Biology subjects

Vincent, E.

Publications and source records attributed to Vincent, E..

6 recordsLinked to original sources

bioRxiv2026

Directed evolution of a thermostable family-A DNA polymerase into a reverse transcriptase for 2'-modified RNA

RNA-based therapeutics increasingly rely on nuclease-resistant xeno-nucleic acids (XNAs), but engineering polymerases capable of reading and amplifying these non-canonical substrates remains a major challenge. Natural DNA polymerases cannot process C2' sugar modifications, and the complete structural determinants governing this limitation remain poorly understood, limiting rational design strategies. In this study, we apply directed evolution methods to Thermus thermophilus DNA polymerase using a microfluidic implementation of the Compartmentalized Self-Replication (CSR). We identify an engineered variant, termed E5, capable of reverse transcribing C2'-modified RNA, a previously inaccessible activity for this enzyme. Next-generation sequencing allow us to track evolutionary trajectories across nine rounds of evolution. Through an integrated analysis combining statistical methods, protein language models, and crystallographic data, we reveal unknown epistatic networks controlling polymerase function. This large-scale experimental dataset (>196,000 thermostable enzyme variants) provides a robust basis for predicting sequence-to-function relationships, accelerating development of enzymes for aptamer discovery and synthetic biology applications.

synthetic biology↗

bioRxiv2026

Polymerase Evolution Enables Access to Glycosylated Xeno-Nucleic Acids with Expanded Chemical Functionality

Chemical modifications expand the functional repertoire of nucleic acids, but the extent to which complex glycans can be integrated into genetic polymers remains largely unexplored. Glycosylated nucleic acids, including glycoRNAs and bacteriophage genomes, are emerging as key mediators of host-pathogen interactions, yet their synthetic accessibility is severely limited by the inability of polymerases to process substrates bearing simultaneous base and backbone modifications. Here we report the directed evolution of a family B DNA polymerase that enables the synthesis of glycosylated xeno-nucleic acids (XNAs). Using fluorescence-activated droplet sorting, we identified an engineered polymerase, termed G2, that efficiently incorporates nucleotides bearing mono- to trisaccharide base-appendages together with C2 substitutions, yielding XNAs containing up to 30% glycosylated bases. Deep sequencing of >93,000 chimeras reveals that this activity arises from epistatic networks concentrated within catalytic domains. These results establish a route to glycan-encoded nucleic acids and advance polymerase engineering for chemically expanded therapeutics.

synthetic biology↗

bioRxiv2021

Ret loss-of-function decreases enteric neural crest progenitor proliferation and restricts developmental fate potential during enteric nervous system development

The receptor tyrosine kinase gene RET plays a critical role in the fate specification of enteric neural crest-derived cells (ENCDCs) during enteric nervous system (ENS) development. Pathogenic RET loss of function (LoF) alleles are associated with Hirschsprung disease (HSCR), which is marked by aganglionosis of the gastrointestinal (GI) tract. ENCDCs invade the developing GI tract, proliferate, migrate caudally, and differentiate into all of the major ENS cell types. Although the major phenotypic consequences and the underlying transcriptional changes from Ret LoF in the developing ENS have been described, its cell type and state-specific effects are unknown. Consequently, we performed single- cell RNA sequencing (scRNA-seq) on an enriched population of ENCDCs isolated from the developing GI tract of Ret null heterozygous and homozygous mouse embryos at embryonic day (E)12.5 and E14.5. We demonstrate four significant findings: (1) Ret-expressing ENCDCs are a heterogeneous population composed of ENS progenitors as well as glial and neuronal committed cells; (2) neurons committed to a predominantly inhibitory motor neuron developmental trajectory are not produced under Ret LoF, leaving behind a mostly excitatory motor neuron developmental program; (3) HSCR-associated and Ret gene regulatory network genes exhibit distinct expression patterns across Ret-expressing ENCDC with their expression impacted by Ret LoF; and (4) Ret deficiency leads to precocious differentiation and reduction in the number of proliferating ENS precursors. Our results support a model in which Ret contributes to multiple distinct cellular phenotypes associated with the proper development of the ENS, including the specification of inhibitory neuron subtypes, cell cycle dynamics of ENS progenitors, and the developmental timing of neuronal and glial commitment. Summary StatementRet LoF affects proper development of the mouse ENS through multiple distinct cellular phenotypes including restriction of neuronal fate potential, disruption of ENCDC migration, and modulation of progenitor proliferation rate.

developmental biology↗

bioRxiv2021

Prenatal immune stress induces a prolonged blunting of microglia activation that impacts striatal connectivity

Recent studies suggested that microglia, the primary brain immune cells, can affect circuit connectivity and neuronal function1-3. Microglia infiltrate the neuroepithelium early in embryonic development and are maintained in the brain throughout adulthood4,5. Several maternal environmental factors, such as aberrant microbiome, immune activation, and poor nutrition, can influence prenatal brain development6-8. Nevertheless, it is unknown how changes in the prenatal environment instruct the developmental trajectory of infiltrating microglia, which in turn affect brain development and function. Here we show that after maternal immune activation (MIA) microglia from the offspring have a long-lived decrease in immune reactivity (blunting) across the developmental trajectory. The blunted immune response was concomitant with changes in the chromatin accessibility and reduced transcription factor occupancy of the open chromatin. Single cell RNA sequencing revealed that MIA does not induce a distinct subpopulation but rather decreases the contribution to inflammatory microglia states. Prenatal replacement of MIA microglia with physiological infiltration of naive microglia ameliorated the immune blunting and restored a decrease in presynaptic vesicle release probability onto dopamine receptor type-two medium spiny neurons, indicating that aberrantly formed microglia due to an adverse prenatal environment impacts the long-term microglia reactivity and proper striatal circuit development.

neuroscience↗

bioRxiv2020

Neural crest-derived neurons are replaced by a newly identified mesodermal lineage in the post-natal and aging enteric nervous system

The enteric nervous system (ENS), a collection of neural cells contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the neural crest and remains largely unchanged thereafter. Here, we show that the lineage composition of maturing ENS changes with time, with a decline in the canonical lineage of neural-crest derived neurons and their replacement by a newly identified lineage of mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility which can be reversed by GDNF supplementation. Transcriptomic analyses of human gut tissues show reduced GDNF-RET signaling in patients with intestinal dysmotility which is associated with reduction in neural crest-derived neuronal markers and concomitant increase in transcriptional patterns specific to mesoderm-derived neurons. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS.

neuroscience↗

bioRxiv2018

Hypothesis-free analysis of deep vein thrombosis aetiology: a Mendelian randomization study

BackgroundDeep vein thrombosis (DVT) is the formation of a thrombus/clot in the deep veins, when part of this clot breaks off it can travel to the lungs, resulting in pulmonary embolism. These two conditions together are known as venous thromboembolism (VTE), a leading cause of death and disability worldwide. Despite the prevalence of VTE, we do not fully understand what causes it and it is often overlooked as a major public health problem. Confirming and identifying risk factors associated with DVT is likely to lead to a reduction in the incidence, morbidity and mortality of VTE especially where these risk factors are modifiable. We can do this, by exploiting the availability of summary genetic data from genome-wide association studies (GWAS) of numerous phenotypes, including DVT, which permits hypothesis-free causal inference.\n\nObjectivesTo identify novel risk factors for DVT and to assess the causality of factors previously shown to be associated with DVT.\n\nMethodsTwo-sample Mendelian randomization (MR) was performed using summarised genetic data. Inverse variance weighted (IVW) estimates were calculated and validated by additional methods more robust to horizontal pleiotropy (MR Egger, simple mode, weighted mode, and weighted median). Bidirectional and heterogeneity sensitivity analyses were performed to further evaluate our findings.\n\nResultsForty-seven exposures passed an exposure-exposure correlation-adjusted Bonferroni P-value threshold (5.43E-05). These included previously hypothesised risk factors for DVT (e.g. body mass index, varicose veins, height, hyperthyroidism) and novel associations (e.g. prospective memory, basal metabolic rate).\n\nConclusionOur analyses confirmed causal associations of risk factors previously associated with DVT and highlighted several novel risk factors for the disease. Our study demonstrates the utility of using a hypothesis free Mendelian randomization approach for the identification of novel disease risk factors.

epidemiology↗