A Novel Mouse Model for Non-Invasive Single Marker Tracking of Mammary Stem Cells In Vivo Reveals Stem Cell Dynamics throughout Pregnancy

Mammary stem cells (MaSCs) play essential roles for the development of the mammary gland and its remodeling during pregnancy. However, the precise localization of MaSCs in the mammary gland and their regulation during pregnancy is unknown. Here we report a transgenic mouse model for luciferase-based single marker detection of MaSCs in vivo that we used to address these issues. Single transgene expressing mammary epithelial cells were shown to reconstitute mammary glands in vivo while immunohistochemical staining identified MaSCs in basal and luminal locations, with preponderance towards the basal position. By quantifying luciferase expression using bioluminescent imaging, we were able to track MaSCs non-invasively in individual mice over time. Using this model to monitor MaSC dynamics throughout pregnancy, we found that MaSCs expand in both total number and percentage during pregnancy and then drop down to or below baseline levels after weaning. However, in a second round of pregnancy, this expansion was not as extensive. These findings validate a powerful system for the analysis of MaSC dynamics in vivo, which will facilitate future characterization of MaSCs during mammary gland development and breast cancer.     

Association between periodontitis and common variants in the promoter of the interleukin-6 gene

We recently reported an association between interleukin-6 (IL6) polymorphisms (SNPs) and haplotypes and aggressive periodontitis (AgP). The aim of this study was to investigate this association in a larger cohort of subjects, affected by either aggressive or chronic periodontitis. Five IL6 SNPs were analyzed in 765 subjects (167 generalized aggressive periodontitis, 57 localized aggressive, 310 chronic periodontitis and 231 periodontally healthy). Among Caucasians (n = 454) there were moderate associations for ?1363T allele (p = 0.011) and for ?174GG and ?1363GG genotypes with diagnosis of periodontitis (respectively, p = 0.044, OR = 1.6, 95% CI = 1.0–2.4, and p = 0.017, OR = 1.8, 95% CI = 1.1–2.8, adjusted for age, gender and smoking). Haplotypes containing the ?174G>C, ?1363G>T and ?1480C>G polymorphisms were associated with diagnosis of periodontitis (p = 0.02). Subgroup analysis by disease phenotype showed associations for the localized AgP (LAgP) group and ?1480C>G and ?6106A>T SNPs (p = 0.007 and 0.010, respectively). Among Caucasians the genotypes IL6 ?1480 CC and ?6106 TT increased the adjusted OR for LAgP (OR = 3.09 and 2.27, respectively). This study supports the hypothesis that IL6 polymorphisms and haplotypes are moderately associated with periodontitis, possibly acting through influencing tissue levels of IL6. This association is stronger for LAgP than for other periodontal disease phenotypes.     

Effectiveness of repeated autumn and spring fires for understorey restoration in weed‐invaded temperate eucalypt woodlands

Question. Can strategic burning, targeting differing ecological characteristics of native and exotic species, facilitate restoration of native understorey in weed‐invaded temperate grassy eucalypt woodlands? Location. Gippsland Plains, eastern Victoria, Australia. Methods. In a replicated, 5‐year experimental trial, the effects of repeated spring or autumn burning were evaluated for native and exotic plants in a representative, degraded Eucalyptus tereticornis grassy woodland. Treatments aimed to reduce seed banks and modify establishment conditions of exotic annual grasses, and to exhaust vegetative reserves of exotic perennial grasses. Treatments were applied to three grassland patch types, dominated by the native grass Austrodanthonia caespitosa, ubiquitous exotic annuals, or the common exotic perennial grass Paspalum dilatatum. Results. The dominant native grass Austrodanthonia caespitosa and native forbs were resilient to repeated fires, and target exotic annuals and perennials were suppressed differentially by autumn and spring fires. Exotic annuals were also suppressed by drought, reducing the overall treatment effects but indicating important opportunities for restoration. The initially sparse exotic geophyte Romulea rosea increased in cover with fire and the impact of this species on native forbs requires further investigation. There was minimal increase in diversity of subsidiary natives with fire, probably owing to lack of propagules. Conclusions. While fire is often considered to increase ecosystem invasibility, our study showed that strategic use of fire, informed by the relative responses of available native and exotic taxa, is potentially an effective step towards restoration of weed‐invaded temperate eucalypt woodlands.     

Moisture Sorption Characteristics of Starchy Products: Oat Flour and Rice Flour

Moisture sorption isotherms for oat flour and rice flour were determined at 5, 23, and 45 °C using a gravimetric technique in an a _w range of 0.08–0.98. The results obtained showed that, for a _w values lower than around 0.75, in both flours, the sorption capacity decreased with increasing temperature, while the opposite behavior was observed at a _w greater than this value. It was found that the experimental data in the water activity range considered were better represented by the four-parameter Peleg equation. The monolayer water contents for both materials were determined by the BET and GAB models. The net isosteric heats of sorption were estimated using the Clausius–Clapeyron equation. For both materials, the monolayer water content and the isosteric heat of sorption were found to decrease with increasing temperature and increasing moisture content, respectively.     

Vernalization-Mediated VIN3 Induction Overcomes the LIKE-HETEROCHROMATIN PROTEIN1/POLYCOMB REPRESSION COMPLEX2-Mediated Epigenetic Repression

VERNALIZATION INSENSITIVE3 (VIN3) induction by vernalization is one of the earliest events in the vernalization response of Arabidopsis (Arabidopsis thaliana). However, the mechanism responsible for vernalization-mediated VIN3 induction is poorly understood. Here, we show that the constitutive repression of VIN3 in the absence of the cold is due to multiple repressive components, including a transposable element-derived sequence, LIKE-HETEROCHROMATIN PROTEIN1 and POLYCOMB REPRESSION COMPLEX2. Furthermore, the full extent of VIN3 induction by vernalization requires activating complex components, including EARLY FLOWERING7 and EARLY FLOWERING IN SHORT DAYS. In addition, we observed dynamic changes in the histone modifications present at VIN3 chromatin during the course of vernalization. Our results show that the induction of VIN3 includes dynamic changes at the level of chromatin triggered by long-term cold exposure.The transition from vegetative growth to reproductive growth is one of major developmental transitions in the life cycle of plants. Flowering plants have evolved to maximize the reproductive success by optimizing the timing of flowering. The onset of floral transition in flowering plants is affected by various environmental cues, including changing daylength and temperature. Plants use such environment cues to monitor seasonal changes and determine the timing of flowering. In temperate climates, the winter season imposes a prolonged period of cold to plants. In many plant species, exposure to prolonged period of cold provides competence to flower in the following spring through the process known as vernalization (for review, see Sung and Amasino, 2005; Dennis and Peacock, 2007).While most lab strains of Arabidopsis (Arabidopsis thaliana) do not require vernalization treatment to flower rapidly, many naturally occurring accessions of Arabidopsis flower very late unless vernalized (Clarke and Dean, 1994; Lee and Amasino, 1995; Michaels and Amasino, 1999; Gazzani et al., 2003). In Arabidopsis, the vernalization requirement is conferred by two dominant genes, FRIGIDA (FRI) and FLOWERING LOCUS C (FLC; Lee et al., 1993; Clarke and Dean, 1994; Michaels and Amasino, 1999; Sheldon et al., 1999; Johanson et al., 2000). FLC encodes a MADS box DNA binding protein that functions as a repressor of the floral integrators, FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) (Michaels and Amasino, 1999; Sheldon et al., 1999; Lee et al., 2000; Samach et al., 2000; Hepworth et al., 2002; Helliwell et al., 2006; Searle et al., 2006). FLC antagonizes the effect of CONSTANS (CO) by directly binding to regulatory regions within FT and SOC1. It appears that FRI contributes to the vernalization requirement solely by activating FLC. FRI encodes a protein with unknown biochemical function (Johanson et al., 2000). Vernalization results in the stable repression of FLC (Michaels and Amasino, 1999; Bastow et al., 2004; Sung and Amasino, 2004) so that floral integrators can be activated when the photoperiod pathway activates CO (Parcy, 2005). Thus, vernalization renders plants to be competent to flower upon exposure to inductive photoperiods in winter annuals and biennials.Other than FRI, another group of genes involved in FLC activation have been identified from screens for early flowering in certain genotypes or photoperiod conditions. They include EARLY FLOWERING5 (ELF5), ELF7, ELF8, VERNALIZATION INDEPENDENCE3 (VIP3), VIP4, EARLY FLOWERING IN SHORT DAYS4 (ESD4), PHOTOPERIOD INDEPENDENT EARLY1 (PIE1), EARLY FLOWERING IN SHORT DAYS (EFS), and ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1)/ATX2/ARABIDOPSIS TRITHORAX-RELATED7 (ATXR7; Reeves et al., 2002; Zhang and van Nocker, 2002; Noh and Amasino, 2003; He et al., 2004; Noh et al., 2004; Oh et al., 2004; He and Amasino, 2005; Kim et al., 2005; Zhao et al., 2005; Choi et al., 2007; Saleh et al., 2008; Tamada et al., 2009). Some of these genes encode proteins with chromatin modification functions, including components of RNA Polymerase II-associated factor 1 (PAF1) complex (VIP3, VIP4, ELF7, and ELF8), a Histone H3 Lys-36 methyltransferase (EFS), a Histone H3 Lys-4 methyltransferase (ATX1, ATX2, and ATXR7), and a SWR1-related nucleosome remodeling factor (PIE1).Mitotically stable repression of FLC by vernalization is also achieved by chromatin modifications (Michaels and Amasino, 1999; Bastow et al., 2004; Sung and Amasino, 2004). FLC mRNA expression is repressed during the course of cold exposure, and several repressive histone marks accumulate at FLC chromatin, including methylations at Histone H3 Lys-9 (H3K9) and Histone H3 Lys-27 (H3K27). The accumulation of histone modifications at FLC chromatin depends on the activity of chromatin remodeling complexes. During the course of cold exposure, POLYCOMB REPRESSION COMPLEX2 (PRC2), which has H3K27 methyltransferase activity, is enriched at FLC chromatin (Wood et al., 2006; De Lucia et al., 2008) and establishes the stable repression of FLC through H3K27 methylation. PRC2 biochemically copurifies with members of the VERNALIZATION INSENSITIVE3 (VIN3) family of proteins, including VIN3, VIN3-LIKE1 (VIL1)/VERNALIZATION5 (VRN5), and VIL2/VERNALIZATION LIKE1 (VEL1; Wood et al., 2006; De Lucia et al., 2008).The vernalization response involves two phases. The first is a cold perception that measures the cumulative time of exposure to cold. Vernalization requires cold exposure over the course of weeks rather than minutes or hours. The second phase is essentially the output of the cold perception. When a sufficient duration of cold has been perceived, a series of changes of gene expression ensue, ultimately leading to the epigenetic repression of FLC. VIN3, which is a repressive chromatin-remodeling component, is induced only after a sufficient duration of cold has been perceived. One of the early molecular events in the vernalization response is the induction of VIN3 by prolonged cold exposure. Upstream of VIN3, there must be a biochemical mechanism to sense cold. However, nothing is known about the upstream event. The induction of VIN3 by cold is unique in that VIN3 induction takes several days of cold, unlike many cold-induced genes, which are induced within hours of cold exposure (Thomashow, 2001). Furthermore, VIN3 mRNA expression is quickly rerepressed once plants are moved to warm temperature.Interestingly, the induction of VIN3 also involves changes in active histone marks at VIN3 chromatin, including Histone H3 acetylation, Histone H4 acetylation, and Histone H3 Lys-4 trimethylation (H3K4me3; Finnegan et al., 2005; Bond et al., 2009). However, no chromatin remodeling complexes have been identified to have roles in those changes at VIN3 chromatin.Here, we show that VIN3 is in a constitutively silenced state, which is mediated by the presence of a transposable element (TE)-derived sequence in its promoter region and by the components of repressive complexes, including PRC2 and LHP1. In addition, the full extent of VIN3 induction by vernalization requires components of activating complexes, including PAF1 and EFS. Thus, VIN3 expression is under the influence of chromatin level regulators. Furthermore, VIN3 chromatin is in a transiently bivalent state when VIN3 mRNA is induced, having both a repressive histone mark and an active histone mark at VIN3 chromatin. Our results show that VIN3 is under a constitutively repressed state, which is transiently relieved from repression only when sufficient cold is provided.     

Antimony uptake and toxicity in sunflower and maize growing in SbIII and SbV contaminated soil

Using pot experiments, we investigated the uptake of antimony (Sb) by sunflower (Helianthus annuus L. cv. Iregi), and maize (Zea mays L. cv. Magister) in two different soils, a potting mix and an agricultural soil. In one treatment Sb was added to the experimental soils as KSb(OH)₆ (“Sb^V-treatment”) and in the other as Sb₂O₃ (“Sb^III-treatment”). Soluble soil Sb concentrations were linearly related to the applied Sb rates, ranging from 0.02 (controls) to 175 mg L^?1 soil solution. Accumulation of Sb tended to be slightly higher in the Sb^V treatment in sunflower, while no difference in Sb uptake between the two Sb treatments was found in maize. The half maximal effective concentration (EC₅₀) values derived from the dose-response curves were higher for the Sb^V than for the Sb^III treatment when they were related to soluble soil Sb concentrations, but differences became insignificant when they were related to shoot Sb concentrations. Maize was substantially more sensitive to Sb toxicity than sunflower, indicating physiological differences in Sb tolerance between the two plant species. Our results show that on soils with high Sb contamination, as often found in shooting ranges, plants may suffer from Sb toxicity.