Clustering and Annotation of T Cell Receptor Repertoires.

Immunological protection against a wide variety of pathogens is largely mediated by the diverse and dynamic T cell receptor (TCR) repertoire, a crucial component of the adaptive immune system. An encounter with infectious agents stimulates specific T cells to initiate a direct immune response to combat intruders. Hence, the TCR repertoire may conceal crucial information regarding current and past infections and might assist in the development and monitoring of vaccines. To unlock its knowledge, we describe a computational workflow involving both supervised and unsupervised machine learning techniques to analyze and annotate full TCR repertoire data. The method is explained using data from a published yellow fever virus (YFV) vaccination study in healthy individuals. The TCR repertoire of one individual is studied before and 2 weeks after vaccination, using an efficient clustering method and identification of YFV-specific TCRs.

Smart genetically-encoded biosensors for the chemical monitoring of living systems.

Genetically-encoded biosensors provide the all-optical and non-invasive visualization of dynamic biochemical events within living systems, which has allowed the discovery of profound new insights. Twenty-five years of biosensor development has steadily improved their performance and has provided us with an ever increasing biosensor repertoire. In this feature article, we present recent advances made in biosensor development and provide a perspective on the future direction of the field.

Comparative Assessment of NMR Probes for the Experimental Description of Protein Folding Pathways with High-Pressure NMR.

Multidimensional NMR intrinsically provides multiple probes that can be used for deciphering the folding pathways of proteins: NH amide and CαHα groups are strategically located on the backbone of the protein, while CH3 groups, on the side-chain of methylated residues, are involved in important stabilizing interactions in the hydrophobic core. Combined with high hydrostatic pressure, these observables provide a powerful tool to explore the conformational landscapes of proteins. In the present study, we made a comparative assessment of the NH, CαHα, and CH3 groups for analyzing the unfolding pathway of ∆+PHS Staphylococcal Nuclease. These probes yield a similar description of the folding pathway, with virtually identical thermodynamic parameters for the unfolding reaction, despite some notable differences. Thus, if partial unfolding begins at identical pressure for these observables (especially in the case of backbone probes) and concerns similar regions of the molecule, the residues involved in contact losses are not necessarily the same. In addition, an unexpected slight shift toward higher pressure was observed in the sequence of the scenario of unfolding with CαHα when compared to amide groups.

High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias.

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins-AlkB and FTO-biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.