Rolle, K. | Search bioRxiv

bioRxiv2024

Transcriptome-wide analysis of circRNA and RBP profiles and their molecular and clinical relevance for GBM

Glioblastoma (GBM) is the most aggressive and lethal type of glioma, characterized by aberrant expression of non-coding RNAs including circular RNAs (circRNAs). They might impact cellular processes by interacting with other molecules - like microRNAs or RNA-binding proteins (RBPs). The diagnostic value of circRNAs and circRNAs/RBPs complexes is still largely unknown. To explore circRNAs and RBPs transcripts expression in GBM, we performed and further analyzed RNA-seq data from GBM patients primary and recurrent tumor samples. We identified circRNAs differentially expressed in primary tumors, the circRNA progression markers in recurrent GBM samples as well as the expression profile of RBP transcripts. Subsequent analysis allowed us to generate a comprehensive catalog of circRNA-RBP interactions regarding both the RBPs sequestration by circRNA as well as the RBPs involvement in circRNA biogenesis. Furthermore, we demonstrated the clinical potential of circRNAs and RBPs in GBM and proposed them as the stratification markers in the de novo assembled tumor subtypes. Therefore, our transcriptome-wide study specified circRNA-RBP interactions that could play a significant regulatory role in gliomagenesis and GBM progression.

cancer biology↗

bioRxiv2022

Self-pressurized rapid freezing at arbitrary cryoprotectant concentrations

Self-pressurized rapid freezing (SPRF) has been proposed as a simple alternative to traditional high pressure freezing (HPF) protocols for vitrification of biological samples in electron microscopy and cryopreservation applications. Both methods exploit the circumstance that the melting point of ice reaches a minimum when subjected to pressure of around 210 [MPa], however, in SPRF its precise quantity depends on sample properties and hence, is generally unknown. In particular, cryoprotective agents (CPAs) are expected to be a factor; though eschewed by many SPRF experiments, vitrification of larger samples notably cannot be envisaged without them. Thus, in this study, we address the question of how CPA concentration affects pressure inside sealed capillaries, and how to design SPRF experiments accordingly. By embedding a fiber-optic probe in samples and performing Raman spectroscopy after freezing, we first present a direct assessment of pressure buildup during SPRF, enabled by the large pressure sensitivity of the Raman shift of hexagonal ice. Choosing dimethyl sulfoxide (DMSO) as a model CPA, this approach allows us to demonstrate that average pressure drops to zero when DMSO concentrations of 15 wt % are exceeded. Since a trade-off between pressure and DMSO concentration represents an impasse with regards to vitrification of larger samples, we introduce a sample architecture with two chambers, separated by a partition that allows for equilibration of pressure but not DMSO concentrations. We show that pressure and concentration in the fiber-facing chamber can be tuned independently, and present differential scanning calorimetry (DSC) data supporting the improved vitrification performance of two-chamber designs.

biophysics↗