Formulation of In Situ Chemically Cross-Linked Hydrogel Depots for Protein Release: From the Blob Model Perspective

The fast release rate and the undesirable covalent binding are two major problems often encountered in formulating in situ chemically cross-linked hydrogel as protein release depot, particularly when prolonged release over months is desirable. In this study, we applied the De Gennes’ blob theory to analyze and tackle these two problems using a vinylsulfone-thiol (VS-SH) reaction based in situ hydrogel system. We showed that the simple scaling relation ξ_b ≈ <i>R</i>_g(<i>c</i>/<i>c</i>*)^{−<i>v</i>/(3<i>v</i>−1)} is applicable to the in situ hydrogel and the mesh size estimated from the precursor polymer parameters is a reasonable match to experimental results. On the other hand, as predicted by the theory and confirmed by experiments, the drug diffusion within hydrogel depends mainly on polymer concentration but not the degree of modification (DM). The covalent binding was found to be caused by the mismatch of location between the reactive groups and the entanglement points. The mismatch and, thus, the protein binding were minimized by increasing the DM and concentration of the SH polymer relative to the VS polymer, as predicted by theory. Using these principles, an in situ hydrogel system for the controlled release of an antiangiogenic antibody therapeutics bevacizumab for 3 months was developed

One-Step “Click” Method for Generating Vinyl Sulfone Groups on Hydroxyl-Containing Water-Soluble Polymers

One-Step “Click” Method for Generating Vinyl Sulfone Groups on Hydroxyl-Containing Water-Soluble Polymer

<i>Arabidopsis</i> AL PHD-PRC1 Complexes Promote Seed Germination through H3K4me3-to-H3K27me3 Chromatin State Switch in Repression of Seed Developmental Genes

<div><p>Seed germination and subsequent seedling growth define crucial steps for entry into the plant life cycle. For those events to take place properly, seed developmental genes need to be silenced whereas vegetative growth genes are activated. Chromatin structure is generally known to play crucial roles in gene transcription control. However, the transition between active and repressive chromatin states during seed germination is still poorly characterized and the underlying molecular mechanisms remain largely unknown. Here we identified the <i>Arabidopsis</i> PHD-domain H3K4me3-binding ALFIN1-like proteins (ALs) as novel interactors of the Polycomb Repressive Complex 1 (PRC1) core components AtBMI1b and AtRING1a. The interactions were confirmed by diverse <i>in vitro</i> and <i>in vivo</i> assays and were shown to require the AL6 N-terminus containing PAL domain conserved in the AL family proteins and the AtRING1a C-terminus containing RAWUL domain conserved in animal and plant PRC1 ring-finger proteins (including AtRNIG1a/b and AtBMI1a/b). By T-DNA insertion mutant analysis, we found that simultaneous loss of AL6 and AL7 as well as loss of AtBMI1a and AtBMI1b retards seed germination and causes transcriptional derepression and a delayed chromatin state switch from H3K4me3 to H3K27me3 enrichment of several seed developmental genes (<i>e.g. ABI3</i>, <i>DOG1</i>, <i>CRU3</i>, <i>CHO1</i>). We found that AL6 and the PRC1 H3K27me3-reader component LHP1 directly bind at <i>ABI3</i> and <i>DOG1</i> loci. In light of these data, we propose that AL PHD-PRC1 complexes, built around H3K4me3, lead to a switch from the H3K4me3-associated active to the H3K27me3-associated repressive transcription state of seed developmental genes during seed germination. Our finding of physical interactions between PHD-domain proteins and PRC1 is striking and has important implications for understanding the connection between the two functionally opposite chromatin marks: H3K4me3 in activation and H3K27me3 in repression of gene transcription.</p></div

Functional characterization of <i>AL</i> genes.

<p>(A) Tissue-specificity of <i>AL</i> gene expression. Relative expression levels of <i>AL1</i>, <i>AL2</i>, <i>AL5</i>, <i>AL6</i>, and <i>AL7</i> were determined by quantitative RT-PCR in different plant organs. Leaves: rosette leaves from 4-week-old plants; Buds: floral buds before anthesis; Flowers: flowers at anthesis; Seeds: dry seeds. Data represent means ± SD of three biological replicates. (B) <i>AL6</i> and <i>AL7</i> genomic structure and T-DNA insertion mutants. Genes are schematically represented by black boxes for exons, black lines for introns and dashed boxes for untranslated regions. Triangles indicate T-DNA insertion sites and arrowheads indicate RT-PCR primer positions. Relative expression levels of <i>AL6</i> and <i>AL7</i> in Col-0 and in <i>al6</i> and <i>al7</i> mutants are shown as means ± SD of three biological replicates. (C) Representative seed germination images of Col-0, <i>al6 al7</i> double mutant, and the double mutant complemented by the <i>AL6</i> promoter driving <i>GFP-AL6</i> fusion gene (<i>+pAL6:GFP-AL6</i>). Images were taken five days after stratification from plates containing MS media or MS supplemented with 100 mM NaCl (MS+NaCl). (D) Germination rate of Col-0, double mutants <i>al6 al7</i> and <i>Atbmi1a Atbmi1b</i>, and the quadruple mutant <i>al6 al7 Atbmi1a Atbmi1b</i> plated on MS (top graph), MS supplemented with 200 mM mannitol (middle graph) or with 100 mM NaCl (bottom graph). Data represent average germination percentages ± SD of three biological replicates, each >60 seeds, observed daily for 12 days after stratification.</p

LHP1 and AL6 binding at <i>ABI3</i> and <i>DOG1</i> chromatin.

<p>Relative enrichments of LHP1-myc and GFP-AL6 proteins were analyzed at the five regions (a to e) of <i>ABI3</i> (A) and <i>DOG1</i> (B) loci. Transgenic seeds/seedlings expressing <i>LHP1-myc</i> or <i>GFP-AL6</i> were analyzed at 24 or 72 hours after stratification (HAS) by ChIP using anti-myc or anti-GFP antibodies. Samples in the absence of antibodies serve as negative controls (mock). Values were normalized to internal controls (relative to input and to <i>TUB2</i>). Data represent means ± SD of three biological replicates.</p

A proposed model for AL PHD-PRC1 complexes in silencing seed developmental genes during seed germination.

<p>ALs, <i>via</i> their highly conserved PHD domains, bind H3K4me3 of chromatin, triggering the recruitment of PRC1 components BMI1 and RING1 <i>via</i> AL-AtBMI1, AL-AtRING1, and AtBMI1-AtRING1 physical interactions. Next, two possible pathways (1 and 2) can lead to stable repressive chromatin state formation. In the first case (1), PRC2 is recruited <i>via</i> its subunit CLF interaction with AtRING1 and deposits H3K27me3, favoring further LHP1 recruitment <i>via</i> H3K27me3-LHP1 binding. In the second case (2), LHP1 is first recruited <i>via</i> its interaction with AtRING1 or AtBMI1, and then PRC2 is recruited <i>via</i> its subunit MSI1 interaction with LHP1 and deposits H3K27me3. In both cases, H3K27me3-LHP1 and PRC2 MSI1-LHP1 interactions form a positive loop in H3K27me3 enrichment. This hypothetic model can explain how seed developmental genes (<i>e.g. ABI3</i>, <i>DOG1</i>) are switched from active transcription to a stably repressed state, which is necessary for timely seed germination and proper seedling growth and development.</p

Interactions of ALs and PRC1 ring-finger proteins in yeast two-hybrid assay.

<p>(A) Schematic representation of full-length and truncated AtRING1a and AL6 proteins. The conserved domains PAL, PHD, RING and RAWUL are indicated. (B) Yeast two-hybrid assays. Yeast cultures co-expressing the indicated protein combinations from pGADT7 and pGBKT7 were plated as a 1∶10 dilution from left to right onto SD-LTA selective media. Growth of yeast cells indicates positive protein-protein interaction.</p

Relative expression levels of seed developmental genes in Col-0, <i>al6 al7</i> and <i>Atbmi1a Atbmi1b</i>.

<p>Relative expression levels of <i>ABI3</i>, <i>DOG1</i>, <i>CRU1</i>, <i>CRU3</i>, <i>PER1</i> and <i>CHO1</i> were analyzed by quantitative RT-PCR using seeds/seedlings at 0, 24 and 72 hours after stratification. Data represent means ± SD of three biological replicates.</p

Additional file 1: of Policy development and challenges of global mental health: a systematic review of published studies of national-level mental health policies

Appendix A to E. (DOC 230 kb

SDG2-Mediated H3K4 Methylation Is Required for Proper <em>Arabidopsis</em> Root Growth and Development

<div><p>Trithorax group (TrxG) proteins are evolutionarily conserved in eukaryotes and play critical roles in transcriptional activation via deposition of histone H3 lysine 4 trimethylation (H3K4me3) in chromatin. Several <i>Arabidopsis</i> TrxG members have been characterized, and among them SET DOMAIN GROUP 2 (SDG2) has been shown to be necessary for global genome-wide H3K4me3 deposition. Although pleiotropic phenotypes have been uncovered in the <i>sdg2</i> mutants, <i>SDG2</i> function in the regulation of stem cell activity has remained largely unclear. Here, we investigate the <i>sdg2</i> mutant root phenotype and demonstrate that <i>SDG2</i> is required for primary root stem cell niche (SCN) maintenance as well as for lateral root SCN establishment. Loss of SDG2 results in drastically reduced H3K4me3 levels in root SCN and differentiated cells and causes the loss of auxin gradient maximum in the root quiescent centre. Elevated DNA damage is detected in the <i>sdg2</i> mutant, suggesting that impaired genome integrity may also have challenged the stem cell activity. Genetic interaction analysis reveals that <i>SDG2</i> and <i>CHROMATIN ASSEMBLY FACTOR-1</i> act synergistically in root SCN and genome integrity maintenance but not in telomere length maintenance. We conclude that SDG2-mediated H3K4me3 plays a distinctive role in the regulation of chromatin structure and genome integrity, which are key features in pluripotency of stem cells and crucial for root growth and development.</p></div