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This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids. • Paraformaldehyde fixation of spheroids • Antigen retrieval treatment of spheroids • Embedding in freezing medium and cryosectioning

Temporal molecular changes in ageing mammalian organs are of relevance to disease aetiology because many age-related diseases are linked to changes in the transcriptional and epigenetic machinery that regulate gene expression. We performed quantitative proteome analysis of chromatin-enriched protein extracts to investigate the dynamics of the chromatin proteomes of the mouse brain, heart, lung, kidney, liver, and spleen at 3, 5, 10, and 15 months of age. Each organ exhibited a distinct chromatin proteome and sets of unique proteins. The brain and spleen chromatin proteomes were the most extensive, diverse, and heterogenous among the six organs. The spleen chromatin proteome appeared static during the lifespan, presenting a young phenotype that reflects the permanent alertness state and important role of this organ in physiological defence and immunity. We identified a total of 5928 proteins, including 2472 nuclear or chromatin-associated proteins across the six mouse organs. Up to 3125 proteins were quantified in each organ, demonstrating distinct and organ-specific temporal protein expression timelines and regulation at the post-translational level. Bioinformatics meta-analysis of these chromatin proteomes revealed distinct physiological and ageing-related features for each organ. Our results demonstrate the efficiency of organelle-specific proteomics for in vivo studies of a model organism and consolidate the hypothesis that chromatin-associated proteins are involved in distinct and specific physiological functions in ageing organs.

Non-alcoholic fatty liver disease affects one-fourth of the world’s population. Central to the disease progression is lipid accumulation in the liver, followed by inflammation, fibrosis and cirrhosis. The underlying mechanism behind the early stages of the disease is poorly understood. We have exposed human hepatic HepG2/C3A cells-based spheroids to 65 μM oleic acid and 45 μM palmitic acid and employed proteomics and lipidomics analysis to investigate their effect on hepatocytes. The treatment successfully induced in vivo hallmarks of NAFLD, as evidenced by intracellular lipid accumulation and increased ATP levels. Quantitative lipidome analysis revealed an increase in ceramides, LPC and saturated triglycerides and a decrease in the ratio of PC/PE, similar to the changes observed in patients’ liver biopsies. The proteomics analysis combined with qPCR showed increased epithelial to mesenchymal transition (EMT) signalling. Activation of EMT was further validated by transcriptomics in TGF-β treated spheroids, where an increase in mesenchymal cell markers (N-cadherin and collagen expression) was found. Our study demonstrates that this model system thus closely echoes several of the clinical features of non-alcoholic fatty liver disease and can be used to investigate the underlying molecular changes occurring in the condition.

Abstract Background Sample preparation is a critical process for proteomic studies. Many efficient and reproducible sample preparation methods have been developed for mass spectrometry-based proteomic analysis of human and animal tissues or cells, but no attempt has been made to evaluate these protocols for plants. We here present an LC–MS/MS-based proteomics study of barley leaf aimed at optimization of methods to achieve efficient and unbiased trypsin digestion of proteins prior to LC–MS/MS based sequencing and quantification of peptides. We evaluated two spin filter-aided sample preparation protocols using either sodium dodecyl-sulphate or sodium deoxycholate (SDC), and three in-solution digestion (ISD) protocols using SDC or trichloroacetic acid/acetone precipitation. Results The proteomics workflow identified and quantified up to 1800 barley proteins based on sequencing of up to 6900 peptides per sample. The two spin filter-based protocols provided a 12–38% higher efficiency than the ISD protocols, including more proteins of low abundance. Among the ISD protocols, a simple one-step reduction and S-alkylation method (OP-ISD) was the most efficient for barley leaf sample preparation; it identified and quantified 1500 proteins and displayed higher peptide-to-protein inference ratio and higher average amino acid sequence coverage of proteins. The two spin filter-aided sample preparation protocols are compatible with TMT labelling for quantitative proteomics studies. They exhibited complementary performance as about 30% of the proteins were identified by either one or the other protocol, but also demonstrated a positive bias for membrane proteins when using SDC as detergent. Conclusions We provide detailed protocols for efficient plant protein sample preparation for LC–MS/MS-based proteomics studies. Spin filter-based protocols are the most efficient for the preparation of leaf samples for MS-based proteomics. However, a simple protocol provides comparable results although with different peptide digestion profile.

Although corporate social responsibility is a permanent feature of activities of many companies, only a few companies can show a strategic approach and use complex solutions. It is emphasized that factors which could cause a positive change are institutional incentives such as tax benefits, and a bigger involvement of stakeholders, mostly employees. As far as providing hygienic working conditions is concerned, one must focus on better connecting occupational health and safety (OHS) with positive targets of the company, i.e. health promotion, efficiency increase and business excellence. It is becoming all the more important since the results of European research point out that the main source of stress at work is its psychosocial layer. Problems such as work-related stress, mobbing or discrimination are commonly considered to be the main challenges for OHS. The aim of the article is to emphasize that protecting the employees from psychosocial factors is not only necessitated by the law, but also is an ethical issue and a part of social responsibility of business. The article presents author's prevention model of psychosocial risk which can be implemented in any enterprises. It also shows examples of good practices of psychosocial risk reduction based on the cases presented in the literature.

Chlorination of tyrosine is a commonly known effect/consequence of myeloperoxidase activity at sites of inflammation, and detection of 3-chlorotyrosine has been used as biomarker for inflammatory diseases. However, few studies have addressed site specific chlorination in proteins, and no methods for large scale chloroproteomics studies have yet been published.In this study, we present an optimized mass spectrometry based protocol to identify and quantify chlorinated peptides from single proteins modified by HOCl (100 and 500 μM, within estimated pathophysiological levels), at a high level of sensitivity and accuracy. Particular emphasis was placed on 1) sensitive and precise detection of modification sites, 2) the avoidance of loss or artefactual creation of modifications, 3) accurate quantification of peptide abundance and reduction of missing values problem, 4) monitoring the dynamics of modification in samples exposed to different oxidant concentrations and 5) development of guidelines for verification of chlorination sites assignment.A combination of an optimised sample preparation protocol, and improved data analysis approaches have allowed identification of 33 and 15 chlorination sites in laminin and fibronectin, respectively, reported in previous manuscripts [1,2]. The method was subsequently tested on murine basement membrane extract, which contains high levels of laminin in a complex mixture. Here, 10 of the major chlorination sites in laminin were recapitulated, highlighting the utility of the method in detecting damage in complex samples. Keywords: Protein chlorination, Hypochlorous acid, Myeloperoxidase, Inflammation, Extracellular matrix proteins, Mass spectrometry, Proteomics