Biology subjects

Kashani, M.

Publications and source records attributed to Kashani, M..

2 recordsLinked to original sources

bioRxiv2024

On the same side: The immune regulatory protein Vista and its ligands interact in cis

VISTA, an essential immune checkpoint regulatory protein, regulates peripheral T-cell quiescence and tolerance. Despite its potential as a target for anti-tumor and autoimmune disease therapies, uncertainty regarding VISTAs binding mode and membrane orientation has hindered these developments. Contrary to the prevailing paradigm, we found using cell aggregation assays that VISTA cannot interact with its ligands in trans (between cells). Using MST and flow cytometry, we showed that soluble VISTA binds to its ligands, suggesting that VISTAs membrane orientation restricts trans interactions. In contrast, split luciferase complementation assays showed that VISTA interacts with its ligands in cis. We propose that a disulfide bond bends VISTAs Ig domain towards the membrane in an orientation that prevents trans while enabling cis interactions. Co-expression data analysis from the cancer genome atlas showed a strong correlation between VISTA and its ligand, PSGL-1, consistent with our in-vitro cis interaction data. Our findings reveal VISTAs binding mechanism and suggest an intrinsic inhibition signaling pathway independent of additional cells. Importantly, our experimental framework provides a platform for identifying novel VISTA-targeted therapeutics.

immunology↗

bioRxiv2024

Amplified DNA Heterogeneity Assessment with Oxford Nanopore Sequencing Applied to Cell Free Expression Templates

In this work, Oxford Nanopore sequencing is tested as an accessible method for quantifying heterogeneity of amplified DNA. This method enables rapid quantification of deletions, insertions, and substitutions, the probability of each mutation error, and their locations in the replicated sequences. Amplification techniques tested were conventional polymerase chain reaction (PCR) with varying levels of polymerase fidelity (OneTaq, Phusion, and Q5) as well as rolling circle amplification (RCA) with Phi29 polymerase. Plasmid amplification using bacteria was also assessed. By analyzing the distribution of errors in a large set of sequences for each sample, we examined the heterogeneity and mode of errors in each sample. This analysis revealed that Q5 and Phusion polymerases exhibited the lowest error rates observed in the amplified DNA. As a secondary validation, we analyzed the emission spectra of sfGFP fluorescent proteins synthesized with amplified DNA using cell free expression. Error-prone polymerase chain reactions confirmed the dependency of reporter protein emission spectra peak broadness to DNA error rates. The presented nanopore sequencing methods serve as a roadmap to quantify the accuracy of other gene amplification techniques, as they are discovered, enabling more homogenous cell-free expression of desired proteins.

bioinformatics↗