α9α10 nicotinic acetylcholine receptors (nAChRs) putatively exist at different stoichiometries. We systematically investigated the molecular determinants of α-conotoxins Vc1.1, RgIA#, and PeIA inhibition at hypothetical stoichiometries of the human α9α10 nAChR. Our results suggest that only Vc1.1 exhibits stoichiometric-dependent inhibition at the α9α10 nAChR. The hydrogen bond between N154 of α9 and D11 of Vc1.1 at the α9(+)-α9(-) interface is responsible for the stoichiometric-dependent potency of Vc1.1.

α-Conotoxins preferentially antagonize muscle and neuronal nicotinic acetylcholine receptors (nAChRs). Native α-conotoxins have two disulfide links, CI–CIII and CII–CIV, and owing to the inherent properties of disulfide bonds, α-conotoxins have been systematically engineered to improve their chemical and biological properties. In this study, we explored the possibility of simplifying the disulfide framework of α-conotoxins Vc1.1, BuIA, ImI, and AuIB, by introducing [C2H,C8F] modification to the CI–CIII bond. We therefore explored the possibility of using hydrophobic packing of standard amino acid side chains to replace disulfide bonds as an alternative strategy to nonnatural amino acid cross-links. The impact of CI–CIII disulfide bond replacement on the conformation of the α-conotoxins was investigated using molecular dynamics (MD) simulations and nuclear magnetic resonance chemical shift index study. Two-electrode voltage clamp techniques and MD simulations were used to study the impact of disulfide bond deletion on the activities of the peptides at human neuronal nAChRs. All disulfide-deleted variants except ImI[C2H,C8F] had reduced potency for inhibiting nAChRs. Our results suggest that the CI–CIII disulfide bond is important to stabilize the secondary structure of α-conotoxins as well as their interaction with neuronal nAChR targets. Results from this study enrich our understanding of the function of the CI–CIII disulfide bond and are useful in guiding future structural engineering of the α-conotoxins.

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Nicotinic acetylcholine receptors (nAChRs) belong to the Cys-loop receptor family and are important drug targets for the treatment of neurological diseases. However, the precise determinants of the binding efficacies of ligands for these receptors are unclear. Therefore, in this study, the binding energy profiles of various ligands (full agonists, partial agonists, and antagonists) were quantified by docking those ligands with structural ensembles of the α7 nAChR exhibiting different degrees of C-loop closure. This approximate treatment of interactions suggested that full agonists, partial agonists, and antagonists of the α7 nAChR possess distinctive binding energy profiles. Results from docking revealed that ligand binding efficacy may be related to the capacity of the ligand to stabilize conformational states with a closed C loop.

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Currently, milk quality is judge on its health and hygienic parameters equally as its composition. Raw milk is a rich medium for the growth and proliferation of different pathogenic microorganisms such food-safety hazard may incorporate into the dairy chain, not only affect its safety and quality but also cause various milk borne diseases. Objective: To evaluate the microbiological assessment of raw milk accessible in Karachi with the intention to develop risk tracking strategies and its mitigation plan in future. Methods: Total 270 raw milk samples were collected from different sites throughout the dairy supply chains in each district of the city. Microbiological assessment of raw milk was carried out by following the bacteriological analytical manual (BAM, FDA) protocol. Results: The raw milk safety and quality at the dairy farms were satisfied, except raw milk storage tanks samples. The bacterial count boosted in almost all the samples collected during its transportation, higher aerobic and pathogenic load was observed in the retail shop’s samples however, maximum counts were noticed in the selling point milk samples. None of the milk samples contained other pathogens like Listeria or Salmonella. Conclusions: The milk quality of the supply chain, retail shops and storage tanks at dairy farms were un-satisfied based on different microbiological parameters.

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