36 research outputs found
Regioselective Annulation of Aryl Sulfonamides with Allenes through Cobalt-Promoted CβH Functionalization
The
development of an efficient method for the construction of
biologically relevant sultams is described, which represents the first
case of cobalt-promoted CβH/NβH functionalization of
sulfonamides with allenes. This newly developed annulation reaction
demonstrated good functional group tolerance and excellent regioselectivity.
Both terminal monosubstituted allenes and internal disubstituted allenes
can be employed to give the desired sultams in good yields. This strategy
can be successfully used to build a unique sultam library with novel
structural diversity
Epigenetic Modifications Unlock the Milk Protein Gene Loci during Mouse Mammary Gland Development and Differentiation
<div><h3>Background</h3><p>Unlike other tissues, development and differentiation of the mammary gland occur mostly after birth. The roles of systemic hormones and local growth factors important for this development and functional differentiation are well-studied. In other tissues, it has been shown that chromatin organization plays a key role in transcriptional regulation and underlies epigenetic regulation during development and differentiation. However, the role of chromatin organization in mammary gland development and differentiation is less well-defined. Here, we have studied the changes in chromatin organization at the milk protein gene loci (casein, whey acidic protein, and others) in the mouse mammary gland before and after functional differentiation.</p> <h3>Methodology/Principal Findings</h3><p>Distal regulatory elements within the casein gene cluster and whey acidic protein gene region have an open chromatin organization after pubertal development, while proximal promoters only gain open-chromatin marks during pregnancy in conjunction with the major induction of their expression. In contrast, other milk protein genes, such as alpha-lactalbumin, already have an open chromatin organization in the mature virgin gland. Changes in chromatin organization in the casein gene cluster region that are present after puberty persisted after lactation has ceased, while the changes which occurred during pregnancy at the gene promoters were not maintained. In general, mammary gland expressed genes and their regulatory elements exhibit developmental stage- and tissue-specific chromatin organization.</p> <h3>Conclusions/Significance</h3><p>A progressive gain of epigenetic marks indicative of open/active chromatin on genes marking functional differentiation accompanies the development of the mammary gland. These results support a model in which a chromatin organization is established during pubertal development that is then poised to respond to the systemic hormonal signals of pregnancy and lactation to achieve the full functional capacity of the mammary gland.</p> </div
Summary of chromatin organization in the Elf5 region.
<p>(<b>A</b>) ChIP-seq for H3K4me2 in liver (red), lactating mammary glands (blue) and mammary epithelial cells isolated from 12 week virgin mammary glands (black). Black bars at the top of the Liver, Lactating MG, and Virgin MG panels show the H3K4me2 enriched regions identified by MACS. <b>CpG</b>: genomic locations of CpG sites in the 5β² flanking region, exon1 & part of intron 1 (β1418 to +189); DMR indicates the differentially modified region analyzed. (<b>B</b>) Table: shows average levels of DNA methylation (%) at developmental stages for individual CpG sites in region analyzed by SEQUENOM mass-array. (<b>C</b>) Scatter plot of the changes in DNA methylation levels at different time points during mammary gland development and differentiation at DMR in Elf5 promoter: different developmental time points are depicted with different filled symbols. <i><u>Mammary epithelial cell enriched fractions (MEC</u>)</i>, filled symbols: Virgin (2.5_3V: 2.5β3 week old virgin female, filled circle; 3.5β4V, 3.5β4 week old virgin, filled square; 5.5_6V: 5.5β6 week old female virgin, filled downward pointing triangle; 8_15V; 8β15 week old virgin, filled upward pointing triangle); Pregnancy (7P: 7day pregnant, filled diamond; 16P: 16 days pregnant (whole tissue), half vertically filled circle); Lactation (8L; 8 day lactation (whole tissue), half horizontally filled circle); Inv (>28 day after lactation) 5-pointed star; AMV: Age Matched Virgin (Virgin animal same age as >28 day involuted animal), filled hexagonal. <i><u>Non-MEC cell fraction of the mammary gland</u></i> (Non-MEC, open symbols): 2.5_3: 2.5β3 week old virgin female, open circle; 3.5β4V, 3.5β4 week old virgin, open square; 5.5_6: 5.5β6 week old female virgin, open downward pointing triangle; 8_15; 8β15 Week virgin, open upward pointing triangle; Pregnancy (7P: 7day pregnant, open diamond); Inv (>28 day after lactation, 8 pointed star); AMV: Age Matched Virgin (Virgin animal same age as >28 day involuted animal), open hexagonal); or <i><u>Non-mammary tissue</u></i> (hexagonal with star): Liver.</p
H3K4me2 ChIP-seq and RT-PCR results for genes expressed in virgin mammary gland.
<p>(<b>AβB</b>): ChIP-seq reads for H3K4me2 in liver tissue (<b>Liver</b>, red), lactating mammary glands (<b>Lactating MG</b>, blue) and mammary epithelial cells isolated from 12 week virgin mammary glands (<b>Virgin MG</b>, black). (<b>CβD</b>): Q-RT-PCR of (<b>C</b>) Amphiregulin (Areg) and (<b>D</b>) Progesterone receptor PgR at different developmental stages in MEC isolated from mammary gland tissue of 13 week virgin (13wV), 3 days pregnant (3dP) or whole mammary tissue from 16.5 day pregnant (16.dP Mg) day 1 and day8 lactation (1dL Mg and 8dL MG) as well as liver from an 8d lactating animal (8dL Lvr). Real-time-RT-PCR data are normalized to Keratine 18 and expressed relative to 13wV MEC.</p
Alpha-lactalbumin (Lalba) and extracellular proteinase inhibitor (Expi) developmental chromatin organization.
<p>ChIP-seq reads for H3K4me2 in liver tissue (<b>Liver</b>, red), mammary epithelial cells isolated from 12 week virgin mammary glands (<b>Virgin MEC</b>, black), and lactating mammary glands (<b>Lactating MG</b>, blue). In the bottom panel, black arrows indicate location and transcriptional direction of the Lalba and Expi genes.</p
Chromatin markers in the casein gene cluster region in different tissues and developmental stages.
<p>This illustration is a close-up of the CSN region from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone-0053270-g001" target="_blank">Figure 1</a>. (<b>A</b>) ChIP-seq reads for H3K4me2 in MEC isolated from virgin mammary glands at 12 weeks of age, staged at diestrus (<b>Virgin MG</b>, black), in lactating mammary glands (<b>Lactating MG</b>, blue) and in liver tissue (<b>Liver</b>, red). A summary of peaks identified by MAC and SICER are at the top of each of the Virgin MG, Lactating MG, and Liver panels. In the evolutionary conserved region (<b>ECR</b>) panel, ECRs are shown in green; ECR1, 6 16 and 21 are indicated in pink. In the bottom panel, the locations of the casein genes (black) and non-casein genes (gray) are indicated by solid arrows denoting the direction of transcription. (<b>B</b>) Summary of markers of epigenetic regulation in the CSN region. (<b><i>H3Ac</i></b>) Summary of results of ChIP for H3-acetylation on lactating mammary gland (<b>Lact</b>, blue) and liver tissue (<b>Lvr</b>, red). Sites investigated are indicated by rectangles: filled rectangles, H3AC enriched; open rectangle, non-enriched (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270.s003" target="_blank">figure S3</a>). (<b><i>DHS</i></b>) Summary of DNaseI Hypersensitivity analysis in MEC isolated from from virgin mammary glands at 12 weeks of age, staged at diestrus (<b>Virg</b>, black), in lactating mammary gland (<b>Lact</b>, blue) and liver tissue (<b>Lvr</b>, red). Sites investigated are indicated by rectangles: filled rectangle, DHS; open rectangle, non-DHS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270.s004" target="_blank">Fig. S4</a>). (<b><i>DNAme</i></b>) Summary of results of DNA methylation analysis on lactating mammary gland (<b>Lact</b>) and liver tissue (<b>Lvr</b>) (see also Fig. 5). Levels of DNA methylation are based on Fig. 5A and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270.s009" target="_blank">Table S2</a> and are color coded as follows: 0β20% lightest blue, 21β40% light blue, 41β60% blue, 61β80% dark blue and 80β100% black; open rectangles: not analyzed. (<b><i>K9me2</i></b>) Summary of ChIP-chip for H3K9me2, a histone modification associated with closed chromatin, on liver tissue from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270-Wen1" target="_blank">[62]</a>; red filled rectangle indicates enrichment of H3K9me2 in liver tissue.</p
DNA methylation levels during mammary gland development and differentiation at casein gene promoters.
<p>(<b>A, B</b>) DNA methylation levels (%) for Beta-casein promoter (Csn2) (average of 4 CpG sites in 326 bp), (<b>C,D</b>) Alpha s2b casein promoter (Csn1s2b) (average of 2CpG sites in 300 bp) and (<b>E,F</b>) Kappa casein promoter <i>(Csn3)</i> (average of 5 CpG sites in 588 bp) were determined by bisulfite cloning and sequencing. <i><u>Mammary epithelial Cell enriched fractions (MEC</u>)</i>: Virgin (8_15V; 8β15 week old virgins,); Pregnancy (7P: 7day pregnant females); Inv (>28 day after lactation); AMV: Age Matched Virgin (Virgin animals same age as >28 day involuted animals). <i><u>Whole tissue</u>:</i> pregnancy (16P: 16 days pregnant); Lactation (8L; 8 day lactation). <i><u>Non-mammary gland</u>: none-MEC cell fraction of the mammary gland</i> (8_15; 8β15 Week old virgins); Pregnancy (7P: 7day pregnant females); Inv (>28 day after lactation); AMV: Age Matched Virgin (Virgin animals same age as >28 day involuted animals)); <i>or <u>non-mammary tissue</u></i>: Liver. (<b>A,C,E</b>) Overview of Csn2 (<b>A</b>), Csn1s2b (<b>C</b>) and Csn3 (<b>E</b>) promoter and CpG sites analyzed, Tabular representation of DNA methylation levels (%) at individual CpG sites in analyzed region, and DNA methylation levels (%) heatmap representation (yellow 0%-dark blue 100%);. (<b>B, D, F</b>) Methylation status at CpG sites in Bisulfite-sequence clones for mammary gland or non-mammary gland tissue (yellow 0% methylation, dark blue 100% methylation): (2β6 individual preps (mammary gland MEC (8-15wV 8β15, 7dP, INV, AMV) or Non-Mammary gland epithelial cells (8-15wV_s, 8-15_s, 7dP_s, INV_s, AMV_s)) and 4β6 individual animals (16dp, 8dL, Lvr), 5β10 clones/DNA prep), Csn2 (C), Csn1s2b (E), Csn3 (F).</p
Chromatin organization at genomic region around Whey Acidic Protein gene (Wap).
<p>Summary of markers of chromatin organization aligned to the Wap region in mouse genome assembly mm9 in the UCSC Genome Browser. (<b>AβB</b>) ChIP-seq reads for H3K4me2 in liver tissue (<b>Liver</b>, red), lactating mammary gland (<b>Lactating MG</b>, blue) and mammary epithelial cells isolated from 12 week virgin mammary glands (<b>Virgin MEC</b>, black). <b>ECR</b>: genomic locations of DHS conserved in mouse and rabbit <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270-Millot1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270-Millot2" target="_blank">[54]</a>, CpG island is indicated by dark green bar. (<b>B</b>) Close-up of Wap region from (<b>A</b>). <b>H3Ac</b>: Summary of H3Ac-ChIP results for Wap promoter and HSS2 (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270.s003" target="_blank">Fig. S3</a>) in lactating mammary gland (<b>Lact</b>, blue) and Liver tissue (<b>liver</b>, red): closed rectangle indicates enrichment of H3Ac at site, open rectangle indicates lack of enrichment at site. <b>DHS</b>: Summary of DHS results for rabbit from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270-Millot1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053270#pone.0053270-Millot2" target="_blank">[54]</a>. Closed rectangle indicates presence of DHS, open rectangle indicates absence of DHS.</p
Summary of ChIP-seq for H3K4me2 at the casein gene cluster region and flanking regions.
<p>ChIP-seq reads aligned to mouse gene assembly mm9 in the UCSC Genome Browser for H3K4me2 enrichment in liver tissue (red), lactating mammary gland (blue) and mammary epithelial cells (MEC) isolated from 12 week old virgin animals in diestrus (black). The bottom panel displays the locations of annotated genes.</p
Flow-Induced Structured Phase in Nonionic Micellar Solutions
In
this work, we consider the flow of a nonionic micellar solution
(precursor) through an array of microposts, with focus on its microstructural
and rheological evolution. The precursor contains polyoxyethylene(20)
sorbitan monooleate (Tween-80) and cosurfactant monolaurin (ML). An
irreversible flow-induced structured phase (NI-FISP) emerges after
the nonionic precursor flows through the hexagonal micropost arrays,
when subjected to strain rates βΌ10<sup>4</sup> s<sup>β1</sup> and strain βΌ10<sup>3</sup>. NI-FISP consists of close-looped
micellar bundles and multiconnected micellar networks as evidenced
by transmission electron microscopy (TEM) and cryo-electron microscopy
(cryo-EM). We also conduct small-angle neutron scattering (SANS) measurements
in both precursor and NI-FISP to illustrate the structural transition.
We propose a potential mechanism for the NI-FISP formation that relies
on the micropost arrays and the flow kinematics in the microdevice
to induce entropic fluctuations in the micellar solution. Finally,
we show that the rheological variation from a viscous precursor solution
to a viscoelastic micellar structured phase is associated with the
structural evolution from the precursor to NI-FISP