SFRP-4 abrogates Wnt-3a-induced β-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a

Broadband dual-comb hyperspectral imaging and adaptable spectroscopy with programmable frequency combs

We explore the advantages of a free-form dual-comb spectroscopy (DCS) platform based on time-programmable frequency combs for real-time, penalty-free apodized scanning. In traditional DCS, the fundamental spectral resolution, which equals the comb repetition rate, can be excessively fine for many applications. While the fine resolution is not itself problematic, it comes with the penalty of excess acquisition time. Post-processing apodization (windowing) can be applied to tailor the resolution to the sample, but only with a deadtime penalty proportional to the degree of apodization. The excess acquisition time remains. With free-form DCS, this deadtime is avoided by programming a real-time apodization pattern that dynamically reverses the pulse periods between the dual frequency combs. In this way, one can tailor the spectrometer's resolution and update rate to different applications without penalty. We show operation of a free-form DCS system where the spectral resolution is varied from the intrinsic fine resolution of 160 MHz up to 822 GHz by applying tailored real-time apodization. Because there is no deadtime penalty, the spectral signal-to-noise ratio increases linearly with resolution by 5000x over this range, as opposed to the square root increase observed for postprocessing apodization in traditional DCS. We explore the flexibility to change resolution and update rate to perform hyperspectral imaging at slow camera frame rates, where the penalty-free apodization allows for optimal use of each frame. We obtain dual-comb hyperspectral movies at a 20 Hz spectrum update rate with broad optical spectral coverage of over 10 THz

Antibody-mediated PCSK9 neutralization worsens outcome after bare-metal stent implantation in mice

AIMS Despite advances in pharmacotherapy and device innovation, in-stent restenosis (ISR) and stent thrombosis (ST) remain serious complications following percutaneous coronary intervention (PCI) procedure with stent implantation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme involved in plasma cholesterol homeostasis and recently emerged as a therapeutic target for hypercholesterolemia. Antibody-based PCSK9 inhibition is increasingly used in different subsets of patients, including those undergoing PCI. However, whether PCSK9 inhibition affects outcome after stent implantation remains unknown. METHODS AND RESULTS 12 to 14 weeks old C57Bl/6 mice underwent carotid artery bare-metal stent implantation. Compared to sham intervention, stent implantation was associated with increased expression of several inflammatory mediators, including PCSK9. The increase in PCSK9 protein expression was confirmed in the stented vascular tissue, but not in plasma. To inhibit PCSK9, alirocumab was administered weekly to mice before stent implantation. After 6 weeks, histological examination revealed increased intimal hyperplasia in the stented segment of alirocumab-treated animals compared to controls. In vitro, alirocumab promoted migration and inhibited the onset of senescence in primary human vascular smooth muscle cells (VSMC). Conversely, it blunted the migration and increased the senescence of endothelial cells (EC). CONCLUSION Antibody-based PCSK9 inhibition promotes in-stent intimal hyperplasia and blunts vascular healing by increasing VSMC migration, while reducing that of EC. This effect is likely mediated, at least in part, by a differential effect on VSMC and EC senescence. The herein-reported data warrant additional investigations concerning the use of PCSK9 inhibitors in patients undergoing PCI with stent implantation

Synchronization of Distant Optical Clocks at the Femtosecond Level

The use of optical clocks/oscillators in future ultra-precise navigation, gravitational sensing, coherent arrays, and relativity experiments will require time comparison and synchronization over terrestrial or satellite free-space links. Here we demonstrate full unambiguous synchronization of two optical timescales across a free-space link. The time deviation between synchronized timescales is below 1 fs over durations from 0.1 s to 6500 s, despite atmospheric turbulence and kilometer-scale path length variations. Over several days, the time wander is 40 fs peak-to-peak. Our approach relies on the two-way reciprocity of a single-spatial-mode optical link, valid to below 225 attoseconds across a turbulent 4-km path. This femtosecond level of time-frequency transfer should enable optical networks using state-of-the-art optical clocks/oscillators.Comment: 19 pages, 9 figure