The testosterone-dependent and independent transcriptional networks in the hypothalamus of Gpr54 and Kiss1 knockout male mice are not fully equivalent.

BACKGROUND: Humans and mice with loss of function mutations in GPR54 (KISS1R) or kisspeptin do not progress through puberty, caused by a failure to release GnRH. The transcriptional networks regulated by these proteins in the hypothalamus have yet to be explored by genome-wide methods. RESULTS: We show here, using 1 million exon mouse arrays (Exon 1.0 Affymetrix) and quantitative polymerase chain reaction (QPCR) validation to analyse microdissected hypothalamic tissue from Gpr54 and Kiss1 knockout mice, the extent of transcriptional regulation in the hypothalamus. The sensitivity to detect important transcript differences in microdissected RNA was confirmed by the observation of counter-regulation of Kiss1 expression in Gpr54 knockouts and confirmed by immunohistochemistry (IHC). Since Gpr54 and Kiss1 knockout animals are effectively pre-pubertal with low testosterone (T) levels, we also determined which of the validated transcripts were T-responsive and which varied according to genotype alone. We observed four types of transcriptional regulation (i) genotype only dependent regulation, (ii) T only dependent regulation, (iii) genotype and T-dependent regulation with interaction between these variables, (iv) genotype and T-dependent regulation with no interaction between these variables. The results implicate for the first time several transcription factors (e.g. Npas4, Esr2), proteases (Klk1b22), and the orphan 10-transmembrane transporter TMEM144 in the biology of GPR54/kisspeptin function in the hypothalamus. We show for the neuronal activity regulated transcription factor NPAS4, that distinct protein over-expression is seen in the hypothalamus and hippocampus in Gpr54 knockout mice. This links for the first time the hypothalamic-gonadal axis with this important regulator of inhibitory synapse formation. Similarly we confirm TMEM144 up-regulation in the hypothalamus by RNA in situ hybridization and western blot. CONCLUSIONS: Taken together, global transcriptional profiling shows that loss of GPR54 and kisspeptin are not fully equivalent in the mouse hypothalamus.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

ZNF703 is a common Luminal B breast cancer oncogene that differentially regulates luminal and basal progenitors in human mammary epithelium.

The telomeric amplicon at 8p12 is common in oestrogen receptor-positive (ER+) breast cancers. Array-CGH and expression analyses of 1172 primary breast tumours revealed that ZNF703 was the single gene within the minimal amplicon and was amplified predominantly in the Luminal B subtype. Amplification was shown to correlate with increased gene and protein expression and was associated with a distinct expression signature and poor clinical outcome. ZNF703 transformed NIH 3T3 fibroblasts, behaving as a classical oncogene, and regulated proliferation in human luminal breast cancer cell lines and immortalized human mammary epithelial cells. Manipulation of ZNF703 expression in the luminal MCF7 cell line modified the effects of TGFβ on proliferation. Overexpression of ZNF703 in normal human breast epithelial cells enhanced the frequency of in vitro colony-forming cells from luminal progenitors. Taken together, these data strongly point to ZNF703 as a novel oncogene in Luminal B breast cancer

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Regulatory mechanisms governing mammary epithelial and progenitor cell growth

The processes involved in mammary gland development are intimately linked with those that drive breast oncogenesis. Regulation of growth, tissue polarity and genome stability are a few of the factors the maintain homeostasis in breast epithelium and prevent malignant progression. In this work, a series of clonal 184-hTERT cell lines were generated that modeled the in vitro growth characteristics of bi-potent mammary progenitor cells; they were dependent upon fibroblasts for low-density growth and formed dual-lineage acini in 3D culture. These lines were subsequently used in a genome-wide siRNA screen to identify the factors that regulate fibroblast-driven epithelial cell growth. Fibroblasts constitute the majority of cells within stroma, which plays a major role in supporting mammary progenitor cell growth. From this screen, 49 surface and secreted factors were identified that putatively transduce the signals emanating from the fibroblasts that are required for epithelial cell growth. These factors were more potent than any of the previously described growth factor receptors. When assessed in primary tissue, Gpr39, Scarb2, Ntn1, Efna4, Nptx1, and Ctnna1 were found to have the greatest effect on overall progenitor cell growth, while SerpinH1 differentially suppressed luminal progenitor cells, and Nkain4 and Kcnj5 differentially suppressed bi-potent progenitor cells. Further profiling of these lines identified the planar cell polarity protein Celsr1 as differentially regulated under fibroblast-dependent conditions. Silencing of Celsr1 increased the number of bi-potent progenitor cells detected in the colonyforming assay. Furthermore, it induced branching morphogenesis within normally spherical acini and disrupted the apical polarity of these structures in 3D culture. Within this system, Celsr1 is suspected of signaling through Shisa4. This is the first description of a noncanonical Celsr1 interactor. Finally, a curious variant line was identified amongst the collection of 184-hTERT cells generated for this work. This line harbours mitotic spindle and cell cycle checkpoint defects, and rapidly gains chromosome 20 during passaging. Through an elimination process, de novo promoter hypomethylation and subsequent overexpression of CENPI was identified as likely being responsible for this phenotype. This is the first description of CENPI deregulation and one of a few descriptions of gene promoter hypomethylation resulting in genome instability.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofGraduat

A co-culture genome-wide RNAi screen with mammary epithelial cells reveals transmembrane signals required for growth and differentiation

Introduction: The extracellular signals regulating mammary epithelial cell growth are of relevance to understanding the pathophysiology of mammary epithelia, yet they remain poorly characterized. In this study, we applied an unbiased approach to understanding the functional role of signalling molecules in several models of normal physiological growth and translated these results to the biological understanding of breast cancer subtypes. Methods: We developed and utilized a cytogenetically normal clonal line of hTERT immortalized human mammary epithelial cells in a fibroblast-enhanced co-culture assay to conduct a genome-wide small interfering RNA (siRNA) screen for evaluation of the functional effect of silencing each gene. Our selected endpoint was inhibition of growth. In rigorous postscreen validation processes, including quantitative RT-PCR, to ensure on-target silencing, deconvolution of pooled siRNAs and independent confirmation of effects with lentiviral short-hairpin RNA constructs, we identified a subset of genes required for mammary epithelial cell growth. Using three-dimensional Matrigel growth and differentiation assays and primary human mammary epithelial cell colony assays, we confirmed that these growth effects were not limited to the 184-hTERT cell line. We utilized the METABRIC dataset of 1,998 breast cancer patients to evaluate both the differential expression of these genes across breast cancer subtypes and their prognostic significance. Results: We identified 47 genes that are critically important for fibroblast-enhanced mammary epithelial cell growth. This group was enriched for several axonal guidance molecules and G protein–coupled receptors, as well as for the endothelin receptor PROCR. The majority of genes (43 of 47) identified in two dimensions were also required for three-dimensional growth, with HSD17B2, SNN and PROCR showing greater than tenfold reductions in acinar formation. Several genes, including PROCR and the neuronal pathfinding molecules EFNA4 and NTN1, were also required for proper differentiation and polarization in three-dimensional cultures. The 47 genes identified showed a significant nonrandom enrichment for differential expression among 10 molecular subtypes of breast cancer sampled from 1,998 patients. CD79A, SERPINH1, KCNJ5 and TMEM14C exhibited breast cancer subtype–independent overall survival differences. Conclusion: Diverse transmembrane signals are required for mammary epithelial cell growth in two-dimensional and three-dimensional conditions. Strikingly, we define novel roles for axonal pathfinding receptors and ligands and the endothelin receptor in both growth and differentiation.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofReviewedFacult

Impact of Post Manufacturing Handling of Protein-Based Biologic Drugs on Product Quality and User Centricity

This article evaluates the current gaps around the impact of post-manufacturing processes on the product qualities of protein-based biologics, with a focus on user centricity. It includes the evaluation of the regulatory guidance available, describes a collection of scientific literature and case studies to showcase the impact of post-manufacturing stresses on product and dosing solution quality. It also outlines the complexity of clinical handling and the need for communication, and alignment between drug providers, healthcare professionals, users, and patients. Regulatory agencies provide clear expectations for drug manufacturing processes, however, guidance supporting post-product manufacturing handling is less defined and often misaligned. This is problematic as the pharmaceutical products experience numerous stresses and processes which can potentially impact drug quality, safety and efficacy. This article aims to stimulate discussion amongst pharmaceutical developers, health care providers, device manufacturers, and public researchers to improve these processes. Patients and caregivers’ awareness can be achieved by providing relevant educational material on pharmaceutical product handling. This project has received funding from the Innovative MedicinesInitiative 2 Joint Undertaking (JU) under grant agreement N°101007939 (RealHOPE). This Joint Undertaking receives support fromthe European Union’s Horizon 2020 research and innovation programme and EFPIA.</p

Impact of Post Manufacturing Handling of Protein-Based Biologic Drugs on Product Quality and User Centricity

This article evaluates the current gaps around the impact of post-manufacturing processes on the product qualities of protein-based biologics, with a focus on user centricity. It includes the evaluation of the regulatory guidance available, describes a collection of scientific literature and case studies to showcase the impact of post-manufacturing stresses on product and dosing solution quality. It also outlines the complexity of clinical handling and the need for communication, and alignment between drug providers, healthcare professionals, users, and patients. Regulatory agencies provide clear expectations for drug manufacturing processes, however, guidance supporting post-product manufacturing handling is less defined and often misaligned. This is problematic as the pharmaceutical products experience numerous stresses and processes which can potentially impact drug quality, safety and efficacy. This article aims to stimulate discussion amongst pharmaceutical developers, health care providers, device manufacturers, and public researchers to improve these processes. Patients and caregivers' awareness can be achieved by providing relevant educational material on pharmaceutical product handling.</p

Epithelial-Mesenchymal Plasticity Induced by Discontinuous Exposure to TGFβ1 Promotes Tumour Growth

Transitions between epithelial and mesenchymal cellular states (EMT/MET) contribute to cancer progression. We hypothesize that EMT followed by MET promotes cell population heterogeneity, favouring tumour growth. We developed an EMT model by on and off exposure of epithelial EpH4 cells (E-cells) to TGFβ1 that mimics phenotypic EMT (M-cells) and MET. We aimed at understanding whether phenotypic MET is accompanied by molecular and functional reversion back to epithelia by using RNA sequencing, immunofluorescence (IF), proliferation, wound healing, focus formation and mamosphere formation assays as well as cell xenografts in nude mice. Phenotypic reverted epithelial cells (RE-cells) obtained after MET induction presented epithelial morphologies and proliferation rates resembling E cells. However, the RE transcriptomic profile and IF staining of epithelial and mesenchymal markers revealed a uniquely heterogeneous mixture of cell subpopulations with a high self-renewal ability. RE cell heterogeneity was stably maintained for long periods after TGFβ1 removal both in vitro and in large tumours derived from the nude mice. Overall, we show that phenotypic reverted epithelial cells (RE cells) do not return to the molecular and functional epithelial state and present mesenchymal features related to aggressiveness and cellular heterogeneity that favour tumour growth in vivo. This work strengthens epithelial cell reprogramming and cellular heterogeneity fostered by inflammatory cues as a tumour growth-promoting factor in vivo.AlumniNon UBCReviewedFacultyResearcherOthe