BRAF(V600) inhibition alters the microRNA cargo in the vesicular secretome of malignant melanoma cells

The BRAF inhibitors vemurafenib and dabrafenib can be used to treat patients with metastatic melanomas harboring BRAF(V600) mutations. Initial antitumoral responses are often seen, but drug-resistant clones with reactivation of the MEK-ERK pathway soon appear. Recently, the secretome of tumor-derived extracellular vesicles (EVs) has been ascribed important functions in cancers. To elucidate the possible functions of EVs in BRAF-mutant melanoma, we determined the RNA content of the EVs, including apoptotic bodies, microvesicles, and exosomes, released from such cancer cells after vemurafenib treatment. We found that vemurafenib significantly increased the total RNA and protein content of the released EVs and caused significant changes in the RNA profiles. RNA sequencing and quantitative PCR show that cells and EVs from vemurafenib-treated cell cultures and tumor tissues harvested from cell-derived and patient-derived xenografts harbor unique miRNAs, especially increased expression of miR-211-5p. Mechanistically, the expression of miR-211-5p as a result of BRAF inhibition was induced by increased expression of MITF that regulates the TRPM1 gene resulting in activation of the survival pathway. In addition, transfection of miR-211 in melanoma cells reduced the sensitivity to vemurafenib treatment, whereas miR-211-5p inhibition in a vemurafenib resistant cell line affected the proliferation negatively. Taken together, our results show that vemurafenib treatment induces miR-211-5p up-regulation in melanoma cells both in vitro and in vivo, as well as in subsets of EVs, suggesting that EVs may provide a tool to understand malignant melanoma progression.1114sciescopu

S-Phase Favours Notch Cell Responsiveness in the Drosophila Bristle Lineage

We have studied cell sensitivity to Notch pathway signalling throughout the cell cycle. As model system, we used the Drosophila bristle lineage where at each division N plays a crucial role in fate determination. Using in vivo imaging, we followed this lineage and activated the N-pathway at different moments of the secondary precursor cell cycle. We show that cells are more susceptible to respond to N-signalling during the S-phase. Thus, the period of heightened sensitivity coincided with the period of the S-phase. More importantly, modifications of S-phase temporality induced corresponding changes in the period of the cell's reactivity to N-activation. Moreover, S-phase abolition was correlated with a decrease in the expression of tramtrack, a downstream N-target gene. Finally, N cell responsiveness was modified after changes in chromatin packaging. We suggest that high-order chromatin structures associated with the S-phase create favourable conditions that increase the efficiency of the transcriptional machinery with respect to N-target genes

Notch and Prospero Repress Proliferation following Cyclin E Overexpression in the Drosophila Bristle Lineage

Understanding the mechanisms that coordinate cell proliferation, cell cycle arrest, and cell differentiation is essential to address the problem of how “normal” versus pathological developmental processes take place. In the bristle lineage of the adult fly, we have tested the capacity of post-mitotic cells to re-enter the cell cycle in response to the overexpression of cyclin E. We show that only terminal cells in which the identity is independent of Notch pathway undergo extra divisions after CycE overexpression. Our analysis shows that the responsiveness of cells to forced proliferation depends on both Prospero, a fate determinant, and on the level of Notch pathway activity. Our results demonstrate that the terminal quiescent state and differentiation are regulated by two parallel mechanisms acting simultaneously on fate acquisition and cell cycle progression

Charm and Bottom Semileptonic Decays

We review the present status of theoretical attempts to calculate the semileptonic charm and bottom decays and then present a calculation of these decays in the light--front frame at the kinematic point $q^2=0$. This allows us to evaluate the form factors at the same value of $q^2$, even though the allowed kinematic ranges for charm and bottom decays are very different. Also, at this kinematic point the decay is given in terms of only one form factor $A_{0}(0)$. For the ratio of the decay rates given by the E653 collaboration we show that the determination of the ratio of the Cabibbo--Kobayashi--Maskawa (CKM) matrix elements is consistent with that obtained from the unitarity constraint. At present, though, the unitarity method still has greater accuracy. Since comparisons of the semileptonic decays into $\rho$ and either electrons or muons will be available soon from the E791 Fermilab experiment, we also look at the massive muon case. We show that for a range of $q^2$ the $SU(3)_F$ symmetry breaking is small even though the contributions of the various helicity amplitudes becomes more complicated. For $B$ decays, the decay $B \rightarrow K^{*} \ell \bar{\ell}$ at $q^2=0$ involves an extra form factor coming from the photon contribution and so is not amenable to the same kind of analysis, leaving only the decay $B \rightarrow K^{*}\nu \bar{\nu}$ as a possibility. As the mass of the decaying particle increases we note that the $SU(3)$ symmetry becomes badly broken at $q^2=0$.Comment: Latex, 19 pages, two figures are attached, a minor change in the manuscript related to thi

A role for core planar polarity proteins in cell contact-mediated orientation of planar cell division across the mammalian embryonic skin

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017. Supplementary information accompanies this paper at doi:10.1038/s41598-017-01971-2.The question of how cell division orientation is determined is fundamentally important for understanding tissue and organ shape in both healthy or disease conditions. Here we provide evidence for cell contact-dependent orientation of planar cell division in the mammalian embryonic skin. We propose a model where the core planar polarity proteins Celsr1 and Frizzled-6 (Fz6) communicate the long axis orientation of interphase basal cells to neighbouring basal mitoses so that they align their horizontal division plane along the same axis. The underlying mechanism requires a direct, cell surface, planar polarised cue, which we posit depends upon variant post-translational forms of Celsr1 protein coupled to Fz6. Our hypothesis has parallels with contact-mediated division orientation in early C. elegans embryos suggesting functional conservation between the adhesion-GPCRs Celsr1 and Latrophilin-1. We propose that linking planar cell division plane with interphase neighbour long axis geometry reinforces axial bias in skin spreading around the mouse embryo body.Peer reviewe

Correction of human phospholamban R14del mutation associated with cardiomyopathy using targeted nucleases and combination therapy

published_or_final_versio

Van Gogh and Frizzled Act Redundantly in the Drosophila Sensory Organ Precursor Cell to Orient Its Asymmetric Division

Drosophila sensory organ precursor cells (SOPs) divide asymmetrically along the anterior-posterior (a-p) body axis to generate two different daughter cells. Planar Cell Polarity (PCP) regulates the a-p orientation of the SOP division. The localization of the PCP proteins Van Gogh (Vang) and Frizzled (Fz) define anterior and posterior apical membrane domains prior to SOP division. Here, we investigate the relative contributions of Vang, Fz and Dishevelled (Dsh), a membrane-associated protein acting downstream of Fz, in orienting SOP polarity. Genetic and live imaging analyses suggest that Dsh restricts the localization of a centrosome-attracting activity to the anterior cortex and that Vang is a target of Dsh in this process. Using a clone border assay, we provide evidence that the Vang and fz genes act redundantly in SOPs to orient its polarity axis in response to extrinsic local PCP cues. Additionally, we find that the activity of Vang is dispensable for the non-autonomous polarizing activity of fz. These observations indicate that both Vang and Fz act as cues for downstream effectors orienting the planar polarity axis of dividing SOPs

Dynamics of Endoreplication during Drosophila Posterior Scutellar Macrochaete Development

Endoreplication is a variant type of DNA replication, consisting only of alternating G1 and S phases. Many types of Drosophila tissues undergo endoreplication. However, the timing and the extent to which a single endocycling macrochaete undergoes temporally programmed endoreplication during development are unclear. Here, we focused on the dynamics of endoreplication during posterior scutellar (pSC) macrochaete development. Quantitative analyses of C values in shaft cells and socket cells revealed a gradual rise from 8C and 4C at 8 hours after pupal formation (APF) to 72C and 24C at 29 hours APF, respectively. The validity of the values was further confirmed by the measurement of DNA content with a confocal laser microscope. BrdU incorporation assays demonstrated that shaft cells undergo four rounds of endoreplication from 18 to 29.5 hours APF. In contrast, socket cells undergo two rounds of endoreplication during the same period. Statistical analyses showed that the theoretical C values, based on BrdU assays, nearly coincide with the actually measured C values in socket cells, but not in shaft cells after 22 hours APF. These analyses suggest that socket cells undergo two rounds of endoreplication. However, the mechanism of endoreplication in the shaft cells may change from 22 hours APF, suggesting the possibility that shaft cells undergo two or four rounds of endoreplication during the periods. We also found that the timing of endoreplication differs, depending on the type of macrochaete. Moreover, endocycling in shaft cells of both the left and right sides of pSC bristle lineages occurs in the same pattern, indicating that the process is synchronized for specific types of macrochaete. Our findings suggest that endocycling in macrochaete cell lineages can be a model for understanding mechanisms of endoreplication at the single-cell level