Institute of Molecular Biotechnology

Field Asymmetric Ion Mobility Spectrometry (FAIMS) is an indispensable tool in single-cell and low-input proteomics. Here, we show that tuning FAIMS resolution via electrode temperature modulation improves sensitivity. We demonstrate that lowering FAIMS resolution broadens the compensation voltage window, thereby increasing ion transmission. This significantly improves ion counts, quantitative precision and peptide identifications by 25-34%. These results were mirrored when analyzing single cell samples.

Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.

Traumatic brain injury (TBI) causes sex-specific memory deficits, yet the underlying mechanisms are not fully understood. Using a mouse TBI model, we investigated the role of reactive astrocytes in sex-specific outcome. TBI provoked long-term contextual memory impairment in males and ovariectomized females, but not in intact females. The synthetic steroid tibolone preserved memory and cFos+ neuronal density in the hippocampus of ovariectomized females. Hormone deprivation upregulated astrocytic GFAP and S100B, reduced Homer1, and impaired myelin phagocytosis by astrocytes in females. These effects were counteracted by tibolone. In Four-Core-Genotype mice, memory loss correlated with reduced astrocytic myelin uptake and neuronal activity in XX males and XY female animals. Astrocyte transplantation showed that female astrocytes exhibit superior myelin clearance capacity, especially in female brain environments, though they outperform male astrocytes in both sex contexts. These findings identify astrocyte-mediated myelin phagocytosis as a key mechanism for memory preservation after TBI, governed by both hormonal and chromosomal sex factors.

Lytic polysaccharide monooxygenases catalyse the H 2 O 2 -dependent cleavage of polysaccharides. An optical H 2 O 2 sensor allows for real-time monitoring of enzyme activity and avoids the determination of oxidised reaction products.

Neutrophils are vital innate immune cells shown to infiltrate glioblastomas, however we currently lack the molecular understanding of their functional states within the tumour niche. Given that neutrophils are known to display a prominent discordance between mRNA and protein abundance, we developed ultra-sensitive mini-bulk and single cell proteomic (SCP) workflows to study the heterogeneity of peripheral blood and tumour associated neutrophils (TAN) from patients with glioblastoma. Mini-bulk analysis enabled a deeper protein coverage of circulating immature, mature and TAN populations, defining signatures of maturity and demonstrating that TANs resemble mature circulating neutrophils. Analysis of the SCP data results in the detection of >1100 proteins from a single TAN providing a detailed characterization of neutrophil subsets in glioblastoma. Our approach shows evidence of pathogenic and anti-tumorigenic clusters and discovers cell states invisible to scRNAseq, opening new opportunities to selectively target pro-tumoural neutrophil states.

The nuclear export of mRNA is an important step in eukaryotic gene expression1. Despite recent molecular insights into how newly transcribed mRNAs are packaged into ribonucleoprotein complexes (mRNPs)2,3, the subsequent events that govern mRNA export are poorly understood. Here we uncover the molecular basis underlying key events of human mRNA export, including the remodelling of mRNP-bound transcription-export complexes (TREX), the formation of export-competent mRNPs, the docking of mRNPs at the nuclear pore complex (NPC), and the release of mRNPs at the NPC to initiate their export. Our biochemical and structural data show that the ATPase UAP56 (also known as DDX39) acts as a central molecular switch that directs nucleoplasmic mRNPs from TREX to NPC-anchored TREX-2 complexes through its ATP-gated mRNA-binding cycle. Collectively, these findings establish a mechanistic framework for a general and evolutionarily conserved mRNAexport pathway.

Overlap extension PCR (OE-PCR) is a simple technique allowing for the construction of linear expression cassettes for direct integration into the Komagataella phaffii genome. This strategy enables the assembly and amplification of expression cassettes of interest that are genetically unstable or cause physiological problems in Escherichia coli and accelerates the path from gene to yeast transformation. As a typical example, here we describe the cloning of the Secale cereale (rye) gene Sec2, coding for the prolamin 75kγ-secalin. This gene proved to be unstable in circular plasmids in E. coli; in silico analysis suggested a loop-out-prone secondary structure of the DNA due to repetitive sequences. In three individual reactions, overlapping DNA fragments of the expression cassette and the gene of interest were amplified, followed by a two-step PCR protocol joining these three fragments to a linear expression cassette. After purification, the linear expression construct was directly used for the transformation of K. phaffii.

Abstract This study explores protein‐loaded, semipermeable polymer capsules for potential applications in drug delivery and biomedical analytics. Several proteins were loaded into mesoporous silica particles and encapsulated by layer‐by‐layer (LbL) assembling of oppositely charged polyelectrolytes. The LbL capsules are characterized using confocal laser scanning microscopy (CLSM). A novel protocol for the removal of the silica core under mild conditions (e.g., pH 7, room temperature, low ionic strength) is developed, providing a less harsh approach than existing methods. The functionality of the encapsulated biomolecules after the dissolution of the silica core is shown. By adjusting the pore size corresponding to the protein molecular weight, a homogeneous biomolecule distribution within the silica templates and the LbL capsules is achieved. The LbL coating can be performed without penetration into the protein‐loaded cores, preventing complexation and inactivation of the biomolecules. To assess the functionality of the protein‐loaded capsules, assays are conducted using streptavidin – biotin interactions, and anti‐streptomycin antibodies (aSM‐1), which bind streptomycin. The activity of the encapsulated proteins and the permeability of the LbL membrane for low‐molecular compounds are confirmed. A homogeneous displacement immunoassay is created using different fluorescence labeled antibodies and streptomycin.

PIWI-clade Argonaute proteins and their associated PIWI-interacting RNAs (piRNAs) are essential guardians of genome integrity, silencing transposable elements through distinct nuclear and cytoplasmic pathways. Nuclear PIWI proteins direct heterochromatin formation at transposon loci, while cytoplasmic PIWIs cleave transposon transcripts to initiate piRNA amplification. Both processes rely on target RNA recognition by PIWI-piRNA complexes, yet how this leads to effector recruitment is unclear. Here, we show that target engagement triggers formation of complexes, termed PIWI∗-comprising a PIWI protein, a piRNA-target duplex, a GTSF family protein, and Maelstrom-that serve as molecular platforms recruiting downstream effectors. In Drosophila, nuclear Piwi∗ engages the SFiNX complex to establish heterochromatin, while cytoplasmic Aubergine∗ complexes recruit the helicase Spindle-E to promote piRNA biogenesis. Evolutionary analysis reveals that PIWI∗ formation is conserved across metazoans, uncovering an ancient mechanism coupling piRNA-guided target recognition to effector function. These findings define a unifying molecular principle for PIWI-mediated silencing across cellular compartments.