RBFOX2 regulated EYA3 isoforms partner with SIX4 or ZBTB1 to control transcription during myogenesis.

Alternative splicing is a prevalent gene-regulatory mechanism, with over 95% of multi-exon human genes estimated to be alternatively spliced. Here, we describe a tissue-specific, developmentally regulated, highly conserved, and disease-associated alternative splicing event in exon 7 of the eyes absent homolog 3 (Eya3) gene. We discovered that EYA3 expression is vital to the proliferation and differentiation of myoblasts. Genome-wide transcriptomic analysis and mass spectrometry-based proteomic studies identified SIX homeobox 4 (SIX4) and zinc finger and BTB-domain containing 1 (ZBTB1), as major transcription factors that interact with EYA3 to dictate gene expression. EYA3 isoforms differentially regulate transcription, indicating that splicing aids in temporal control of gene expression during muscle cell differentiation. Finally, we identified RNA-binding fox-1 homolog 2 (RBFOX2) as the main regulator of EYA3 splicing. Together, our findings illustrate the interplay between alternative splicing and transcription during myogenesis.

Experiences with accreditation for radon measurement laboratories.

The adoption of the Council Directive 2013/59/EURATOM into Austrian national law generated new challenges for businesses, authorities and measurement services. The law defines radon priority regions in which all employers are obliged to hire an authorised radon-monitoring service to determine radon activity concentration at workplaces in basements and on ground floors. In this paper, an overview of our experiences with the process of becoming an accredited and authorised radon-monitoring body using integrating and time-resolved radon measurement equipment is given. The main challenges to overcome, such as determination of measurement uncertainty, conducting metrologically traceable calibration of the track-etch detector system, information not covered by ISO 11665-1, ISO 11665-4 and ISO 11665-5, availability of proficiency tests, etc., are described. This paper aims to be a guideline for laboratories seeking accreditation in the field of radon activity concentration measurements.

Radionuclide metrology methods and analysis of the Joint Danube Survey 4 sediment samples.

This low-level radionuclide metrology research work was carried out within the Joint Danube Survey 4 (JDS4), coordinated in 2018-2020 by the International Commission for the Protection of the Danube River (ICPDR). The gamma-emitting radionuclides of the sediment samples were analysed by low-level gamma-ray spectrometry. The activity concentration of 90Sr was determined by liquid scintillation counting (LSC) after isolating the radio-strontium using a new radiochemical separation method. The results of the radiometric analysis of 90Sr, 137Cs and naturally occurring radionuclides 40K, 210Pb, 226Ra, 228Ra in recent riverbed sediment are presented and discussed focused on public health.

The synaptosome-associated protein 23 (SNAP23) is necessary for proper myogenesis.

Vesicle-mediated transport is necessary for maintaining cellular homeostasis and proper signaling. The synaptosome-associated protein 23 (SNAP23) is a member of the SNARE protein family and mediates the vesicle docking and membrane fusion steps of secretion during exocytosis. Skeletal muscle has been established as a secretory organ; however, the role of SNAP23 in the context of skeletal muscle development is still unknown. Here, we show that depletion of SNAP23 in C2C12 mouse myoblasts reduces their ability to differentiate into myotubes as a result of premature cell cycle exit and early activation of the myogenic transcriptional program. This effect is rescued when cells are seeded at a high density or when cultured in conditioned medium from wild type cells. Proteomic analysis of collected medium indicates that SNAP23 depletion leads to a misregulation of exocytosis, including decreased secretion of the insulin-like growth factor 1 (IGF1), a critical protein for muscle growth, development, and function. We further demonstrate that treatment of SNAP23-depleted cells with exogenous IGF1 rescues their myogenic capacity. We propose that SNAP23 mediates the secretion of specific proteins, such as IGF1, that are important for achieving proper differentiation of skeletal muscle cells during myogenesis. This work highlights the underappreciated role of skeletal muscle as a secretory organ and contributes to the understanding of factors necessary for myogenesis.

SET domain containing 2 (SETD2) influences metabolism and alternative splicing during myogenesis.

Epigenetic regulatory mechanisms are increasingly recognized as crucial determinants of cellular specification and differentiation. During muscle cell differentiation (myogenesis), extensive remodelling of histone acetylation and methylation occurs. Several of these histone modifications aid in the expression of muscle-specific genes and the silencing of genes that block lineage commitment. Therefore, the identification of new epigenetic regulatory mechanisms is of high interest. Still, the functional relevance of numerous histone modifications during myogenesis remain completely uncertain. In this study, we focus on the function of H3K36me3 and its epigenetic writer, SET domain containing 2 (SETD2), in the context of muscle cell differentiation. We first observed that SETD2 expression increases during myogenesis. Targeted depletion of SETD2 in undifferentiated (myoblasts) and differentiated (myotubes) muscle cells reduced H3K36me3 levels and induced profound changes in gene expression and slight alterations in alternative splicing, as determined by deep RNA-sequencing analysis. Enzymes that function in metabolic pathways were upregulated in response to SETD2 depletion. Furthermore, we demonstrated that upregulation of several glycolytic enzymes was associated with an increase in intracellular pyruvate levels in SETD2-depleted cells, indicating a novel role for SETD2 in metabolic programming during myogenesis. Together, our results provide new insight into the signalling pathways controlled by chromatin-modifying enzymes and their associated histone modifications during muscle cell differentiation.

Low-Level Radon Activity Concentration-A MetroRADON International Intercomparison.

An international comparison of continuous monitors measuring radon activity concentration was performed to validate the traceability of the European radon calibration facilities. It was carried out by comparing the secondary standards used by these previous facilities, ranging from 100 Bq·m-3 to 300 Bq·m-3. Secondary standards were individually compared to a secondary reference device previously calibrated in a reference radon atmosphere traceable to a primary standard. The intercomparison was organized by the National Institute for Nuclear, Chemical, and Biological Protection (SUJCHBO) in the period from October 2019 to April 2020 within the European Metrology Program for Innovation and Research (EMPIR), JRP-Contract 16ENV10 MetroRADON. Eight European laboratories participated in this study. The results of the experiment are presented and discussed.

Alternative splicing regulation of membrane trafficking genes during myogenesis.

Alternative splicing transitions occur during organ development, and, in numerous diseases, splicing programs revert to fetal isoform expression. We previously found that extensive splicing changes occur during postnatal mouse heart development in genes encoding proteins involved in vesicle-mediated trafficking. However, the regulatory mechanisms of this splicing-trafficking network are unknown. Here, we found that membrane trafficking genes are alternatively spliced in a tissue-specific manner, with striated muscles exhibiting the highest levels of alternative exon inclusion. Treatment of differentiated muscle cells with chromatin-modifying drugs altered exon inclusion in muscle cells. Examination of several RNA-binding proteins revealed that the poly-pyrimidine tract binding protein 1 (PTBP1) and quaking regulate splicing of trafficking genes during myogenesis, and that removal of PTBP1 motifs prevented PTBP1 from binding its RNA target. These findings enhance our understanding of developmental splicing regulation of membrane trafficking proteins which might have implications for muscle disease pathogenesis.

The Metrological Traceability, Performance and Precision of European Radon Calibration Facilities.

An interlaboratory comparison for European radon calibration facilities was conducted to evaluate the establishment of a harmonized quality level for the activity concentration of radon in air and to demonstrate the performance of the facilities when calibrating measurement instruments for radon. Fifteen calibration facilities from 13 different European countries participated. They represented different levels in the metrological hierarchy: national metrology institutes and designated institutes, national authorities for radiation protection and participants from universities. The interlaboratory comparison was conducted by the German Federal Office for Radiation Protection (BfS) and took place from 2018 to 2020. Participants were requested to measure radon in atmospheres of their own facilities according to their own procedures and requirements for metrological traceability. A measurement device with suitable properties was used to determine the comparison values. The results of the comparison showed that the radon activity concentrations that were determined by European calibration facilities complying with metrological traceability requirements were consistent with each other and had common mean values. The deviations from these values were normally distributed. The range of variation of the common mean value was a measure of the degree of agreement between the participants. For exposures above 1000 Bq/m3, the variation was about 4% for a level of confidence of approximately 95% (k=2). For lower exposure levels, the variation increased to about 6%.

It's not just a phase: function and characteristics of RNA-binding proteins in phase separation.

Biomolecular condensates that form via phase separation are increasingly regarded as coordinators of cellular reactions that regulate a wide variety of biological phenomena. Mounting evidence suggests that multiple steps of the RNA life cycle are organized within RNA-binding protein-rich condensates. In this Review, we discuss recent insights into the influence of phase separation on RNA biology, which has implications for basic cell biology, the pathogenesis of human diseases and the development of novel therapies.

Comparing calculated and experimental activity and dose values obtained from image-based quantification of 90Y SPECT/CT Data.

PURPOSE: Selective internal radiation therapy (SIRT) is a treatment for various kinds of liver tumours by injecting 90Y bearing microspheres into the liver vessels. To perform meaningful post-treatment dosimetry, quantitative imaging is performed. METHODS: This work uses a Monte-Carlo based reconstruction software with scatter and attenuation correction and collimator modelling that allows the quantification of 90Y bremsstrahlung SPECT/CT data. A dataset comprising 17 patients and measurements on a Jaszczak phantom, a NEMA IEC Body phantom and an anthropomorphic liver phantom are analysed and activities and dose values are acquired. These measured values are compared with applied activities and pre-treatment calculations, allowing to assess the quality of the SPECT reconstruction. A detailed uncertainty budget is presented, including uncertainties of the dose calibrator, the count rate, non-included interactions and other factors. RESULTS: The applied method is validated by finding measurements repeatable within the given uncertainty, and it is shown the influence of various parameters on the reconstruction process is negligible. Furthermore, activities and doses measured in the phantoms show good agreement with calculated values, if they are corrected for partial volume effects. CONCLUSIONS: The strict observation of metrological requirements and the creation of an uncertainty budget increase the reliability and traceability of this novel approach to 90Y dosimetry. It gives an example of successful voxel-based dosimetry based on quantitative 90Y SPECT/CT image data.

An innovative quick method for tracable measurement of radon 222 in drinking water.

In this work, a quick and simple low-level radionuclide metrology technique for the traceable measurement of Rn-222 activity concentration in drinking water using a 0.56 l ionisation chamber operating in spectrometric pulse mode has been developed, tested, verified and applied to 16 water samples successfully. The impact of essential influencing factors on the result has been investigated, discussed and considered in the uncertainty budget of the measurement method. Finally, the new method has been assessed regarding the applicability on the EU Council Directive 2013/51.

RNA processing in skeletal muscle biology and disease.

RNA processing encompasses the capping, cleavage, polyadenylation and alternative splicing of pre-mRNA. Proper muscle development relies on precise RNA processing, driven by the coordination between RNA-binding proteins. Recently, skeletal muscle biology has been intensely investigated in terms of RNA processing. High throughput studies paired with deletion of RNA-binding proteins have provided a high-level understanding of the molecular mechanisms controlling the regulation of RNA-processing in skeletal muscle. Furthermore, misregulation of RNA processing is implicated in muscle diseases. In this review, we comprehensively summarize recent studies in skeletal muscle that demonstrated: (i) the importance of RNA processing, (ii) the RNA-binding proteins that are involved, and (iii) diseases associated with defects in RNA processing.

Enhancement of BDNF Expression and Memory by HDAC Inhibition Requires BET Bromodomain Reader Proteins.

Histone deacetylase (HDAC) inhibitors may have therapeutic utility in multiple neurological and psychiatric disorders, but the underlying mechanisms remain unclear. Here, we identify BRD4, a BET bromodomain reader of acetyl-lysine histones, as an essential component involved in potentiated expression of brain-derived neurotrophic factor (BDNF) and memory following HDAC inhibition. In in vitro studies, we reveal that pharmacological inhibition of BRD4 reversed the increase in BDNF mRNA induced by the class I/IIb HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). Knock-down of HDAC2 and HDAC3, but not other HDACs, increased BDNF mRNA expression, whereas knock-down of BRD4 blocked these effects. Using dCas9-BRD4, locus-specific targeting of BRD4 to the BDNF promoter increased BDNF mRNA. In additional studies, RGFP966, a pharmacological inhibitor of HDAC3, elevated BDNF expression and BRD4 binding to the BDNF promoter, effects that were abrogated by JQ1 (an inhibitor of BRD4). Examining known epigenetic targets of BRD4 and HDAC3, we show that H4K5ac and H4K8ac modifications and H4K5ac enrichment at the BDNF promoter were elevated following RGFP966 treatment. In electrophysiological studies, JQ1 reversed RGFP966-induced enhancement of LTP in hippocampal slice preparations. Last, in behavioral studies, RGFP966 increased subthreshold novel object recognition memory and cocaine place preference in male C57BL/6 mice, effects that were reversed by cotreatment with JQ1. Together, these data reveal that BRD4 plays a key role in HDAC3 inhibitor-induced potentiation of BDNF expression, neuroplasticity, and memory.SIGNIFICANCE STATEMENT Some histone deacetylase (HDAC) inhibitors are known to have neuroprotective and cognition-enhancing properties, but the underlying mechanisms have yet to be fully elucidated. In the current study, we reveal that BRD4, an epigenetic reader of histone acetylation marks, is necessary for enhancing brain-derived neurotrophic factor (BDNF) expression and improved memory following HDAC inhibition. Therefore, by identifying novel epigenetic regulators of BDNF expression, these data may lead to new therapeutic targets for the treatment of neuropsychiatric disorders.

Modulation of alternative splicing of trafficking genes by genome editing reveals functional consequences in muscle biology.

Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.

Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations.

Advancements in NORM metrology - Results and impact of the European joint research project MetroNORM.

The results of the three years European Metrology Research Programme's (EMRP) joint research project 'Metrology for processing materials with high natural radioactivity' (MetroNORM) are presented. In this project, metrologically sound novel instruments and procedures for laboratory and in-situ NORM activity measurements have been developed. Additionally, standard reference materials and sources for traceable calibration and improved decay data of natural radionuclides have been established.

Application of an artificial neural network for evaluation of activity concentration exemption limits in NORM industry.

NORM emits many different gamma energies that have to be analysed by an expert. Alternatively, artificial neural networks (ANNs) can be used. These mathematical software tools can generalize "knowledge" gained from training datasets, applying it to new problems. No expert knowledge of gamma-ray spectrometry is needed by the end-user. In this work an ANN was created that is able to decide from the raw gamma-ray spectrum if the activity concentrations in a sample are above or below the exemption limits.

Study of particular problems appearing in NORM samples and recommendations for best practice gamma-ray spectrometry.

In this paper spectral interference effects for selected gamma-emitting radionuclides of the natural decay series and 40K in selected NORM samples are studied. Recommendations for the choice of γ-lines and the consideration of possible spectral interferences are provided. Special attention is given to the radon tightness of the sample containers. A simple and sensitive method for the estimation of the 222Rn leakage of sample containers is introduced. The applied polystyrene sample containers show 222Rn leakages lower than 1%.