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Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine–antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds. [ABSTRACT FROM AUTHOR]

The discovery of concretions of Chang 7 shale formations in the Ordos basin has increased interest in the exploration of lacustrine carbonate genesis in these basins. In this paper, these concretions were sampled and used in major, trace, and isotopic geochemistry tests. We used a microscope to investigate these concretions, and the results showed that the concretions consisted of carbonate rocks, the calcite was hydrothermal calcite, and obvious hydrothermal activity was present in the Yanchang period. We used seismic data to interpret the faults, and we determined that tectonic activity was relatively frequent in the middle–late Triassic period and that the faults were channels for hydrothermal upwelling. During the middle–late Triassic period, tectonic movement of the basin occurred, and synsedimentary faults developed in the Yanchang Formation. As deep hydrothermal gushers rose through faults and fractures, they carried particles upward through the deep limestone strata. When the hydrothermal gushers reached the lake bottom, the particles precipitated and eventually formed concretions via diagenesis. [ABSTRACT FROM AUTHOR]