Szymon Stoma

Last Name

Stoma

First Name

Szymon

ORCID

0000-0001-5127-8553

Organisational unit

02891 - ScopeM / ScopeM

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Publications 1 - 5 of 5

The U2-spliceosome and its interactors regulate the levels and activity of the LDL receptor in humans
Zanoni, Paolo; Othman, Alaa; Meier, Roger; et al. (2020)
Atherosclerosis
Background and Aims: The low-density lipoprotein receptor (LDLR) in the liver is the major determinant of LDL-cholesterol levels in humans. The discovery of novel genes that regulate the activity of LDLR could lead to the identification of pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNAi screen for fluorescent LDL uptake in Huh-7 hepatocarcinoma cells and validated our top hit genes in vitro, ex vivo as well as in population genetics datasets. Results: In our genome-wide RNAi screen, the knock-down of 54 genes led to a significant inhibition of LDL uptake. Fifteen of these genes encode for proteins involved in splicing, especially components or interactors of the U2-spliceosome. Knocking down 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The transcript is translated into an LDLR fragment, which lacks 88% of the full length LDLR and is detectable in cells and their medium upon overexpression, but neither in non-transfected cells nor in human plasma. The intron 3 retention transcript is expressed in considerable amounts in human liver and in blood cells. Its expression correlates with plasma LDL-cholesterol and age and increases after bariatric surgery. Single nucleotide polymorphisms and rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Conclusions: We identified a novel mechanism of post-transcriptional regulation of LDLR activity in humans.
How Modifications of Corneal Cross-Linking Protocols Influence Corneal Resistance to Enzymatic Digestion and Treatment Depth
Kowalska, Malwina; Mischi, Elisa; Stoma, Szymon; et al. (2023)
Translational Vision Science & Technology
Purpose: The purpose of this study was to determine the effects of the Photoactivated Chromophore for Keratitis Corneal Cross-Linking (PACK-CXL) protocol modifications on corneal resistance to enzymatic digestion and treatment depth. Methods: Eight hundred one ex vivo porcine eyes were randomly divided into groups of 12 to 86 corneas, treated with various epi-off PACK-CXL modifications, including acceleration (30 > 2 minutes, 5.4 J/cm2), increased fluence (5.4 > 32.4 J/cm2), deuterium oxide (D2O) supplementation, different carrier types (dextran versus hydroxypropyl methylcellulose [HPMC]), increased riboflavin concentration (0.1 > 0.4%), and riboflavin replenishment during irradiation (yes/no). Control group eyes did not receive PACK-CXL. A pepsin digestion assay was used to determine corneal resistance to enzymatic digestion. A phalloidin fluorescent imaging assay was used to determine the PACK-CXL treatment effect depth. Differences between groups were evaluated using a linear model and a derivative method, respectively. Results: PACK-CXL significantly increased corneal resistance to enzymatic digestion compared to no treatment (P < 0.03). When compared to a 10 minute, 5.4 J/cm2 PACK-CXL protocol, fluences of 16.2 J/cm2 and higher increased corneal resistance to enzymatic digestion by 1.5- to 2-fold (P < 0.001). Other protocol modifications did not significantly change corneal resistance. A 16.2 J/cm2 fluence also increased collagen compaction in the anterior stroma, whereas omitting riboflavin replenishment during irradiation increased PACK-CXL treatment depth. Conclusions: Increasing fluence will likely optimize PACK-CXL treatment effectiveness. Treatment acceleration reduces treatment duration without compromising effectiveness. Translational Relevance: The generated data help to optimize clinical PACK-CXL settings and direct future research efforts.
Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome
Zanoni, Paolo; Panteloglou, Grigorios; Othman, Alaa; et al. (2022)
Circulation Research
Background: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. Conclusions: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.
Long-term tracking of budding yeast cells in brightfield microscopy: CellStar and the Evaluation Platform
Versari, Cristian; Stoma, Szymon; Batmanov, Kirill; et al. (2017)
Journal of the Royal Society. Interface
With the continuous expansion of single cell biology, the observation of the behaviour of individual cells over extended durations and with high accuracy has become a problem of central importance. Surprisingly, even for yeast cells that have relatively regular shapes, no solution has been proposed that reaches the high quality required for long-term experiments for segmentation and tracking (S&T) based on brightfield images. Here, we present CellStar, a tool chain designed to achieve good performance in long-term experiments. The key features are the use of a new variant of parametrized active rays for segmentation, a neighbourhood-preserving criterion for tracking, and the use of an iterative approach that incrementally improves S&T quality. A graphical user interface enables manual corrections of S&T errors and their use for the automated correction of other, related errors and for parameter learning. We created a benchmark dataset with manually analysed images and compared CellStar with six other tools, showing its high performance, notably in long-term tracking. As a community effort, we set up a website, the Yeast Image Toolkit, with the benchmark and the Evaluation Platform to gather this and additional information provided by others.
Detecting variation in starch granule size and morphology by high-throughput microscopy and flow cytometry
Thieme, Mercedes; Hochmuth, Anton; Ilse, Theresa Elisabeth; et al. (2023)
Carbohydrate Polymers
Starch forms semi-crystalline, water-insoluble granules, the size and morphology of which vary according to biological origin. These traits, together with polymer composition and structure, determine the physicochemical properties of starch. However, screening methods to identify differences in starch granule size and shape are lacking. Here, we present two approaches for high-throughput starch granule extraction and size determination using flow cytometry and automated, high-throughput light microscopy. We evaluated the practicality of both methods using starch from different species and tissues and demonstrated their effectiveness by screening for induced variation in starch extracted from over 10,000 barley lines, yielding four with heritable changes in the ratio of large A-granules to small B-granules. Analysis of Arabidopsis lines altered in starch biosynthesis further demonstrates the applicability of these approaches. Identifying variation in starch granule size and shape will enable identification of trait-controlling genes for developing crops with desired properties, and could help optimise starch processing.

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Szymon Stoma

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