Jovic, M. | Search bioRxiv

bioRxiv2025

EXPRESSION OF ANO1 IN HUMAN GASTROINTESTINAL TRACT DURING EMBRYONIC AND FETAL DEVELOPMENT

Anoctamin 1 (ANO1, TMEM16A) is a transmembrane protein belonging to the ANO family, with a role in the formation of calcium-activated chloride channels (CaCC). It is included in the regulation of physiological processes such as muscle contraction, gastrointestinal motility, secretion, and electrical excitability. Also, recent data suggest that ANO1 is a specific marker for interstitial cells of Cajal (ICC). The aim of the paper was to examine the spatial and temporal distribution of ANO1 in the stomach, small intestine, and large intestine during embryofetal development as a potential marker for the differentiation of ICC and smooth muscle cells. As a material, we used 2 human embryos and samples from 21 human fetuses. The tissue samples were routinely processed into paraffin blocks, and 5 {micro}m-thick sections were immunostained for ANO1. Our results show that ANO1 appears during embryonic development (8th week), and its expression continues through the fetal stages. Epithelial, endothelial and ICC cells consistently expressed ANO1 in all examined samples. Smooth muscle cells showed strong expression in muscularis propria, however by the 25th week this immunopositivity was absent from outer muscle layers in stomach and large intestine. In conclusion, ANO1 can be considered as a reliable marker for following the differentiation of SMC and ICC during embryonic and fetal development.

developmental biology↗

bioRxiv2024

de Novo Sequencing of Antibodies for Identification of Neutralizing Antibodies in Human Plasma Post SARS-CoV-2 Vaccination

We present a method for sequencing polyclonal IgG enriched from human plasma, employing a combination of de novo sequencing, proteomics, bioinformatics, protein separation, sequencing, and peptide separations. Our study analyzes a single patients IgG antibody response triggered by the Moderna Spikevax mRNA COVID-19 vaccine. From the sequencing data of the natural polyclonal response to vaccination, we generated 12 recombinant antibodies. Six derived recombinant antibodies, including four generated with de novo sequencing, exhibited similar or higher binding affinities than the original natural polyclonal antibody. Our neutralization tests revealed that the six antibodies possess neutralizing capabilities against the target antigen. This research provides insights into sequencing polyclonal IgG antibodies while highlighting the effectiveness and potential of our approach in generating recombinant antibodies with robust binding affinity and neutralization capabilities. Our proposed approach is an advancement in characterizing the IgG response by directly investigating the circulating pool of IgG without relying exclusively on the B-cell repertoire or population. This is crucial as the B-cell analysis may not accurately represent the circulating antibodies. Interestingly, a large proportion (80 to 90%) of the human antibody sequences generated against SARS-CoV-2 in the literature have been derived solely from B-cell analysis. Therefore, the ability to offer a different perspective is crucial in gaining a comprehensive understanding of the IgG response. Significance StatementWe investigate human IgG targeting the receptor binding domain using de novo proteomics. The peripheral B-cell repertoire may not adequately cover all the circulating IgG for human IgG sequencing. Our approach overcomes this limitation by using a de novo protein sequencing on top of standard proteomics. We obtained distinct de novo sequences, showcasing our methods potential. The recombinant proteins we generate possess traits comparable to or surpassing the naturally occurring polyclonal antibodies (pAbs). This study highlights similarities and differences between IgG populations in blood and circulating B-cells, which is crucial for future biologics development.

immunology↗