations, we conducted reverse MR analysis. Multivariate Mendelian randomization analysis (MVMR) was then employed to verify the independence of the causal associations. Finally, we performed SNP annotation on the instrumental variables of independent gut microbiota and acne vulgaris to determine the genes where these genetic variations are located. We also explored the biological functions of these genes through enrichment analysis.ResultThe IVW method of forward MR identified nine gut microbes with a causal relationship with acne vulgaris (p ConclusionOur study found independent causal relationships between four gut microbes and acne vulgaris, and revealed a genetic association between acne vulgaris patients and gut microbiota. Consider preventing and treating acne vulgaris by interfering with the relative content of these four gut microbes.</p