The perspectives of parents, general practitioners, and community pharmacists about treating an ill child in primary care: a comparative study.

As children are unable to make health-related decisions themselves, parents play a central role in consultations with healthcare providers. Parents' perspectives are therefore the focus of this study. Our first aim was to determine parents' expectations of a healthcare visit with a general practitioner and a community pharmacist. The second aim was to determine the general practitioners' and community pharmacists' perspectives about consultations with children. An observational cross-sectional study was conducted in April and May 2018. We developed three questionnaires: one for parents, one for general practitioners, and one for community pharmacists. The questionnaire for parents was only available through an online platform. The healthcare providers were questioned face-to-face and through an online platform. The study included 380 respondents. Parents considered prescribing or proposing medication the least important action by a general practitioner or community pharmacist, respectively. As well, parents expect information in most cases from both healthcare providers. The questionnaire for general practitioners and community pharmacists revealed that prescribing or proposing medication was regarded the least important action.Conclusion: Considering parents' expectations for a consultation with a general practitioner or community pharmacist, there is a substantial resemblance with the healthcare providers' perspective.What is Known:• The previous studies focusing on parents' perspectives were carried out in a hospital setting or focused on a specific disorder.• Parents consider reassurance and advice from their general practitioner to be very important; the treatment is considered less important.What is New:• Parents considered for both general practitioners' and community pharmacists' verbal information, answers to their questions, and reassurance as more important than receiving pharmacological treatment, while general practitioners and community pharmacists consider prescribing/proposing medication and providing written information as less important.• The expectations of the different groups (parents in relation to not only the healthcare providers but also the general practitioners and community pharmacists compared to each other) know a great resemblance.

A large scale mass spectrometry-based histone screening for assessing epigenetic developmental toxicity.

Toxicoepigenetics is an emerging field that studies the toxicological impact of compounds on protein expression through heritable, non-genetic mechanisms, such as histone post-translational modifications (hPTMs). Due to substantial progress in the large-scale study of hPTMs, integration into the field of toxicology is promising and offers the opportunity to gain novel insights into toxicological phenomena. Moreover, there is a growing demand for high-throughput human-based in vitro assays for toxicity testing, especially for developmental toxicity. Consequently, we developed a mass spectrometry-based proof-of-concept to assess a histone code screening assay capable of simultaneously detecting multiple hPTM-changes in human embryonic stem cells. We first validated the untargeted workflow with valproic acid (VPA), a histone deacetylase inhibitor. These results demonstrate the capability of mapping the hPTM-dynamics, with a general increase in acetylations as an internal control. To illustrate the scalability, a dose-response study was performed on a proof-of-concept library of ten compounds (1) with a known effect on the hPTMs (BIX-01294, 3-Deazaneplanocin A, Trichostatin A, and VPA), (2) classified as highly embryotoxic by the European Centre for the Validation of Alternative Methods (ECVAM) (Methotrexate, and All-trans retinoic acid), (3) classified as non-embryotoxic by ECVAM (Penicillin G), and (4) compounds of abuse with a presumed developmental toxicity (ethanol, caffeine, and nicotine).

An interactive mass spectrometry atlas of histone posttranslational modifications in T-cell acute leukemia.

The holistic nature of omics studies makes them ideally suited to generate hypotheses on health and disease. Sequencing-based genomics and mass spectrometry (MS)-based proteomics are linked through epigenetic regulation mechanisms. However, epigenomics is currently mainly focused on DNA methylation status using sequencing technologies, while studying histone posttranslational modifications (hPTMs) using MS is lagging, partly because reuse of raw data is impractical. Yet, targeting hPTMs using epidrugs is an established promising research avenue in cancer treatment. Therefore, we here present the most comprehensive MS-based preprocessed hPTM atlas to date, including 21 T-cell acute lymphoblastic leukemia (T-ALL) cell lines. We present the data in an intuitive and browsable single licensed Progenesis QIP project and provide all essential quality metrics, allowing users to assess the quality of the data, edit individual peptides, try novel annotation algorithms and export both peptide and protein data for downstream analyses, exemplified by the PeptidoformViz tool. This data resource sets the stage for generalizing MS-based histone analysis and provides the first reusable histone dataset for epidrug development.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

An experimental design to extract more information from MS-based histone studies.

Histone-based chromatin organization paved the way for eukaryotic genome complexity. Because of their key role in information management, the histone posttranslational modifications (hPTM), which mediate their function, have evolved into an alphabet that has more letters than there are amino acids, together making up the "histone code". The resulting combinatorial complexity is manifold higher than what is usually encountered in proteomics. Consequently, a considerably bigger part of the acquired MSMS spectra remains unannotated to date. Adapted search parameters can dig deeper into the dark histone ion space, but the lack of false discovery rate (FDR) control and the high level of ambiguity when searching combinatorial PTMs makes it very hard to assess whether the newly assigned ions are informative. Therefore, we propose an easily adoptable time-lapse enzymatic deacetylation (HDAC1) of a commercial histone extract as a quantify-first strategy that allows isolating ion populations of interest, when studying e.g. acetylation on histones, that currently remain in the dark. By adapting search parameters to study potential issues in sample preparation, data acquisition and data analysis, we stepwise managed to double the portion of annotated precursors of interest from 10.5% to 21.6%. This strategy is intended to make up for the lack of validated FDR control and has led to several adaptations of our current workflow that will reduce the portion of the dark histone ion space in the future. Finally, this strategy can be applied with any enzyme targeting a modification of interest.

Histone Sample Preparation for Bottom-Up Mass Spectrometry: A Roadmap to Informed Decisions.

Histone-based chromatin organization enabled eukaryotic genome complexity. This epigenetic control mechanism allowed for the differentiation of stable gene-expression and thus the very existence of multicellular organisms. This existential role in biology makes histones one of the most complexly modified molecules in the biotic world, which makes these key regulators notoriously hard to analyze. We here provide a roadmap to enable fast and informed selection of a bottom-up mass spectrometry sample preparation protocol that matches a specific research question. We therefore propose a two-step assessment procedure: (i) visualization of the coverage that is attained for a given workflow and (ii) direct alignment between runs to assess potential pitfalls at the ion level. To illustrate the applicability, we compare four different sample preparation protocols while adding a new enzyme to the toolbox, i.e., RgpB (GingisREX®, Genovis, Lund, Sweden), an endoproteinase that selectively and efficiently cleaves at the c-terminal end of arginine residues. Raw data are available via ProteomeXchange with identifier PXD024423.

Cov-MS: A Community-Based Template Assay for Mass-Spectrometry-Based Protein Detection in SARS-CoV-2 Patients.

Rising population density and global mobility are among the reasons why pathogens such as SARS-CoV-2, the virus that causes COVID-19, spread so rapidly across the globe. The policy response to such pandemics will always have to include accurate monitoring of the spread, as this provides one of the few alternatives to total lockdown. However, COVID-19 diagnosis is currently performed almost exclusively by reverse transcription polymerase chain reaction (RT-PCR). Although this is efficient, automatable, and acceptably cheap, reliance on one type of technology comes with serious caveats, as illustrated by recurring reagent and test shortages. We therefore developed an alternative diagnostic test that detects proteolytically digested SARS-CoV-2 proteins using mass spectrometry (MS). We established the Cov-MS consortium, consisting of 15 academic laboratories and several industrial partners to increase applicability, accessibility, sensitivity, and robustness of this kind of SARS-CoV-2 detection. This, in turn, gave rise to the Cov-MS Digital Incubator that allows other laboratories to join the effort, navigate, and share their optimizations and translate the assay into their clinic. As this test relies on viral proteins instead of RNA, it provides an orthogonal and complementary approach to RT-PCR using other reagents that are relatively inexpensive and widely available, as well as orthogonally skilled personnel and different instruments. Data are available via ProteomeXchange with identifier PXD022550.

Comprehensive histone epigenetics: A mass spectrometry based screening assay to measure epigenetic toxicity.

Evidence of the involvement of epigenetics in pathologies such as cancer, diabetes, and neurodegeneration has increased global interest in epigenetic modifications. For nearly thirty years, it has been known that cancer cells exhibit abnormal DNA methylation patterns. In contrast, the large-scale analysis of histone post-translational modifications (hPTMs) has lagged behind because classically, histone modification analysis has relied on site specific antibody-based techniques. Mass spectrometry (MS) is a technique that holds the promise to picture the histone code comprehensively in a single experiment. Therefore, we developed an MS-based method that is capable of tracking all possible hPTMs in an untargeted approach. In this way, trends in single and combinatorial hPTMs can be reported and enable prediction of the epigenetic toxicity of compounds. Moreover, this method is based on the use of human cells to provide preliminary data, thereby omitting the need to sacrifice laboratory animals. Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort. Still, this novel toxicoepigenetic assay and the data it generates holds great potential for, among others, pharmaceutical industry, food science, clinical diagnostics and, environmental toxicity screening. •There is a growing interest in epigenetic modifications, and more specifically in histone post-translational modifications (hPTMs).•We describe an MS-based workflow that is capable of tracking all possible hPTMs in an untargeted approach that makes use of human cells.•Improving the workflow and the user-friendliness in order to become a high throughput, easily applicable, toxicological screening assay is an ongoing effort.