Vemurafenib and dabrafenib downregulates RIPK4 level

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1) - a suppressor of the BRAF/MEK/ERK pathway - via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors

In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental signalling

STUDY QUESTION How does maternal cigarette smoking disturb development of the human fetal ovary?<p></p> SUMMARY ANSWER Maternal smoking increases fetal estrogen titres and dysregulates several developmental processes in the fetal ovary.<p></p> WHAT IS KNOWN ALREADY Exposure to maternal cigarette smoking during gestation reduces human fetal ovarian cell numbers, germ cell proliferation and subsequent adult fecundity.<p></p> STUDY DESIGN, SIZE, DURATION The effects of maternal cigarette smoking on the second trimester human fetal ovary, fetal endocrine signalling and fetal chemical burden were studied. A total of 105 fetuses were studied, 56 from mothers who smoked during pregnancy and 49 from those who did not.<p></p> PARTICIPANTS/MATERIALS, SETTING METHODS Ovary, liver and plasma samples were collected from electively terminated, normally progressing, second trimester human fetuses. Circulating fetal hormones, levels of 73 fetal ovarian transcripts, protein localization, density of oocytes/primordial follicles and levels of 16 polycyclic aromatic hydrocarbons (PAHs) in the fetal liver were determined.<p></p> MAIN RESULTS AND THE ROLE OF CHANCE Circulating fetal estrogen levels were very high and were increased by maternal smoking (ANOVA, P = 0.055–0.004 versus control). Smoke exposure also dysregulated (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.046–0.023) four fetal ovarian genes (cytochrome P450 scc [CYP11A1], NOBOX oogenesis homeobox [NOBOX], activator of apoptosis harakiri [HRK], nuclear receptor subfamily 2, group E, member 1 [NR2E1]), shifted the ovarian Inhibin βA/inhibin α ratio (NHBA/INHA) transcript ratio in favour of activin (ANOVA, P = 0.049 versus control) and reduced the proportion of dominant-negative estrogen receptor 2 (ERβ: ESR2) isoforms in half the exposed fetuses. PAHs, ligands for the aryl hydrocarbon receptor (AHR), were increased nearly 6-fold by maternal smoking (ANOVA, P = 0.011 versus control). A fifth transcript, COUP transcription factor 1 (nuclear receptor subfamily 2, group F, member 1: NR2F1, which contains multiple AHR-binding sites), was both significantly increased (ANOVA, P = 0.026 versus control) and dysregulated by (two-way ANOVA, smoking versus gestation weeks interaction, P = 0.021) maternal smoking. NR2F1 is associated with repression of FSHR expression and smoke-exposed ovaries failed to show the normal increase in FSHR expression during the second trimester. There was a significantly higher number of DEAD (Asp-Glu-Ala-Asp) box polypeptide 4 (DDX4) VASA-positive (ANOVA, P = 0.016 versus control), but not POU domain, class 1, transcription factor 1 (POU5F1) OCT3/4-positive, oocytes in smoke-exposed fetuses and this matched with a significantly higher number of primordial follicles (ANOVA, P = 0.024 versus control).<p></p> LIMITATIONS, REASONS FOR CAUTION The effects of maternal smoking on establishment of the maximum fetal primordial follicle pool cannot be reliably studied in our population since the process is not completed until 28 weeks of gestation and normal fetuses older than 21 weeks of gestation are not available for study. Our data suggest that some fetal ovaries are affected by smoke exposure while others are not, indicating that additional studies, with larger numbers, may show more significant effects.<p></p> WIDER IMPLICATIONS OF THE FINDINGS Fetal exposure to chemicals in cigarette smoke is known to lead to reduced fecundity in women. Our study suggests, for the first time, that this occurs via mechanisms involving activation of AHR, disruption of inhibin/activin and estrogen signalling, increased exposure to estrogen and dysregulation of multiple molecular pathways in the exposed human fetal ovary. Our data also suggest that alterations in the ESR2 positive and dominant negative isoforms may be associated with reduced sensitivity of some fetuses to increased estrogens and maternal smoking

GDF9 is Transiently Expressed in Oocytes before Follicle Formation in the Human Fetal Ovary and is Regulated by a Novel NOBOX Transcript

During human fetal ovary development, the process of primordial follicle formation is immediately preceded by a highly dynamic period of germ cell and somatic cell reorganisation. This is regulated by germ-cell specific transcription regulators, by the conserved RNA binding proteins DAZL and BOLL and by secreted growth factors of the TGFβ family, including activin βA: these all show changing patterns of expression preceding follicle formation. In mice, the transcription factor Nobox is essential for follicle formation and oocyte survival, and NOBOX regulates the expression of GDF9 in humans. We have therefore characterised the expression of GDF9 in relation to these known key factors during follicle formation in the human fetal ovary. mRNA levels of GDF9, BMP15 and NOBOX were quantified by qRT-PCR and showed dramatic increases across gestation. GDF9 protein expression was localised by immunohistochemistry to the same population of germ cells as those expressing activin βA prior to follicle formation but did not co-localise with either BOLL or DAZL. A novel NOBOX isoform was identified in fetal ovary that was shown to be capable of up-regulating the GDF9 promoter in reporter assays. Thus, during oogenesis in humans, oocytes go through a dynamic and very sharply demarcated sequence of changes in expression of these various proteins, even within individual germ cell nests, likely to be of major functional significance in determining selective germ cell survival at this key stage in ovarian development. Transcriptional variation may contribute to the range of age of onset of POI in women with NOBOX mutations

Application of infrared thermography technique to the thermal assessment of multiple thermal bridges and windows

A major contribution to the global trend in reducing energy consumption can be made by improving the thermal performance of buildings. Minimization of heat loss via the building envelope is key to maximizing building energy efficiency. The building envelope contains different types of thermal bridging that must be accounted for while assessing the overall building envelope thermal performance. Multiple thermal bridges commonly occur and the distance between them determines the degree to which they interact thermally. To avoid overestimation of the linear thermal transmittance, it is important to account for interaction effects. Complex multiple thermal bridging occurs in window systems. The thermal performance of windows depends not only on the window performance itself but also on its installation into the wall. This study demonstrates an application of the quantitative infrared thermography technique to evaluate the heat lost via multiple thermal bridging. It is shown that using this methodology, the heat loss via multiple thermal bridges can be easily estimated in an existing building envelope, without any knowledge of its internal structure or material properties. For windows, it is demonstrated that jointly assessing the additional heat loss through the window and due to the installation of the window into the wall is a practical way to determine the actual heat loss caused by the presence of a window. A window thermal transmittance or M-value is introduced to quantify the total additional heat loss through the building element due to the presence of the window. The methodology was validated against experimental measurements taken on different specimens in a hot box device. Results from the thermographic analysis also co-related well with results from finite element heat transfer and computational fluid dynamics simulations. (C) 2018 Elsevier B.V. All rights reserved.The authors wish to thank the College of Engineering and Informatics, National University of Ireland Galway for providing a Postgraduate Scholarship for the first author. Cracow University of Technology CUT, especially the Head of the Institute, Prof. Jacek Schnotale, and the technician, Eng. Mariusz Rusiecki, for access to the hot box facility and constant assistance during the testing. SIP Energy Ltd., Athenry, Co. Galway, in particular John Moylan, for supplying the test specimens. Enterprise Ireland for Innovation Voucher IV-2014-4203.peer-reviewed2020-03-1

Application of infrared thermography technique to the thermal assessment of multiple thermal bridges and windows

A major contribution to the global trend in reducing energy consumption can be made by improving the thermal performance of buildings. Minimization of heat loss via the building envelope is key to maximizing building energy efficiency. The building envelope contains different types of thermal bridging that must be accounted for while assessing the overall building envelope thermal performance. Multiple thermal bridges commonly occur and the distance between them determines the degree to which they interact thermally. To avoid overestimation of the linear thermal transmittance, it is important to account for interaction effects. Complex multiple thermal bridging occurs in window systems. The thermal performance of windows depends not only on the window performance itself but also on its installation into the wall. This study demonstrates an application of the quantitative infrared thermography technique to evaluate the heat lost via multiple thermal bridging. It is shown that using this methodology, the heat loss via multiple thermal bridges can be easily estimated in an existing building envelope, without any knowledge of its internal structure or material properties. For windows, it is demonstrated that jointly assessing the additional heat loss through the window and due to the installation of the window into the wall is a practical way to determine the actual heat loss caused by the presence of a window. A window thermal transmittance or M-value is introduced to quantify the total additional heat loss through the building element due to the presence of the window. The methodology was validated against experimental measurements taken on different specimens in a hot box device. Results from the thermographic analysis also co-related well with results from finite element heat transfer and computational fluid dynamics simulations. (C) 2018 Elsevier B.V. All rights reserved.The authors wish to thank the College of Engineering and Informatics, National University of Ireland Galway for providing a Postgraduate Scholarship for the first author. Cracow University of Technology CUT, especially the Head of the Institute, Prof. Jacek Schnotale, and the technician, Eng. Mariusz Rusiecki, for access to the hot box facility and constant assistance during the testing. SIP Energy Ltd., Athenry, Co. Galway, in particular John Moylan, for supplying the test specimens. Enterprise Ireland for Innovation Voucher IV-2014-4203.peer-reviewed2020-03-1