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Prostate cancer progression is driven by androgen receptor (AR) activity, which is a target for therapeutic approaches. Enzalutamide is an AR inhibitor that prolongs the survival of patients with advanced prostate cancer. However, resistance mechanisms arise and impair its efficacy. One of these mechanisms is the expression of AR-V7, a constitutively active AR splice variant. The Mediator complex is a multisubunit protein that modulates gene expression on a genome-wide scale. MED12 and cyclin-dependent kinase (CDK)8, or its paralog CDK19, are components of the kinase module that regulates the proliferation of prostate cancer cells. In this study, we investigated how MED12 and CDK8/19 influence cancer-driven processes in prostate cancer cell lines, focusing on AR activity and the enzalutamide response. We inhibited MED12 expression and CDK8/19 activity in LNCaP (AR+, enzalutamide-sensitive), 22Rv1 (AR-V7+, enzalutamide-resistant), and PC3 (AR−, enzalutamide-insensitive) cells. Both MED12 and CDK8/19 inhibition reduced cell proliferation in all cell lines, and MED12 inhibition reduced proliferation in the respective 3D spheroids. MED12 knockdown significantly inhibited c-Myc protein expression and signaling pathways. In 22Rv1 cells, it consistently inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. CDK8/19 inhibition significantly decreased PSA secretion in LNCaP and 22Rv1 cells and, when combined with enzalutamide, additively reduced proliferation in 22Rv1 cells. Our study revealed that MED12 and CDK8/19 regulate AR activity and that their inhibition may modulate response to enzalutamide in prostate cancer. [ABSTRACT FROM AUTHOR]

Estrogen receptor α is commonly used in synthetic biology to control the activity of genome editing tools. The activating ligands, estrogens, however, interfere with various cellular processes, thereby limiting the applicability of this receptor. Altering its ligand preference to chemicals of choice solves this hurdle but requires adaptation of unspecified ligand‐interacting residues. Here, we provide a solution by combining rational protein design with multi‐site‐directed mutagenesis and directed evolution of stably integrated variants in Saccharomyces cerevisiae. This method yielded an estrogen receptor variant, named TERRA, that lost its estrogen responsiveness and became activated by tamoxifen, an anti‐estrogenic drug used for breast cancer treatment. This tamoxifen preference of TERRA was maintained in mammalian cells and mice, even when fused to Cre recombinase, expanding the mammalian synthetic biology toolbox. Not only is our platform transferable to engineer ligand preference of any steroid receptor, it can also profile drug‐resistance landscapes for steroid receptor‐targeted therapies. [ABSTRACT FROM AUTHOR]

In advanced prostate cancer (PC), in particular after acquisition of resistance to androgen receptor (AR) signaling inhibitors (ARSI), upregulation of AR splice variants compromises endocrine therapy efficiency. Androgen receptor splice variant‐7 (ARV7) is clinically the most relevant and has a distinct 3′ untranslated region (3′UTR) compared to the AR full‐length variant, suggesting a unique post‐transcriptional regulation. Here, we set out to evaluate the applicability of the ARV7 3′UTR as a therapy target. A common single nucleotide polymorphism, rs5918762, was found to affect the splicing rate and thus the expression of ARV7 in cellular models and patient specimens. Serine/arginine‐rich splicing factor 9 (SRSF9) was found to bind to and increase the inclusion of the cryptic exon 3 of ARV7 during the splicing process in the alternative C allele of rs5918762. The dual specificity protein kinase CLK2 interferes with the activity of SRSF9 by regulating its expression. Inhibition of the Cdc2‐like kinase (CLK) family by the small molecules cirtuvivint or lorecivivint results in the decreased expression of ARV7. Both inhibitors show potent anti‐proliferative effects in enzalutamide‐treated or ‐naive PC models. Thus, targeting aberrant alternative splicing at the 3′UTR of ARV7 by disturbing the CLK2/SRSF9 axis might be a valuable therapeutic approach in late stage, ARSI‐resistant PC. [ABSTRACT FROM AUTHOR]

Purpose: To analyze the interaction between aflibercept and galectin-1 and evaluate the plasma levels of galectin-1 and vascular endothelial growth factor (VEGF)-A after intravitreal injection of aflibercept in patients with diabetic retinopathy (DR). Methods: Interaction of galectin-1 with aflibercept was determined via immunoprecipitation. Seventeen patients with type 2 diabetes and diabetic macular edema (DME) were each treated with a single intravitreal injection of aflibercept (2.0 mg, 50 µL) monthly for three consecutive months. Plasma galectin-1 and VEGF-A levels were measured just before an injection was administered, 1 week after the first injection, and 2 months after the last injection. Nineteen age- and sex-matched healthy participants served as controls. Results: Irrespective of the tested galectin-1 concentration, 24% of added galectin-1 was precipitated by aflibercept. Baseline plasma concentrations of galectin-1 were 22.0 and 23.0 ng/mL in the control and aflibercept-treated groups, respectively. Systemic galectin-1 levels increased to 27.0 and 24.0 ng/mL at 7 days and 4 weeks, respectively, after treatment. At week 8, plasma galectin-1 levels significantly increased to 36.0 ng/mL. This level persisted for 20 weeks. Systemic VEGF-A levels significantly reduced to below the minimum detectable dose in 16 DME patients at 7 days after treatment. This level persisted for 4 weeks. Plasma VEGF-A levels were reduced at weeks 8 (p = 0.099) and 20 (p = 0.023). Decreased plasma VEGF-A levels were observed in all patients after treatment. Conclusion: We confirmed that physiological aflibercept levels precipitate galectin-1 inin vitroassays. Additionally, systemic upregulation of galectin-1 might be induced by intravitreal aflibercept, which may be relevant in the clinical outcomes of DR treatment. [ABSTRACT FROM PUBLISHER]

Ageing of bitumen leads to increased stiffness and brittleness. Thus, bituminous bound pavements become more prone to failure by low-temperature and fatigue cracking. Therefore, the ageing behaviour of bitumen has a crucial impact on durability, as well as recyclability of pavements. To assess ageing of bitumen, the rolling thin film oven test and pressure ageing vessel are standardised methods for short-term and long-term ageing in the lab. For lab-ageing of hot mix asphalt (HMA), various methods have been developed in the last decades. This paper presents a study on the potential of employing a highly oxidant gas for simulating the long-term oxidative ageing of asphalt mix specimens in the lab. Based on the results, an optimised lab-ageing procedure (Viennese Ageing Procedure – VAPro) for compacted HMA specimens to assess mix performance of long-term lab-aged specimens is developed. Thus, it is possible to optimise mix design not only for short-term performance but to take into account effects of oxidative ageing during its in-service life. VAPro is based on a triaxial cell with forced flow of a gaseous oxidant agent through the specimen. The oxidant agent is enriched in ozone and nitric oxides to increase the rate of oxidation. It is shown by stiffness tests of unaged and lab-aged specimens, as well as by Dynamic Shear Rheometer tests of recovered binder from aged specimens that asphalt mixes can be long-term aged at moderate temperatures (+60°C) and within 4 days and a flow rate of 1 l/min by applying VAPro. Thus, an ageing procedure is at hand that can simulate long-term ageing at conditions that are representative of conditions that occur in the field within an efficient amount of time. [ABSTRACT FROM AUTHOR]

Due to the urgent need for new prostate cancer (PCa) therapies, the role of androgen receptor (AR)-interacting proteins should be investigated. In this study we aimed to address whether the AR coactivator nuclear receptor coactivator 1 (NCOA1) is involved in PCa progression. Therefore, we tested the effect of long-term NCOA1 knockdown on processes relevant to metastasis formation. [³H]-thymidine incorporation assays revealed a reduced proliferation rate in AR-positive MDA PCa 2b and LNCaP cells upon knockdown of NCOA1, whereas AR-negative PC3 cells were not affected. Furthermore, Boyden chamber assays showed a strong decrease in migration and invasion upon NCOA1 knockdown, independently of the cell line's AR status. In order to understand the mechanistic reasons for these changes, transcriptome analysis using cDNA microarrays was performed. Protein kinase D1 (PRKD1) was found to be prominently up-regulated by NCOA1 knockdown in MDA PCa 2b, but not in PC3 cells. Inhibition of PRKD1 reverted the reduced migratory potential caused by NCOA1 knockdown. Furthermore, PRKD1 was negatively regulated by AR. Immunohistochemical staining of PCa patient samples revealed a strong increase in NCOA1 expression in primary tumors compared with normal prostate tissue, while no final conclusion could be drawn for PRKD1 expression in tumor specimens. Thus, our findings directly associate the AR/NCOA1 complex with PRKD1 regulation and cellular migration and support the concept of therapeutic inhibition of NCOA1 in PCa. [ABSTRACT FROM AUTHOR]

When it comes to describe the mechanical behaviour of hot mix asphalt (HMA), the change of the mechanical properties over time due to environmental impacts known as ageing has to be considered. Hardening and embrittlement of bitumen lead to a reduced resistance against cryogenic cracks and premature formation of fatigue cracks in bituminous layers. Within this work, the microstructure of bitumen is investigated by conducting static shear creep tests on artificially composed bitumen with asphaltene contents varying between 0 vol-% and 26.71 vol-% as well as on a paving grade bitumen 70/100. Both are considered in unaged and laboratory-aged (rolling thin-film oven test + pressure ageing vessel) conditions to be able to identify and describe ageing effects. While the properties of the considered material phases of bitumen (identical to saturates, aromatics, resins and asphaltenes (SARA) fractions) seem to remain unaffected, an increase of the asphaltene content due to ageing can be observed. A micromechanical model is proposed that allows a prediction of the consequences of these microstructural changes on the mechanical behaviour of bitumen. Atomic force microscopy and environmental scanning electron microscopy images confirm a composition consisting of a micelle-like structure in a contiguous matrix, a structural representative volume element concept based on SARA fractions is suggested, extending an existing multiscale model for HMA. A very good accordance between model predictions and experimental results indicates the model's ability to reproduce as well as to describe the effects related to ageing. [ABSTRACT FROM PUBLISHER]

Simple Summary: In this review, we will look into the existing methods to study treatment resistance to androgen receptor targeted therapies in prostate cancer. This will encompass both the different existing preclinical models as well as the role specific models have played in the current understanding of resistance mechanisms. Prostate cancer is an androgen-driven tumor. Different prostate cancer therapies consequently focus on blocking the androgen receptor pathway. Clinical studies reported tumor resistance mechanisms by reactivating and bypassing the androgen pathway. Preclinical models allowed the identification, confirmation, and thorough study of these pathways. This review looks into the current and future role of preclinical models to understand resistance to androgen receptor-targeted therapies. Increasing knowledge on this resistance will greatly improve insights into tumor pathophysiology and future treatment strategies in prostate cancer. [ABSTRACT FROM AUTHOR]