Biology subjects

Petrova, V.

Publications and source records attributed to Petrova, V..

12 recordsLinked to original sources

bioRxiv2025

Studying cis-regulatory heterogeneity in single-cells at allelic resolution

Single-cell RNA-seq data from F1 hybrids provides a unique framework for dissecting complex regulatory phenomena, but allelic measurements are limited by technical noise. Here, we present ASPEN, a statistical method for modeling allelic mean and variance in single-cell transcriptomic data from F1 hybrids. ASPEN uses a sensitive mapping pipeline and adaptive shrinkage to distinguish allelic imbalance and variance in single cells. Through extensive simulation based on sparse droplet-based single-cell data, ASPEN demonstrates improved sensitivity and control of false discoveries compared to existing approaches. Applied to mouse brain organoids and T cells, ASPEN identifies genes with incomplete X inactivation, stochastic monoallelic expression, and significant deviations in allelic variance. This reveals reduced variance in essential cellular pathways, and increased variance in neurodevelopmental and immune-specific genes.

bioinformatics↗

bioRxiv2025

Gearing up Golden Braid assembly for plant synthetic genomics with RepTiles

We have developed RepTiles, a system for generating traces of random DNA for synthetic genomics applications. RepTiles reduced the burden associated with the manual design of long DNA from standard biological parts. We are applying RepTiles to the construction of random DNA segments that will form part of a neochromosome in Physcomitrium patens. RepTiles has a base DNA collection of 52 1.7 kb chemically synthesised Phytobricks mini-chunks. We provide a user-friendly web application that facilitates the design of DNA assemblies based on the base collection. The system generates assembly plans for generating chunks using Golden Braid. The resulting chunks can then be assembled into megachunks using Transformation Associated Recombination cloning. It thus supports the generation of ultra-long synthetic DNA from phytobricks, contributing to the vision of synthetic plant genomics from modular parts to complete genomes.

synthetic biology↗

bioRxiv2025

Lipid nanoparticle delivery of siRNA to dorsal root ganglion neurons to treat pain

Sensory neurons within the dorsal root ganglion (DRG) are the primary trigger of pain, relaying activity about noxious stimuli from the periphery to the central nervous system; however, targeting DRG neurons for pain management has remained a clinical challenge. Here, we demonstrate the use of lipid nanoparticles (LNPs) for effective intrathecal delivery of small interfering RNA (siRNA) to DRG neurons, achieving potent silencing of the transient receptor potential vanilloid 1 (TRPV1) ion channel that is predominantly expressed in nociceptor sensory neurons. This leads to a reversible interruption of heat-, capsaicin-, and inflammation-induced nociceptive conduction, as observed by behavioral outputs. Our work provides a proof-of-concept for intrathecal siRNA therapy as a novel and selective analgesic modality.

neuroscience↗

bioRxiv2024

A mouse organoid platform for modeling cerebral cortex development and cis-regulatory evolution in vitro

Natural selection has shaped the gene regulatory networks that orchestrate the development of the neocortex, leading to diverse neocortical structure and function across mammals, but the molecular and cellular mechanisms driving phenotypic changes have proven difficult to characterize. Here, we develop a reproducible protocol to generate neocortical organoids from mouse epiblast stem cells (EpiSCs) that gives rise to diverse cortical cell types, including distinct classes of excitatory neurons (pre-plate, deep-layer, and upper-layer) and glia (oligodendrocyte precursor cells, myelinating oligodendrocytes, astrocytes, ependymal cells). Cortical organoids develop with similar kinetics to the mouse cortex in vivo and begin to exhibit features of maturation in glia and neuronal cell types relatively rapidly compared to human brain organoids. Using this new protocol, we generated cortical organoids from F1 hybrid EpiSCs derived from crosses between standard laboratory mice (C57BL/6J) and four wild-derived mouse strains from distinct sub-species spanning [~]1M years of evolutionary divergence. This allowed us to comprehensively map cis-acting transcriptional regulatory variation across developing cortical cell types using scRNA-seq. We identify hundreds of genes that exhibit dynamic allelic imbalances during cortical neurogenesis, providing the first insight into the developmental mechanisms underpinning changes in cortical structure and function between mouse strains. These experimental methods and cellular resources represent a powerful new platform for investigating mechanisms of gene regulation in the developing cerebral cortex.

developmental biology↗

bioRxiv2024

Satellite glial contact enhances differentiation and maturation of human induced sensory neurons

Sensory neurons generated from induced pluripotent stem cells (iSNs) are used to model human peripheral neuropathies, however current differentiation protocols produce sensory neurons with an embryonic phenotype. Peripheral glial cells contact sensory neurons early in development and contribute to formation of the canonical pseudounipolar morphology, but these signals are not encompassed in current iSN differentiation protocols. Here, we show that terminal differentiation of iSNs in co-culture with rodent Dorsal Root Ganglion satellite glia (rSG) advances their differentiation and maturation. Co-cultured iSNs develop a pseudounipolar morphology through contact with rSGs. This transition depends on semaphorin-plexin guidance cues and on glial gap junction signaling. In addition to morphological changes, iSNs terminally differentiated in co-culture exhibit enhanced spontaneous action potential firing, more mature gene expression, and increased susceptibility to paclitaxel induced axonal degeneration. Thus, iSNs differentiated in coculture with rSGs provide a better model for investigating human peripheral neuropathies.

neuroscience↗

bioRxiv2024

Macrophages protect against sensory axon degeneration in diabetic neuropathy

Diabetic peripheral neuropathy (DPN) is a common complication of diabetes, causing sensory loss and debilitating neuropathic pain1,2. Although the onset and progression of DPN have been linked with dyslipidemia and hyperglycemia3, the contribution of inflammation in the pathogenesis of DPN has not been investigated. Here, we use a High Fat High Fructose Diet (HFHFD) to model DPN and the diabetic metabolic syndrome in mice. Diabetic mice develop persistent heat hypoalgesia after three months, but a reduction in epidermal skin innervation only manifests at 6 months. Using single-cell sequencing, we find that CCR2+ macrophages infiltrate the sciatic nerves of diabetic mice well before axonal degeneration is detectable. We show that these infiltrating macrophages share gene expression similarities with nerve crush-induced macrophages4 and express neurodegeneration-associated microglia marker genes5 although there is no axon loss or demyelination. Inhibiting this macrophage recruitment in diabetic mice by genetically or pharmacologically blocking CCR2 signaling results in a more severe heat hypoalgesia and accelerated skin denervation. These findings reveal a novel neuroprotective recruitment of macrophages into peripheral nerves of diabetic mice that delays the onset of terminal axonal degeneration, thereby reducing sensory loss. Potentiating and sustaining this early neuroprotective immune response in patients represents, therefore, a potential means to reduce or prevent DPN.

neuroscience↗

bioRxiv2022

Enhancer turnover in cancer and species evolution are associated with DNA replication timing

Enhancers are fast-evolving genomic sequences that control spatiotemporal gene expression patterns. By examining enhancer turnover across mammalian species and in multiple tissue types, we uncovered a relationship between the emergence of novel enhancers and genome organization as a function of germline DNA replication time. While enhancers are most abundant in euchromatic regions, new enhancers emerged almost twice as often in late compared to early germline replicating regions, independent of transposable elements. Using a sequence model, we demonstrate that new enhancers are enriched for mutations that alter transcription factor (TF) binding. Recently evolved enhancers appeared to be mostly neutrally evolving and enriched in eQTLs. They also show more tissue specificity than conserved enhancers, and the TFs that bind to these elements, as inferred by binding sequences, also show increased tissue-specific gene expression. We find a similar relationship with DNA replication time in cancer, suggesting that these observations may be time-invariant principles of genome evolution. Our work underscores that genome organization has a profound impact in shaping mammalian gene regulation.

genomics↗

bioRxiv2022

Towards resolution of the intron retention paradox in breast cancer

After many years of neglect in the field of alternative splicing, the importance of intron retention (IR) in cancer has come into focus following landmark discoveries of aberrant IR patterns in cancer. Many solid and liquid tumours are associated with drastic increases in IR and such patterns have been pursued as both biomarkers and therapeutic targets. Paradoxically, breast cancer (BrCa) is the only tumour type in which IR is reduced compared to adjacent normal breast tissue. In this study, we have conducted a pan-cancer analysis of IR with emphasis on BrCa and its subtypes. We explored mechanisms that could cause aberrant and pathological IR and clarified why normal breast tissue has unusually high IR. Strikingly, we found that reduced IR in BrCa can be largely attributed to normal breast tissue having the highest occurrence of IR events compared to other healthy tissues. Our analyses suggest that low numbers of IR events in breast tumours are associated with poor prognosis, particularly in the luminal B subtype. Interestingly, we found that IR frequencies negatively correlate with cell proliferation in BrCa cells, i.e. rapidly dividing tumour cells have the lowest number of IR events. Aberrant RNA binding protein (RBP) expression and changes in tissue composition are among the causes of low IR in BrCa. Our results suggest that IR should be considered for therapeutic manipulation in BrCa patients with aberrantly low IR levels and that further work is needed to understand the cause and impact of high IR in other tumour types.

cancer biology↗

bioRxiv2021

Integrin-driven Axon Regeneration in the Spinal Cord Activates a Distinctive CNS Regeneration Program

The peripheral branch of sensory dorsal root ganglion (DRG) neurons regenerates readily after injury unlike their central branch in the spinal cord. However extensive regeneration and reconnection of sensory axons in the spinal cord can be driven by the expression of 9 integrin and its activator kindlin- 1(9k1), which enable axons to interact with tenascin-C. To elucidate the mechanisms and downstream pathways affected by activated integrin expression and central regeneration, we conducted transcriptomic analyses of DRG sensory neurons transduced with 9k1, and controls, with and without axotomy of the central branch. Expression of 9k1 without the central axotomy led to upregulation of a known PNS regeneration program, including many genes associated with peripheral nerve regeneration. Coupling 9k1 treatment with dorsal root axotomy led to extensive central axonal regeneration and caused expression of a distinctive CNS regeneration program, including genes associated with ubiquitination, autophagy, endoplasmic reticulum, trafficking, and signalling. Pharmacological inhibition of these processes blocked the regeneration of axons from DRGs and human iPS-derived sensory neurons, validating their causal contributions. This CNS regeneration- associated program showed little correlation with either embryonic development or PNS regeneration programs. Potential transcriptional drivers of this CNS program coupled to regeneration include Mef2a, Runx3, E2f4, Tfeb, Yy1. Signalling from integrins primes sensory neurons for regeneration, but their axon growth in the CNS is associated with a distinctive program that differs from that involved in PNS regeneration.

neuroscience↗

bioRxiv2021

Chromatin accessibility regulates intron retention in a cell type-specific manner

Dynamic intron retention (IR) in vertebrate cells is of widespread biological importance. Aberrant IR is associated with numerous human diseases including cancer. Despite consistent reports demonstrating intrinsic sequence features that predispose introns to become retained, conflicting findings about cell type-specific IR regulation demand a systematic analysis in a controlled experimental setting. We integrated matched transcriptomics and epigenetics data (including DNA methylation, nucleosome occupancy, histone modifications) from primary human myeloid and lymphoid cells. Using machine learning we trained two complementary models to determine the role of epigenetic factors in the regulation of IR. We show that increased chromatin accessibility contributes substantially to the retention of introns in a cell-specific manner. We also confirm that intrinsic characteristics of introns are key for them to evade splicing. With mounting reports linking pathogenic alterations to RNA processing, our findings may have profound implications for the design of therapeutic approaches targeting aberrant splicing.

bioinformatics↗

bioRxiv2020

Protrudin functions as a scaffold in the endoplasmic reticulum to support axon regeneration in the adult central nervous system

Adult mammalian central nervous system axons have intrinsically poor regenerative capacity, so axonal injury has permanent consequences. One approach to enhancing regeneration is to increase the axonal supply of growth molecules and organelles. We achieved this by expressing the adaptor molecule Protrudin which is normally found at low levels in non-regenerative neurons. Elevated Protrudin expression enabled robust central nervous system regeneration both in vitro in primary cortical neurons and in vivo in the injured adult optic nerve. Protrudin overexpression facilitated the accumulation of endoplasmic reticulum, integrins and Rab11 endosomes in the distal axon, whilst removing Protrudins endoplasmic reticulum localization, kinesin-binding or phosphoinositide-binding properties abrogated the regenerative effects. These results demonstrate that Protrudin promotes regeneration by functioning as a scaffold to link axonal organelles, motors and membranes, establishing important roles for these cellular components in mediating regeneration in the adult central nervous system.

neuroscience↗

bioRxiv2019

Distinct microbial and immune niches of the human colon

Gastrointestinal microbiota and immune cells interact closely and display regional specificity, but little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterisation of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady-state. We describe distinct T helper cell activation and migration profiles along the colon and characterise the transcriptional adaptation trajectory of T regulatory cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from caecum to sigmoid colon, and link this to the increasing number of reactive bacterial species.

immunology↗