P-glycoprotein (Pgp, ABCB1) is a member of one of the largest families of active transporter proteins called ABC transporters. Thanks to its expression in tissues with barrier functions and its broad substrate spectrum, it is an important determinant of the absorption, metabolism and excretion of many drugs. Pgp and/or some other drug transporting ABC proteins (e.g., ABCG2, MRP1) are overexpressed in nearly all cancers and cancer stem cells by which cancer cells become resistant against many drugs. Thus, Pgp inhibition might be a strategy for fighting against drug-resistant cancer cells. Previous studies have shown that certain polyphenols interact with human Pgp. We tested the effect of 15 polyphenols of sour cherry origin on the basal and verapamil-stimulated ATPase activity of Pgp, calcein-AM and daunorubicin transport as well as on the conformation of Pgp using the conformation sensitive UIC2 mAb. We found that quercetin, quercetin-3-glucoside, narcissoside and ellagic acid inhibited the ATPase activity of Pgp and increased the accumulation of calcein and daunorubicin by Pgp-positive cells. Cyanidin-3O-sophoroside, catechin, naringenin, kuromanin and caffeic acid increased the ATPase activity of Pgp, while they had only a weaker effect on the intracellular accumulation of fluorescent Pgp substrates. Several tested polyphenols including epicatechin, trans-ferulic acid, oenin, malvin and chlorogenic acid were ineffective in all assays applied. Interestingly, catechin and epicatechin behave differently, although they are stereoisomers. We also investigated the effect of quercetin, naringenin and ellagic acid added in combination with verapamil on the transport activity of Pgp. In these experiments, we found that the transport inhibitory effect of the tested polyphenols and verapamil was additive or synergistic. Generally, our data demonstrate diverse interactions of the tested polyphenols with Pgp. Our results also call attention to the potential risks of drug–drug interactions (DDIs) associated with the consumption of dietary polyphenols concurrently with chemotherapy treatment involving Pgp substrate/inhibitor drugs.

Background and Purpose: Cell penetrating peptides are promising tools for delivery of cargo into cells, but factors limiting or facilitating their cellular uptake are largely unknown. We set out to study the effect of the biophysical properties of the cell membrane on the uptake of penetratin, a cell penetrating peptide.Experimental Approach: Using labelling with pH‐insensitive and pH‐sensitive dyes, the kinetics of cellular uptake and endo‐lysosomal escape of penetratin were studied by flow cytometry.Key Results: We report that escape of penetratin from acidic endo‐lysosomal compartments is retarded compared with its total cellular uptake. The membrane dipole potential, known to alter transmembrane transport of charged molecules, is shown to be negatively correlated with the concentration of penetratin in the cytoplasmic compartment. Treatment of cells with therapeutically relevant concentrations of atorvastatin, an inhibitor of HMG‐CoA reductase and cholesterol synthesis, significantly increased endosomal escape of penetratin in two different cell types. This effect of atorvastatin correlated with its ability to decrease the membrane dipole potential.Conclusion and Implications: These results highlight the importance of the dipole potential in regulating cellular uptake of cell penetrating peptides and suggest a clinically relevant way of boosting this process.

Cell-penetrating peptides represent an emerging class of carriers capable of effective cellular delivery. This work demonstrates the preparation and investigation of efficient CPPs. We have already shown that the presence of 4-((4-(dimethylamino)phenyl)azo)benzoic acid (Dabcyl) and Trp greatly increase the uptake of oligoarginines. This work is a further step in that direction. We have explored the possibility of employing unnatural, aromatic amino acids, to mimic Trp properties and effects. The added residues allow the introduction of aromaticity, not as a side-chain group, but rather as a part of the sequence. The constructs presented exceptional internalization on various cell lines, with an evident structure–activity relationship. The CPPs were investigated for their entry mechanisms, and our peptides exploit favorable pathways, yet one of the peptides relies highly on direct penetration. Confocal microscopy studies have shown selectivity towards the cell lines, by showing diffuse uptake in FADU cells, while vesicular uptake takes place in SCC-25 cell line. These highly active CPPs have proved their applicability in cargo delivery by successfully delivering antitumor drugs into MCF-7 and MDA-MB-231 cells. The modifications in the sequences allow the preparation of short yet highly effective constructs able to rival the penetration of well-known CPPs such as octaarginine (Arg8).

Fusarium verticillioides poses a high food safety risk worldwide due to its mycotoxin production. Successful control of Fusaria may rely on promising biocontrol agents, including yeasts. Although the fission yeast Schizosaccharomyces pombe tolerated Fusarium mycotoxins well, including zearalenone, T2, deoxynivalenol, and fumonisins (FUMs), it did not significantly inhibit the growth of F. verticillioides. Meanwhile fumonisin B1 (FB1) supplementation did not decrease S. pombe cell density in submerged liquid cultures, the colony-forming capability of the yeast was reduced. RNA sequencing showed that S. pombe genes involved in cell adhesion and flocculation were downregulated after FB1 exposure. In addition, the expression of several hydrolase genes was also altered. In co-cultures with F. verticillioides , genes encoding oxidoreductases and hydrolases and those linked to purine nucleotide metabolisms were downregulated, while the expression of genes involved in membrane and transport processes was increased. The expression of several F. verticillioides genes also changed after co-cultivation. Oxidoreductase, transmembrane transport, and purine metabolism genes were upregulated under co-culturing; meanwhile, hydrolase genes, together with carbon metabolism and polysaccharide catabolism genes, were downregulated. Co-cultivation also decreased fumonisin production via the downregulation of genes FUM19 , FUM21 , and FvATFA encoding the fumonisin transporter, a local Zn(II)2Cys6-type transcriptional regulator and an important global regulator bZIP-type transcription factor, respectively. Although further experiments should clarify the mechanism of the fission yeast-elicited inhibition of fumonisin production, these results may pave the way for the development and implementation of novel, innovative approaches to control mycotoxin production by F. verticillioides in the feed and food chain. Key points: • 0.5 ppm FB1 reduced the colony-forming ability of S. pombe and caused transcriptional changes. • Expression of transport and hydrolase genes changed in yeast during co-cultivation with mold. • Two FUM cluster genes and FvATFA were downregulated in Fusarium co-cultured with S. pombe.

Fusarium graminaerum causes Fusarium Head Blight (FHB) on wheat, reduces yield, and contaminates food and feed. It is therefore of paramount importance to control its growth or convert its harmful mycotoxins. This study aimed to find yeasts with biocontrol activity against F. graminearum, and to identify genes with potential detoxifying activities, using microbiological, molecular methods and bioinformatics. Co-cultivation tests showed that Schizosaccharomyces japonicus was able to inhibit the growth of F. graminearum. Transcriptomic analysis of the yeast cells co-cultured with F. graminearum highlighted differentially expressed genes (DEGs) encoding various enzymes, such as oxidoreductases, transferases, hydrolases, or genes involved in transmembrane transport. Three trichothecene-3-O-acetyltransferase homologous genes, which can convert trichothecenes to less toxic forms, were also among them. A database search showed that several yeast species contained this gene, including S. japonicus, which unexpectedly had seven copies. Real-time PCR analysis and mycotoxin tolerance tests confirmed that some of these genes could be induced by deoxynivalenol (DON), and S. japonicus had stronger DON tolerance than the related S. pombe, whose genome did not contain such a gene. This study is the first to report the biocontrol efficacy of S. japonicus against F. graminearum and the identification of its potential detoxification genes, offering promising new avenues for biotechnological applications in food safety.

/Summary: Homer proteins are modular scaffold molecules that constitute an integral part of the protein network within the postsynaptic density. Full-length Homer1 forms a large homotetramer via a long coiled coil region, and can interact with proline-rich target sequences with its globular EVH1 domain. Here we report an atomistic model of the full-length Homer1 tetramer along with the NMR solution structure of its EVH1 domain. Compared to the already available EVH1 structures, our NMR ensemble exhibits subtle differences, mostly in and around its partner binding region, suggesting the presence of ligand-induced conformational transitions. Molecular dynamics simulations of the long coiled coil reveal distinct regions with different stability and flexibility, with the N-terminal part of the coiled coil exhibiting the largest motions. Interestingly, this segment is highly conserved, pointing to the functional relevance of the observed dynamical features. Our results indicate previously unexplored aspects of the flexibility of the full-length Homer1 tetramer that might contribute to the dynamic rearrangements of the postsynaptic protein network linked to its functional transitions.