Accuracy of wind observations from open-ocean buoys: Correction for flow distortion

The comparison of equivalent neutral winds obtained from (a) four WHOI buoys in the subtropics and (b) scatterometer estimates at those locations reveals a root-mean-square (RMS) difference of 0.56-0.76 m/s. To investigate this RMS difference, different buoy wind error sources were examined. These buoys are particularly well suited to examine two important sources of buoy wind errors because: (1) redundant anemometers and a comparison with numerical flow simulations allow us to quantitatively assess flow distortion errors, and (2) one-minute sampling at the buoys allows us to examine the sensitivity of buoy temporal sampling/averaging in the buoy-scatterometer comparisons. The inter-anemometer difference varies as a function of wind direction relative to the buoy wind vane and is consistent with the effects of flow distortion expected based on numerical flow simulations. Comparison between the anemometers and scatterometer winds supports the interpretation that the inter-anemometer disagreement, which can be up to 5% of the wind speed, is due to flow distortion. These insights motivate an empirical correction to the individual anemometer records and subsequent comparison with scatterometer estimates show good agreement

Charge-State-Dependent Collision-Induced Dissociation Behaviors of RNA Oligonucleotides via High-Resolution Mass Spectrometry

Mass spectrometry (MS)-based analysis of RNA oligonucleotides (oligos) plays an increasingly important role in the development of RNA therapeutics and epitranscriptomics research. However, MS fragmentation behaviors of RNA oligomers are understood insufficiently. Herein, we characterized the negative-ion-mode fragmentation behaviors of 26 synthetic RNA oligos containing four to eight nucleotides using collision-induced dissociation (CID) on a high-resolution, accurate-mass instrument. We found that in CID spectra acquired under the normalized collision energy (NCE) of 35%, approximately 70% of the total peak intensity was attributed to sequencing ions (a-B, a, b, c, d, w, x, y, z), around 25% of the peak intensity came from precursor ions that experienced complete or partial loss of a nucleobase in the form of either a neutral or an anion, and the remainder were internal ions and anionic nucleobases. The top five sequencing ions were the y, c, w, a-B, and a ions. Furthermore, we observed that CID fragmentation behaviors of RNA oligos were significantly impacted by their precursor charge. Specifically, when the precursors had a charge from 1– to 5–, the fractional intensity of sequencing ions decreased, while that of precursors that underwent either neutral or charged losses of a nucleobase increased. Additionally, we found that RNA oligos containing 3′-U tended to produce precursors with HNCO and/or NCO– losses, which presumably corresponded to isocyanic acid and cyanate anion, respectively. These findings provide valuable insights for better comprehending the mechanism behind RNA fragmentation by MS/MS, thereby facilitating the future automated identification of RNA oligos based on their CID spectra in a more efficient manner

Two conserved SQ/TQ motifs in the N-terminal region of Crb2 are essential for Chk1 recruitment and activation.

<p>(A) Sequence alignment of <i>S. pombe</i> Crb2 and its orthologs from three other fission yeast species revealed two conserved neighboring SQ/TQ motifs in the N-terminal region of Crb2. The positions of the two motifs in <i>S. pombe</i> Crb2 are labeled on top. (B) Mutations in Crb2 SQ/TQ cluster resulted in DNA damage hypersensitivity. Fivefold serial dilutions of cells were spotted on YES plates and incubated at 30°C. Photos were taken 2 d later for untreated, UV-treated, IR-treated and CPT-containing plates. The HU-containing plates were photographed 3 d later. Strains used were LD195, LD346, DY377, DY369, DY370 and DY371. (C) DNA damage-induced Chk1 phosphorylation is defective in Crb2 SQ/TQ cluster mutants. Cells were untreated or treated with 20 µM CPT for 2 h. Cell lysates were separated on SDS-PAGE and probed with an anti-Myc antibody by immunoblotting. Strains used were DY377, LD195, DY369, DY370 and DY371. (D) Mutations in Crb2 SQ/TQ cluster diminished Chk1 foci but not Crb2 foci. Cells expressing Chk1-GFP and CFP-Crb2 were challenged with S-phase IR treatment as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g001" target="_blank">Figure 1A</a> and examined by fluorescence microscopy. Arrows indicate dim Chk1 foci in <i>crb2-T73A</i> and <i>crb2-S80A</i> cells. Strains used were DY6503, DY6504, DY6505 and DY6506. Bar, 5 µm.</p

A model of how Crb2 mediates Chk1 activation.

<p>In step 1, DSB formation induces the phosphorylation of H2A (<b>γ</b>-H2A) on surrounding chromatin. Upon DSB resection, Rad3 and 9-1-1 are recruited to single-stranded DNA and single-strand/double-strand junction, respectively. Rad4/Cut5 is also recruited via binding to Rad9. In step 2, through its interactions with modified histones and Rad4/Cut5, Crb2 relocalizes to the DSB and becomes phosphorylated at the SQ/TQ cluster by Rad3. In step 3, phosphorylated SQ/TQ cluster interacts with Chk1 and promotes its phosphorylation by Rad3. In step 4, the activated Chk1 molecule leaves the DSB to fulfill its effector function and allows further rounds of Chk1 activation to occur.</p

Fusing the Crb2(67–85) peptide with homologous recombination protein Rad22 can bypass Crb2 and Rad9 for Chk1 recruitment and activation.

<p>(A) Rad22-Crb2(67–85) can recruit Chk1 to DSBs in the absence of Crb2 and Rad9, but not when the SQ/TQ motifs are mutated, or in the absence of Rad3. Cells were treated with 80 Gy IR and examined by fluorescence microscopy after 1.5 h. Strains used were DY6536, DY6534, DY6535, DY6989 and DY6538. Bar, 5 µm. (B and C) The Crb2(67–85) peptide fused with Rad22 is phosphorylated by Rad3 upon DNA damage treatment. Cells expressing Flag-tagged Rad22 or Flag-tagged Rad22-Crb2(67–85) fusion protein were untreated or treated with 20 µM CPT for 2 h. Rad22 was immunoprecipitated using anti-Flag beads, eluted by boiling in SDS loading buffer, and immunoblotted with anti-Flag or anti-phospho-SQ/TQ antibody. Strains used were DY6561, DY6562 and DY6565. (D) The Crb2(67–85) peptide fused with Rad22 interacts with Chk1 in a manner dependent on Rad3 kinase and the SQ/TQ motifs. Cells expressing Myc-tagged Chk1 were treated with 320 Gy IR. Rad22 was immunoprecipitated using anti-Flag beads, and Chk1 was detected by immunoblotting with anti-Myc antibody. Strains used were DY6561, DY6562, DY6563 and DY6565. (E) DNA damage sensitivity of <i>crb2Δ</i> can be partially rescued by expressing a Rad22-Crb2(67–85) fusion protein. Spot assay was performed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002817#pgen-1002817-g002" target="_blank">Figure 2B</a>. Strains used were DY6539, DY6540, DY6541, DY6543, DY6536, DY6534 and DY6535. (F) Rad22-Crb2(67–85) can rescue the checkpoint defect of <i>crb2Δ</i> in a manner dependent on the SQ/TQ motifs and Chk1, but not Rad9. The indicated mutants in a <i>cdc25-22</i> background were synchronized at late G2 phase by incubating at 35.5°C for 2.5 h. Following 80 Gy IR treatment, cultures were returned to the permissive temperature of 25°C. Mitosis was monitored by staining cells with Hoechst and Calcofluor dyes. Strains used were DY6551, DY6550, DY6552, DY6553, DY6555 and DY6554.</p

The effect of <i>cgt-1/3</i> double RNAi on the localization patterns of hTAC::GFP.

<p>Confocal images of intestinal cells of wild type worms treated with control RNAi (A, A’), <i>cgt-1/3</i> double RNAi (B, B’) and <i>sgk-1(ok538)</i> mutant animals treated with control RNAi (C, C’), <i>cgt-1/3</i> double RNAi (D, D’) expressing hTAC::GFP. The basolateral membranes were indicated by arrows. Scale bars: 5 μm.</p

MIG-14::GFP was delivered to early lysosomes marked by RAB-7.

<p>Confocal images of the wild type (A, A’, A”) and <i>sgk-1(null)</i> (B, B’, B”) intestinal cells expressing MIG-14::GFP (A, B) or mCHERRY::RAB-7 (A’, B’). Insets show magnified areas (× 2.5). Scale bars: 5 μm. Quantitation of RAB-7-positive ring-like vesicles with or without MIG-14::GFP puncta inside (C). *** <i>P</i> value <0.001, ** <i>P</i> value <0.01 (Student’s <i>t</i>-test).</p