Although the caecum is the most important fermentation chamber in non-ruminant animals, the epithelial responses evoked by microbially produced short-chain fatty acids such as propionate are not well understood. Short-circuit currents (I_sc) across rat caecum were measured in Ussing chamber experiments, where anionic conditions, transport mechanisms and underlying signaling pathways regarding propionate-induced I_sc (I_Prop) were analyzed. These findings were complemented with immunofluorescence and RT-PCR. I_Prop was dependent on the simultaneous presence of Cl^- and HCO₃^-. This contrasts with the I_sc evoked by the cAMP- or Ca²⁺-dependent secretagogues, forskolin and carbachol, where replenishment of Cl^- was sufficient to induce a strong increase in I_sc. Blocking the cystic fibrosis transmembrane regulator (CFTR) with CFTR_inh-172 inhibited I_Prop in the oral caecum, whereas in the aboral caecum the additional presence of 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of anion exchangers, was necessary to reduce I_Prop significantly. Since propionate can activate free-fatty acid receptors type 2 (coupled to G_q and G_i proteins) and 3 (coupled to G_i proteins), downstream signaling was further elucidated. Inhibition of G_qα, phospholipase C, inositol-3,4,5-trisphosphate receptors, or phosphatidyl inositol kinases reduced I_Prop significantly. Kinases of the 'with no lysine' family (WNK), known to mediate the switch between Cl^- to HCO₃^- secretion in other cells, are also involved in propionate-induced anion secretion, since I_Prop was reduced by panWNK and WNK1 inhibition. Propionate stimulates anion secretion, probably in order to buffer the caecal luminal content, by a mechanism exhibiting a unique anion dependency different from that of other secretagogues.

Human cryptosporidiosis caused by the zoonotic apicomplexan parasite Cryptosporidium parvum represents a neglected and re-emerging poverty-related disease. C. parvum possesses minimalistic metabolic capacities and highly depends on its intestinal epithelial host cell for intracellular replication. Based on previous results showing that glycolysis and glutaminolysis inhibition diminished C. parvum replication in vitro, we here investigated the impact of the olive oil component oleocanthal on C. parvum infection in HCT-8 cells under physioxia (5% O₂) and hyperoxia (21% O₂). Oleocanthal targets a broad spectrum of regulatory molecules, amongst which mTOR represents a master regulator of glycolysis and glutaminolysis. Using a host cell pre-treatment as well as a pre- and post-infection treatment protocol, 5 µM oleocanthal reduced C. parvum infection rates between 51% and 94%. Host cellular metabolic conversion rates linked oleocanthal-induced inhibition of C. parvum infection with an impairment in glutaminolysis, representing an important metabolic pathway in intestinal cells. The principal involvement of mTOR in C. parvum inhibition was confirmed by another mTOR-inhibitor (PP242, 0.5 µM), which also reduced C. parvum infection by 70–77%. Given that oleocanthal is not a selective mTOR inhibitor, we assume that this compound drives a multi-target-based inhibition of asexual C. parvum replication, amongst which mTOR is addressed.

Delineating the constituents and structural composition of RNA-associated protein complexes is essential to mapping the molecular machinery driving RNA metabolism and its impact on cellular function. Here, we present a comprehensive dataset of RNA-dependent proteins and complexes in the phylogenetically distant yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe . Using R-DeeP—a density gradient-based method that uses quantitative mass spectrometry to profile protein sedimentation in the presence and absence of RNA—we introduce an RNA dependence index (RDI) as a descriptive framework for RNA dependence, enabling the robust comparative analysis of RNA dependence across proteins in both species and relative to existing data from their human counterparts. This identifies a conserved core of RNA-dependent proteins shared across both yeasts, alongside distinct, organism-specific adaptations in complex behaviour. The data further support the analysis of co-sedimentation behaviour of protein complexes with known RNA-directed functions. For instance, we find that the five subunits of the S. cerevisiae THO complex only co-sediment in the absence of RNA, pointing to an underappreciated structural modularity of the well-characterized pentameric complex. The two datasets, available at https://yeast-r-deep.computational.bio , provide a resource for hypothesis-driven research in RNA biology and establish R-DeeP as a broadly applicable tool for comparative analysis of RNA-protein interactions.

Cellular adaptation to stress requires extensive remodeling of RNA metabolism. The rewiring of the RNA-protein interaction landscape and the assembly of biomolecular condensates are central to this response, yet the global stress-induced reorganization of macromolecular RNA-protein assemblies remains incompletely characterized. Here, we apply R-DeeP, a density gradient approach coupled to quantitative mass spectrometry, to profile the RNA-dependent sedimentation behaviour of the Saccha-romyces cerevisiae proteome during heat and salt stress. We demonstrate that stress triggers a profound, stress-specific reorganization of the RNA interactome. Many proteins undergo pronounced, selective shifts into high-molecular weight fractions, indicative of aggregation. RNase treatment markedly exacerbates this aggregation, consistent with the model that RNA acts as a general chaperone that keeps proteins with intrinsically disordered regions soluble. Strikingly, our data demonstrate that this protection is compartmentalised. Nuclear proteins are partially shielded from stress-induced aggregation. This protection coincides with the nuclear accumulation of RNA caused by a block of RNA export during stress, and RNase treatment abolishes this protection. Nuclear RNA retention may therefore act as a buffer that actively increases the local chaperoning capacity of the nucleus. In contrast, cytoplasmic proteins, including translation factors and ribonucleoprotein granule components, exhibit increased aggregation under stress. Fluorescence microscopy confirmed that increased biochemical aggregation in density gradients reflects changes in assembly state in vivo. For many stress-induced aggregators, these changes are visible as spatial reorganisation in stressed cells, such as the formation of foci. Our findings suggest that cells use subcellular RNA distribution to tune the condensation and/or aggregation threshold of proteins in a compartment-specific manner.