Genetically engineered multicistronic allele of Pmel yielding highly specific CreERT2-mediated recombination in the melanocyte lineage.

Genetic approaches that allow lineage tracing are essential to our future understanding of melanocytes and melanoma. To date, the approaches used to label melanocytes in mice have relied on random integration of transgenes driven by the promoters of the Tyrosinase and Dopachrome tautomerase genes, knock-in to the Dopachrome tautomerase locus or knock-in to the Mlana locus in a bacterial artificial chromosome. These strategies result in expression in other tissues such as telencephalon and other cell types such as nerves. Here we used homologous recombination in mouse embryonic stem cells to generate a targeted multicistronic allele of the Pmel locus that drives melanocyte-specific expression of CreERT2, nuclear localised H2B-Cerulean and membrane localised marcks-mKate2 allowing live imaging of melanocytes and activation of other conditional alleles. We combined this allele with R26R-EYFP mice allowing induction of EYFP expression on administration of tamoxifen or its metabolite 4-OHT. The fluorescent proteins H2B-Cerulean and marcks-mKate2 label the cell nucleus and plasma membrane respectively allowing live imaging and FACS isolation of melanoblasts and melanocytes as well as serving to provide an internal control allowing estimation of recombination efficiency after administration of tamoxifen. We demonstrate the utility of the transgene in embryonic and adult tissues.

Fingerprinting of skin cells by live cell Raman spectroscopy reveals melanoma cell heterogeneity and cell-type-specific responses to UVR.

Raman spectroscopy is an emerging dermatological technique with the potential to discriminate biochemically between cell types in a label-free and non-invasive manner. Here, we use live single-cell Raman spectroscopy and principal component analysis (PCA) to fingerprint mouse melanoblasts, melanocytes, keratinocytes and melanoma cells. We show the differences in their spectra are attributable to biomarkers in the melanin biosynthesis pathway and that melanoma cells are a heterogeneous population that sit on a trajectory between undifferentiated melanoblasts and differentiated melanocytes. We demonstrate the utility of Raman spectroscopy as a highly sensitive tool to probe the melanin biosynthesis pathway and its immediate response to ultraviolet (UV) irradiation revealing previously undescribed opposing responses to UVA and UVB irradiation in melanocytes. Finally, we identify melanocyte-specific accumulation of ß-carotene correlated with a stabilisation of the UVR response in lipids and proteins consistent with a ß-carotene-mediated photoprotective mechanism. In summary, our data show that Raman spectroscopy can be used to determine the differentiation status of cells of the melanocyte lineage and describe the immediate and temporal biochemical changes associated with UV exposure which differ depending on cell type, differentiation status and competence to synthesise melanin. Our work uniquely applies Raman spectroscopy to discriminate between cell types by biological function and differentiation status while they are growing in culture. In doing so, we demonstrate for the first time its utility as a tool with which to probe the melanin biosynthesis pathway.

Melanins as Sustainable Resources for Advanced Biotechnological Applications.

Melanins are a class of biopolymers that are widespread in nature and have diverse origins, chemical compositions, and functions. Their chemical, electrical, optical, and paramagnetic properties offer opportunities for applications in materials science, particularly for medical and technical uses. This review focuses on the application of analytical techniques to study melanins in multidisciplinary contexts with a view to their use as sustainable resources for advanced biotechnological applications, and how these may facilitate the achievement of the United Nations Sustainable Development Goals.

Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier-Mediated Process.

The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( Vmax = 3299 pmol/million cells/min, Km = 35.9 µM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.

Innovative organotypic in vitro models for safety assessment: aligning with regulatory requirements and understanding models of the heart, skin, and liver as paradigms.

The development of improved, innovative models for the detection of toxicity of drugs, chemicals, or chemicals in cosmetics is crucial to efficiently bring new products safely to market in a cost-effective and timely manner. In addition, improvement in models to detect toxicity may reduce the incidence of unexpected post-marketing toxicity and reduce or eliminate the need for animal testing. The safety of novel products of the pharmaceutical, chemical, or cosmetics industry must be assured; therefore, toxicological properties need to be assessed. Accepted methods for gathering the information required by law for approval of substances are often animal methods. To reduce, refine, and replace animal testing, innovative organotypic in vitro models have emerged. Such models appear at different levels of complexity ranging from simpler, self-organized three-dimensional (3D) cell cultures up to more advanced scaffold-based co-cultures consisting of multiple cell types. This review provides an overview of recent developments in the field of toxicity testing with in vitro models for three major organ types: heart, skin, and liver. This review also examines regulatory aspects of such models in Europe and the UK, and summarizes best practices to facilitate the acceptance and appropriate use of advanced in vitro models.

Statin regulated ERK5 stimulates tight junction formation and reduces permeability in human cardiac endothelial cells.

The MEKK3/MEK5/ERK5 signaling axis is required for cardiovascular development in vivo. We analyzed the physiological role of ERK5 in cardiac endothelial cells and the consequence of activation of this kinase by the statin class of HMG Co-A reductase inhibitor drugs. We utilized human cardiac microvascular endothelial cells (HCMECs) and altered ERK5 expression using siRNA mediated gene silencing or overexpression of constitutively active MEK5 and ERK5 to reveal a role for ERK5 in regulating endothelial tight junction formation and cell permeability. Statin treatment of HCMECs stimulated activation of ERK5 and translocation to the plasma membrane resulting in co-localization with the tight junction protein ZO-1 and a concomitant reduction in endothelial cell permeability. Statin mediated activation of ERK5 was a consequence of reduced isoprenoid synthesis following HMG Co-A reductase inhibition. Statin pretreatment could overcome the effect of doxorubicin in reducing endothelial tight junction formation and prevent increased permeability. Our data provide the first evidence for the role of ERK5 in regulating endothelial tight junction formation and endothelial cell permeability. Statin mediated ERK5 activation and the resulting decrease in cardiac endothelial cell permeability may contribute to the cardioprotective effects of statins in reducing doxorubicin-induced cardiotoxicity.

RCAN1.4 regulates VEGFR-2 internalisation, cell polarity and migration in human microvascular endothelial cells.

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the calcineurin pathway in cells. It is expressed as two isoforms in vertebrates: RCAN1.1 is constitutively expressed in most tissues, whereas transcription of RCAN1.4 is induced by several stimuli that activate the calcineurin-NFAT pathway. RCAN1.4 is highly upregulated in response to VEGF in human endothelial cells in contrast to RCAN1.1 and is essential for efficient endothelial cell migration and tubular morphogenesis. Here, we show that RCAN1.4 has a role in the regulation of agonist-stimulated VEGFR-2 internalisation and establishment of endothelial cell polarity. siRNA-mediated gene silencing revealed that RCAN1 plays a vital role in regulating VEGF-mediated cytoskeletal reorganisation and directed cell migration and sprouting angiogenesis. Adenoviral-mediated overexpression of RCAN1.4 resulted in increased endothelial cell migration. Antisense-mediated morpholino silencing of the zebrafish RCAN1.4 orthologue revealed a disrupted vascular development further confirming a role for the RCAN1.4 isoform in regulating vascular endothelial cell physiology. Our data suggest that RCAN1.4 plays a novel role in regulating endothelial cell migration by establishing endothelial cell polarity in response to VEGF.

Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability.

Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes.