Biology subjects

Pustka, V.

Publications and source records attributed to Pustka, V..

3 recordsLinked to original sources

bioRxiv2025

Human Serum Albumin as a Hidden Source of Variability in IVF Culture Media

Soluble proteins present in spent culture media (SCM) have been proposed as potential indicators of embryo viability. However, results across studies remain inconsistent, and the identity of reliable protein biomarkers is still unclear. To evaluate the potential of secretome profiling for non-invasive embryo assessment, we applied state-of-the-art mass spectrometry to analyze the protein composition of SCM from individual successful and unsuccessful human embryo cultures, alongside corresponding controls cultured under identical conditions. Surprisingly, both untargeted (nanoLC-TIMS-TOF-MS/MS) and targeted (UHPLC-QqQ-MS/MS) proteomic analyses consistently detected multiple human proteins in unconditioned media, indicating that these signals are not of embryonic origin. Undeclared proteins were also identified in media sampled before their first use, with considerable variability observed across production batches. Supplementation experiments demonstrated that this variability originates from the addition of plasma-derived human serum albumin, which introduces a range of contaminating proteins. In contrast, recombinant albumin did not contribute detectable serum proteins. These findings indicate that the protein background in commercial IVF media is an overlooked variable, not only compromising the reproducibility and interpretation of secretome analyses in research, but also contributing to unstandardized culture conditions in clinical IVF practice. While this study evaluated 13 production lots of monophasic media from two different manufacturers, the results underscore a broader need for transparency and standardization in IVF media composition. The adoption of fully chemically defined IVF media, free from purified serum components, would support more consistent embryo culture conditions and advance the discovery of biomarkers in SCM.

cell biology↗

bioRxiv2025

Arabidopsis seed stored mRNAs and translation regulation during post-harvest ripening and imbibition.

Seed germination marks the critical transition from dormancy to active growth, driven by environmental cues and water availability. This study explores translational regulation during germination by comparing two Arabidopsis thaliana accessions with contrasting dormancy phenotypes: Columbia (Col), which germinates readily, and Cape Verde Islands (Cvi), which exhibits deep dormancy. Using sucrose gradient centrifugation, we isolated monosomal and polysomal fractions from freshly harvested (FH), after-ripened (AR), and imbibed (IM) seeds. RNA-seq analysis revealed stage- and genotype-specific gene expression, with Col IM seeds displaying the highest number of expressed genes. We identified [~]14,000 mRNAs in FH seeds, increasing to 19,000 in Col and 17,000 in Cvi upon imbibition. Of these, 9,000 were shared, while 3,000 were accession-specific in monosomes. Enrichment analysis highlighted molecular pathways associated with translation and dormancy release. Analysis of RNA modifications identified N1-methyladenosine (m1A) as the predominant modification, with Col seeds exhibiting higher m1A levels than Cvi, peaking at three months post-harvest. m6A sequencing revealed distinct modification patterns between accessions, with the highest abundance of m6A-modified transcripts in IM seeds. Positional analysis of m6A peaks suggested a link to differential gene expression between Col and Cvi. Proteomic analysis identified [~]15,000 proteins, with translation-related proteins enriched in IM seeds. Notable differences between Col and Cvi were observed in both monosomal and polysomal fractions. RNA-binding proteins exhibited similar profiles in FH and AR stages but diverged significantly in IM seeds. Col-specific proteins were enriched in 40S ribosomes, processing bodies, and RNA-binding complexes. These findings provide new insights into the molecular and translational dynamics underlying seed germination, advancing our understanding of dormancy release and early seedling establishment.

plant biology↗

bioRxiv2024

An iPSC-derived bio-inspired scaffold modelling the structure and the effects of extracellular matrix in cardiac fibrosis

Cardiac fibrosis occurs following insults to the myocardium and is characterized by the abnormal accumulation of non-compliant extracellular matrix (ECM), which compromises cardiomyocyte contractile activity and eventually leads to heart failure. This phenomenon is driven by the differentiation of cardiac fibroblasts (cFbs) into myofibroblasts and results in changes in ECM biochemical, structural and mechanical properties. The lack of predictive in vitro models of heart fibrosis has so far hampered the search for innovative treatments. Here, we devised a single-step decellularization protocol to obtain and thoroughly characterize the biochemical and micro-mechanical properties of the ECM secreted by activated cFbs differentiated from human induced pluripotent stem cells (iPSCs). We activated iPSC-derived cFbs to the myofibroblast phenotype by tuning basic fibroblast growth factor (bFGF) and transforming growth factor beta 1 (TGF-{beta}1) signalling and confirmed that activated cells acquired key features of myofibroblast phenotype, like SMAD2/3 nuclear shuttling, the formation of aligned alpha-smooth muscle actin (-SMA)-rich stress fibres and increased focal adhesions (FAs) assembly. Next, we used Mass Spectrometry, nanoindentation, scanning electron and confocal microscopy to unveil the characteristic composition and the visco-elastic properties of the abundant, collagen-rich ECM deposited by cardiac myofibroblasts in vitro. Finally, we demonstrated that the fibrotic ECM activates mechanosensitive pathways in iPSC-derived cardiomyocytes, impacting on their shape, sarcomere alignment, phenotype, and calcium handling properties. We thus propose human bio-inspired decellularized matrices as animal-free, isogenic cardiomyocyte culture substrates recapitulating key pathophysiological changes occurring at the cellular level during cardiac fibrosis.

cell biology↗