Biology subjects

Hesselson, D.

Publications and source records attributed to Hesselson, D..

9 recordsLinked to original sources

bioRxiv2025

Examining Selection Dynamics and Limitations in Multi-round Protein Selection of High Diversity Libraries

Proteins and peptides underpin essential biological functions and technological applications, from targeting disease-relevant interactions to providing broad enzymatic activities. However, engineering molecules with desired properties remains difficult, owing to complex sequence-structure-function relationships and the lack of data on specific systems. Experimental selection strategies, including directed evolution, phage display, and mRNA display, address this challenge by leveraging high diversity libraries and iterative enrichment under defined selection pressures. This allows for the identification of candidates without requiring extensive prior knowledge, and can generate extensive datasets for use in machine learning. While many selection systems exist, comparisons across different selection approaches are hindered by the lack of a unifying analytical framework. Here, we present a set of broadly applicable analyses for assessing selection dynamics in multi-round or multi-condition experiments, ranging from position level analysis of sequence properties to full sequence space mappings through protein language model embeddings. Using the toolset to analyze a variety of different datasets in parallel, we explore the potential effects of diversity, coverage, and reproducibility, offering generalizable insights to guide experimental design, interpretation, and troubleshooting across protein and peptide discovery platforms.

molecular biology↗

bioRxiv2024

A chimeric viral platform for directed evolution in mammalian cells

Directed evolution (DE) is a process of mutation and artificial selection to breed biomolecules with new or improved activity1,2. DE platforms are primarily prokaryotic or yeast-based, and stable mutagenic mammalian systems have been challenging to establish and apply3. To this end, we developed PROTein Evolution Using Selection (PROTEUS), a new platform that uses chimeric virus-like vesicles (VLVs) to enable extended mammalian DE campaigns without loss of system integrity. This platform is stable and can generate sufficient diversity for DE in mammalian systems. Using PROTEUS, we altered the doxycycline responsiveness of tetracycline-controlled transactivators, generating a more sensitive TetON-4G tool for gene regulation. PROTEUS is also compatible with intracellular nanobody evolution, and we use it to design a DNA damage-responsive anti-p53 nanobody. Overall, PROTEUS is an efficient and stable platform to direct evolution of biomolecules within mammalian cells.

molecular biology↗

bioRxiv2023

P-Selectin promotes SARS-CoV-2 interactions with platelets and the endothelium

COVID-19 causes a clinical spectrum of acute and chronic illness and host / virus interactions are not completely understood1,2. To identify host factors that can influence SARS-CoV-2 infection, we screened the human genome for genes that, when upregulated, alter the outcome of authentic SARS-CoV-2 infection. From this, we identify 34 new genes that can alter the course of infection, including the innate immune receptor P-selectin, which we show is a novel SARS-CoV-2 spike receptor. At the cellular level expression of P-selectin does not confer tropism for SARS-CoV-2, instead it acts to suppress infection. More broadly, P-selectin can also promote binding to SARS-CoV-2 variants, SARS-CoV-1 and MERS, acting as a general spike receptor for highly pathogenic coronaviruses. P-selectin is expressed on platelets and endothelium3, and we confirm SARS-CoV-2 spike interactions with these cells are P-selectin-dependent and can occur under shear flow conditions. In vivo, authentic SARS-CoV-2 uses P-selectin to home to airway capillary beds where the virus interacts with the endothelium and platelets, and blocking this interaction can clear vascular-associated SARS-CoV-2 from the lung. Together we show for the first time that coronaviruses can use the leukocyte recruitment system to control tissue localization, and this fundamental insight may help us understand and control highly pathogenic coronavirus disease progression.

immunology↗

bioRxiv2022

The VEGAS platform is not suitable for mammalian directed evolution

Directed evolution uses cycles of gene diversification and selection to generate proteins with novel properties. While traditionally directed evolution is performed in prokaryotic systems, recently a mammalian directed evolution system (viral evolution of genetically actuating sequences, or "VEGAS") has been described. Here we report that the VEGAS system has major limitations precluding its use for directed evolution. The primary technical issue with the VEGAS system is an immediate contamination with "cheater" particles that bypass directed evolution circuits. By sequencing we find these cheater particles contain Sindbis structural genes instead of the intended directed evolution target transgene. These cheaters outcompete the VEGAS transgenes within 2 rounds of transduction but cannot themselves activate synthetic circuits that drive expression of Sindbis structural genes, preventing directed evolution campaigns. Similar results have been obtained in independent labs. Taken together, the VEGAS system does not work as described and, without significant redesign to suppress cheaters, cannot be used for mammalian directed evolution campaigns.

synthetic biology↗

bioRxiv2021

LRRC15 suppresses SARS-CoV-2 infection and controls collagen production

Although ACE2 is the primary receptor for SARS-CoV-2 infection, a systematic assessment of host factors that regulate binding to SARS-CoV-2 spike protein has not been described. Here we use whole genome CRISPR activation to identify host factors controlling cellular interactions with SARS-CoV-2. Our top hit was a TLR-related cell surface receptor called leucine-rich repeat-containing protein 15 (LRRC15). LRRC15 expression was sufficient to promote SARS-CoV-2 Spike binding where they form a cell surface complex. LRRC15 mRNA is expressed in human collagen-producing lung myofibroblasts and LRRC15 protein is induced in severe COVID-19 infection where it can be found lining the airways. Mechanistically, LRRC15 does not itself support SARS-CoV-2 infection, but fibroblasts expressing LRRC15 can suppress both pseudotyped and authentic SARS-CoV-2 infection in trans. Moreover, LRRC15 expression in fibroblasts suppresses collagen production and promotes expression of IFIT, OAS, and MX-family antiviral factors. Overall, LRRC15 is a novel SARS-CoV-2 spike-binding receptor that can help control viral load and regulate antiviral and antifibrotic transcriptional programs in the context of COVID-19 infection.

cell biology↗

bioRxiv2020

Characterization of cell-cell communication in COVID-19 patients

COVID-19 patients display a wide range of disease severity, ranging from asymptomatic to critical symptoms with high mortality risk. Our ability to understand the interaction of SARS-CoV-2 infected cells within the lung, and of protective or dysfunctional immune responses to the virus, is critical to effectively treat these patients. Currently, our understanding of cell-cell interactions across different disease states, and how such interactions may drive pathogenic outcomes, is incomplete. Here, we developed a generalizable workflow for identifying cells that are differentially interacting across COVID-19 patients with distinct disease outcomes and use it to examine five public single-cell RNA-seq datasets with a total of 85 individual samples. By characterizing the cell-cell interaction patterns across epithelial and immune cells in lung tissues for patients with varying disease severity, we illustrate diverse communication patterns across individuals, and discover heterogeneous communication patterns among moderate and severe patients. We further illustrate patterns derived from cell-cell interactions are potential signatures for discriminating between moderate and severe patients.

systems biology↗

bioRxiv2020

Developmental accumulation of gene body and transposon non-CpG methylation in the zebrafish brain

DNA methylation predominantly occurs at CG dinucleotides in vertebrate genomes; however, non-CG methylation (mCH) is also detectable in vertebrate tissues, most notably in the nervous system. In mammals it is well established that mCH is targeted to CAC trinucleotides by DNMT3A during nervous system development where it is enriched in gene bodies and associated with transcriptional repression. However, the conservation of developmental mCH accumulation and its deposition by DNMT3A is largely unexplored and has yet to be functionally demonstrated in other vertebrates. In this study, by analyzing DNA methylomes and transcriptomes of zebrafish brains, we identified enrichment of mCH at CAC trinucleotides (mCAC) at defined transposon motifs as well as in developmentally downregulated genes associated with developmental and neural functions. We further generated and analyzed DNA methylomes and transcriptomes of developing zebrafish larvae and demonstrated that, like in mammals, mCH accumulates during post-embryonic brain development. Finally, by employing CRISPR/Cas9 technology, we unraveled a conserved role for Dnmt3a enzymes in developmental mCAC deposition. Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development.

genomics↗

bioRxiv2020

Low Repeatability of Aversive Learning in Zebrafish (Danio rerio)

Aversive learning - avoiding certain situations based on negative experiences - can profoundly increase fitness in animal species. The extent to which this cognitive mechanism could evolve depends upon individual differences in aversive learning being stable through time, and heritable across generations, yet no published study has quantified the stability of individual differences in aversive learning using the repeatability statistic, R (also known as the intra-class correlation). We assessed the repeatability of aversive learning by conditioning approximately 100 zebrafish (Danio rerio) to avoid a colour cue associated with a mild electric shock. Across eight different colour conditions zebrafish did not show consistent individual differences in aversive learning (R = 0.04). Within conditions, when zebrafish were twice conditioned to the same colour, blue conditioning was more repeatable than green conditioning (R = 0.15 and R = 0.02). In contrast to the low repeatability estimates for aversive learning, zebrafish showed moderately consistent individual differences in colour preference during the baseline period (i.e. prior to aversive conditioning; R ~ 0.45). Overall, aversive learning responses of zebrafish were weak and variable (difference in time spent near the aversive cue <6 seconds per minute), but individual differences in learning ability did not explain substantial variability. We speculate that either the effect of aversive learning was too weak to quantify consistent individual differences, or directional selection might have eroded additive genetic variance. Finally, we discuss how confounded repeatability assays and publication bias could have inflated average estimates of repeatability in animal behaviour publications. Summary StatementZebrafish exhibit low repeatability (intra-class correlation) in an aversive learning assay possibly due to past selection pressure exhausting genetic variance in this learning trait.

animal behavior and cognition↗

bioRxiv2020

A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~400 non-synonymous coding variants which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilises the new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. Similar to A20-deficient mice, A20-deficient zebrafish are hyper-responsive to inflammatory triggers and exhibit spontaneous early lethality. While ectopic addition of human A20 rescued A20-null zebrafish from lethality, missense mutations at two conserved A20 residues, S381A and C243Y reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a C243Y mutation that associates with human autoimmune disease. These data reveal an evolutionarily conserved role for A20, but also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant TNFAIP3 gene variants. This approach could be utilised to investigate genetic variation for other conserved genes.

genetics↗