14 research outputs found
Probing the conformation of ISWI-type chromatin remodeling enzymes by an integrative structural approach
Rapid purification of recombinant histones.
The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method "rapid histone purification" (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants
Digital Interventions for Common Mental Disorders in Low- and Middle-Income Countries: A Systematic Review and Meta-Analysis
Background:In low-resource settings, e-mental health may substantially increase access to evidence-based interventions for common mental disorders. We conducted a systematic literature search to identify randomised trials examining the effects of digital interventions with or without therapeutic guidance compared to control conditions in individuals with anxiety and/or depression symptoms in low- and middle-income countries (LMICs).Methods:The main outcome was the reduction in symptoms at the post-test. Secondary outcomes included improvements in quality of life and longer-term effects (≥20 weeks post-randomisation). The effect size Hedges’ g was calculated using the random effects model.Results:A total of 21 studies (23 comparisons) with 5.296 participants were included. Digital interventions were more effective than controls in reducing symptoms of common mental disorders at the post-test (g = −0.89, 95% confidence interval [CI] −1.26 to −0.52, p < 0.001; NNT = 2.91). These significant effects were confirmed when examining depressive (g = −0.77, 95% CI −1.11; −0.44) and anxiety symptoms separately (g = −1.02, 95% CI −1.53 to −0.52) and across all other sensitivity analyses. Digital interventions also resulted in a small but significant effect in improving quality of life (g = 0.32, 95% CI 0.19 to 0.45) at the post-test. Over the longer term, the effects were smaller but remained significant for all examined outcomes. Heterogeneity was moderate to high in all analyses. Subgroup and meta-regression analyses did not result in significant outcomes in any of the examined variables (e.g., guided vs. unguided interventions).Conclusions:Digital interventions, with or without guidance, may effectively bridge the gap between treatment supply and demand in LMICs. Nevertheless, more studies are needed to draw firm conclusions regarding the magnitude of the effects of digital interventions
Histone purification by cation exchange chromatography.
<p>The whole cell extract from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone-0104029-g002" target="_blank">Figure 2</a> containing solubilized <i>Drosophila</i> H2B (SN) was filtered and applied to cation exchange chromatography under denaturing conditions. (A) Equivalent amounts of the filtered whole cell extract (Input) and the flow-through fraction of the cation exchange column were analyzed by SDS-PAGE. Most H2B bound to the chromatography resin. (B) H2B was eluted by a NaCl gradient as indicated. Fractions 4–8 were pooled and processed further as described in the main text.</p
Side-by-side comparison of histone purities.
<p>Histones were purified according to the RHP protocol (RHP) and according to a published protocol that started with the preparation of inclusion bodies (IB; ref. 5). Both purification procedures started from the same amount of bacteria that were grown on the same day. (A) SDS-PAGE analysis. H2A showed the weakest overexpression (Fig. S1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>) and is consequently the least pure. M: protein marker. (B) Purities.</p
Histone purification strategies.
<p>Schematic depiction of the workflow of (A) the conventional histone purification method according to Luger and coworkers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Luger2" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Luger3" target="_blank">[3]</a> and (B) our RHP protocol. For further details see the main text. Footnotes indicate variations and simplifications of the initial protocol. * The gel filtration step was successfully omitted in simplified purification schemes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Clapier1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Vary1" target="_blank">[7]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. # These steps can be replaced by dilution into or dialysis against SAU 200 buffer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Clapier1" target="_blank">[5]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. ‡ To remove possible DNA contaminations, it was suggested to filter the sample through an anion exchange resin prior to applying it to the cation exchange chromatography <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Vary1" target="_blank">[7]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Gelbart1" target="_blank">[9]</a>. § Anion exchange filtering and cation exchange chromatography can be combined. See note in step 3.2 and Figure S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>.</p
Quality control of the histone octamers.
<p>Stoichiometry and purity of the octamers assembled from the histones purified according to the RHP method outlined in the main text were analyzed by SDS-PAGE (RHP). An octamer preparation assembled from histones purified from inclusion bodies according to published protocols was loaded in parallel (IB; ref. 5). The asterisk marks a contamination that is present to a lesser extent in RHP octamers.</p
Histone extraction.
<p>Whole cell extracts were prepared under denaturing conditions from bacteria expressing <i>Drosophila</i> H2B by French Press and sonication. Cell debris and residual insoluble material were pelleted by centrifugation. Efficiency of the histone extraction was analyzed on Coomassie-stained SDS gels by loading equivalent amounts of the supernatant containing the solubilized histones (SN) and the corresponding pellet fraction (P). Most H2B was present in the supernatant.</p
Histone octamer assembly.
<p>Histones purified according to the RHP protocol were assembled into octamers. The elution profile of the size exclusion chromatography column is depicted (upper panel). The protein content of selected elution fractions was analysed by SDS-PAGE (lower panel and Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029.s001" target="_blank">File S1</a>). Octamers eluted with a tailing shoulder, which contained a contaminating protein (asterisk).</p
Extinction coefficients of <i>Drosophila</i> histones at 280 nm.
<p>*Molecular weights do not include the initial methionine.</p>#<p>The extinction coefficients were calculated using the ProtParam tool with water as solvent (Swiss Institute of Bioinformatics; <a href="http://web.expasy.org/protparam/" target="_blank">http://web.expasy.org/protparam/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104029#pone.0104029-Artimo1" target="_blank">[17]</a>.</p