Anreiter, I. | Search bioRxiv

bioRxiv2026

A novel High-Throughput Ligase-Independent Mapping method to detect Viral Integration Sites

Integrated DNA elements are central to virology, functional genomics, and gene therapy, but current insertion-site mapping methods often rely on restriction digestion, ligation, or complex sequencing workflows that introduce bias and limit recovery. Here, we present Terminal Mapping, a high-throughput method that identifies host-insert junctions without restriction enzymes or DNA ligation. The workflow combines linear amplification from a known terminal sequence, enrichment of single-stranded products, terminal transferase-mediated poly-A tailing, and PCR amplification for Illumina or Oxford Nanopore sequencing. Applied to Jurkat T cells transduced with HIV-1- and SIVmac251-derived vectors, Terminal Mapping recovered more HIV-1 insertion sites than inverse PCR, reproduced known integration biases, and showed improved robustness with long-read sequencing. It revealed shared but quantitatively distinct HIV-1 and SIVmac251 hotspots, as well as substantial differences between Jurkat cell sources. Comparison of 5' and 3' LTR-derived reads also provided an internal control for unintegrated viral DNA. Terminal Mapping therefore offers a rapid and flexible platform for profiling integrated genetic elements across vectors and cellular contexts.

genomics↗

bioRxiv2025

Methylphenidate exposure alters brain gene expression and induces transgenerational DNA methylation changes in Poecilia reticulata guppies

Chronic exposure to stimulants is known to affect behavioral phenotypes and epigenetic profiles intergenerationally. We have previously shown that chronic methylphenidate hydrochloride (MPH) exposure in male guppies (Poecilia reticulata) induces persistent, paternally transmitted behavioral changes across multiple unexposed generations. Here, we investigated the underlying epigenetic signatures of this transgenerational behavioral inheritance. Building on our previous study, which used composite behavioral scores, we investigates the transgenerational inheritance patters of individual behaviors and found a robust, male-lineage-dependent increase in swimming across unexposed G2-G4 female offspring. Our molecular analysis showed that that chronic MPH exposure in G1 males alters brain gene expression, with 76 differentially expressed genes including genes with developmental and locomotory functions, and an over-representation of long non-coding RNAs. Furthermore, we found that unexposed G4 descendants from MPH-treated lineages exhibit widespread changes in brain DNA methylation, including genes related to developmental and swimming behavior. Interestingly, we found one lnRNA, LOC103476631, as both differentially expressed in G1 males and differentially methylated in G4 fish, providing a compelling candidate for ncRNA-directed DNA methylation as a mechanism for the observed transgenerational epigenetic inheritance of swimming behavior. Author SummaryDrugs used to treat attention-deficit/hyperactivity disorder, such as methylphenidate, are widely prescribed to children and adolescents during key stages of brain development. We previously showed that exposing male guppies to a low, chronic dose of methylphenidate changed anxiety-like behavior not only in the treated fish but also in several generations of their unexposed descendants. In this study, we asked how such long-lasting behavioral effects might be recorded in the brain. We first confirmed that a simple measure of swimming behavior remains altered in descendants of treated males across multiple generations. We then examined brains from the directly exposed fathers and from great-grand-offspring that were never exposed to the drug. In fathers, we found changes in the activity of genes, including many long non-coding RNAs, a class of genes that has been linked to transgenerational inheritance. In great-grand-offspring, we found significantly altered DNA methylation profiles, an epigenetic mark that has also been associated with transgenerational inheritance. Together, our findings suggest that developmental exposure to a commonly used stimulant can leave a stable molecular memory in the brain that persists across generations.

genetics↗

bioRxiv2022

Glial control of sphingolipid levels sculpts diurnal remodeling of sleep circuitry

Structural plasticity in the brain often necessitates dramatic remodeling of neuronal processes and attendant reorganization of the cytoskeleton and membranes. While cytoskeletal restructuring has been studied extensively, how lipids might orchestrate structural plasticity remains unclear. We show that specific glial cells in Drosophila produce Glucocerebrosidase (GBA) to locally catabolize sphingolipids. Sphingolipid accumulation drives lysosomal dysfunction, causing gba1b mutants to harbor protein aggregates that cycle across circadian time and are regulated by neural activity, the circadian clock, and sleep. While the vast majority of membrane lipids are stable across the day, a specific subset, highly enriched in sphingolipids, cycles daily in a gba1b-dependant fashion. In parallel, circadian clock neurons remodel their neurites, growing and shrinking across the day to shape circadian behavior. Remarkably, this neuronal remodeling relies on a cycle of temporally offset sphingolipid biosynthesis and catabolism. Thus, dynamic sphingolipid regulation by glia enables diurnal circuit remodeling and proper circadian behavior.

neuroscience↗