Using computational methods, we constructed a model of the cardiac ion channel hERG in complex with BeKm-1, a scorpion toxin. We identified the crucial role of the toxin residue Arg20 and validated it by in silico and in vitro mutagenesis. The BeKm-1^R20K mutant showed dramatically reduced activity, suggesting the significance of Arg20 for channel binding. Our model aids future drug design attempts.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (K_v11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG–BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1^R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid β in the brain. Human apoE4 isoform is a risk factor for apoE-related diseases, although only one or two residues are different in other isoforms. Here, we generated novel anti-apoE monoclonal antibodies and constructed a sandwich ELISA system to selectively detect the apoE4 isoform.

Apolipoprotein E (apoE) is a regulator of lipid metabolism, cholesterol transport, and the clearance and aggregation of amyloid β in the brain. The three human apoE isoforms apoE2, apoE3, and apoE4 only differ in one or two residues. Nevertheless, the functions highly depend on the isoform types and lipidated states. Here, we generated novel anti-apoE monoclonal antibodies (mAbs) and obtained an apoE4-selective mAb whose epitope is within residues 110–117. ELISA and bio-layer interferometry measurements demonstrated that the dissociation constants of mAbs are within the nanomolar range. Using the generated antibodies, we successfully constructed sandwich ELISA systems, which can detect all apoE isoforms or selectively detect apoE4. These results suggest the usability of the generated anti-apoE mAbs for selective detection of apoE isoforms.

Our study showed that bile-mediated modulation of bacterial metabolism involves induction of various metabolic processes, e.g., anaerobic respiration dependent on nitrate. We observed that the activation of the nitrate metabolism-related genes fnr and narL is notably higher in the bile-tolerant WT strain compared to the bile-sensitive ΔcspE strain. Consequently, the WT strain displays lower amounts of reactive oxygen species and higher survival compared to the ΔcspE strain during bile stress.

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyze the adaptive responses to bile in two S. Typhimurium strains: wild type (WT) and a mutant lacking cold shock protein E (ΔcspE). CspE is an RNA chaperone which is crucial for survival of S. Typhimurium during bile stress. This study identifies transcriptional responses in bile-tolerant WT and bile-sensitive ΔcspE. Upregulation of several genes involved in nitrate metabolism was observed, including fnr, a global regulator of nitrate metabolism. Notably, Δfnr was susceptible to bile stress. Also, complementation with fnr lowered reactive oxygen species and enhanced the survival of bile-sensitive ΔcspE. Importantly, intracellular nitrite amounts were highly induced in bile-treated WT compared to ΔcspE. Also, the WT strain pre-treated with nitrate displayed better growth with bile. These results demonstrate that nitrate-dependent metabolism promotes adaptation of S. Typhimurium to bile.

Nanog and Oct4 are the key regulatory genes that govern the developmental dynamics of embryonic stem cells to trophectoderm and primitive endoderm by maintaining specific steady-state expression patterns. Herein, we hypothesize stepwise switching and mushroom-like bifurcation dynamics for Oct4 and Nanog, respectively, that align well with the existing experimental findings and shed light on fate-determination events.

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors—Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.