Gastrointestinal absorption and metabolism of apple polyphenols ex vivo by the pig intestinal mucosa in the Ussing chamber

Polyphenols contained in food have various positive effects on human health. The absorption and metabolism of polyphenols in the intestinal tract needs to be studied to estimate these effects. The Ussing chamber technique was used to investigate the transport behavior of apple polyphenols through pig small intestinal mucosa, which served as a model for human gastrointestinal mucosa. The identities and concentrations of polyphenols and their metabolites in the half-chambers (luminal and basolateral) within an incubation period of 4 h were determined by HPLC–MS/MS and HPLC–DAD (DAD = diode-array detection). Flux values were also measured. It was found that 5-caffeoylquinic acid and caffeic acid were absorbed and translocated to the basolateral side (1.9 and 3.7%, respectively), but other compounds, including glycosides of phloretin and quercetin, were observed without translocation. A Ussing chamber utilizing pig small intestinal mucosa is a suitable model for assessing the effect of apple polyphenols on mucosal integrity and nutrition absorption across porcine mucosa.     

The mutational dynamics of the SARS-CoV-2 virus in serial passages in vitro

Since its outbreak in 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) keeps surprising the medical community by evolving diverse immune escape mutations in a rapid and effective manner. To gain deeper insight into mutation frequency and dynamics, we isolated ten ancestral strains of SARS-CoV-2 and performed consecutive serial incubation in ten replications in a suitable and common cell line and subsequently analysed them using RT-qPCR and whole genome sequencing. Along those lines we hoped to gain fundamental insights into the evolutionary capacity of SARS-CoV-2 in vitro. Our results identified a series of adaptive genetic changes, ranging from unique convergent substitutional mutations and hitherto undescribed insertions. The region coding for spike proved to be a mutational hotspot, evolving a number of mutational changes including the already known substitutions at positions S:484 and S:501. We discussed the evolution of all specific adaptations as well as possible reasons for the seemingly inhomogeneous potential of SARS-CoV-2 in the adaptation to cell culture. The combination of serial passage in vitro with whole genome sequencing uncovers the immense mutational potential of some SARS-CoV-2 strains. The observed genetic changes of SARS-CoV-2 in vitro could not be explained solely by selectively neutral mutations but possibly resulted from the action of directional selection accumulating favourable genetic changes in the evolving variants, along the path of increasing potency of the strain. Competition among a high number of quasi-species in the SARS-CoV-2 in vitro population gene pool may reinforce directional selection and boost the speed of evolutionary change.     

Innate immunity: the worm fights back

Innate immunity is an evolutionarily ancient defense system that enables animals and plants to resist invading microorganisms. Recent studies have demonstrated the existence of innate immune responses in Caenorhabditis elegans.     

A densely interconnected genome-wide network of microRNAs and oncogenic pathways revealed using gene expression signatures

MicroRNAs (miRNAs) are important components of cellular signaling pathways, acting either as pathway regulators or pathway targets. Currently, only a limited number of miRNAs have been functionally linked to specific signaling pathways. Here, we explored if gene expression signatures could be used to represent miRNA activities and integrated with genomic signatures of oncogenic pathway activity to identify connections between miRNAs and oncogenic pathways on a high-throughput, genome-wide scale. Mapping >300 gene expression signatures to >700 primary tumor profiles, we constructed a genome-wide miRNA-pathway network predicting the associations of 276 human miRNAs to 26 oncogenic pathways. The miRNA-pathway network confirmed a host of previously reported miRNA/pathway associations and uncovered several novel associations that were subsequently experimentally validated. Globally, the miRNA-pathway network demonstrates a small-world, but not scale-free, organization characterized by multiple distinct, tightly knit modules each exhibiting a high density of connections. However, unlike genetic or metabolic networks typified by only a few highly connected nodes ("hubs"), most nodes in the miRNA-pathway network are highly connected. Sequence-based computational analysis confirmed that highly-interconnected miRNAs are likely to be regulated by common pathways to target similar sets of downstream genes, suggesting a pervasive and high level of functional redundancy among coexpressed miRNAs. We conclude that gene expression signatures can be used as surrogates of miRNA activity. Our strategy facilitates the task of discovering novel miRNA-pathway connections, since gene expression data for multiple normal and disease conditions are abundantly available.     

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).