In depth compositional analysis of ceramic (Bi2O3)0.75(Er2O3)0.25 by AES and XPS

The chemical composition of dense ceramics of erbia-stabilized δ-Bi2O3 was analyzed by Auger electron spectroscopy (AES) depth profiling using Ar+ ion sputtering. The relative sensitivity factors (rsf) and sputter rates of bismuth and erbium in this material have been determined by electron probe microanalysis (EPMA) and chemical analysis. These results, supplemented by data from angle resolved X-ray photoelectron spectroscopy (ARXPS), shows a bismuth enrichment at the surface. Evidence has been found for reduction of the bismuth-oxide at the outermost part of the surface layer

Novel chitosan-modified surfaces to study stem cell receptors and signaling

Stem cells have great potential for understanding early development, treating human diseases, tissue trauma and early phase drug discovery. The factors that control the regulation of stem cell survival, proliferation, migration and differentiation are still emerging. Some evidences now exist demonstrating the potent effects of various receptors and G-protein coupled receptor (GPCR)-ligands on the biology of stem cells. In this context, we developed novel synthetic surfaces to study stem cell receptors and their signaling, with a particular interest in the biocompatible material chitosan. These synthetic surfaces will present a series of small molecules that are anticipated to act both as agonists/ or antagonists and targeting agents of protein receptors and/or growth factor receptors located at the surface of our stem cells of choice. A special interest was given to extracellular receptor-ligand interactions and how they enhance and/ or inhibit their cellular responses, as well as how they activate/silence their connected pathway(s)

CRISPR-Cas9 screen identifies mechanisms of BET bromodomain inhibitor sensitivity including manganese

BET Bromodomain Inhibitors hold promise as therapeutic agents in inflammation and cancer but clinical studies show adverse side-effects at high, sustained dose. Clinical success requires further mechanistic understanding of inhibition of BET bromodomains and biomarkers to optimize efficacious dosing. To uncover the mechanisms of sensitivity and resistance to BETi, we employed a whole-genome CRISPR-Cas9 proliferation screen using colorectal cancer cells. We identify the mTOR signaling pathway as a key determinant of BETi sensitivity and that two Ca/Mn transporters mediate resistance. This later finding led to the discovery that extracellular manganese regulates sensitivity to BETi and that exposure of cells to BETi dose dependently increases intracellular manganese concentration. Our results describe new molecular pathways mediating BETi action and suggest several potential avenues for biomarker discovery

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has a significant role in regulating cell proliferation and growth, thus controlling organ size and regeneration. The Hippo pathway regulates two highly conserved, transcription co-activators YAP and TAZ. The upstream regulators of the Yap-Hippo pathway have not been fully characterized. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that allow activation of the YAP transcription co-activator at high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene). The effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

Activation of Yap Directed Transcription by Knock-down of Conserved Cellular Functions

The Yap-Hippo pathway has been shown to have a significant role in regulating cell proliferation and growth and hence controlling organ size and regeneration, as well as the differentiation of stem cells. These cell fate decisions are regulated by the Hippo pathway through the action of two, highly conserved, transcription co-activators YAP and TAZ. However, the upstream regulators of the Yap-Hippo pathway have not been fully characterized and may vary in different cell types and organs. The aim of this study was to use a siRNA screen, in a liver biliary cell line, to identify regulators of the Yap-Hippo pathway that when knocked-down allow activation of the YAP transcription co-activator at high cell density. This project used a commercially available library of siRNAs purchased from Qiagen that consisted of four siRNAs targeting seven thousand genes of the “druggable” genome. The library was screened using a reverse transfection protocol with a biliary derived cell line which was grown to high cell density. Activation of the Yap transcription co-activator was monitored using a high content, image based assay that measured the intracellular localization of native YAP protein. Active siRNAs were identified and further validated by quantification of CYR61 mRNA levels (a known Yap target gene) following knock-down and the effect of compounds targeting the putative gene targets identified as hits was also used for further validation. A number of validated hits reveal basic aspects of Yap-Hippo biology; such as components of the nuclear pore, by which YAP cytoplasmic/nuclear shuttling occurs, or how proteasomal degradation regulates intracellular YAP concentrations, which then alter YAP localization and transcription. Such results highlight how targeting conserved cellular functions can lead to validated activity in phenotypic assays

Mir-210 promotes sensory hair cell formation in the organ of Corti

Background: Hearing loss is the most common sensory defect with several hundred million people worldwide having hearing disorders. In most cases, the cause of hearing loss is related to the degeneration and death of hair cells and their associated spiral ganglion neurons. However, despite this knowledge, relatively few studies have reported regeneration of the auditory system. Significant gaps remain in our understanding of the molecular mechanisms underpinning auditory function, including the factors required for sensory cell regeneration. Recently, the identification of transcriptional activators and repressors of hair cell fate has been augmented by the discovery of microRNAs (miRNAs) associated with hearing loss. As miRNAs are central players of differentiation and cell fate, identification of miRNAs and their gene targets may reveal new pathways for hair cell regeneration and thereby provide new avenues for the treatment of hearing loss. Results: In order to identify new genetic elements enabling regeneration of sensory hair cells in the inner ear, next-generation miRNA sequencing (miRSeq) was used to identify the most prominent microRNAs expressed in the mouse embryonic inner ear cell line UB/OC-1 during differentiation towards a hair cell like phenotype. Based on miRSeq we selected eight most differentially expressed miRNAs for further characterization. Of those, miR-210 knock-down in vitro resulted in hair cell marker expression in UB-OC1, whereas ectopic expression of miR-210 resulted in new hair cell formation in cochlear explants. By using a lineage tracing mouse model, we identified transdifferentiation of supporting epithelial cells as the likely mechanism for new hair cell formation. Potential miR-210 targets were predicted in silico and identified experimentally using a miR-trap approach. Conclusion: MiRSeq followed by ex vivo validation revealed miR-210 as a novel factor driving transdifferentiation of supporting epithelial cells to sensory hair cells. Our data suggest that miR-210 might be a potential new factor for hearing loss therapy. Moreover, identification of inner ear pathways regulated by miR-210 identified novel drug targets for the treatment of hearing loss

Combined deletion of Lgr4 and Lgr5 impairs embryonic mouse development

Lgr4 and Lgr5 proteins are known markers of adult and embryonic tissue stem cells in various organs. However, the role of these proteins in propagating and maintaining individual tissue stem cell compartments is still controversial. While it was reported that Lgr4 is dispensable for normal embryonic gut development, Lgr4 deletion functionally impaired maintenance of the postnatal and adult intestinal crypt stem cell compartment. Furthermore, concomitant deletion of Lgr4 in Lgr5-null embryos was able to rescue their perinatal lethality, whereas combined deletion of Lgr4 and Lgr5 in adult mice exacerbated the latter phenotype, suggesting antagonistic or complementary functions of both receptors, respectively. While the effects of Lgr4 deletion during embryonic skin and kidney development have been reported, combined deletion of Lgr4 and Lgr5 has not been studied to date. To elucidate the functions of Lgr4 and Lgr5 during intestinal crypt development and to study their role in developing kidney and skin, we generated homozygous mice lacking either Lgr4 (Lgr4KO), Lgr5 (Lgr5KO) or both receptors (Lgr4/5dKO). Lgr4 deletion resulted in loss of Lgr5+ intestinal stem cells and impaired proliferation in the developing gut of E16.5 mice, a phenotype that was not further increased nor ameliorated by combined deletion of Lgr4 and Lgr5 (Lgr4/5dKO). In skin, E16.5 Lgr4KO and Lgr4/5dKO mice displayed impaired proliferation of basal cell progenitors accompanied by reduced epidermal thickness and reduced numbers of hair follicles. In contrast to E16.5 Lgr4KO mice, Lgr4/5dkO mice did neither show dilated kidney tubules nor cysts. However, E16.5 Lgr4/5dKO mice showed impaired kidney cell proliferation which was not observed in Lgr4KO mice. In summary, our data show that combined deletion of Lgr4 and Lgr5 impairs embryonic development with a dominant role of Lgr4 and support a complementary rather than an antagonistic function for both receptors

High Throughput Screening using iPSC-derived neuronal progenitors to identify compounds counteracting epigenetic gene silencing in Fragile X Syndrome

Fragile X Syndrome (FXS) is the most common form of inherited mental retardation. The underlying cause of the disease is the lack of Fragile X mental retardation protein (FMRP) which regulates synapsis. Its absence is caused in the majority of cases by epigenetic silencing of the FMR1 gene by DNA methylation triggered by an abnormal CGG repeat expansion (>200) in the untranslated 5’UTR. Today, no specific therapy exists for FXS and current treatments are only directed to improve behavioral symptoms. Neuronal progenitors derived from FXS patient-induced pluripotent stem cells (iPSCs) represent a unique model to study the disease and develop assays for large scale drug discovery screens since they conserve the FMR1 gene silenced within the disease context. We have established a high content imaging assay to run the first large-scale phenotypic screen aimed to identify compounds that reactivate the silenced FMR1 gene. A set of 50000 compounds was tested including modulators of several epigenetic targets. We describe an integrated drug discovery model comprising iPS generation, culture scale-up and quality control and screening with a very sensitive high content imaging assay assisted by single cell image analysis and multi-parametric data analysis based on machine learning algorithms. The screening identified several compounds that induced a weak expression of FMRP and thus, sets the basis for further large scale screens to find candidate drugs or targets tackling the underlying mechanism of FXS with potential for therapeutic intervention

Chemical genetic approach identifies microtubule affinity-regulating kinase 1 as a leucine-rich repeat kinase 2 substrate

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the most common cause of autosomal dominant forms of Parkinson’s disease. LRRK2 is a modular, multi-domain protein containing two enzymatic domains, including a kinase domain, as well as several protein-protein interaction domains, pointing to a role in cellular signaling, however knowledge of LRRK2’s upstream regulators and downstream effectors is incomplete. In this study, we used a chemical genetics approach to identify LRRK2 substrates from mouse brain. To this end, we first generated a catalytically active LRRK2 protein capable of employing bulky bio-orthogonal ATPγS analogues (M1947A/G2019S). This engineered, bioactive and functional LRRK2 kinase was used to thiophosphorylate putative substrates from brain extracts of LRRK2 KO mice. Isolation of thiophosphorylated peptides led to the identification of putative LRRK2 substrates. Several of these are involved in the regulation of microtubule (MT) dynamics including MAP/Microtubule Affinity-Regulating Kinase 1 (MARK1). MARK1 is a serine/threonine kinase known to phosphorylate microtubule-binding proteins such as Tau, MAP2 and MAP4 at KXGS motifs leading to MT destabilization. In vitro kinase assays and metabolic labelling experiments in living cells confirmed MARK1 as a LRRK2 substrate. In addition, we show that LRRK2 and MARK1 are interacting in eukaryotic cells. Taken together, we used a chemical genetic approach to identify LRRK2 substrates in the context of a complex cellular environment and have identified and validated MARK1 as a substrate. Our findings contribute to the identification of physiological LRRK2 substrates, and point to a potential mechanism explaining the reported effects of LRRK2 on neurite morpholog

Screening of Intestinal Crypt Organoids: A Simple Readout for Complex Biology

Oral and intestinal mucositis is a debilitating, often dose limiting side effect of radiation treatment. A mouse model of mucositis, induced by gamma irradiation, leads to weight loss and tissue damage, similar to that observed in patients. This model reflects the human ailment as it responds to keratinocyte growth factor (KGF), the standard of care treatment. Culturing of intestinal crypt organoids derived from primary cells allowed the development of a 3D assay to monitor the effect of treatments of intestinal epithelium to radiation-induced damage. This in vitro assay closely resembles the mouse model as KGF and Roof Plate-Specific Spondin-1 (RSPO1) enhanced the recovery of crypt organoids following radiation. Screening identified tool compounds that increased the survival of organoids post radiation. Repeated testing of these compounds revealed that the organoids changed their response over time. To investigate this adaptive behavior, intestinal organoid cultures were studied over time. Samples of organoids at various time points were used to prepare mRNA for unbiased transcriptome analyses. This expression profiling revealed a number of genes and pathways that were modulated over time, providing a rationale for the altered sensitivity of the intestinal crypt organoid cultures. This report describes the development of an in vitro assay that reflects the response of disease to therapeutic treatment. The assay was miniaturized and used to identify bioactive tool compounds, which served as probes to interrogate the patho-physiology of organoids over prolonged culture conditions. In vitro disease models based on primary 3D cell cultures represent valuable tools to identify potential drug targets and bioactive hits