Genetic and epigenetic determinants of reactivation of Mecp2 and the inactive X chromosome in neural stem cells.

Rett syndrome may be treated by reactivating the silent copy of Mecp2 from the inactive X chromosome in female cells. Most studies that model Mecp2 reactivation have used mouse fibroblasts rather than neural cells, which would be critical for phenotypic reversal, and rely on fluorescent reporters that lack adequate sensitivity. Here, we present a mouse model based on a dual bioluminescent and fluorescent reporter to assess the level of reactivation of Mecp2 and the inactive X chromosome by treating neural stem cells with 5-azacytidine and Xist knockdown. We show that reactivation of Mecp2 and other X-linked genes correlates with CpG density, with distance from escapees, and, very strongly, with the presence of short interspersed nuclear elements. In addition, X-linked genes reactivated in neural stem cells overlap substantially with early reactivating genes by induced pluripotent stem cell reprogramming of fibroblasts or neuronal progenitors, indicating that X chromosome reactivation follows similar paths regardless of the technique or cell type used.

Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling.

Urinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

Molecular underpinnings of enzalutamide resistance

Prostate cancer (PCa) is among the most common adult malignancies, and the second leading cause of cancer-related death in men. As PCa is hormone dependent, blockade of the androgen receptor (AR) signaling is an effective therapeutic strategy for men with advanced metastatic disease. The discovery of enzalutamide, a compound that effectively blocks the AR axis and its clinical application has led to a significant improvement in survival time. However, the effect of enzalutamide is not permanent, and resistance to treatment ultimately leads to development of lethal disease, for which there currently is no cure. This review will focus on the molecular underpinnings of enzalutamide resistance, bridging the gap between the preclinical and clinical research on novel therapeutic strategies for combating this lethal stage of prostate cancer.

Automated spherical aberration correction in scanning confocal microscopy.

Mismatch between the refractive indexes of immersion media and glass coverslips introduces spherical aberrations in microscopes especially for high numerical aperture objectives. This contribution demonstrates an automated adjustment of the coverslip correction collar in scanning confocal microscopy to compensate for spherical aberrations due to coverslip thickness mismatch. With a motorized coverslip correction collar, the adjustment procedure consists of xz image scans, image processing, correction quality evaluation, the mismatch estimation, and eventually the optimal adjustment of the correction collar. For fast correction with less photodamage, coarse-fine Gaussian fitting algorithms are proposed and evaluated with various specimen for their estimation accuracy. The benefits of the proposed automated correction are demonstrated for various coverslips with biological specimens, showing the optimized resolution of the confocal microscope.

Cross-resistance between taxanes and new hormonal agents abiraterone and enzalutamide may affect drug sequence choices in metastatic castration-resistant prostate cancer.

INTRODUCTION: Treatment options for patients with metastatic castration-resistant prostate cancer (mCRPC) have expanded in recent years with the introduction of cabazitaxel, abiraterone and enzalutamide. With new systemic therapies available, the optimal treatment sequence of these drugs in mCRPC becomes increasingly important. As shown recently, patients who had previously been treated with abiraterone showed impaired responses to docetaxel, suggesting clinical cross-resistance [1]. In the present study, we aimed to identify cross-resistance between taxanes (docetaxel and cabazitaxel) and the new hormonal agents abiraterone and enzalutamide. As a potential mechanism for cross-resistance, we investigated the effects on androgen receptor (AR) nuclear translocation of these compounds. METHODS: To identify cross-resistance, we determined the effects of docetaxel, cabazitaxel, abiraterone and enzalutamide on cell viability in prostate cancer cell lines with acquired resistance to abiraterone and enzalutamide. Time-lapse confocal microscopy was used to study the dynamics of AR nuclear translocation. RESULTS: We observed impaired efficacy of docetaxel, cabazitaxel and enzalutamide in the abiraterone-resistant cell line, compared to the non-resistant cell line, providing evidence for in vitro cross-resistance. Impaired efficacy of docetaxel, cabazitaxel and abiraterone was observed in the enzalutamide-resistant cell line. Furthermore, docetaxel and cabazitaxel inhibited AR nuclear translocation, which was also observed for abiraterone and enzalutamide. CONCLUSIONS: In conclusion we found substantial preclinical evidence for cross-resistance between the taxanes docetaxel and cabazitaxel, and AR targeting agents abiraterone and enzalutamide. Since these compounds all interfere with AR-signalling, this strongly suggests a common mechanism of action, and thus a potential mechanism for cross-resistance in mCRPC.

Variable loss of functional activities of androgen receptor mutants in patients with androgen insensitivity syndrome.

Androgen receptor (AR) mutations in androgen insensitivity syndrome (AIS) are associated with a variety of clinical phenotypes. The aim of the present study was to compare the molecular properties and potential pathogenic nature of 8 novel and 3 recurrent AR variants with a broad variety of functional assays. Eleven AR variants (p.Cys177Gly, p.Arg609Met, p.Asp691del, p.Leu701Phe, p.Leu723Phe, p.Ser741Tyr, p.Ala766Ser, p.Arg775Leu, p.Phe814Cys, p.Lys913X, p.Ile915Thr) were analyzed for hormone binding, transcriptional activation, cofactor binding, translocation to the nucleus, nuclear dynamics, and structural conformation. Ligand-binding domain variants with low to intermediate transcriptional activation displayed aberrant Kd values for hormone binding and decreased nuclear translocation. Transcriptional activation data, FxxFF-like peptide binding and DNA binding correlated well for all variants, except for p.Arg609Met, p.Leu723Phe and p.Arg775Leu, which displayed a relatively higher peptide binding activity. Variants p.Cys177Gly, p.Asp691del, p.Ala766Ser, p.Phe814Cys, and p.Ile915Thr had intermediate or wild type values in all assays and showed a predominantly nuclear localization in living cells. All transcriptionally inactive variants (p.Arg609Met, p.Leu701Phe, p.Ser741Tyr, p.Arg775Leu, p.Lys913X) were unable to bind to DNA and were associated with complete AIS. Three variants (p.Asp691del, p.Arg775Leu, p.Ile915Thr) still displayed significant functional activities in in vitro assays, although the clinical phenotype was associated with complete AIS. The data show that molecular phenotyping based on 5 different functional assays matched in most (70%) but not all cases.

Functional analysis of novel androgen receptor mutations in a unique cohort of Indonesian patients with a disorder of sex development.

Mutations in the androgen receptor (AR) gene, rendering the AR protein partially or completely inactive, cause androgen insensitivity syndrome, which is a form of a 46,XY disorder of sex development (DSD). We present 3 novel AR variants found in a cohort of Indonesian DSD patients: p.I603N, p.P671S, and p.Q738R. The aim of this study was to determine the possible pathogenic nature of these newly found unclassified variants. To investigate the effect of these variants on AR function, we studied their impact on transcription activation, AR ligand-binding domain interaction with an FxxLF motif containing peptide, AR subcellular localization, and AR nuclear dynamics and DNA-binding. AR-I603N had completely lost its transcriptional activity due to disturbed DNA-binding capacity and did not show the 114-kDa hyperphosphorylated AR protein band normally detectable after hormone binding. The patient with AR-I603N displays a partial androgen insensitivity syndrome phenotype, which is explained by somatic mosaicism. A strongly reduced transcriptional activity was observed for AR-Q738R, together with diminished interaction with an FxxLF motif containing peptide. AR-P671S also showed reduced transactivation ability, but no change in DNA- or FxxLF-binding capacity and interferes with transcriptional activity for as yet unclear reasons.

Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching.

To study protein-protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor-acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein-protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP-YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV-induced single-strand DNA damage. ERCC1 and XPF form a heterodimeric 5' endonuclease in nucleotide excision repair. FRET between ERCC1-GFP and XPF-YFP occurs with an efficiency of 30%.

Multiple object tracking in molecular bioimaging by Rao-Blackwellized marginal particle filtering.

Time-lapse fluorescence microscopy imaging has rapidly evolved in the past decade and has opened new avenues for studying intracellular processes in vivo. Such studies generate vast amounts of noisy image data that cannot be analyzed efficiently and reliably by means of manual processing. Many popular tracking techniques exist but often fail to yield satisfactory results in the case of high object densities, high noise levels, and complex motion patterns. Probabilistic tracking algorithms, based on Bayesian estimation, have recently been shown to offer several improvements over classical approaches, by better integration of spatial and temporal information, and the possibility to more effectively incorporate prior knowledge about object dynamics and image formation. In this paper, we extend our previous work in this area and propose an improved, fully automated particle filtering algorithm for the tracking of many subresolution objects in fluorescence microscopy image sequences. It involves a new track management procedure and allows the use of multiple dynamics models. The accuracy and reliability of the algorithm are further improved by applying marginalization concepts. Experiments on synthetic as well as real image data from three different biological applications clearly demonstrate the superiority of the algorithm compared to previous particle filtering solutions.